Insights into Molecular and Cellular Functions of the Golgi Calcium/Manganese-Proton Antiporter TMEM165.

The Golgi compartment performs a number of crucial roles in the cell. However, the exact molecular mechanisms underlying these actions are not fully defined. Pathogenic mutations in genes encoding Golgi proteins may serve as an important source for expanding our knowledge. For instance, mutations in the gene encoding Transmembrane protein 165 (TMEM165) were discovered as a cause of a new type of congenital disorder of glycosylation (CDG). Comprehensive studies of TMEM165 in different model systems, including mammals, yeast, and fish uncovered the new realm of Mn2+ homeostasis regulation. TMEM165 was shown to act as a Ca2+/Mn2+:H+ antiporter in medial- and trans-Golgi network, pumping the metal ions into the Golgi lumen and protons outside. Disruption of TMEM165 antiporter activity results in defects in N- and O-glycosylation of proteins and glycosylation of lipids. An impaired glycosylation of TMEM165-CDG arises from lack of Mn2+ within the Golgi. Nevertheless, Mn2+ insufficiency in the Golgi is compensated by the activity of the ATPase SERCA2. TMEM165 turnover has also been found to be regulated by Mn2+ cytosolic concentration. Besides causing CDG, recent investigations have demonstrated the functional involvement of TMEM165 in several other pathologies including cancer and mental health disorders. This systematic review summarizes the available information on TMEM165 molecular structure, cellular function, and its roles in health and disease.

Endothelial Extracellular Vesicles Enriched in microRNA-34a Predict New-Onset Diabetes in Coronavirus Disease 2019 (COVID-19) Patients: Novel Insights for Long COVID Metabolic Sequelae.

Emerging evidence indicates that the relationship between coronavirus disease 2019 (COVID-19) and diabetes is 2-fold: 1) it is known that the presence of diabetes and other metabolic alterations poses a considerably high risk to develop a severe COVID-19; 2) patients who survived a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection have an increased risk of developing new-onset diabetes. However, the mechanisms underlying this association are mostly unknown, and there are no reliable biomarkers to predict the development of new-onset diabetes. In the present study, we demonstrate that a specific microRNA (miR-34a) contained in circulating extracellular vesicles released by endothelial cells reliably predicts the risk of developing new-onset diabetes in COVID-19. This association was independent of age, sex, body mass index (BMI), hypertension, dyslipidemia, smoking status, and D-dimer. SIGNIFICANCE STATEMENT: We demonstrate for the first time that a specific microRNA (miR-34a) contained in circulating extracellular vesicles released by endothelial cells is able to reliably predict the risk of developing diabetes after having contracted coronavirus disease 2019 (COVID-19). This association was independent of age, sex, body mass index (BMI), hypertension, dyslipidemia, smoking status, and D-dimer. Our findings are also relevant when considering the emerging importance of post-acute sequelae of COVID-19, with systemic manifestations observed even months after viral negativization (long COVID).

Infiltrating macrophages amplify doxorubicin-induced cardiac damage: role of catecholamines.

BACKGROUND: The functional contribution of non-myocyte cardiac cells, such as inflammatory cells, in the setup of heart failure in response to doxorubicin (Dox) is recently becoming of growing interest. OBJECTIVES: The study aims to evaluate the role of macrophages in cardiac damage elicited by Dox treatment. METHODS: C57BL/6 mice were treated with one intraperitoneal injection of Dox (20 mg/kg) and followed up for 5 days by cardiac ultrasounds (CUS), histological, and flow cytometry evaluations. We also tested the impact of Dox in macrophage-depleted mice. Rat cardiomyoblasts were directly treated with Dox (D-Dox) or with a conditioned medium from cultured murine macrophages treated with Dox (M-Dox). RESULTS: In response to Dox, macrophage infiltration preceded cardiac damage. Macrophage depletion prevents Dox-induced damage, suggesting a key role of these cells in promoting cardiotoxicity. To evaluate the crosstalk between macrophages and cardiac cells in response to DOX, we compared the effects of D-Dox and M-Dox in vitro. Cell vitality was lower in cardiomyoblasts and apoptosis was higher in response to M-Dox compared with D-Dox. These events were linked to p53-induced mitochondria morphology, function, and autophagy alterations. We identify a mechanistic role of catecholamines released by Dox-activated macrophages that lead to mitochondrial apoptosis of cardiac cells through ß-AR stimulation. CONCLUSIONS: Our data indicate that crosstalk between macrophages and cardiac cells participates in cardiac damage in response to Dox.

Accumulation of α-synuclein mediates podocyte injury in Fabry nephropathy.

Current therapies for Fabry disease are based on reversing intracellular accumulation of globotriaosylceramide (Gb3) by enzyme replacement therapy (ERT) or chaperone-mediated stabilization of the defective enzyme, thereby alleviating lysosomal dysfunction. However, their effect in the reversal of end-organ damage, like kidney injury and chronic kidney disease, remains unclear. In this study, ultrastructural analysis of serial human kidney biopsies showed that long-term use of ERT reduced Gb3 accumulation in podocytes but did not reverse podocyte injury. Then, a CRISPR/Cas9-mediated α-galactosidase knockout podocyte cell line confirmed ERT-mediated reversal of Gb3 accumulation without resolution of lysosomal dysfunction. Transcriptome-based connectivity mapping and SILAC-based quantitative proteomics identified α-synuclein (SNCA) accumulation as a key event mediating podocyte injury. Genetic and pharmacological inhibition of SNCA improved lysosomal structure and function in Fabry podocytes, exceeding the benefits of ERT. Together, this work reconceptualizes Fabry-associated cell injury beyond Gb3 accumulation, and introduces SNCA modulation as a potential intervention, especially for patients with Fabry nephropathy.

Mitochondrial microRNAs Are Dysregulated in Patients with Fabry Disease.

Fabry disease (FD) is a lysosomal storage disorder caused by mutations in the gene for α-galactosidase A, inducing a progressive accumulation of globotriaosylceramide (GB3) and its metabolites in different organs and tissues. GB3 deposition does not fully explain the clinical manifestations of FD, and other pathogenetic mechanisms have been proposed, requiring the identification of new biomarkers for monitoring FD patients. Emerging evidence suggests the involvement of mitochondrial alterations in FD. Here, we propose mitochondrial-related microRNAs (miRs) as potential biomarkers of mitochondrial involvement in FD. Indeed, we demonstate that miRs regulating different aspects of mitochondrial homeostasis including expression and assembly of respiratory chain, mitogenesis, antioxidant capacity, and apoptosis are consistently dysregulated in FD patients. Our data unveil a novel noncoding RNA signature of FD patients, indicating mitochondrial-related miRs as new potential pathogenic players and biomarkers in FD. SIGNIFICANCE STATEMENT: This study demonstrates for the first time that a specific signature of circulating mitochondrial miRs (mitomiRs) is dysregulated in FD patients. MitomiRs regulating fundamental aspects of mitochondrial homeostasis and fitness, including expression and assembly of the respiratory chain, mitogenesis, antioxidant capacity, and apoptosis are significantly dysregulated in FD patients. Taken together, these new findings introduce mitomiRs as unprecedented biomarkers of FD and point at mitochondrial dysfunction as a novel potential mechanistic target for therapeutic approaches.

The discovery and development of IP3 receptor modulators: an update.

Introduction: Inositol 1,4,5-trisphosphate receptors (IP3Rs) are intracellular calcium (Ca2+) release channels located on the endoplasmic/sarcoplasmic reticulum. The availability of the structure of the ligand-binding domain of IP3Rs has enabled the design of compatible ligands, but the limiting step remains their actual effectiveness in a biological context.Areas covered: This article summarizes the compelling literature on both agonists and antagonists targeting IP3Rs, emphasizing their strengths and limitations. The main challenges toward the discovery and development of IP3 receptor modulators are also described.Expert opinion: Despite significant progress in recent years, the pharmacology of IP3R still has major drawbacks, especially concerning the availability of specific antag onists. Moreover, drugs specifically targeting the three different subtypes of IP3R are especially needed.

Exploiting GRK2 Inhibition as a Therapeutic Option in Experimental Cancer Treatment: Role of p53-Induced Mitochondrial Apoptosis.

The involvement of GRK2 in cancer cell proliferation and its counter-regulation of p53 have been suggested in breast cancer even if the underlying mechanism has not yet been elucidated. Furthermore, the possibility to pharmacologically inhibit GRK2 to delay cancer cell proliferation has never been explored. We investigated this possibility by setting up a study that combined in vitro and in vivo models to underpin the crosstalk between GRK2 and p53. To reach this aim, we took advantage of the different expression of p53 in cell lines of thyroid cancer (BHT 101 expressing p53 and FRO cells, which are p53-null) in which we overexpressed or silenced GRK2. The pharmacological inhibition of GRK2 was achieved using the specific inhibitor KRX-C7. The in vivo study was performed in Balb/c nude mice, where we treated BHT-101 or FRO-derived tumors with KRX-C7. In our in vitro model, FRO cells were unaffected by GRK2 expression levels, whereas BHT-101 cells were sensitive, thus suggesting a role for p53. The regulation of p53 by GRK2 is due to phosphorylative events in Thr-55, which induce the degradation of p53. In BHT-101 cells, the pharmacologic inhibition of GRK2 by KRX-C7 increased p53 levels and activated apoptosis through the mitochondrial release of cytochrome c. These KRX-C7-mediated events were also confirmed in cancer allograft models in nude mice. In conclusion, our data showed that GRK2 counter-regulates p53 expression in cancer cells through a kinase-dependent activity. Our results further corroborate the anti-proliferative role of GRK2 inhibitors in p53-sensitive tumors and propose GRK2 as a therapeutic target in selected cancers.

miR-7 Regulates GLP-1-Mediated Insulin Release by Targeting ß-Arrestin 1.

Glucagon-like peptide-1 (GLP-1) has been shown to potentiate glucose-stimulated insulin secretion binding GLP-1 receptor on pancreatic ß cells. ß-arrestin 1 (ßARR1) is known to regulate the desensitization of GLP-1 receptor. Mounting evidence indicates that microRNAs (miRNAs, miRs) are fundamental in the regulation of ß cell function and insulin release. However, the regulation of GLP-1/ßARR1 pathways by miRs has never been explored. Our hypothesis is that specific miRs can modulate the GLP-1/ßARR1 axis in ß cells. To test this hypothesis, we applied a bioinformatic approach to detect miRs that could target ßARR1; we identified hsa-miR-7-5p (miR-7) and we validated the specific interaction of this miR with ßARR1. Then, we verified that GLP-1 was indeed able to regulate the transcription of miR-7 and ßARR1, and that miR-7 significantly regulated GLP-1-induced insulin release and cyclic AMP (cAMP) production in ß cells. Taken together, our findings indicate, for the first time, that miR-7 plays a functional role in the regulation of GLP-1-mediated insulin release by targeting ßARR1. These results have a decisive clinical impact given the importance of drugs modulating GLP-1 signaling in the treatment of patients with type 2 diabetes mellitus.

Inositol 1,4,5-Trisphosphate Receptors in Human Disease: A Comprehensive Update.

Inositol 1,4,5-trisphosphate receptors (ITPRs) are intracellular calcium release channels located on the endoplasmic reticulum of virtually every cell. Herein, we are reporting an updated systematic summary of the current knowledge on the functional role of ITPRs in human disorders. Specifically, we are describing the involvement of its loss-of-function and gain-of-function mutations in the pathogenesis of neurological, immunological, cardiovascular, and neoplastic human disease. Recent results from genome-wide association studies are also discussed.

A Novel Small Peptide Inhibitor of NFκB, RH10, Blocks Oxidative Stress-Dependent Phenotypes in Cancer.

BACKGROUND: The RH domain of GRK5 is an effective modulator of cancer growth through the inhibition of NFκB activity. The aim of this study was to identify the minimum effective sequence of RH that is still able to inhibit tumor growth and could be used as a peptide-based drug for therapy. METHODS: Starting from the RH sequence, small peptides were cloned and tested in KAT-4 cells. The effects on NFκB signaling and its dependent phenotypes were evaluated by Western blot, TUNEL assay, proliferation assay, and angiogenesis in vitro. In vivo experiments were performed in KAT-4 xenografts in Balb/c nude mice. RESULTS: A minimum RH ten amino acids long sequence (RH10) was able to interact with IκB, to increase IκB levels, to induce apoptosis, to inhibit KAT4-cell proliferation, NFκB activation, ROS production, and angiogenesis in vitro. In vivo, the peptide inhibited tumor growth in a dose-dependent manner. We also tested its effects in combination with chemotherapeutic drugs and radiotherapy. RH10 ameliorated the antitumor responses to cisplatin, doxorubicin, and ionizing radiation. CONCLUSION: Our data propose RH10 as a potential peptide-based drug to use for cancer treatment both alone or in combination with anticancer therapies.

GRK2 moderates the acute mitochondrial damage to ionizing radiation exposure by promoting mitochondrial fission/fusion.

The modern understanding of the G protein-coupled receptor kinase 2 has grown towards the definition of a stress protein, for its ability to rapidly compartmentalize within the cell in response to acute stimulation. Also, mitochondria can be regulated by GRK2 localization. We show that Ionizing Radiation (IR) exposure acutely damages mitochondria regarding mass, morphology, and respiration, with recovery in a framework of hours. This phenomenon is actively regulated by GRK2, whose overexpression results to be protective, and reciprocally, deletion accelerates degenerative processes. The regulatory effects of the kinase involve a new interactome that includes binding HSP90 and binding and phosphorylation of the key molecules involved in the process of mitochondrial fusion and recovery: MFN-1 and 2.

Parathyroid Hormone Causes Endothelial Dysfunction by Inducing Mitochondrial ROS and Specific Oxidative Signal Transduction Modifications.

Vitamin D deficiency contributes to cardiovascular risk (CVR), with hyperparathyroidism advocated as a putative mechanism. Indeed, mounting evidence supports the hypothesis that parathyroid hormone (PTH) impairs endothelial function, even though mechanisms are not fully elucidated. The present study was designed to verify in vitro the ability of sustained exposure to PTH to cause endothelial dysfunction, exploring the underlying mechanisms. In bovine aortic endothelial cells (BAECs), we evaluated the effects of PTH exposure (0.1 nM-24 hours) on both endothelial response to vasodilators, such as bradykinin (Bk (30 nM)) and acetylcholine (Ach (1 µM)), and angiogenic competence. Pretreatment with PTH impaired endothelial response to Bk but not to Ach, in terms of cytosolic calcium fluxes and NO production. In order to explore the underlying mechanisms, we assessed the production of total and mitochondrial ROS (tROS and mROS, respectively) in response to PTH (at 1 and 3 hours). PTH increased ROS generation, to an extent high enough to determine oxidation of Bk receptor B2. Conversely, the oxidation levels of M1 and M3 Ach receptors were not affected by PTH. A mROS selective scavenger (MitoTEMPO (5 µM)) restored the endothelial responsiveness to Bk while the well-known antioxidant properties of vitamin D (100 nM) failed to counteract PTH-mediated oxidative stress. PTH determined mitochondrial calcium fluxes ([Ca2+]mt) and the mitochondrial calcium uniporter inhibitor Ru360 (10 µM) reduced mROS production and prevented the PTH-mediated endothelial dysfunction. Angiogenic competence was evaluated as tubular formations in the endothelial Matrigel assay and showed a significant impairment in PTH-pretreated cells (0.1 nM-24 hours), despite the increase in VEGF transcriptional levels. VEGFR2 oxidation occurred in response to PTH, suggesting that even the impairment of angiogenesis was due to the ROS surge. These results indicate that PTH affects endothelial function through ROS production, driven by mitochondrial calcium overload. PTH-induced oxidative stress might act as signaling modifiers, altering specific pathways (Bk and VEGF) and preserving others (Ach).

Cardiac Nonmyocyte Cell Functions and Crosstalks in Response to Cardiotoxic Drugs.

The discovery of the molecular mechanisms involved in the cardiac responses to anticancer drugs represents the current goal of cardio-oncology research. The oxidative stress has a pivotal role in cardiotoxic responses, affecting the function of all types of cardiac cells, and their functional crosstalks. Generally, cardiomyocytes are the main target of research studies on cardiotoxicity, but recently the contribution of the other nonmyocyte cardiac cells is becoming of growing interest. This review deals with the role of oxidative stress, induced by anticancer drugs, in cardiac nonmyocyte cells (fibroblasts, vascular cells, and immune cells). The alterations of functional interplays among these cardiac cells are discussed, as well. These interesting recent findings increase the knowledge about cardiotoxicity and suggest new molecular targets for both diagnosis and therapy.

Functional Role of Mitochondria in Arrhythmogenesis.

Growing evidence indicate that mitochondria play a functional role in arrhythmogenesis. We report here the molecular mechanisms underlying the action of these highly dynamic organelles in the regulation of cell metabolism, action potential and, overall, heart excitability. In particular, we examine the role of cardiac mitochondria in linking metabolism and cell excitability. The importance of the main mitochondrial channels is evaluated as well, including the recently identified calcium uniporter. Promises and pitfalls of potential therapeutic strategies targeting mitochondrial pathways are also assessed.