As the newly synthesized protein enters thelumen of the endoplasmic reticulum, the newly synthesized protein comes acrossa lot of proteins that assists in series of modifications. Some of the proteinacts as a chaperon and some of them helps them in correct folding and releasedfrom the endoplasmic reticulum.
One of the protein that helps in correctfolding is Protein disulfide isomerase. Protein disulphide isomerase (PDI), is an enzyme found in the lumen ofendoplasmic reticulum and it catalyzes the disulfide bond formation, reductionand isomerization of proteins that has been newly synthesized. It ensuresdisulfides bond connect to proper cysteines and makes sure correct foldingtakes place without any improper interactions.
If there are incorrect arrangements ofdisulphides it rearranges. It belongs to the thioredoxin super family of redoxproteins. All member of PDI family have thioredoxin-like domain structurecharacterized by the BaBaBaBBa fold.
PDI is organized into four thioredoxin-likedomain abb’a’. The a and a’ domain ofPDI are homologous to thioredoxin and each contain an independent active sitesand each has two cysteines in the sequence WCGHCK which is also mostly referredas CXXC motif. The two cysteines cycle between the dithoil and disulfideoxidation states. The a and a’ domainreacts with thiols of newly synthesized proteins to confer disulfideoxidoreducatase. The active site of a and a’ is the site where the disuphidesare introduced to protein substrates. These active sites are linked by the band b’ domain. The b and b’ domain are inactive domain and have a similarsequence to each other.
The b and b’ domain does not contain catalyticallyactive cysteines but they appear to act as spacer and structural, and are ofteninvolved with protein substrate or substrate recruitment. The b and b’ domainis the non-catalytic domain and have a lower sequence identity compared to thecatalytic 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.
Themajority of proteins found in ER are dedicated to protein folding process. Modificationof protein begins from the moment translation starts and the modificationcontinues as it enters endoplasmic lumen until the very last moment, as itexists the ER. Most of mammalian secretory and membrane are imported into theER cotranslationally. Most protein are targeted to the ER by signal sequenceand the timing of cleavage of the signal proteins depends on protein but commonlywithin the first ~25 amino acids of protein. As the newly synthesized polypeptide startsto emerge from the ribosome, they have amino acid sequence, called a signal sequence,which is at the amino terminus of polypeptide chain. Signal sequence isrecognized by signal recognition particle (SRP) and binds to signal sequence aswell as ribosome forming a complex. WhenSRP binds to signal sequence, it slows the translation in a process known aselongation arrest and guides the whole complex to translocon composed of theSec61 ??? complex in the ER membrane. Once it reaches thetranslocon, cotranslational translocation of the newly synthesized polypeptidechain into the ER lumen occurs.
The protein concentration in ER lumen is veryhigh giving the newly synthesised chain many opportunities for interaction withthe proteins. It is at this stage, polypeptidesfirst encounter PDI, which assist in protein folding. Protein folding and formation of disulphide bondshelps the newly synthesized polypeptide to mature and function. The disulphide bonds are important for thestability of a final protein structure.
If there is any mispairing of cysteineresidues, this will have an impact in achieving their native conformation andwill lead to misfolding. According to classic experiment, formation ofdisulphide bonds is spontaneous process and the newly synthesized polypeptideitself is sufficient to achieve the correct folding in vitro. But when it iscompared with other aspect of protein folding, disulphide linked folding areslow and it is due to depending on the redox reaction.
This led toconsideration that disulphide-linked folding is assisted in vivo. Disulphide bonds are critical and theformation process involves oxidation of protein thiols to form disulphide bondsand as well as to rearrange non-native disulphide bonds. For the disulphide bond to form, the environment needsto be highly optimized for oxidative protein folding. If the environment is toreducing, disulphide bond won’t form and if the environment is too oxidizing,it can lead to polypeptides being trapped in misoxidized misfolded states. Accordingto one study which involved combination of genetic and biochemical studiesusing the yeast Saccharomyces cerevisiae and more recently mammalian and plantsystem, it has revealed the proteins involved and how they function and assistin protein folding process. Genetic screens in yeast identified a ER membraneassociated protein Ero1p which is involved in oxidative folding. Ero1 is a ERoxidoreductin which are found in the luminal face of the ER membrane.
Ero1 andPDI are important in pathway to forming protein disulphide bond formation ineukaryotic endoplasmic reticulum. When PDIdonates disulphide bond to newly synthesized polpeptides, it becomes reduced.Ero1 re-oxidises the PDI. Ero1 generates disulphide bond bonds and transfers itto soluble disulphide carrier PDI that then passes it to newly synthesized proteins.This results in transfer of electrons knows as a series of directthiol-disulfide exchange reaction. Electron is transferred from substrateprotein to PDI to Ero1. There are twotypes of ERO1 isoforms in human, Ero1-alpha and Ero1-Beta and they both lack a COOH-terminal tail which is made upof ~127 amino acids required by yeast protein for membrane association. Yeastonly encodes a single Ero1p.
Ero1-alpha is widely expressed and Ero1-beta isabundantly expressed. In yeast, there are four homologues of PDI, which areEUG1, MPD1, MPD2 and EPS1. Although in early context it stated, PDI interactswith Ero1, not all homologue of PDI interacts with Ero1. Ero1 being able tointeract with several PDI variants but also at the same time not being able tointeract with rest of the PDI homologue suggest that Ero1 can discriminatebetween PDI and its homologues.
In yeast PDI1 is the only essential gene out ofhomologues. Rest of the homologues which are EUG1,MPD1,MPD2 and EPS1 are nonessential. However when thesenonessential homologues are overexpressed, they have been found to suppress theharm caused by deletion of PDI1. In a experiment, MPD1 which is a yeast genewas isolated and characterized. The MPD1 had a single disulphide isomeraseactive site.
According to results MPD1 was not necessary for growth but howeveroverexpression of MPD1 gene showed suppression of the maturation defect ofcarboxypeptidase Y which is caused by PDI1 deletion. Mutation within the Ero1can have a lot of impact on the oxidative pathway.If there is a mutation inERO1, it makes reductant DTT sensitive and the accumulation of proteins thatnormally have disulphide bonds is reduced in the ER.. If the Ero1 isn’tregulated properly it could generate high concentration of hydrogen peroxide,which can have impact on cell viability. Also, if the ERO1 in yeast isoverexpressed due to mutant Ero1, it inhibits cell growth and in the humanmutant Ero1 can result in an unfolded protein response.
Also there are many PDI like protein and out of themall ERp72 and ERp57 are expressed at similar levels as PDI. They both have aCxxC sequences within their thioredoxin domain like PDI and they both wereregulated in cells separately and each case the expression of ERp72 and ERp57was unaffected indicating that expression of ERp72 and ERp57 can be reducedefficiently and specifically like PDI. ERp57 is the closet known homologue ofPDI.
ERp57 interacts with lectin chaperone, calreticullin and calnexin toassist protein modification. Calreticullin and calnexin are lectin chaperonethat bind to the monoglucosylated glycan, which are on newly synthesizedglycoproteins. PDI binds directly to substrate for reducatase or isomeraseactivities whereas ERp57 doesn’t bind directly. To bind to substrate it needsto associate with calreticullin to catalyse.
Yeast belongs to fungal family and yeast has beentargeted a lot for research regarding its protein mechanism. All fungal wallsare similarly structured. One fifth of the yeast genome are for thebiosynthesis of yeast cell wall. Yeast cell is made up of fibrous and gel likecarbohydrate polymers, which forms a tensile and strong core scaffold in whichmore variety of proteins and other superficial components are added to makethem strong but still flexible and cell wall that is chemically diverse. Thereare layers on top of layers and are covalently attached beta-(1-3) glucan which is branched which consistsof 3 to 4% interchain and chitin.
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