[Show abstract][Hide abstract] ABSTRACT: Protein folding within the endoplasmic reticulum is assisted by molecular chaperones and folding catalysts that include members of the protein disulfide isomerase and peptidyl-prolyl isomerase families. In this report, we examined the contributions of the cyclophilin subset of peptidyl-prolyl isomerases to protein folding and identified cyclophilin C as an ER cyclophilin in addition to cyclophilin B. Using albumin and transferrin as models of cis-proline-containing proteins in human hepatoma cells, we found that combined knockdown of cyclophilins B and C delayed transferrin secretion but surprisingly resulted in more efficient oxidative folding and secretion of albumin. Examination of the oxidation status of ER protein disulfide isomerase family members revealed a shift to a more oxidized state. This was accompanied by a >5-fold elevation in the ratio of oxidized to total glutathione. This "hyperoxidation" phenotype could be duplicated by incubating cells with the cyclophilin inhibitor cyclosporine A, a treatment that triggered efficient ER depletion of cyclophilins B and C by inducing their secretion to the medium. To identify the pathway responsible for ER hyperoxidation, we individually depleted several enzymes that are known or suspected to deliver oxidizing equivalents to the ER: Ero1αβ, VKOR, PRDX4 or QSOX1. Remarkably, none of these enzymes contributed to the elevated oxidized to total glutathione ratio induced by cyclosporine A treatment. These findings establish cyclophilin C as an ER cyclophilin, demonstrate the novel involvement of cyclophilins B and C in ER redox homeostasis and suggest the existence of an additional ER oxidative pathway that is modulated by ER cyclophilins.
[Show abstract][Hide abstract] ABSTRACT: The transfer of oxidizing equivalents from the endoplasmic reticulum (ER) oxidoreductin (Ero1) oxidase to protein disulfide isomerase is an important pathway leading to disulfide formation in nascent proteins within the ER. However, Ero1-deficient mouse cells still support oxidative protein folding, which led to the discovery that peroxiredoxin IV (PRDX4) catalyzes a parallel oxidation pathway. To identify additional pathways, we used RNA interference in human hepatoma cells and evaluated the relative contributions to oxidative protein folding and ER redox homeostasis of Ero1, PRDX4, and the candidate oxidants quiescin-sulfhydryl oxidase 1 (QSOX1) and vitamin K epoxide reductase (VKOR). We show that Ero1 is primarily responsible for maintaining cell growth, protein secretion, and recovery from a reductive challenge. We further show by combined depletion with Ero1 that PRDX4 and, for the first time, VKOR contribute to ER oxidation and that depletion of all three activities results in cell death. Of importance, Ero1, PRDX4, or VKOR was individually capable of supporting cell viability, secretion, and recovery after reductive challenge in the near absence of the other two activities. In contrast, no involvement of QSOX1 in ER oxidative processes could be detected. These findings establish VKOR as a significant contributor to disulfide bond formation within the ER.
Molecular biology of the cell 04/2012; 23(11):2017-27. DOI:10.1091/mbc.E12-02-0102 · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this chapter we present the evidence that calnexin (CNX) and calreticulin (CRT) function as molecular chaperones to assist
in the folding and subunit assembly of the majority of Asn-linked glycoproteins that pass through the endoplasmic reticulum.
Mechanistic insights into how this function is accomplished have been provided through diverse approaches which include interfering
with the recognition of glycoproteins through CNX/CRT’s lectin site, expression of CNX/CRT and model substrates in heterologous
systems, gene disruption, and reconstitution of function with purified components in vitro. Furthermore, the domain organization
and locations of functional sites have been revealed through mutagenesis and the recent determination of the structure of
the ER luminal domain of CNX and a portion of CRT. The controversial issue of whether CNX/CRT function solely as lectins or
also as “classical” chaperones that recognize the unfolded polypeptide portion of glycoproteins is presented and the evidence
supporting current models is discussed in detail.
[Show abstract][Hide abstract] ABSTRACT: Calreticulin and calnexin are key components in maintaining the quality control of glycoprotein folding within the endoplasmic reticulum. Although their lectin function of binding monoglucosylated sugar moieties of glycoproteins is well documented, their chaperone activity in suppressing protein aggregation is less well understood. Here, we use a series of deletion mutants of calreticulin to demonstrate that its aggregation suppression function resides primarily within its lectin domain. Using hydrophobic peptides as substrate mimetics, we show that aggregation suppression is mediated through a single polypeptide binding site that exhibits a K(d) for peptides of 0.5-1 μM. This site is distinct from the oligosaccharide binding site and differs from previously identified sites of binding to thrombospondin and GABARAP (4-aminobutyrate type A receptor-associated protein). Although the arm domain of calreticulin was incapable of suppressing aggregation or binding hydrophobic peptides on its own, it did contribute to aggregation suppression in the context of the whole molecule. The high resolution x-ray crystal structure of calreticulin with a partially truncated arm domain reveals a marked difference in the relative orientations of the arm and lectin domains when compared with calnexin. Furthermore, a hydrophobic patch was detected on the arm domain that mediates crystal packing and may contribute to calreticulin chaperone function.
[Show abstract][Hide abstract] ABSTRACT: Calreticulin and calnexin are key components in maintaining the quality control of glycoprotein folding within the endoplasmic
reticulum. While their lectin function of binding monoglucosylated sugar moieties of glycoproteins is well documented, their
chaperone activity in suppressing protein aggregation is less well understood. Here, we use a series of deletion mutants of
calreticulin to demonstrate that its aggregation suppression function resides primarily within its lectin domain. Using hydrophobic
peptides as substrate mimetics, we show that aggregation suppression is mediated through a single polypeptide binding site
that exhibits a Kd for peptides of 0.5-1 μM. This site is distinct from the oligosaccharide binding site and differs from
previously identified sites of binding to thrombospondin and GABARAP. Although the arm domain of calreticulin was incapable
of suppressing aggregation or binding hydrophobic peptides on its own, it did contribute to aggregation suppression in the
context of the whole molecule. The high resolution X-ray crystal structure of calreticulin with a partially truncated arm
domain reveals a marked difference in the relative orientations of the arm and lectin domains when compared to calnexin. Furthermore,
a hydrophobic patch was detected on the arm domain that mediates crystal packing and may contribute to calreticulin's chaperone
Journal of Biological Chemistry 06/2011; · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Calnexin is an integral membrane protein of the endoplasmic reticulum (ER) that binds transiently to a wide array of newly synthesized membrane and secretory proteins. It also exhibits prolonged binding to misfolded or incompletely folded proteins. Recent studies have demonstrated that calnexin functions as a molecular chaperone to facilitate the folding and assembly of proteins in the ER. It is also a component of the quality control system that prevents proteins from progressing along the secretory pathway until they have acquired proper tertiary or quaternary structure. Most proteins that are translocated into the ER are glycosylated at Asn residues, and calnexin's interactions are almost exclusively restricted to proteins that possess this posttranslational modification. The preference for glycoproteins resides in calnexin's ability to function as a lectin with specificity for the GlC1Man9GlcNAc2 oligosaccharide, an early intermediate in the processing of Asn-linked oligosaccharides. Calnexin also has the capacity to bind to polypeptide segments of unfolded glycoproteins. Available evidence suggests that calnexin utilizes its lectin property during initial capture of a newly synthesized glycoprotein and that subsequent association (and chaperone function) is mediated through polypeptide interactions. Unlike other molecular chaperones that are soluble proteins, calnexin is an intrinsic component of the ER membrane. Its unique ability to capture unfolded glycoproteins through their large oligosaccharide moieties may have evolved as a means to overcome accessibility problems imposed by being constrained within a lipid bilayer.Key words: protein folding, molecular chaperones, calnexin, quality control, endoplasmic reticulum.
[Show abstract][Hide abstract] ABSTRACT: Protein folding within the endoplasmic reticulum occurs in conjunction with a complex array of molecular chaperones and folding catalysts that assist the folding process as well as function in quality control processes to monitor the outcome. In this review, we summarize recent advances in the calnexin/calreticulin chaperone system that is directed primarily toward Asn-linked glycoproteins, as well as the protein disulfide isomerase family of enzymes that catalyze disulfide formation, reduction, and isomerization. We highlight issues related to function and substrate specificity as well as the functional interplay between the two systems.
Current opinion in cell biology 11/2010; 23(2):157-66. DOI:10.1016/j.ceb.2010.10.011 · 8.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To examine the relationship between protein disulfide isomerase family members within the mammalian endoplasmic reticulum, PDI, ERp57, ERp72, and P5 were depleted with high efficiency in human hepatoma cells, either singly or in combination. The impact was assessed on the oxidative folding of several well-characterized secretory proteins. We show that PDI plays a predominant role in oxidative folding because its depletion delayed disulfide formation in all secretory proteins tested. However, the phenotype was surprisingly modest suggesting that other family members are able to compensate for PDI depletion, albeit with reduced efficacy. ERp57 also exhibited broad specificity, overlapping with that of PDI, but with preference for glycosylated substrates. Depletion of both PDI and ERp57 revealed that some substrates require both enzymes for optimal folding and, furthermore, led to generalized protein misfolding, impaired export from the ER, and degradation. In contrast, depletion of ERp72 or P5, either alone or in combination with PDI or ERp57 had minimal impact, revealing a narrow substrate specificity for ERp72 and no detectable role for P5 in oxidative protein folding.
Molecular biology of the cell 09/2010; 21(18):3093-105. DOI:10.1091/mbc.E10-04-0356 · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The calnexin cycle is a process by which glycosylated proteins are subjected to folding cycles in the endoplasmic reticulum lumen via binding to the membrane protein calnexin (CNX) or to its soluble homolog calreticulin (CRT). CNX and CRT specifically recognize monoglucosylated Glc(1)Man(9)GlcNAc(2) glycans, but the structural determinants underlying this specificity are unknown. Here, we report a 1.95-Å crystal structure of the CRT lectin domain in complex with the tetrasaccharide α-Glc-(1→3)-α-Man-(1→2)-α-Man-(1→2)-Man. The tetrasaccharide binds to a long channel on CRT formed by a concave β-sheet. All four sugar moieties are engaged in the protein binding via an extensive network of hydrogen bonds and hydrophobic contacts. The structure explains the requirement for glucose at the nonreducing end of the carbohydrate; the oxygen O(2) of glucose perfectly fits to a pocket formed by CRT side chains while forming direct hydrogen bonds with the carbonyl of Gly(124) and the side chain of Lys(111). The structure also explains a requirement for the Cys(105)-Cys(137) disulfide bond in CRT/CNX for efficient carbohydrate binding. The Cys(105)-Cys(137) disulfide bond is involved in intimate contacts with the third and fourth sugar moieties of the Glc(1)Man(3) tetrasaccharide. Finally, the structure rationalizes previous mutagenesis of CRT and lays a structural groundwork for future studies of the role of CNX/CRT in diverse biological pathways.
[Show abstract][Hide abstract] ABSTRACT: Class I molecules of the major histocompatibility complex play a vital role in cellular immunity, reporting on the presence of viral or tumor-associated antigens by binding peptide fragments of these proteins and presenting them to cytotoxic T cells at the cell surface. The folding and assembly of class I molecules is assisted by molecular chaperones and folding catalysts that comprise the general ER quality control system which also monitors the integrity of the process, disposing of misfolded class I molecules through ER associated degradation (ERAD). Interwoven with general ER quality control are class I-specific components such as the peptide transporter TAP and the tapasin-ERp57 chaperone complex that supply peptides and monitor their loading onto class I molecules. This ensures that at the cell surface class I molecules will possess mainly optimal peptides with a long half-life. In this review we discuss these processes as well as a number of strategies that viruses have evolved to subvert normal class I assembly within the ER and thereby evade immune recognition by cytotoxic T cells.
Seminars in Cell and Developmental Biology 07/2010; 21(5):512-9. DOI:10.1016/j.semcdb.2009.12.013 · 6.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The presentation of antigenic peptides by class I molecules of the major histocompatibility complex begins in the endoplasmic reticulum (ER) where the co-ordinated action of molecular chaperones, folding enzymes and class I-specific factors ensures that class I molecules are loaded with high-affinity peptide ligands that will survive prolonged display at the cell surface. Once assembled, class I molecules are released from the quality-control machinery of the ER for export to the plasma membrane where they undergo dynamic endocytic cycling and turnover. We review recent progress in our understanding of class I assembly, anterograde transport and endocytosis and highlight some of the events targeted by viruses as a means to evade detection by cytotoxic T cells and natural killer cells.
[Show abstract][Hide abstract] ABSTRACT: The production of erythrocytes requires the massive synthesis of red cell-specific proteins including hemoglobin, cytoskeletal proteins, as well as membrane glycoproteins glycophorin A (GPA) and anion exchanger 1 (AE1). We found that during the terminal differentiation of human CD34(+) erythroid progenitor cells in culture, key components of the endoplasmic reticulum (ER) protein translocation (Sec61alpha), glycosylation (OST48), and protein folding machinery, chaperones BiP, calreticulin (CRT), and Hsp90 were maintained to allow efficient red cell glycoprotein biosynthesis. Unexpected was the loss of calnexin (CNX), an ER glycoprotein chaperone, and ERp57, a protein-disulfide isomerase, as well as a major decrease of the cytosolic chaperones, Hsc70 and Hsp70, components normally involved in membrane glycoprotein folding and quality control. AE1 can traffic to the cell surface in mouse embryonic fibroblasts completely deficient in CNX or CRT, whereas disruption of the CNX/CRT-glycoprotein interactions in human K562 cells using castanospermine did not affect the cell-surface levels of endogenous GPA or expressed AE1. These results demonstrate that CNX and ERp57 are not required for major glycoprotein biosynthesis during red cell development, in contrast to their role in glycoprotein folding and quality control in other cells.
[Show abstract][Hide abstract] ABSTRACT: ERp57 is a thiol oxidoreductase that catalyzes disulfide formation in heavy chains of class I histocompatibility molecules. It also forms a mixed disulfide with tapasin within the class I peptide loading complex, stabilizing the complex and promoting efficient binding of peptides to class I molecules. Since ERp57 associates with the lectin chaperones calnexin and calreticulin, it is thought that ERp57 requires these chaperones to gain access to its substrates. To test this idea, we examined class I biogenesis in cells lacking calnexin or calreticulin or that express an ERp57 mutant that fails to bind to these chaperones. Remarkably, heavy chain disulfides formed at the same rate in these cells as in wild type cells. Moreover, ERp57 formed a mixed disulfide with tapasin and promoted efficient peptide loading in the absence of interactions with calnexin and calreticulin. These findings suggest that ERp57 has the capacity to recognize its substrates directly in addition to being recruited through lectin chaperones. We also found that calreticulin could be recruited into the peptide loading complex in the absence of interactions with both ERp57 and substrate oligosaccharides, demonstrating the importance of its polypeptide binding site in substrate recognition. Finally, by inactivating the redox-active sites of ERp57, we demonstrate that its enzymatic activity is dispensable in stabilizing the peptide loading complex and in supporting efficient peptide loading. Thus, ERp57 appears to play a structural rather than catalytic role within the peptide loading complex.
[Show abstract][Hide abstract] ABSTRACT: Calnexin is a Ca2+-binding transmembrane chaperone of the endoplasmic reticulum that recognizes Glc1Man5-9GlcNAc2 oligosaccharides on folding glycoproteins as well as non-native elements of the polypeptide backbone. This latter mode of recognition enables calnexin to suppress the aggregation of both glycosylated and nonglycosylated substrates. The luminal portion of calnexin (S-Cnx) consists of two domains, a globular lectin domain and an extended arm domain. To understand the function of these domains during the interaction of calnexin with non-native protein conformers, we tested deletion mutants of S-Cnx for their abilities to suppress the aggregation of nonglycosylated firefly luciferase. The arm domain alone exhibited no capacity to suppress aggregation. However, stepwise truncation of the arm domain in S-Cnx resulted in a progressive reduction in aggregation suppression potency to the point where the globular domain alone exhibited 25% potency. To characterize the polypeptide-binding site, we used hydrophobic peptides that were competitors of the ability of S-Cnx to suppress luciferase aggregation. Direct binding experiments revealed a single site of peptide binding in the globular domain (Kd = 0.9 microm) at a location distinct from the lectin site. Progressive truncation of the arm domain in S-Cnx had no effect on the binding of small peptides but reduced the binding affinity of S-Cnx for large, non-native protein substrates. Because protein substrates exhibited no binding to the isolated arm domain, our findings support a model in which calnexin suppresses aggregation through a polypeptide-binding site in its globular domain, with the arm domain enhancing aggregation suppression by sterically constraining large substrates.
[Show abstract][Hide abstract] ABSTRACT: Dying tumour cells can elicit a potent anticancer immune response by exposing the calreticulin (CRT)/ERp57 complex on the cell surface before the cells manifest any signs of apoptosis. Here, we enumerate elements of the pathway that mediates pre-apoptotic CRT/ERp57 exposure in response to several immunogenic anticancer agents. Early activation of the endoplasmic reticulum (ER)-sessile kinase PERK leads to phosphorylation of the translation initiation factor eIF2alpha, followed by partial activation of caspase-8 (but not caspase-3), caspase-8-mediated cleavage of the ER protein BAP31 and conformational activation of Bax and Bak. Finally, a pool of CRT that has transited the Golgi apparatus is secreted by SNARE-dependent exocytosis. Knock-in mutation of eIF2alpha (to make it non-phosphorylatable) or BAP31 (to render it uncleavable), depletion of PERK, caspase-8, BAP31, Bax, Bak or SNAREs abolished CRT/ERp57 exposure induced by anthracyclines, oxaliplatin and ultraviolet C light. Depletion of PERK, caspase-8 or SNAREs had no effect on cell death induced by anthracyclines, yet abolished the immunogenicity of cell death, which could be restored by absorbing recombinant CRT to the cell surface.
The EMBO Journal 02/2009; 28(5):578-90. DOI:10.1038/emboj.2009.1 · 10.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Calreticulin is a molecular chaperone of the endoplasmic reticulum that uses both a lectin site specific for Glc(1)Man(5-9)GlcNAc(2) oligosaccharides and a polypeptide binding site to interact with nascent glycoproteins. The latter mode of substrate recognition is controversial. To examine the relevance of polypeptide binding to protein folding in living cells, we prepared lectin-deficient mutants of calreticulin and examined their abilities to support the assembly and quality control of mouse class I histocompatibility molecules. In cells lacking calreticulin, class I molecules exhibit inefficient loading of peptide ligands, reduced cell surface expression and aberrantly rapid export from the endoplasmic reticulum. Remarkably, expression of calreticulin mutants that are completely devoid of lectin function fully complemented all of the class I biosynthetic defects. We conclude that calreticulin can use nonlectin-based modes of substrate interaction to effect its chaperone and quality control functions on class I molecules in living cells. Furthermore, pulse-chase coimmunoisolation experiments revealed that lectin-deficient calreticulin bound to a similar spectrum of client proteins as wild-type calreticulin and dissociated with similar kinetics, suggesting that lectin-independent interactions are commonplace in cells and that they seem to be regulated during client protein maturation.
Molecular biology of the cell 07/2008; 19(6):2413-23. DOI:10.1091/mbc.E07-10-1055 · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Calnexin is a membrane-bound chaperone of the endoplasmic reticulum (ER) that participates in the folding and quality control of newly synthesized glycoproteins. Binding to glycoproteins occurs through a lectin site with specificity for Glc1Man9GlcNAc2 oligosaccharides as well as through a polypeptide binding site that recognizes non-native protein conformations. The latter interaction is somewhat controversial because it is based on observations that calnexin can suppress the aggregation of non-glycosylated substrates at elevated temperature or at low calcium concentrations, conditions that may affect the structural integrity of calnexin. Here, we examine the ability of calnexin to interact with a non-glycosylated substrate under physiological conditions of the ER lumen. We show that the soluble ER luminal domain of calnexin can indeed suppress the aggregation of non-glycosylated firefly luciferase at 37 degrees C and at the normal resting ER calcium concentration of 0.4 mM. However, gradual reduction of calcium below the resting level was accompanied by a progressive loss of native calnexin structure as assessed by thermal stability, protease sensitivity, intrinsic fluorescence, and bis-ANS binding. These assays permitted the characterization of a single calcium binding site on calnexin with a Kd = 0.15 +/- 0.05 mM. We also show that the suppression of firefly luciferase aggregation by calnexin is strongly enhanced in the presence of millimolar concentrations of ATP and that the Kd for ATP binding to calnexin in the presence of 0.4 mM calcium is 0.7 mM. ATP did not alter the overall stability of calnexin but instead triggered the localized exposure of a hydrophobic site on the chaperone. These findings demonstrate that calnexin is a potent molecular chaperone that is capable of suppressing the aggregation of substrates through polypeptide-based interactions under conditions that exist within the ER lumen.
[Show abstract][Hide abstract] ABSTRACT: ERp57 is a thiol oxidoreductase of the endoplasmic reticulum that appears to be recruited to substrates indirectly through its association with the molecular chaperones calnexin and calreticulin. However, its functions in living cells have been difficult to demonstrate. During the biogenesis of class I histocompatibility molecules, ERp57 has been detected in association with free class I heavy chains and, at a later stage, with a large complex termed the peptide loading complex. This implicates ERp57 in heavy chain disulfide formation, isomerization, or reduction as well as in the loading of peptides onto class I molecules. In this study, we show that ERp57 does indeed participate in oxidative folding of the heavy chain. Depletion of ERp57 by RNA interference delayed heavy chain disulfide bond formation, slowed folding of the heavy chain alpha(3) domain, and caused slight delays in the transport of class I molecules from the endoplasmic reticulum to the Golgi apparatus. In contrast, heavy chain-beta(2)-microglobulin association kinetics were normal, suggesting that the interaction between heavy chain and beta(2) -microglobulin does not depend on an oxidized alpha(3) domain. Likewise, the peptide loading complex assembled properly, and peptide loading appeared normal upon depletion of ERp57. These studies demonstrate that ERp57 is involved in disulfide formation in vivo but do not support a role for ERp57 in peptide loading of class I molecules. Interestingly, depletion of another thiol oxidoreductase, ERp72, had no detectable effect on class I biogenesis, consistent with a specialized role for ERp57 in this process.
[Show abstract][Hide abstract] ABSTRACT: Calnexin and calreticulin are related proteins that comprise an ER chaperone system that ensures the proper folding and quality control of newly synthesized glycoproteins. The specificity for glycoproteins is conferred by a lectin site that recognizes an early oligosaccharide processing intermediate on the folding glycoprotein, Glc1Man9GlcNAc2. In addition, calnexin and calreticulin possess binding sites for ATP, Ca2+, non-native polypeptides and ERp57, an enzyme that catalyzes disulfide bond formation, reduction and isomerization. Recent studies have revealed the locations of some of these ligand-binding sites and have provided insights into how they contribute to overall chaperone function. In particular, the once controversial non-native-polypeptide-binding site has now been shown to function both in vitro and in cells. Furthermore, there is clear evidence that ERp57 participates in glycoprotein biogenesis either alone or in tandem with calnexin and calreticulin.
[Show abstract][Hide abstract] ABSTRACT: MHC class I molecules bind cytosolically derived peptides within the endoplasmic reticulum (ER) and present them at the cell surface to cytotoxic T cells. A major focus of our laboratory has been to understand the functions of the diverse proteins involved in the intracellular assembly of MHC class I molecules. These include the molecular chaperones calnexin and calreticulin, which enhance the proper folding and subunit assembly of class I molecules and also retain assembly intermediates within the ER; ERp57, a thiol oxidoreductase that promotes heavy chain disulfide formation and proper assembly of the peptide loading complex; tapasin, which recruits class I molecules to the TAP peptide transporter and enhances the loading of high affinity peptide ligands; and Bap31, which is involved in clustering assembled class I molecules at ER exit sites for export along the secretory pathway. This review describes our contributions to elucidating the functions of these proteins; the combined effort of many dedicated students and postdoctoral fellows.
Immunologic Research 02/2006; 35(1-2):151-62. DOI:10.1385/IR:35:1:151 · 3.10 Impact Factor