M.

Prasad Naidu
MSc Medical Biochemistry, Ph.D,.
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
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.
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.
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
Induction
p53 Gene Structure Map
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.
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.
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.

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).
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.
(Sites of most common Ras mutations)
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 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.
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).

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
protein
trafficking
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.