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Protein Mutations in Disease. Lecture 11, Medical Biochemistry. 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.

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protein mutations in disease

Protein Mutations in Disease

Lecture 11, Medical Biochemistry

lecture 11 outline
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
protein mutations
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 other environmental factors. Some of these forms of mutations can be directly inherited, especially the first three mechanisms. Environmental mutations can be acquired as germ-line mutations in the parent and passed on to offspring, 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.
p53 tumor suppressor
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 nuclear phosphoprotein 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 cell survival and cell death.
p53 tumor suppressor functions
p53 Tumor Suppressor Functions
  • In 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 block allows 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 death, 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 from potentially progressing towards uncontrolled, cancerous growth.

p53 Function: Cell Cycle

Regulation and Apoptosis


p53 mutations
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 binding domains. 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.
p53 mutation structure function concepts
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 with hemoglobin, 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 from binding DNA and activating p53 responsive genes.
p21 ras oncogene
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 extracellular signalling 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 a permanently active form of Ras. This can lead to constant cellular growth or division signals that contribute to the unregulated growth of tumor cells.

Schematic of the central

role Ras plays in the

response to multiple

signalling pathways.

Ras with altered

activity due to

mutations can cause

many diverse cellular

and genetic effects,

most of which are

not desirable.

regulation of ras activity
Regulation of Ras Activity
  • The biological activity of Ras is dependent on the form of guanine nucleotide 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).
ras mutations activation
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.
ras gene structure map
Ras Gene Structure Map

(Sites of most common Ras mutations)

mutant ras structure function concepts
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.
cystic fibrosis
Cystic Fibrosis
  • Cystic Fibrosis is an autosomal recessive genetic disorder of the secretory processes of all exocrine glands that affects both mucus secreting and sweat glands 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.
cftr mutations
CFTR Mutations
  • In CF, the primary defect has been attributed to abnormal regulation of epithelial 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 phenylalanine residue at position 508, termed DF508 (deleted Phe-508).
cftr mutation effects
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 chloride transport and the disease complications associated with it.

Normal secreted

and membrane



cftr structure function concepts
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.