Interplay between myotubularins and Ca2+ homeostasis.

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.

Interplay between calcium and endoplasmic reticulum stress.

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.

Cisplatin Toxicity Is Mediated by Direct Binding to Toll-Like Receptor 4 through a Mechanism That Is Distinct from Metal Allergens.

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.

Loss of the fructose transporter SLC2A5 inhibits cancer cell migration.

Metastasis is the primary cause of cancer patient death and the elevation of SLC2A5 gene expression is often observed in metastatic cancer cells. Here we evaluated the importance of SLC2A5 in cancer cell motility by silencing its gene. We discovered that CRISPR/Cas9-mediated inactivation of the SLC2A5 gene inhibited cancer cell proliferation and migration in vitro as well as metastases in vivo in several animal models. Moreover, SLC2A5-attenuated cancer cells exhibited dramatic alterations in mitochondrial architecture and localization, uncovering the importance of SLC2A5 in directing mitochondrial function for cancer cell motility and migration. The direct association of increased abundance of SLC2A5 in cancer cells with metastatic risk in several types of cancers identifies SLC2A5 as an important therapeutic target to reduce or prevent cancer metastasis.

Calreticulin and the Heart.

Calreticulin is an endoplasmic Ca2+ binding protein and molecular chaperone. As a cardiac embryonic gene, calreticulin is essential for heart development. The protein supports Ca2+-dependent signaling events that are critical to cardiomyocyte differentiation and cardiogenesis. The increased expression of calreticulin and endoplasmic reticulum/sarcoplasmic reticulum Ca2+ capacity produces cardiomyocytes with enhanced efficiency, and detrimental mechanical stretching of cardiac fibroblasts, leading to cardiac pathology. Deletion of the calreticulin gene in adult cardiomyocytes results in left ventricle dilation, an impaired electrocardiogram, and heart failure. These observations indicate that a well-adjusted endoplasmic reticulum and calreticulin-dependent Ca2+ pool in cardiomyocytes are critical for the maintenance of proper cardiac function.

Calcium signaling and endoplasmic reticulum stress.

Cellular homeostasis is essential for healthy functioning of cells and tissues as well as proper organ development and maintenance. A disruption in cellular homeostasis triggers stress responses including the unfolded protein response (UPR), an endoplasmic reticulum (ER) stress coping response. There is increasing evidence that Ca2+ signaling plays a pivotal role in stress responses, as Ca2+ is involved many cellular activities. The ER is the main Ca2+ storage organelle and the source of Ca2+ for intracellular signaling. The ER is equipped with a variety of stress sensors and contains many Ca2+ handling proteins that support a role for Ca2+ in stress sensing and in coordinating strategies required to cope with cellular stress. Maintenance of ER Ca2+ homeostasis is therefore vital in sustaining cellular functions especially during times of cellular stress. Here we focus on selected aspects of ER Ca2+ homeostasis, its links to ER stress, and activation of the ER stress coping response.

Selective enhancement of cardiomyocyte efficiency results in a pernicious heart condition.

Transgenic mice with selective induction of calreticulin transgene expression in cardiomyocytes (CardiacCRT+) were analyzed. CardiacCRT+ cardiomyocytes showed increased contractility and Ca2+ transients. Yet, in vivo assessment of cardiac performance, and ischemic tolerance of CardiacCRT+ mice demonstrated right ventricle dilation and reduced cardiac output, increased QT interval and decreased P amplitude. Paradoxically, ex vivo working hearts from CardiacCRT+ mice showed enhanced ischemic cardio-protection and cardiac efficiency. Under aerobic conditions, CardiacCRT+ hearts showed less efficient cardiac function than sham control hearts due to an increased ATP production from glycolysis relative to glucose oxidation. During reperfusion, this inefficiency was reversed, with CardiacCRT+ hearts exhibiting better functional recovery and increased cardiac efficiency compared to sham control hearts. On the other hand, mechanical stretching of isolated cardiac fibroblasts activated the IRE1α branch of the unfolded protein response pathway as well as induction of Col1A2 and TGFß gene expression ex vivo, which were all suppressed by tauroursodeoxycholic acid.

Two pools of IRE1α in cardiac and skeletal muscle cells.

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.

Endoplasmic reticulum and the microRNA environment in the cardiovascular system 1.

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.

Tauroursodeoxycholic acid attenuates cyclosporine-induced renal fibrogenesis in the mouse model.

Chronic exposure to cyclosporine causes nephrotoxicity and organ damage. Here we show that cyclosporine nephrotoxicity in vivo is associated with the activation of the unfolded protein response (UPR) pathway to initiate tissue fibrosis. We demonstrate that cyclosporine therapy activated the IRE1α branch of the unfolded protein response (UPR) and stimulated the TGFß1 signaling pathway in the kidneys of male mice. Co-administration of the proteostasis promoter tauroursodeoxycholic acid (TUDCA) with cyclosporine inhibited the UPR pathway in the kidneys of treated male mice as well as decreased the development of renal fibrogenesis.

Cyclosporine A binding to COX-2 reveals a novel signaling pathway that activates the IRE1α unfolded protein response sensor.

Cyclosporine, a widely used immunosuppressant in organ transplantation and in treatment of various autoimmune diseases, activates the unfolded protein response (UPR), an ER stress coping response. In this study we discovered a new and unanticipated cyclosporine-dependent signaling pathway, with cyclosporine triggering direct activation of the UPR. COX-2 binds to and activates IRE1α, leading to IRE1α splicing of XBP1 mRNA. Molecular interaction and modeling analyses identified a novel interaction site for cyclosporine with COX-2 which caused enhancement of COX-2 enzymatic activity required for activation of the IRE1α branch of the UPR. Cyclosporine-dependent activation of COX-2 and IRE1α in mice indicated that cyclosporine-COX-2-IRE1α signaling pathway was functional in vivo. These findings identify COX-2 as a new IRE1α binding partner and regulator of the IRE1α branch of the UPR pathway, and establishes the mechanism underlying cytotoxicity associated with chronic cyclosporine exposure.

Interactome Screening Identifies the ER Luminal Chaperone Hsp47 as a Regulator of the Unfolded Protein Response Transducer IRE1α.

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.

Endoplasmic reticulum in health and disease: the 12th International Calreticulin Workshop, Delphi, Greece.

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.

Fatty acid binding protein (Fabp) 5 interacts with the calnexin cytoplasmic domain at the endoplasmic reticulum.

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.

Inhibition of the Unfolded Protein Response Mechanism Prevents Cardiac Fibrosis.

BACKGROUND: Cardiac fibrosis attributed to excessive deposition of extracellular matrix proteins is a major cause of heart failure and death. Cardiac fibrosis is extremely difficult and challenging to treat in a clinical setting due to lack of understanding of molecular mechanisms leading to cardiac fibrosis and effective anti-fibrotic therapies. The objective in this study was to examine whether unfolded protein response (UPR) pathway mediates cardiac fibrosis and whether a pharmacological intervention to modulate UPR can prevent cardiac fibrosis and preserve heart function. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate here that the mechanism leading to development of fibrosis in a mouse with increased expression of calreticulin, a model of heart failure, stems from impairment of endoplasmic reticulum (ER) homeostasis, transient activation of the unfolded protein response (UPR) pathway and stimulation of the TGFß1/Smad2/3 signaling pathway. Remarkably, sustained pharmacologic inhibition of the UPR pathway by tauroursodeoxycholic acid (TUDCA) is sufficient to prevent cardiac fibrosis, and improved exercise tolerance. CONCLUSIONS: We show that the mechanism leading to development of fibrosis in a mouse model of heart failure stems from transient activation of UPR pathway leading to persistent remodelling of cardiac tissue. Blocking the activation of the transiently activated UPR pathway by TUDCA prevented cardiac fibrosis, and improved prognosis. These findings offer a window for additional interventions that can preserve heart function.

Ces3/TGH Deficiency Attenuates Steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease in developed countries. NAFLD describes a wide range of liver pathologies from simple steatosis to nonalcoholic steatohepatitis (NASH) and cirrhosis. NASH is distinguished from simple steatosis by inflammation, cell death and fibrosis. In this study we found that mice lacking triacylglycerol hydrolase (TGH, also known as carboxylesterase 3 or carboxylesterase 1d) are protected from high-fat diet (HFD) - induced hepatic steatosis via decreased lipogenesis, increased fatty acid oxidation and improved hepatic insulin sensitivity. To examine the effect of the loss of TGH function on the more severe NAFLD form NASH, we ablated Tgh expression in two independent NASH mouse models, Pemt(-/-) mice fed HFD and Ldlr(-/-) mice fed high-fat, high-cholesterol Western-type diet (WTD). TGH deficiency reduced liver inflammation, oxidative stress and fibrosis in Pemt(-/-) mice. TGH deficiency also decreased NASH in Ldlr(-/-) mice. Collectively, these findings indicate that TGH deficiency attenuated both simple hepatic steatosis and irreversible NASH.

The Endoplasmic Reticulum Chaperone Calnexin Is a NADPH Oxidase NOX4 Interacting Protein.

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.

Calreticulin secures calcium-dependent nuclear pore competency required for cardiogenesis.

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.

Unfolding the complexities of ER chaperones in health and disease: report on the 11th international calreticulin workshop.

The 11th International Calreticulin workshop was held May 15-18, 2015 at New York University School of Medicine-Langone Medical Center, New York. The meeting highlighted many of the new discoveries in the past 2 years involving the important role of molecular chaperones in physiological and pathological processes. Crucial to the understanding of these disease processes was the role of chaperones in maintaining quality control of protein processing in the endoplasmic reticulum, the importance of Ca(2) regulation acting through its action in stress-related diseases, and the trafficking of glycoproteins to the cell surface. Central to maintaining healthy cell physiology is the correct ER-associated protein degradation of specific misfolded proteins. Information on different mechanisms involved in the degradation of misfolded proteins was revealed. This was a landmark meeting for the chaperone field in terms of new insights into their roles in physiology. These insights included the unfolded protein response, innate/adaptive immunity, tissue repair, the functions of calreticulin/chaperones from the cell surface, and extracellular environment. Diseases included neurodegenerative disorders, prion disease, autoimmunity, fibrosis-related disease, the host immune response to cancer, and hematologic diseases associated with calreticulin mutations. The 12th calreticulin workshop is planned for the spring of 2017 in Delphi, Greece.

Systems biology surveillance decrypts pathological transcriptome remodeling.

BACKGROUND: Pathological cardiac development is precipitated by dysregulation of calreticulin, an endoplasmic reticulum (ER)-resident calcium binding chaperone and critical contributor to cardiogenesis and embryonic viability. However, pleiotropic phenotype derangements induced by calreticulin deficiency challenge the identification of specific downstream transcriptome elements that direct proper cardiac formation. Here, differential transcriptome navigation was used to diagnose high priority calreticulin domain-specific gene expression changes and decrypt complex cardiac-specific molecular responses elicited by discrete functional regions of calreticulin. METHODS: Wild type (WT), calreticulin-deficient (CALR(-/-)), and calreticulin truncation variant (CALR(-/-)-NP and CALR(-/-)-PC) pluripotent stem cells were used to investigate molecular remodeling underlying a model of cardiopathology. Bioinformatic deconvolution of isolated transcriptomes was performed to identify predominant expression trends, gene ontology prioritizations, and molecular network features characteristic of discrete cell types. RESULTS: Stem cell lines with wild type (WT), calreticulin-deficient (CALR(-/-)) genomes, as well as calreticulin truncation variants exclusively expressing either the chaperoning (CALR(-/-)-NP) or the calcium binding (CALR(-/-)-PC) domain exhibited characteristic molecular signatures determined by unsupervised agglomerative clustering. Kohonen mapping of RNA expression changes identified transcriptome dynamics that segregated into 12 discrete gene expression meta-profiles which were enriched for regulation of Eukaryotic Initiation Factor 2 (EIF2) signaling. Focused examination of domain-specific gene ontology remodeling revealed a general enrichment of Cardiovascular Development in the truncation variants, with unique prioritization of "Cardiovascular Disease" exclusive to the cohort of down regulated genes of the PC truncation variant. Molecular cartography of genes that comprised this cardiopathological category revealed uncharacterized and novel gene relationships, with identification of Pitx2 as a critical hub within the topology of a CALR(-/-) compromised network. CONCLUSIONS: Diagnostic surveillance, through an algorithm that integrates pluripotent stem cell transcriptomes with advanced high throughput assays and computational bioinformatics, revealed collective gene expression network changes that underlie differential phenotype development. Stem cell transcriptomes provide a deep collective molecular index that reflects ad hoc robustness of the pluripotent gene network. Remodeling events such as monogenic lesions provide a background by which high priority candidate disease effectors and regulators can be identified, demonstrated here by a molecular profiling algorithm that decrypts pluripotent wild type versus disrupted genomes.