Introduction

vCells interact with extracellular material to form

defined tissues.

vThese interactions are crucial to the formation of

Epithelial tissue and Connective tissue, which are
key to various cellular activities.

1
 Introduction: Overview of cell organization into tissues

An overview of how cells are organized into tissues and how they interact with one another
and with their extracellular environment. Dermis: Deeper layer of skin which consists of a
type of connective tissues 2
 The Extracellular Space

Electron micrograph: apical

Basal surface of an ectodermal surface of an epithelial cell from
cell of an early chick embryo the lining of the intestine
vThe Glycocalyx (GC) (cell coat) is formed from carbohydrate projections from the
plasma membrane & is very prominent eg in digestive tract .
vGlycocalyx mediates cell-cell & cell-substratum interactions & provide mechanical
protection to cells, serves as a barrier to particles moving towards the plasma
membrane & bind important regulatory factors that act on the cell surface.
vGC and outer BM (Basement Membrane) interact with one another
The glycocalyx is a Glycoprotein-Polysaccharide covering that surrounds the cell membranes
of some bacteria, epithelia and other cells. 3
 The Extracellular Space

Scanning electron micrograph of a colony of ECM of a single chondrocyte made visible

cartilage cells & secreted extracellular materials by adding a suspension of red blood cells

vThe Extra Cellular Matrix (ECM) is an organized network beyond the plasma
membrane. It often plays a regulatory role in determining shape and activities of the cell.
The thickness of ECM is evident (Upper right fig) by the clear space (arrowhead) that is
not penetrated by red blood cells (RBC); Chondrocytes: Cartilage cells

4
 The Extracellular Space: The basement membrane

Glomerular Basement Membrane (GBM);

Scanning electron micrograph Capillary Lumen (CL); podocyte of tubule,
of human skin collagen staining

v ECM: contains the basement membrane (basal lamina), a continuous

sheet (50-200nM thick) that underlies epithelial tissue and surrounds blood
vessels.
Ø Helps maintain cell attachment.
Ø Serves as substratum for cell migration.
Ø Forms a barrier to macromolecules.
5
 The Extracellular Space: Organization of the ECM

Organized network of extracellular materials outside plasma membrane, provides support

and determines shape and activity of cell. Unlike most proteins inside the cell which are
compact, globular, ECM proteins (Collagens etc) are extended and fibrous in nature
6
 The Extracellular Space: The structure of Collagen
v Collagens – fibrous
glycoproteins found only in Collagen molecule:
the ECM. triple helix of three
ØCollagen is the most helical alpha chains
abundant protein in the
human body (25% of all Collagen I molecules
body proteins). become aligned in
Ø28 types in human: staggered rows
Fibrillar (I, II, III, V, XI)
and NonFibrillar (Rest);
(90%~Fibrillar Type I) EM of human
collagen fibrils after
ØProvide high tensile
metal shadowing
strength.
ØEach collagen is restricted
Atomic force
to particular locations in the
micrograph
body.
of a collagen
ØAll collagens are a trimer fibril surface
of polypeptide chains
wound around each other.
7
 The Extracellular Space: Collagens

Corneal stroma:
vCollagens
layers of collagen
Ø Provide the insoluble fibrils of uniform
framework that determines
diameter and
mechanical properties of the
matrix. spacing arranged
at right angles
Ø Abnormalities in collagen
formation lead to serious
disorders.
vNot all collagens form fibrils. Type IV collagen
vCollagen type IV is non- network: basement
fibrillar, and is restricted to the membrane from
basement membrane. human amniotic
tissue shows an
irregular, polygonal
lattice

8
 The Extracellular Space: Collgens

vCollagen-based diseases
v Fibril collagens
Ø Type I (Osteogenesis Corneal stroma:
Imperfect : Fragile bones) layers of collagen
Ø Type II (Dwarfism) fibrils of uniform
Ø Ehler-Danlos diameter and
Syndrome:Hyperflexibility spacing arranged
Ø Fibrosis: Overproduction of at right angles
collagen in lung (Pulmonary
Fibrosis) or liver (Cirrhosis)
v Non-fibrillar (Type IV)
Type IV collagen
Ø Alpert Syndrome:Kidney network: basement
disease of the glomerular
membrane from
basement membrane
human amniotic
tissue shows an
irregular, polygonal
lattice

9
 The Extracellular Space
Structures of Proteoglycan Complex

Schematic
representations of a
single proteoglycan,
repeating
disaccharide structure
of GAGs, and linkage
to Hyaluronic acid to
form a giant complex

v Proteoglycans – Protein-Polysaccharide Complex, with a core

protein attached to GlycosAminoGlycans (GAGs).
Ø Have a repeating disaccharide structure.
Ø Negatively charged GAGs attract lots of cations which in turn attract water
forming a porous, hydrated gel. 10
 Proteoglycan Complex
Hyaluronic acid chain

Protein chain

Chondroitin sulphate chain

(Common name: GAG or
Glycosamino Glycan)

Keratan / Heparan Sulphate chain

(Common name: GAG or Glycosamino
Glycan)

11
GlycosAminoGlycans (GAG) / Mucopolysaccharides
v Long unbranched polysaccharides containing a repeating unit of a disaccharide.

v The repeating unit (except for keratin) consists of an amino sugar (N-Acetyl
glucosamine or N-Acetyl galactosamine along with a uronic sugar (with COOH
group).

v Glycosaminoglycans are highly polar and attract water. They are therefore useful
to the body as a lubricant or as a shock absorber.

12
 The Extracellular Space
Structure of a proteoglycan complex
vProteoglycans
Ø Cross-linked into large matrix by
hyaluronic acid (nonsulfated GAG)
Ø Resist crushing forces; cushion cells
Ø Provide binding sites for growth
hormones to protect from proteases,
regulate diffusion of small signaling
molecules in developing embryo
Ø Cartilage matrix contains abut 30
keratin sulfate and 100 chondroitin
Electron micrograph of a proteoglycan
sulfate chains
complex isolated from cartilage matrix.

13
The Extracellular Space
Structure of Fibronectin

vFibronectin (Fn) – a
linear array of distinct
polypeptides giving it a
modular structure.
Ø Each polypeptide is about
30 Fn modules.
Ø Fn modules are found in
other proteins too.
Ø Fn has binding sites for
other components of the
ECM.
Ø Fn guides migrating cells
during embryogenesis.

Human fibronectin molecule consists of two similar polypeptides joined by disulfide bonds.
They are organized into several larger functional units, each containing one or more binding
sites for a component of the ECM or for the surface of cells.
14
 The Extracellular Space
Fibronectin Role in Embryonic Development

Young chick embryo section treated with

fibronectin fluorescent Ab (Antibody).
Present as fibrils in the basement
membranes (dark red sites) beneath the
embryonic epithelia to provide a substratum
over which cells migrate.

The role of fibronectin in the formation Neural crest cells exclusively adhere to strips
of the embryonic salivary gland of fibronectin coating the culture dish surface

15
 The Extracellular Space
Cell migration during embryogenesis

A 10 day old mouse embryo section

shows primordial germ cells (green)
A summary of some of the cellular migrating along a tract of laminin
traffic occurring during mammalian (red) from the dorsal mesentery to the
development developing gonad.

16
 The Extracellular Space: Laminins

vLaminins – extracellular
glycoproteins consisting of
three polypeptide chains
linked by disulfide bonds.
Ø More than 15 types found
Ø Help cell migration during
development.
Ø Components of basement
membranes lining tissues.
Ø Domains for interaction
with other proteins
(proteoglycans)
A model of the basement membrane scaffold.
Basement membranes contain two network-
forming molecules, collagen IV (pink), and
laminin (light blue) that are connected by
entactin molecules (purple).

17
Laminins

18
 The Extracellular Space: ECM

vDynamic Properties of the ECM

Ø The ECM can be stretched during tension.
Ø Not static, constant remodeling by degradation and reconstruction.
Ø ECM materials degraded by Matrix Metallo-Proteinases (MMPs) (A
Family of Proteolytic Enzymes).
Ø MMPs possibly involved in tissue remodeling, embryonic cell migration,
wound healing and formation of blood vessels.
Ø Disease states associated with MMPs
q Arthritis, Tumor progression, Blood clots, and Heart attacks.
Ø MMPs regulated by TIMPs (Tissue Inhibitor of Metallo-Proteinases.

19
Interactions of Cells with Extracellular Materials (ECM)

EMs and ribbon drawings of

the extracellular domains of
an integrin (avb3) in the
“bent”/inactive and
“upright”/active
conformation. Changes
driven by divalent metal ions.

vIntegrins (Receptors) – Family of membrane proteins composed of

heterodimers with a and b subunits.
Ø Have a major role in integrating extracellular and intracellular environments.
Ø Another role is adhesion of cells to their substratum or other cells.

20
Interactions of Cells with Extracellular Materials
Model of Integrin Activation

Inside-out signaling: Talin binding to

cytoplasmic tails separates a and b
chains, inducing conformational
change that allows Integrin to bind
extracellular matrix ligands

vIntegrins – Family of membrane proteins composed of heterodimers with

a and b subunits.
Ø Have a major role in integrating extracellular and intracellular environments.
Ø Another role is adhesion of cells to their substratum or other cells.

21
 Interactions of Cells with Extracellular Materials

vIntegrins
ØLinkage between
integrins and their
ligands mediates
adhesion between cells
and their environment.
ØBinding of proteins to
integrins is facilitated by
the tripeptide segment
“Arg-Gly-Asp” (or
“RGD”)

22
 Interactions of Cells with Extracellular Materials
v “RGD” required for
platelet aggregation.
v Fibrinogen binds to
Integrin in “gluing”
platelets together Blood clots form
when platelets
v Clot Buster Drugs adhere to one
Ø RGD peptide acts as another through
competitive inhibitor to fibrinogen bridges
Fibrinogen/Integrin that bind to the
interaction. platelet-integrins
Ø “Aggrastat”
(a RGD peptide)
Ø ReoPro (anti-RGDAb)
Ab: Antibody

23
Fibrinogen: Soluble large complex glycoprotein converted to Fibrin by Thrombin enzyme
 Interactions of Cells with Extracellular Materials
Focal Adhesions (FA)
vIntegrins
Ø Cytoplasmic domains of integrins contain
binding sites for a variety of cytoplasmic
proteins.
Ø Integrins make the connection between
the ECM and the cytoskeleton.
vFocal adhesions – scattered, discrete
sites for cell adhesion to their substratum
(underlying layer/substance) in vitro.
Ø They may act as a type of sensory
structure.
Ø Are also implicated in cell locomotion. Focal adhesions are sites where cells adhere to their
substratum and send signals to the cell interior
 Interactions of Cells with Extracellular Materials
Cell Matrix or Focal Adhesions (FA): Kinetics and Forces
(“FA” is a Large Macromolecular Assembly)

Steps in the process of cell spreading. Scanning

electron micrographs showing mouse fibroblasts at
successive times during attachment and spreading on
glass coverslips. Cells were fixed after 30 minutes, 60
minutes, 2 hours, and 24 hours of attachment.

Focal adhesions generate

force that deforms collagen
substratum grid pattern

25
 Interactions of Cells with Extracellular Materials
Hemidesmosomes

Hemidesmosomes: Electron micrograph and

schematic showing binding of intermediate filaments.

vHemidesmosomes – basal attachments of epithelial cells to the

basement membrane in vivo.
Ø Contain a dense plaque with filaments consisting of keratin.
Ø Keratin filaments are linked to the ECM by membrane-spanning integrins.
 Interactions of Cells with Extracellular Materials
Hemidesmosomes

Hemidesmosomes: Electron micrograph and

schematic showing binding of intermediate filaments.

vHuman diseases:
Ø Bullous pemphigoid, Autoimmune disease (Antibodies produced against
plaque proteins).
Ø Epidermolysis bullosa, Genetic disease for Hemidesmosomal proteins.
Interaction of Cells with Other Cells
Structure of a desmosome
Electron
micrograph of
a desmosome
from newt
epidermis.

Model: molecular
architecture of a
desmosome

Desmosomes – disk-shaped adhesive junctions

between cells.
Found in a variety of tissues (areas of mechanical
stress, e.g. skin, gum, cervix).
Contain cadherins that link the two cells across a
narrow gap.
Cadherins of desmosomes have different domain
structures: desmogleins and desmocollins.

28
vHemi-desmosomes join cells to the basal
membrane (ONE cytoplasmic plug)

vDesmosomes join cells together (involve TWO

cytoplasmic plugs)

29
Interaction of Cells with Other Cells

• Cells have surface-recognition sites that maintain

organization
• Experimental demonstrations of cell-cell recognition

Light micrograph: mixed pre-cartilage Ectoderm and mesoderm from an early

cells from a chick limb and chick heart amphibian embryo re-associate after
ventricle cells re-sort based on cell type initial random dissociation
 Interaction of Cells with Other Cells
vSelectins - family of integral
membrane glycoproteins that bind to
sugars on the surface of cells.
Ø Contain a small cytoplasmic domain, a
single membrane-spanning domain,
and a large extracellular segment.
Ø Three types:
§ E-selectin - on endothelial cells.
(Endothelial leukocyte Adhesion)
§ P-selectin - on platelets and
endothelial cells.
§ (Platelet Activation dependent)
§ L-selectin - on white blood cells.
§ (Lymphocyte Endothelial Cell
interaction)

Schematic of the three selectins and their ligand

All Selectins (a) recognize and bind to a similar carbohydrate ligand at the ends of oligosaccharide chains
on glycoproteins, such as the one shown in (b). (c) The detailed structure of the carbohydrate ligand. The
terminal Fucose and Sialic acid moieties are particularly important in selectin recognition, and the N-
acetylglucosamine moiety is often sulfated.
 The Human Perspective:
The Role of Cell Adhesion in Inflammation and Metastasis

Movement of neutrophils during inflammation

vInflammation is a response to infection or injury but can be triggered inappropriately.

vInflammatory response:
Ø Recruitment of leukocytes to site of injury.
Ø Neutrophils (type of white blood cells) attach to P- and E-selectins.
Ø Neutrophils start to “roll” along wall of vessel.
The Human Perspective:
The Role of Cell Adhesion in Inflammation and Metastasis

v Cancer is the result of abnormal cell

proliferation.
v The spread of a tumor to other parts of the
body is called metastasis.
v Metastatic cells have special cell adhesion
properties:
Ø Are less adhesive.
Ø Are able to penetrate several barriers.
Ø Are able to invade normal tissues.

Steps leading to metastatic spread

 Interaction of Cells with Other Cells

vImmunoglobulin superfamily
(IgSF) – most proteins are involved
in immune functions.
Ø Most IgSF molecules mediate
interaction of lymphocytes with cells
required or immune response.
Ø However, some members mediate
adhesion between non-immune cells:
Ø VCAM (vascular cell-adhesion
molecule)
Ø NCAM (neural cell adhesion
molecule)
Ø L1 (neural development)

Cell–cell adhesion from homotypic

interactions of two L1 molecules
through Ig domains at the N-termini.
 Interaction of Cells with Other Cells

vCadherins –glycoproteins that mediate

Ca2+-dependent cell-cell adhesion.
vJoin cells of similar types to one another by
preferential binding to the same cadherin
present at the surface of the neighboring
cell
v30 known families, Commons are:
Ø E-cadherin (Epithelial)
Ø N-cadherin (Neural)
Ø P-cadherin (Placental)
v Involved in transmitting signals from the ECM
to the cytoplasm.
v Mediate many of the changes in adhesive
contacts during embryonic development by
forming Epithelial-Mesenchymal Transition
(EMT). Schematic of two adhering cells due to
v During embryogenesis, cells gain and lose interactions between cadherins projecting
specific adhesive properties. from the plasma membrane of each cell
 Gap Junctions and Plasmodesmata: Mediating
Intercellular Communication

• Gap junctions –
sites between animal Electron micrograph of a section through a gap junction
cells for intercellular perpendicular to the plane of the two adjacent membranes
communication.
• Composed entirely
of membrane
protein
connexin.
• Connexins are Schematic model
organized into a of a gap junction
complex called showing the
connexon. arrangement of
six connexin
subunits to form
a connexon
 Gap Junctions and Plasmodesmata:
Mediating Intercellular Communication

vGap junctions –
sites between animal Atomic force
cells for intercellular microscopy of the
communication. extracellular surface of
a connexon in open and
ØComposed closed conformations
entirely of
membrane protein
connexin.
Freeze-fracture replica
ØConnexins are of a gap junction
organized into a plaque showing the
complex called large numbers of
connexon. connexons and their
high concentration.
Tight Junctions:
Sealing the Extracellular Space

Freeze-fracture replica showing the E Scanning electron micrograph of the apical

face of the plasma membrane of one of surface of an epithelium showing the
the cells in a region of a tight junction. encircling nature of the tight junctions

vTight junctions (TJs) – specialized contacts between epithelial cells.

Ø Located at very apical end of the junctional complex between adjacent cells.
Ø Serve as barrier to free diffusion of water and solutes from the extracellular
compartment.
Ø Some are permeable to specific ions or solutes.
 Interaction of Cells with Other Cells
Intercellular junction complex

Diagram showing
the junctional
complex on the
lateral surfaces of
a simple columnar
epithelial cell

vAdherens Junctions and Desmosomes: Anchoring Cells to Other Cells

Ø Adherens junctions – they form “belts” near apical surface called junctional complex.
Ø Cells of an adherens junction held together by calcium-dependent linkages.
 Junctions between Cells
1 Tight junction (Claudin & other proteins)
2 Adherens Junctions (Cadherins the
transmembrane catenins bound to actins)
3 Desmosome (+Hemidesmosome) (Linked
to intermmitant filaments of keratins) via
Cadherin type proteins
4 Gap junction (6 copies of transmembrane
proteins called Connexins).
 Gap Junctions
Mediating Intercellular Communication

vA new type of communication has been

discovered – Tunneling Nanotubes.
ØIt has been observed in cells growing in
culture.
ØCells connected to one another by a thin
tubular process capable of carrying
materials between the cytoplasm of the
neighboring cells.
ØProcesses, which are only about 100 nm
in diameter, are supported by an internal
actin “skeleton.”
ØCan transmit viral particles and prions.

Scanning electron micrograph:

Tunneling nanotubes between
neuroendocrine cells
 Synopsis
vThe extracellular space extends outward from the outer surface
of the plasma membrane and contains a variety of secreted
materials that influence cell behavior.
vThe major components of extracellular matrices include collagens,
proteoglycans, and a variety of proteins, such as fibronectin, and
laminin.
vIntegrins are cell-surface receptors that mediate interactions
between cells and their substratum.
vCells attach to their substratum by means of cell-surface
specializations such as focal adhesions and hemidesmosomes.
vThe adhesion of cells to other cells is mediated by several distinct
families of integral membrane proteins, including selectins,
integrins, cadherins, and members of the immunoglobulin
superfamily (IgSF).
Synopsis

vStrong adhesions between cells are facilitated by the

formation of specialized adherans junctions and
desmosomes.
vTight junctions are specialized sites of contact that block
solutes from diffusing between the cells in an epithelium.
vGap junctions are specialized sites of communication
between adjoining cells in animals.
FROM GENES TO PROTEINS
http://youtube/erOP76_qLWA

44
FROM GENES TO PROTEINS

4
5 45
Relationship between Genes & Proteins

ØGenes store information for producing all cellular proteins.

ØEarly observation suggested a direct relationship between genes and
proteins.

ØA. Garrod studied the relationship between a specific gene, a

specific enzyme, and a metabolic condition (Alcaptonuria).

ØG. Beadle and E. Tatum formulated the “one gene–one

enzyme” hypothesis.

4
The Relationship Between Genes and Proteins
It was first demonstrated by Scottish Physician A Garrod (1908)
who noted a rare inherited disease called Alcaptonuria where
urine turns dark upon exposure to air due to lack of an enzyme in
their blood that oxidized Homogentisic acid (HA) (a compound
formed during breakdown of Phe and Tyr). As HA also called
Melanic acid accumulated and excreted in the urine it turned dark
due to oxidation by air.

Garrod had discovered the

relationship between a genetic defect,
a specific enzyme, and a specific
metabolic condition. He called such
diseases “Inborn Errors of
HA (Homogentisic acid), Metabolism.” This finding remains
Melanic acid, unnoticed for decades
Chemical Name: 2,5-Dihydroxy-
phenylacetic acid 47
Gene Directs The Production of Enzyme

Experiment with Neurospora (Bread mold) 1940

Beadle and Tatum (Caltech, USA):

Irradiated 1000 cells of Neurospora. Two of the cells

(due to mutation) was found unable to grow in minimal
medium that lacked the essential compounds known to
be synthesized by the organism. They found a genetic
mutant in Neurospora that grows in minimal medium
only when it is supplemented with a particular
coenzyme (Pantothenic acid of coenzyme-A).

48
Gene Directs The Production of Enzyme

49
Relationship Between Genes & Proteins

• Beadle and Tatum’s

hypothesis was later
modified to “one gene-one
polypeptide chain”
• Mutation in a single gene
causes a single substitution
in an amino acid sequence
of a single protein.

If a spore can’t grow in minimal medium but can with

supplemented Pantothenic acid, it concludes an enzymatic
deficiency that prevents pantothenic acid production 50
 Relationship Between Genes & Proteins
Flow of Information

• An Overview of the Flow of

Information through the Cell
• Messenger RNA (mRNA) is
an intermediate between a gene
and a polypeptide.
• Transcription is the process by
which RNA is synthesised from
a DNA template in the nucleus
• Translation is the process by
which proteins are synthesized
in the cytoplasm using
information encoded by mRNA
template.

Overview of the flow of

information in eukaryotes
51
 Mechanism By Which Specific Polypeptide Chain Is
Generated
Transcription Translation
Gene (DNA) mRNA Protein

The momentous discovery of mRNA was made in 1961 by François

Jacob and Jacques Monod of the Pasteur Institute in Paris, Sydney
Brenner of the University of Cambridge and Matthew Meselson of the
California Institute of Technology.

A messenger RNA is assembled as a complementary copy

of one of the two DNA strands that make up a gene. The
synthesis of an RNA from a DNA template is called
TRANSCRIPTION. Because its nucleotide sequence is
complementary to that of the gene from which it is
transcribed, the mRNA retains the same information as the
gene itself. 52

52
Gene (A coding segment of DNA present in Chromosome)

- Unwinding of double helix DNA

Transcription Required machinery:
- RNA Polymerase (Enzyme)
- Transcription factors

mRNA (messenger RNA) (End product)

Required machinery:
Translation - Ribosomal RNA (rRNA)
- Transfer RNA (tRNA)
- Ribosome (Enzyme)

Polypeptide/Protein (End product)

53
The three roles of RNA in protein synthesis

Messenger RNA (mRNA) is translated into protein by the joint action

of transfer RNA (tRNA) & ribosome, which is composed of numerous
proteins & two major ribosomal RNA (rRNA) molecules.
54
Relationship between Genes & Proteins: Classes of RNA

vThere are three classes of RNA in a

cell: mRNA, ribosomal RNA
(rRNA), and transfer RNA (tRNA).
vrRNA recognizes other molecules,
provide structural support, and help
ribosome to catalyze the chemical
reaction in which amino acids are
linked to one another.
vtRNAs are required to translate
information in the mRNA code into
amino acids. 2-Dimensional structure of a
Bacterial ribosomal RNA
- Extensive base-pairing between different regions of the single strand.
- The expanded section shows the base sequence of a stem and loop, including a nonstandard
base pair (G-U) and a modified nucleotide, methyl-Adenosine (mA). One of the helices is
shaded differently because it plays an important role in ribosome. 55
An Overview of Transcription & Translation in
Prokaryotic and Eukaryotic Cells

56
Transcription & Translation in
Prokaryotic Cells

57
 Transcription in Bacteria (Prokaryotic Cell)
- Bacteria (E. coli) contains a single RNA
polymerase composed of 5 subunits that form
a core enzyme. If this enzyme from bacterial
cells is added to a solution of bacterial DNA
molecules and ribonucleoside triphosphates,
the enzyme binds to the DNA and synthesizes
RNA.
- The RNA molecules thus produced are not the
same as those found within cells since the
enzyme is attached to random sites in the
DNA, sites that it would normally have ignored
in vivo.
- If, however, a purified accessory polypeptide
called “Sigma Factor” (s) is added to the
RNA polymerase before it attaches to DNA,
transcription begins at selected locations.
Attachment of s factor increases the enzyme’s
affinity for promoter sites in DNA.
58 58
 The elements of a promoter region in the DNA of the E. coli
Consensus Consensus sequence
sequence (Pribnow box)

Pribnow box

§ The regulatory sequences required for initiation of transcription are located at -35 &
-10 base pairs from the site at which transcription begins.
• The initiation site marks the boundary between + and - sides of the gene.
• Bacterial Promoters are located in the region of a DNA strand just preceding the initiation
site of RNA synthesis.
• Those portions of the DNA preceding the initiation site (toward the 3’ end of the template)
are said to be “Upstream” from that site. Those portions of the DNA succeeding it (toward
the 5’ end of the template) are said to be “Downstream” from that site.
• Two consensus sequences “TTGACA” and “TATAAT” (Pribnow box) are essential part of
a promoter site on DNA for transcription. Sigma Factor” binds to the latter

“Promoters are the sites in DNA that bind RNA polymerase” 59

Transcription & Translation in
Eukaryotic Cells

60
Transcription & RNA Processing in Eukaryotic Cells
RNA Polymerase: (Discovered in 1969 by Robert Roeder, U Washington) It
binds to DNA and incorporates nucleotides into a strand of RNA whose
sequence is complementary to one of the DNA strands (template). Eukaryotic
cells have 3 distinct transcribing enzymes in their cell nuclei. Each of
these enzymes is responsible for synthesizing a different group of RNAs

61
 The Machinery for Transcription in
Eukaryotic cells

v All eukaryotic mRNA precursors are synthesized

by RNA polymerase II, an enzyme composed of a
dozen different subunits that is remarkably
conserved from yeast to mammals.

v RNA polymerase II binds the promoter region

with the help of a number of General
Transcription Factors (GTFs) to form a Pre-
Initiation Complex (PIC).

62
A comparison of prokaryotic and eukaryotic RNA
polymerase structure
RNA polymerase II (RNAPII) one of 3 major eukaryotic
nuclear RNA polymerases (Within Red Box).

The surface structures of RNA polymerases from each of the

three domains of life: Bacteria, Archaea and and Eukaryotes 63
 Transcription: RNA Polymerase
vPromoter region = Site on DNA to which RNA polymerase binds
prior to initiation of transcription, determines which strand is
template (anti-sense)
vLarge and variable, 100 - 1000 base pairs (bp) long
vCore promoter elements:
- TATA element (Consensus sequence, TATAAA)
• Recognized by transcription factor TATA-binding
protein (TBP)
- B recognition element (BRE)
- Recognized by transcription factor TFIIB
- Initiator element (INR)
- Recognized by TBP-Associated Factors (TAF) 1, 2
- Downstream promoter element (DPE)
- Recognized by TBP-Associated Factors (TAF) 1, 2

64
Transcription: RNA Polymerase

65 65
Transcription: Pre-Initiation Complex

Pre-Initiation
Complex (PIC):

• Helps position RNA

polymerase II over gene
transcription start sites
• Denatures the DNA
• Positions the DNA in
the RNA polymerase II
active site for
transcription

66
Transcription: Pre-initiation Complex

1) TFIID (TBP + TAFs) binds

to TATA box

2) Binding of TFIIA and TFIIB

to complex.
• TFIIB provides specific
binding site for RNA Pol II

67
Transcription: Pre-initiation Complex

3) The RNA Pol II – TFIIF

complex binds to TFIIB in
the PIC

4) TFIIE and TFIIH bind to

the PIC
• TFIIH contains 3
enzymatic subunits
• Kinase Phosphorylates
RNA Pol II
• Helicases Unwind DNA
at promoter start site
• Hydrolysis of ATP by TFIIH
can form the transcription
bubble (13 bp) open
formation

68
Transcription: Pre-initiation Complex

Transcription initiated by
phosphorylation of Carboxyl-terminal
domain of RNA Pol-II

• 7 amino acid (aa) repeating domain of RNA

Pol II subunit (Tyr-Ser-Pro-Thr-Ser-Pro-Ser)
• becomes phosphorylated by TFIIH
• Ser-5 triggers uncoupling of
RNA Pol II from PIC and promotes
elongation

• TFIID remains bound to TATA and

can initiate formation of additional PIC

69
Transcription : Elongation
• Transcription Bubble: unwound section of DNA of
approximately 13 bp regions
• DNA in front of RNA Pol are unwound, compensatory positive
supercoils (Chapter 10)
• DNA behind RNA Pol are rewound and negative supercoils are
present (Chapter 10)
• DNA-RNA hybrid: ~8-9 bp, stabilizes the elongation complex

70
Transcription: Elongation

• Complementary nucleotides are

incorporated by RNA Pol II in a
5’ 3’ direction

• Incoming Adenosine-triphosphate
pairs with the Thymine containing
nucleotide of the template (H-bonds)

• 3`OH of the previous nucleotide

sugar binds to the 5’ α-phosphate
of incoming nucleoside triphosphate

• Pyrophosphate (PPi) is released

and hydrolyzed to 2 inorganic
phosphates (Pi). This releases a
large amount of energy irreversible

71
 Transcription: Elongation

• Elongation Transcription Factors:

• > 50 components involved in elongation
• P-TEFb: phosphorylates the CTD
at Ser-2 leading to productive
elongation and required RNA
modifications
• TFIIF: weakens interactions
between RNA Pol II and nonspecific
binding sites of DNA, suppressing
transient pausing of the polymerase
• TFIIS: stimulates elongation, RNA
Pol II moving after pauses, proofread
& correction of mistaken nucleotides
72
Transcription: Termination
• In bacterial cells: well defined termination sequences
• In eukaryotic cells: No well-defined sequence, not well understood
• 3’ end of mRNA determined by series of processing steps
• Cleavage of the new transcript followed by template-independent
addition of Adenine (A) at its new 3' end Polyadenylation

73
Difference between DNA and RNA in chemical structure
DNA (2’-Deoxyribo-Nucleic Acid)

* The sugar and base

alone are called a
Nucleoside.

* Adding a phosphate (or

Phosphoester bond
more than one phosphate)
to a nucleoside creates a
Nucleotide.
Glycosidic bond

Formation of Nucleotide by Removal of Water. The

carbon atoms in 2’ deoxyribose are labeled in Red. dAMP:
Deoxy Adenosine MonoPhosphate 74
 DNA STRUCTURE

Amino - Imino and Keto - Enol tautomerism. (a)

Cytosine is usually in the amino form but rarely forms
the imino configuration. (b) Guanine is usually in the
keto form but is rarely found in the enol configuration.

75
Structure of double helix DNA

76
DNA IS RIGHT HANDED HELIX

77
 RNA STRUCTURE
v RNA Contains Ribose and Uracil and is Usually Single-
Stranded unlike DNA

The figure shows the structure of the backbone of RNA, composed

of alternating phosphate and ribose moieties. 78
 v G:U Base Pair. The structure shows
hydrogen bonds that allow base pairing to occur
between Guanine and Uracil

v RNA Chains Fold Back on Themselves to

Form Local Regions of Double Helix Similar
to A Form of DNA

Double Helical Characteristics of RNA. In an RNA molecule having regions of

complementary sequences, the intervening (non-complementary) stretches of RNA may
become “looped out” to form one of the structures illustrated in the figure. (a) Hairpin
(b) Bulge (c) Loop 79
 TRANSLATION

v It requires the participation of dozens of various components

including the “Ribosomes” which are nonspecific components of
the translation machinery. These complex, cytoplasmic “machines”
can be programmed, to translate the information encoded by any
mRNA. Ribosomes contain both protein and RNA. The RNAs of
a ribosome are called ribosomal RNAs (rRNAs), and like mRNAs,
each is transcribed from one of the DNA strands of a gene.

v Transfer RNAs (tRNAs) constitute a third major class of RNA

that is required to translate the information in the mRNA
nucleotide code into the amino acid “Alphabet” of a polypeptide.

v Many RNAs fold into a complex three-dimensional shape, which

is markedly different from one type of RNA to another.
- Until recently, the way in which genes control cells was summed up
by a simple formula: DNA makes RNA which makes protein, and
proteins are the cellular workhorses that carry out all the crucial tasks.
-“Small RNAs” do not code for proteins, but instead exercise control
over those RNAs that do.
-They provide a further clue to understanding the mysterious 98% of
the human genome that doesn't direct the production of proteins.

Eukaryotic cells make a host of other RNAs (small) (besides m, r

and t-RNA) which also play vital roles in cellular metabolism.

- Small nuclear (sn)

- Small nucleolar (sno)
- Small (short) interfering (si)
- Micro (mi)
81
MicroRNAs (miRNAs) These are 22-24 nucleotides in length, and down-
regulate gene expression by attaching themselves to messenger RNAs
(mRNAs) and preventing them from being translated into proteins.
Short interfering RNAs (siRNAs) These RNAs are around 22 nucleotides
in length and mediate the recently discovered phenomenon of RNA
interference (RNAi). miRNAs and siRNAs are created in the same way and
both mediate the down-regulation of gene expression. But siRNAs work by
binding to specific mRNAs and labeling them for destruction by enzymes
called Endonucleases.
Small nucleolar RNAs (snoRNAs) snoRNAs modify ribosomal RNAs
(rRNAs) by orchestrating the cleavage of the long pre-rRNA into its
functional subunits (18S, 5.8S and 28S molecules). snoRNAs also add
finishing modifications to the rRNA subunits.
Small nuclear RNAs (snRNAs) These are constituents of the spliceosome,
the cellular machinery that helps to produce mRNA by removing the non-
coding regions (introns) of genes and piecing together the coding regions
(exons) to be translated into proteins. Some of these snRNAs have been
shown to be the functional enzymes in the splicing reaction. 82
 UNDERSTANDING TRANSCRIPTION
RNA polymerase (RNAp) (Enzyme)
DNA RNA

§ One DNA strand serves as the template

§ The site of DNA to which RNAp binds is called “Promoter”
§ For this binding, one needs additional proteins called
“Transcription factors”
§ Promoter contains information that determines which of the two
DNA strands is transcribed & the site at which transcription begins.
§ RNAp moves along template DNA strand from 3’ to 5’,
unwounding
§ A complimentary strand of mRNA grows from 5’ to 3’
83
Summary: Chain elongation Mechanism during Transcription
RNA polymerase moves along the template DNA strand from 3’-5’ .
As the polymerase progresses, the DNA is temporarily unwound and
the polymerase assembles a complementary strand of RNA that
grows from 5’ to 3’ direction. RNA polymerase catalyzes the reaction
(n = no of nucleotide)

RNAn + NTP RNAn+1 + PPi

In which (ribo)Nucleoside Tri-Phosphate substrates (NTPs) are
cleaved into nucleoside monophosphate as they are polymerized into
a covalent chain
Pyrophosphatase enzyme
PPi 2Pi + Free energy
The released energy makes the incorporation of nucleotides
irreversible. As the polymerase moves along the DNA template, it in-
corporates complementary nucleotides into the growing RNA chain.
Once the polymerase has moved past a particular stretch of DNA, the
DNA double helix reforms 84
 Genetic Code and its properties
v One of the first models of the genetic code was presented by the physicist
George Gamow, who proposed that each amino acid in a polypeptide was
encoded by three sequential nucleotides. In other words, the code words,
or codons, for amino acids were nucleotide triplets.

v There are 4 possible one-letter words, 16 (42) possible two-letter words,

and 64 (43) possible three-letter words. Because there are 20 different
amino acids (words) that have to be specified, codons must contain at least
3 successive nucleotides (letters). The triplet nature of the code was soon
verified in a number of insightful genetic experiments conducted by Francis
Crick, Sydney Brenner, and colleagues at Cambridge University.6

v The genetic code is the set of rules by which information encoded in

genetic material (DNA or mRNA sequences) is translated into proteins
(amino acid sequences) by living cells.
85 85
Genetic Code

(Nirenberg & Khorana, 1968) 86 86

 Features of the Genetic Code

v It transfers information from mRNA to proteins with high fidelity

v It is redundant/degenerate: 61 mRNA triplets code for 20 amino acids
v Contains START (AUG) and STOP (UAA, UAG and UGA) codons
v Most codons for a given amino acid differ only in the last (third) base
of the triplet (Exceptions: Leu, Arg, Ser)
v The Genetic Code is nearly universal: correspondence between a
nucleotide triplet and an amino acid is identical from viruses to
mammals. The rare exceptions are mitochondria and unicellular
protozoa.
v The universality of the Genetic Code is a result of strong evolutionary
pressure: a change in a single codon would alter nearly every protein
made by an organism.

87
Example

Met (Start)

Thr

Glu

Leu

Arg

Ser

STOP

m
Peptide

88
 CHAPTER 12
The Cell Nucleus and the Control of Gene
Expression

v Genes operate as part of interacting networks. This map displays the

functional interactions of approximately 1700 genes from the budding yeast
S. cerevisiae. There are 170,000 gene-gene interactions. It is a great
challenge to determine which of these genes participated in a particular
cellular process or acted in the same organelle.
v To study this number of genes, researchers had to examine ~5.4 million
different combinations of mutant genes spanning all biological processes.
8
Keys

v Here, we will explore some of the ways in

which cells control gene expression and
thereby ensure that certain proteins are
synthesized, while others are not.

v First, we will describe the structure and

properties of eukaryotic cell nucleus where
most of the regulatory machinery is housed

9
0
Eukaryotic cell

91
 Control of Gene Expression in Eukaryotes:
Structure and Function of Cell Nucleus

The cell nucleus.

EM (Electron Micrograph)
of an interphase HeLa cell
nucleus (left) and
schematic drawing of
major components (right).

vUndistinguished morphology
vThe contents of the nucleus are enclosed by the Nuclear Envelope that forms the
boundary between Nucleus and Cytoplasm..
vA typical nondividing Nucleus consists of:
§ Chromosomes present as highly extended nucleoprotein fibers (Chromatin)
§ One or more Nucleoli, which are irregularly shaped electron-dense structures
that function in the synthesis of ribosomal RNA and the assembly of
ribosomes
§ Nucleoplasm as the fluid where solutes are dissolved.
§ The Nuclear Matrix, which is the protein-containing fibrillary network. 9
 Control of Gene Expression in Eukaryotes
The nuclear envelope
vNuclear Envelope/Membrane)
§ The Nuclear Envelope is a
structure that divides the nucleus
from its cytoplasm.
§ It consists of two membranes
separated by a nuclear space.
§ The two membranes are fused at
sites forming a nuclear pore.
§ The inner surface of the nuclear
envelope is lined by the Nuclear
Lamina.
§ Contains around 60 distinct
transmembrane proteins.

9
Nuclear Envelope and Nuclear Pore Complex (NPC)

The nuclear envelope. Schematic drawing (top) & EM of

an onion root tip cell (bottom)

94
Control of Gene Expression in Eukaryotes
The nuclear lamina

Nucleus stained for EM: metal-shadowed Micrographs of fibroblast nuclei from a

nuclear lamina (Red) and nuclear envelope of a patient with HGPS (Hutchinson-
nuclear matrix (Green) Xenopus oocyte Gilford Progeria Syndrome), (bottom)
or a healthy subject (top).

§ The Nuclear Lamina is a 30-100 nM thick dense fibrillary

structure inside the nucleus of eukaryotic cell.

§ It is made up of intermediate filaments & membrane proteins.

95
 The Nuclear Lamina

v Supports the nuclear envelope and it is composed of Lamin

proteins.
v Integrity of nuclear Lamina regulated by
phosphorylation/dephosphorylation.
v Human conditions / disease:
§ Lamin A/C mutation gives Hutchinson-Gilford Progeria
Syndrome,
§ Lamin B mutation causes Leukodystrophy (loss of Myelin -
insulating layer or sheath that forms around nerves)
§ Lamin A mutation also leads to Muscular Dystrophy
(weakened muscle)

96
Nuclear lamins form a filament meshwork-the nuclear lamina (white)
- along the inside of the nuclear membrane (purple; shown with a
nuclear pore). Nuclear lamina constitutes a scaffold that interacts with
chromatin [a protein (red)-DNA (blue) complex] and influences
nuclear functions such as transcriptional regulation.
v Hutchinson-Gilford Progeria Syndrome: A genetic premature
aging disease due to improper processing of lamin-A/C proteins due
to mutation
v Leukodystrophy (Krabbe disease): Progressive nervous system
disorder due to myelin loss
v Muscular Dystrophy: Progressive muscle weakness and loss 97
 Control of Gene Expression in Eukaryotes
The Nuclear Pore Complex (NPC)

Movement of materials through the

nuclear pore: EM (frog oocyte) after
injection with gold particles coated with
nuclear protein (left, middle) and EM of an
insect cell showing the movement of
granular material (ribosomal subunit, right)

v Structure of Nuclear Pore Complex and

its Role in Nucleocytoplasmic Trafficking
§ Proteins and RNA are transported in and
out of the nucleus.
§ Nuclear pores contain the Nuclear Pore
Complex (NPC) that appears to fill the
pore like a stopper.
§ NPC is composed of ~30 proteins called
Nucleoporins

98
 Control of Gene Expression in Eukaryotes
The Nuclear Pore Complex (NPC)

Scanning electron micrographs (SEM) of the nuclear

pore complex from an amphibian oocyte. Cytoplasmic
(a) and nuclear (b) faces of the nuclear envelope
complex. Nuclear face shows NPC distribution and
intact patches of the Nuclear Lamina (NEL) (c).

v Structure of Nuclear Pore Complex and its

Role in Nucleocytoplasmic Trafficking
§ Proteins and RNA are transported in and out
of the nucleus.
§ Nuclear pores contain the Nuclear Pore
Complex (NPC) that appears to fill the pore
like a stopper.
§ NPC is composed of ~30 proteins called
Nucleoporins.
 Control of Gene Expression in Eukaryotes
The nuclear pore complex (NPC)

Model of a vertebrate nuclear

pore complex (NPC). The
structure consists of several
parts, including a scaffold that
anchors the complex to the
nuclear envelope, a cytoplasmic
and a nuclear ring, a nuclear
basket, and eight cytoplasmic
filaments.

vStructure of Nuclear Pore Complex and its Role in Nucleocytoplasmic

Trafficking
§ Huge complex (15-30X mass of ribosome) that exhibits octagonal symmetry.
§ Channel: 20-to 30-nm-wide
§ FG (Phenylalanine-Glycine) domains of nucleoporins form a hydrophobic
sieve that blocks the diffusion of larger macromolecules (>40,000 Daltons)
10
Control of Gene Expression in Eukaryotes
Importing proteins

Importing proteins into the nucleus. Steps in nuclear protein import (left). Gold particle-
nucleoplasmin injection into frog oocytes shows binding to cytoplasmic filaments (right)
v Proteins synthesized in the cytoplasm are targeted for the nucleus by the Nuclear
Localization Signal (NLS) (eg. Pro-Lys-Lys-Lys-Arg-Lys-Val) having basic residues.
Other types of NLS are also described
§ Proteins with an NLS bind to an NLS receptor (Importin a/b).
§ Conformation of the NPC changes as the protein passes through.
§ RNAs move through NPCs as RNPs (RiboNucleoProteins) (Protein-RNA
complex) via transport receptors and carry NES (Nuclear Export Signals) to pass.

Importin: Hetero-dimeric import receptor; Ran: RAs-related Nuclear protein,

also known as GTP-binding nuclear protein. 101
 Characteristics of Nucleus
v Unlike the plasma membrane, which prevents passage of
macromolecules between the cytoplasm and the extracellular
space, the nuclear envelope is a hub of activity for the movement
of RNAs and proteins in both directions between the nucleus
and cytoplasm. The replication and transcription of genetic
material within the nucleus require the participation of proteins
made in the cytoplasm and transported across the nuclear envelope.
v Conversely, the mRNAs, tRNAs, and ribosomal subunits that
are manufactured in the nucleus must be transported through the
nuclear envelope in the opposite direction.
v To appreciate the magnitude of the traffic between the two major
cellular compartments, consider a HeLa cell, which is estimated to
contain about 10 million ribosomes. To support its growth, a single
HeLa cell nucleus must import approximately 560,000 ribosomal
proteins and export ~ 14,000 ribosomal subunits/min.
102
 The Nuclear Envelope
v The separation of a cell’s genetic material from the
surrounding cytoplasm is most important feature that
distinguishes Eukaryotes from Prokaryotes. This
makes the appearance of the nuclear envelope a
landmark in biology. The nuclear envelope consists of
two cellular membranes arranged parallel to one
another and separated by 10-50 nm.
v The membranes of the nuclear envelope serve as a
controlled barrier that keeps ions, solutes, and
macromolecules from passing freely between the
nucleus and cytoplasm.
v The average mammalian cell contains several thousand
nuclear pores. 103
 Chromosomes
v Chromosomes are composed of DNA and associated protein,
which together is called chromatin. The orderly packaging of
eukaryotic DNA depends on Histone, a remarkable group of small
proteins that possess an unusually high content of the basic amino
acids Arg and Lys. Histones are divided into 5 classes, which can
be distinguished by their Arg/Lys ratio. Histones bind with the
backbone of DNA: Same for all organisms.

v It was presumed that the proteins associated with the DNA were
providing the protection against enzymatic degradation. In 1974,
using the data from nuclease digestion and other types of
information, Roger Kornberg, then at Harvard University,
proposed an entirely new structure for chromatin. Kornberg
proposed that DNA and histones are organized into repeating
subunits, called Nucleosomes which represent the lowest level of
chromosome organization. 104
Note: Chromosomes are condensed / compact chromatins (which are untangled or lose)
 Control of Gene Expression in Eukaryotes
Chromosomes and Chromatin
vChromosomes and Chromatin
§ Packaging the Genome
§ Chromosomes consist of
chromatin fibers, composed of
DNA and associated proteins.
§ Each chromosome contains a
single, continuous DNA
molecule.
vNucleosomes: The Lowest Level of
Chromosome Organization
§ The protein component of
chromosomes include Histones,
a group of highly conserved
proteins.
§ Histones have a high content of
basic amino acids (Arginine
and Lysine). https://www.youtube.com/watch?v=TvOcAosqxrM

105
 Control of Gene Expression in Eukaryotes
Chromosomes and Chromatin

vDNA and histones are

organized into repeating H4
subunits called
nucleosomes. H2B H3 Nucleosomal
H2A organization of
vEach nucleosome includes a chromatin:
core particle of supercoiled H2A
Schematic diagram
DNA and histone H1 (top) and EM of
serving as a linker. H3
H2B H4 Drosophila cell
vDNA is wrapped around the nucleus with
core complex. nucleosomes along
vThe histone core complex DNA strand
consists of two molecules (bottom)
each of H2A, H2B, H3, and
H4 forming an octamer.

106
 Control of Gene Expression in Eukaryotes
Chromosomes and Chromatin

v DNA and histones are

organized into repeating
subunits called
nucleosomes.
v Each nucleosome includes
a core particle of
supercoiled DNA and
histone H1 serving as a
linker.
v DNA is wrapped around 3D structure of a
the core complex. nucleosome from X-ray
crystallography. Core
v The histone core particle at two views (top)
complex consists of two
and schematic of half of a
molecules each of H2A, core particle (side)
H2B, H3, and H4
forming an octamer.
 Control of Gene Expression in Eukaryotes
Higher Levels of Chromatin Structure

vHigher Levels of Chromatin

Structure
§ A 30-nm filament is another
level of chromatin
packaging, maintained by
histone H1.
§ Chromatin filaments are
organized into large
supercoiled loops.
§ The presence of loops in
chromatin can be seen:
Ø In mitotic chromosomes
form which histones have
been extracted.
Ø In meiotic lampbrush
chromosomes from
amphibian oocytes. 30-nm fiber: EM of a fiber (left) and
two packaging models (middle, right).
 Control of Gene Expression in Eukaryotes
Higher Levels of Chromatin Structure

vHigher Levels of Chromatin

Structure
§ A 30-nm filament is another
level of chromatin packaging,
maintained by histone H1.
§ Chromatin filaments are
organized into large
supercoiled loops.
§ The presence of loops in
chromatin can be seen:
Ø In mitotic chromosomes
form which histones have
been extracted.
Chromatin loops: a higher level of
Ø In meiotic lampbrush
chromosomes from chromatin structure. EM: of a mitotic
amphibian oocytes. chromosome (left) and model for cohesin
protein in maintaining loops (right)

109
 Control of Gene Expression in Eukaryotes
Higher Levels of Chromatin Structure

vHigher Levels of Chromatin

Structure
§ A nucleus 10 mm in
diameter can pack 200,000
times this length of DNA
Levels of
within its boundaries.
organization of
§ Packing ratio of the DNA chromatin.
in nucleosomes is
approximately 7:1.
§ Assembly of the 30-nm
fiber increases the DNA-
packing ratio to 40:1.
§ Mitotic chromosomes
represent the ultimate in
chromatin compactness
with a ratio of 10,000:1.

110
Correlation between Transcriptional Activity & Histone Acetylation
§ Histone Acetylation interferes with the interaction
between DNA and the nucleosomes, leading to less
compact, more transcriptionally active chromatin.
§ Histone Deacetylases remove acetyl groups,
causing chromatin to become condensed and
transcriptionally silenced.
§ Histone deacetylase inhibitors (HDI) cause
hyperacetylation of histones, which increases gene
expression

This metaphase chromosome spread has been labeled with fluorescent antibodies to acetylated
histone H4, which fluoresce green. It is evident that all of the chromosomes except the
inactivated X stain brightly with the antibody against the acetylated histone.

Removal of the acetyl groups from H3 and H4 histones is among the initial steps in
conversion of “Euchromatin” (less condense) into “Heterochromatin” (more
condensed). The correlation between transcriptional repression and histone
deacetylation can be seen by comparing the inactive, heterochromatic X chromosome
of female cells, which contains deacetylated histones, to the active, euchromatic X
chromosome, whose histones exhibit a normal level of acetylation. 11
 Control of Gene Expression in Eukaryotes
The Structure of a Mitotic Chromosome (Human) (Total: 46; 22 Autosomes with
identical pairs, two sex chromosomes XX (Female), XY (Male)

vThe Structure of a Mitotic

Chromosome
§ A karyotype (describes the
chromosome count) is a
preparation of homologous pairs
ordered according to size.
• The pattern on a karyotic may be
used to screen chromosomal
abnormalities.

The stained chromosomes of a human

male arranged in a karyotype
112
Regulation of Gene Expression in
Prokaryotes (eg. Bacteria)

113
Control of Gene Expression in Bacteria

vBacterial cells selectively express

genes to use the available resources
effectively.
§ The presence of Lactose in the
medium indicates the synthesis
of the enzyme β-
Galactosidase.
§ The presence of Tryptophan
in the medium represses the
genes that encode enzymes for
Tryptophan synthesis.

The kinetics of β-galactosidase induction

in E. coli: mRNA and protein induction

114
 Control of Gene Expression in Bacteria
The Bacterial Operon

Organization of a bacterial
operon. Enzymes in a metabolic
pathway are encoded by a series
of structural genes that reside in a
contiguous array within the
bacterial chromosome.

vThe Bacterial Operon

q An operon is a functional complex of genes containing the information for
enzymes of a metabolic pathway. It includes:
§ Structural genes – Code for the enzymes and are translated from a single
mRNA that is usually polycistronic (encodes for more than one protein).
§ Promoter – Where the RNA polymerase binds.
§ Operator – Site next to promoter where the regulatory protein can bind
§ Regulatory – the repressor protein
Bacterial Gene
regulation by operons

vThe lac Operon

q It is an inducible operon,
which is turned on in the
presence of lactose
(inducer).
§ The lac operon contains
three structural genes.
§ Lactose binds to the
repressor, changing its
conformation and
making it unable to bind
to the operator.
§ A repressor protein can
bind to the operator and
prevent transcription in
the absence of lactose.
Bacterial Gene
regulation by operons

vThe trp Operon

§ It is a repressible
operon, which is
turned off in the
presence of
tryptophan.
§ The trp operon
repressor is active
only when it is
bound to a
corepressor such as
tryptophan.
 Control of Gene Expression in Bacteria
The lac Operon

Nucleotide sequence of binding sites in the control region of the lac operon

vThe lac Operon: catabolite repression

§ The lac repressor exerts negative control.
§ The glucose effect is an example of positive control.
§ Cyclic AMP (cAMP) acts by binding to a cAMP receptor protein (CRP).
§ Binding of CRP-cAMP to the lac control region changes the conformation of DNA
thus allowing RNA polymerase to transcribe the lac operon.
 Control of Gene Expression in Bacteria
Riboswitches

vRiboswitches
§ A number of bacterial mRNAs can bind to a small metabolite in their
5’ untranslated region, which in turn alters the gene involved in the
production of such metabolite.
§ These mRNAs are called riboswitches because they undergo a
conformational change and can suppress gene expression.
§ Most riboswitches suppress gene expression by blocking either
termination of transcription or initiation of translation.
§ Riboswitches allow bacteria to regulate gene expression in
response to some metabolites.
§ Given that they act without the participation of protein cofactors,
riboswitches are likely another legacy from an ancestral RNA world.

119
Control of Gene expression in
Eukaryotes

120
 Regulation of gene expression in eukaryotic
cells occurs primarily at three distinct levels

1. Transcriptional-level control: mechanisms determine

whether a particular gene can be transcribed and if so, how
often.
2. Translational-level control: mechanisms determine
whether a particular mRNA is actually translated and if so,
how often and for how long period.
3. Processing-level control: mechanisms determine the
path by which the primary mRNA transcript (pre-mRNA) is
processed into a mature messenger RNA that can be
translated into a polypeptide.

121
Transcription Factors in Regulation of Gene Expression

Transcriptional control is orchestrated by a large number

of proteins, called Transcription Factors (TFs) which
bind with DNA. Two kinds:

General Transcription Factors that bind at core promoter

sites in association with RNA polymerase.

Sequence-specific Transcription Factors that bind to

various regulatory sites of particular genes. This latter
group of transcription factors can act either as
transcriptional activators that stimulate transcription
of the adjacent gene or as transcriptional repressors
that inhibit its transcription. 122
FACTORS INFLUENCING ACTIVITY OF TRANSCRIPTION FACTORS

TFs bound at distant sites can influence gene expression.

Transcriptional activators bound at upstream enhancers influence
gene expression through interaction with co-activators.

1. Transcriptional Activation from Poised (Rapid action)

Polymerases

2. Coactivators that alter Chromatin Structure

3. Chromatin (Histone) Acetylation (Transcriptional Enhancer)

4. DNA Methylation This simple chemical modification is

thought to serve as an epigenetic mark or “tag” that allows certain
regions of the DNA to be identified and utilized differently from
other regions. (In mammals, the methyl-Cytosine is most
common) 123

123
 Transcriptional control
Transcription factors

vThe Role of Transcription Factors in Determining a Cell’s

Phenotype
§ Embryonic stem (ES) cells are:
Ø Capable of indefinite self-renewal
Ø Pluripotent, capable of differentiating into all of the different types of
cells.
§ The importance of transcription factors in ES cells was demonstrated when
these factors were introduced and shown to reprogram these cells.
§ Introducing a combination of genes encoding only four specific transcription
factors (Oct4, Sox2, Myc, and Klf4) was sufficient to reprogram the
fibroblasts and convert them into undifferentiated cells that behaved like ES
cells.
§ The induced pluripotent cells, or iPS cells, that were generated in these early
experiments were capable of dividing indefinitely in culture and of
differentiating into all of the various types of the body’s cells.

124
 Transcriptional control
Transcription factors

vThe Structure of Transcription Factors

§ Transcription factors contain a DNA-
binding domain and an activation domain.
§ Many transcription factors can bind a
protein of identical or similar structure to
form a dimer.
vTranscription Factor Motifs
§ The DNA-binding domains of most
transcription factors have related structures
(motifs) that interact with DNA sequences.
§ Most motifs contain a segment that binds
to the major groove of the DNA.

Interaction between dimeric

glucocorticoid receptor (GR) and DNA,
with zinc ion co-factor (purple spheres)
 Transcriptional control
Transcription factors

vTranscription factor motifs

§ The zinc finger motif –
the zinc ion of each Complex between
GLI (has five zinc
finger is held in place by fingers) and DNA.
two cysteines and two Each fingers is
histidines. colored differently.
§ The helix-loop-helix Inset: structure of a
single zinc finger.
(HLH) motif – has two
α-helical segments
separated by a loop.
§ The leucine zipper motif
– has a leucine at every
seventh amino acid of
an α-helix. A model of TFIIIA bound to
the DNA of the 5S RNA gene
126
 Transcriptional control
Transcription factors
vTranscription factor motifs
§ The zinc finger motif –
the zinc ion of each
finger is held in place
by two cysteines and
two histidines.
§ The helix-loop-helix
(HLH) motif – has two
α-helical segments
separated by a loop.
§ The leucine zipper
motif – has a leucine at MyoD is a dimeric bHLH transcription factor for
every seventh amino muscle cell differentiation. (Left): Basic (red)
acid of an α-helix. and HLH regions (brown) are shown. The DNA
bases bound are indicated (yellow). (Right):
Sketch of the MyoD complex
 Transcriptional control
Transcription factors
vTranscription factor motifs
§The zinc finger motif – AP-1
the zinc ion of each Fos
Jun
finger is held in place by
two cysteines and two
histidines.
§The helix-loop-helix
(HLH) motif – has two
α-helical segments
separated by a loop.
§The leucine zipper
motif – has a leucine at
every seventh amino AP-1, a bZIP transcription factor, is a
acid of an α-helix. heterodimer between Fos (red) and Jun
(blue) that plays a role in cell proliferation

128
 Transcriptional control
Transcription factors
Increasing the DNA-binding
specificities of transcription
factors through
dimerization
The human genome encodes
approximately 118 different
bHLH monomers

§ bHLH and HLH-containing transcription factors play a key role in

the differentiation of certain tissues.
§ bHLH and HLH-containing transcription factors also participate in
the control of cell proliferation and cancer.
§ Heterodimerization greatly expands the diversity of regulatory
factors that can be generated from a limited number of polypeptides
 Control of Gene Expression in Eukaryotes: The Histone Code

Histones can
be modified
by addition
of Methyl,
Acetyl,
phosphate &
other groups

v The Histone Code and Formation of Heterochromatin

§ The histone code hypothesis states that the activity of a chromatin region depends
on the degree of chemical modification of histone tails.
§ Histone tail modifications influence chromatin in two ways:
q Serve as docking sites to recruit nonhistone proteins.
q Alter the way histones of neighboring nucleosomes interact.
130
 Histone Code

- Phosphorylation (Lys): Mostly Gene Activation

- Ubiquitination (Lys): Gene Suppresion

- Acetylation by HAT (Histone Acetyl Transferase)

- Deacetylation by HDAC (Histone Deacetylase) 131
 Control of Gene Expression in Eukaryotes
Histone modification
Deacetylation by Histone deacetylase (HDAC)
Methylation by Histone methyltransferase (HMT)

vRemoval of the acetyl groups from H3 and H4

histones is among the initial steps in
conversion of Euchromatin into
Heterochromatin.
vHistone deacetylation is accompanied by
methylation of H3K9 histone
methyltransferase (SUV39H1 in humans.
vMethylated H3K9 binds to proteins with a
chromodomain, for example heterochromatic
protein 1 (HP1)
vOnce HP1 is bound to the histone tails, HP1-
HP1 interactions facilitate chromatin
packaging into a heterochromatin state,

132
Model of possible events during the formation of heterochromatin
 Control of Gene Expression in Eukaryotes
Telomeres
vTelomeres
§ The end of each chromosome is
called a telomere and is
distinguished by a set of repeated
sequences.
§ New repeats are added by a
telomerase, a reverse transcriptase
that synthesizes DNA from a DNA
template.
§ Telomeres are required for the
complete replication of the
chromosome because they protect
the ends from being degraded.
§ Telomerase activity is thought to
have major effects on cell life.
The end-replication problem: Generation
of single stranded overhangs that shorten
DNA

133
 Telomeres/Telomerase
Each chromosome contains a single, continuous, double-stranded DNA molecule.
The tips of each DNA molecule are composed of an unusual stretch of repeated
sequences that, together with a group of specialized proteins, forms a cap at each end
of the chromosome called a Telomere. Human telomeres contain the sequence
TTAGGG
AATCCC
repeated from about 500 to 5000 times. Unlike most repeated sequences that vary
considerably from species to species, the same telomere sequence is found throughout
the vertebrates, and similar sequences are found in most other organisms. This
similarity in sequence suggests that telomeres have a conserved function in
diverse organisms. A number of DNA-binding proteins have been identified that
bind specifically to the telomere sequence and are essential for telomere function.

In situ hybridization of a DNA probe containing the

sequence TTAGGG, which localizes to the telomeres
of human chromosomes

Telomerase is a ribonucleoprotein that is an enzyme

which adds DNA sequence repeats "TTAGGG" in all
vertebrates) to the 3' end of DNA strands in the
13
 Control of Gene Expression in Eukaryotes
Epigenetics
• Epigenetics: There’s More to Inheritance than DNA
• Epigenetic inheritance depends on factors other than DNA sequences.
• X-chromosome inactivation is an example, since the two X chromosomes can have
identical DNA sequences, but one is inactivated and the other is not.
• An epigenetic state can usually be reversed; X chromosomes, for example, are
reactivated prior to formation of gametes.
• Differences in disease susceptibility and longevity between genetically identical
twins may be due, in part, to epigenetic differences that appear between the twins
as they age.
• Parental histones determine the chemical modifications found in the newly
synthesized histones.
• As heterochromatin is replicated, a histone methyltransferase labels the newly
synthesized H3 molecules added into the daughter nucleosomes.
• Euchromatic regions tend to contain acetylated H3 tails, a modification transmitted
from parental chromatin to progeny chromatin.

13
Not all genes are active at all times. DNA methylation is one of
several epigenetic mechanisms that cells use to control gene
expression
There are many ways that gene expression is controlled in eukaryotes,
but methylation of DNA (not to be confused with histone methylation)
is a common epigenetic signaling tool that cells use to lock genes in the
"off" position. In recent decades, researchers have learned a great deal
about DNA methylation, including how it occurs and where it occurs,
and they have also discovered that methylation is an important
component in numerous cellular processes, including embryonic
development, genomic imprinting, X-chromosome inactivation, and
preservation of chromosome stability. Given the many processes in
which methylation plays a part, it is perhaps not surprising that
researchers have also linked errors in methylation to a variety of
devastating consequences, including several human diseases.
Mammals tend to have fairly globally distributed CpG
methylation patterns
136
This diagram shows a representative region of genomic DNA in a normal cell. The region
contains repeat-rich, hypermethylated pericentromeric heterochromatin and an actively
transcribed tumor suppressor gene (TSG) associated with a hypomethylated CpG island
(indicated in red). In tumor cells, repeat-rich heterochromatin becomes hypomethylated, and
this contributes to genomic instability (a hallmark of tumor cells) through increased mitotic
recombination events. De novo methylation of CpG islands also occurs in cancer cells, and it
can result in the transcriptional silencing of growth-regulatory genes. These changes in
methylation are early events in tumorigenesis. (Reproduced from Robertson, K., DNA
methylation and human disease, Nature Reviews Genetics 6, 597-561 137
 Translational control

A model for the mechanism of translational activation of mRNAs following

fertilization of a Xenopus egg. mRNAs are maintained in the cytoplasm in an
inactive state by their short poly(A) tails and a bound inhibitory protein Maskin.

vThe Control of mRNA Translation

§ Several important processes depend on mRNAs that were synthesized at a
previous time and stored in the cytoplasm in an inactive state.
§ Other mechanisms influence the rate of translation of specific mRNAs through
proteins that recognize specific elements in the UTRs of those mRNAs.
§ Example: mRNA that codes for ferritin.
 Translational control
Translation

vThe Control of mRNA Translation

§ When iron concentrations are low,
iron regulatory protein (IRP) binds to
the iron-response element (IRE) to
prevent translation.
§ When iron becomes available, it binds
to the IRP, changing its conformation
and causing it to dissociate from the
IRE, allowing the translation of the
mRNA to form ferritin.

The control of ferritin

mRNA translation
139
 Translational control
mRNA stability

vThe Control of mRNA Stability

§ The lifetimes of eukaryotic mRNA vary widely.
q Fos mRNA (cell cycle-related) is 10-30 minutes.
q Hemoglobin mRNA is greater than 24 hours.
§ Poly(A) tail length may influence the longevity of mRNA.
q As an mRNA remains in the cytoplasm, its poly(A) tail tends to be
reduced.
q When the tail is about 30 A residues, the tail is shortened.
§ Certain destabilizing proteins in the 3’ UTR may affect the rate of poly(A)
tail shortening.
q Globin mRNA 3’ UTR contains CCUCCU repeats that serve as
binding sites for stabilizing proteins.
q Short-lived mRNA 3’ UTRs have AU-rich regions that destabilize it.
q Introduction of a destabilizing sequence into the globin mRNA 3’
UTR shifts the half-life from 10 hours to 90 minutes.

140
 Translational control
microRNAs

vThe Role of MicroRNAs in

Translational-level Control
§ miRNAs act by binding to
site in the 3’UTR of their
target mRNAs.
§ Suppress gene expression
by either promoting
deadenylation and
degradation, inhibiting the
initiation of translation,
inhibiting elongation, or
possibly activating
degradation of nascent
peptides.

miRNA mediated gene silencing.

miRNAs pair with sequence elements
within the 3’ UTR of target mRNAs
141
Post-translational Control
Determining Protein Stability

vThe factors that control a

protein’s lifetime are not
well understood.
vProtein stability may be
determined by the amino
acids on the N-terminus.
vDegradation of proteins is
carried out within hollow,
cylindrical Proteasomes.
Proteasome structure and function. High-
resolution EM of an isolated Drosophila
proteasome (left) and model of a proteasome
based on high-resolution electron microscopy
and X-ray crystallography (right)
 Post-translational Control:
Determining Protein Stability
Cap: removes
chain

vProteasomes recognize
proteins linked to ubiquitin.
vUbiquitin is transferred by
ubiquitin ligases to proteins Lys
being degraded.
vOnce polyubiquitinated, a
protein is recognized by the
cap of the proteasome.
vOnce degraded, the
component amino acids are
ATP
released back into the b: protease dependent
cytosol.
Proteasome-mediated degradation: 1, protein
is ubiquitinated; 2, protein binds to proteasome
cap; 3, unfolded polypeptide enters proteasome;
4/5, catalytic β subunits degrades protein
 PROCESSING-LEVEL CONTROL
Alternative splicing (or differential splicing) regulates gene
expression at the level of RNA processing and provides a
mechanism by which a single gene can encode two or more related
proteins.
It is a process by which the exons of the RNA produced by
transcription of a gene are reconnected in multiple ways during
RNA splicing (modification of nascent pre-mRNA or precursor or
immature mRNA).

144
 Chromatin
Cells devised an ingenious packaging system. The double
stranded DNA helix wraps around histones (major) and other
proteins. The resulting DNA-protein complex is called
chromatin.
Chromatid
During initial phase of cell division, the DNA is replicated,
producing two identical copies of DNA, which are connected to
each other at the centromere. This replicated X-like structure is
now called a sister chromatid pair. A chromatid is thus just
one of the strands.

Chromosome
Each eukaryotic cell nucleus contains the DNA molecule which
is packaged into rod/thread-like structures called chromosomes.
Each chromosome is made up of DNA tightly coiled many times
around histone (major) and other proteins that support its
structure. It contains the crucial genetic information.

Nucleosome
The repeating structural units of chromatin, each consisting of
approximately 200 base pairs of DNA wound around a protein
core composed of the histones H2A, H2B, H3, and H4
145
 How many chromosomes are there in human?
In humans, each cell normally contains 23 pairs of chromosomes, for a total of 46.
22 of these pairs (Autosomes) look the same in both males and females. The 23rd
pair (Sex chromosomes) differ between males and females. Females have two
copies of X-chromosome and males have one X and one Y chromosome.

146
How many chromosomes are there in human?
v In humans, each cell normally contains 23 pairs of
chromosomes, for a total of 46.

v 22 of these pairs (Autosomes) look the same in

both males and females.

v The 23rd pair (Sex chromosomes) differ between

males and females.

v Females have two copies of X-chromosome and

males have one X and one Y chromosome.

1
4
 Chromosomes in human
Karyotype
It is a test to examine chromosome in cells, to help identify genetic problems
/diseases. It counts the number of chromosomes & looks for their structural changes.

Numbered mainly
according to the size

Nearly every cell in a person’s body has the same DNA. Most DNA is located in the
cell nucleus (where it is called nuclear DNA), but a small amount of DNA can also
be found in the mitochondria (called mitochondrial DNA). 1
4
Genetic Disease

vDisease caused by one or combinations of variations

in the genome of an individual.

vDevelopment of any disease is under the

influence of both environmental and genetic factors,
with the latter playing a significant role.

1
4
 Genetic Diseases

1. Genetic Abnormalities/Variabilities
Alteration in the DNA/gene

(a) Monogenic diseases (Single gene mutation)

(b) Polygenic complex diseases (Multiple gene
mutations)

2. Chromosomal
Aberrations/Anomalies/Abnormalities

(a) Defect in chromosome number

(Numerical defect)
(b) Structure defect in chromosome
1
5
Genetic Abnormalities (Variabilities)
vUsually occur due to errors during recombination,
replication or repair of DNA.
vPoint mutation- Single ‘letter’ of genetic code is changed
vDeletion / Insertion (Addition) mutation- Addition or loss
of genetic material.
vAddition could be New Addition or Substitution (one for
another)
vLoss or deletion could be single or a segment
v Frame-shift mutation- Addition / Deletion of any number
of nucleotides that is not a multiple of three (more
commonly 1 or 2), causing a change in the reading frame
15
Genetic Abnormalities/Variabilities

The Genetic mutation can be Somatic, Germline or De Novo

1. Somatic mutation: Affects tissue or group of tissues and here

mutation or change to DNA takes place after fertilization.

vIf the reproductive cells are not affected, the

variation is not transmitted to offspring

2. Germline mutation: Present in all cells of the body because

genetic variation was originally present in the sperm and /or egg.

vGermline mutation can be transmitted to offspring

152
Genetic Abnormalities / Variabilities

3. De novo mutation / variation:

vOccurs in an egg or sperm cell, or immediately after
fertilization
vNeither parent possess mutation ie here mutation occurs in
the gametes of one or both parents or more rarely it
occurred in a post-zygotic mutation

• Affected child has a mutation in every cell, but has no

family history of the disorder.

153
 Consequence of Mutations on
functional properties

v Loss of Function Mutation- Reduces normal gene

effect, severe version called null mutation

v Gain of Function Mutation- Constitutively

activates or augments (enhances) normal function
of a gene

v No change in Function Mutation- Does not affect

the functional property of the gene.

154
 Genetics and disease
Genetic mutations

Single/Multi
Gene Mutations

Chromosomal
Abnormalities

155
Mutations

Nonsense Mutation- Causes

Transcription to arrest prematurely,
produces truncated protein

Missense Mutation- Encodes

abnormal amino acid, phenotypic
consequences depending upon
structural consequences of
substitution

156
Frameshift Mutations
Addition / Deletion of any number of nucleotides that
is not a multiple of three, causing a change in the
reading frame

157
Monogenic Disorders
vSingle gene defect / disorders
vMendelian disorders: Mutations involving single gene
follow one of three patterns of inheritance:

vAutosomal dominant,
vAutosomal recessive, or
vX-linked.

vDisorder pertaining to or influenced by a single gene as an

inherited characteristic.
v >10,000 human diseases are monogenic
ØGlobal prevalence of monogenic disorders at birth = 1%
ØIn Canada, monogenic disorders make up ~40% of the
work of hospital based pediatric practice.

158
 Mendelian Disorders

vThe gene loci are now known for nearly all of the Mendelian Disorders
vMendelian disorders:
§ 40 cancer syndromes
§ 50 cardiovascular diseases
§ 29 diabetes subtypes

159
Review of Genetics Inheritance
Each cell:
• 2 meters of DNA
• 25,000 genes à code for proteins
• 46 chromosomes
• 44 autosomal chromosomes
(22 pairs)
• 2 sex chromosomes
(1 pair; X and/or Y)

160
 Genetics and disease
Single Gene Disorders

1) Autosomal Dominant

v Transmission of a
dominant allele
v 50% chance of being
affected
v Disease appears in every
generation
v Males and females equally
affected

161
Genetics and disease
Single Gene Disorders

1) Autosomal Dominant

Case Study:
Carl is 25 yrs old and is married to Susan who is pregnant
with their first child. Over the past decade Carl’s mother has
demonstrated dramatic mood swings and declining dementia-
like symptoms, which they attributed to menopause and older
age. More recently his mother has had major difficulty in
walking. In the past couple of months Carl’s brother John, 30,
has started showing evidence of paranoia and hallucinations in
addition to a generalized lack of coordination.

What is happening in this family?

162
 Genetics and disease
Single Gene Disorders
1) Autosomal Dominant
Huntington’s Disease:
v Inherited brain disorder
v Mutations in the “HTT (Huntingtin)” gene à neuronal huntingtin protein
v Each child of a parent with HD has a 50% chance of inheriting
v Symptoms
§ Personality disturbancesà depression, apathy, irritability, anxiety,
obsessive behaviour
§ Cognitive loss à inability to focus, plan, recall or make decisions,
impaired insight
§ Physical deterioration à weight loss, involuntary movements,
diminished coordination, difficulty walking, talking, swallowing
§ Leads to complete incapacitation and, eventually, death
v Symptoms usually appear between 30-45yrs
v Can begin at any age (infancy to old age)
v No cure
v ~ 1/7,000 Canadians affected
163
 Huntington’s Disease
It is a “Trinucleotide Repeat Disorder” which is caused by the length of a
repeated section of a gene exceeding a normal range. The HTT gene contains a
sequence of 3 DNA bases - Cytosine-Adenine-Guanine (CAG) - repeated
multiple times (i.e. ... CAGCAGCAG ...), known as a trinucleotide repeat. CAG
is the 3-letter genetic codon for the amino acid Glutamine, so a series of them
results in the production of a chain of glutamine known as a Polyglutamine tract
and the repeated part of the gene, the Poly-Q region.

164
 Genetics and disease
Single Gene Disorders

2) Autosomal Recessive
v Disease manifests when
individual is homozygous
for the defective allele
v Parents are carriers; they
do not have the disease
v Child has a 25% chance
of being affected
v Recessive allele appears
more frequently in close
intermarriages

165
 Recessive Alleles
Phenotype expressed when both alleles are the same.
(aa)
One dominant allele and one recessive allele, the
trait is not expressed because it is overshadowed by
the dominant allele.
Heterozygous individual is said to be a carrier for
that trait. (Aa)
Parents are carriers; they - do not have the disease
Child has a 25% chance of being affected

Examples
Cystic fibrosis: Difficulty in breathing, frequent lung infection.
Sickle cell anemia: Abnormality in red blood cell shape, blocks blood flow
Tay-Sachs disease: Destroys nerve cells in the brain and spinal chord
Phenylketonuria: (PKU): Can lead to intellectual disability, seizures and
other serious medical problems due to the accumulation of Phenyl ketone
made from accumulated Phe. 166
 Genetics and disease
Single Gene Disorders
2) Autosomal Recessive
Cystic Fibrosis:
v Child inherits two defective copies of the gene, one
from each parent
v Mutation in Cystic Fibrosis Transmembrane
Conductance Regulator (CFTR) gene
Ø Protein required to regulate the components of
sweat, digestive fluids, and mucus
v Symptoms
Ø Difficulty breathing
Ø Wet, rattling cough
Ø Severe, chronic lung infections
Ø Permanent lung damage disease
Ø Difficulty digesting food à failure to grow
v 1/3,600 children born in Canada
v 1/25 Canadians is a CF carrier
167
Genetics and disease
Single Gene Disorders

3) Sex-linked

v Defective gene on X
chromosome
v Defective X on male is
unmasked and the trait is
expressed
v Female is carrier for the
disease; heterozygous
v Male transmits the
defective allele to his
daughters

168
 Genetics and disease
Single Gene Disorders
3) Sex-linked
Color blindness:
v Inability/decreased ability to see color,
or perceive color differences

§ Red/Green discrimination

v Mutations in genes that produce photo-

pigments à located on X-chromosome
v 1 in 10 males affected by some form of
color blindness
v Uncommon in females à second X
chromosome

169
 Genetics and disease
Chromosomal abnormalities
vChromosomal Diseases: Result from a chromosome abnormality.
Cause problems with a person's growth/ development/ body functions
vChromosome Abnormality: (a) Numerical (number): missing,
extra, (Anueploidy) or (b) Structural/Irregular portion of
chromosomal DNA
(a) Numerical anomalies: Monosomy, Trisomy
Monosomy: Presence of only one chromosome instead of a pair
Trisomy: Presence of 3 copies instead of normal 2.
(b) Structural anomalies:
Deletions, Duplications, Translocations, Inversions, Insertions
vError in cell division following meiosis or mitosis
§ Germ cells: all cells of body affected
§ Somatic cells: “Mosaicism” (2 or more populations of cells with
different genotypes)
170
 Genetics and disease
Chromosomal Abnormalities

Types of Chromosomal Abnormalities:

1. Gamete Nondisjunction
• Autosomal chromosomes
• Sex chromosomes
Numerical
2. Zygote Nondisjunction

3. Chromosome deletions
4. Chromosome duplications
Structural
5. Chromosome translocations

Non-disjunction: Failure of the chromosome to equally separate

during meiosis (anaphase of cell division)
 Genetics and disease
Chromosomal Abnormalities
Gamete non-disjunction of autosomal chromosomes:
vAutosomal chromosomes (1-22)
v~0.2-0.5% of living newborns
vMost incompatible with fetal survival
Ø Monosomy not compatible with fetal survival
vTrisomies for all chromosomes have been reported
Ø Only 3 types (Chromosome numbers 13, 18 and 21) reported to
result in live births
v~ 30-50% of spontaneous abortions (miscarriages) result of
anueploidy
vNondisjunction in oocyte germ cell at least 2X more common than
sperm germ cell
vTrisomies for all chromosomes have been reported
172
 Genetics and disease
Chromosomal Abnormalities 173
Gamete nondisjunction of autosomal chromosomes:
100,000 PREGNANCIES
15,000 spontaneous abortions (15%) 85,000 live births (85%)

Trisomy
1 0 0

2 159 0

3 53 0

4 95 0

5 0 0

6–12 561 0

13 128
17 (~10%)
14 275 0

15 318 0

16 1229 0

17 10 0

18 223
13 (~10%)
19–20 52 0

21 350
113 (~80%)
22 424 0

173
Karyotype of 47, XX+16 (trisomy 16), most common trisomy
associated with spontaneous abortion

174
Karyotype of 69,XXY (triploidy), common finding in spontaneous
abortion

175
 Genetics and disease
Chromosomal Abnormalities
Gamete nondisjunction of autosomal chromosomes:

Trisomy 13 - Patau
syndrome
• Left palate, Close-set eyes,
Decreased muscle tone,
Severe intellectual disability,
Seizures, Skeletal
abnormalities, Microcephaly,
Congenital heart defects
• More than 80% of children
die in the first year of life

47, XX+13

176
 Genetics and disease
Chromosomal Abnormalities
Gamete nondisjunction of autosomal chromosomes:

Trisomy 18 – Edwards
syndrome
• 3X more common in girls
• Low birth weight, mental
delay, microcephaly,
congenital heart
abnormalities, kidney defects
• More than 50% of children
die in the first week of life

47,XX+18
177
 Genetics and disease
Chromosomal Abnormalities

Gamete nondisjunction of autosomal chromosomes:

Trisomy 21 – Downs
syndrome
• Most common trisomy
• positively correlated to
maternal age
• Distinctive facial features,
developmental and social
delays, eye problems,
congenital heart anomalies
• Can live independent and
productive lives well into
adulthood
47,XX+21
178
Down syndrome or trisomy 21
Genetic disorder caused by the
presence of all or part of an extra
21st chromosome

Impairment of cognitive ability

Impairment of physical growth
Distinctive facial appearance
Developmental disabilities

Higher risk for:

Congenital heart defects
Gastroesophageal reflux disease
Recurrent ear infections
Obstructive sleep apnea
Thyroid dysfunctions

179
 Genetics and disease
Chromosomal Abnormalities

Gamete nondisjunction of sex chromosomes:

vFailure of proper separation of sex chromosomes (X, Y) in
germ cells
v~0.2-0.3% of living newborns
vPhenotypes less severe than autosomal nondisjunction
• X chromosome inactivation
• Y chromosomes have low gene content
vCommon signs/symptoms:
• Delay in onset of puberty
• Primary or secondary amenorrhea
• Infertility
• Ambiguous genitalia

180
 Genetics and disease
Chromosomal Abnormalities

Gamete nondisjunction of sex chromosomes:

Klinefelter's Syndrome (XXY):

v External genetalia = male, but
testicles are atrophic
v No sperm production à sterile
v Body configuration somwhat femal,
possible breast hypertrophy
v Intelligance usually subnormal
v Function relatively well in society
v ~1/500-1000 males born in Canada

47,XXY181
 Genetics and disease
Chromosomal Abnormalities
Gamete nondisjunction of sex chromosomes:

Turner's Syndrome (X0):

v ~ 50% result from nondisjunction
v Incomplete X chromosome
(20%) or mosaic expression
(30%)
v External genetalia = female
v Uterus is small, ovaries only
contain fibrous tissue à sterile
v Secondary sex characteristics
underdevelopped
v Short stature, broad neck and
chest
v Cardiovascular malformations
v ~1/2,000 females born in Canada

45,X0 182
 Genetics and disease
Chromosomal Abnormalities
Gamete nondisjunction of sex chromosomes:
Triple X Syndrome (Super-female):
v ~ 70% result from nondisjunction
• Mosaic expression (30%)
v Phenotype is subtle and can be
variable
• Tall stature at adolescence, normal
sexual development/puberty, are
fertile, no/minor mental deficiencies,
may have learning disabilities and or
problems with motor coordination
v Associated with advanced maternal
age
• 1/1,400 females born in Canada 47,XXX183
 Genetics and disease
Chromosomal Abnormalities
Gamete nondisjunction of sex chromosomes:
XYY Syndrome (Super-male):
• Phenotype is usually normal, many
males do not know
• Increased growth velocity from
early childhood, severe acne in
some cases, some learning
disabilities, normal sexual
development, normal fertility
• Associated with advanced
maternal age
•~1/1,000 males born in Canada
47,XYY
 Genetics and disease
Chromosomal Abnormalities
3) Chromosome deletions
vChromosomes deletion = part of a chromosome has been deleted
due to aberrant meiosis
vCan occur on any chromosome, at any allele, and can be any
size (large or small)
vResults of deletion depends on where the deletion is and what genes
are missing
-Embryos with significant deletions do not develop to term

185
 Genetics and disease
Chromosomal abnormalities

3) Chromosome Deletions

Cri Du Chat Syndrome:

vDeletion on short arm of chromosome 5
vDefect in larynx àhigh-pitched “cat-cry”
vIntellectual disability, delayed
development, microcephaly, low birth
weight, and hypotonia in infancy
vDistinctive facial features à widely set
eyes, low-set ears, small jaw, rounded face
vSome children are born with a heart defect
vCan lead to death in childhood
Genetics and disease
Chromosomal Abnormalities
4) Chromosome duplications
vChromosome duplication = part of a
chromosome is repeated due to aberrant
meiosis
vPhenotype due to altered gene dosage
• Genes present in 3 copies à partial
trisomy
• Phenotype usually less severe than
deletion
vTandem duplication à duplicated section is
adjacent to the original
vDisplaced duplication à duplicated sections
are separated by non-duplicated regions
 Genetics and disease
Chromosomal abnormalities

4) Chromosome Duplications
Charcot-Marie-Tooth Disease (CMT):

v70-80% CMT cases à duplication of large region

on short arm of chromosome 17
vmultiple copies of peripheral myelin protein 22
(PMP22)
vAbnormal myelin production
vProgressive loss of muscle tissue and touch
sensation across various parts of the body.
Breathing, hearing and vision can be affected in
some individuals
• Onset à late childhood/early
adulthood
vSymptoms and progression of the disease can vary

188
 Genetics and disease
Chromosomal Abnormalities

5) Chromosome Translocations
vPiece of one chromosome becomes attached to
another chromosome and vice-versa
Balanced translocation:
vIn a somatic cell à no real loss or gain of
genetic material therefore little effect on
function
vA gene fusion protein may be created when the
translocation joins two otherwise separated genes
• increases likelihood of malignancy
vProcess not entirely clear
• May occur following breakage of
chromosomal DNA during normal
process of transcription

189
 Genetics and disease
Chromosomal Abnormalities
5) Chromosome translocations
Chronic Myelogenous Leukemia (CML):

v Philadelphia Chromosome à
portion of Chromosome 22 is
translocated to Chromosome 9
v Fusion of a protein kinase gene
(ABL; Ch9) with a portion of a
BCR gene (Ch22)
v Novel fusion protein retains
catalytic properties involved in
cell proliferation of protein kinase
but no longer be easily regulated
by cell à Malignancy

ABL: stands for "Abelson", the name of a leukemia

virus which carries a similar protein
BCR: Breakdown Cluster Region
190
 Diagnosis of Genetic Diseases
vEarly diagnosis is critical to prevention and treatment of
genetic diseases

vPre-natal Diagnosis
ØAmniocentesis
ØChorionic Villous Sampling (Placental tissue)

vPost-natal Diagnosis
ØNewborn Blood Sampling
§ 28 conditions à metabolic disorder, endocrine disorders, blood
disorders, cystic fibrosis
§ http://www.cadth.ca/products/environmental-scanning/environmental-
scans/newborn-screening

191
 Diagnosis of Genetic Diseases

vAmniocentesis
• Amniotic fluid withdrawn (14th – 18th
week)
• can detect ~ 200 genetic diseases

vChorionic villous sampling

• Removing chorionic villi cells from
placental (8th-10th week)
• gives results earlier in the pregnancy

192
Diagnosis of Genetic Diseases

193
 Gene Therapy
vGene therapy (genetic engineering) = Insertion, Alteration, or
Removal of gene within an individual's cells or tissues to treat
disease
vLimitations:
• Longevity of new gene integration
• Multiple copies of gene insertion
• Immune response to viral vectors
• Multi-gene disorders
• Mutagenesis
• Long term outcomes unclear

http://www.youtube.com/watch?v=Ez560GnkSrE&feature=s
hare&list=TLjSkTSUlmwTk

194
 Course HSS 2305 B
Molecular Mechanism of Disease

Protein Post-Translation Modification

(PTM) and implications to Disease

19
 Eukaryotic Cell
(Outside: Extracellular); Within the cell: Intracellular)

Cellular membrane (nonporous) (double walled composed of bilayer of phospholipid)

Cytosol (contains many individual granules/vesicles/compartments such as ER, Golgi,

mitochondria, Lysosome, endosome etc) + Nucleus (contains double layer membrane which
has pores that is covered with several thousand Nuclear Pores complexes, lamin or
heterochromatin proteins.

Inside nucleus, chromosome reside that contains double strand DNA bound to histone core
and covered with chromatin

(DNA unwinds and DNA replication occurs with the help of DNA-polymerase enzymes),
DNA (genes) is converted into mRNA (Transcription) via pre-mRNA (Transcriptional
Processing)

mRNA makes proteins (Translation) using tRNA (that transports) and rRNA (the molecular
component of ribosome) that catalyze the conversion of mRNA to proteins/polypeptides

The protein thus synthesized is not fully functionally active.

PTM (Post-Translation Modification of Proteins) that leads to diverse proteome 196

Protein Posttranslational Modifications: The Chemistry of Proteome Diversifications
Christopher T. Walsh,* Sylvie Garneau-Tsodikova, and Gregory J. Gatto, Jr. Review; Angew.
Chem. Int. Ed. 2005, 44, 7342 -7372

197
 Post Translation Modifications (PTM) of Proteins
** Modification of protein structure after its synthesis from mRNA in ribosome

** This occurs during its transit to the cell surface through ER, Golgi and other organelles

** Constitutive pathway where proteins are produced without any regulation

** Regulated pathway where proteins are synthesized and stored in secretory granules.
They are released in a regulated fashion

198
The diversity of distinct covalent forms of proteins
(the proteome) greatly exceeds the number of
proteins predicted by DNA coding capacities owing
to directed posttranslational modifications. Enzymes
dedicated to such protein modifications include 500
Human Protein Kinases, 150 Protein
Phosphatases, and 500 Proteases. An understanding
of the scope and pattern of the many posttranslational
modifications in eukaryotic cells provides insight into
the function and dynamics of proteome compositions.

199
Proteins are made up of a-amino acids linked to each other by amide (peptide) bond
R1 R2

NH2 C* CO-OH H2N C * CO-OH

H H
a-Amino acid #1 a-Amino acid #2
-H2O
R1 R2

NH2 C CO HN C CO-OH
Di-peptide
H H R3

-H2O
Side chains H2N C * CO-OH a-Amino acid #3
H
R1 R2 R3
NH2 C CO HN C CO HN C * CO-OH
H H H Tri-peptide 20
 There are 20 natural amino acids that comprise all proteins
All are laevo-rotatory forms except Gly which exists only in one form

1. Alanine (Ala) (A) 11. Leucine (Leu) (L)

2. Arginine (Arg) (R) 12. Lysine (Lys) (K)
3. Aspartic acid (Asp) (D) 13. Methionine (Met) (M)
4. Asparagine (Asn) (N) 14. Proline (Pro) (P)
5. Cysteine (Cys) (C) 15. Phenyl alanine (Phe) (F)
6. Glutamic acid (Glu) (E) 16. Serine (Ser) (S)
7. Glutamine (Gln) (Q) 17. Threonine (Thr) (T)
8. Glycine (Gly) (G) 18. Tryptophan (Trp) (W)
9. Histidine (His) (H) 19. Tyrosine (Tyr) (Y)
10. Isoleucine (Ile) (I) 20. Valine (Val) (V)

201
Amino acids that are not modified during
Post Translational Modification of Proteins

Leu
Ile
Val
Ala
Phe

These amino acids contain mainly alkyl or un-

substituted phenyl ring which do not have any
reactive functional group
202
TWO TYPES OF POSTTRANSLATIONAL MODIFICATIONS OF
PROTEINS
1) Covalent modification of a nucleophilic amino acid side chain by an
electrophilic agent
2) Cleavage (Break) of a protein backbone at a specific peptide bond.

203
 Major Types of Post Translational Protein Modifications
1. Phosphorylation (Addition of Phosphoryl PO3 group)
2. Acylation/Acetylation (Addition of Acyl/Acetyl CH3-CO- group)
3. Lipidation (Addition of Fatty acid eg Myristic/Palmitic acid)
4. Glycosylation (Addition of carbohydrate or sugar moiety)
5. Oxidation (Addition of oxygen or hydroxyl group)
6. Alkylation (Addition of alkyl such as CH3 group)
7. Prenylation (Addition of isoprenoid (C5) eg. Farnesyl group
8. Ubiquitination (Addition of ubiquitin - a 76 amino acid protein)
9. S-S bond linkage (Linking free SH of two / more even no of Cys)
10. Sulphation (Addition of Sulphuryl SO3 group)
11. Nitration (Addition of Nitro or NO2 group) 20
4
 Rare Types of Post Translational Protein Modifications
(A selected few)

12. Citrullination
13. Glycylation / Glutamylation / Tyrosination
14. Iodination
15. Pyroglutamic acid formation
16. Trans-glutamination

Ref: Basak et al. Post-translational Protein Modifications with The Emphasis On Rare Types and
Their Implications. Current Medicinal Chemistry, 23 (7), 714-745, 2016.

205
 1. Phosphorylation (Addition of Phosphoryl group : PO3
group)

Phosphorylated forms of amino acid side chains in proteins: phospho-Ser (pS);

phospho-Thr (pT); phospho-Tyr (pY); phospho-His (pHis); phospho-Asp (pAsp). 206
 1. Phosphorylation
Most common: via Ser/Thr/Tyr side chain OH

Phosphate is added by Phosphorylating enzyme (Kinases)

- 518 human protein kinases
- 478 belong to a single family (similar catalytic domain)

Protein phosphorylation can

- Regulate protein functional activity
- Direct localization
- Signal Transduction Protein Signaling
- Cell Cycle
- Protein Trafficking
- Involved in almost all cellular processes

20
Kinase Description

AGC Containing PKA, PKG, PKC families

CAMK Calcium/calmodulin-dependent protein
kinase
CK1 Casein kinase 1
CMGC Containing CDK, MAPK, GSK3, CLK
families
STE Homologs of yeast Sterile 7, Sterile 11,
Sterile 20 kinases
TK Tyrosine kinase
TKL Tyrosine kinase-like

- 7 Distinct Types
- The kinase dendrograms show the sequence similarity between kinase catalytic
domains: the distance along the branches between two kinases is proportional to the
divergence between their sequences.
- Except TK all other kinases eg as Protein Kinase A (PKA) phosphorylate Ser/Thr
- AGC means these are PK of A, G or C family
- CMGC named after some family members
- STE; Homologs of the yeast STE7, STE11 and STE20 genes 208
20
 1. Phosphorylation

Phosphate group is removed from phospho protein by the action of

De-phosphorylating enzyme (called Phosphatases):

- 4 main types (Sequence, Structure and Catalytic function)

- ~150 human phosphatases
- Phosphoprotein phosphatase (PPP) family (largest)
- Protein phosphatase Mg2+/Mn2+-dependent (PPM) family
- Protein Tyr phosphatase (PTP) family
- Aspartate-based protein phosphatase (APP) family

209
 An Example of Effect of phosphorylation
P53 protein: A tumor suppressor protein
Mdm2: A negative regulator of p53
Mdm2
p53

210
 2. Acylation (Acetylation)
(Addition of R-CO- group where R= alkyl group like CH3)

Amino acids affected

Lys
Arg
Enzyme KAT/HAT/NAT: Lysine Acetyl Transferase / Histone Acetyl
Transferase / N-Acetyl Transferase;
HDAC: Histone Deacetylase (Removes acetyl group)

211
 Functional role of Acetylation
Histone Acetylation:
- Acetylation increases gene expression
-Activation of transcription

p53 Acetylation:
- p53 is a tumor suppressor gene
- Its activity is increased upon acetylation (via Lys: three sites)
- When all 3 sites of acetylation are blocked, p53 loses its tumor
suppressing property

212
 Histone Acetylation Regulates Gene Expression
HAT: Histone Acetyl Transferase; HDAC: Histone De-Acetylase)

21 213
3
 3. Lipidation (Addition of Fatty acid)
Example of Fatty acids: Palmitic Acid (Palmitoylation) and Myristic
acid (Myristoylation). Palmitoylation occurs mainly on the Cys side
chain via its SH group and ocassionaly via Ser/Thr side chain OH
group (Mainly membrane proteins)

Myristoylation happens to N-terminal of Gly-containing proteins

(of nascent type that is generated in situ).

+ NH2-Gly-containing protein
+ Enzyme:N-Myristoyl Transferase (NMT) (likes G-X-X-S/T)
or N Palmitoyl Transferase (NPT)
NH-CH2-CO -protein

NH-CH2-CO-protein 214
Myristoylation/Palmitoylation:
As protein synthesis is initiated with N-terminal Met residues, cotranslational hydrolysis of the
Met1–Gly2 bond by Methionine Aminopeptidase is a prerequisite for such modifications

215
Functional role of Fatty Acid Lipidation of Proteins

Palmitoylation is linked to the modification of particularly G-

protein coupled receptors and their cognate G-proteins, where it is
thought to have an important regulatory function.

It also regulates receptor phosphorylation and desensitization as

well as in G-protein membrane translocation

Myristoylation allows for weak protein-protein and protein-

lipid interactions and plays an essential role in membrane
targeting, Protein:Protein interactions and regulates signal
transduction pathways.
- Viral replication
- Oncogenesis
216
 4. Glycosylation (Glycation)

3 types
- N-Glycosylation via Asn side chain CO-NH2 group
which contains the motif:

Ser/Thr-X-Asn
- O-Glycosylation via Ser side chain OH group

- C-Glycosylation via Trp residue

Enzymes involved:
Glycosyl transferase (add sugar)
Glycosidase (remove sugar)
217
Arrows show
some typical
points of
attachment to
other sugar units
or protein side
chains (Ser or
Thr side chain or
Asn amide side
chains)

218
 5. Oxidation
(Addition of Oxygen or Hydroxyl group)

Pro

Lys

Asn

The above amino acids in proteins have been

shown to undergo oxidation during PTM
219
 5. Protein Hydroxylation
Enzyme-mediated hydroxylation that occur at non-nucleophilic sites in aminoacyl
side chains to generate 3-OH-Pro, 4-OH-Pro, 5-OH-Lys and 3-OH-Asn

Example: Collagen:
Enzyme involved is FeII-
dependent Mono-Oxygenase:
which led to
3-OH-Pro
4-OH-Pro
5-OH-Lys
3-OH-Asn
in collagen leading to its proper
maturation for fiber and muscle
action

220
 5. Protein hydroxylation

a) Hydroxylation of Pro and Asn residues in the HIF (Hypoxia Inducible Factor)-1a
subunit (low oxygen); b) interaction of the HO-Pro564 residue of HIF with the E3
ligase that will catalyze polyubiquitination of HIF. a-KG = Alpha keto glutaric acid
221
 6. Alkylation
Addition of an alkyl chain, the simplest being Methylation

Methylation occurs on Lys and Arg side chains

Methyl Tranferase Enzyme is

required

222
6. N-Methylations
Whereas C-, O-, and S methylations of protein side chains are known, the reactions of
most contemporary interest are the N-methylations of Lys and Arg side chains,
particularly on the same histone tails that are acetylated. 7 of the first 36 residues,
Arg2,17,26 and Lys4,9,27,36 of Histone H3 are known to be methylated by a family of
histone methyltransferases

N-methylations can occur at Arg2,17,26 and Lys4,9,27,36 of histone H3. Lys18 and Lys27 can
be acetylated and Ser10 and Ser28 phosphorylated.
Most cases, DNA methylation: Turn off genes 22
3
The size and hydrophobicity differences between monomethyl- and trimethyl
substituents on Lys side chains of Histone H3 enable selective recruitment of
proteins involved in transcriptional control. For example, trimethyl-Lys9 in H3
recruits partner protein HP1 by binding to its chromodomain as part of
transcription factor and co-activator protein complex assemblies

224
 6. Protein Alkylation
3 common alkyl groups transferred are the methyl (C1) (eg histone methylations
of Lys and Arg side chains or the C15 and C20 isoprenyl (Farnesyl and Geranyl
geranyl) groups

Alkyl groups transferred to protein side chains: the methyl group from S-
adenosylmethionine (SAM) is transferred most often to Lys and Arg side chains (although O-,
S-, and C-methylations of protein side chains are known); the two isoprenyl units transferred
by Protein Prenyltransferases to Cys side chains are the C15 (farnesyl) and the C20
(geranylgeranyl) groups from the corresponding prenyl diphosphate substrates. 225
 7. Prenylation
(Addition of isoprene derived farnesyl (C15) or geranyl geranyl (C20)
group
Prenylation
- It requires the action of two enzymes, Farnesyl
Transferase and Geranylgeranyltransferase
- The target protein contains the motif C-a-a-X, where a =
aliphatic amino acid
- Role in attachment to membrane
- Protein-Protein bending

Prenyl/Isopentinyl group
226
 8. Ubiquitination
(Addition of 76 amino acid protein Ubiquitin)
Addition of ubiquitin protein takes place via its C-terminal
Gly-Gly carboxyl group to amino terminal of Lys of specific
protein
Following attachment with Ubiquitin, the modified protein (i) is
directed to Proteosome machinary for degradation (ii) undergoes
change in cellular location, (iii) is affected in terms of its activity
and (iv) undergoes change in protein interactions.

MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG-COOH

Amino acid sequence of Ubiquitin protein

22
7
The ubiquitylation system
8. Ubiquitination system and mechanism (three ubiquitin
activating enzymes, E1, E2 and E3 are involved)

228
Recognition of Ub-tagged protein for chaperoning to proteasomes where the Ub
tag is retrieved by hydrolysis of the isopeptide link to the target protein; the
target protein is unfolded and threaded into the chamber of the proteasome. 22
a) 3D trace of the 76-
residue ubiquitin:
Lys29,48,63 side
chains on different
faces of ubiquitin
offer different
surfaces for tandem
conjugation of
growing
polyubiquityl
chains;
b) structure of a
tetraubiquityl unit,
the minimum chain
length to direct
polyubiquitylated
proteins to the
proteasome.

230
 9. Disulphide
(S-S bond formation via two free Cys residues within a protein chain)

Protein chain containing

two Cys residues linked
by S-S bond
CH2-S S -CH2

NH-CH-CO NH-CH-CO
Cys Cys

S-S bonded cyclic protein.

It can also lead to cross linking of one Cys of one protein to another
Cys of another protein 23
 9. S-S Bond Formation
One of the two main types of linkages serve to cross-link proteins, or portions of proteins,
covalently. By far the more common are disulfide links from oxidation of Cys residue thiolate
side chains. The cytoplasmic and nuclear compartments in eukaryotic cells are reducing
microenvironments, as reflected in the 100:1 ratio of the redox-active tripeptide glutathione
in reduced (GSH) to oxidized (GSSG) state. The high reducing ratio is maintained by the high
levels of NAD(P)H and enzymes, such as glutathione reductase and thioredoxin reductase.

Oxidation of thiolate side chains

of Cys:
a) oxidation of dithiols to
disulfides (for example via
sulfenic acid intermediates)
and reversible reduction
back to dithiols by
glutathione reductase
action;
b) Oxidation of Cys-S side chain
to S-nitrosyl-Cys by nitric
oxide (CNO).

Regeneration of the dithiol forms is mediated by thiol– disulfide interchange using reduced
glutathione or the lowmolecular-weight dithiol protein thioredoxin (TSH).The oxidized GSSG or
TSST are recycled at the expense of NADPH oxidation by thioredoxin reductase and glutathione
reductases. 23
 10. Sulphation

Transfer of 4 SO3 groups from PAPS to the phenolate oxygen atoms of 4 side
chains of Tyr residues at the N-terminal region of the CCR5 (Chemokine)
receptor during its passage through the secretory compartments to the cell
surface. Upon sulphation CCR5 becomes more active as a co-receptor to HIV.
233
 11. Nitration

Nitration has been found to occur in the aromatic ring of

Tyr residues (2 and/or 4 positions) of protein molecules.
It is a substitution of aromatic hydrogen by NO2 group.
Such type of modification has been observed in many
proteins of physiological interest, thereby imparting
crucial biological activity to the protein in question.

Consequences
Protein self aggregation
(Implicated in Dementia)
234
COMPETITION BETWEEN PTMs OF OPPOSING EFFECT
There is good evidence for competition between PTMs, with opposing functional
consequences for the target proteins. Two such examples involving competition between
ubiquitination and acetylation are 1) SMAD7 protein in TGFb signal transduction
pathway and 2) 5 Lys side chains near the C-terminus of the transcription factor p53.
The Lys e-NH2 residues can be acetylated or ubiquitinated and then extended to
polyubiquitin chains, leading to proteolytic removal of p53 or SMAD7. The acetylations
block ubiquitylations and consequently lengthen the lifetime of the proteins in cells.

235
Multistep modification of the Ras GTPase
The consecutive 4-step
modification of the C-termini
of Ras proteins
1) S-prenylation,
2) S-palmitoylation of
neighboring cysteine
residues,
3) Specific endoproteolytic
cleavage to reveal one of
the cysteines as the new C-
terminus
4) Methylation of that new C-
terminal carboxylate—
comprise the integrated
maturation process that
moves modified Ras to
membranes to dock with
its upstream protein kinase
partners
23
7 237
A FEW SELECTED
RARE “PTM’s”

238
 12. Citrullination
(also called Deimination)
§ Amino acid involved: Arginine

§ Gene expression, Cell differentiation of epithelial

terminus, Autoimmune disorders, Rheumatoid arthritis
and Cellular apoptosis (Cancer).

239
 13. Glycylation / Glutamylation / Tyrosination

§ Addition of Gly, Glu or Tyr to amino terminal of a Protein via

their carboxylic acid groups.

§ Possible role:
§ Spermiogenesis, Chromatin remodeling, Ciliary assembly,
Neurodegeneration and Cancer

240
 14. Iodination
§ Tyrosine (Iodine substituting for aromatic H)

• Thyroid function and Hormone release, Grave’s

disease & Thyrotoxicosis of nodular goiter.

241
 15. Pyroglutamic Acid

§ Cyclization of N-terminal Glutamic acid to form

Pyroglutamic acid

§ Possible role in dementia such as Alzheimer’s Disease,

May function in Glutamate storage and acts to oppose the
action of Glutamate

242
 16. Trans-glutamination
Formation of an isopeptide bond between γ-carboxamide groups
( -(C=O)NH2 ) of glutamine residue side chains and the ε-amino
groups ( -NH2 ) of lysine residue side chains with subsequent
release of ammonia (NH3).

Transglutaminase enzyme
Gln-(C=O)-NH2 + NH2-Lys → Gln-(C=O)-NH-Lys + NH3

Lead to deficiency of factor XIII (a rare genetic condition) predisposes to hemorrhage;

243
 PTM site:

http://www.geneinfinity.org/sp/sp_proteinptmodifs.html

244
Introduction
DNA Replication

ØReproduction is a property of all organisms.

ØDNA duplicates by a process called DNA
Replication.
ØThe DNA replication machinery is also used for
DNA repair.

2
4
5
 DNA Replication
q Organisms duplicate by asexual or sexual
reproduction

q Cells duplicate by cellular division

q The genetic material duplicates by DNA

replication

The machinery that replicates DNA is also

called into action in another capacity:

q To repair the genetic material after any damage

24
6
 DNA Replication

qThe two strands of the double

helix are held together by
hydrogen bonds between the
bases.
qWatson and Crick envisioned
that replication occurred by
gradual separation of the strands The original
of the double helix (zipper). Watson-Crick
qDNA replication takes place by proposal for the
separation of the strands of the replication of a
double helix, and synthesis of double-helical
two daughter strands molecule of DNA
complementary to the two
parental templates.

24
7
DNA Replication
Three alternate schemes of replication

Semiconservative: each
daughter duplex contains one
strand from the parent
structure

vSemiconservative Replication
q DNA replication is called semiconservative because half of the parent
structure is retained in each of the daughter duplexes.
q This model of DNA replication took over the other two models previously
considered: conservative and dispersive.

Progeny: Off-spring or descendent 24

8
 DNA Replication
Three alternate schemes of replication

Conservative: distinct
separation and
segregation of parental
and daughter strands.

vConservative Replication
q In conservative replication, the two original strands would remain together (after
serving as templates), as would the two newly synthesized strands.
q As a result, one of the daughter duplexes would contain only parental DNA, while
the other daughter duplex would contain only newly synthesized DNA.

24
9
DNA Replication
Three alternate schemes of replication

Dispersive: daughter duplexes

would contain strands that were
composites of old and new DNA.

vDispersive Replication
q In dispersive replication, the parental strands would be broken into fragments,
and the new strands would be synthesized in short segments.
q Old fragments and new segments would be joined together to form a complete
strand.

25
0
 DNA Replication
Validation of semiconservative replication

vThe Meselson and

Stahl experiments
supported the semi-
conservative model
of replication in
Bacterial cells.

vSemiconservative
replication was later
demonstrated in
Eukaryotic cells as
well.

DNA was extracted from bacteria at different stages, mixed with a concentrated CsCl soln and centrifuged
to equilibrium at high speed in an ultracentrifuge. Cs+1 ions have sufficient atomic mass to be affected by
the centrifugal force, and they form a density gradient during the centrifugation period with the lowest
concentration (lowest density) of Cs at the top of the tube and the greatest concentration (highest density) at
the bottom of the tube. https://www.youtube.com/watch?v=UvGCXtX5MAM 25
 In Meselson-Stahl experiment, the density of a DNA molecule is
directly proportional to the % 15N or 14N atoms it contains. If replication
is semi-conservative, one would expect that the density of DNA
molecules would decrease during culture in 14N-containing medium in
the manner shown in the upper set of centrifuge tubes. After one
generation, all DNA molecules would be 15N-14N hybrids, and their
buoyant density would be halfway between that expected for totally
heavy and totally light DNA.
As replication continued beyond the first generation, the newly
synthesized strands would continue to contain only light isotopes, and
two types of duplexes would appear in the gradients: those containing
15N–14N hybrids and those containing only 14N.
As the time of growth in the light medium continued, a greater and
greater percentage of the DNA molecules present would be light.
However, as long as replication continued semi-conservatively, the
original heavy parental strands would remain intact and present in hybrid
DNA molecules that occupy smaller and smaller % of total DNA
252
 DNA Replication
Validation of semiconservative replication

q Cultured mammalian
cells were allowed to
undergo replication in
Bromo-deoxyUridine
(BrdU), a compound
that is incorporated into
DNA in place of
Thymidine.
q After one round of
replication in BrdU,
both chromatids of each
chromosome contained Experimental demonstration of
semiconservative DNA replication
BrdU. in eukaryotes: Schematic diagram
and micrograph of mitotic
(J Taylor, Columbia U) chromosomes

25
 DNA Replication
Validation of semiconservative replication

q After two rounds of

replication in BrdU, one
chromatid of each
chromosome was
composed of two BrdU-
containing strands,
whereas the other
chromatid was a hybrid
consisting of a BrdU-
containing strand and a
Thymidine-containing Experimental demonstration
of semiconservative DNA
strand. replication in eukaryotes:
Schematic diagram and
micrograph of mitotic
chromosomes
BrdU:Beominated Deoxy
Ribonucleic Acid 25
 DNA Replication
Replication in bacteria

vReplication in Bacterial Cells

q Temperature-sensitive (ts) mutants

were used to identify the genes of
replication.
q Replication can be studied using in
vitro systems reconstituted from
purified cellular compounds.

vReplication Forks and Bidirectional

Replication

q Replication starts at the origin site,

where a number of proteins bind to
initiate replication.
q Replication proceeds bidirectionally.
q Replication forks are points where a
pair of replicating segments come
together and join the nonreplicated
segments.
Model of a bacterial chromosome
undergoing bidirectional replication
25
 DNA Replication
The unwinding problem

Unwinding strands cause torsional stress: Toposiomerase or Gyrase breaks

unseparated portion becomes more tightly and rejoins tightly coiled strand ahead of
wound. replication and thereby relieves tension.

v Unwinding the Duplex and Separating the Strands

§ Tension is built up as DNA begins unwinding , and the DNA becomes positively supercoiled.
§ DNA gyrase (Topoisomerase II) relieves the tension by changing the DNA into negatively
super-coiled DNA; uses ATP hydrolysis.

v Topoisomerases: enzymes that regulate the over-winding or under-winding of DNA. If left

unabated, this tension would eventually halt DNA replication Type I: Transient break in
one strand; Type II: Transient break in both strands of a DNA duplex. 25
6
 DNA Replication
Properties of DNA polymerase
v DNA polymerase enzyme is required for synthesis of new DNA strands by
assembling nucleotides.
v For Bacteria: 5 DNA Polymerases I, II, III, IV, & V
v “DNA Pol-III” (Also called “Holoenzyme”) is the major one

vThe Properties of DNA Polymerases

§ DNA polymerase is responsible for
synthesizing new DNA strands from a
DNA template.
§ DNA polymerase requires a primer
which provides the 3’ hydroxyl
terminus on which to add new
nucleotides.
§ Polymerization occurs in the 5’-to-3’
direction.
§ None of the three main DNA
polymerases in bacteria can initiate
DNA chains. Templates (right) and non-templates
(left) for DNA polymerase activity
25
 DNA Replication
Properties of DNA polymerase

Incorporation of
nucleotides onto
the 3’ end of a
growing strand
by DNA
polymerase,
which requires
Mg+2 ion for the
reaction.
P=Phosphate
group; S=Sugar
group

Ø During the polymerization reaction, the - OH group at the 3’ end of the primer carries out a
nucleophilic attack on the 5’ -phosphate of the incoming nucleoside triphosphate.

Ø The polymerase molecules responsible for construction of the two new strands of DNA both
move in a 3’-to-5’ direction along the template, and both construct a chain that grows from its
5’-P terminus
25
 DNA Polymerases
v Enzymes that synthesize new DNA strands
v Isolated by Kornberg (Sr) in 1950s (DNA polymerase I) from bacteria. It required
the presence of DNA and all 4 Deoxy-ribonucleosides Triphosphates [dATP (dA),
dGTP (dG), dTTP (dT) and dCTP (dC)] and also the original DNA strands that
serve as templates for the polymerization reaction .
v Single-stranded DNA circle cannot serve as a template for DNA polymerase because the
enzyme cannot initiate the formation of a DNA strand. Rather, it can only add nucleotides
to the 3’ OH terminus of an existing strand. The strand that provides the necessary 3’ OH
is called a primer. All DNA polymerases have these same two basic requirements.

259
 DNA Replication
Semi-discontinuous Replication

The two strands of a double

helix are synthesized by a
different sequence of events,
one growing toward the
replication fork and the other
growing away from it.

vSemi-discontinuous Replication
§ Both daughter strands are synthesized simultaneously.
§ The leading strand (in the direction of the replication fork movement) is
synthesized continuously.
§ The lagging strand (in the opposite direction of the replication fork movement) is
synthesized discontinuously.
26
 The strand that grows toward the fork can be
constructed by the continuous addition of nucleotides
to its 3’ end.

But how is the other strand synthesized ??

In fact, the strand that grows away from the

replication fork is synthesized in a discontinuous
manner, that is as fragments.

261
v Before the synthesis of a fragment can be initiated, a suitable
stretch of template must be exposed by movement of the
replication fork. Once initiated, each fragment grows away from
the replication fork toward the 5‘ end of a previously synthesized
fragment to which it is subsequently linked.
v Thus, the two newly synthesized strands of the daughter duplexes
are synthesized by very different processes. The strand that is
synthesized continuously is called the Leading Strand because its
synthesis continues as the replication fork advances. The strand
that is synthesized discontinuously is called the Lagging Strand
because initiation of each fragment must wait for the parental
strands to separate and expose additional template
v Both strands are synthesized simultaneously, so that the terms
leading and lagging may not be appropriate as first thought.
Because one strand is synthesized continuously and the other
discontinuously, replication is said to be Semi-discontinuous.
Discovered by Reiji Okazaki of Nagoya University, Japan 262
v If bacteria were incubated in [3H]-T for a few seconds and immediately
killed, most of the radioactivity could be found as part of small DNA
fragments 1000 to 2000 nucleotides.
v In contrast, if cells were incubated in the labeled DNA precursor for a minute or
two, most of the incorporated radioactivity became part of much larger DNA
molecules. These results indicated that a portion of the DNA was constructed in
small segments (later called Okazaki fragments) that were rapidly linked to
longer pieces that had been synthesized previously. The enzyme that joins the
Okazaki fragments into a continuous strand is called DNA ligase.

v How does the synthesis of each of these fragments begin when none of the DNA
polymerases are capable of strand initiation? Further studies revealed that
initiation is not accomplished by a DNA polymerase but, rather, by a distinct
type of RNA polymerase, called “Primase”, that constructs a short primer
composed of RNA, not DNA.

v The leading strand, whose synthesis begins at the origin of replication, is also
initiated by a Primase molecule. The short RNAs synthesized by the primase at
the 5 ‘-end of the leading strand and the 5’-end of each Okazaki fragment serve
as the required primer for the synthesis of DNA by a DNA polymerase.
263
The use of short RNA fragments as removable primers in initiating synthesis of each
Okazaki fragment of the lagging strand: The major steps are indicated in the drawing and
discussed in the text. The role of various accessory proteins in these activities is indicated.
 The Machinery Operating at the Replication Fork

v Replication involves more than incorporating nucleotides. Un-winding the

duplex and separating the strands require the aid of two proteins that bind to
the DNA, a Helicase (or DNA unwinding enzyme) and Single-Stranded
DNA-Binding (SSB) proteins.
v DNA helicases unwind a DNA duplex in a reaction that uses energy released by
ATP hydrolysis to move along one of the DNA strands, breaking the hydrogen
bonds that hold the two strands together and exposing the single-stranded
DNA templates.

v The Helicase moves along the DNA, catalyzing the ATP-driven unwinding
of the duplex. As the DNA is unwound, the strands are prevented from
reforming the duplex by SSB proteins.
v The Primase associated with the Helicase synthesizes the RNA primers (~
10 nucleotides in length) that begin each “Okazaki fragment”.
v The RNA primers are subsequently removed.

265
 DNA Replication
Replication fork machinery

The role of DNA

helicase, SSB
proteins, and primase
at the replication fork

vThe Machinery Operating at the Replication Fork

Ø Helicase and single-stranded DNA-binding (SSB) proteins unwind the parental
duplex and separate the two strands.
Ø Primase and helicase form a “primosome”, which processively moves along the
lagging-strand template.
Ø A single replisome synthesizes both leading and lagging strands.

26
6
 DNA Replication
DNA polymerase III (Holoenzyme)
(Major DNA-Polymerase for Bacteria)

Schematic representation of DNA

polymerase III holoenzyme organized
into several distinct components.

v As long as it is attached to a “β sliding clamp” (A ring shaped protein complex) ,

DNA polymerase can move processively from one nucleotide to the next.
v The assembly of the β clamp around the DNA requires a Clamp Loader, which is part of
the DNA polymerase III holoenzyme.
v Replisome: Consists of the “holoenzyme, helicase, SSBs, and primase”.
26
 DNA Polymerase for Eukaryotics

• Family X DNA Polymerases: Beta, Lambda, Sigma, and Mu

(MAJOR)

• Family B DNA Polymerases: Alpha, Delta, and Epsilon

• Family Y Polymerases: Eta, Iota, and Kappa

• Two Sub unit containing Polymerases: Rev1 and Zeta

268
 DNA Replication
Eukaryotic replication fork

vThe Eukaryotic Replication Fork

Ø Replication activities are similar in eukaryotes and prokaryotes.
Ø There are several DNA polymerases in eukaryotes.
Ø Eukaryotic DNA polymerases elongate in the 5’-to-3’ direction and require a
primer; some have 3’-to-5’ exonuclease activity.
26
 DNA Replication
High fidelity
High Fidelity means that DNA polymerase has a very low error rate in incorporating
correct nucleotides into the newly synthesized DNA strand during replication

v Ensuring High Fidelity during DNA

Replication
Ø The error rate of incorporation of an
incorrect nucleotide during DNA
replication is the spontaneous
mutation rate.
Ø Incorporation of a particular
nucleotide onto the end of growing
strand depends upon the geometry of
the base pair.
Ø Among the four possible incoming
nucleoside triphosphates, only one
forms a proper geometric fit with the
template, producing either an A-T or
a G-C base pair that can fit into a Geometry of proper (Top) and
binding pocket within the enzyme. mismatched (Bottom) base pairs. A-T and
G-C pairs have nearly identical geometry
(Size & Shape) 27
 DNA damage

The magnitude of spontaneous DNA alterations, or lesions, can

be appreciated from the estimate that each cell of a warm-
blooded mammal loses approximately 10,000 bases per day!
Failure to repair such lesions produces permanent
alterations, or mutations, in the DNA.

If the mutation occurs in a cell destined to become a

gamete, the genetic alteration may be passed on to the next
generation.

Mutations also have effects in somatic cells (i.e., cells that are
not in the germ line): they can interfere with transcription
and replication, lead to the malignant transformation of a
cell, or speed the process by which an organism ages.
271
DNA REPAIR
v Cells have arsenal for repairing damaged DNA

v Both prokaryotic and eukaryotic cells possess a variety of proteins that patrol vast
stretches of DNA, searching for subtle chemical modifications of DNA duplex.

v Humans, possess enzymes that can directly repair damage from cancer-producing
alkylating agents. Most repair systems require that a damaged section of the DNA
be excised and selectively removed.

v One of the great virtues of the DNA duplex is that each strand contains the information
required for constructing its partner. Consequently, if one or more nucleotides is
removed from one strand, the complementary strand can serve as a template for
reconstruction of the duplex.

v The repair of DNA damage in eukaryotic cells is complicated by the relative

inaccessibility of DNA within the folded chromatin fibers of the nucleus.

v As in the case of transcription, DNA repair involves the participation of

chromatin-reshaping machines, such as the histone modifying enzymes and
nucleosome remodeling complexes
272
 DNA DAMAGE
Types of damages and agents responsible

1. Breakage of backbone of DNA with Ionizing Radiation (eg.

X-ray, Gamma ray, Radioactive particles)
2. Structural alteration of bases of DNA by Reactive Chemicals
some of which may be produced by the cell itself (Superoxide
[O2]•−, Peroxides (O2-2), Hydroxyl radicals (OH), etc
3. When subjected to Ultraviolet Radiation, adjacent
pyrimidines on a DNA strand have a tendency to interact with
one another to form a covalent dimer
4. Even absorption of Thermal Energy generated by
metabolism is sufficient to split “A” and “G” bases from their
attachment to the sugars of the DNA backbone
27
3
 DNA DAMAGE
Nucleotide adduct upon UV light exposure

A pyrimidine dimer formed within a DNA duplex by UV irradiation

27
 Nucleotide Excision Repair (NER)
v NER operates by a cut-and- patch mechanism that removes a
variety of bulky lesions, including pyrimidine dimers and
nucleotides to which various chemical groups have become
attached.

v Two distinct NER pathways can be distinguished:

q A Transcription-Coupled Pathway in which the repair of a

template strand is thought to occur as the DNA is being
transcribed, and the presence of the lesion may be signaled by a
stalled RNA polymerase.

q Global Genomic Pathway slower and less efficient corrects DNA

strands in the remainder of the genome.
27
Nucleotide Excision Repair (NER)
(1) Damage recognition in the Global
Pathway is mediated by an XPC
(linked to Xeroderma Pigmentosum,
Complementation) - containing protein
complex (DNA repair protein),
whereas damage recognition in the
Transcription-Coupled Pathway is
thought to be mediated by a stalled
RNA polymerase in conjunction with a
CSB (linked to Cockayne Syndrome:
form of dwarfism) protein (DNA repair
protein);
(2) DNA strand separation (by XPB and
XPD proteins, two helicase subunits of
TFIIH- a huge protein);
(3) Incision by XPG on the 3’ side and the
XPF–ERCC1 complex on the 5‘ side)
(4) Excision,
(5) DNA repair synthesis
276
Base Excision Repair (BER) Step-1 (Recognition)

A separate excision repair system

operates to remove altered nucleotides
generated by reactive chemicals
present in the diet or produced by Step-2 (Cleavage of base)
metabolism. BER is initiated by a
DNA glycosylase (specific for altered
base) that recognizes the alteration Step-3 (Cleavage of sugar
phosphate bond by
endonuclease + DNA
C, Due to removal of polymerase )
amino group to form U
Step-4 (Removal of
sugar)
8-oxo-G, Due to
oxidation by O-
free radical Step-5 (Addition correct
nucleotide)

3-Me-A, Due to
Alkylation Step-6 (Joining)
277
 Base Excision Repair (BER)

Detecting damaged bases during BER

In Step 1, a DNA glycosylase (hOGG1: Human 8-Oxo Guanine Glycosylase) is
inspecting a nucleotide that is paired to a cytosine. In Step 2, the nucleotide is flipped out of
the DNA duplex. In this case, the base is an oxidized version of G, 8-oxoG, and it is able to
fit into the active site of the enzyme (Step 3) where it is cleaved from its attached sugar. The
subsequent steps in BER were shown previously. In Step 4, the extruded base is a normal G,
which is unable to fit into the active site of the glycosylase and is returned to the base stack.
Failure to remove oxoG would have resulted in a G-to-T mutation.

278
 Mis-Match Repair (MMR)

vCells can remove mismatched bases that are incorporated by the

DNA polymerase and escape the enzyme’s proof reading
exonuclease.

vA mismatched base pair causes a distortion in the geometry of

the double helix that can be recognized by a repair enzyme.

vBut how does the enzyme “recognize” which member of the

mismatched pair is the incorrect nucleotide? for a mismatch to be
re-paired after the DNA polymerase has moved past a site, it is
important that the repair system distinguish the newly
synthesized strand, which contains the incorrect nucleotide,
from the parental strand, which contains the correct nucleotide.
279
 Double-Strand Breakage (DSB) Repair
X-rays/gamma rays/radioactive particles are ionizing radiation since they generate
ions while passing through matter. Gamma rays pass through our bodies every minute.
These can collide with a fragile DNA and may break its both helix strands. This
may also be caused by certain chemicals, including “bleomycin” etc used in cancer
chemotherapy, and free radicals produced by normal cellular metabolism.
(a) In this simplified model of double-strand
break repair, the lesion (Step 1) is detected by a
heterodimeric, ring-shaped protein called Ku,
that binds to the broken ends of the DNA (Step
2). The DNA-bound Ku recruits another protein,
called DNA-PKcs, which is the catalytic subunit
of a DNA-dependent protein kinase (Step 3).
Most of the substrates phosphorylated by this
protein kinase have not been identified. These
proteins bring the ends of the broken DNA
together in such a way that they can be joined by
DNA ligase IV to re-generate an intact DNA
duplex (Step 4). The NHEJ pathway may also
nvolve the activities of nucleases and polymerases
(not shown) and is more error prone than is the
homologous recombination pathway of DSB
repair. 28
 The Consequences of DNA Repair Deficiencies
(1) Rare genetic disorder, XERODERMA PIGMENTOSUM (XP).
Patients with XP possess a deficient Nucleotide Excision Repair
(NER) system that cannot remove segments of DNA damaged by UV
radiation. As a result, persons are highly sensitive to sunlight; even
limited exposure can produce large numbers of dark-pigmented
spots.

(2) Cancer is one of the consequences of double-strand DNA breaks (by

Ionizing Radiations) that have either gone unrepaired or repaired
incorrectly. The most serious environmental hazard in this regard is
Rn, a radioactive isotope gas formed during the disintegration of
222

Uranium. A significant fraction of lung-cancer deaths in nonsmokers is

probably due to radon exposure.

(3) Cockayne syndrome (CS) is an inherited disorder characterized by

acute sensitivity to light, neurological dysfunction due to
Demyelination of neurons, and dwarfism, but no evident increase in
the frequency of skin cancer. Most cases of CS can be traced to a
mutation in one of two genes, either CSA or CSB, which are thought to
be involved in coupling transcription to DNA repair. Defect in “NER”
system.

(4) Premature (or accelerated) aging

DNA REPLICATION: SUMMARY

282 282
 Presentation Format
Background on Neurological disorders (NDs)

Alzheimer’s Disease (AD)

v Background
Ø Symptoms
Ø Statistics
Ø Diagnosis

v Understanding the disease

Ø Proteins/Enzymes implicated
Ø Research
Ø Biomarkers/Diagnostic
Ø Possible intervention strategy
Ø Treatment
283

http://www.med.harvard.edu/AANLIB/home.html
 Human Brain

3-1/2 lb (approx) of grey & white soft tissue

As many neurons in brain as there are stars in the milky way galaxy

Despite the glow from recent advances in brain science, we still find
ourselves squinting in the dark

However we are now beginning to grasp the crucial of brain function and
the “mysteries of neuroscience”

Even partial answers to some key questions could restructure our

understanding about this organ that defines who we are.
284
 Human Brain
Frontal Lobes Temporal Lobes
Planning Recognizing
Organizing
/ processing sound
Problem solving
Memory Understanding
Impulse control / producing speech
Decision making Various aspects of memory
Selective attention
Controlling our behavior
/ Emotions

Occipital Lobes Parietal Lobes

Receive/ process Integrate sensory information from
visual information Brain Stem various parts of the body
Sense of shapes and colors Breathing Contain the primary sensory cortex,
Heart rate which controls sensation (touch, hot
Cerebellum Blood pressure or cold, pain)
Balance Swallowing Up-down sense, Bumping into
Movement things while walking
Coordination 285
Dementia refers to a cluster of symptoms
caused by changes in brain function.
Memory loss is the hallmark of
dementia, but personality and behavior
changes are also common.
Difficulties with one or more following activities

Learning & retaining new information

Handling complex tasks

Ability to reason

Spatial ability and orientation

Language

Behavior 286
Alzheimer’s Disease:
Progression in the
eye of an artist

287
Most common NDs (~23)
Alzheimer’s Disease (AD): [loss of memory/cognition, visual hallucinations,
delusions/ depression, neurons death]
(Proteins: Amyloid and Tau)
Parkinson’s Disease (PD): [Loss of spontaneous movement, rigidity, muscle
stiffness, tremors and movement resistance]
(Protein: Parkin)
Lewis body Dementia (a variant of AD/PD): [abnormal structures in brain,
see things that are not there, 10% neurons die, most do not function]
(Protein: a-Synuclein)
Huntington’s Disease (HD): [uncontrolled movements, loss of intellectual
faculties, emotional disturbance, nerve cells/neurons degeneration, a familial]
(Protein: Polyglutamine protein >35).
Mad Cow Disease/Creutzfeldt-Jakob Disease (CJD): [holes in the
brain, spongiform encephalopathies, unsteady] (Prion protein)
ALS or Amyotrophic Lateral Sclerosis (Lou Gehrig’s Disease):
[Progressively paralyzed due to degeneration of the upper and lower motor neurons in
the brain and spinal cord. 80% die within 2-5 years]
Multiple System Atrophy (MSA)-Picks Disease: 288
 Common feature: Insoluble protein aggregates in brain

The electron microscope pictures showing the structures of various types of protein
aggregates found in the post mortal brain tissues of patients with neurological diseases.
289
Alzheimer’s Disease (AD)
Also called Senile Dementia Alzheimer's Type (SDAT), named after Alois
Alzheimer who found in 1906 abnormal “clumps" and “tangled bundles of fibers” in
the brain tissue of a woman with mental illness. These clumps and fibers are now
known as amyloid senile plaques (SPs) and neurofibrillary tangles (NFTs) found in
neocortex and hippocampus.

Symptoms:
Decline of cognitive functions like memory, thinking, comprehension,
calculation, language, learning capacity, judgment, decision-making ability,
attention, personality.

It is differentiated from age-related normal decline of cognitive functions which

is much less, gradual and leads to milder disabilities.

It develops and progresses rapidly. If it is slow, it will likely continue on a slow

pace.

The diagnosis of AD is based on characteristic symptoms. But it can only be

confirmed by microscopic examination of a sample of brain tissue after death.
290
 Neuropathology of AD in a patient’s brain

A: Brain cortex (low power). B: Amyloid plaque, central core-fibrils, surrounded by a

halo of rod-shaped dystrophic nerve (fibrils). C: Silver-stain neurofibrilary tangles.
D: Silver-stain neurofibrilary tangles (enlarged) 291

SS Sisidia et. al. Nature Reviews,3,281-290,2002

Progression of AD with
significant tissue loss

29
2
Two Proteins in AD

Amyloid beta (Ab) peptide derived from its precursor

protein called Amyloid Precursor Protein (APP). Ab
aggregate is present in plaque or clumps found in the
brain of AD patients

Tau protein (ι) also found as aggregate in the brain of

AD patients. This is present as fibrils around the plaque

29
3
Crucial events involved in AD-initiation and
disease progression

Production of Ab peptide from its precursor protein

(Amyloid Precurser Protein or APP)

Aggregation or self-association of Ab peptide to form

highly insoluble precipitate

Cleavage of tau protein to form a slightly truncated

(shorter) form found in high amount in AD

Hyper and / or Abnormal phosphorylation of tau protein

leading to aggregation and fibril (fine rod like structure)
formation

29
4
 Amyloid beta (Ab) Production
from APP in the brain

Three endoproteolytic enzymes are crucial

b Secretase
a Secretase
g Secretase

29
5
Amyloid Precursor Protein (APP: 770 amino acids long)

Beta Secretase Alpha Secretase Gamma Secretase

Enzyme Enzyme Enzyme: Two sites
within membrane

CH2O : Carbohydrates; SO4: Sulphate, PO4: Phosphate, KPI:

Kunitz Protease Inhibitory domain; Signal: Signal Peptide domain 29
6
 APP cleavage: Pathological cleavage or
Amyloidogenic Pathway
TM
1 18 671 687 711/713 770
H2 N COOH
Ab (40-42 aa)

b - secretase g - secretase
18 671 711/713 770

sAPP-b
Ab (40-42 aa)
TM: Transmembrane;
s: Soluble; aa: Amino acid
: Signal Peptide domain

29
7
Prog. Neurobiol. 2003, 70(1):1-32, Nature, 1999, 399, Suppl June 24
 Normal Cleavage of APP (Non pathological or
non-Amyloidogenic Pathway)
TM 711/713
1 18 671 687 770

H2 N COOH
Ab peptide

a - secretase g - secretase
18 687 711/713 770

sAPP- a
P3
(24-26 aa)
sAPPα plays important role in (i) neuronal plasticity/survival, (ii) protective against excitotoxicity; (iii)
regulates neural stem cell proliferation’ (iv) important for early CNS development and (v) is neuroprotective

Nature, 1999, Vol. 399. Supp. June 24 29

Mutations in APP
v More than 50 mutations were
detected which enhance APP
cleavage by beta-secretase

29
Ab 42-peptide and APP mutations

672 membrane 713

DAEFRHDSG YEVHHQKLVF FAEDVGSNKG AIIGLMVGGV VIA

Wild type (control)

b a g
661 718
IKTEEISEVK M DAEFRHDSG YEVHHQK LVF FAEDVGSNKG AIIGLMVGGV VIA TVIVI

Swedish mutant Membrane anchored

661 718
IKTEEISEVNL DAEFRHDSG YEVHHQK LVF FAEDVGSNKG AIIGLMVGGV VIA TVIVI

Enhanced cleavage APP Processing 30

By the enzyme 0
Possible steps in the formation of Amyloid Plaque

1. Formation of A-beta (40/42 mer) peptide in soluble

form from APP
ØEnhanced of b and / or g Secretase activities
ØLess a Secretase activity
ØCauses ?? (eg Mutation in APP, etc)

2. Self association of A-beta

Ø Formation of highly insoluble aggregates
Ø Potential causes ?? (Many)

30
 Potential Causes for Ab-aggregation
vHigh content of b-sheet structure
vHigh levels of metal ions (Copper, Zinc, Aluminum, Iron)
vExposure to neurotoxic chemicals
vPolyanions (negatively charged molecules eg Heparin)
vNon-ionic hydrophilic polymers (eg Dextran, Polyethylene glycol
etc)
vBacterial infection
vpH changes (low pH + low ions promote aggregation)
vCholesterol/lipid level (high level promote aggregation)
vFree radicals or Oxidation species (Reactive Oxygen Species or
ROS) (high level lead to aggregation)

30
 Identication of Secretases implicated in APP processing

v a-Secretase: ADAM-10, TACE/Tumor Necrosis Factor a-

Converting Enzyme (a member of ADAM family).

v b-Secretase: BACE-1, also called ASP-2 or Memapsin-2 (Most

Likely): (Transmembrane Aspartyl Protease)
Another BACE-2 (ASP-1, Memapsin-1 or DRAP) (Less likely)
[BACE: Basic Amino Acid Cleaving Enzyme]

v g-Secretase: Presenilin-l (?) (Produces A-beta 42)

v e1/2-Secretase: ??? (Produces A-beta 43, A-beta 40 two other
forms of Amyloid peptide)

In BACE-1 knock out mice: 90% reduction in Ab formation

* Two inhibitors of BACE-1: Reduce plaque formation
* X-ray structure of BACE-1complexed with inhibitor 30
Ann. Neurol. 2000, 47:249-253 3
3-Dimensional model structure
of Ab peptide

30
4
 3D model of Ab-42 in aqueous medium showing the presence of
High sheet content

Highly hydrophobic
region (middle part)
responsible for its self
assembly property
Internal
water

1DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA42

30
Helix (a; 3,10 and pi) v Protein Secondary structure is
analyzed by Circular Dichroism (CD)
spectrum study
b-sheet (3 antiparallel) CD spectral pattern of peptides with pure Helix,
Beta-sheet, Turn or Random structure

Turn
Antiparallel b-sheets

Ellipticity (mdeg)
Loop
Turn

Coiled-coil

Helix
b-hair pin (anti-parallel)
Other (coil etc)

Random
Wave length (nm) 30
6
 Mass spectra of Ab-42 peptide at 7 days in buffer pH 7.8
1DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA42 (MW ~ 4500 Da)

10000 20000 30000 40000

9,047 Full mass spectrum Mono 4,500, Sesqui 6,750

4,514 Di 9,000, Tri 13,500
Tetra 18,000, Penta 22,500
40 Hexa 27,000 Hepta 31,500
Octa 36,000 Nona 40,500
Deca 45,000 Undeca 49,500
Peak Intensity

13,699
20 18,271
22,800 27,385

20000 30000 40000

10000 Mass/charge
20000 30000 40000
10000 20000 30000 40000
13,699
12
22,800 Expanded Mass Spectrum
10 27,385
31,857
36,440

20000
20000 30000
30000 40000
40000 30
 Metal ion theory
vCu+2, Zn+2 , Al+3, Fe+3, Hg+2, and even Ca+2 are thought to play
important roles in Abeta-aggregation.

vAmong these ions, most studies indicate a connection between

accumulation of Cu+2 / Zn+2 and amyloid aggregation.

vStudies indicated that His-rich domain of Ab + other residues

(shown by circle) possess a strong affinity towards Cu+2. A model
structure of Ab (1-28) bound to copper has been accomplished.

Ab42
1DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA42

Molecular Weight (MW) = 4,500 Da

30
8
Examination of mortal brain tissues of AD patients indicated
a much higher level of oxidising metal ions such as
Copper (0.46-1.76 mg),
Zinc (0.12-0.93 mg),
Iron (0.24-3.51 mg) per mg tissue,
Aluminum (?) & Mercury (?)

Normal level (per mg tissue):

~ 0.004 mg (Copper)
~ 0.0722 mg (Zinc)
~ 0.019 mg (Iron)

Lovella MA et al J of Neurol Sci, 158 (1), 47-52, 1998

Rajan et al Life Sciences, 18 (4), 423-431, 1976
Gaeta,, A and Hider, RC, British J of Pharmacol 146 (8) 1041–1059, 2005 30
9
 Monomer
Ab1-28
Model of
coordination
sites of Cu+2 (1
Dimer
Ab 1-28 or 2 atoms
shown with
arrow) bound
to Ab 1-28
peptide
Square planar

Ab(1-42) : 1DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA42

31
Ab 1-28 0
Ab-42 peptide contains following important structural features

(i) (16K---A21) important for fibrilogenesis

(ii) (17LVFF20) hydrophobic core essential for self

aggregation of full-length b-amyloid peptide

(iii) (16KLVFF20) binds to full length Ab and prevents its

assembly into amyloid fibrils

(iv) 1D----K28 contains metal binding property that may

initiate metal induced aggregation
1 11 21 31 41
Ab-42 DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA
31
1
Various regions of APP, NT:Growth factor, CuBD: Cys-rich Cu binding, Acidic
domain; KP1: Kunitz Serine Protease 1 Inhibitory, OX2: Microglial inhibitory
peptide; Carbohydrate, TM: Transmembrane, Cyto: Cytosolic tail.
APP

31
2
Cu+2 binding of APP-N-terminal segment containing His147, His151,
Tyr168 and Met170

v APP Knock Out mice - Elevated free Cu ion level

v APP over-expressed mice - Reduced free Cu ion level
Cu+2
v APP - a major regulator of neuronal Cu homeostasis
v Cu-binding regulates Ab production
JBC, 2003, 278, 17401-17407 31
 Hypothesis

Model outlining the mechanism and biological consequences of CuBD-Cu

interaction:

(i) APP binds Cu+2 via CuBD, (ii) CuBD reduces Cu(II) to Cu(I), (iii) Dimerization
occurs, (iv) APP-Cu (I) complex promotes processing via non Ab pathway, (v)
Alternately APP-Cu(I) act as a Cu transporter to transport Cu (I) away from the tissue
for excretion via liver
31
4
Polyphenols are strong antioxidants that are
likely to prevent formation of amyloid aggregates
by protecting the peptides from oxidative
modification and damage and ultimately from
polymerization.

31
5
 Cholesterol connection
Cholesterol has been implicated in Abeta aggregation. The
fibrillogenesis of Ab(1-42) in the presence of cholesterol has been
investigated using aqueous suspensions of microcrystalline
cholesterol and cholesteryl acetate, globular particles of cholesteryl
oleate and other cholesterol derivatives.

In all cases, with the exception of cholesteryl oleate, considerable

potentiation of long smooth helical fibril formation noted, compared
to 20h 370C control samples containing Ab(1-42) alone.

31
6
 Lipid connection

Lipid membrane interaction of Ab is essential for its

neurotoxicity. Previous study revealed that membrane
insertion may provide a possible pathway by which Ab
prevents itself from aggregation and fibril formation. The
results show that Ab is surface active and can insert into
lipid monolayers.

31
7
 Neurotoxic
Chemicals
Cycasin
BMAA hepatotoxic & carcinogenic
glycoside of methyl
bN-methyl amino L-alanine azoxymethanol (MAM)
- Present in soil-dwelling Cyanobacteria
- Concentrate in plants and flying bats in Guam. The bats are traditional delicacy of the
indigenous CHAMORRO people, who suffer a high rate of a dementia-related disorders
(ALS-PARKINSONIUM).
- BMAA has been found in high level the brains of affected individuals.

Cox, PA et al (2005) Diverse taxa of cyanobacteria produc Je beta-N-methylamino-L-

alanine, a neurotoxic amino acid. Proc Natl Acad Sci USA. 102(14):5074-78.

Mortal brain tissues from 13 ALS, 12 AD, 8 HD patients vs 12 age-matched non-

neurological controls

-2-Fold increase in BMAA (ALS AND AD BUT NOT HD) but much less than the
brain tissues of Chamorro people
- BMMA triggers neuro-degeneration and possibly protein aggregation.
(Ref; Pablo et al Proc Nat Acad Sci, USA, 2009, 120 (4) 216-225. 31
8
CYANO BACTERIA (BLUE GREEN ALGAE) PRESENT ON OCEAN FLOOR

31
 CHAMORRO TRIBES

Amyotrophic Lateral Sclerosis-Parkinson Dementia Complex (ALS-PDC)

v In the 1950s, ALS/PDC prevalence ratios & death rates for Chamorro residents of Guam and
Rota were 50–100 x that of developed countries.
v No heritable or viral factors were found.
v A subsequent decline of ALS/PDC after 1955 led to the search for environmental agents.
v In addition to eating the CYCAD seeds directly, BMAA may have found its way into human
diets by way of Fruit Bats, a Chamorro delicacy. These bats feed on cycad seeds and
concentrate the toxin in their flesh. Three museum specimen bats, collected in Guam in
contained hundreds of times more BMAA gm/gm than cycad seeds
v Use of CYCAD seeds in food had decreased as the Chamorro became more Americanized.
v 400 per 100,000 to 22 per 100,000, since the bat population decreased. 32
0
The male cone of the most commonly grown species, The female cone of Cycas revoluta begins as a central
Cycas revoluta, which bears pollen pineapple-like form, then once pollinated, red seeds mature the
cone splits and a new whorl of foliage appears out the middle 32
 Reactive oxygen species (ROS)
Molecules like Hydrogen peroxide
Ions like the Hypochlorite ion
Radicals like the hydroxyl radical
The hydroxyl ion
Superoxide anion
Ferryl radical
Alkoxyl radical

H2O2, OCl−, OH-, OH−, O2−, Ferryl and Alkoxyl,

Reactive oxygen species in living systems: Source,
biochemistry, and role in human disease
B Halliwell, The American J of Med, 91, (3), Supplement 3, pS14-22, 1991

ROS Antioxidants

32
2
 Tau protein in AD

The Microtubule-Associated Protein (MAP) or Neurofibrillary

Tangle (NFT) or Paired Helical Filament (PHF) protein, more
commonly called “Tau” protein, mostly found in the axons of
neurons, in the cytosol and in association with plasma membrane
components is another hallmark protein of AD-pathology.

32
3
 Human Tau protein

vMicrotubules (MTs) play an important role in the maintenance of cell shape, cell
division, axonal transport, secretion, and receptor activity, and maintain their
functions with the help of microtubule-associated proteins (MAPs).
vMicrotubule associated protein Tau (MAPT) exist in human brain in 6
isoforms. The major form is 441 amino acid long.
vTau is composed of a 3 or 4 microtubule binding domains (MTBD), each
consisting of 31 or 32 amino acids.
vTau mutations (particularly in MT regions) have been associated with AD, Pick's
disease, and other dementias.
vIn AD brain, it dissociates from axonal MTs and aggregates abnormally to form
insoluble PHF which is linked to neuro-degeneration
vHyper/abnormal phosphorylations as well as Tyr-nitration

32
4
Tau-phosphorylation and MT-binding

32
 hTau (major form)

MT-1
PR-2

PR-3
PR-1

MT-2

MT-3
NP-1

NP-2

NP-3
MT-4
1 40 75 201 244 275 306 337 369 425 441

Microtubule Helix– blue

Alpha helix: 17.5%- blue (MT) Binding Sheet– red
Extended beta strand: 15.2% - red domains
Tau201-240 Coil-- purple
Random coil: 67.4%- purple
1 2 3 4

NP: ; PR: Phospho Rich domain (3 domains 1, 2, 3); MT: Microtubule

binding domain (4 domains 1, 2, 3, 4); 32
6
 Tau Protein and AD
v Tau-aggregation mediates via one or more of its 4 microtubule (MT) binding domains
(MT1, 2, 3, and 4) which is promoted by increased and/or abnormal phosphorylation at the
N-Terminal Phospho-Rich (PR) domain. More phosphorylated tau leads to enhanced
fibril formation and rapid progression.
v MT2 and MT3 peptides have high self-aggregation ability.
v There are 25 Ser/Thr phosphorylation sites some of which are implicated in tau
aggregation.
v Most Ser/Thr sites found in vivo are phosphorylated by Proline Dependent Kinases
(PDKs).
v Tau-nitration (via Tyr residue) through the action of Peroxy Nitrite (PN) is also linked to its
self aggregation.
v Cleavage by caspase-3 at (D-M-V-D) or Asp-Met-Val-Asp421 leads to more toxic product
and more neuronal cell death

PN
32
 Summary of AD pathogenesis (Ab path)

Amyloid protein precursor (APP), 770 amino acid Furin activates

Cleavages by Bace (b-secretase) enzyme Activates BACE
+ g1/2-secretase enzyme
AD Intervention Options
Soluble amyloid peptide (Ab42/40)
Ø Bace inhibitors
Specific agents/conditions Ø Secretase inhibitors
e.g. High Cu, Zn, Fe ions, Low pH, Ø Furin inhibitors
High sheet structure, High cholesterol/Lipid
Oxidising condition Ø Metal chelators
Ø Sheet breakers
Ab42/40) self-aggregation Ø Cholesterol/Lipid lowering
agents
Cross linking, Ø Antioxidents (polyphenols)
Ab binding agents
Ø Cross linking agents
Insoluble amyloid plaque, Ø Ab antibody
toxic to neuronal cells 32
 Summary of AD mechanism (Tau path)
Tau protein

Hyper or abnormal Cleavage by Caspase-3

phosphorylation by enzyme

Enhanced interaction N-terminal cleaved

Enhanced fibril fragment Tau (1-420 aa)
formation with Ab and aggregation)
(leads to neuronal
AD Intervention Options apoptosis)

v Caspase 3 inhibitor
v Anti-phosphorylating agent
v Phosphorylation regulator 32
9
Emerging strategies for AD intervention using
protein/enzyme as targets

Inhibitors against b/g sceretase

Inhibitors against Furin enzyme which activates BACE1
Anti-aggregating agents (metal chelators)
b-sheet blockers
Agents preventing tau phosphorylation
Anti-fibril agents
Ab-aggregate clearing agents

33
0
Other Key Molecules in AD pathology

v Acetyl Cholinesterase enzyme

v NMDA (N-Methyl D-Aspartate) Receptor

33
1
 Acetyl Cholinesterase and AD

Acetyl Choline (Ach) (Neurotransmitter: a chemical

messenger required for memory, judgement, thinking)
Acetyl Cholinesterase enzyme (AChE)

Acetic acid and Choline

Cholinergic neurons are the predominant class which degenerate in the early stages of AD
leading to a significant decrease of ACh. Certain brain cells release Ach which helps deliver
messages to other cells.

v AChE inhibitors (Galantamine, from plant; Rivastigmine and Donepezil) are approved drugs
for AD. Tacrine (1993) is rarely prescribed now because of side effects.

33
2
 NMDA receptor
Memantin/Namenda

NMDA
33
3
Bio markers for early detection of AD
Biomarkers From Recent Study

v Increased phospho-Tau level compared

Sample Source
to Total Tau (ratio), combined with
Invasive reduced Ab42 in CSF but not in plasma
(i) CSF or serum: Most Sensitive AD diagnostic
(Cerebrospinal fluid) marker
Very painful, Requires
lumber puncture
v Ratio of Ab42/Ab40 in CSF (High
Non-invasive indicates AD)
(i) Blood serum
(ii) Saliva v pTau/Total Tau ratio in saliva (High
(iii) Urine indicates AD)
(iv) Brain imaging

v Ratio of pTau181/Ab42 (High for AD)

33
Galasko and Golde Alzheimer’s Research & Therapy 2013, 5:10
4
 DRUGS FOR AD
CLIOQUINOL
(IODO CHLORO
HYDROXY QUINOLINE)

* Normally used to treat ear infections/indigestion (antiinfective agent, >100 years).

* Absorb Zn and Cu atoms that concentrate in the brains of Alzheimer’s sufferers
before dementia sets in and arrest onset of dementia.

v Plasma Ab42 level declined in treatment group, Zn up, Cu unchanged,

normal values, Zn = 9.4 mmol/L, Cu = 13 mmol/L
v Retained significantly more mental capacity
v Only 1.4% decline in their mental faculties
v Compare 8.9% decline with placebo treatment
v Twice as effective as the AD drug Donepezil

Prevents Zn from building up on the surface of brain. In mice it reversed the disease
Long term effect: May damage peripheral nerves and the nerves in the eye
33
Arch. Neurol. 60, 1685-1691, 2003 5
DRUGS FOR AD

v “Galantamine”
A basic natural product present in plant
used to treat for memory loss;
A powerful Cu-chelating agent

“Azurin”
Cu-binding blue protein from bacteria
(MW 14kDa), slows down Ab
aggregation

Both are “Neuro-protective agents”

Improves neuronal function

33
6