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Lecture 11 Outline

Four examples of protein mutations that lead to

altered function and disease complications will

be discussed:

1. Sickle Cell Anemia

2. p53 Tumor Suppressor

3. Ras p21 Oncogene.

4. Cystic Fibrosis Transporter

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Protein Mutations Mutations to genes, and hence the resulting protein products of these genes,

can arise by many different mechanisms. These include 1) gene deletions, 2)frameshift mutations, 3) point mutations, or 4) damage to DNA, for example,by carcinogens, ultraviolet light and other forms of radiation, plus otherenvironmental factors. Some of these forms of mutations can be directlyinherited, especially the first three mechanisms. Environmental mutationscan be acquired as germ-line mutations in the parent and passed on tooffspring, or these can be acquired as somatic mutations (such as cancer).

Not all of these mutations result in identifiable defects in proteins, and

obviously a gene deletion will lead to a complete absence of a protein.

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p53 Tumor Suppressor

Mutations in the p53 tumor suppressor gene are found in over

50% of all human cancers, and it is the most prevalent mutation

found in human cancers. p53 is a tetrameric nuclearphosphoprotein found at low levels in normal cells, however

following DNA damage due to irradiation or other DNA

damaging treatments, the levels of p53 quickly increase. The

increased levels of p53 function in two distinct pathways of cellsurvival and cell death.

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p53 Tumor Suppressor FunctionsIn cells that are early in the cell cycle when damaged (at G1), p53 triggers a

checkpoint that blocks further progression through the cell cycle. This blockallows the cell time to repair the damaged DNA before progressing into the DNA

replication phase (S-phase) of the cycle. If the damaged cell had already been

committed to cell division (G2-M), then p53 acts to trigger a program of cell deat

termed apoptosis. Essentially, p53 acts to save cells that can be repaired, but also

triggers death of cells that have too much damage and prevents them frompotentially progressing towards uncontrolled, cancerous growth.

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p53 Function: Cell Cycle

Regulation and Apoptosis

Induction

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Genes Activated by p53

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p53 Gene Structure Map

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p53 Mutations p53 is able to regulate these processes by its capacity to bind to DNA and regulate

transcription of genes involved in apoptosis and cell cycle control. The most common

form of p53 mutations are single amino acid substitutions within the DNA bindingdomains. These mutations prevent p53 from binding DNA, and they still allow the

mutated subunit to bind with normal p53 monomers and prevent their DNA binding

functions. This form of mutation is termed dominant negative. The consequence for

cells carrying mutant p53 genes is that the normal target genes are not activated and the

cell no longer responds to growth regulation following DNA damage. This is why p53

is referred to as a tumor suppressor protein.

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Summary of p53 Functions

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p53 Mutation Structure/Function

Concepts

The main biochemical concept is the dominant

negative protein interaction that mutant p53 has

with other normal p53 monomers. As withhemoglobin, this highlights the importance of

subunit interactions in a multimeric protein: one

amino acid change in the DNA binding domains of

one p53 monomer can prevent the tetramer frombinding DNA and activating p53 responsive genes.

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p21 Ras Oncogene Ras is an example of a monomeric guanine nucleotide binding protein. It is a

plasma membrane protein that is a central regulatory point between extracellularsignalling molecules and their receptors, and intracellular mitogen activating

protein kinase (MAP kinase) pathways that are responsible for transmitting the

signal to the nucleus. Thus, activation of Ras directly results in the transmittance

of mitogenic signals to the nucleus. In most normal situations, this is a transient

activation event. Mutations in Ras found in different types of cancer result in apermanently active form of Ras. This can lead to constant cellular growth or

division signals that contribute to the unregulated growth of tumor cells.

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Schematic of the central

role Ras plays in the

response to multiplesignalling pathways.

Ras with altered

activity due to

mutations can causemany diverse cellular

and genetic effects,

most of which are

not desirable.

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Regulation of Ras Activity

The biological activity of Ras is dependent on the form of guaninenucleotide that is bound to it: GTP, active; GDP, inactive. Ras

interacts with two accessory protein, one termed GEF (guanine-

nucleotide exchange protein) and the other termed GAP (GTPase

activating protein). GEF acts to promote exchange of GDP bound in

the active-site of inactive Ras with GTP. The active Ras-GTP form is

inactivated by interaction with GAP which promotes the hydrolysis of

GTP to GDP (making Ras inactive).

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Ras Mutations: Activation

Most mutations characterized for Ras result in stabilization of the

GTP-bound, active form of Ras. Some mutations accomplish this by

decreasing the GTPase activity and increasing the nucleotide exchange

rate (loading of GTP), or by decreasing GTPase activity and

decreasing interactions with GAP (GTPase activating protein).

Mutated versions of the three known human Ras genes are found in

30% of all human cancers, but it varies with tumor type. Ras mutations

are highly prevalent in pancreatic (90%), lung (40%) and colorectal

(50%) carcinomas, but are rarely mutated in breast, ovarian and

cervical cancers.

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Ras Gene Structure Map

(Sites of most common Ras mutations)

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Mutant Ras Structure/Function

Concepts The mutant Ras examples highlight how mutations can

affect and modulate protein activity. These types of

mutations are unique in that they disrupt protein-protein

interactions, and change catalytic and binding activities

in the active site. It also highlights the importance of

transient protein-protein interactions in the mediation of

extracellular signalling pathways.

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Cystic Fibrosis

Cystic Fibrosis is an autosomal recessive genetic

disorder of the secretory processes of all exocrine

glands that affects both mucus secreting and sweatglands throughout the body. The primary physiological

defect is disregulation of chloride ion transport. The

clinical features of the disorder include recurrent

pulmonary infections, pancreatic insufficiency,

malnutrition, intestinal obstruction and male infertility.

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CFTR Mutations

In CF, the primary defect has been attributed to abnormal regulation ofepithelial chloride transport due to mutations in the cystic fibrosis

transmembrane conductance regulator (CFTR) gene. The protein product of

the CFTR gene has been shown to be a cyclic-AMP regulated chloride ion

transporter in the plasma membrane. Over 70% of the identified mutations

in the CFTR gene result in a protein that is lacking a critical phenylalanineresidue at position 508, termed F508 (deleted Phe-508).

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Proposed Structure of CFTR

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CFTR Mutation Effects Deletion of F508 results in a protein that can no longer fold properly, and it

is not translocated out of the endoplasmic reticulum (ER) to the Golgi

appartus due to incomplete glycosylation. This results in the protein being

targeted for degradation rather than transport to the cell surface where it

normally functions. Other mutations in CFTR have been found in the

nucleotide binding domain or in the membrane spanning domain responsible

for chloride ion conductance. These still result in malfunctioning chloridetransport and the disease complications associated with it.

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Normal secreted

and membrane

protein

trafficking

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Normal vs Mutant CFTR

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CFTR Structure/Function

Concepts Protein conformation is an important recognition factor for processing

and transport of membrane proteins from their site of synthesis in the

ER to the plasma membrane or other organelles. For CFTR, the missing

Phe-508 leads to a conformational change in the protein that prevents

normal glycosylation and transport out of the ER. Ironically, if this

mutant form of CFTR is expressed by itself and assayed in artificial

systems, the protein will still function to translocate chloride ions.

Thus, this mutation does not affect function, but rather critical structural

determinants responsible for correct protein localization.