ORGANELLE MARKERS - Endoplasmic Reticulum

ERp57

ERp57 (also known as Grp58), a member of the thioredoxin superfamily, is an ER protein with disulfide isomerase activity. It has been shown to increase after oncogenic transformation and to be a component of the MHC Class I peptide-loading complex.

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Grp78

Grp78, a 78 kDa glucose-regulated protein, also known as BiP or immunoglobulin heavy chain binding protein, is a stress-response
protein which is induced by agents or conditions that adversely affect endoplasmic reticulum (ER) function . This protein is essential for
the proper glycosylation, folding and assembly of many membrane bound and secreted proteins . Grp78 is critical for maintenance of cell homeostasis and the prevention of apoptosis . Grp78 protein levels have been shown to be a reliable biomarker of hypoglycermia as well as serving a neuroprotective function in neurons exposed to glutamate and oxidative stress .Grp78 levels are reduced in the brains of Alzheimer.s Disease patients and decreased expression of Grp78 is found associated with missense mutations in the human presenilin-1 (PS1) gene . The induction of the Grp78 protein has been associated with the development of drug-resistance to antitumor drugs.

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ERp72

Endoplasmic reticulum protein (ERp) 72, a 72 kDa protein localized to the endoplasmic reticulum, is a member of the protein disulfide isomerase (PDI) family of proteins . It contains three repeats of the thioredoxin-like regions that is postulated to represent three independently acting catalytic domains (CGHC) of PDI activity. ERp72 contains the sequence at its carboxyl terminus which serves as its ER retention signal. Together with other ER resident proteins such as BiP, GRP94, and PDI, they serve as the molecular chaperones for proper folding of newly translocated and glycolsylated proteins such as thyroglobulin (Tg) and human chorionic gonadotropin (hCG)-b. ERp72 expression is regulated by the level of misfolding proteins in the ER, as the amount of ERp72 increased in response to epithelial ischemia, a condition that perturbs the maturation of secretory proteins.

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Glucosidase II

a-glucosidase II is a 110 kDa glycosylated endoplasmic reticulum resident protein that is involved in trimming of glucose from newly synthesized glycosylated proteins in the endoplasmic reticulum (ER). Together with an 80 kDa ß subunit, these polypeptides remove two a-1,3-linked glucose from high mannose oligosaccharides linked to asparagine residue on glycoprotein . a-glucosidase II mediated glucose trimming is necessary for the interaction of substrates such as influenza virus hemagglutinin (HA) , MHC Class I , and T-cell antigen receptor (TCR) with calnexin, an ER resident chaperon that is specific for monoglucosylated glycoproteins.

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Glucose-related protein

Glucose-regulated protein 94, also known as Grp94 or gp96, is an abundant resident endoplasmic reticulum (ER) lumenal stress protein which together with cytosolic Hsp90 belongs to the Hsp90 family of molecular chaperones. Grp94 and other resident soluble proteins of the ER such as members of the Ca(2+) binding protein subfamily (CaBP), CaBPI and CaBP2 as well as calreticulin, possesses the COOH-terminal tetrapeptide Lys-Asp-Glu-Leu (KDEL) which is a sorting signal that is thought to lead to the retention of these proteins in the pre-Golgi compartments. Grp94 expression is upregulated by stress conditions such as glucose starvation and heat shock, which promote protein misfolding or unfolding. In addition to a homeostatic role in protein folding and assembly, Grp94 can function in the intracellular trafficking of peptides from the extracellular space to the MHC class I antigen processing pathway of antigen presentation cells. Grp94
and Hsp90 share high sequence identity and presumably identical adenosine nucleotide-dependent modes of regulation. But earlier data suggests that Hsp90 and Grp94 may differ in their nucleotide binding properties. The Nterminal domain of eukaryotic Hsp90 proteins contains a conserved adenosine nucleotide binding pocket which also serves as the binding site for the Hsp90 inhibitors geldanamycin and radicicol. However, the molecular basis for adenosine nucleotide-dependent regulation of Grp94 remains to be established. Recent data has identified a ligand dependent regulation of Grp94 function and suggest a model whereby Grp94 function is regulated through a ligand-dependent conversion of Grp94 from an inactive to an active conformation.

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Hsp25, Hsp27

Mouse Hsp25, human Hsp27, and aß-crystallin are part of a diverse family of small heat shock proteins (sHsps) which are produced in all organisms. The overall homology between the different sHsps is low and they are grouped together based on conserved sequences in the C-terminal half of the protein and short conserved phenylalanine-rich stretches near the N terminus. Mammalian sHsps are expressed constitutively under physiological conditions but stress factors such as heat shock induce a strong up-regulation of protein levels by 10-20-fold to maximum concentrations of 0.1% of the cellular protein. They function as chaperone-like proteins by binding unfolded polypeptides and preventing uncontrolled protein aggregation. sHsps all share the striking feature of forming high molecular weight oligomeric complexes of variable size. The quaternary structure of sHsps is essential for their function and regulation of activity but its basic properties are still rather poorly understood. Data indicates that Hsp25 is a dynamic tetramer of tetramers with a unique ability to refold and reassemble into its active quaternary structure after denaturation. Current studies demonstrate that Hsp25 helps facilitate the glutathione-redox cycle by enhancing glutathione utilization and maintaining the cellular glutathione pool in favor of the reduced states.

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PDI

The mammalian protein disulphide-isomerase (PDI) family encompasses several highly divergent proteins which are involved in the processing and maturation of secretory proteins in the ER by catalyzing the rearrangement of disulphide bonds. PDI, which is an abundant protein of the ER (>400uM), has a carboxy-terminal
retention signal sequence, KDEL, similar to that of BiP and Grp94. The PDI proteins are characterized by the presence of one or more domains of ~95-110 amino acids related to the cytoplasmic protein thioredoxin. All but the PDI-D subfamily are composed entirely of repeats of such domains, with at least one domain containing and one domain lacking a redox-active-Cys-Xaa-Xaa-Cystetrapeptide.

In addition to their roles as redox catalysts and isomerases, PDI proteins have other functions such as peptide binding, cell adhesion and perhaps chaperone activities. Platelet surface thiols and disulphides play an important role in platelet responses. Catalytically active PDI is found on platelet surfaces where it has been demonstrated to mediate platelet aggregation and secretion possible by reducing disulfide bonds thus leading to exposure of fibrinogen receptors in platelets.

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TAPI

TAP1, a 70kDa transmembrane protein, and TAP2 are two structurally related subunits of the transporter associated with antigen processing (TAP). The TAP complex is a member of the ATP binding cassette (ABC) family of transmembrane transporters. TAP1 and TAP2 each contain an N-terminal transmembrane region and Cterminal nucleotide binding domains (NBD). TAP1 and TAP2 form a complex in the endoplasmic reticulum (ER) membrane with the NBD oriented in the cytosol. The TAP transporter is an essential component of the MHC class I antigen presentation pathway by binding peptides in its cytosolic part and subsequently translocating the peptides into the lumen of the endoplasmic reticulum (ER) where assembly of MHC class I and pepide takes place. Assembly of MHC class I-ß2-micorglobulin (ß2-m) dimers in the ER involves 2 chaperones, calnexin which interacts with free class I heavy (H) chains and calreticulin which binds human class I-ß2 dimers prior to peptide loading. Calreticulin remains associated with at least a subset of class I molecules when they in turn bind to TAP . Polymorphic differences in MHC class I H chains can results in quantitative as well as qualitative differences in how they interact with peptide, ß2-m, calnexin, calreticulin, ERp57, TAP and Tapasin, a subunit of the TAP complex which binds to both TAP1 and MHC class I. Data obtained with Tapasin deletion mutants revealed that binding to TAP is mediated by the C-terminal region and that the N-terminal region is required to stabilize the MHC class I loading complex. The Tapasin gene is centromeric of HLA-DP locus between the HSET and HKE1.5 genes and within 500 kbp of the transporters associated with antigen processing, TAP1 and TAP2 genes. The localization of these genes within such a short distance of each other on the chromosome implies some regulatory or functional
significance.

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UDP-glucose

UDP-glucose: glycoprotein glucosyltransferase (UGGT) is a 170 kDa lumenal endoplasmic reticulum (ER) resident protein that catalyzes monoglucosylation of high mannose oligosaccharides. UGGT catalyzes the transfer of glucose from UDP-glucose to Man7-9GlcNAc2-moiety of unfolded glycoproteins in a Ca2+ dependent reaction. UGGT binds unfolded glycoprotein via two elements on its substrate: a N-acetylglucosamine unit on the oligosaccharide and hydrophobic amino acids that are exposed in the denatured conformation. It is postulated that UGGT serves as a sensor that is able to distinguish between folded and denatured protein in the ER.

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