As the newly synthesized protein enters the
lumen of the endoplasmic reticulum, the newly synthesized protein comes across
a lot of proteins that assists in series of modifications. Some of the protein
acts as a chaperon and some of them helps them in correct folding and released
from the endoplasmic reticulum. One of the protein that helps in correct
folding is Protein disulfide isomerase. Protein disulphide isomerase  (PDI), is an enzyme found in the lumen of
endoplasmic reticulum and it catalyzes the disulfide bond formation, reduction
and isomerization of proteins that has been newly synthesized. It ensures
disulfides bond connect to proper cysteines and makes sure correct folding
takes place without any improper interactions.  If there are incorrect arrangements of
disulphides it rearranges. It belongs to the thioredoxin super family of redox
proteins. All member of PDI family have thioredoxin-like domain structure
characterized by the BaBaBaBBa fold. PDI is organized into four thioredoxin-like
domain abb’a’.  The a and a’ domain of
PDI are homologous to thioredoxin and each contain an independent active sites
and each has two cysteines in the sequence WCGHCK which is also mostly referred
as CXXC motif. The two cysteines cycle between the dithoil and disulfide
oxidation states. The a and a’  domain
reacts with thiols of newly synthesized proteins to confer disulfide
oxidoreducatase. The active site of a and a’ is the site where the disuphides
are introduced to protein substrates. These active sites are linked by the b
and b’ domain. The b and b’ domain are inactive domain and have a similar
sequence to each other. The b and b’ domain does not contain catalytically
active cysteines but they appear to act as spacer and structural, and are often
involved with protein substrate or substrate recruitment. The b and b’ domain
is the non-catalytic domain and have a lower sequence identity compared to the
catalytic domain a and a’. PDI structure also has  a short interdomain region between b’ and a’
domains known as the x-linker.


In eukaryotic, protein folding,
modification and quality control occurs in the endoplasmic reticulum. The
majority of proteins found in ER are dedicated to protein folding process. Modification
of protein begins from the moment translation starts and the modification
continues as it enters endoplasmic lumen until the very last moment, as it
exists the ER. Most of mammalian secretory and membrane are imported into the
ER cotranslationally. Most protein are targeted to the ER by signal sequence
and the timing of cleavage of the signal proteins depends on protein but commonly
within the first ~25 amino acids of protein.

As the newly synthesized polypeptide starts
to emerge from the ribosome, they have amino acid sequence, called a signal sequence,
which is at the amino terminus of polypeptide chain. Signal sequence is
recognized by signal recognition particle (SRP) and binds to signal sequence as
well as ribosome forming a complex.  When
SRP binds to signal sequence, it slows the translation in a process known as
elongation arrest and guides the whole complex to translocon composed of the
Sec61 ??? complex in the ER membrane. Once it reaches the
translocon, cotranslational translocation of the newly synthesized polypeptide
chain into the ER lumen occurs. The protein concentration in ER lumen is very
high giving the newly synthesised chain many opportunities for interaction with
the proteins.  It is at this stage, polypeptides
first encounter PDI, which assist in protein folding.


Protein folding and formation of disulphide bonds
helps the newly synthesized polypeptide to mature and function.  The disulphide bonds are important for the
stability of a final protein structure. If there is any mispairing of cysteine
residues, this will have an impact in achieving their native conformation and
will lead to misfolding. According to classic experiment, formation of
disulphide bonds is spontaneous process and the newly synthesized polypeptide
itself is sufficient to achieve the correct folding in vitro. But when it is
compared with other aspect of protein folding, disulphide linked folding are
slow and it is due to depending on the redox reaction. This led to
consideration that disulphide-linked folding is assisted in vivo.  Disulphide bonds are critical and the
formation process involves oxidation of protein thiols to form disulphide bonds
and as well as to rearrange non-native disulphide bonds.


For the disulphide bond to form, the environment needs
to be highly optimized for oxidative protein folding. If the environment is to
reducing, disulphide bond won’t form and if the environment is too oxidizing,
it can lead to polypeptides being trapped in misoxidized misfolded states. According
to one study which involved combination of genetic and biochemical studies
using the yeast Saccharomyces cerevisiae and more recently mammalian and plant
system, it has revealed the proteins involved and how they function and assist
in protein folding process. Genetic screens in yeast identified a ER membrane
associated protein Ero1p which is involved in oxidative folding. Ero1 is a ER
oxidoreductin which are found in the luminal face of the ER membrane. Ero1 and
PDI are important in pathway to forming protein disulphide bond formation in
eukaryotic endoplasmic reticulum.  When PDI
donates disulphide bond to newly synthesized polpeptides, it becomes reduced.
Ero1 re-oxidises the PDI. Ero1 generates disulphide bond bonds and transfers it
to soluble disulphide carrier PDI that then passes it to newly synthesized proteins.
This results in transfer of electrons knows as a series of direct
thiol-disulfide exchange reaction. Electron is transferred from substrate
protein to PDI to Ero1.


 There are two
types of ERO1 isoforms in human, Ero1-alpha 
and Ero1-Beta and they both lack a COOH-terminal tail which is made up
of ~127 amino acids required by yeast protein for membrane association. Yeast
only encodes a single Ero1p. Ero1-alpha is widely expressed and Ero1-beta is
abundantly expressed.  


In yeast, there are four homologues of PDI, which are
EUG1, MPD1, MPD2 and EPS1. Although in early context it stated, PDI interacts
with Ero1, not all homologue of PDI interacts with Ero1. Ero1 being able to
interact with several PDI variants but also at the same time not being able to
interact with rest of the PDI homologue suggest that Ero1 can discriminate
between PDI and its homologues. In yeast PDI1 is the only essential gene out of
homologues. Rest of the homologues which are EUG1,MPD1,MPD2 and EPS1 are non
essential.  However when these
nonessential homologues are overexpressed, they have been found to suppress the
harm caused by deletion of PDI1. In a experiment, MPD1 which is a yeast gene
was isolated and characterized. The MPD1 had a single disulphide isomerase
active site. According to results MPD1 was not necessary for growth but however
overexpression of MPD1 gene showed suppression of the maturation defect of
carboxypeptidase Y which is caused by PDI1 deletion. Mutation within the Ero1
can have a lot of impact on the oxidative pathway.If there is a mutation in
ERO1, it makes reductant DTT sensitive and the accumulation of proteins that
normally have disulphide bonds is reduced in the ER.. If the Ero1 isn’t
regulated properly it could generate high concentration of hydrogen peroxide,
which can have impact on cell viability. Also, if the ERO1 in yeast is
overexpressed due to mutant Ero1, it inhibits cell growth and in the human
mutant Ero1 can result in an unfolded protein response.


Also there are many PDI like protein and out of them
all ERp72 and ERp57 are expressed at similar levels as PDI. They both have a
CxxC sequences within their thioredoxin domain like PDI and they both were
regulated in cells separately and each case the expression of ERp72 and ERp57
was unaffected indicating that expression of ERp72 and ERp57 can be reduced
efficiently and specifically like PDI. ERp57 is the closet known homologue of
PDI. ERp57 interacts with lectin chaperone, calreticullin and calnexin to
assist protein modification. Calreticullin and calnexin are lectin chaperone
that bind to the monoglucosylated glycan, which are on newly synthesized
glycoproteins. PDI binds directly to substrate for reducatase or isomerase
activities whereas ERp57 doesn’t bind directly. To bind to substrate it needs
to associate with calreticullin to catalyse.


Yeast belongs to fungal family and yeast has been
targeted a lot for research regarding its protein mechanism. All fungal walls
are similarly structured. One fifth of the yeast genome are for the
biosynthesis of yeast cell wall. Yeast cell is made up of fibrous and gel like
carbohydrate polymers, which forms a tensile and strong core scaffold in which
more variety of proteins and other superficial components are added to make
them strong but still flexible and cell wall that is chemically diverse. There
are layers on top of layers and are covalently attached  beta-(1-3) glucan which is branched which consists
of 3 to 4% interchain and chitin. GLucan and chitin assemble into microfibrils
by forming a intrachain hydrogen bonds which form a basket like scaffold around
the cell. The internal hydrostatic pressure caused on the wall by cytoplasm and
membrane are resisted by the exoskeleton




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