Protein Mutations in Disease

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Protein Mutations in Disease

• Lecture 11, Medical Biochemistry

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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 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.

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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 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.

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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.

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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 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.

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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 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.

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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 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.

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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.
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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).

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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 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.

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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).

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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
  chloride transport 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.