Vesicle-associated Membrane Protein-associated Protein Research Articles

Arabidopsis ORP2A mediates ER–autophagosomal membrane contact sites and regulates PI3P in plant autophagy

Autophagy is an intracellular degradation system for cytoplasmic constituents which is mediated by the formation of a double-membrane organelle termed the autophagosome and its subsequent fusion with the lysosome/vacuole. The formation of the autophagosome requires membrane from the endoplasmic reticulum (ER) and is tightly regulated by a series of autophagy-related (ATG) proteins and lipids. However, how the ER contacts autophagosomes and regulates autophagy remain elusive in plants. In this study, we identified and demonstrated the roles of Arabidopsis oxysterol-binding protein-related protein 2A (ORP2A) in mediating ER-autophagosomal membrane contacts and autophagosome biogenesis. We showed that ORP2A localizes to both ER-plasma membrane contact sites (EPCSs) and autophagosomes, and that ORP2A interacts with both the ER-localized VAMP-associated protein (VAP) 27-1 and ATG8e on the autophagosomes to mediate the membrane contact sites (MCSs). In ORP2A artificial microRNA knockdown (KD) plants, seedlings display retarded growth and impaired autophagy levels. Both ATG1a and ATG8e accumulated and associated with the ER membrane in ORP2A KD lines. Moreover, ORP2A binds multiple phospholipids and shows colocalization with phosphatidylinositol 3-phosphate (PI3P) invivo. Taken together, ORP2A mediates ER-autophagosomal MCSs and regulates autophagy through PI3P redistribution.

Large Oncosome-Loaded VAPA Promotes Bone-Tropic Metastasis of Hepatocellular Carcinoma Via Formation of Osteoclastic Pre-Metastatic Niche.

Tumor-derived extracellular vesicles (EVs) function as critical mediators in selective modulation of the microenvironment of distant organs to generate a pre-metastatic niche that facilitates organotropic metastasis. Identifying the organ-specific molecular determinants of EVs can develop potential anti-metastatic therapeutic targets. In the current study, large oncosomes (LOs), atypically large cancer-derived EVs, are found to play a crucial role in facilitating bone-tropic metastasis of hepatocellular carcinoma (HCC) cells by engineering an osteoclastic pre-metastatic niche and establishing a vicious cycle between the osteoclasts and HCC cells. Transmembrane protein, VAMP-associated protein A (VAPA), is significantly enriched on LOs surface via direct interaction with LOs marker αV-integrin. VAPA-enriched LOs-induced pre-metastatic education transforms the bone into a fertile milieu, which supports the growth of metastatic HCC cells. Mechanically, LOs-delivered VAPA integrates to plasma membrane of osteoclasts and directly interacts with and activates neural Wiskott-Aldrich syndrome protein (N-WASP) via dual mechanisms, consequently resulting in ARP2/3 complex-mediated reorganization of actin cytoskeleton in osteoclasts and osteoclastogenesis. Importantly, treatment with N-WASP inhibitor 187-1-packaged LOs (LOs/187-1) dramatically abolishes the inductive effect of VAPA-enriched LOs on pre-metastatic niche formation and precludes HCC bone metastasis. These findings reveal a plausible mechanism for bone-tropism of HCC and can represent a potential strategy to prevent HCC bone metastasis.

Involvement of a Cluster of Basic Amino Acids in Phosphorylation-Dependent Functional Repression of the Ceramide Transport Protein CERT.

Ceramide transport protein (CERT) mediates ceramide transfer from the endoplasmic reticulum to the Golgi for sphingomyelin (SM) biosynthesis. CERT is inactivated by multiple phosphorylation at the serine-repeat motif (SRM), and mutations that impair the SRM phosphorylation are associated with a group of inherited intellectual disorders in humans. It has been suggested that the N-terminal phosphatidylinositol 4-monophosphate [PtdIns(4)P] binding domain and the C-terminal ceramide-transfer domain of CERT physically interfere with each other in the SRM phosphorylated state, thereby repressing the function of CERT; however, it remains unclear which regions in CERT are involved in the SRM phosphorylation-dependent repression of CERT. Here, we identified a previously uncharacterized cluster of lysine/arginine residues that were predicted to be located on the outer surface of a probable coiled-coil fold in CERT. Substitutions of the basic amino acids in the cluster with alanine released the SRM-dependent repression of CERT activities, i.e., the synthesis of SM, PtdIns(4)P-binding, vesicle-associated membrane protein-associated protein (VAP) binding, ceramide-transfer activity, and localization to the Golgi, although the effect on SM synthesis activity was only partially compromised by the alanine substitutions, which moderately destabilized the trimeric status of CERT. These results suggest that the basic amino acid cluster in the coiled-coil region is involved in the regulation of CERT function.

Therapeutic interventions of Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS) is a non-demyelinating neurodegenerative disease mostly found in adults between 40 to 60 years old. This disease is usually prevalent in males, however it’s irrespective to the different genders. ALS is progressive and within 2-5 years of diagnosis ulimately ends with death. The majority of ALS cases is sporadic (90%) and is recorded without any defined aetiology. The other 10-12% of cases is recognized from mutations in more than 20 genes. The genes reported to cause ALS are Superoxide Dismutase 1 (SOD1), TAR DNA Binding Protein (TDP), Fused in Sarcoma, (FUS), Chromosome 9 Open Reading Frame 72 (c9orf72) and Vesicle-Associated Membrane-ProteinAssociated Protein B (VAPB). Furthermore, abnormal lipid metabolism with higher LDL/HDL ratio was reported in ALS patients. The aetiology of ALS is shown in the schematic diagram below (Figure 1) Due to the multi-nature of ALS causative factors and symptoms, there is no specific therapy for ALS today. However, this paper will touch on potential therapies that are in practice or may come up in the future. The goal is to maintain and improve the function of motor neuron, the well-being and quality of life for ALS patients. Until then, we have to rely on the symptomatic treatment and rehabilitative measures to support the patient’s quality of life.

Activity-dependent endoplasmic reticulum Ca2+ uptake depends on Kv2.1-mediated endoplasmic reticulum/plasma membrane junctions to promote synaptic transmission.

The endoplasmic reticulum (ER) forms a continuous and dynamic network throughout a neuron, extending from dendrites to axon terminals, and axonal ER dysfunction is implicated in several neurological disorders. In addition, tight junctions between the ER and plasma membrane (PM) are formed by several molecules including Kv2 channels, but the cellular functions of many ER-PM junctions remain unknown. Recently, dynamic Ca2+ uptake into the ER during electrical activity was shown to play an essential role in synaptic transmission. Our experiments demonstrate that Kv2.1 channels are necessary for enabling ER Ca2+ uptake during electrical activity, as knockdown (KD) of Kv2.1 rendered both the somatic and axonal ER unable to accumulate Ca2+ during electrical stimulation. Moreover, our experiments demonstrate that the loss of Kv2.1 in the axon impairs synaptic vesicle fusion during stimulation via a mechanism unrelated to voltage. Thus, our data demonstrate that a nonconducting role of Kv2.1 exists through its binding to the ER protein VAMP-associated protein (VAP), which couples ER Ca2+ uptake with electrical activity. Our results further suggest that Kv2.1 has a critical function in neuronal cell biology for Ca2+ handling independent of voltage and reveals a critical pathway for maintaining ER lumen Ca2+ levels and efficient neurotransmitter release. Taken together, these findings reveal an essential nonclassical role for both Kv2.1 and the ER-PM junctions in synaptic transmission.

VAP Proteins - From Organelle Tethers to Pathogenic Host Interactors and Their Role in Neuronal Disease.

Vesicle-associated membrane protein (VAMP)-associated proteins (VAPs) are ubiquitous ER-resident tail-anchored membrane proteins in eukaryotic cells. Their N-terminal major sperm protein (MSP) domain faces the cytosol and allows them to interact with a wide variety of cellular proteins. Therefore, VAP proteins are vital to many cellular processes, including organelle membrane tethering, lipid transfer, autophagy, ion homeostasis and viral defence. Here, we provide a timely overview of the increasing number of VAPA/B binding partners and discuss the role of VAPA/B in maintaining organelle-ER interactions and cooperation. Furthermore, we address how viruses and intracellular bacteria hijack VAPs and their binding partners to induce interactions between the host ER and pathogen-containing compartments and support pathogen replication. Finally, we focus on the role of VAP in human disease and discuss how mutated VAPB leads to the disruption of cellular homeostasis and causes amyotrophic lateral sclerosis.

CircVAPA promotes small cell lung cancer progression by modulating the miR-377-3p and miR-494-3p/IGF1R/AKT axis

BackgroundMultiple lines of evidence have demonstrated that circular RNAs (circRNAs) play oncogenic or tumor-suppressive roles in various human cancers. Nevertheless, the biological functions of circRNAs in small cell lung cancer (SCLC) are still elusive.MethodsCircVAPA (annotated as hsa_circ_0006990) was identified by mining the circRNA profiling dataset of six paired SCLC tissues and the RNA-seq data of serum samples from 36 SCLC patients and 118 healthy controls. The circVAPA expression level was evaluated using quantitative real-time PCR in SCLC cells and tissues. Cell viability, colony formation, cell cycle and apoptosis analysis assays and in vivo tumorigenesis were used to reveal the biological roles of circVAPA. The underlying mechanism of circVAPA was investigated by Western blot, RNA pulldown, RNA immunoprecipitation, dual-luciferase reporter assay and rescue experiments.ResultsWe revealed that circVAPA, derived from exons 2-4 of the vesicle-associated membrane protein-associated protein A (VAPA) gene, exhibited higher expression levels in SCLC cell lines, clinical tissues, and serum from SCLC patients than the controls, and facilitated SCLC progression in vitro and in vivo. Mechanistically, circVAPA activated the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway by modulating the miR-377-3p and miR-494-3p/insulin-like growth factor 1 receptor (IGF1R) axis to accelerate SCLC progression. Furthermore, circVAPA depletion markedly enhanced the inhibitory effects of BMS-536924, an IGF1R kinase inhibitor in cellular and xenograft mouse models.ConclusionsCircVAPA promotes SCLC progression via the miR-377-3p and miR-494-3p/IGF1R/AKT axis. We hope to develop clinical protocols of combinations of circVAPA inhibition and BMS-536924 addition for treating SCLC with circVAPA upregulation.

Hyperosmotic Stress Induces Phosphorylation of CERT and Enhances Its Tethering throughout the Endoplasmic Reticulum

The ceramide transport protein (CERT) delivers ceramide from the endoplasmic reticulum (ER) to the Golgi apparatus, where ceramide is converted to sphingomyelin (SM). The function of CERT is regulated in two distinct phosphorylation-dependent events: multiple phosphorylations in a serine-repeat motif (SRM) and phosphorylation of serine 315 residue (S315). Pharmacological inhibition of SM biosynthesis results in an increase in SRM-dephosphorylated CERT, which serves as an activated form, and an enhanced phosphorylation of S315, which augments the binding of CERT to ER-resident VAMP-associated protein (VAP), inducing the full activation of CERT to operate at the ER–Golgi membrane contact sites (MCSs). However, it remains unclear whether the two phosphorylation-dependent regulatory events always occur coordinately. Here, we describe that hyperosmotic stress induces S315 phosphorylation without affecting the SRM-phosphorylation state. Under hyperosmotic conditions, the binding of CERT with VAP-A is enhanced in an S315 phosphorylation-dependent manner, and this increased binding occurs throughout the ER rather than restrictedly at the ER–Golgi MCSs. Moreover, we found that de novo synthesis of SM with very-long acyl chains preferentially increases via a CERT-independent mechanism under hyperosmotic-stressed cells, providing an insight into a CERT-independent ceramide transport pathway for de novo synthesis of SM.

Caspar, an adapter for VAPB and TER94, modulates the progression of ALS8 by regulating IMD/NFκB-mediated glial inflammation in a Drosophila model of human disease.

Amyotrophic lateral sclerosis (ALS) is a fatal, late-onset, progressive motor neurodegenerative disorder. A key pathological feature of the disease is the presence of heavily ubiquitinated protein inclusions. Both the unfolded protein response and the ubiquitin–proteasome system appear significantly impaired in patients and animal models of ALS. We have studied cellular and molecular mechanisms involved in ALS using a vesicle-associated membrane protein-associated protein B (VAPB/ALS8) Drosophila model [Moustaqim-Barrette, A., Lin, Y.Q., Pradhan, S., Neely, G.G., Bellen, H.J. and Tsuda, H. (2014) The ALS 8 protein, VAP, is required for ER protein quality control. Hum. Mol. Genet., 23, 1975–1989], which mimics many systemic aspects of the human disease. Here, we show that VAPB, located on the cytoplasmic face of the endoplasmic reticulum membrane, interacts with Caspar, an orthologue of human fas associated factor 1 (FAF1). Caspar, in turn, interacts with transitional endoplasmic reticulum ATPase (TER94), a fly orthologue of ALS14 (VCP/p97, valosin-containing protein). Caspar overexpression in the glia extends lifespan and also slows the progression of motor dysfunction in the ALS8 disease model, a phenomenon that we ascribe to its ability to restrain age-dependent inflammation, which is modulated by Relish/NFκB signalling. Caspar binds to VAPB via an FFAT motif, and we find that Caspar’s ability to negatively regulate NFκB signalling is not dependent on the VAPB:Caspar interaction. We hypothesize that Caspar is a key molecule in the pathogenesis of ALS. The VAPB:Caspar:TER94 complex appears to be a candidate for regulating both protein homeostasis and NFκB signalling, with our study highlighting a role for Caspar in glial inflammation. We project human FAF1 as an important protein target to alleviate the progression of motor neuron disease.

Antibody Characterization Report for Vesicle-associated membrane protein-associated protein B/C (VAPB)

Antibody Characterization Report for Vesicle-associated membrane protein-associated protein B/C (VAPB)

Sequence requirements of the FFAT-like motif for specific binding to VAP-A are revealed by NMR.

The endoplasmic reticulum transmembrane protein vesicle-associated membrane protein-associated protein (VAP) plays a central role in the formation and function of membrane contact sites (MCS) through its interactions with proteins. The major sperm protein (MSP) domain of VAP binds to a variety of sequences which are referred to as FFAT-like motifs. In this study, we investigated the interactions of eight peptides containing FFAT-like motifs with the VAP-A MSP domain (VAP-AMSP ) by solution NMR. Six of eight peptides are specifically bound to VAP-A. Furthermore, we found that the RNA-dependent RNA polymerase of severe acute respiratory syndrome coronavirus 2 has an FFAT-like motif which specifically binds to VAP-AMSP as well as other FFAT-like motifs. Our results will contribute to the discovery of new VAP interactors.

Amyotrophic Lateral Sclerosis (ALS): Stressed by Dysfunctional Mitochondria-Endoplasmic Reticulum Contacts (MERCs).

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease for which there is currently no cure. Progress in the characterization of other neurodegenerative mechanisms has shifted the spotlight onto an intracellular structure called mitochondria-endoplasmic reticulum (ER) contacts (MERCs) whose ER portion can be biochemically isolated as mitochondria-associated membranes (MAMs). Within the central nervous system (CNS), these structures control the metabolic output of mitochondria and keep sources of oxidative stress in check via autophagy. The most relevant MERC controllers in the ALS pathogenesis are vesicle-associated membrane protein-associated protein B (VAPB), a mitochondria-ER tether, and the ubiquitin-specific chaperone valosin containing protein (VCP). These two systems cooperate to maintain mitochondrial energy output and prevent oxidative stress. In ALS, mutant VAPB and VCP take a central position in the pathology through MERC dysfunction that ultimately alters or compromises mitochondrial bioenergetics. Intriguingly, both proteins are targets themselves of other ALS mutant proteins, including C9orf72, FUS, or TDP-43. Thus, a new picture emerges, where different triggers cause MERC dysfunction in ALS, subsequently leading to well-known pathological changes including endoplasmic reticulum (ER) stress, inflammation, and motor neuron death.

Role of Vesicle-Associated Membrane Protein-Associated Proteins (VAP) A and VAPB in Nuclear Egress of the Alphaherpesvirus Pseudorabies Virus.

The molecular mechanism affecting translocation of newly synthesized herpesvirus nucleocapsids from the nucleus into the cytoplasm is still not fully understood. The viral nuclear egress complex (NEC) mediates budding at and scission from the inner nuclear membrane, but the NEC is not sufficient for efficient fusion of the primary virion envelope with the outer nuclear membrane. Since no other viral protein was found to be essential for this process, it was suggested that a cellular machinery is recruited by viral proteins. However, knowledge on fusion mechanisms involving the nuclear membranes is rare. Recently, vesicle-associated membrane protein-associated protein B (VAPB) was shown to play a role in nuclear egress of herpes simplex virus 1 (HSV-1). To test this for the related alphaherpesvirus pseudorabies virus (PrV), we mutated genes encoding VAPB and VAPA by CRISPR/Cas9-based genome editing in our standard rabbit kidney cells (RK13), either individually or in combination. Single as well as double knockout cells were tested for virus propagation and for defects in nuclear egress. However, no deficiency in virus replication nor any effect on nuclear egress was obvious suggesting that VAPB and VAPA do not play a significant role in this process during PrV infection in RK13 cells.

CircVAPA promotes the proliferation, migration and invasion of oral cancer cells through the miR-132/HOXA7 axis.

ObjectiveTo study the relationship between the circular RNA vesicle-associated membrane protein-associated protein A (circVAPA) and the pathogenesis of oral squamous cell carcinoma.MethodsThe expression of circVAPA was detected by RT-qPCR. In vitro loss-of-function experiments were performed in Cal-27 cells. The malignant phenotype of cells was evaluated by cell counting kit-8, clone formation and transwell assays. Luciferase reporter assays were used to assess the circVAPA/miR-132/homeobox A (HOXA) regulatory axis.ResultscircVAPA expression was significantly increased in oral cancer tissues and cells. The overall survival and progression-free survival of patients with oral cancer who exhibited high circVAPA expression were significantly shorter compared with those with low expression. circVAPA expression was closely related to tumor size, TNM stage and distant metastasis. circVAPA knockdown reduced the proliferation, invasion and migration of Cal-27 cells. MiR-132 was identified as a target of circVAPA in Cal-27 cells. Cotransfection with si-circVAPA and miR-132 inhibitor reversed the inhibitory effect of circVAPA knockdown on cell malignant phenotypes. HOXA7 was further identified as a downstream target of miR-132.ConclusioncircVAPA is highly expressed in oral cancer, and its abnormal expression might affect the proliferation, invasion and migration of oral cancer cells by modulating the miR-132/HOXA7 signaling axis.

Pathomechanisms of ALS8: altered autophagy and defective RNA binding protein (RBP) homeostasis due to the VAPB P56S mutation

Mutations in RNA binding proteins (RBPs) and in genes regulating autophagy are frequent causes of familial amyotrophic lateral sclerosis (fALS). The P56S mutation in vesicle-associated membrane protein-associated protein B (VAPB) leads to fALS (ALS8) and spinal muscular atrophy (SMA). While VAPB is primarily involved in the unfolded protein response (UPR), vesicular trafficking and in initial steps of the autophagy pathway, the effect of mutant P56S-VAPB on autophagy regulation in connection with RBP homeostasis has not been explored yet. Examining the muscle biopsy of our index ALS8 patient of European origin revealed globular accumulations of VAPB aggregates co-localised with autophagy markers LC3 and p62 in partially atrophic and atrophic muscle fibres. In line with this skin fibroblasts obtained from the same patient showed accumulation of P56S-VAPB aggregates together with LC3 and p62. Detailed investigations of autophagic flux in cell culture models revealed that P56S-VAPB alters both initial and late steps of the autophagy pathway. Accordingly, electron microscopy complemented with live cell imaging highlighted the impaired fusion of accumulated autophagosomes with lysosomes in cells expressing P56S-VAPB. Consistent with these observations, neuropathological studies of brain and spinal cord of P56S-VAPB transgenic mice revealed signs of neurodegeneration associated with altered protein quality control and defective autophagy. Autophagy and RBP homeostasis are interdependent, as demonstrated by the cytoplasmic mis-localisation of several RBPs including pTDP-43, FUS, Matrin 3 which often sequestered with P56S-VAPB aggregates both in cell culture and in the muscle biopsy of the ALS8 patient. Further confirming the notion that aggregation of the RBPs proceeds through the stress granule (SG) pathway, we found persistent G3BP- and TIAR1-positive SGs in P56S-VAPB expressing cells as well as in the ALS8 patient muscle biopsy. We conclude that P56S-VAPB-ALS8 involves a cohesive pathomechanism of aberrant RBP homeostasis together with dysfunctional autophagy.

Microtubule-independent movement of the fission yeast nucleus.

ABSTRACTMovement of the cell nucleus typically involves the cytoskeleton and either polymerization-based pushing forces or motor-based pulling forces. In the fission yeast Schizosaccharomyces pombe, nuclear movement and positioning are thought to depend on microtubule polymerization-based pushing forces. Here, we describe a novel, microtubule-independent, form of nuclear movement in fission yeast. Microtubule-independent nuclear movement is directed towards growing cell tips, and it is strongest when the nucleus is close to a growing cell tip, and weakest when the nucleus is far from that tip. Microtubule-independent nuclear movement requires actin cables but does not depend on actin polymerization-based pushing or myosin V-based pulling forces. The vesicle-associated membrane protein (VAMP)-associated proteins (VAPs) Scs2 and Scs22, which are critical for endoplasmic reticulum–plasma membrane contact sites in fission yeast, are also required for microtubule-independent nuclear movement. We also find that in cells in which microtubule-based pushing forces are present, disruption of actin cables leads to increased fluctuations in interphase nuclear positioning and subsequent altered septation. Our results suggest two non-exclusive mechanisms for microtubule-independent nuclear movement, which may help illuminate aspects of nuclear positioning in other cells.

Amyotrophic lateral sclerosis-associated Vap33 is required for maintaining neuronal dendrite morphology and organelle distribution in Drosophila.

VAMP-associated protein (VAP) is an endoplasmic reticulum (ER) membrane protein that functions as a tethering protein at the membrane contact sites between the ER and various intracellular organelles. Mutations such as P56S in human VAPB cause neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). However, VAP functions in neurons are poorly understood. Here, we utilized Drosophila olfactory projection neurons with a mosaic analysis with a repressible cell marker (MARCM) to analyze the neuronal function of Vap33, a Drosophila ortholog of human VAPB. In vap33 null mutant clones, the dendrites of projection neurons exhibited defects in the maintenance of their morphology. The subcellular localization of the Golgi apparatus and mitochondria were also abnormal. These results indicate that Vap33 is required for neuronal morphology and organelle distribution. Additionally, to examine the impact of ALS-associated mutations in neurons, we overexpressed human VAPB-P56S in vap33 null mutant clones (mosaic rescue experiments) and found that, in aged flies, human VAPB-P56S expression caused mislocalization of Bruchpilot, a presynaptic protein. These results implied that synaptic protein localization and ER quality control may be affected by disease mutations. We provide insights into the physiological and pathological functions of VAP in neurons.

The Novel ALG-2 Target Protein CDIP1 Promotes Cell Death by Interacting with ESCRT-I and VAPA/B.

Apoptosis-linked gene 2 (ALG-2, also known as PDCD6) is a member of the penta-EF-hand (PEF) family of Ca2+-binding proteins. The murine gene encoding ALG-2 was originally reported to be an essential gene for apoptosis. However, the role of ALG-2 in cell death pathways has remained elusive. In the present study, we found that cell death-inducing p53 target protein 1 (CDIP1), a pro-apoptotic protein, interacts with ALG-2 in a Ca2+-dependent manner. Co-immunoprecipitation analysis of GFP-fused CDIP1 (GFP-CDIP1) revealed that GFP-CDIP1 associates with tumor susceptibility gene 101 (TSG101), a known target of ALG-2 and a subunit of endosomal sorting complex required for transport-I (ESCRT-I). ESCRT-I is a heterotetrameric complex composed of TSG101, VPS28, VPS37 and MVB12/UBAP1. Of diverse ESCRT-I species originating from four VPS37 isoforms (A, B, C, and D), CDIP1 preferentially associates with ESCRT-I containing VPS37B or VPS37C in part through the adaptor function of ALG-2. Overexpression of GFP-CDIP1 in HEK293 cells caused caspase-3/7-mediated cell death. In addition, the cell death was enhanced by co-expression of ALG-2 and ESCRT-I, indicating that ALG-2 likely promotes CDIP1-induced cell death by promoting the association between CDIP1 and ESCRT-I. We also found that CDIP1 binds to vesicle-associated membrane protein-associated protein (VAP)A and VAPB through the two phenylalanines in an acidic tract (FFAT)-like motif in the C-terminal region of CDIP1, mutations of which resulted in reduction of CDIP1-induced cell death. Therefore, our findings suggest that different expression levels of ALG-2, ESCRT-I subunits, VAPA and VAPB may have an impact on sensitivity of anticancer drugs associated with CDIP1 expression.

Sphingomyelin Is Essential for the Structure and Function of the Double-Membrane Vesicles in Hepatitis C Virus RNA Replication Factories.

Some plus-stranded RNA viruses generate double-membrane vesicles (DMVs), one type of the membrane replication factories, as replication sites. Little is known about the lipid components involved in the biogenesis of these vesicles. Sphingomyelin (SM) is required for hepatitis C virus (HCV) replication, but the mechanism of SM involvement remains poorly understood. SM biosynthesis starts in the endoplasmic reticulum (ER) and gives rise to ceramide, which is transported from the ER to the Golgi by the action of ceramide transfer protein (CERT), where it can be converted to SM. In this study, inhibition of SM biosynthesis, either by using small-molecule inhibitors or by knockout (KO) of CERT, suppressed HCV replication in a genotype-independent manner. This reduction in HCV replication was rescued by exogenous SM or ectopic expression of the CERT protein, but not by ectopic expression of nonfunctional CERT mutants. Observing low numbers of DMVs in stable replicon cells treated with a SM biosynthesis inhibitor or in CERT-KO cells transfected with either HCV replicon or with constructs that drive HCV protein production in a replication-independent system indicated the significant importance of SM to DMVs. The degradation of SM of the in vitro-isolated DMVs affected their morphology and increased the vulnerability of HCV RNA and proteins to RNase and protease treatment, respectively. Poliovirus, known to induce DMVs, showed decreased replication in CERT-KO cells, while dengue virus, known to induce invaginated vesicles, did not. In conclusion, these findings indicated that SM is an essential constituent of DMVs generated by some plus-stranded RNA viruses.IMPORTANCE Previous reports assumed that sphingomyelin (SM) is essential for HCV replication, but the mechanism was unclear. In this study, we showed for the first time that SM and ceramide transfer protein (CERT), which is in the SM biosynthesis pathway, are essential for the biosynthesis of double-membrane vesicles (DMVs), the sites of viral replication. Low numbers of DMVs were observed in CERT-KO cells transfected with replicon RNA or with constructs that drive HCV protein production in a replication-independent system. HCV replication was rescued by ectopic expression of the CERT protein, but not by CERT mutants, that abolishes the binding of CERT to vesicle-associated membrane protein-associated protein (VAP) or phosphatidylinositol 4-phosphate (PI4P), indicating new roles for VAP and PI4P in HCV replication. The biosynthesis of DMVs has great importance to replication by a variety of plus-stranded RNA viruses. Understanding of this process is expected to facilitate the development of diagnosis and antivirus.

Uptake and Fate of Extracellular Membrane Vesicles: Nucleoplasmic Reticulum-Associated Late Endosomes as a New Gate to Intercellular Communication.

Extracellular membrane vesicles (EVs) are emerging as new vehicles in intercellular communication, but how the biological information contained in EVs is shared between cells remains elusive. Several mechanisms have been described to explain their release from donor cells and the initial step of their uptake by recipient cells, which triggers a cellular response. Yet, the intracellular routes and subcellular fate of EV content upon internalization remain poorly characterized. This is particularly true for EV-associated proteins and nucleic acids that shuttle to the nucleus of host cells. In this review, we will describe and discuss the release of EVs from donor cells, their uptake by recipient cells, and the fate of their cargoes, focusing on a novel intracellular route wherein small GTPase Rab7+ late endosomes containing endocytosed EVs enter into nuclear envelope invaginations and deliver their cargo components to the nucleoplasm of recipient cells. A tripartite protein complex composed of (VAMP)-associated protein A (VAP-A), oxysterol-binding protein (OSBP)-related protein-3 (ORP3), and Rab7 is essential for the transfer of EV-derived components to the nuclear compartment by orchestrating the particular localization of late endosomes in the nucleoplasmic reticulum.