GRP75-dependent mitochondria-ER contacts ensure cell survival during early mouse thymocyte development.

Mitochondria and endoplasmic reticulum contacts (MERCs) control multiple cellular processes, including cell survival and differentiation. Based on the observations that MERCs were specifically enriched in the CD4-CD8- double-negative (DN) stage, we studied their role in early mouse thymocyte development. We found that T cell-specific knockout of Hspa9, which encodes GRP75, a protein that mediates MERC formation by assembling the IP3R-GRP75-VDAC complex, impaired DN3 thymocyte viability and resulted in thymocyte developmental arrest at the DN3-DN4 transition. Mechanistically, GRP75 deficiency induced mitochondrial stress, releasing mitochondrial DNA (mtDNA) into the cytosol and triggering the type I interferon (IFN-I) response. The IFN-I pathway contributed to both the impairment of cell survival and DN3-DN4 transition blockage, while increased lipid peroxidation (LPO) played a major role downstream of IFN-I. Thus, our study identifies the essential role of GRP75-dependent MERCs in early thymocyte development and the governing facts of cell survival and differentiation in the DN stage.

Circulating mortalin in blood and activation of the alternative complement pathway as risk indicators in COVID-19 infection.

Background: Mortalin/GRP75 is a ubiquitous mitochondrial chaperone related to the cytosolic heat shock protein 70. It protects cells from various types of damages and from senescence. Our goal was to determine whether COVID-19 patients have circulating mortalin in their blood and to assess its prognostic value in anticipating disease severity. Methods: Mortalin was determined by ELISA in the sera of 83 COVID-19 patients enrolled in the study. Patients were categorized into 4 groups: critical patients who died (FATAL) or required intensive care and survived (ICU), patients of mild severity (hospitalized but not critical) who required nasal oxygen support (HOSP+O2), and patients who did not need oxygen therapy (HOSP). Results: The mortalin concentration in the serum of all COVID-19 patients in the cohort was 194-2324 pg/mL. A comparison of the mortalin levels by peak severity among the various patient groups showed a highly significant difference between the HOSP and FATAL groups and a significant difference between the HOSP and the ICU groups. COVID-19 patients who eventually failed to survive had at hospitalization a markedly higher level of mortalin in their sera. Cox regression analysis revealed a high mortality hazard (HR=3.96, p<0.01) in patients with high mortalin circulating levels (above the median, ≥651 pg/mL). This was confirmed in survival curve analysis (Kaplan-Meier; p=0.0032, log-rank test). Mortalin remained an independent predictor of mortality even after adjusting for age and sex or various complement activation products. Complement activation data collected in an earlier study in the same cohort was compared regarding the mortalin levels. Patients with higher circulating mortalin levels also had higher levels of complement C3a but reduced levels of properdin. Discussion: This is the first report on circulating mortalin in COVID-19 patients. Higher mortalin levels were associated with more severe illnesses and a higher risk of death. We claim that quantifying the blood levels of mortalin and activated complement proteins will provide important information on the prognosis of COVID-19 patients and will serve as a useful tool for guiding their clinical management and treatment.

Ip3r-Grp75-Vdac and Relevant Ca2+ Signaling Regulate Dietary Palmitic Acid-Induced De Novo Lipogenesis by Mitochondria-Associated ER Membrane (MAM) Recruiting Seipin in Yellow Catfish.

BACKGROUND: The mitochondria-associated endoplasmic reticulum membrane (MAM) is the central hub for endoplasmic reticulum and mitochondria functional communication. It plays a crucial role in hepatic lipid homeostasis. However, even though MAM has been acknowledged to be rich in enzymes that contribute to lipid biosynthesis, no study has yet investigated the exact role of MAM on hepatic neutral lipid synthesis. OBJECTIVES: To address these gaps, this study investigated the systemic control mechanisms of MAM on neutral lipids synthesis by recruiting seipin, focusing on the role of the inositol trisphosphate receptor-1,4,5(Ip3r)-75 kDa glucose-regulated protein (Grp75)-voltage-dependent anion channel (Vdac) complex and their relevant Ca2+ signaling in this process. METHODS: To this end, a model animal for lipid metabolism, yellow catfish (Pelteobagrus fulvidraco), were fed 6 different diets containing a range of palmitic acid (PA) concentrations from 0-150 g/kg in vivo for 10 wk. In vitro, experiments were also conducted to intercept the MAM-mediated Ca2+ signaling in isolated hepatocytes by transfecting them with si-mitochondrial calcium uniporter (mcu). Because mcu was placed in the inner mitochondrial membrane (IMM), si-mcu cannot disrupt MAM's structural integrity. RESULTS: 1. Hepatocellular MAM subproteome analysis indicated excessive dietary PA intake enhanced hepatic MAM structure joined by activating Ip3r-Grp75-Vdac complexes. 2. Dietary PA intake induced hepatic neutral lipid accumulation through MAM recruiting Seipin, which activated lipid droplet biogenesis. Our findings also revealed a previously unidentified mechanism whereby MAM-recruited seipin and controlled hepatic lipid homeostasis, depending on Ip3r-Grp75-Vdac-controlled Ca2+ signaling and not only MAM's structural integrity. CONCLUSIONS: These results offer a novel insight into the MAM-recruited seipin in controlling hepatic lipid synthesis in a MAM structural integrity-dependent and Ca2+ signaling-dependent manner, highlighting the critical contribution of MAM in maintaining hepatic neutral lipid homeostasis.

Potential targets for the treatment of MI: GRP75-mediated Ca2+ transfer in MAM.

After myocardial infarction (MI), there is a notable disruption in cellular calcium ion homeostasis and mitochondrial function, which is believed to be intricately linked to endoplasmic reticulum (ER) stress. This research endeavors to elucidate the involvement of glucose regulated protein 75 (GRP75) in post-MI calcium ion homeostasis and mitochondrial function. In MI rats, symptoms of myocardial injury were accompanied by an increase in the activation of ER stress. Moreover, in oxygen-glucose deprivation (OGD)-induced cardiomyocytes, it was confirmed that inhibiting ER stress exacerbated intracellular Ca2+ disruption and cell apoptosis. Concurrently, the co-localization of GRP75 with IP3R and VDAC1 increased under ER stress in cardiomyocytes. In OGD-induced cardiomyocytes, knockdown of GRP75 not only reduced the Ca2+ levels in both the ER and mitochondria and improved the ultrastructure of cardiomyocytes, but it also increased the number of contact points between the ER and mitochondria, reducing mitochondria associated endoplasmic reticulum membrane (MAM) formation, and decreased cell apoptosis. Significantly, knockdown of GRP75 did not affect the protein expression of PERK and hypoxia-inducible factor 1α (HIF-1α). Transcriptome analysis of cardiomyocytes revealed that knockdown of GRP75 mainly influenced the molecular functions of sialyltransferase and IP3R, as well as the biosynthesis of glycosphingolipids and lactate metabolism. The complex interaction between the ER and mitochondria, driven by the GRP75 and its associated IP3R1-GRP75-VDAC1 complex, is crucial for calcium homeostasis and cardiomyocyte's adaptive response to ER stress. Modulating GRP75 could offer a strategy to regulate calcium dynamics, diminish glycolysis, and thereby mitigate cardiomyocyte apoptosis.

Targeting GRP75 with a Chlorpromazine Derivative Inhibits Endometrial Cancer Progression Through GRP75-IP3R-Ca2+-AMPK Axis.

Tumors often overexpress glucose-regulated proteins, and agents that interfere with the production or activity of these proteins may represent novel cancer treatments. The chlorpromazine derivative JX57 exhibits promising effects against endometrial cancer with minimal extrapyramidal side effects; however, its mechanisms of action are currently unknown. Here, glucose-regulated protein 75 kD (GRP75) is identified as a direct target of JX57 using activity-based protein profiling and loss-of-function experiments. The findings show that GRP75 is necessary for the biological activity of JX57, as JX57 exhibits moderate anticancer properties in GRP75-deficient cancer cells, both in vitro and in vivo. High GRP75 expression is correlated with poor differentiation and poor survival in patients with endometrial cancer, whereas the knockdown of GRP75 can significantly suppress tumor growth. Mechanistically, the direct binding of JX57 to GRP75 impairs the structure of the mitochondria-associated endoplasmic reticulum membrane and disrupts the endoplasmic reticulum-mitochondrial calcium homeostasis, resulting in a mitochondrial energy crisis and AMP-activated protein kinase activation. Taken together, these findings highlight GRP75 as a potential prognostic biomarker and direct therapeutic target in endometrial cancer and suggest that the chlorpromazine derivative JX57 can potentially be a new therapeutic option for endometrial cancer.

By regulating the IP3R/GRP75/VDAC1 complex to restore mitochondrial dynamic balance, selenomethionine reduces lipopolysaccharide-induced neuronal apoptosis.

Selenium (Se), as one of the essential trace elements, plays an anti-inflammatory, antioxidation, and immune-enhancing effect in the body. In addition, Se can also improve nervous system damage induced by various factors. Earlier studies have described the important role of mitochondrial dynamic imbalance in lipopolysaccharide (LPS)-induced nerve injury. The inositol 1,4,5-triphosphate receptor (IP3R)/glucose-regulated protein 75 (GRP75)/voltage-dependent anion channel 1 (VDAC1) complex is considered to be the key to regulating mitochondrial dynamics. However, it is not clear whether Selenomethionine (SeMet) has any influence on the IP3R/GRP75/VDAC1 complex. Therefore, the aim of this investigation was to determine whether SeMet can alleviate LPS-induced brain damage and to elucidate the function of the IP3R/GRP75/VDAC1 complex in it. We established SeMet and/or LPS exposure models in vivo and in vitro using laying hens and primary chicken nerve cells. We noticed that SeMet reversed endoplasmic reticulum stress (ERS) and the imbalance in mitochondrial dynamics and significantly prevented the occurrence of neuronal apoptosis. We made this finding by morphological observation of the brain tissue of laying hens and the detection of related genes such as ERS, the IP3R/GRP75/VDAC1 complex, calcium signal (Ca2+), mitochondrial dynamics, and apoptosis. Other than that, we also discovered that the IP3R/GRP75/VDAC1 complex was crucial in controlling Ca2+ transport between the endoplasmic reticulum and the mitochondrion when SeMet functions as a neuroprotective agent. In summary, our results revealed the specific mechanism by which SeMet alleviated LPS-induced neuronal apoptosis for the first time. As a consequence, SeMet has great potential in the treatment and prevention of neurological illnesses (like neurodegenerative diseases).

The SIRT3-ATAD3A axis regulates MAM dynamics and mitochondrial calcium homeostasis in cardiac hypertrophy.

Mitochondria are energy-producing organelles that are mobile and harbor dynamic network structures. Although mitochondria and endoplasmic reticulum (ER) play distinct cellular roles, they are physically connected to maintain functional homeostasis. Abnormal changes in this interaction have been linked to pathological states, including cardiac hypertrophy. However, the exact regulatory molecules and mechanisms are yet to be elucidated. Here, we report that ATPase family AAA-domain containing protein 3A (ATAD3A) is an essential regulator of ER-mitochondria interplay within the mitochondria-associated membrane (MAM). ATAD3A prevents isoproterenol (ISO)-induced mitochondrial calcium accumulation, improving mitochondrial dysfunction and ER stress, which preserves cardiac function and attenuates cardiac hypertrophy. We also find that ATAD3A is a new substrate of NAD+-dependent deacetylase Sirtuin 3 (SIRT3). Notably, the heart mitochondria of SIRT3 knockout mice exhibited excessive formation of MAMs. Mechanistically, ATAD3A specifically undergoes acetylation, which reduces self-oligomerization and promotes cardiac hypertrophy. ATAD3A oligomerization is disrupted by acetylation at K134 site, and ATAD3A monomer closely interacts with the IP3R1-GRP75-VDAC1 complex, which leads to mitochondrial calcium overload and dysfunction. In summary, ATAD3A localizes to the MAMs, where it protects the homeostasis of ER-mitochondria contacts, quenching mitochondrial calcium overload and keeping mitochondrial bioenergetics unresponsive to ER stress. The SIRT3-ATAD3A axis represents a potential therapeutic target for cardiac hypertrophy.

Restoration of p53 functions by suppression of mortalin-p53 sequestration: an emerging target in cancer therapy.

Functional inactivation of wild-type p53 is a major trait of cancerous cells. In many cases, such inactivation occurs by either TP53 gene mutations or due to overexpression of p53 binding partners. This review focuses on an overexpressed p53 binding partner called mortalin, a mitochondrial heat shock protein that sequesters both wild-type and mutant p53 in malignant cells due to changes in subcellular localization. Clinical evidence suggests a drastic depletion of the overall survival time of cancer patients with high mortalin expression. Therefore, mortalin-p53 sequestration inhibitors could be game changers in improving overall survival rates. This review explores the consequences of mortalin overexpression and challenges, status and strategies for accelerating drug discovery to suppress mortalin-p53 sequestration.

Vitamin E succinate mediated apoptosis by juxtaposing endoplasmic reticulum and mitochondria.

Vitamin E succinate (VES) is an esterified form of natural α-tocopherol, has turned out to be novel anticancer agent. However, its anticancer mechanisms have not been illustrated. Previously, we reported VES mediated Ca2+ release from the endoplasmic reticulum (ER) causes mitochondrial Ca2+ overload, leading to mitochondrial depolarization and apoptosis. Here, we elucidated the mechanism of VES-induced Ca2+ transfer from ER to mitochondria by investigating the role of VES in ER-mitochondria contact formation. Transmission electron microscopic observation confirms VES mediated ER-mitochondria contact while fluorescence microscopic analysis revealed that VES increased mitochondria-associated ER membrane (MAM) formation. Pre-treatment with the inositol 1,4,5-triphosphate receptor (IP3R) antagonist 2-aminoethyl diphenylborinate (2-APB) decreased VES-induced MAM formation, suggesting the involvement of VES-induced Ca2+ efflux from ER in MAM formation. The ER IP3R receptor is known to interact with voltage-dependent anion channels (VDAC) via the chaperone glucose-regulated protein 75 kDa (GRP75) to bring ER and mitochondria nearby. Although we revealed that VES treatment does not affect GRP75 protein level, it increases GRP75 localization in the MAM. In addition, the inhibition of Ca2+ release from ER by 2-APB decreases GRP75 localization in the MAM, suggesting the possibility of Ca2+-induced conformational change of GRP75 that promotes formation of the IP3R-GRP75-VDAC complex and thereby encourages MAM formation. This study identifies the mechanism of VES-induced enhanced Ca2+ transfer from ER to mitochondria, which causes mitochondrial Ca2+ overload leading to apoptosis.

IP3R at ER-Mitochondrial Contact Sites: Beyond the IP3R-GRP75-VDAC1 Ca2+ Funnel.

Membrane contact sites (MCS) circumvent the topological constraints of functional coupling between different membrane-bound organelles by providing a means of communication and exchange of materials. One of the most characterised contact sites in the cell is that between the endoplasmic reticulum and the mitochondrial (ERMCS) whose function is to couple cellular Ca2+ homeostasis and mitochondrial function. Inositol 1,4,5-trisphosphate receptors (IP3Rs) on the ER, glucose-regulated protein 75 (GRP 75) and voltage-dependent anion channel 1 (VDAC1) on the outer mitochondrial membrane are the canonical component of the Ca2+ transfer unit at ERMCS. These are often reported to form a Ca2+ funnel that fuels the mitochondrial low-affinity Ca2+ uptake system. We assess the available evidence on the IP3R subtype selectivity at the ERMCS and consider if IP3Rs have other roles at the ERMCS beyond providing Ca2+. Growing evidence suggests that all three IP3R subtypes can localise and regulate Ca2+ signalling at ERMCS. Furthermore, IP3Rs may be structurally important for assembly of the ERMCS in addition to their role in providing Ca2+ at these sites. Evidence that various binding partners regulate the assembly and Ca2+ transfer at ERMCS populated by IP3R-GRP75-VDAC1, suggesting that cells have evolved mechanisms that stabilise these junctions forming a Ca2+ microdomain that is required to fuel mitochondrial Ca2+ uptake.

SIRT3 ameliorates diabetes-associated cognitive dysfunction via regulating mitochondria-associated ER membranes.

BACKGROUND: Diabetes is associated with an increased risk of cognitive decline and dementia. These diseases are linked with mitochondrial dysfunction, most likely as a consequence of excessive formation of mitochondria-associated membranes (MAMs). Sirtuin3 (SIRT3), a key mitochondrial NAD+-dependent deacetylase, is critical responsible for mitochondrial functional homeostasis and is highly associated with neuropathology. However, the role of SIRT3 in regulating MAM coupling remains unknown. METHODS: Streptozotocin-injected diabetic mice and high glucose-treated SH-SY5Y cells were established as the animal and cellular models, respectively. SIRT3 expression was up-regulated in vivo using an adeno-associated virus in mouse hippocampus and in vitro using a recombinant lentivirus vector. Cognitive function was evaluated using behavioural tests. Hippocampus injury was assessed using Golgi and Nissl staining. Apoptosis was analysed using western blotting and TUNEL assay. Mitochondrial function was detected using flow cytometry and confocal fluorescence microscopy. The mechanisms were investigated using co-immunoprecipitation of VDAC1-GRP75-IP3R complex, fluorescence imaging of ER and mitochondrial co-localisation and transmission electron microscopy of structural analysis of MAMs. RESULTS: Our results demonstrated that SIRT3 expression was significantly reduced in high glucose-treated SH-SY5Y cells and hippocampal tissues from diabetic mice. Further, up-regulating SIRT3 alleviated hippocampus injuries and cognitive impairment in diabetic mice and mitigated mitochondrial Ca2+ overload-induced mitochondrial dysfunction and apoptosis. Mechanistically, MAM formation was enhanced under high glucose conditions, which was reversed by genetic up-regulation of SIRT3 via reduced interaction of the VDAC1-GRP75-IP3R complex in vitro and in vivo. Furthermore, we investigated the therapeutic effects of pharmacological activation of SIRT3 in diabetic mice via honokiol treatment, which exhibited similar effects to our genetic interventions. CONCLUSIONS: In summary, our findings suggest that SIRT3 ameliorates cognitive impairment in diabetic mice by limiting aberrant MAM formation. Furthermore, targeting the activation of SIRT3 by honokiol provides a promising therapeutic candidate for diabetes-associated cognitive dysfunction. Overall, our study suggests a novel role of SIRT3 in regulating MAM coupling and indicates that SIRT3-targeted therapies are promising for diabetic dementia patients.

ß-carotene targets IP3R/GRP75/VDAC1-MCU axis to renovate LPS-induced mitochondrial oxidative damage by regulating STIM1.

Endoplasmic reticulum (ER) and mitochondria are the main sites for the storage and regulation of Ca2+ homeostasis. An imbalance of Ca2+ homeostasis can cause ER stress and mitochondrial dysfunction, thereby inducing apoptosis. The store-operated calcium entry (SOCE) is the main channel for extracellular calcium influx. Mitochondria-associated endoplasmic reticulum (MAM) is an important agent for Ca2+ transfer from the ER to the mitochondria. Therefore, regulation of SOCE and MAMs has potential therapeutic value for disease prevention and treatment. In this study, bovine mammary epithelial cells (BMECs) and mice were used as models to explore the mechanisms of ß-carotene to relieve ER stress and mitochondrial dysfunction. BAPTA-AM, EGTA (Ca2+ inhibitor), and BTP2 (SOCE channel inhibitor) alleviated ER stress and mitochondrial oxidative damage induced by increased intracellular Ca2+ levels after lipopolysaccharide (LPS) stimulation. Furthermore, inhibition of ER stress by 4-PBA (ER stress inhibitor), 2-APB (IP3R inhibitor), and ruthenium red (mitochondrial calcium uniporter (MCU) inhibitor) restored mitochondrial function by reducing mitochondrial ROS. Our data also confirm that ß-carotene targeted STIM1 and IP3R channels to repair LPS-induced ER stress and mitochondrial disorders. Consistent with the in vitro study, in vito experiments in mice further showed that ß-carotene attenuated LPS-induced ER stress and mitochondrial oxidative damage by inhibiting the expression of STIM1 and ORAI1, and reducing the level of Ca2+ in mouse mammary glands. Therefore, ER stress-mitochondrial oxidative damage mediated by the STIM1-ER-IP3R/GRP75/VDAC1-MCU axis plays an vital role in the development of mastitis. Our results provided novel ideas and therapeutic targets for the prevention and treatment of mastitis.

Clinical Relevance of Mortalin in Ovarian Cancer Patients.

Background: Ovarian cancer (OC) is the most lethal malignancy of the female reproductive tract. Consequently, a better understanding of the malignant features in OC is pertinent. Mortalin (mtHsp70/GRP75/PBP74/HSPA9/HSPA9B) promotes cancer development, progression, metastasis, and recurrence. Yet, there is no parallel evaluation and clinical relevance of mortalin in the peripheral and local tumor ecosystem in OC patients. Methods: A cohort of 92 pretreatment women was recruited, including 50 OC patients, 14 patients with benign ovarian tumors, and 28 healthy women. Blood plasma and ascites fluid-soluble mortalin concentrations were measured by ELISA. Mortalin protein levels in tissues and OC cells were analyzed using proteomic datasets. The gene expression profile of mortalin in ovarian tissues was evaluated through the analysis of RNAseq data. Kaplan-Meier analysis was used to demonstrate the prognostic relevance of mortalin. Results: First, we found upregulation of local mortalin in two different ecosystems, i.e., ascites and tumor tissues in human OC compared to control groups. Second, abundance expression of local tumor mortalin is associated with cancer-driven signaling pathways and worse clinical outcome. Third, high mortalin level in tumor tissues, but not in the blood plasma or ascites fluid, predicts worse patient prognosis. Conclusions: Our findings demonstrate a previously unknown mortalin profile in peripheral and local tumor ecosystem and its clinical relevance in OC. These novel findings may serve clinicians and investigators in the development of biomarker-based targeted therapeutics and immunotherapies.

Hepatitis C virus replication requires integrity of mitochondria-associated ER membranes.

Background & Aims: Chronic HCV infection causes cellular stress, fibrosis and predisposes to hepatocarcinogenesis. Mitochondria play key roles in orchestrating stress responses by regulating bioenergetics, inflammation and apoptosis. To better understand the role of mitochondria in the viral life cycle and disease progression of chronic hepatitis C, we studied morphological and functional mitochondrial alterations induced by HCV using productively infected hepatoma cells and patient livers. Methods: Biochemical and imaging assays were used to assess localization of cellular and viral proteins and mitochondrial functions in cell cultures and liver biopsies. Cyclophilin D (CypD) knockout was performed using CRISPR/Cas9 technology. Viral replication was quantified by quantitative reverse-transcription PCR and western blotting. Results: Several HCV proteins were found to associate with mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), the points of contact between the ER and mitochondria. Downregulation of CypD, which is known to disrupt MAM integrity, reduced viral replication, suggesting that MAMs play an important role in the viral life cycle. This process was rescued by ectopic CypD expression. Furthermore, HCV proteins were found to associate with voltage dependent anion channel 1 (VDAC1) at MAMs and to reduce VDAC1 protein levels at MAMs in vitro and in patient biopsies. This association did not affect MAM-associated functions in glucose homeostasis and Ca2+ signaling. Conclusions: HCV proteins associate specifically with MAMs and MAMs play an important role in viral replication. The association between viral proteins and MAMs did not impact Ca2+ signaling between the ER and mitochondria or glucose homeostasis. Whether additional functions of MAMs and/or VDAC are impacted by HCV and contribute to the associated pathology remains to be assessed. Impact and implications: Hepatitis C virus infects the liver, where it causes inflammation, cell damage and increases the long-term risk of liver cancer. We show that several HCV proteins interact with mitochondria in liver cells and alter the composition of mitochondrial subdomains. Importantly, HCV requires the architecture of these mitochondrial subdomains to remain intact for efficient viral replication.

GRP75 as a functional element of cholix transcytosis.

Cholix (Chx) is secreted by non-pandemic strains of Vibrio cholerae in the intestinal lumen. For this exotoxin to induce cell death in non-polarized cells in the intestinal lamina propria, it must traverse the epithelium in the fully intact form. We identified host cell elements in polarized enterocytes associated with Chx endocytosis and apical to basal (A→B) vesicular transcytosis. This pathway overcomes endogenous mechanisms of apical vesicle recycling and lysosomal targeting by interacting with several host cell proteins that include the 75 kDa glucose-regulated protein (GRP75). Apical endocytosis of Chx appears to involve the single membrane spanning protein TMEM132A, and interaction with furin before it engages GRP75 in apical vesicular structures. Sorting within these apical vesicles results in Chx being trafficked to the basal region of cells in association with the Lectin, Mannose Binding 1 protein LMAN1. In this location, Chx interacts with the basement membrane-specific heparan sulfate proteoglycan perlecan in recycling endosomes prior to its release from this basal vesicular compartment to enter the underlying lamina propria. While the furin and LMAN1 elements of this Chx transcytosis pathway undergo cellular redistribution that are reflective of the polarity shifts noted for coatamer complexes COPI and COPII, GRP75 and perlecan fail to show these dramatic rearrangements. Together, these data define essential steps in the A→B transcytosis pathway accessed by Chx to reach the intestinal lamina propria where it can engage and intoxicate certain non-polarized cells.

The Vibrio cholerae exotoxin protein cholix interacts with a number of host cell proteins, including GRP75, to facilitate its vesicular transcytosis across polarized intestinal epithelial cells following apical endocytosis.

GRP75 Modulates Endoplasmic Reticulum-Mitochondria Coupling and Accelerates Ca2+-Dependent Endothelial Cell Apoptosis in Diabetic Retinopathy.

Endoplasmic reticulum (ER) and mitochondrial dysfunction play fundamental roles in the pathogenesis of diabetic retinopathy (DR). However, the interrelationship between the ER and mitochondria are poorly understood in DR. Here, we established high glucose (HG) or advanced glycosylation end products (AGE)-induced human retinal vascular endothelial cell (RMEC) models in vitro, as well as a streptozotocin (STZ)-induced DR rat model in vivo. Our data demonstrated that there was increased ER-mitochondria coupling in the RMECs, which was accompanied by elevated mitochondrial calcium ions (Ca2+) and mitochondrial dysfunction under HG or AGE incubation. Mechanistically, ER-mitochondria coupling was increased through activation of the IP3R1-GRP75-VDAC1 axis, which transferred Ca2+ from the ER to the mitochondria. Elevated mitochondrial Ca2+ led to an increase in mitochondrial ROS and a decline in mitochondrial membrane potential. These events resulted in the elevation of mitochondrial permeability and induced the release of cytochrome c from the mitochondria into the cytoplasm, which further activated caspase-3 and promoted apoptosis. The above phenomenon was also observed in tunicamycin (TUN, ER stress inducer)-treated cells. Meanwhile, BAPTA-AM (calcium chelator) rescued mitochondrial dysfunction and apoptosis in DR, which further confirmed of our suspicions. In addition, 4-phenylbutyric acid (4-PBA), an ER stress inhibitor, was shown to reverse retinal dysfunction in STZ-induced DR rats in vivo. Taken together, our findings demonstrated that DR fueled the formation of ER-mitochondria coupling via the IP3R1-GRP75-VDAC1 axis and accelerated Ca2+-dependent cell apoptosis. Our results demonstrated that inhibition of ER-mitochondrial coupling, including inhibition of GRP75 or Ca2+ overload, may be a potential therapeutic target in DR.

ER, Mitochondria, and ISR Regulation by mt-HSP70 and ATF5 upon Procollagen Misfolding in Osteoblasts.

Cellular response to protein misfolding underlies multiple diseases. Collagens are the most abundant vertebrate proteins, yet little is known about cellular response to misfolding of their procollagen precursors. Osteoblasts (OBs)-the cells that make bone-produce so much procollagen that it accounts for up to 40% of mRNAs in the cell, which is why bone bears the brunt of mutations causing procollagen misfolding in osteogenesis imperfecta (OI). The present study of a G610C mouse model of OI by multiple transcriptomic techniques provides first solid clues to how OBs respond to misfolded procollagen accumulation in the endoplasmic reticulum (ER) and how this response affects OB function. Surprisingly, misfolded procollagen escapes the quality control in the ER lumen and indirectly triggers the integrated stress response (ISR) through other cell compartments. In G610C OBs, the ISR is regulated by mitochondrial HSP70 (mt-HSP70) and ATF5 instead of their BIP and ATF4 paralogues, which normally activate and regulate ISR to secretory protein misfolding in the ER. The involvement of mt-HSP70 and ATF5 together with other transcriptomic findings suggest that mitochondria might initiate the ISR upon disruption of ER-mitochondria connections or might respond to the ISR activated by a yet unknown sensor.

IP3R1/GRP75/VDAC1 complex mediates endoplasmic reticulum stress-mitochondrial oxidative stress in diabetic atrial remodeling.

RATIONALE: Endoplasmic reticulum (ER) stress and mitochondrial dysfunction are important mechanisms of atrial remodeling, predisposing to the development of atrial fibrillation (AF) in type 2 diabetes mellitus (T2DM). However, the molecular mechanisms underlying these processes especially their interactions have not been fully elucidated. OBJECTIVE: To explore the potential role of ER stress-mitochondrial oxidative stress in atrial remodeling and AF induction in diabetes. METHODS AND RESULTS: Mouse atrial cardiomyocytes (HL-1 cells) and rats with T2DM were used as study models. Significant ER stress was observed in the diabetic rat atria. After treatment with tunicamycin (TM), an ER stress agonist, mass spectrometry (MS) identified several known ER stress and calmodulin proteins, including heat shock protein family A (HSP70) member [HSPA] 5 [GRP78]) and HSPA9 (GRP75, glucose-regulated protein 75). In situ proximity ligation assay indicated that TM led to increased protein expression of the IP3R1-GRP75-VDAC1 (inositol 1,4,5-trisphosphate receptor 1-glucose-regulated protein 75-voltage-dependent anion channel 1) complex in HL-1 cells. Small interfering RNA silencing of GRP75 in HL-1 cells and GRP75 conditional knockout in a mouse model led to impaired calcium transport from the ER to the mitochondria and alleviated mitochondrial oxidative stress and calcium overload. Moreover, GRP75 deficiency attenuated atrial remodeling and AF progression in Myh6-Cre+/Hspa9flox/flox + TM mice. CONCLUSIONS: The IP3R1-GRP75-VDAC1 complex mediates ER stress-mitochondrial oxidative stress and plays an important role in diabetic atrial remodeling.

Preserving mitochondrial function by inhibiting GRP75 ameliorates neuron injury under ischemic stroke.

Ischemic stroke is a life­threatening disease, which is closely related to neuron damage during ischemia. Mitochondrial dysfunction is essentially involved in the pathophysiological process of ischemic stroke. Mitochondrial calcium overload contributes to the development of mitochondrial dysfunction. However, the underlying mechanisms of mitochondrial calcium overload are far from being fully revealed. In the present study, middle cerebral artery obstruction (MCAO) was performed in vivo and oxygen and glucose deprivation (OGD) in vitro. The results indicated that both MCAO and OGD induced significant mitochondrial dysfunction in vivo and in vitro. The mitochondria became fragmented under hypoxia conditions, accompanied with upregulation of the heat shock protein 75 kDa glucose­regulated protein (GRP75). Inhibition of GRP75 was able to effectively ameliorate mitochondrial calcium overload and preserve mitochondrial function, which may provide evidence for further translational studies of ischemic diseases.

Notch1 Modulation of Cellular Calcium Regulates Mitochondrial Metabolism and Anti-Apoptotic Activity in T-Regulatory Cells.

As the major hub of metabolic activity and an organelle sequestering pro-apoptogenic intermediates, mitochondria lie at the crossroads of cellular decisions of death and survival. Intracellular calcium is a key regulator of these outcomes with rapid, uncontrolled uptake into mitochondria, activating pro-apoptotic cascades that trigger cell death. Here, we show that calcium uptake and mitochondrial metabolism in murine T-regulatory cells (Tregs) is tuned by Notch1 activity. Based on analysis of Tregs and the HEK cell line, we present evidence that modulation of cellular calcium dynamics underpins Notch1 regulation of mitochondrial homeostasis and consequently anti-apoptotic activity. Targeted siRNA-mediated ablations reveal dependency on molecules controlling calcium release from the endoplasmic reticulum (ER) and the chaperone, glucose-regulated protein 75 (Grp75), the associated protein Voltage Dependent Anion Channel (VDAC)1 and the Mitochondrial Calcium Uniporter (MCU), which together facilitate ER calcium transfer and uptake into the mitochondria. Endogenous Notch1 is detected in immune-complexes with Grp75 and VDAC1. Deficits in mitochondrial oxidative and survival in Notch1 deficient Tregs, were corrected by the expression of recombinant Notch1 intracellular domain, and in part by recombinant Grp75. Thus, the modulation of calcium dynamics and consequently mitochondrial metabolism underlies Treg survival in conditions of nutrient stress. This work positions a key role for Notch1 activity in these outcomes.