Cardiac-Specific Suppression of Valosin-Containing Protein Induces Progressive Heart Failure and Premature Mortality Correlating with Temporal Dysregulations in mTOR Complex 2 and Protein Phosphatase 1.

Valosin-containing protein (VCP), an ATPase-associated protein, is emerging as a crucial regulator in cardiac pathologies. However, the pivotal role of VCP in the heart under physiological conditions remains undetermined. In this study, we tested a hypothesis that sufficient VCP expression is required for cardiac development and physiological cardiac function. Thus, we generated a cardiac-specific VCP knockout (KO) mouse model and assessed the consequences of VCP suppression on the heart through physiological and molecular studies at baseline. Our results reveal that homozygous KO mice are embryonically lethal, whereas heterozygous KO mice with a reduction in VCP by ~40% in the heart are viable at birth but progressively develop heart failure and succumb to mortality at the age of 10 to 12 months. The suppression of VCP induced a selective activation of the mammalian target of rapamycin complex 1 (mTORC1) but not mTORC2 at the early age of 12 weeks. The prolonged suppression of VCP increased the expression (by ~2 folds) and nuclear translocation (by >4 folds) of protein phosphatase 1 (PP1), a key mediator of protein dephosphorylation, accompanied by a remarked reduction (~80%) in AKTSer473 phosphorylation in VCP KO mouse hearts at a later age but not the early stage. These temporal molecular alterations were highly associated with the progressive decline in cardiac function. Overall, our findings shed light on the essential role of VCP in the heart under physiological conditions, providing new insights into molecular mechanisms in the development of heart failure.

Valosin-Containing Protein (VCP): A Review of Its Diverse Molecular Functions and Clinical Phenotypes.

In this review we examine the functionally diverse ATPase associated with various cellular activities (AAA-ATPase), valosin-containing protein (VCP/p97), its molecular functions, the mutational landscape of VCP and the phenotypic manifestation of VCP disease. VCP is crucial to a multitude of cellular functions including protein quality control, endoplasmic reticulum-associated degradation (ERAD), autophagy, mitophagy, lysophagy, stress granule formation and clearance, DNA replication and mitosis, DNA damage response including nucleotide excision repair, ATM- and ATR-mediated damage response, homologous repair and non-homologous end joining. VCP variants cause multisystem proteinopathy, and pathology can arise in several tissue types such as skeletal muscle, bone, brain, motor neurons, sensory neurons and possibly cardiac muscle, with the disease course being challenging to predict.

Tau filaments with the chronic traumatic encephalopathy fold in a case of vacuolar tauopathy with VCP mutation D395G.

Dominantly inherited mutation D395G in the gene encoding valosin-containing protein causes vacuolar tauopathy, a type of behavioural-variant frontotemporal dementia, with marked vacuolation and abundant filamentous tau inclusions made of all six brain isoforms. Here we report that tau inclusions were concentrated in layers II/III of the frontotemporal cortex in a case of vacuolar tauopathy. By electron cryomicroscopy, tau filaments had the chronic traumatic encephalopathy (CTE) fold. Tau inclusions of vacuolar tauopathy share this cortical location and the tau fold with CTE, subacute sclerosing panencephalitis and amyotrophic lateral sclerosis/parkinsonism-dementia complex, which are believed to be environmentally induced. Vacuolar tauopathy is the first inherited disease with the CTE tau fold.

TurboID-Based IRE1 Interactome Reveals Participants of the Endoplasmic Reticulum-Associated Protein Degradation Machinery in the Human Mast Cell Leukemia Cell Line HMC-1.2.

The unfolded protein response is an intricate system of sensor proteins in the endoplasmic reticulum (ER) that recognizes misfolded proteins and transmits information via transcription factors to either regain proteostasis or, depending on the severity, to induce apoptosis. The main transmembrane sensor is IRE1α, which contains cytoplasmic kinase and RNase domains relevant for its activation and the mRNA splicing of the transcription factor XBP1. Mast cell leukemia (MCL) is a severe form of systemic mastocytosis. The inhibition of IRE1α in the MCL cell line HMC-1.2 has anti-proliferative and pro-apoptotic effects, motivating us to elucidate the IRE1α interactors/regulators in HMC-1.2 cells. Therefore, the TurboID proximity labeling technique combined with MS analysis was applied. Gene Ontology and pathway enrichment analyses revealed that the majority of the enriched proteins are involved in vesicle-mediated transport, protein stabilization, and ubiquitin-dependent ER-associated protein degradation pathways. In particular, the AAA ATPase VCP and the oncoprotein MTDH as IRE1α-interacting proteins caught our interest for further analyses. The pharmacological inhibition of VCP activity resulted in the increased stability of IRE1α and MTDH as well as the activation of IRE1α. The interaction of VCP with both IRE1α and MTDH was dependent on ubiquitination. Moreover, MTDH stability was reduced in IRE1α-knockout cells. Hence, pharmacological manipulation of IRE1α-MTDH-VCP complex(es) might enable the treatment of MCL.

Age-dependent dynamics of neuronal VAPBALS inclusions in the adult brain.

Amyotrophic Lateral Sclerosis (ALS) is a relentlessly progressive and fatal disease, caused by the degeneration of upper and lower motor neurons within the brain and spinal cord in the ageing human. The dying neurons contain cytoplasmic inclusions linked to the onset and progression of the disease. Here, we use a Drosophila model of ALS8 (VAPP58S) to understand the modulation of these inclusions in the ageing adult brain. The adult VAPP58S fly shows progressive deterioration in motor function till its demise 25 days post-eclosion. The density of VAPP58S-positive brain inclusions is stable for 5-15 days of age. In contrast, adding a single copy of VAPWT to the VAPP58S animal leads to a large decrease in inclusion density with concomitant rescue of motor function and lifespan. ER stress, a contributing factor in disease, shows reduction with ageing for the disease model. Autophagy, rather than the Ubiquitin Proteasome system, is the dominant mechanism for aggregate clearance. We explored the ability of Drosophila Valosin-containing protein (VCP/TER94), the ALS14 locus, which is involved in cellular protein clearance, to regulate age-dependent aggregation. Contrary to expectation, TER94 overexpression increased VAPP58S punctae density, while its knockdown led to enhanced clearance. Expression of a dominant positive allele, TER94R152H, further stabilised VAPP58S puncta, cementing roles for an ALS8-ALS14 axis. Our results are explained by a mechanism where autophagy is modulated by TER94 knockdown. Our study sheds light on the complex regulatory events involved in the neuronal maintenance of ALS8 aggregates, suggesting a context-dependent switch between proteasomal and autophagy-based mechanisms as the larvae develop into an adult. A deeper understanding of the nucleation and clearance of the inclusions, which affect cellular stress and function, is essential for understanding the initiation and progression of ALS.

Valosin-containing protein acts as a target and mediator of S-nitrosylation in the heart through distinct mechanisms.

S-nitrosylation (SNO) is an emerging paradigm of redox signaling protecting cells against oxidative stress in the heart. Our previous studies demonstrated that valosin-containing protein (VCP), an ATPase-associated protein, is a vital mediator protecting the heart against cardiac stress and ischemic injury. However, the molecular regulations conferred by VCP in the heart are not fully understood. In this study, we explored the potential role of VCP in cardiac protein SNO using multiple cardiac-specific genetically modified mouse models and various analytical techniques including biotin switch assay, liquid chromatography, mass spectrometry, and western blotting. Our results showed that cardiac-specific overexpression of VCP led to an overall increase in the levels of SNO-modified cardiac proteins in the transgenic (TG) vs. wild-type (WT) mice. Mass spectrometry analysis identified mitochondrial proteins involved in respiration, metabolism, and detoxification as primary targets of SNO modification in VCP-overexpressing mouse hearts. Particularly, we found that VCP itself underwent SNO modification at a specific cysteine residue in its N-domain. Additionally, our study demonstrated that glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a key enzyme in glycolysis, also experienced increased SNO in response to VCP overexpression. While deletion of inducible nitric oxide synthase (iNOS) in VCP TG mice did not affect VCP SNO, it did abolish SNO modification in mitochondrial complex proteins, suggesting a dual mechanism of regulation involving both iNOS-dependent and independent pathways. Overall, our findings shed light on post-translational modification of VCP in the heart, unveiling a previously unrecognized role for VCP in regulating cardiac protein SNO and offering new insights into its function in cardiac protection.

Characterization of MYBL1 Gene in Triple-Negative Breast Cancers and the Genes' Relationship to Alterations Identified at the Chromosome 8q Loci.

The MYBL1 gene is a strong transcriptional activator involved in events associated with cancer progression. Previous data show MYBL1 overexpressed in triple-negative breast cancer (TNBC). There are two parts to this study related to further characterizing the MYBL1 gene. We start by characterizing MYBL1 reference sequence variants and isoforms. The results of this study will help in future experiments in the event there is a need to characterize functional variants and isoforms of the gene. In part two, we identify and validate expression and gene-related alterations of MYBL1, VCIP1, MYC and BOP1 genes in TNBC cell lines and patient samples selected from the Breast Invasive Carcinoma TCGA 2015 dataset available at cBioPortal.org. The four genes are located at chromosomal regions 8q13.1 to 8q.24.3 loci, regions previously identified as demonstrating a high percentage of alterations in breast cancer. We identify alterations, including changes in expression, deletions, amplifications and fusions in MYBL1, VCPIP1, BOP1 and MYC genes in many of the same patients, suggesting the panel of genes is involved in coordinated activity in patients. We propose that MYBL1, VCPIP1, MYC and BOP1 collectively be considered as genes associated with the chromosome 8q loci that potentially play a role in TNBC pathogenesis.

Vcp overexpression and leucine supplementation extend lifespan and ameliorate neuromuscular junction phenotypes of a SOD1G93A-ALS mouse model.

Many genes with distinct molecular functions have been linked to genetically heterogeneous amyotrophic lateral sclerosis (ALS), including SuperOxide Dismutase 1 (SOD1) and Valosin-Containing Protein (VCP). SOD1 converts superoxide to oxygen and hydrogen peroxide. VCP acts as a chaperon to regulate protein degradation and synthesis and various other cellular responses. Although the functions of these two genes differ, in the current report we show that overexpression of wild-type VCP in mice enhances lifespan and maintains the size of neuromuscular junctions (NMJs) of both male and female SOD1G93A mice, a well-known ALS mouse model. Although VCP exerts multiple functions, its regulation of ER formation and consequent protein synthesis has been shown to play the most important role in controlling dendritic spine formation and social and memory behaviors. Given that SOD1 mutation results in protein accumulation and aggregation, it may direct VCP to the protein degradation pathway, thereby impairing protein synthesis. Since we previously showed that the protein synthesis defects caused by Vcp deficiency can be improved by leucine supplementation, to confirm the role of the VCP-protein synthesis pathway in SOD1-linked ALS, we applied leucine supplementation to SOD1G93A mice and, similar to Vcp overexpression, we found that it extends SOD1G93A mouse lifespan. In addition, the phenotypes of reduced muscle strength and fewer NMJs of SOD1G93A mice are also improved by leucine supplementation. These results support the existence of crosstalk between SOD1 and VCP and suggest a critical role for protein synthesis in ASL. Our study also implies a potential therapeutic treatment for ALS.

Valosin-containing-protein pathogenic variant p.R487H in Parkinson's disease.

We describe a 66-year-old woman with Parkinson's disease, carrying a known pathogenic missense variant in the Valosin-containing-protein (VCP) gene. She responded excellently to L-dopa, had no cognitive or motoneuronal dysfunction. Laboratory analyses and MRI were unremarkable. Genetic testing revealed a heterozygous variant in VCP(NM_007126.5), chr9 (GRCh3 7):g.35060820C > T, c.1460G > A p.Arg487His (p.R487H).

Remote respiratory resistance exercise training improves respiratory function in individuals with VCP multisystem proteinopathy.

Valosin-containing protein (VCP) disease is an autosomal dominant multisystem proteinopathy associated with hereditary inclusion body myopathy, Paget disease of bone, and frontotemporal dementia. Myopathy frequently results in respiratory muscle weakness, leading to early mortality due to respiratory failure. We investigated the effects of a remotely administered inspiratory muscle training program in individuals with VCP disease. Nine adults with VCP mutation-positive familial myopathy without evidence of dementia were recruited for a 40-week remotely administered study. Baseline performance was established during the first 8 weeks, followed by 32 weeks of inspiratory muscle training. The primary outcome was maximum inspiratory pressure (MIP). The secondary and exploratory endpoints included spirometry, grip strength, Inclusion Body Myopathy Functional Rating Scale (IBMFRS), Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS), timed up and go, and six-minute walk test (6MWT). During the treatment phase, MIP increased significantly by a weekly mean of 0.392cm. H2O (p=0.023). In contrast, grip strength and ALSFRS significantly decreased by 0.088 lbs. (p=0.031) and 0.043 points (p=0.004) per week, respectively, as expected from the natural progression of this disease. A remotely administered inspiratory muscle training program is therefore feasible, safe, and well-tolerated in individuals with VCP disease and results in improved inspiratory muscle strength.

The autophagy of stress granules.

Our understanding of stress granule (SG) biology has deepened considerably in recent years, and with this, increased understanding of links has been made between SGs and numerous neurodegenerative diseases. One of the proposed mechanisms by which SGs and any associated protein aggregates may become pathological is based upon defects in their autophagic clearance, and so the precise processes governing the degradation of SGs are important to understand. Mutations and disease-associated variants implicated in amyotrophic lateral sclerosis, Huntington's disease, Parkinson's disease and frontotemporal lobar dementia compromise autophagy, whilst autophagy-inhibiting drugs or knockdown of essential autophagy proteins result in the persistence of SGs. In this review, we will consider the current knowledge regarding the autophagy of SG.

The mechanism of VCP-mediated metastasis of osteosarcoma based on cell autophagy and the EMT pathway

Objective Study of the molecular mechanisms of metastasis is still the research focus for osteosarcoma (OS) prevention. This study investigates the mechanism of valosin-containing protein (VCP) promoting OS metastasis in vitro through autophagy and epithelial–mesenchymal transition (EMT). Methods Different cell lines of osteosarcoma (143B and MG63) were adopted in this study. The level of VCP expression in osteosarcoma cells was changed, and the level of autophagy and the progression of the epithelial–mesenchymal transition (EMT) were observed. Then autophagy and EMT in OS cells were changed artificially, and proliferation and migration ability were observed. Results The expression of LC3II/I was decreased, but the insolubilized P62 protein expression was increased in the VCP inhibiting group and the autophagy inhibitor treatment group. Simultaneously, E-cadherin protein expression increased while N-cadherin protein expression decreased in the VCP inhibiting group but increased in the TGF-β1 treatment group. In addition, suppressing VCP can cause a decrease in Transforming Growth Factor β1 (TGF-β1), smad2, smad3, phosphorylated smad2 (p-smad2), and phosphorylated smad3 (p-smad3). Autophagy inhibitors and agonists have no significant effect on the migration and invasion of OS cells but can significantly affect the ability of cells to resist anoikis. EMT inhibitors and agonists have a proportional effect on the migration and invasion of OS cells. Conclusion VCP is likely to promote the migration and invasion of OS cells by inducing EMT, possibly via TGF-β1/smad2/3 signaling pathway. In this process, VCP-mediated autophagy may contribute to successful distant metastasis of tumor cells indirectly (AU)

Expression of valosin-containing protein in epithelial ovarian cancer tissue and its clinical significance / 吉林大学学报(医学版)

Objective: To observe the expression of valosin-containing protein CVCP) in the epithelial ovarian cancer (EOC) tissue and its relationships with the clinicopathological features of the EOC patients∗ and to provide the basis for the molecular treatment of EOC. Methods: The expressions of VCP in 94 EOC tissue samples, 13 ovarian borderline tumor tissue samples, 36 ovarian benign tumor tissue samples and 8 normal ovarian tissue samples were measured by immunohistochemical method. The relationships between the VCP expression and the clinicopathological parameters (age, FIGO stage, pathological type, histological grade, lymph node metastasis or not, ascites or not, preoperative CA125 level) of the EOC patients were analyzed. The expressions of VCP in human ovarian epithelial HOSEPIC cells and human EOC SKOV-3 cells were detected by immunofluorescence technique and Western blotting method. The SKOV-3 cells were divided into control group (without CB-5083) and treatment group (with CB-5083)∗ the migration rates of the SKOV-3 cells in two groups were analyzed by scratch experiment, and the clone formation rates of the SKOV-3 cells in two groups were analyzed by plate clone formation experiment. The expressions of VCP in the cells in two groups were analyzed by immunofluorescence technique. The expression levels of VCP, NF-kB/P65» IieBa∗ and p-Iiefta proteins in the SKOV-3 cells in two groups were detected by Western blotting method. Results: The positive expression rates of VCP in EOC, borderline ovarian tumor, benign ovarian tumor and normal ovarian tissues had significant difference ( P0. 05). The VCP expression level in the SKOV-3 cells was higher than that in HOSEpiC cells ( P<0. 05). The migration rate of the SKOV-3 cells in treatment group was lower than that in control group ( P<0.05), the cloning formation rate of the SKOV-3 cells in treatment group was lower than that in control group ( P<0. 05). The expression levels of VCP and NF-kB/P65 proteins of the SKOV-3 cells in treatment group were lower than those in control group (P'<0. 05) ∗ and the expression level of p-IieBa protein was higher than that in control group ( P< 0. 05). Conclusion: VCP is highly expressed in the EOC tissue and cells∗ and high-expression VCP can promot the tumor migration and proliferation.

Involvement of Peripheral and Central Nervous Systems in a Valosin-Containing Protein Mutation

BACKGROUND: Inclusion-body myopathy with Paget's disease of the bone and frontotemporal dementia (IBMPFD) is a rare, late-onset autosomal disorder arising from missense mutations in a gene coding for valosin-containing protein. CASE REPORT: We report the case of a man carrying the previously described p.Arg159His mutation, who had an unusual axonal sensorimotor neuropathy as the first clinical manifestation of IBMPFD, and for whom diagnosis only became clear 8 years later when the patient developed frontotemporal dementia. CONCLUSIONS: Peripheral neuropathy is a rare manifestation of IBMPFD. This underdiagnosed disorder should be considered when a patient develops dementia or has signs of Paget's disease.

miRNA-129-5p regulates the targets of the VCP gene expression in human osteosarcoma cells / 医学研究生学报

Objective High expression of the valosin-containing protein ( VCP) gene can enhance the metastasis of osteosar-coma via the AKT/PI3K/NF-KappaB/MMP-9 signaling pathway, but the molecular mechanisms underlying the up-regulation of VCP in osteosarcoma cells remains unknown .This study aimed to determine whether miRNA-129-5p can regulate the VCP expression and its targets in human osteosarcoma cells . Methods The microRNA target-predicting software TargetScanhuman 6.2 ( http://www.tar-getscan.org/) was used to predict the possible targets of miRNA-129-5p on the VCP gene.Then, two recombinant gene report vectors containing the wild VCP gene 3′UTR ( psi-VCP vector ) and mutant VCP gene 3′UTR ( psi-VCPmut vector ) were constructed , se-quenced, and identified.The human osteosarcoma U2-OS cells were co-transfected with miRNA-129-5p mimic and psi-VCP vector or psi-VCPmut vector, respectively.A non-specificity mimic transfection served as negative control , and the luciferase activity was detec-ted in each group. Results The software prediction showed only one conserved function site of miRNA-129-5p on the VCP gene 3′UTR163-169 bp.Luciferase activity was significantly lower in the psi-VCP vector +miRNA-129-5p transfection group (15.529 ± 1.902) than in the VCP control group (21.781 ±0.854), VCP mutation experimental group (19.978 ±1.377), and VCP mutation control group (21.952 ±1.516) (P<0.05), with no remarkable difference between the VCP mutation control and VCP control groups (P=0.276). Conclusion miRNA-129-5p can probably regulate the targets of the VCP gene in human osteosarcoma U 2-OS cells.

Cloning and expression of Schistosoma japonicum VCP gene and its mRNA expression levels in different stages / 中国血吸虫病防治杂志

Objective To prokaryotically express the valosin-containing protein(VCP)of Schistosoma japonicum,and ana-lyze its VCP mRNA expressions in the cercaria,schistosomulum,adult worm(female and male worms)and egg. Methods RNA of S. japonicum eggs were extracted,and reversely transcribed into cDNA. The VCP gene of S. japonicum was amplified by using polymerase chain reaction(PCR),and subcloned into the prokaryotically expressed vector pET15b. The recombined plasmid was transformed into BL21 cells,and the expression of the target gene was induced with isopropyl-beta-D-thiogalactopyranoside (IPTG). The recombinant protein was yielded through the purification of inclusion body,and identified by using sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). The RNA(s)of cercaria,schistosomulum,female adult worm,male adult worm,and egg of S. japonicum were extracted,digested with DNase,purified,and reversely transcribed into cDNA. The mRNA expressions of the VCP gene in various developmental stages of S. japonicum were determined by using fluorescence-based quantitative real-time PCR. Results The VCP gene of S. japonicum was yielded by PCR amplification,and the recombinant pro-tein was obtained through recombinant plasmid expression and purification of inclusion body. The highest VCP mRNA expression in S. japonicum cercaria was detected by the fluorescence-based quantitative real-time PCR,while low expressions were found in the schistosomulum,egg,female and male adult worms. Conclusion The recombinant protein encoded by the VCP gene of S. ja-ponicum is successfully obtained,and the VCP mRNA expression is determined in various developmental stages of S. japonicum.