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Marek Michalak - One of the best experts on this subject based on the ideXlab platform.
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Calreticulin inhibits commitment to adipocyte differentiation
The Journal of cell biology, 2015Co-Authors: Eva Szabo, Marek Michalak, Shairaz Baksh, Yuanyuan Qiu, Michal OpasAbstract:Calreticulin, an endoplasmic reticulum (ER) resident protein, affects many critical cellular functions, including protein folding and calcium homeostasis. Using embryonic stem cells and 3T3-L1 preadipocytes, we show that Calreticulin modulates adipogenesis. We find that Calreticulin-deficient cells show increased potency for adipogenesis when compared with wild-type or Calreticulin-overexpressing cells. In the highly adipogenic crt−/− cells, the ER lumenal calcium concentration was reduced. Increasing the ER lumenal calcium concentration led to a decrease in adipogenesis. In Calreticulin-deficient cells, the calmodulin–Ca2+/calmodulin-dependent protein kinase II (CaMKII) pathway was up-regulated, and inhibition of CaMKII reduced adipogenesis. Calreticulin inhibits adipogenesis via a negative feedback mechanism whereby the expression of Calreticulin is initially up-regulated by peroxisome proliferator–activated receptor γ (PPARγ). This abundance of Calreticulin subsequently negatively regulates the expression of PPARγ, lipoprotein lipase, CCAAT enhancer–binding protein α, and aP2. Thus, Calreticulin appears to function as a Ca2+-dependent molecular switch that regulates commitment to adipocyte differentiation by preventing the expression and transcriptional activation of critical proadipogenic transcription factors.
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Disrupted WNT Signaling in Mouse Embryonic Stem Cells in the Absence of Calreticulin
Stem Cell Reviews and Reports, 2014Co-Authors: Jody Groenendyk, Marek MichalakAbstract:The role of endoplasmic reticulum (ER) homeostasis and protein quality control in the regulation of WNT signaling is not understood. Here we provide evidence for a role of Calreticulin in the regulation of WNT signaling. We show that a deficiency in Calreticulin disrupted WNT signaling, and prevented cell cycle progression via the miR-302 microRNA family. These effects were dependent on the Ca^2+ buffering capacity of Calreticulin, as the protein is important in regulating ER Ca^2+ release and activation of Ca^2+-dependent kinase and phosphatase cascades (including c-Src, Akt, and PTP1B). We also show that Calreticulin plays a role in the secretion and ER retention of WNT3a, thereby affecting downstream WNT signaling. In Calreticulin-deficient ES cells, the WNT and miR-302 dependent maintenance of the naïve ES cell state and the transition to primed pluripotency transition were lost, preventing cells from undergoing accurate differentiation. Together, these findings demonstrate unexpected roles of Calreticulin and ER Ca^2+ homeostasis/signaling in the canonical WNT signaling pathway.
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Calreticulin signaling in health and disease.
The international journal of biochemistry & cell biology, 2012Co-Authors: Wen-an Wang, Jody Groenendyk, Marek MichalakAbstract:Abstract Calreticulin is an endoplasmic reticulum Ca 2+ binding chaperone that has multiple functions inside and outside of the endoplasmic reticulum. It is involved in the quality control of newly synthesized proteins and glycoproteins, interacting with various other endoplasmic reticulum chaperones, specifically calnexin and ER protein of 57-kDa in the Calreticulin/calnexin cycle. Calreticulin also plays a crucial role in regulating intracellular Ca 2+ homeostasis, associating Calreticulin with a wide variety of signaling processes, such as cardiogenesis, adipocyte differentiation and cellular stress responses. The role of Calreticulin outside of the endoplasmic reticulum is also extensive, including functions in wound healing and immunity. Therefore, Calreticulin has important implications in health and disease. Signaling facts - Calreticulin controls calcineurin activity and NF-AT- and MEF2C-dependent expression of cardiac genes through InsP 3 R mediated ER Ca 2+ release. - Calreticulin deficiency up-regulates the CAMKII-dependent signaling pathway. - Calreticulin over-expression decreases adipogenic factors (lipoprotein lipase, αP2, PPAR and C/EBPα). - Cell surface Calreticulin signals pre-apoptotic cellular clearance by binding to LDL-receptor-related protein (LRP) on phagocytes.
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Calreticulin a multi process calcium buffering chaperone of the endoplasmic reticulum
Biochemical Journal, 2009Co-Authors: Marek Michalak, Jody Groenendyk, Eva Szabo, Leslie I Gold, Michal OpasAbstract:Calreticulin is an ER (endoplasmic reticulum) luminal Ca2+-buffering chaperone. The protein is involved in regulation of intracellular Ca2+ homoeostasis and ER Ca2+ capacity. The protein impacts on store-operated Ca2+ influx and influences Ca2+-dependent transcriptional pathways during embryonic development. Calreticulin is also involved in the folding of newly synthesized proteins and glycoproteins and, together with calnexin (an integral ER membrane chaperone similar to Calreticulin) and ERp57 [ER protein of 57 kDa; a PDI (protein disulfide-isomerase)-like ER-resident protein], constitutes the 'Calreticulin/calnexin cycle' that is responsible for folding and quality control of newly synthesized glycoproteins. In recent years, Calreticulin has been implicated to play a role in many biological systems, including functions inside and outside the ER, indicating that the protein is a multi-process molecule. Regulation of Ca2+ homoeostasis and ER Ca2+ buffering by Calreticulin might be the key to explain its multi-process property.
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Transcriptional control of the Calreticulin gene in health and disease.
The international journal of biochemistry & cell biology, 2008Co-Authors: Yuanyuan Qiu, Marek MichalakAbstract:Abstract Calreticulin is a multifunctional Ca2+ binding chaperone in the endoplasmic reticulum and expression of the protein is tightly regulated at the transcriptional level. There are two Calreticulin genes, named Calreticulin-1 and Calreticulin-2 gene. The Calreticulin-1 promoter contains a number of putative binding sites for transcription factors including tissue specific factors. Direct regulation of the Calreticulin-1 promoter by several of these factors has been confirmed experimentally including Nkx2.5, MEF2C, GATA6, PPAR, COUP-TF1 and Evi-1 factors. Studies on Calreticulin-deficient mice and transgenic animal models indicate that Calreticulin is critical for cardiac development and that expression of the protein must be tightly regulated during cardiogenesis. Moreover, differential expression of Calreticulin has been associated with several diseases, including neurodegenerative problems, cancers, autoimmune diseases and wound healing. Understanding the mechanisms responsible for the regulation of expression of Calreticulin may contribute to the treatment of many diverse diseases.
Michal Opas - One of the best experts on this subject based on the ideXlab platform.
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Calreticulin regulates a switch between osteoblast and chondrocyte lineages derived from murine embryonic stem cells
Journal of Biological Chemistry, 2020Co-Authors: Carlos Pilquil, Zahra Alvandi, Michal OpasAbstract:Calreticulin is a highly conserved, ubiquitous Ca2+-buffering protein in the endoplasmic reticulum that controls transcriptional activity of various developmental programs and also of embryonic stem cell (ESC) differentiation. Calreticulin activates calcineurin, which dephosphorylates and induces the nuclear import of the osteogenic transcription regulator nuclear factor of activated T cells 1 (NFATC1). We investigated whether Calreticulin controls a switch between osteogenesis and chondrogenesis in mouse ESCs through NFATC1. We found that in the absence of Calreticulin, intranuclear transport of NFATC1 is blocked and that differentiation switches from osteogenic to chondrogenic, a process that could be mimicked by chemical inhibition of NFAT translocation. Glycogen synthase kinase 3β (GSK3β) deactivation and nuclear localization of β-catenin critical to osteogenesis were abrogated by Calreticulin deficiency or NFAT blockade. Chemically induced GSK3β inhibition bypassed the Calreticulin/calcineurin axis and increased osteoblast output from both control and Calreticulin-deficient ESCs, while suppressing chondrogenesis. Calreticulin-deficient ESCs or cells treated with an NFAT blocker had enhanced expression of dickkopf WNT-signaling pathway inhibitor 1 (Dkk1), a canonical Wnt pathway antagonist that blocks GSK3β deactivation. The addition of recombinant mDKK1 switched osteogenic ESC differentiation toward chondrogenic differentiation. The results of our study indicate a role for endoplasmic reticulum calcium signaling via Calreticulin in the differentiation of ESCs to closely associated osteoblast or chondrocyte lineages.
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Calreticulin inhibits commitment to adipocyte differentiation
The Journal of cell biology, 2015Co-Authors: Eva Szabo, Marek Michalak, Shairaz Baksh, Yuanyuan Qiu, Michal OpasAbstract:Calreticulin, an endoplasmic reticulum (ER) resident protein, affects many critical cellular functions, including protein folding and calcium homeostasis. Using embryonic stem cells and 3T3-L1 preadipocytes, we show that Calreticulin modulates adipogenesis. We find that Calreticulin-deficient cells show increased potency for adipogenesis when compared with wild-type or Calreticulin-overexpressing cells. In the highly adipogenic crt−/− cells, the ER lumenal calcium concentration was reduced. Increasing the ER lumenal calcium concentration led to a decrease in adipogenesis. In Calreticulin-deficient cells, the calmodulin–Ca2+/calmodulin-dependent protein kinase II (CaMKII) pathway was up-regulated, and inhibition of CaMKII reduced adipogenesis. Calreticulin inhibits adipogenesis via a negative feedback mechanism whereby the expression of Calreticulin is initially up-regulated by peroxisome proliferator–activated receptor γ (PPARγ). This abundance of Calreticulin subsequently negatively regulates the expression of PPARγ, lipoprotein lipase, CCAAT enhancer–binding protein α, and aP2. Thus, Calreticulin appears to function as a Ca2+-dependent molecular switch that regulates commitment to adipocyte differentiation by preventing the expression and transcriptional activation of critical proadipogenic transcription factors.
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Calreticulin a multi process calcium buffering chaperone of the endoplasmic reticulum
Biochemical Journal, 2009Co-Authors: Marek Michalak, Jody Groenendyk, Eva Szabo, Leslie I Gold, Michal OpasAbstract:Calreticulin is an ER (endoplasmic reticulum) luminal Ca2+-buffering chaperone. The protein is involved in regulation of intracellular Ca2+ homoeostasis and ER Ca2+ capacity. The protein impacts on store-operated Ca2+ influx and influences Ca2+-dependent transcriptional pathways during embryonic development. Calreticulin is also involved in the folding of newly synthesized proteins and glycoproteins and, together with calnexin (an integral ER membrane chaperone similar to Calreticulin) and ERp57 [ER protein of 57 kDa; a PDI (protein disulfide-isomerase)-like ER-resident protein], constitutes the 'Calreticulin/calnexin cycle' that is responsible for folding and quality control of newly synthesized glycoproteins. In recent years, Calreticulin has been implicated to play a role in many biological systems, including functions inside and outside the ER, indicating that the protein is a multi-process molecule. Regulation of Ca2+ homoeostasis and ER Ca2+ buffering by Calreticulin might be the key to explain its multi-process property.
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Embryonic stem cell-derived cardiomyogenesis: a novel role for Calreticulin as a regulator.
Stem cells (Dayton Ohio), 2009Co-Authors: Sylvia Papp, Ewa Dziak, Michal OpasAbstract:A role for Calreticulin, an endoplasmic reticulum (ER)-resident, Ca2+-binding chaperone, has recently emerged in the context of cardiomyogenesis. We previously proposed Calreticulin to be a novel cardiac fetal gene, because Calreticulin knockout causes embryonic lethality in mice as a result of cardiac defects, it is transiently activated during heart development, and heart-targeted overexpression of constitutively active calcineurin in Calreticulin-null mice rescues the lethal phenotype. Calreticulin affects Ca2+ homeostasis and expression of adhesion-related genes. Using cardiomyocytes derived from both Calreticulin-null and wild-type embryonic stem (ES) cells, we show here that cardiomyogenesis from Calreticulin-null ES cells is accelerated but deregulated, such that the myofibrils of Calreticulin-null cardiomyocytes become disorganized and disintegrate with time in culture. We have previously shown that the disorganization of the actin cytoskeleton in Calreticulin-null cells may be explained, at least in part, by the downregulation of adhesion proteins, implying that Calreticulin ablation causes adhesion-related defects. Here, upon examination of adhesion proteins, we found that vinculin is downregulated in Calreticulin-null cardiomyocytes. We also found c-Src activity to be higher in Calreticulin-null cardiomyocytes than in wild-type cardiomyocytes, and c-Src activity is affected by both Calreticulin and [Ca2+]. Finally, we show that Calreticulin and calsequestrin, the major Ca2+ storage proteins of the ER and sarcoplasmic reticulum, respectively, exhibit alternate distributions. This suggests that Calreticulin may have a housekeeping role to play in mature cardiomyocytes as well as during cardiomyogenesis. We propose here that Calreticulin, an ER Ca2+ storage protein, is a crucial regulator of cardiomyogenesis whose presence is required for controlled cardiomyocyte development from ES cells. STEM CELLS 2009;27:1507–1515
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Calreticulin and focal-contact-dependent adhesion.
Biochemistry and cell biology = Biochimie et biologie cellulaire, 2009Co-Authors: Maria Villagomezm. Villagomez, Sylvia Papp, Eva Szabo, Alexey Podchekoa. Podcheko, Tianshu Fengt. Feng, Michal OpasAbstract:Cell adhesion is regulated by a variety of Ca2+-regulated pathways that depend on Ca2+-binding proteins. One such protein is Calreticulin, an ER-resident protein. Calreticulin signalling from within the ER can affect processes outside the ER, such as expression of several adhesion-related genes, most notably vinculin and fibronectin. In addition, changes in the expression level of Calreticulin strongly affect tyrosine phosphorylation of cellular proteins, which is known to affect many adhesion-related functions. While Calreticulin has been localized to cellular compartments other than the ER, it appears that only the ER-resident Calreticulin affects focal-contact-dependent adhesion. In contrast, Calreticulin residing outside the ER may be involved in contact disassembly and other adhesion phenomena. Here, we review the role of Calreticulin in focal contact initiation, stabilization, and turnover. We propose that Calreticulin may regulate cell–substratum adhesion by participating in an “ER-to-nucleus” sign...
Malini Raghavan - One of the best experts on this subject based on the ideXlab platform.
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The C-Terminal Acidic Region of Calreticulin Mediates Phosphatidylserine Binding and Apoptotic Cell Phagocytosis
Journal of immunology (Baltimore Md. : 1950), 2016Co-Authors: Sanjeeva J. Wijeyesakere, Sukhmani Bedi, David Anh Huy Huynh, Malini RaghavanAbstract:Calreticulin is a calcium-binding chaperone that is normally localized in the endoplasmic reticulum. Calreticulin is detectable on the surface of apoptotic cells under some apoptosis-inducing conditions, where it promotes the phagocytosis and immunogenicity of dying cells. However, the precise mechanism by which Calreticulin, a soluble protein, localizes to the outer surface of the plasma membrane of dying cells is unknown, as are the molecular mechanisms that are relevant to Calreticulin-induced cellular phagocytosis. Calreticulin comprises three distinct structural domains: a globular domain, an extended arm-like P-domain, and a C-terminal acidic region containing multiple low-affinity calcium binding sites. We show that Calreticulin, via its C-terminal acidic region, preferentially interacts with phosphatidylserine (PS) compared with other phospholipids and that this interaction is calcium dependent. Additionally, exogenous Calreticulin binds apoptotic cells via a higher-affinity calcium-dependent mode that is acidic region dependent. Exogenous Calreticulin also binds live cells, including macrophages, via a second, lower-affinity P-domain and globular domain-dependent, but calcium-independent binding mode that likely involves its generic polypeptide binding site. Truncation constructs lacking the acidic region or arm-like P-domain of Calreticulin are impaired in their abilities to induce apoptotic cell phagocytosis by murine peritoneal macrophages. Taken together, the results of this investigation provide the first molecular insights into the phospholipid binding site of Calreticulin as a key anchor point for the cell surface expression of Calreticulin on apoptotic cells. These findings also support a role for Calreticulin as a PS-bridging molecule that cooperates with other PS-binding factors to promote the phagocytosis of apoptotic cells.
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glycan dependent and independent interactions contribute to cellular substrate recruitment by Calreticulin
Journal of Biological Chemistry, 2013Co-Authors: Sanjeeva J. Wijeyesakere, Syed Monem Rizvi, Malini RaghavanAbstract:Calreticulin is an endoplasmic reticulum chaperone with specificity for monoglucosylated glycoproteins. Calreticulin also inhibits precipitation of nonglycosylated proteins and thus contains generic protein-binding sites, but their location and contributions to substrate folding are unknown. We show that Calreticulin binds glycosylated and nonglycosylated proteins with similar affinities but distinct interaction kinetics. Although both interactions involve the glycan-binding site or its vicinity, the arm-like proline-rich (P-) domain of Calreticulin contributes to binding non/deglycosylated proteins. Correspondingly, ensemble FRET spectroscopy measurements indicate that glycosylated and nonglycosylated proteins induce "open" and "closed" P-domain conformations, respectively. The co-chaperone ERp57 influences substrate-binding kinetics and induces a closed P-domain conformation. Together with analysis of the interactions of Calreticulin with cellular proteins, these findings indicate that the recruitment of monoglucosylated proteins to Calreticulin is kinetically driven, whereas the P-domain and co-chaperone contribute to stable substrate binding. Substrate sequestration in the cleft between the glycan-binding site and P-domain is a likely mechanism for Calreticulin-assisted protein folding.
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Glycan-dependent and -independent interactions contribute to cellular substrate recruitment by Calreticulin.
Journal of Biological Chemistry, 2013Co-Authors: Sanjeeva J. Wijeyesakere, Syed Monem Rizvi, Malini RaghavanAbstract:Abstract Calreticulin is an endoplasmic reticulum (ER) chaperone with specificity for monoglucosylated glycoproteins. Calreticulin also inhibits precipitation of non-glycosylated proteins and thus contains generic protein binding sites, but their location and contributions to substrate folding are unknown. We show that Calreticulin binds glycosylated and non-glycosylated proteins with similar affinities but distinct interaction kinetics. While both interactions involve the glycan binding site or its vicinity, the arm-like proline-rich (P-) domain of Calreticulin contributes to binding non/de-glycosylated proteins. Correspondingly, ensemble FRET spectroscopy measurements indicate that glycosylated and non-glycosylated proteins induce open and closed P-domain conformations respectively. The co-chaperone ERp57 influences substrate-binding kinetics and induces a closed P-domain conformation. Together with analysis of the interactions of Calreticulin with cellular proteins, these findings indicate that the recruitment of monoglucosylated proteins to Calreticulin is kinetically driven, while the P-domain and co-chaperone contribute to stable substrate binding. Substrate sequestration in the cleft between the glycan binding site and P-domain is a likely mechanism for Calreticulin-assisted protein folding.
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Calreticulin in the immune system: ins and outs
Trends in immunology, 2012Co-Authors: Malini Raghavan, Sanjeeva J. Wijeyesakere, Larry Robert Peters, Natasha Del CidAbstract:Calreticulin is a calcium-binding chaperone that has several functions in the immune response. In the endoplasmic reticulum (ER), Calreticulin facilitates the folding of major histocompatibility complex (MHC) class I molecules and their assembly factor tapasin, thereby influencing antigen presentation to cytotoxic T cells. Although Calreticulin is normally ER-resident, it is found at the cell surface of living cancer cells and dying cells. Here, Calreticulin promotes cellular phagocytic uptake. In tumor vaccine models, drugs that induce cell surface Calreticulin confer enhanced tumor protection in an extracellular Calreticulin-dependent manner. Much remains to be understood about the roles of Calreticulin in these distinct functions. Further investigations are important towards advancing basic knowledge of glycoprotein-folding pathways, and towards developing new cancer therapeutic strategies.
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Calreticulin is a thermostable protein with distinct structural responses to different divalent cation environments
Journal of Biological Chemistry, 2011Co-Authors: Sanjeeva J. Wijeyesakere, Ari Gafni, Malini RaghavanAbstract:Calreticulin is a soluble calcium-binding chaperone of the endoplasmic reticulum (ER) that is also detected on the cell surface and in the cytosol. Calreticulin contains a single high affinity calcium-binding site within a globular domain and multiple low affinity sites within a C-terminal acidic region. We show that the secondary structure of Calreticulin is remarkably thermostable at a given calcium concentration. Rather than corresponding to complete unfolding events, heat-induced structural transitions observed for Calreticulin relate to tertiary structural changes that expose hydrophobic residues and reduce protein rigidity. The thermostability and the overall secondary structure content of Calreticulin are impacted by the divalent cation environment, with the ER range of calcium concentrations enhancing stability, and calcium-depleting or high calcium environments reducing stability. Furthermore, magnesium competes with calcium for binding to Calreticulin and reduces thermostability. The acidic domain of Calreticulin is an important mediator of calcium-dependent changes in secondary structure content and thermostability. Together, these studies indicate interactions between the globular and acidic domains of Calreticulin that are impacted by divalent cations. These interactions influence the structure and stability of Calreticulin, and are likely to determine the multiple functional activities of Calreticulin in different subcellular environments.
Gunnar Houen - One of the best experts on this subject based on the ideXlab platform.
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Chemical and thermal unfolding of Calreticulin.
Protein and peptide letters, 2013Co-Authors: Karen Duus, Nanna Larsen, T. A.t. Tran, Esin Güven, Lars Kobberoee Skov, C. Jespersgaard, Michael Gajhede, Gunnar HouenAbstract:Calreticulin is a soluble endoplasmic reticulum chaperone, which has a relatively low melting point due to its remarkable structure with a relatively high content of flexible structural elements. Using far ultraviolet circular dichroism (CD) spectroscopy and a fluorescent dye binding thermal shift assay, we have investigated the chemical and thermal stability of Calreticulin. When the chemical stability of Calreticulin was assessed, a midpoint for Calreticulin unfolding was calculated to 3.0M urea using CD data at 222 nm. Using the fluorescent dye binding thermal shift assay, Calreticulin was found to obtain a molten structure in urea concentrations between 1-1.5 M urea, and to unfold/aggregate at high and low pH values. The results demonstrated that the fluorescent dye binding assay could measure the thermal stability of Calreticulin in aqueous buffers with results comparable to melting points obtained by other techniques.
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The interactions of Calreticulin with immunoglobulin G and immunoglobulin Y
Biochimica et Biophysica Acta, 2011Co-Authors: Karen Mai Møllegaard, Karen Duus, Sofie Dietz Træholt, Morten Thaysen-andersen, Angelina S. Palma, Ten Feizi, Paul R. Hansen, Peter Højrup, Gunnar HouenAbstract:Abstract Calreticulin is a chaperone of the endoplasmic reticulum (ER) assisting proteins in achieving the correctly folded structure. Details of the binding specificity of Calreticulin are still a matter of debate. Calreticulin has been described as an oligosaccharide-binding chaperone but data are also accumulating in support of Calreticulin as a polypeptide binding chaperone. In contrast to mammalian immunoglobulin G (IgG), which has complex type N -glycans, chicken immunoglobulin Y (IgY) possesses a monoglucosylated high mannose N -linked glycan, which is a ligand for Calreticulin. Here, we have used solid and solution-phase assays to analyze the in vitro binding of Calreticulin, purified from human placenta, to human IgG and chicken IgY in order to compare the interactions. In addition, peptides from the respective immunoglobulins were included to further probe the binding specificity of Calreticulin. The experiments demonstrate the ability of Calreticulin to bind to denatured forms of both IgG and IgY regardless of the glycosylation state of the proteins. Furthermore, Calreticulin exhibits binding to peptides (glycosylated and non-glycosylated) derived from trypsin digestion of both immunoglobulins. Additionally, Calreticulin peptide binding was examined with synthetic peptides covering the IgG Cγ2 domain demonstrating interaction with approximately half the peptides. Our results show that the dominant binding activity of Calreticulin in vitro is toward the polypeptide moieties of IgG and IgY even in the presence of the monoglucosylated high mannose N -linked oligosaccharide on IgY.
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the chaperone and potential mannan binding lectin mbl co receptor Calreticulin interacts with mbl through the binding site for mbl associated serine proteases
FEBS Journal, 2008Co-Authors: Rasmus Pagh, Karen Duus, Paul R. Hansen, Peter Højrup, Inga Laursen, Julie Mangor, Nicole M Thielens, Gerard J Arlaud, Leif Kongerslev, Gunnar HouenAbstract:The chaperone Calreticulin has been suggested to function as a C1q and collectin receptor. The interaction of Calreticulin with mannan-binding lectin (MBL) was investigated by solid-phase binding assays. Calreticulin showed saturable and time-dependent binding to recombinant MBL, provided that MBL was immobilized on a solid surface or bound to mannan on a surface. The binding was non-covalent and biphasic with an initial salt-sensitive phase followed by a more stable salt-insensitive interaction. For plasma-derived MBL, known to be complexed with MBL-associated serine proteases (MASPs), no binding was observed. Interaction of Calreticulin with recombinant MBL was fully inhibited by recombinant MASP-2, MASP-3 and MAp19, but not by the MASP-2 D105G and MAp19 Y59A variants characterized by defective MBL binding ability. Furthermore, MBL point mutants with impaired MASP binding showed no interaction with Calreticulin. Comparative analysis of MBL with complement component C1q, its counterpart of the classical pathway, revealed that they display similar binding characteristics for Calreticulin, providing further indication that Calreticulin is a common co-receptor/chaperone for both proteins. In conclusion, the potential MBL co-receptor Calreticulin binds to MBL at the MASP binding site and the interaction may involve a conformational change in MBL.
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Interaction of Calreticulin with amyloid beta peptide 1-42.
Protein and peptide letters, 2008Co-Authors: Gunnar Houen, Karen Duus, Paul R. HansenAbstract:The interaction of Calreticulin with amyloid beta (Aβ) was investigated using solid phase and solution binding assays. Calreticulin bound Aβ 1-42 in a time and concentration dependent fashion. The binding was optimal at pH 5 and was stimulated by Ca2+ and inhibited by Zn2+ at pH 7. Interaction took place through the hydrophobic C-terminus of Aβ 1- 42 and the polypeptide binding site of Calreticulin. The results are discussed in the light of a reported role of Calreticulin as a cell surface scavenger receptor.
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Polypeptide binding properties of the chaperone Calreticulin
European journal of biochemistry, 2000Co-Authors: Charlotte Sværke Jørgensen, Peter Højrup, Niels H. H. Heegaard, Arne Holm, Gunnar HouenAbstract:Calreticulin is a highly conserved eukaryotic ubiquitious protein located mainly in the endoplasmic reticulum. Two major characteristics of Calreticulin are its chaperone activity and its lectin properties, but its precise function in intracellular protein and peptide processing remains to be elucidated. We have investigated the interactions of human Calreticulin with denatured ovalbumin, proteolytic digests of ovalbumin, and different available peptides by solid phase assays, size-exclusion chromatography, capillary electrophoresis, and MS. The results show that Calreticulin interacts better with unfolded ovalbumin than with native ovalbumin, that Calreticulin strongly binds components in proteolytic digests of denatured ovalbumin, and that Calreticulin interacts strongly with certain synthetic peptides.
Kimberly Burns - One of the best experts on this subject based on the ideXlab platform.
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Endoplasmic Reticulum Form of Calreticulin Modulates Glucocorticoid-sensitive Gene Expression
The Journal of biological chemistry, 1996Co-Authors: Marek Michalak, Nasrin Mesaeli, Kimberly Burns, Christi Andrin, Greta H. Jass, Jody L. Busaan, Michal OpasAbstract:Calreticulin is a ubiquitously expressed Ca2+-binding protein of the endoplasmic reticulum (ER), which inhibits DNA binding in vitro and transcriptional activation in vivo by steroid hormone receptors. Transient transfection assays were carried out to investigate the effects of different intracellular targeting of Calreticulin on transactivation mediated by glucocorticoid receptor. BSC40 cells were transfected with either Calreticulin expression vector (ER form of Calreticulin) or Calreticulin expression vector encoding Calreticulin minus leader peptide, resulting in cytoplasmic localization of the recombinant protein. Transfection of BSC40 cells with Calreticulin expression vector encoding the ER form of the protein led to 40-50% inhibition of the dexamethasone-sensitive stimulation of luciferase expression. However, in a similar experiment, but using the Calreticulin expression vector encoding cytoplasmic Calreticulin, dexamethasone-stimulated activation of the luciferase reporter gene was inhibited by only 10%. We conclude that the ER, but not cytosolic, form of Calreticulin is responsible for inhibition of glucocorticoid receptor-mediated gene expression. These effects are specific to Calreticulin, since overexpression of the ER lumenal proteins (BiP, ERp72, or calsequestrin) has no effect on glucocorticoid-sensitive gene expression. The N domain of Calreticulin binds to the DNA binding domain of the glucocorticoid receptor in vitro; however, we show that the N+P domain of Calreticulin, when synthesized without the ER signal sequence, does not inhibit glucocorticoid receptor function in vivo. Furthermore, expression of the N domain of Calreticulin and the DNA binding domain of glucocorticoid receptor as fusion proteins with GAL4 in the yeast two-hybrid system revealed that Calreticulin does not interact with glucocorticoid receptor under these conditions. We conclude that Calreticulin and glucocorticoid receptor may not interact in vivo and that the Calreticulin-dependent modulation of the glucocorticoid receptor function may therefore be due to a Calreticulin-dependent signaling from the ER.
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Interaction of Calreticulin with Protein Disulfide Isomerase
The Journal of biological chemistry, 1995Co-Authors: Shairaz Baksh, Kimberly Burns, Christi Andrin, Marek MichalakAbstract:Abstract We report here that Calreticulin interacts with protein disulfide isomerase (PDI). The PDI-Calreticulin complex can be dissociated by Zn-iminodiacetate-substituted Sepharose-agarose chromatography, suggesting that these interactions may be Zn dependent. Direct interaction between Calreticulin and PDI is also documented by Calreticulin affinity chromatography. PDI was the only pancreatic microsomal protein retained on the Calreticulin affinity column. Calreticulin and PDI were identified by their NH2-terminal amino acid sequence analysis, mobilities in SDS-polyacrylamide gel electrophoresis, binding of Ca, and their reactivity with specific antibodies. Using glutathione S-transferase-Calreticulin fusion proteins, we show that PDI interacts strongly with the P-domain and only weakly with the N-domain of Calreticulin. Expression of Calreticulin domains and PDI as fusion proteins with GAL4 in the yeast two-hybrid system revealed that Calreticulin interacted with PDI also under normal cellular conditions. Interaction with PDI required only the NH2-terminal region of the N-domain (amino acid residues 1-83) and the P-domain (amino acid residues 150-240) of Calreticulin. Importantly, interaction between Calreticulin and PDI led to the modulation of their activities. In the presence of PDI, Calreticulin does not bind Ca with high affinity. Calreticulin or the N-domain of Calreticulin inhibited PDI ability to refold scrambled RNase A.
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Interactions of Calreticulin with proteins of the endoplasmic and sarcoplasmic reticulum membranes
FEBS letters, 1993Co-Authors: Kimberly Burns, Marek MichalakAbstract:The ability of [125I]Calreticulin to bind to membrane fractions isolated from different muscle and non-muscle tissues was examined by a protein overlay technique. Specific [125AI]Calreticulin binding proteins were detected in rat liver smooth and rough endoplasmic reticulum and Golgi, in canine pancreatic microsomes, and in rabbit skeletal muscle sarcoplasmic reticulum. These proteins were confined only to membranes that contain Calreticulin; they were not found in rat liver mitochondria or cytosol. [125I]Calreticulin binds to a 50-kDa protein and a number of lower Mr (20,000–38,000) endoplasmic reticulum membrane proteins and to 30-kDa protein in skeletal muscle sarcoplasmic reticulum. Full-length Calreticulin and the carboxyl-terminal region (C-domain) of the protein both competed with [125I]Calreticulin for binding to the membrane proteins. Binding of [125I]Calreticulin to pancreatic microsomes was also partially inhibited by the N-domain and to a lesser extent by the P-domain of the protein. We conclude that Calreticulin interacts with the endoplasmic reticulum membrane proteins mainly through its carboxyl-terminal domain and that the endoplasmic and sarcoplasmic reticulum membranes may contain different Calreticulin binding proteins.
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Calreticulin in T-lymphocytes : identification of Calreticulin in T-lymphocytes and demonstration that activation of T cells correlates with increased levels of Calreticulin mRNA and protein
The Journal of biological chemistry, 1992Co-Authors: Kimberly Burns, C D Helgason, R. C. Bleackley, Marek MichalakAbstract:Abstract Ca2+ is an essential second messenger for T cell activation, but the exact mechanisms of its action are poorly understood. The cytosolic Ca2+ concentration is significantly increased upon the stimulation of T cells with either mitogen, cross-linking antibodies, or their cognate ligands. In this study, expression of Calreticulin, a major Ca(2+)-binding (storage), KDEL protein of the endoplasmic reticulum was examined in resting and concanavalin A (ConA)-stimulated mouse and human T-lymphocytes. Both resting, mouse and human lymphocytes contain very low levels of Calreticulin mRNA and protein. Mouse splenocytes stimulated with ConA exhibited an induction in Calreticulin mRNA which peaked by Day 4. A 5-fold increase in the immunoreactive Calreticulin protein band was also observed in the cells during this period of stimulation. Similarly when human lymphocytes were cultured with ConA a significant increase in the levels of the Calreticulin mRNA and protein was observed. The peak of Calreticulin mRNA was observed at Day 1 rather than Day 4 as seen for the mouse. These results clearly demonstrate the presence of Calreticulin, a Ca(2+)-binding protein originally characterized in muscle tissue, in activated T-lymphocytes. Furthermore, we show that expression of Calreticulin correlates with T-lymphocyte activation. Our results suggest that Calreticulin may be involved in the signaling pathway for the induction of Ca(2+)-dependent processes and may represent one regulatory mechanism operating in activation of T-lymphocytes.
