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David B. Williams - One of the best experts on this subject based on the ideXlab platform.
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In Vitro Assays of the Functions of Calnexin and Calreticulin, Lectin Chaperones of the Endoplasmic Reticulum
Methods in molecular biology (Clifton N.J.), 2006Co-Authors: Breanna S. Ireland, Monika Niggemann, David B. WilliamsAbstract:Calnexin and calreticulin are molecular chaperones of the endoplasmic reticulum (ER) whose folding-promoting functions are directed predominantly toward aspargine-linked glycoproteins. This is a consequence of Calnexin and calreticulin being lectins with specificity for the early oligosaccharide (OS)-processing intermediate, Glc1Man9GlcNAc2. In addition, they interact with non-native conformers of glycoprotein polypeptide chains to prevent aggregation and recruit the thiol oxidoreductase ERp57 to catalyze glycoprotein disulfide formation/isomerization. In vitro assays of these functions have contributed greatly to our understanding of how Calnexin and calreticulin promote glycoprotein folding. This chapter describes the isolation of Glc1Man9GlcNAc2 OS, as well as the assay used to measure OS binding. Furthermore, details are provided of assays that detect ERp57 binding by Calnexin and calreticulin, as well as the abilities of these chaperones to suppress the aggregation of non-native protein substrates.
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lectin deficient Calnexin is capable of binding class i histocompatibility molecules in vivo and preventing their degradation
Journal of Biological Chemistry, 2004Co-Authors: Michael R Leach, David B. WilliamsAbstract:Abstract Calnexin is a membrane-bound lectin of the endoplasmic reticulum (ER) that binds transiently to newly synthesized glycoproteins. By interacting with oligosaccharides of the form Glc1Man9GlcNAc2, Calnexin enhances the folding of glycoprotein substrates, retains misfolded variants in the ER, and in some cases participates in their degradation. Calnexin has also been shown to bind polypeptides in vivo that do not possess a glycan of this form and to function in vitro as a molecular chaperone for nonglycosylated proteins. To test the relative importance of the lectin site compared with the polypeptide-binding site, we have generated six Calnexin mutants defective in oligosaccharide binding using site-directed mutagenesis. Expressed as glutathione S-transferase fusions, these mutants were still capable of binding ERp57, a thiol oxidoreductase, and preventing the aggregation of a nonglycosylated substrate, citrate synthase. They were, however, unable to bind Glc1 Man9GlcNAc2 oligosaccharide and were compromised in preventing the aggregation of the monoglucosylated substrate jack bean α-mannosidase. Two of these mutants were then engineered into full-length Calnexin for heterologous expression in Drosophila cells along with the murine class I histocompatibility molecules Kb and Db as model glycoproteins. In this system, lectin site-defective Calnexin was able to replace wild type Calnexin in forming a complex with Kb and Db heavy chains and preventing their degradation. Thus, at least for class I molecules, the lectin site of Calnexin is dispensable for some of its chaperone functions.
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Localization of the lectin, ERp57 binding, and polypeptide binding sites of Calnexin and calreticulin
The Journal of biological chemistry, 2002Co-Authors: Michael R Leach, David Y Thomas, Myrna F. Cohen-doyle, David B. WilliamsAbstract:Abstract Calnexin and calreticulin are membrane-bound and soluble chaperones, respectively, of the endoplasmic reticulum (ER) which interact transiently with a broad spectrum of newly synthesized glycoproteins. In addition to sharing substantial sequence identity, both Calnexin and calreticulin bind to monoglucosylated oligosaccharides of the form Glc1Man5–9GlcNAc2, interact with the thiol oxidoreductase, ERp57, and are capable of acting as chaperones in vitro to suppress the aggregation of non-native proteins. To understand how these diverse functions are coordinated, we have localized the lectin, ERp57 binding, and polypeptide binding sites of Calnexin and calreticulin. Recent structural studies suggest that both proteins consist of a globular domain and an extended arm domain comprised of two sequence motifs repeated in tandem. Our results indicate that the primary lectin site of Calnexin and calreticulin resides within the globular domain, but the results also point to a much weaker secondary site within the arm domain which lacks specificity for monoglucosylated oligosaccharides. For both proteins, a site of interaction with ERp57 is centered on the arm domain, which retains ∼50% of binding compared with full-length controls. This site is in addition to a Zn2+-dependent site located within the globular domain of both proteins. Finally, Calnexin and calreticulin suppress the aggregation of unfolded proteins via a polypeptide binding site located within their globular domains but require the arm domain for full chaperone function. These findings are integrated into a model that describes the interaction of glycoprotein folding intermediates with Calnexin and calreticulin.
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the lectin chaperone Calnexin utilizes polypeptide based interactions to associate with many of its substrates in vivo
Journal of Biological Chemistry, 2001Co-Authors: Ursula Danilczyk, David B. WilliamsAbstract:Abstract Calnexin and calreticulin are molecular chaperones of the endoplasmic reticulum that bind to newly synthesized glycoproteins in part through a lectin site specific for monoglucosylated (Glc1Man7–9GlcNAc2) oligosaccharides. In addition to this lectin-oligosaccharide interaction, in vitro studies have demonstrated that Calnexin and calreticulin can bind to polypeptide segments of both glycosylated and nonglycosylated proteins. However, the in vivo relevance of this latter interaction has been questioned. We examined whether polypeptide-based interactions occur between Calnexin and its substrates in vivo using the glucosidase inhibitor castanospermine or glucosidase-deficient cells to prevent the formation of monoglucosylated oligosaccharides. We show that if care is taken to preserve weak interactions, the block in lectin-oligosaccharide binding leads to the loss of some Calnexin-substrate complexes, but many others remain readily detectable. Furthermore, we demonstrate that Calnexin is capable of associating in vivo with a substrate that completely lacks Asn-linked oligosaccharides. The binding of Calnexin to proteins that lack monoglucosylated oligosaccharides could not be attributed to nonspecific adsorption nor to its inclusion in protein aggregates. We conclude that both lectin-oligosaccharide and polypeptide-based interactions occur between Calnexin and diverse proteins in vivo and that the strength of the latter interaction varies substantially between protein substrates.
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Functional Relationship between Calreticulin, Calnexin, and the Endoplasmic Reticulum Luminal Domain of Calnexin
The Journal of biological chemistry, 2000Co-Authors: Ursula Danilczyk, Myrna F. Cohen-doyle, David B. WilliamsAbstract:Calnexin is a membrane protein of the endoplasmic reticulum (ER) that functions as a molecular chaperone and as a component of the ER quality control machinery. Calreticulin, a soluble analog of Calnexin, is thought to possess similar functions, but these have not been directly demonstrated in vivo. Both proteins contain a lectin site that directs their association with newly synthesized glycoproteins. Although many glycoproteins bind to both Calnexin and calreticulin, there are differences in the spectrum of glycoproteins that each binds. Using a Drosophila expression system and the mouse class I histocompatibility molecule as a model glycoprotein, we found that calreticulin does possess apparent chaperone and quality control functions, enhancing class I folding and subunit assembly, stabilizing subunits, and impeding export of assembly intermediates from the ER. Indeed, the functions of Calnexin and calreticulin were largely interchangeable. We also determined that a soluble form of Calnexin (residues 1-387) can functionally replace its membrane-bound counterpart. However, when Calnexin was expressed as a soluble protein in L cells, the pattern of associated glycoproteins changed to resemble that of calreticulin. Conversely, membrane-anchored calreticulin bound to a similar set of glycoproteins as Calnexin. Therefore, the different topological environments of Calnexin and calreticulin are important in determining their distinct substrate specificities.
Marek Michalak - One of the best experts on this subject based on the ideXlab platform.
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fatty acid binding protein fabp 5 interacts with the Calnexin cytoplasmic domain at the endoplasmic reticulum
Biochemical and Biophysical Research Communications, 2017Co-Authors: Joanna Jung, Jody Groenendyk, Marek Michalak, Dukgyu Lee, Jessica Wang, Luis B AgellonAbstract:Calnexin is a type 1 integral endoplasmic reticulum membrane molecular chaperone with an endoplasmic reticulum luminal chaperone domain and a highly conserved C-terminal domain oriented to the cytoplasm. Fabp5 is a cytoplasmic protein that binds long-chain fatty acids and other lipophilic ligands. Using a yeast two-hybrid screen, immunoprecipitation, microscale thermophoresis analysis and cellular fractionation, we discovered that Fabp5 interacts with the Calnexin cytoplasmic C-tail domain at the endoplasmic reticulum. These observations identify Fabp5 as a previously unrecognized Calnexin binding partner.
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The Endoplasmic Reticulum Chaperone Calnexin Is a NADPH Oxidase NOX4 Interacting Protein.
Journal of Biological Chemistry, 2016Co-Authors: Kim-kristin Prior, Ajay M. Shah, Ilka Wittig, Matthias S. Leisegang, Jody Groenendyk, Norbert Weissmann, Marek Michalak, Pidder Jansen-dürr, Ralf P BrandesAbstract:Within the family of NADPH oxidases, NOX4 is unique as it is predominantly localized in the endoplasmic reticulum, has constitutive activity, and generates hydrogen peroxide (H2O2). We hypothesize that these features are consequences of a so far unidentified NOX4-interacting protein. Two-dimensional blue native (BN) electrophorese combined with SDS-PAGE yielded NOX4 to reside in macromolecular complexes. Interacting proteins were screened by quantitative SILAC (stable isotope labeling of amino acids in cell culture) co-immunoprecipitation (Co-IP) in HEK293 cells stably overexpressing NOX4. By this technique, several interacting proteins were identified with Calnexin showing the most robust interaction. Calnexin also resided in NOX4-containing complexes as demonstrated by complexome profiling from BN-PAGE. The Calnexin NOX4 interaction could be confirmed by reverse Co-IP and proximity ligation assay, whereas NOX1, NOX2, or NOX5 did not interact with Calnexin. Calnexin deficiency as studied in mouse embryonic fibroblasts from Calnexin(-/-)mice or in response to Calnexin shRNA reduced cellular NOX4 protein expression and reactive oxygen species formation. Our results suggest that endogenous NOX4 forms macromolecular complexes with Calnexin, which are needed for the proper maturation, processing, and function of NOX4 in the endoplasmic reticulum.
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Role of cysteine amino acid residues in Calnexin.
Molecular and Cellular Biochemistry, 2011Co-Authors: Helen Coe, Jody Groenendyk, Jeannine D. Schneider, Monika Dabrowska, Joanna Jung, Marek MichalakAbstract:Calnexin is an endoplasmic reticulum protein that has a role in folding newly synthesized glycoproteins. In this study, we used site-specific mutagenesis to disrupt cysteine and histidine amino acid residues in the N- and P-domains of Calnexin and determined whether these mutations impact the structure and function of Calnexin. We identified that disruption of the N-domain cysteines resulted in significant loss of the chaperone activity of Calnexin toward the glycosylated substrate, IgY, while disruption of the P-domain cysteines only had a small impact toward IgY. We observed that wild-type Calnexin as well as the P-domain double cysteine mutant contained an intramolecular disulfide bond which is lost when the N-domain cysteines are mutated. Mutation to the N-domain histidine and N-domain cysteines resulted in increased binding of ERp57. Mutations to the P-domain cysteines further enhanced ERp57 binding to Calnexin. Taken together, these observations indicated that the cysteine residues within Calnexin were important for the structure and function of Calnexin.
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Enhanced clathrin-dependent endocytosis in the absence of Calnexin.
PloS one, 2011Co-Authors: Wen-xin Liu, Marek MichalakAbstract:Calnexin, together with calreticulin, constitute the Calnexin/calreticulin cycle. Calnexin is a type I endoplasmic reticulum integral membrane protein and molecular chaperone responsible for the folding and quality control of newly-synthesized (glyco)proteins. The endoplasmic reticulum luminal domain of Calnexin is responsible for lectin-like activity and interaction with nascent polypeptide chains. The role of the C-terminal, cytoplasmic portion of Calnexin is not clear. Using yeast two hybrid screen and immunoprecipitation techniques, we showed that the Src homology 3-domain growth factor receptor-bound 2-like (Endophilin) interacting protein 1 (SGIP1), a neuronal specific regulator of endocytosis, forms complexes with the C-terminal cytoplasmic domain of Calnexin. The Calnexin cytoplasmic C-tail interacts with SGIP1 C-terminal domains containing the adaptor complexes medium subunit (Adap-Comp-Sub) region. Calnexin-deficient cells have enhanced clathrin-dependent endocytosis in neuronal cells and mouse neuronal system. This is reversed by expression of full length Calnexin or Calnexin C-tail. We show that the effects of SGIP1 and Calnexin C-tail on clathrin-dependent endocytosis are due to modulation of the internalization of the receptor-ligand complexes. Enhanced clathrin-dependent endocytosis in the absence of Calnexin may contribute to the neurological phenotype of Calnexin-deficient mice.
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enhanced clathrin dependent endocytosis in the absence of Calnexin
PLOS ONE, 2011Co-Authors: Wen-xin Liu, Marek MichalakAbstract:Background Calnexin, together with calreticulin, constitute the Calnexin/calreticulin cycle. Calnexin is a type I endoplasmic reticulum integral membrane protein and molecular chaperone responsible for the folding and quality control of newly-synthesized (glyco)proteins. The endoplasmic reticulum luminal domain of Calnexin is responsible for lectin-like activity and interaction with nascent polypeptide chains. The role of the C-terminal, cytoplasmic portion of Calnexin is not clear. Methodology/Principal Findings Using yeast two hybrid screen and immunoprecipitation techniques, we showed that the Src homology 3-domain growth factor receptor-bound 2-like (Endophilin) interacting protein 1 (SGIP1), a neuronal specific regulator of endocytosis, forms complexes with the C-terminal cytoplasmic domain of Calnexin. The Calnexin cytoplasmic C-tail interacts with SGIP1 C-terminal domains containing the adaptor complexes medium subunit (Adap-Comp-Sub) region. Calnexin-deficient cells have enhanced clathrin-dependent endocytosis in neuronal cells and mouse neuronal system. This is reversed by expression of full length Calnexin or Calnexin C-tail. Conclusions/Significance We show that the effects of SGIP1 and Calnexin C-tail on clathrin-dependent endocytosis are due to modulation of the internalization of the receptor-ligand complexes. Enhanced clathrin-dependent endocytosis in the absence of Calnexin may contribute to the neurological phenotype of Calnexin-deficient mice.
David Y Thomas - One of the best experts on this subject based on the ideXlab platform.
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Calnexin Phosphorylation Attenuates the Release of Partially Misfolded α1-Antitrypsin to the Secretory Pathway
The Journal of biological chemistry, 2009Co-Authors: Pamela H. Cameron, David Y Thomas, Eric Chevet, Olivier Pluquet, John J M BergeronAbstract:Calnexin is a type I integral membrane phosphoprotein resident of the endoplasmic reticulum. Its intraluminal domain has been deduced to function as a lectin chaperone coordinating the timing of folding of newly synthesized N-linked glycoproteins of the secretory pathway. Its C-terminal cytosolic oriented extension has an ERK1 phosphorylation site at Ser563 affecting Calnexin association with the translocon. Here we find an additional function for Calnexin phosphorylation at Ser563 in endoplasmic reticulum quality control. A low dose of the misfolding agent l-azetidine 2-carboxylic acid slows glycoprotein maturation and diminishes the extent and rate of secretion of newly synthesized secretory α1-antitrypsin. Under these conditions the phosphorylation of Calnexin is enhanced at Ser563. Inhibition of this phosphorylation by the MEK1 inhibitor PD98059 enhanced the extent and rate of α1-antitrypsin secretion comparable with that achieved by inhibiting α-mannosidase activity with kifunensine. This is the first report in which the phosphorylation of Calnexin is linked to the efficiency of secretion of a cargo glycoprotein.
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Calnexin, an ER Integral Membrane Chaperone in Health and Disease
Calreticulin, 2003Co-Authors: John J M Bergeron, David Y ThomasAbstract:This review discusses the ER protein Calnexin that is related in structure and function to calreticulin. In vivo and in vitro experiments from many laboratories have provided evidence that Calnexin and calreticulin interact transiently with glycoproteins while they are folding in the ER a that this interaction is via a specific Glc1Man9GlcNAc2 glycoform. The structure of Calnexin has recently been determined to 2.9A resolution by X-ray crystallography and has a unique and remarkable structure a globular domain and an extended 140A arm termed the P domain. The P domain recruits a member of the protein disulfide isomerase family, ERp57, that specifically catalyzes disulfide bond exchange on glycoproteins bound to Calnexin. Calnexin links N-glycosylation and protein folding and forms the quality control system for glycoproteins. Mutant glycoproteins are the basis of many human protein trafficking diseases and the ER quality system is responsible for their retention in the ER and their proteolytic degradation in the cytosol.
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Localization of the lectin, ERp57 binding, and polypeptide binding sites of Calnexin and calreticulin
The Journal of biological chemistry, 2002Co-Authors: Michael R Leach, David Y Thomas, Myrna F. Cohen-doyle, David B. WilliamsAbstract:Abstract Calnexin and calreticulin are membrane-bound and soluble chaperones, respectively, of the endoplasmic reticulum (ER) which interact transiently with a broad spectrum of newly synthesized glycoproteins. In addition to sharing substantial sequence identity, both Calnexin and calreticulin bind to monoglucosylated oligosaccharides of the form Glc1Man5–9GlcNAc2, interact with the thiol oxidoreductase, ERp57, and are capable of acting as chaperones in vitro to suppress the aggregation of non-native proteins. To understand how these diverse functions are coordinated, we have localized the lectin, ERp57 binding, and polypeptide binding sites of Calnexin and calreticulin. Recent structural studies suggest that both proteins consist of a globular domain and an extended arm domain comprised of two sequence motifs repeated in tandem. Our results indicate that the primary lectin site of Calnexin and calreticulin resides within the globular domain, but the results also point to a much weaker secondary site within the arm domain which lacks specificity for monoglucosylated oligosaccharides. For both proteins, a site of interaction with ERp57 is centered on the arm domain, which retains ∼50% of binding compared with full-length controls. This site is in addition to a Zn2+-dependent site located within the globular domain of both proteins. Finally, Calnexin and calreticulin suppress the aggregation of unfolded proteins via a polypeptide binding site located within their globular domains but require the arm domain for full chaperone function. These findings are integrated into a model that describes the interaction of glycoprotein folding intermediates with Calnexin and calreticulin.
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The Structure of Calnexin, an ER chaperone involved in quality control of protein folding.
Molecular cell, 2001Co-Authors: Joseph D. Schrag, David Y Thomas, John J M Bergeron, Svetlana Borisova, Michael Hahn, Miroslaw CyglerAbstract:The three-dimensional structure of the lumenal domain of the lectin-like chaperone Calnexin determined to 2.9 A resolution reveals an extended 140 A arm inserted into a beta sandwich structure characteristic of legume lectins. The arm is composed of tandem repeats of two proline-rich sequence motifs which interact with one another in a head-to-tail fashion. Identification of the ligand binding site establishes Calnexin as a monovalent lectin, providing insight into the mechanism by which the Calnexin family of chaperones interacts with monoglucosylated glycoproteins.
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PHOSPHORYLATION BY CK2 AND MAPK ENHANCES Calnexin ASSOCIATION WITH RIBOSOMES
The EMBO journal, 1999Co-Authors: Eric Chevet, David Y Thomas, Pamela H. Cameron, Hetty N. Wong, D. Gerber, Claude Cochet, Ali Fazel, Jennifer N. Gushue, John J M BergeronAbstract:Calnexin was initially identified as an endoplasmic reticulum (ER) type I integral membrane protein, phosphorylated on its cytosolic domain by ER-associated protein kinases. Although the role of the ER luminal domain of Calnexin has been established as a constituent of the molecular chaperone machinery of the ER, less is known about the role of the cytosolic phosphorylation of Calnexin. Analysis by two-dimensional phosphopeptide maps revealed that Calnexin was in vitro phosphorylated in isolated microsomes by casein kinase 2 (CK2) and extracellular-signal regulated kinase-1 (ERK-1) at sites corresponding to those for in vivo phosphorylation. In canine pancreatic microsomes, synergistic phosphorylation by CK2 and ERK-1 led to increased association of Calnexin with membrane-bound ribosomes. In vivo, Calnexin-associated ERK-1 activity was identified by co-immunoprecipitation. This activity was abolished in cells expressing a dominant-negative MEK-1. Activation of ERK-1 in cells by addition of serum led to a 4-fold increase in ribosome-associated Calnexin over unstimulated cells. Taken together with studies revealing Calnexin association with CK2 and ERK-1, a model is proposed whereby phosphorylation of Calnexin leads to a potential increase in glycoprotein folding close to the translocon.
Luis A Rokeach - One of the best experts on this subject based on the ideXlab platform.
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Calnexin regulates apoptosis induced by inositol starvation in fission yeast.
PloS one, 2009Co-Authors: Renée Guérin, Pascale B Beauregard, Alexandre Leroux, Luis A RokeachAbstract:Inositol is a precursor of numerous phospholipids and signalling molecules essential for the cell. Schizosaccharomyces pombe is naturally auxotroph for inositol as its genome does not have a homologue of the INO1 gene encoding inositol-1-phosphate synthase, the enzyme responsible for inositol biosynthesis. In this work, we demonstrate that inositol starvation in S. pombe causes cell death with apoptotic features. This apoptotic death is dependent on the metacaspase Pca1p and is affected by the UPR transducer Ire1p. Previously, we demonstrated that Calnexin is involved in apoptosis induced by ER stress. Here, we show that cells expressing a lumenal version of Calnexin exhibit a 2-fold increase in the levels of apoptosis provoked by inositol starvation. This increase is reversed by co-expression of a Calnexin mutant spanning the transmembrane domain and C-terminal cytosolic tail. Coherently, Calnexin is physiologically cleaved at the end of its lumenal domain, under normal growth conditions when cells approach stationary phase. This cleavage suggests that the two naturally produced Calnexin fragments are needed to continue growth into stationary phase and to prevent cell death. Collectively, our observations indicate that Calnexin takes part in at least two apoptotic pathways in S. pombe, and suggest that the cleavage of Calnexin has regulatory roles in apoptotic processes involving Calnexin.
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A nucleolar protein allows viability in the absence of the essential ER-residing molecular chaperone Calnexin.
Journal of Cell Science, 2009Co-Authors: Pascale B Beauregard, Renée Guérin, Cynthia Turcotte, Susan Lindquist, Luis A RokeachAbstract:In fission yeast, the ER-residing molecular chaperone Calnexin is normally essential for viability. However, a specific mutant of Calnexin that is devoid of chaperone function (Δhcd_Cnx1p) induces an epigenetic state that allows growth of Schizosaccharomyces pombe without Calnexin. This Calnexin-independent (Cin) state was previously shown to be mediated via a non-chromosomal element exhibiting some prion-like features. Here, we report the identification of a gene whose overexpression induces the appearance of stable Cin cells. This gene, here named cif1+ for Calnexin-independence factor 1, encodes an uncharacterized nucleolar protein. The Cin cells arising from cif1+ overexpression (Cincif1 cells) are genetically and phenotypically distinct from the previously characterized CinΔhcd_cnx1 cells, which spontaneously appear in the presence of the Δhcd_Cnx1p mutant. Moreover, cif1+ is not required for the induction or maintenance of the CinΔhcd_cnx1 state. These observations argue for different pathways of induction and/or maintenance of the state of Calnexin independence. Nucleolar localization of Cif1p is required to induce the Cincif1 state, thus suggesting an unexpected interaction between the vital cellular role of Calnexin and a function of the nucleolus.
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A nucleolar protein allows viability in the absence of the essential ER-residing molecular chaperone Calnexin.
Journal of cell science, 2009Co-Authors: Pascale B Beauregard, Renée Guérin, Cynthia Turcotte, Susan Lindquist, Luis A RokeachAbstract:In fission yeast, the ER-residing molecular chaperone Calnexin is normally essential for viability. However, a specific mutant of Calnexin that is devoid of chaperone function (Deltahcd_Cnx1p) induces an epigenetic state that allows growth of Schizosaccharomyces pombe without Calnexin. This Calnexin-independent (Cin) state was previously shown to be mediated via a non-chromosomal element exhibiting some prion-like features. Here, we report the identification of a gene whose overexpression induces the appearance of stable Cin cells. This gene, here named cif1(+) for Calnexin-independence factor 1, encodes an uncharacterized nucleolar protein. The Cin cells arising from cif1(+) overexpression (Cin(cif1) cells) are genetically and phenotypically distinct from the previously characterized Cin(Deltahcd_cnx1) cells, which spontaneously appear in the presence of the Deltahcd_Cnx1p mutant. Moreover, cif1(+) is not required for the induction or maintenance of the Cin(Deltahcd_cnx1) state. These observations argue for different pathways of induction and/or maintenance of the state of Calnexin independence. Nucleolar localization of Cif1p is required to induce the Cin(cif1) state, thus suggesting an unexpected interaction between the vital cellular role of Calnexin and a function of the nucleolus.
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Calnexin is involved in apoptosis induced by endoplasmic reticulum stress in the fission yeast.
Molecular biology of the cell, 2008Co-Authors: Renée Guérin, Geneviève Arseneault, Stéphane Dumont, Luis A RokeachAbstract:Stress conditions affecting the functions of the endoplasmic reticulum (ER) cause the accumulation of unfolded proteins. ER stress is counteracted by the unfolded-protein response (UPR). However, under prolonged stress the UPR initiates a proapoptotic response. Mounting evidence indicate that the ER chaperone Calnexin is involved in apoptosis caused by ER stress. Here, we report that overexpression of Calnexin in Schizosaccharomyces pombe induces cell death with apoptosis markers. Cell death was partially dependent on the Ire1p ER-stress transducer. Apoptotic death caused by Calnexin overexpression required its transmembrane domain (TM), and involved sequences on either side of the ER membrane. Apoptotic death caused by tunicamycin was dramatically reduced in a strain expressing endogenous levels of Calnexin lacking its TM and cytosolic tail. This demonstrates the involvement of Calnexin in apoptosis triggered by ER stress. A genetic screen identified the S. pombe homologue of the human antiapoptotic protein HMGB1 as a suppressor of apoptotic death due to Calnexin overexpression. Remarkably, overexpression of human Calnexin in S. pombe also provoked apoptotic death. Our results argue for the conservation of the role of Calnexin in apoptosis triggered by ER stress, and validate S. pombe as a model to elucidate the mechanisms of Calnexin-mediated cell death.
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The Calnexin-independent state does not compensate for all Calnexin functions in Schizosaccharomyces pombe.
FEMS yeast research, 2007Co-Authors: Cynthia Turcotte, Pascale B Beauregard, Renée Guérin, Antoine E. Roux, Patrick Sénéchal, Fadi Hajjar, Luis A RokeachAbstract:In the yeast Schizosaccharomyces pombe, the molecular chaperone Calnexin (Cnx1p) has been shown to be essential for viability. However, we recently reported that, under certain circumstances, S. pombe cells are able to survive in the absence of Calnexin/Cnx1p, indicating that an inducible pathway can complement the Calnexin/Cnx1p essential function(s). This Calnexin-independent state (Cin) is transmitted by a nonchromosomal proteinaceous element exhibiting several prion-like properties. To assess to what extent the Cin state compensates for the absence of Calnexin/Cnx1p, the Cin strain was further characterized. Cin cells exhibited cell-wall defects, sensitivity to heat shock, as well as higher secretion levels of a model glycoprotein. Together, these results indicate that the Cin state does not compensate for all Calnexin/Cnx1p functions. Reintroduction of plasmid-borne cnx1(+) partially rescued most but not all of the phenotypes displayed by Cin cells. Interestingly, Cin cells in stationary phase exhibited increased levels of caspase activation, and this phenotype was not suppressed by the reintroduction of cnx1(+), suggesting that cells in the Cin state are subjected to a stress other than the absence of Calnexin/Cnx1p.
John J M Bergeron - One of the best experts on this subject based on the ideXlab platform.
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Calnexin Phosphorylation Attenuates the Release of Partially Misfolded α1-Antitrypsin to the Secretory Pathway
The Journal of biological chemistry, 2009Co-Authors: Pamela H. Cameron, David Y Thomas, Eric Chevet, Olivier Pluquet, John J M BergeronAbstract:Calnexin is a type I integral membrane phosphoprotein resident of the endoplasmic reticulum. Its intraluminal domain has been deduced to function as a lectin chaperone coordinating the timing of folding of newly synthesized N-linked glycoproteins of the secretory pathway. Its C-terminal cytosolic oriented extension has an ERK1 phosphorylation site at Ser563 affecting Calnexin association with the translocon. Here we find an additional function for Calnexin phosphorylation at Ser563 in endoplasmic reticulum quality control. A low dose of the misfolding agent l-azetidine 2-carboxylic acid slows glycoprotein maturation and diminishes the extent and rate of secretion of newly synthesized secretory α1-antitrypsin. Under these conditions the phosphorylation of Calnexin is enhanced at Ser563. Inhibition of this phosphorylation by the MEK1 inhibitor PD98059 enhanced the extent and rate of α1-antitrypsin secretion comparable with that achieved by inhibiting α-mannosidase activity with kifunensine. This is the first report in which the phosphorylation of Calnexin is linked to the efficiency of secretion of a cargo glycoprotein.
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Calnexin, an ER Integral Membrane Chaperone in Health and Disease
Calreticulin, 2003Co-Authors: John J M Bergeron, David Y ThomasAbstract:This review discusses the ER protein Calnexin that is related in structure and function to calreticulin. In vivo and in vitro experiments from many laboratories have provided evidence that Calnexin and calreticulin interact transiently with glycoproteins while they are folding in the ER a that this interaction is via a specific Glc1Man9GlcNAc2 glycoform. The structure of Calnexin has recently been determined to 2.9A resolution by X-ray crystallography and has a unique and remarkable structure a globular domain and an extended 140A arm termed the P domain. The P domain recruits a member of the protein disulfide isomerase family, ERp57, that specifically catalyzes disulfide bond exchange on glycoproteins bound to Calnexin. Calnexin links N-glycosylation and protein folding and forms the quality control system for glycoproteins. Mutant glycoproteins are the basis of many human protein trafficking diseases and the ER quality system is responsible for their retention in the ER and their proteolytic degradation in the cytosol.
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The Structure of Calnexin, an ER chaperone involved in quality control of protein folding.
Molecular cell, 2001Co-Authors: Joseph D. Schrag, David Y Thomas, John J M Bergeron, Svetlana Borisova, Michael Hahn, Miroslaw CyglerAbstract:The three-dimensional structure of the lumenal domain of the lectin-like chaperone Calnexin determined to 2.9 A resolution reveals an extended 140 A arm inserted into a beta sandwich structure characteristic of legume lectins. The arm is composed of tandem repeats of two proline-rich sequence motifs which interact with one another in a head-to-tail fashion. Identification of the ligand binding site establishes Calnexin as a monovalent lectin, providing insight into the mechanism by which the Calnexin family of chaperones interacts with monoglucosylated glycoproteins.
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PHOSPHORYLATION BY CK2 AND MAPK ENHANCES Calnexin ASSOCIATION WITH RIBOSOMES
The EMBO journal, 1999Co-Authors: Eric Chevet, David Y Thomas, Pamela H. Cameron, Hetty N. Wong, D. Gerber, Claude Cochet, Ali Fazel, Jennifer N. Gushue, John J M BergeronAbstract:Calnexin was initially identified as an endoplasmic reticulum (ER) type I integral membrane protein, phosphorylated on its cytosolic domain by ER-associated protein kinases. Although the role of the ER luminal domain of Calnexin has been established as a constituent of the molecular chaperone machinery of the ER, less is known about the role of the cytosolic phosphorylation of Calnexin. Analysis by two-dimensional phosphopeptide maps revealed that Calnexin was in vitro phosphorylated in isolated microsomes by casein kinase 2 (CK2) and extracellular-signal regulated kinase-1 (ERK-1) at sites corresponding to those for in vivo phosphorylation. In canine pancreatic microsomes, synergistic phosphorylation by CK2 and ERK-1 led to increased association of Calnexin with membrane-bound ribosomes. In vivo, Calnexin-associated ERK-1 activity was identified by co-immunoprecipitation. This activity was abolished in cells expressing a dominant-negative MEK-1. Activation of ERK-1 in cells by addition of serum led to a 4-fold increase in ribosome-associated Calnexin over unstimulated cells. Taken together with studies revealing Calnexin association with CK2 and ERK-1, a model is proposed whereby phosphorylation of Calnexin leads to a potential increase in glycoprotein folding close to the translocon.
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Conformation-Independent Binding of Monoglucosylated Ribonuclease B to Calnexin
Cell, 1997Co-Authors: André Zapun, David Y Thomas, Stefana M. Petrescu, Pauline M. Rudd, Raymond A. Dwek, John J M BergeronAbstract:Calnexin is a membrane protein of the endoplasmic reticulum that associates transiently with newly synthesized N-linked glycoproteins in vivo. Using defined components, the binding of ribonuclease B (RNase B) Man7-Man9 glycoforms to the luminal domain of Calnexin was observed in vitro only if RNase B was monoglucosylated. Binding was independent of the conformation of the glycoprotein. Calnexin protected monoglucosylated RNase B from the action of glucosidase II and PNGase F but not from that of Endo H, which completely released the protein from Calnexin. These observations directly demonstrate that Calnexin can act exclusively as a lectin.
