ABSTRACT
Maintenance of endoplasmic reticulum (ER) proteostasis is controlled by a dynamic signaling network known as the unfolded protein response (UPR). IRE1α is a major UPR transducer, determining cell fate under ER stress. We used an interactome screening to unveil several regulators of the UPR, highlighting the ER chaperone Hsp47 as the major hit. Cellular and biochemical analysis indicated that Hsp47 instigates IRE1α signaling through a physical interaction. Hsp47 directly binds to the ER luminal domain of IRE1α with high affinity, displacing the negative regulator BiP from the complex to facilitate IRE1α oligomerization. The regulation of IRE1α signaling by Hsp47 is evolutionarily conserved as validated using fly and mouse models of ER stress. Hsp47 deficiency sensitized cells and animals to experimental ER stress, revealing the significance of Hsp47 to global proteostasis maintenance. We conclude that Hsp47 adjusts IRE1α signaling by fine-tuning the threshold to engage an adaptive UPR.
Subject(s)
Endoribonucleases/metabolism , HSP47 Heat-Shock Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Animals , COS Cells , Chlorocebus aethiops , DNA-Binding Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum Stress/physiology , HSP47 Heat-Shock Proteins/physiology , Humans , Mice , Molecular Chaperones/metabolism , Signal Transduction , Stress, Physiological , Transcription Factors/metabolism , Unfolded Protein ResponseABSTRACT
Cisplatin is an effective chemotherapeutic agent, yet its use is limited by several adverse drug reactions, known as cisplatin-induced toxicities (CITs). We recently demonstrated that cisplatin could elicit proinflammatory responses associated with CITs through Toll-like receptor 4 (TLR4). TLR4 is best recognized for binding bacterial lipopolysaccharide (LPS) via its coreceptor, MD-2. TLR4 is also proposed to directly bind transition metals, such as nickel. Little is known about the nature of the cisplatin-TLR4 interaction. Here, we show that soluble TLR4 was capable of blocking cisplatin-induced, but not LPS-induced, TLR4 activation. Cisplatin and nickel, but not LPS, were able to directly bind soluble TLR4 in a microscale thermophoresis binding assay. Interestingly, TLR4 histidine variants that abolish nickel binding reduced, but did not eliminate, cisplatin-induced TLR4 activation. This was corroborated by binding data that showed cisplatin, but not nickel, could directly bind mouse TLR4 that lacks these histidine residues. Altogether, our findings suggest that TLR4 can directly bind cisplatin in a manner that is enhanced by, but not dependent on, histidine residues that facilitate binding to transition metals. SIGNIFICANCE STATEMENT: This work describes how the xenobiotic cisplatin interacts with Toll-like receptor 4 (TLR4) to initiate proinflammatory signaling that underlies cisplatin toxicities, which are severe adverse outcomes in cisplatin treatment. Here, this study provides a mechanistic bridge between cisplatin extracellular interactions with TLR4 and previous observations that genetic and chemical inhibition of TLR4 mitigates cisplatin-induced toxicity.
Subject(s)
Cisplatin , Toll-Like Receptor 4 , Animals , Mice , Allergens , Cisplatin/toxicity , Histidine , Lipopolysaccharides/pharmacology , Lymphocyte Antigen 96/chemistry , Lymphocyte Antigen 96/genetics , Lymphocyte Antigen 96/metabolism , Toll-Like Receptor 4/drug effects , Toll-Like Receptor 4/metabolismABSTRACT
The endoplasmic reticulum (ER) plays a central role in cellular stress responses via mobilization of ER stress coping responses, such as the unfolded protein response (UPR). The inositol-requiring 1α (IRE1α) is an ER stress sensor and component of the UPR. Muscle cells also have a well-developed and highly subspecialized membrane network of smooth ER called the sarcoplasmic reticulum (SR) surrounding myofibrils and specialized for Ca2+ storage, release, and uptake to control muscle excitation-contraction coupling. Here, we describe 2 distinct pools of IRE1α in cardiac and skeletal muscle cells, one localized at the perinuclear ER and the other at the junctional SR. We discovered that, at the junctional SR, calsequestrin binds to the ER luminal domain of IRE1α, inhibiting its dimerization. This novel interaction of IRE1α with calsequestrin, one of the highly abundant Ca2+ handling proteins at the junctional SR, provides new insights into the regulation of stress coping responses in muscle cells.-Wang, Q., Groenendyk, J., Paskevicius, T., Qin, W., Kor, K. C., Liu, Y., Hiess, F., Knollmann, B. C., Chen, S. R. W., Tang, J., Chen, X.-Z., Agellon, L. B., Michalak, M. Two pools of IRE1α in cardiac and skeletal muscle cells.
Subject(s)
Endoribonucleases/metabolism , Muscle Fibers, Skeletal/metabolism , Myocytes, Cardiac/metabolism , Protein Serine-Threonine Kinases/metabolism , Animals , Binding Sites , COS Cells , Calcium Signaling , Calsequestrin/metabolism , Cells, Cultured , Chlorocebus aethiops , Endoribonucleases/chemistry , Mice , Protein Binding , Protein Serine-Threonine Kinases/chemistry , Rabbits , Sarcoplasmic Reticulum/metabolismABSTRACT
Stress responses are important to human physiology and pathology, and the inability to adapt to cellular stress leads to cell death. To mitigate cellular stress and re-establish homeostasis, cells, including those in the cardiovascular system, activate stress coping response mechanisms. The endoplasmic reticulum, a component of the cellular reticular network in cardiac cells, mobilizes so-called endoplasmic reticulum stress coping responses, such as the unfolded protein response. MicroRNAs play an important part in the maintenance of cellular and tissue homeostasis, perform a central role in the biology of the cardiac myocyte, and are involved in pathological cardiac function and remodeling. In this paper, we review a link between endoplasmic reticulum homeostasis and microRNA with an emphasis on the impact on stress responses in the cardiovascular system.
Subject(s)
Cardiovascular System/cytology , Cardiovascular System/metabolism , Endoplasmic Reticulum/metabolism , MicroRNAs/genetics , Animals , Base Sequence , Endoplasmic Reticulum Stress , Homeostasis , Humans , Up-RegulationABSTRACT
Starting from 1994, every 2 years, an international workshop is organized focused on calreticulin and other endoplasmic reticulum chaperones. In 2017, the workshop took place at Delphi Greece. Participants from North and South America, Europe, Asia and Australia presented their recent data and discussed them extensively with their colleagues. Presentations dealt with structural aspects of calreticulin and calnexin, the role of Ca2+ in cellular signalling and in autophagy, the endoplasmic reticulum stress and the unfolded protein response, the role of calreticulin in immune responses. Several presentations focused on the role of calreticulin and other ER chaperones in a variety of disease states, including haemophilia, obesity, diabetes, Sjogren's syndrome, Chagas diseases, multiple sclerosis, amyotrophic lateral sclerosis, neurological malignancies (especially glioblastoma), haematological malignancies (especially essential thrombocythemia and myelofibrosis), lung adenocarcinoma, renal pathology with emphasis in fibrosis and drug toxicity. In addition, the role of calreticulin and calnexin in growth and wound healing was discussed, as well as the possible use of extracellular calreticulin as a marker for certain diseases. It was agreed that the 13th International Calreticulin Workshop will be organized in 2019 in Montreal, Quebec, Canada.
Subject(s)
Amyotrophic Lateral Sclerosis/genetics , Calreticulin/genetics , Endoplasmic Reticulum/genetics , Hemophilia A/genetics , Neoplasms/genetics , Obesity/genetics , Amyotrophic Lateral Sclerosis/immunology , Amyotrophic Lateral Sclerosis/pathology , Animals , Autophagy , Calcium/metabolism , Calnexin/genetics , Calnexin/isolation & purification , Calreticulin/immunology , Endoplasmic Reticulum/immunology , Endoplasmic Reticulum/pathology , Endoplasmic Reticulum Stress , Gene Expression Regulation , Hemophilia A/immunology , Hemophilia A/pathology , Humans , Immunity, Innate , Molecular Chaperones/genetics , Molecular Chaperones/immunology , Neoplasms/immunology , Neoplasms/pathology , Obesity/immunology , Obesity/pathology , Signal Transduction , Unfolded Protein Response , Wound Healing/genetics , Wound Healing/immunologyABSTRACT
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.
Subject(s)
Calnexin/genetics , Endoplasmic Reticulum/metabolism , Fibroblasts/metabolism , NADPH Oxidases/genetics , Animals , Calnexin/antagonists & inhibitors , Calnexin/metabolism , Cell Line , Endoplasmic Reticulum/chemistry , Fibroblasts/cytology , Gene Expression , HEK293 Cells , Humans , Immunoprecipitation , Isotope Labeling , Mice , Mice, Knockout , NADPH Oxidase 4 , NADPH Oxidases/metabolism , Protein Binding , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Reactive Oxygen Species/metabolismABSTRACT
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.
Subject(s)
Calnexin/chemistry , Calnexin/metabolism , Cytoplasm/metabolism , Endoplasmic Reticulum/metabolism , Fatty Acid-Binding Proteins/metabolism , Fibroblasts/metabolism , Neoplasm Proteins/metabolism , Animals , Binding Sites , Cells, Cultured , Cytoplasm/chemistry , Endoplasmic Reticulum/chemistry , Fatty Acid-Binding Proteins/chemistry , Fibroblasts/chemistry , Mice , Neoplasm Proteins/chemistry , Protein Binding , Protein DomainsABSTRACT
The endoplasmic reticulum (ER) is a multifunctional intracellular organelle, a component of the cellular reticular network that allows cells to adjust to a wide variety of conditions. The cardiomyocyte reticular network is the ideal location of sensors for both intrinsic and extrinsic factors that disrupt energy and/or nutrient homeostasis and lead to ER stress, a disturbance in ER function. ER stress has been linked to both physiological and pathological states in the cardiovascular system; such states include myocardial infarction, oxygen starvation (hypoxia) and fuel starvation, ischemia, pressure overload, dilated cardiomyopathy, hypertrophy, and heart failure. The ER stress coping response (e.g., the unfolded protein response) is composed of discrete pathways that are controlled by a collection of common regulatory components that may function as a single entity involved in reacting to ER stress. These corrective strategies allow the cardiomyocyte reticular network to restore energy and/or nutrient homeostasis and to avoid cell death. Therefore, the identities of the ER stress corrective strategies are important targets for the development of therapeutic approaches for cardiovascular and other acquired disorders.
Subject(s)
Cardiovascular Physiological Phenomena , Cardiovascular System/physiopathology , Endoplasmic Reticulum Stress/physiology , Cardiovascular System/cytology , Energy Metabolism/physiology , Homeostasis/physiology , Humans , Myocytes, Cardiac/physiologyABSTRACT
Calreticulin deficiency causes myocardial developmental defects that culminate in an embryonic lethal phenotype. Recent studies have linked loss of this calcium binding chaperone to failure in myofibrillogenesis through an as yet undefined mechanism. The purpose of the present study was to identify cellular processes corrupted by calreticulin deficiency that precipitate dysregulation of cardiac myofibrillogenesis related to acquisition of cardiac phenotype. In an embryonic stem cell knockout model, calreticulin deficit (crt(-/-)) compromised nucleocytoplasmic transport of nuclear localization signal-dependent and independent pathways, disrupting nuclear import of the cardiac transcription factor MEF2C. The expression of nucleoporins and associated nuclear transport proteins in derived crt(-/-) cardiomyocytes revealed an abnormal nuclear pore complex (NPC) configuration. Altered protein content in crt(-/-) cells resulted in remodeled NPC architecture that caused decreased pore diameter and diminished probability of central channel occupancy versus wild type counterparts. Ionophore treatment of impaired calcium handling in crt(-/-) cells corrected nuclear pore microarchitecture and rescued nuclear import resulting in normalized myofibrillogenesis. Thus, calreticulin deficiency alters nuclear pore function and structure, impeding myofibrillogenesis in nascent cardiomyocytes through a calcium dependent mechanism. This essential role of calreticulin in nucleocytoplasmic communication competency ties its regulatory action with proficiency of cardiac myofibrillogenesis essential for proper cardiac development.
Subject(s)
Calreticulin/genetics , Cardiomyopathies/genetics , Muscle Development/genetics , Nuclear Pore/genetics , Active Transport, Cell Nucleus/genetics , Animals , Calcium/metabolism , Calcium Signaling/genetics , Calreticulin/deficiency , Cardiomyopathies/metabolism , Cardiomyopathies/pathology , Cell Differentiation/genetics , Embryonic Stem Cells/metabolism , Embryonic Stem Cells/pathology , Gene Knockout Techniques , Humans , MEF2 Transcription Factors/genetics , Mice , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Myocytes, Cardiac/ultrastructure , Nuclear Pore/metabolism , Nuclear Pore/ultrastructureABSTRACT
Calnexin is a type I integral endoplasmic reticulum (ER) membrane protein, molecular chaperone, and a component of the translocon. We discovered a novel interaction between the calnexin cytoplasmic domain and UBC9, a SUMOylation E2 ligase, which modified the calnexin cytoplasmic domain by the addition of SUMO. We demonstrated that calnexin interaction with the SUMOylation machinery modulates an interaction with protein tyrosine phosphatase 1B (PTP1B), an ER-associated protein tyrosine phosphatase involved in the negative regulation of insulin and leptin signaling. We showed that calnexin and PTP1B form UBC9-dependent complexes, revealing a previously unrecognized contribution of calnexin to the retention of PTP1B at the ER membrane. This work shows that the SUMOylation machinery links two ER proteins from divergent pathways to potentially affect cellular protein quality control and energy metabolism.
Subject(s)
Calnexin/metabolism , Endoplasmic Reticulum/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 1/metabolism , Ubiquitin-Conjugating Enzymes/metabolism , Animals , Blotting, Western , Calnexin/genetics , Dogs , HeLa Cells , Humans , Mice , Mice, Knockout , Microscopy, Confocal , NIH 3T3 Cells , Protein Binding , Protein Tyrosine Phosphatase, Non-Receptor Type 1/genetics , RNA Interference , Sumoylation , Two-Hybrid System Techniques , Ubiquitin-Conjugating Enzymes/geneticsABSTRACT
Influenza is a major cause of morbidity and mortality in immunosuppressed persons, and vaccination often confers insufficient protection. IL-28B, a member of the interferon (IFN)-λ family, has variable expression due to single nucleotide polymorphisms (SNPs). While type-I IFNs are well known to modulate adaptive immunity, the impact of IL-28B on B- and T-cell vaccine responses is unclear. Here we demonstrate that the presence of the IL-28B TG/GG genotype (rs8099917, minor-allele) was associated with increased seroconversion following influenza vaccination (OR 1.99 pâ=â0.038). Also, influenza A (H1N1)-stimulated T- and B-cells from minor-allele carriers showed increased IL-4 production (4-fold) and HLA-DR expression, respectively. In vitro, recombinant IL-28B increased Th1-cytokines (e.g. IFN-γ), and suppressed Th2-cytokines (e.g. IL-4, IL-5, and IL-13), H1N1-stimulated B-cell proliferation (reduced 70%), and IgG-production (reduced>70%). Since IL-28B inhibited B-cell responses, we designed antagonistic peptides to block the IL-28 receptor α-subunit (IL28RA). In vitro, these peptides significantly suppressed binding of IFN-λs to IL28RA, increased H1N1-stimulated B-cell activation and IgG-production in samples from healthy volunteers (2-fold) and from transplant patients previously unresponsive to vaccination (1.4-fold). Together, these findings identify IL-28B as a key regulator of the Th1/Th2 balance during influenza vaccination. Blockade of IL28RA offers a novel strategy to augment vaccine responses.
Subject(s)
Adaptive Immunity/drug effects , B-Lymphocytes/pathology , Influenza A Virus, H1N1 Subtype/immunology , Influenza Vaccines/pharmacology , Influenza, Human/pathology , Interleukins/physiology , T-Lymphocytes/pathology , Adaptive Immunity/immunology , Adaptive Immunity/physiology , Adult , Aged , B-Lymphocytes/drug effects , B-Lymphocytes/metabolism , Cell Proliferation , Female , HLA-DR Antigens/metabolism , Humans , Immunocompromised Host , Immunoglobulin G/metabolism , In Vitro Techniques , Influenza Vaccines/immunology , Influenza, Human/metabolism , Influenza, Human/prevention & control , Interferons , Interleukin-4/metabolism , Interleukins/genetics , Interleukins/pharmacology , Male , Middle Aged , Polymorphism, Single Nucleotide/genetics , Recombinant Proteins/pharmacology , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , Th1 Cells/pathology , Th2 Cells/pathology , Transplant RecipientsABSTRACT
The endoplasmic reticulum (ER) is responsible for many housekeeping functions within the cell and is an important site for pathways that regulates its state of homeostasis. When cellular states perturb ER functions, a phenomenon termed "ER stress" activates a number of pathways to counteract the associated damages; these pathways are together called the unfolded protein response (UPR). The UPR has a dualistic function; it exists to alleviate damage associated with ER stress, however, if this is not possible, then it signals for cell death through apoptosis. Cancer cells are shown to be very resilient under extreme environmental stress and an increasing number of studies have indicated that this may be largely due to an altered state of the UPR. The role of ER stress and the UPR in cancer is still not clear, however many components are involved and may prove to be promising targets in future anti-cancer therapy. This article is part of a Special Issue entitled: Calcium Signaling in Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.
Subject(s)
Endoplasmic Reticulum Stress/genetics , Gene Expression Regulation, Neoplastic , Neoplasm Proteins/genetics , Neoplasms/genetics , Animals , Cell Death , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/metabolism , Homeostasis , Humans , Neoplasm Proteins/metabolism , Neoplasms/metabolism , Signal Transduction , Unfolded Protein Response/geneticsABSTRACT
MOTIVATION: Off-target interactions of a popular immunosuppressant Cyclosporine A (CSA) with several proteins besides its molecular target, cyclophilin A, are implicated in the activation of signaling pathways that lead to numerous side effects of this drug. RESULTS: Using structural human proteome and a novel algorithm for inverse ligand binding prediction, ILbind, we determined a comprehensive set of 100+ putative partners of CSA. We empirically show that predictive quality of ILbind is better compared with other available predictors for this compound. We linked the putative target proteins, which include many new partners of CSA, with cellular functions, canonical pathways and toxicities that are typical for patients who take this drug. We used complementary approaches (molecular docking, molecular dynamics, surface plasmon resonance binding analysis and enzymatic assays) to validate and characterize three novel CSA targets: calpain 2, caspase 3 and p38 MAP kinase 14. The three targets are involved in the apoptotic pathways, are interconnected and are implicated in nephrotoxicity.
Subject(s)
Cyclosporine/chemistry , Immunosuppressive Agents/chemistry , Proteomics/methods , Algorithms , Calpain/chemistry , Calpain/metabolism , Caspase 3/chemistry , Caspase 3/metabolism , Cyclosporine/metabolism , Humans , Immunosuppressive Agents/metabolism , Mitogen-Activated Protein Kinase 14/chemistry , Mitogen-Activated Protein Kinase 14/metabolism , Molecular Docking Simulation , Proteome/chemistry , Signal Transduction , Surface Plasmon ResonanceABSTRACT
The endoplasmic reticulum (ER) supports many cellular processes and performs diverse functions, including protein synthesis, translocation across the membrane, integration into the membrane, folding, and posttranslational modifications including N-linked glycosylation; and regulation of Ca2+ homeostasis. In mammalian systems, the majority of proteins synthesized by the rough ER have N-linked glycans critical for protein maturation. The N-linked glycan is used as a quality control signal in the secretory protein pathway. A series of chaperones, folding enzymes, glucosidases, and carbohydrate transferases support glycoprotein synthesis and processing. Perturbation of ER-associated functions such as disturbed ER glycoprotein quality control, protein glycosylation and protein folding results in activation of an ER stress coping response. Collectively this ER stress coping response is termed the unfolded protein response (UPR), and occurs through the activation of complex cytoplasmic and nuclear signaling pathways. Cellular and ER homeostasis depends on balanced activity of the ER protein folding, quality control, and degradation pathways; as well as management of the ER stress coping response.
Subject(s)
Endoplasmic Reticulum Stress/physiology , Glycoproteins/metabolism , Protein Folding , Protein Processing, Post-Translational/physiology , Proteolysis , Unfolded Protein Response/physiology , Animals , Glycoproteins/genetics , Glycosylation , Humans , Protein Transport/physiologyABSTRACT
The myotubularin family, encompassing myotubularin 1 (MTM1) and 14 myotubularin-related proteins (MTMRs), represents a conserved group of phosphatases featuring a protein tyrosine phosphatase domain. Nine members are characterized by an active phosphatase domain C(X)5R, dephosphorylating the D3 position of PtdIns(3)P and PtdIns(3,5)P2. Mutations in myotubularin genes result in human myopathies, and several neuropathies including X-linked myotubular myopathy and Charcot-Marie-Tooth type 4B. MTM1, MTMR6 and MTMR14 also contribute to Ca2+ signaling and Ca2+ homeostasis that play a key role in many MTM-dependent myopathies and neuropathies. Here we explore the evolving roles of MTM1/MTMRs, unveiling their influence on critical aspects of Ca2+ signaling pathways.
Subject(s)
Calcium Signaling , Calcium , Homeostasis , Protein Tyrosine Phosphatases, Non-Receptor , Humans , Protein Tyrosine Phosphatases, Non-Receptor/metabolism , Protein Tyrosine Phosphatases, Non-Receptor/genetics , Calcium/metabolism , Animals , Myopathies, Structural, Congenital/genetics , Myopathies, Structural, Congenital/metabolism , MutationABSTRACT
STIM1 is an endoplasmic reticulum (ER) membrane Ca(2+) sensor responsible for activation of store-operated Ca(2+) influx. We discovered that STIM1 oligomerization and store-operated Ca(2+) entry (SOC) are modulated by the ER oxidoreductase ERp57. ERp57 interacts with the ER luminal domain of STIM1, with this interaction involving two conserved cysteine residues, C(49) and C(56). SOC is accelerated in the absence of ERp57 and inhibited in C(49) and C(56) mutants of STIM1. We show that ERp57, by ER luminal interaction with STIM1, has a modulatory role in capacitative Ca(2+) entry. This is the first demonstration of a protein involved in ER intraluminal regulation of STIM1.
Subject(s)
Calcium Signaling , Calcium/metabolism , Endoplasmic Reticulum/enzymology , Membrane Glycoproteins/metabolism , Protein Disulfide-Isomerases/metabolism , Animals , Calcium Channels , Cysteine/metabolism , Disulfides/metabolism , Fluorescence Resonance Energy Transfer , Gene Knockdown Techniques , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/deficiency , Mice , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Protein Binding , Protein Disulfide-Isomerases/deficiency , Protein Structure, Tertiary , Stromal Interaction Molecule 1 , Structure-Activity RelationshipABSTRACT
Cellular homeostasis is crucial for the healthy functioning of the organism. Disruption of cellular homeostasis activates endoplasmic reticulum (ER) stress coping responses including the unfolded protein response (UPR). There are three ER resident stress sensors responsible for UPR activation - IRE1α, PERK and ATF6. Ca2+ signaling plays an important role in stress responses including the UPR and the ER is the main Ca2+ storage organelle and a source of Ca2+ for cell signaling. The ER contains many proteins involved in Ca2+ import/export/ storage, Ca2+ movement between different cellular organelles and ER Ca2+ stores refilling. Here we focus on selected aspects of ER Ca2+ homeostasis and its role in activation of the ER stress coping responses.
Subject(s)
Calcium , Protein Serine-Threonine Kinases , Protein Serine-Threonine Kinases/metabolism , Calcium/metabolism , Endoribonucleases/genetics , Endoplasmic Reticulum Stress , Unfolded Protein ResponseABSTRACT
Calnexin is a type I endoplasmic reticulum lectin-like chaperone protein. In this study, we have used site-specific mutagenesis to investigate the functional importance of glutamate E351 found at the tip of the P-domain of calnexin, and tryptophan W428 found in the carbohydrate binding region of the globular domain of the protein. The E351 and W428 calnexin mutants lost the ability to inhibit aggregation of IgY (glycosylated substrate). The E351 mutation led to slightly enhanced ERp57 binding to calnexin, whereas W428 greatly enhanced binding of ERp57 to calnexin. These findings indicate that modification of a residue(s) in the carbohydrate binding region may have a profound effect on the structural and functional properties of the P-domain and consequently on association of calnexin with the folding enzyme ERp57.
Subject(s)
Calnexin/genetics , Calnexin/metabolism , Mutant Proteins/metabolism , Mutation , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/pharmacology , Animals , Binding Sites/genetics , Calcium/chemistry , Calcium/pharmacology , Calnexin/chemistry , Circular Dichroism , Dose-Response Relationship, Drug , Glutamic Acid/genetics , Glutamic Acid/metabolism , Immunoglobulins/chemistry , Immunoglobulins/metabolism , Kinetics , Mice , Mutagenesis, Site-Directed , Mutant Proteins/chemistry , Protein Binding , Protein Conformation/drug effects , Protein Denaturation , Protein Disulfide-Isomerases/metabolism , Surface Plasmon Resonance , Temperature , Tryptophan/genetics , Tryptophan/metabolism , Zinc/chemistry , Zinc/pharmacologyABSTRACT
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.
Subject(s)
Calnexin/chemistry , Cysteine/physiology , Calnexin/genetics , Calnexin/metabolism , Disulfides , Histidine , Humans , Immunoglobulins , Molecular Chaperones , Mutagenesis, Site-Directed , Protein Disulfide-Isomerases/metabolism , Protein TransportABSTRACT
The endoplasmic reticulum (ER) is a multifunctional intracellular organelle supporting many processes required by virtually every mammalian cell, including cardiomyocytes. It performs diverse functions, including protein synthesis, translocation across the membrane, integration into the membrane, folding, posttranslational modification including N-linked glycosylation, and synthesis of phospholipids and steroids on the cytoplasmic side of the ER membrane, and regulation of Ca(2+) homeostasis. Perturbation of ER-associated functions results in ER stress via the activation of complex cytoplasmic and nuclear signaling pathways, collectively termed the unfolded protein response (UPR) (also known as misfolded protein response), leading to upregulation of expression of ER resident chaperones, inhibition of protein synthesis and activation of protein degradation. The UPR has been associated with numerous human pathologies, and it may play an important role in the pathophysiology of the heart. ER stress responses, ER Ca(2+) buffering, and protein and lipid turnover impact many cardiac functions, including energy metabolism, cardiogenesis, ischemic/reperfusion, cardiomyopathies, and heart failure. ER proteins and ER stress-associated pathways may play a role in the development of novel UPR-targeted therapies for cardiovascular diseases.