STAT3 is an oncogenic transcription factor exerting its functions both as a canonical transcriptional activator and as a non-canonical regulator of energy metabolism and mitochondrial functions. While both activities are required for cell transformation downstream of different oncogenic stimuli, they rely on different post-translational activating events, namely phosphorylation on either Y705 (nuclear activities) or S727 (mitochondrial functions). Here, we report the discovery of the unexpected STAT3 localization to the endoplasmic reticulum (ER), from where it modulates ER-mitochondria Ca2+ release by interacting with the Ca2+ channel IP3R3 and facilitating its degradation. The release of Ca2+ is of paramount importance for life/death cell decisions, as excessive Ca2+ causes mitochondrial Ca2+ overload, the opening of the mitochondrial permeability transition pore, and the initiation of the intrinsic apoptotic program. Indeed, STAT3 silencing enhances ER Ca2+ release and sensitivity to apoptosis following oxidative stress in STAT3-dependent mammary tumor cells, correlating with increased IP3R3 levels. Accordingly, basal-like mammary tumors, which frequently display constitutively active STAT3, show an inverse correlation between IP3R3 and STAT3 protein levels. These results suggest that STAT3-mediated IP3R3 downregulation in the ER crucially contributes to its anti-apoptotic functions via modulation of Ca2+ fluxes.

Mutations in the inositol 5-phosphatase OCRL cause Lowe syndrome and Dent's disease. Although OCRL, a direct clathrin interactor, is recruited to late-stage clathrin-coated pits, clinical manifestations have been primarily attributed to intracellular sorting defects. Here we show that OCRL loss in Lowe syndrome patient fibroblasts impacts clathrin-mediated endocytosis and results in an endocytic defect. These cells exhibit an accumulation of clathrin-coated vesicles and an increase in U-shaped clathrin-coated pits, which may result from sequestration of coat components on uncoated vesicles. Endocytic vesicles that fail to lose their coat nucleate the majority of the numerous actin comets present in patient cells. SNX9, an adaptor that couples late-stage endocytic coated pits to actin polymerization and which we found to bind OCRL directly, remains associated with such vesicles. These results indicate that OCRL acts as an uncoating factor and that defects in clathrin-mediated endocytosis likely contribute to pathology in patients with OCRL mutations.

The mitochondrial permeability transition pore (mPTP) has long been known to have a role in mitochondrial calcium (Ca(2+)) homeostasis under pathological conditions as a mediator of the mitochondrial permeability transition and the activation of the consequent cell death mechanism. However, its role in the context of mitochondrial Ca(2+) homeostasis is not yet clear. Several studies that were based on PPIF inhibition or knock out suggested that mPTP is involved in the Ca(2+) efflux mechanism, while other observations have revealed the opposite result. The c subunit of the mitochondrial F1/FO ATP synthase has been recently found to be a fundamental component of the mPTP. In this work, we focused on the contribution of the mPTP in the Ca(2+) efflux mechanism by modulating the expression of the c subunit. We observed that forcing mPTP opening or closing did not impair mitochondrial Ca(2+) efflux. Therefore, our results strongly suggest that the mPTP does not participate in mitochondrial Ca(2+) homeostasis in a physiological context in HeLa cells.

Activation by extracellular ligands of G protein-coupled (GPCRs) and tyrosine kinase receptors (RTKs), results in the generation of second messengers that in turn control specific cell functions. Further, modulation/amplification or inhibition of the initial signalling events, depend on the recruitment onto the plasma membrane of soluble protein effectors.High throughput methodologies to monitor quantitatively second messenger production, have been developed over the last years and are largely used to screen chemical libraries for drug development. On the contrary, no such high throughput methods are yet available for the other aspect of GPCRs regulation, i.e. protein translocation to the plasma membrane, despite the enormous interest of this phenomenon for the modulation of receptor downstream functions. Indeed, to date, the experimental procedures available are either inadequate or complex and expensive.

Potassium channels have been found in the inner mitochondrial membranes of various cells. These channels regulate the mitochondrial membrane potential, the matrix volume and respiration. The activation of these channels is cytoprotective. In our study, the single-channel activity of a large-conductance Ca(2+)-regulated potassium channel (mitoBKCa channel) was measured by patch-clamping mitoplasts isolated from the human astrocytoma (glioblastoma) U-87 MG cell line. A potassium-selective current was recorded with a mean conductance of 290 pS in symmetrical 150 mM KCl solution. The channel was activated by Ca(2+) at micromolar concentrations and by the potassium channel opener NS1619. The channel was inhibited by paxilline and iberiotoxin, known inhibitors of BKCa channels. Western blot analysis, immuno-gold electron microscopy, high-resolution immunofluorescence assays and polymerase chain reaction demonstrated the presence of the BKCa channel β4 subunit in the inner mitochondrial membrane of the human astrocytoma cells. We showed that substrates of the respiratory chain, such as NADH, succinate, and glutamate/malate, decrease the activity of the channel at positive voltages. This effect was abolished by rotenone, antimycin and cyanide, inhibitors of the respiratory chain. The putative interaction of the β4 subunit of mitoBKCa with cytochrome c oxidase was demonstrated using blue native electrophoresis. Our findings indicate possible structural and functional coupling of the mitoBKCa channel with the mitochondrial respiratory chain in human astrocytoma U-87 MG cells.

The voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane mediates metabolic flow, Ca(2+), and cell death signaling between the endoplasmic reticulum (ER) and mitochondrial networks. We demonstrate that VDAC1 is physically linked to the endoplasmic reticulum Ca(2+)-release channel inositol 1,4,5-trisphosphate receptor (IP(3)R) through the molecular chaperone glucose-regulated protein 75 (grp75). Functional interaction between the channels was shown by the recombinant expression of the ligand-binding domain of the IP(3)R on the ER or mitochondrial surface, which directly enhanced Ca(2+) accumulation in mitochondria. Knockdown of grp75 abolished the stimulatory effect, highlighting chaperone-mediated conformational coupling between the IP(3)R and the mitochondrial Ca(2+) uptake machinery. Because organelle Ca(2+) homeostasis influences fundamentally cellular functions and death signaling, the central location of grp75 may represent an important control point of cell fate and pathogenesis.

Malignant mesothelioma (MM) is a very aggressive type of cancer, with a dismal prognosis and inherent resistance to chemotherapeutics. Development and evaluation of new therapeutic approaches is highly needed. Immunosuppressant FTY720, approved for multiple sclerosis treatment, has recently raised attention for its anti-tumor activity in a variety of cancers. However, its therapeutic potential in MM has not been evaluated yet.

Tissue engineering-based bone graft is an emerging viable treatment modality to repair and regenerate tissues damaged as a result of diseases or injuries. The structure and composition of scaffolds should modulate the classical osteogenic pathways in human stem cells. The osteoinductivity properties of the hydroxylapatite-collagen hybrid scaffold named Coll/Pro Osteon 200 were investigated in an in vitro model of human adipose mesenchymal stem cells (hASCs), whereas the clinical evaluation was carried out in maxillofacial patients. Differentially expressed genes (DEGs) induced by the scaffold were analyzed using the Osteogenesis RT2 PCR Array. The osteoinductivity potential of the scaffold was also investigated by studying the alkaline phosphatase (ALP) activity, matrix mineralization, osteocalcin (OCN), and CLEC3B expression protein. Fifty patients who underwent zygomatic augmentation and bimaxillary osteotomy were evaluated clinically, radiologically, and histologically during a 3-year follow-up. Among DEGs, osteogenesis-related genes, including BMP1/2, ALP, BGLAP, SP7, RUNX2, SPP1, and EGFR, which play important roles in osteogenesis, were found to be upregulated. The genes to cartilage condensation SOX9, BMPR1B, and osteoclast cells TNFSF11 were detected upregulated at every time point of the investigation. This scaffold has a high osteoinductivity revealed by the matrix mineralization, ALP activity, OCN, and CLEC3B expression proteins. Clinical evaluation evidences that the biomaterial promotes bone regrowth. Histological results of biopsy specimens from patients showed prominent ossification. Experimental data using the Coll/Pro Osteon 200 indicate that clinical evaluation of bone regrowth in patients, after scaffold implantation, was supported by DEGs implicated in skeletal development as shown in "in vitro" experiments with hASCs.

FAM46C is a non-canonical poly(A) polymerase uniquely mutated in up to 20% of multiple myeloma (MM) patients, implying a tissue-specific tumor suppressor function. Here, we report that FAM46C selectively stabilizes mRNAs encoding endoplasmic reticulum (ER)-targeted proteins, thereby concertedly enhancing the expression of proteins that control ER protein import, folding, N-glycosylation, and trafficking and boosting protein secretion. This role requires the interaction with the ER membrane resident proteins FNDC3A and FNDC3B. In MM cells, FAM46C expression raises secretory capacity beyond sustainability, inducing ROS accumulation, ATP shortage, and cell death. FAM46C activity is regulated through rapid proteasomal degradation or the inhibitory interaction with the ZZ domain of the autophagic receptor p62 that hinders its association with FNDC3 proteins via sequestration in p62+ aggregates. Altogether, our data disclose a p62/FAM46C/FNDC3 circuit coordinating sustainable secretory activity and survival, providing an explanation for the MM-specific oncosuppressive role of FAM46C and uncovering potential therapeutic opportunities against cancer.

Dementia is a neurocognitive disorder characterized by a progressive memory loss and impairment in cognitive and functional abilities. Autophagy and mitophagy are two important cellular processes by which the damaged intracellular components are degraded by lysosomes. To investigate the contribution of autophagy and mitophagy in degenerative diseases, we investigated the serum levels of specific autophagic markers (ATG5 protein) and mitophagic markers (Parkin protein) in a population of older patients by enzyme-linked immunosorbent assay. Two hundred elderly (≥65 years) outpatients were included in the study: 40 (20 F and 20 M) with mild-moderate late onset Alzheimer's disease (AD); 40 (20 F and 20 M) affected by vascular dementia (VAD); 40 with mild cognitive impairment (MCI); 40 (20 F and 20 M) with "mixed" dementia (MD); 40 subjects without signs of cognitive impairment were included as sex-matched controls. Our data indicated that, in serum samples, ATG5 and Parkin were both elevated in controls, and that VAD compared with AD, MCI and MD (all p < 0.01). Patients affected by AD, MD, and MCI showed significantly reduced circulating levels of both ATG5 and Parkin compared to healthy controls and VAD individuals, reflecting a significant down-regulation of autophagy and mitophagy pathways in these groups of patients. The measurement of serum levels of ATG5 and Parkin may represent an easily accessible diagnostic tool for the early monitoring of patients with cognitive decline.

Numerous papers have reported altered expression patterns of Ras and/or ShcA proteins in different types of cancers. Their level can be potentially associated with oncogenic processes. We analyzed samples of pediatric brain tumors reflecting different groups such as choroid plexus tumors, diffuse astrocytic and oligodendroglial tumors, embryonal tumors, ependymal tumors, and other astrocytic tumors as well as tumor malignancy grade, in order to characterize the expression profile of Ras, TrkB, and three isoforms of ShcA, namely, p66Shc, p52Shc, and p46Shc proteins. The main aim of our study was to evaluate the potential correlation between the type of pediatric brain tumors, tumor malignancy grade, and the expression patterns of the investigated proteins.

Hyperreactive platelets are commonly observed in diabetic patients indicating a potential link between glucose homeostasis and platelet reactivity. This raises the possibility that platelets may play a role in the regulation of metabolism. Pancreatic β cells are the central regulators of systemic glucose homeostasis. Here, we show that factor(s) derived from β cells stimulate platelet activity and platelets selectively localize to the vascular endothelium of pancreatic islets. Both depletion of platelets and ablation of major platelet adhesion or activation pathways consistently resulted in impaired glucose tolerance and decreased circulating insulin levels. Furthermore, we found platelet-derived lipid classes to promote insulin secretion and identified 20-Hydroxyeicosatetraenoic acid (20-HETE) as the main factor promoting β cells function. Finally, we demonstrate that the levels of platelet-derived 20-HETE decline with age and that this parallels with reduced impact of platelets on β cell function. Our findings identify an unexpected function of platelets in the regulation of insulin secretion and glucose metabolism, which promotes metabolic fitness in young individuals.

Adaptor protein 2 (AP2) is a major constituent of clathrin-coated pits (CCPs). Whether it is essential for all forms of clathrin-mediated endocytosis (CME) in mammalian cells is an open issue. Here, we demonstrate, by live TIRF microscopy, the existence of a subclass of relatively short-lived CCPs lacking AP2 under physiological, unperturbed conditions. This subclass is retained in AP2-knockout cells and is able to support the internalization of epidermal growth factor receptor (EGFR) but not of transferrin receptor (TfR). The AP2-independent internalization mechanism relies on the endocytic adaptors eps15, eps15L1, and epsin1. The absence of AP2 impairs the recycling of the EGFR to the cell surface, thereby augmenting its degradation. Accordingly, under conditions of AP2 ablation, we detected dampening of EGFR-dependent AKT signaling and cell migration, arguing that distinct classes of CCPs could provide specialized functions in regulating EGFR recycling and signaling.

Alteration of mitochondria-associated membranes (MAMs) has been proposed to contribute to the pathogenesis of Alzheimer's disease (AD). We studied herein the subcellular distribution, the processing, and the protein interactome of the amyloid-β protein precursor (AβPP) and its proteolytic products in MAMs. We reveal that AβPP and its catabolites are present in MAMs in cellular models overexpressing wild type AβPP or AβPP harboring the double Swedish or London familial AD mutations, and in brains of transgenic mice model of AD. Furthermore, we evidenced that both β- and γ-secretases are present and harbor AβPP processing activities in MAMs. Interestingly, cells overexpressing APPswe show increased ER-mitochondria contact sites. We also document increased neutral lipid accumulation linked to Aβ production and reversed by inhibiting β- or γ-secretases. Using a proteomic approach, we show that AβPP and its catabolites interact with key proteins of MAMs controlling mitochondria and ER functions. These data highlight the role of AβPP processing and proteomic interactome in MAMs deregulation taking place in AD.

FBXL2 targets IP3R3 for ubiquitin-mediated degradation to limit Ca2+ flux to mitochondria and, consequently, apoptosis. Efficient replication of hepatitis C virus (HCV) requires geranylgeranylation of FBXL2. Here, we show that the viral protein NS5A forms a trimeric complex with IP3R3 and FBXL2, unmasking IP3R3's degron in the absence of inositol 1,4,5-trisphosphate (IP3) stimulation. FBXL2 knockdown or expression of a stable IP3R3 mutant causes persistent Ca2+ flux and sensitizes cells to apoptosis, resulting in the inhibition of viral replication. Importantly, the effect of FBXL2 silencing is rescued by depleting IP3R3, but not p85β, another established FBXL2 substrate, indicating that the anti-HCV effect of FBXL2 knockdown is largely due to IP3R3 stabilization. Finally, disruption of the FBXL2-NS5A-IP3R3 complex using somatic cell genetics or pharmacologic inhibition results in IP3R3 stabilization and suppression of HCV replication. This study reveals an IP3-independent molecular mechanism through which HCV promotes IP3R3 degradation, thereby inhibiting virus-induced apoptosis and establishing chronic infection.

Disorders of lysosomal physiology have increasingly been found to underlie the pathology of a rapidly growing cast of neurodevelopmental disorders and sporadic diseases of aging. One cardinal aspect of lysosomal (dys)function is lysosomal acidification in which defects trigger lysosomal stress signaling and defects in proteolytic capacity. We have developed a genetically encoded ratiometric probe to measure lysosomal pH coupled with a purification tag to efficiently purify lysosomes for both proteomic and in vitro evaluation of their function. Using our probe, we showed that lysosomal pH is remarkably stable over a period of days in a variety of cell types. Additionally, this probe can be used to determine that lysosomal stress signaling via TFEB is uncoupled from gross changes in lysosomal pH. Finally, we demonstrated that while overexpression of ARL8B GTPase causes striking alkalinization of peripheral lysosomes in HEK293 T cells, peripheral lysosomes per se are no less acidic than juxtanuclear lysosomes in our cell lines.Abbreviations: ARL8B: ADP ribosylation factor like GTPase 8B; ATP: adenosine triphosphate; ATP5F1B/ATPB: ATP synthase F1 subunit beta; ATP6V1A: ATPase H+ transporting V1 subunit A; Baf: bafilomycin A1; BLOC-1: biogenesis of lysosome-related organelles complex 1; BSA: bovine serum albumin; Cos7: African green monkey kidney fibroblast-like cell line; CQ: chloroquine; CTSB: cathepsin B; CYCS: cytochrome c, somatic; DAPI: 4',6-diamidino -2- phenylindole; DIC: differential interference contrast; DIV: days in vitro; DMEM: Dulbecco's modified Eagle's medium;‎ E8: embryonic day 8; EEA1: early endosome antigen 1; EGTA: ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid; ER: endoplasmic reticulum; FBS: fetal bovine serum; FITC: fluorescein isothiocyanate; GABARAPL2: GABA type A receptor associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GOLGA2/GM130: golgin A2; GTP: guanosine triphosphate; HEK293T: human embryonic kidney 293 cells, that expresses a mutant version of the SV40 large T antigen; HeLa: Henrietta Lacks-derived cell; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HRP: horseradish peroxidase; IGF2R/ciM6PR: insulin like growth factor 2 receptor; LAMP1/2: lysosomal associated membrane protein 1/2; LMAN2/VIP36: lectin, mannose binding 2; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; PCR: polymerase chain reaction; PDL: poly-d-lysine; PGK1p: promotor from human phosphoglycerate kinase 1; PIKFYVE: phosphoinositide kinase, FYVE-type zinc finger containing; PPT1/CLN1: palmitoyl-protein thioesterase 1; RPS6KB1/p70: ribosomal protein S6 kinase B1; STAT3: signal transducer and activator of transcription 3; TAX1BP1: Tax1 binding protein 1; TFEB: transcription factor EB; TGN: trans-Golgi network; TGOLN2/TGN46: trans-Golgi network protein 2; TIRF: total internal reflection fluorescence; TMEM106B: transmembrane protein 106B; TOR: target of rapamycin; TRPM2: transient receptor potential cation channel subfamily M member 2; V-ATPase: vacuolar-type proton-translocating ATPase; VPS35: VPS35 retromer complex component.

Mitochondria are dynamic organelles that have essential metabolic activity and are regarded as signalling hubs with biosynthetic, bioenergetics and signalling functions that orchestrate key biological pathways. However, mitochondria can influence all processes linked to oncogenesis, starting from malignant transformation to metastatic dissemination. In this review, we describe how alterations in the mitochondrial metabolic status contribute to the acquisition of typical malignant traits, discussing the most recent discoveries and the many unanswered questions. We also highlight that expanding our understanding of mitochondrial regulation and function mechanisms in the context of cancer cell metabolism could be an important task in biomedical research, thus offering the possibility of targeting mitochondria for the treatment of cancer.

SEPN1-related myopathy (SEPN1-RM) is a muscle disorder due to mutations of the SEPN1 gene, which is characterized by muscle weakness and fatigue leading to scoliosis and life-threatening respiratory failure. Core lesions, focal areas of mitochondria depletion in skeletal muscle fibers, are the most common histopathological lesion. SEPN1-RM underlying mechanisms and the precise role of SEPN1 in muscle remained incompletely understood, hindering the development of biomarkers and therapies for this untreatable disease. To investigate the pathophysiological pathways in SEPN1-RM, we performed metabolic studies, calcium and ATP measurements, super-resolution and electron microscopy on in vivo and in vitro models of SEPN1 deficiency as well as muscle biopsies from SEPN1-RM patients. Mouse models of SEPN1 deficiency showed marked alterations in mitochondrial physiology and energy metabolism, suggesting that SEPN1 controls mitochondrial bioenergetics. Moreover, we found that SEPN1 was enriched at the mitochondria-associated membranes (MAM), and was needed for calcium transients between ER and mitochondria, as well as for the integrity of ER-mitochondria contacts. Consistently, loss of SEPN1 in patients was associated with alterations in body composition which correlated with the severity of muscle weakness, and with impaired ER-mitochondria contacts and low ATP levels. Our results indicate a role of SEPN1 as a novel MAM protein involved in mitochondrial bioenergetics. They also identify a systemic bioenergetic component in SEPN1-RM and establish mitochondria as a novel therapeutic target. This role of SEPN1 contributes to explain the fatigue and core lesions in skeletal muscle as well as the body composition abnormalities identified as part of the SEPN1-RM phenotype. Finally, these results point out to an unrecognized interplay between mitochondrial bioenergetics and ER homeostasis in skeletal muscle. They could therefore pave the way to the identification of biomarkers and therapeutic drugs for SEPN1-RM and for other disorders in which muscle ER-mitochondria cross-talk are impaired.

Clathrin light chains (CLCa and CLCb) are major constituents of clathrin-coated vesicles. Unique functions for these evolutionary conserved paralogs remain elusive, and their role in clathrin-mediated endocytosis in mammalian cells is debated. Here, we find and structurally characterize a direct and selective interaction between CLCa and the long isoform of the actin motor protein myosin VI, which is expressed exclusively in highly polarized tissues. Using genetically-reconstituted Caco-2 cysts as proxy for polarized epithelia, we provide evidence for coordinated action of myosin VI and CLCa at the apical surface where these proteins are essential for fission of clathrin-coated pits. We further find that myosin VI and Huntingtin-interacting protein 1-related protein (Hip1R) are mutually exclusive interactors with CLCa, and suggest a model for the sequential function of myosin VI and Hip1R in actin-mediated clathrin-coated vesicle budding.

Non-alcoholic fatty liver disease (NAFLD), an increasingly prevalent and underdiagnosed disease, is postulated to be caused by hepatic fat mediated pathological mechanisms. Mitochondrial dysfunction is proposed to be involved, but it is not known whether this is a pathological driver or a consequence of NAFLD. We postulate that changes to liver mitochondrial DNA (mtDNA) are an early event that precedes mitochondrial dysfunction and irreversible liver damage. To test this hypothesis, we evaluated the impact of diet on liver steatosis, hepatic mtDNA content, and levels of key mitochondrial proteins. Liver tissues from C57BL/6 mice fed with high fat (HF) diet (HFD) and Western diet (WD, high fat and high sugar) for 16 weeks were used. Steatosis/fibrosis were assessed using haematoxylin and eosin (H&E) Oil Red and Masson's trichome staining and collagen content. Total DNA was isolated, and mtDNA content was determined by quantifying absolute mtDNA copy number/cell using quantitative PCR. Selected mitochondrial proteins were analysed from a proteomics screen. As expected, both HFD and WD resulted in steatosis. Mouse liver contained a high mtDNA content (3617 ± 233 copies per cell), which significantly increased in HFD diet, but this increase was not functional, as indicated by changes in mitochondrial proteins. In the WD fed mice, liver dysfunction was accelerated alongside downregulation of mitochondrial oxidative phosphorylation (OXPHOS) and mtDNA replication machinery as well as upregulation of mtDNA-induced inflammatory pathways. These results demonstrate that diet induced changes in liver mtDNA can occur in a relatively short time; whether these contribute directly or indirectly to subsequent mitochondrial dysfunction and the development of NAFLD remains to be determined. If this hypothesis can be substantiated, then strategies to prevent mtDNA damage in the liver may be needed to prevent development and progression of NAFLD.