VCP/p97 in abnormal protein aggregates, cytoplasmic vacuoles, and cell death, phenotypes relevant to neurodegeneration

Neuronal cell death, abnormal protein aggregates, and cytoplasmic vacuolization are major pathologies observed in many neurodegenerative disorders such as the polyglutamine (polyQ) diseases, prion disease, Alzheimer disease, and the Lewy body diseases, suggesting common mechanisms underlying neurodegeneration. Here, we have identified VCP/p97, a member of the AAA+ family of ATPase proteins, as a polyQ-interacting protein in vitro and in vivo, and report on its characterization. Endogenous VCP co-localized with expanded polyQ (ex-polyQ) aggregates in cultured cells expressing ex-polyQ, with nuclear inclusions in Huntington disease patient brains, and with Lewy bodies in patient samples. Moreover, the expression of VCP mutants with mutations in the 2nd ATP binding domain created cytoplasmic vacuoles, followed by cell death. Very similar vacuoles were also induced by ex-polyQ expression or proteasome inhibitor treatment. These results suggest that VCP functions not only as a recognition factor for abnormally folded proteins but also as a pathological effector for several neurodegenerative phenotypes. VCP may thus be an ideal molecular target for the treatment of neurodegenerative disorders.     

VCP Is an Integral Component of a Novel Feedback Mechanism that Controls Intracellular Localization of Catalase and H2O2 Levels

Catalase is a key antioxidant enzyme that catalyzes the decomposition of hydrogen peroxide (H₂O₂) to water and oxygen, and it appears to shuttle between the cytoplasm and peroxisome via unknown mechanisms. Valosin-containing protein (VCP) belongs to the AAA class of ATPases and is involved in diverse cellular functions, e.g. cell cycle and protein degradation, etc. Here we show that VCP and PEX19, a protein essential for peroxisome biogenesis, interact with each other. Knockdown of either VCP or PEX19 resulted in a predominantly cytoplasmic redistribution of catalase, and loss of VCP ATPase activity also increased its cytoplasmic redistribution. Moreover, VCP knockdown decreased intracellular ROS levels in normal and H₂O₂-treated cells, and an oxidation-resistant VCP impaired the ROS-induced cytoplasmic redistribution of catalase. These observations reveal a novel feedback mechanism, in which VCP can sense H₂O₂ levels, and regulates them by controlling the localization of catalase.     

Flavivirus recruits the valosin-containing protein–NPL4 complex to induce stress granule disassembly for efficient viral genome replication

Flaviviruses are human pathogens that can cause severe diseases, such as dengue fever and Japanese encephalitis, which can lead to death. Valosin-containing protein (VCP)/p97, a cellular ATPase associated with diverse cellular activities (AAA-ATPase), is reported to have multiple roles in flavivirus replication. Nevertheless, the importance of each role still has not been addressed. In this study, the functions of 17 VCP mutants that are reportedly unable to interact with the VCP cofactors were validated using the short-interfering RNA rescue experiments. Our findings of this study suggested that VCP exerts its functions in replication of the Japanese encephalitis virus by interacting with the VCP cofactor nuclear protein localization 4 (NPL4). We show that the depletion of NPL4 impaired the early stage of viral genome replication. In addition, we demonstrate that the direct interaction between NPL4 and viral nonstructural protein (NS4B) is critical for the translocation of NS4B to the sites of viral replication. Finally, we found that Japanese encephalitis virus and dengue virus promoted stress granule formation only in VCP inhibitor-treated cells and the expression of NS4B or VCP attenuated stress granule formation mediated by protein kinase R, which is generally known to be activated by type I interferon and viral genome RNA. These results suggest that the NS4B-mediated recruitment of VCP to the virus replication site inhibits cellular stress responses and consequently facilitates viral protein synthesis in the flavivirus-infected cells.     

Mechanisms of Cdc48/VCP-mediated cell death: from yeast apoptosis to human disease

The evolutionary conserved protein Cdc48/VCP is involved in various cellular processes, such as protein degradation, membrane fusion and chaperone activity. Increased levels of Cdc48/VCP correlate with cancer, whereas Cdc48/VCP at endogenous levels has been proposed to be a pathological effector in protein deposition diseases. Upon mutation Cdc48/VCP triggers the multisystem disorder 'inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia' (IBMPFD). The roles of Cdc48/VCP under these diverse pathological conditions, especially its function in decreased and increased incidences of cell death underlying these diseases, are poorly understood. Mutation of yeast CDC48 (cdc48(S565G)) results in yeast cells demonstrating morphological markers of apoptotic cell death. In other species it has been confirmed that mutations and depletion of Cdc48/VCP cause apoptosis, whereas increased levels of this protein provide an anti-apoptotic effect. This review critically compares mechanisms of Cdc48/VCP-mediated apoptosis observed in yeast and other species. Cdc48/VCP plays a triple role in cell death. At first, loss-of-function of Cdc48/VCP due to mutation or depletion causes ER stress and oxidative stress, triggering apoptosis. Secondly, upon exogenously applied ER stress functional Cdc48/VCP is important in the processing of caspases and plays therewith a pro-apoptotic role. Finally, Cdc48/VCP protects cells from apoptosis through mediating and activating pro-survival signaling pathways, namely Akt and NFkappaB signaling. This complex role in cell death pathways could correspond with the various pathophysiological conditions Cdc48/VCP is involved in.     

Recent advances in p97/VCP/Cdc48 cellular functions

p97/VCP/Cdc48 is one of the best-characterized type II AAA (ATPases associated with diverse cellular activities) ATPases. p97 is suggested to be a ubiquitin-selective chaperone and its key function is to disassemble protein complexes. p97 is involved in a wide variety of cellular activities. Recently, novel functions, namely autophagy and mitochondrial quality control, for p97 have been uncovered. p97 was identified as a causative factor for inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD) and more recently as a causative factor for amyotrophic lateral sclerosis (ALS). In this review, we will summarize and discuss recent progress and topics in p97 functions and the relationship to its associated diseases.     

Structural insight into mutations at 155 position of valosin containing protein (VCP) linked to inclusion body myopathy with Paget disease of bone and frontotemporal Dementia

Mutations in Valosin-containing protein (VCP) have been implicated in the pathology linked to inclusion body myopathy, paget disease of bone and frontotemporal dementia (IBMPFD). VCP is an essential component of AAA-ATPase superfamily involved in various cellular functions. Advanced In-silico analysis was performed using prediction based servers to determine the most deleterious mutation. Molecular dynamics simulation was used to study the protein dynamics at atomic level. Molecular docking was used to study the effect of mutation on ATP/ADP transition in the kinase domain. This ATPase of 806 amino acids has four domains: N-terminal domain, C-terminal domain and two ATPase domains D1 and D2 and each of these domains have a distinct role in its functioning. The mutations in VCP protein are distributed among regions known as hotspots, one such hotspot is codon 155. Three missense mutations reported in this hotspot are R155C, R155H and R155P. Potentiality of the deleteriousness calculated using server based prediction models reveal R155C mutation to be the most deleterious. The atomic insight into the effect of mutation by molecular dynamics simulation revealed major conformational changes in R155C variants ATP binding site in D1 domain. The nucleotide-binding mode at the catalytic pocket of VCP and its three variants at codon 155 showed change in the structure, which affects the ATP–ADP transition kinetics in all the three variants.     

Endolysosomal sorting of ubiquitylated caveolin-1 is regulated by VCP and UBXD1 and impaired by VCP disease mutations

The AAA-ATPase VCP (also known as p97) cooperates with distinct cofactors to process ubiquitylated proteins in different cellular pathways. VCP missense mutations cause a systemic degenerative disease in humans, but the molecular pathogenesis is unclear. We used an unbiased mass spectrometry approach and identified a VCP complex with the UBXD1 cofactor, which binds to the plasma membrane protein caveolin-1 (CAV1) and whose formation is specifically disrupted by disease-associated mutations. We show that VCP-UBXD1 targets mono-ubiquitylated CAV1 in SDS-resistant high-molecular-weight complexes on endosomes, which are en route to degradation in endolysosomes. Expression of VCP mutant proteins, chemical inhibition of VCP, or siRNA-mediated depletion of UBXD1 leads to a block of CAV1 transport at the limiting membrane of enlarged endosomes in cultured cells. In patient muscle, muscle-specific caveolin-3 accumulates in sarcoplasmic pools and specifically delocalizes from the sarcolemma. These results extend the cellular functions of VCP to mediating sorting of ubiquitylated cargo in the endocytic pathway and indicate that impaired trafficking of caveolin may contribute to pathogenesis in individuals with VCP mutations.     

Loss of the novel Vcp (valosin containing protein) interactor Washc4 interferes with autophagy-mediated proteostasis in striated muscle and leads to myopathy in vivo

VCP/p97 (valosin containing protein) is a key regulator of cellular proteostasis. It orchestrates protein turnover and quality control in vivo, processes fundamental for proper cell function. In humans, mutations in VCP lead to severe myo- and neuro-degenerative disorders such as inclusion body myopathy with Paget disease of the bone and frontotemporal dementia (IBMPFD), amyotrophic lateral sclerosis (ALS) or and hereditary spastic paraplegia (HSP). We analyzed here the in vivo role of Vcp and its novel interactor Washc4/Swip (WASH complex subunit 4) in the vertebrate model zebrafish (Danio rerio). We found that targeted inactivation of either Vcp or Washc4, led to progressive impairment of cardiac and skeletal muscle function, structure and cytoarchitecture without interfering with the differentiation of both organ systems. Notably, loss of Vcp resulted in compromised protein degradation via the proteasome and the macroautophagy/autophagy machinery, whereas Washc4 deficiency did not affect the function of the ubiquitin-proteasome system (UPS) but caused ER stress and interfered with autophagy function in vivo. In summary, our findings provide novel insights into the in vivo functions of Vcp and its novel interactor Washc4 and their particular and distinct roles during proteostasis in striated muscle cells.     

Exosome isolation from hemolymph of Korean rhinoceros beetle,Allomyrina dichotoma (Coleoptera: Scarabaeidae)

Exosomes are 30–150 nm vesicles that are secreted from a range of cells. Recently, exosomes have been the subject of considerable research because there is mounting awareness of their diverse functions, including a role in cell–cell communication and presenting pathogens for immune responses. Exosomes contain diverse nucleic acid and protein cargos, derived not only from the organism but also from pathogens, making them suitable for use in disease diagnosis. The Korean rhinoceros beetle, Allomyrina dichotoma (Coleoptera: Scarabaeidae), is commercially reared in Korea for the pet trade and is used in traditional medicine for liver‐related diseases. However, several insect diseases caused by bacteria, fungi and viruses have been reported in A. dichotoma mass‐rearing facilities. Identifying these diseases with accuracy and in a timely manner is of paramount importance. Such diagnosis can be accomplished by identifying the nucleic acid or amino acid fragments from these disease‐causing pathogens in the exosome of A. dichotoma. We isolated exosomes from the hemolymph of A. dichotoma and used them to analyze exosome RNA and proteins. We confirmed the isolation of exosomes through RNA profiling, protein analysis and Western blotting. Our research established a solid foundation for using insect exosome protein and RNA analyses for the accurate diagnosis of insect diseases. To our knowledge, this is the first report of exosome isolation from insect hemolymph.     

Rescue of growth defects of yeast cdc48 mutants by pathogenic IBMPFD-VCPs

VCP/p97/Cdc48 is a hexameric ring-shaped AAA ATPase that participates in a wide variety of cellular functions. VCP is a very abundant protein in essentially all types of cells and is highly conserved among eukaryotes. To date, 19 different single amino acid-substitutions in VCP have been reported to cause IBMPFD (inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia), an autosomal dominant inherited human disease. Moreover, several similar single amino acid substitutions have been proposed to associate with a rare subclass of familial ALS. The mechanisms by which these mutations contribute to the pathogenesis are unclear. To elucidate potential functional differences between wild-type and pathogenic VCPs, we expressed both VCPs in yeast cdc48 mutants. We observed that all tested pathogenic VCPs suppressed the temperature-sensitive phenotype of cdc48 mutants more efficiently than wild-type VCP. In addition, pathogenic VCPs, but not wild-type VCP, were able to rescue a lethal cdc48 disruption. In yeast, pathogenic VCPs, but not wild-type VCP, formed apparent cytoplasmic foci, and these foci were transported to budding sites by the Myo2/actin-mediated transport machinery. The foci formation of pathogenic VCPs appeared to be associated with their suppression of the temperature-sensitive phenotype of cdc48 mutants. These results support the idea that the pathogenic VCP mutations create dominant gain-of-functions rather than a simple loss of functional VCP. Their unique properties in yeast could provide a convenient drug-screening system for the treatment of these diseases.     

Molecular perspectives on p97-VCP: progress in understanding its structure and diverse biological functions

The 97-kDa valosin-containing protein (p97 or VCP) is a type-II AAA ( ATPases associated with a variety of activities) ATPases, which are characterized by possessing two conserved ATPase domains. VCP forms a stable homo-hexameric structure, and this two-tier ring-shaped complex acts as a molecular chaperone that mediates many seemingly unrelated cellular activities. The involvement of VCP in the ubiquitin-proteasome degradation pathway and the identification of VCP cofactors provided us important clues to the understanding of how this molecular chaperone works. In this review, we summarize the reported biological functions of VCP and explore the molecular mechanisms underlying the diverse cellular functions. We discuss the structural and biochemical studies, and elucidate how this sophisticated enzymatic machine converts chemical energy into the mechanical forces required for the chaperone activity.     

The Homozygote VCPR155H/R155H Mouse Model Exhibits Accelerated Human VCP-Associated Disease Pathology

Valosin containing protein (VCP) mutations are the cause of hereditary inclusion body myopathy, Paget''s disease of bone, frontotemporal dementia (IBMPFD). VCP gene mutations have also been linked to 2% of isolated familial amyotrophic lateral sclerosis (ALS). VCP is at the intersection of disrupted ubiquitin proteasome and autophagy pathways, mechanisms responsible for the intracellular protein degradation and abnormal pathology seen in muscle, brain and spinal cord. We have developed the homozygous knock-in VCP mouse (VCP^R155H/R155H) model carrying the common R155H mutations, which develops many clinical features typical of the VCP-associated human diseases. Homozygote VCP^R155H/R155H mice typically survive less than 21 days, exhibit weakness and myopathic changes on EMG. MicroCT imaging of the bones reveal non-symmetrical radiolucencies of the proximal tibiae and bone, highly suggestive of PDB. The VCP^R155H/R155H mice manifest prominent muscle, heart, brain and spinal cord pathology, including striking mitochondrial abnormalities, in addition to disrupted autophagy and ubiquitin pathologies. The VCP^R155H/R155H homozygous mouse thus represents an accelerated model of VCP disease and can be utilized to elucidate the intricate molecular mechanisms involved in the pathogenesis of VCP-associated neurodegenerative diseases and for the development of novel therapeutic strategies.     

Torsins: not your typical AAA+ ATPases

Abstract

Torsin ATPases (Torsins) belong to the widespread AAA+ (ATPases associated with a variety of cellular activities) family of ATPases, which share structural similarity but have diverse cellular functions. Torsins are outliers in this family because they lack many characteristics of typical AAA+ proteins, and they are the only members of the AAA+ family located in the endoplasmic reticulum and contiguous perinuclear space. While it is clear that Torsins have essential roles in many, if not all metazoans, their precise cellular functions remain elusive. Studying Torsins has significant medical relevance since mutations in Torsins or Torsin-associated proteins result in a variety of congenital human disorders, the most frequent of which is early-onset torsion (DYT1) dystonia, a severe movement disorder. A better understanding of the Torsin system is needed to define the molecular etiology of these diseases, potentially enabling corrective therapy. Here, we provide a comprehensive overview of the Torsin system in metazoans, discuss functional clues obtained from various model systems and organisms and provide a phylogenetic and structural analysis of Torsins and their regulatory cofactors in relation to disease-causative mutations. Moreover, we review recent data that have led to a dramatically improved understanding of these machines at a molecular level, providing a foundation for investigating the molecular defects underlying the associated movement disorders. Lastly, we discuss our ideas on how recent progress may be utilized to inform future studies aimed at determining the cellular role(s) of these atypical molecular machines and their implications for dystonia treatment options.     

Pathogenic VCP/TER94 alleles are dominant actives and contribute to neurodegeneration by altering cellular ATP level in a Drosophila IBMPFD model

Inclusion body myopathy with Paget's disease of bone and frontotemporal dementia (IBMPFD) is caused by mutations in Valosin-containing protein (VCP), a hexameric AAA ATPase that participates in a variety of cellular processes such as protein degradation, organelle biogenesis, and cell-cycle regulation. To understand how VCP mutations cause IBMPFD, we have established a Drosophila model by overexpressing TER94 (the sole Drosophila VCP ortholog) carrying mutations analogous to those implicated in IBMPFD. Expression of these TER94 mutants in muscle and nervous systems causes tissue degeneration, recapitulating the pathogenic phenotypes in IBMPFD patients. TER94-induced neurodegenerative defects are enhanced by elevated expression of wild-type TER94, suggesting that the pathogenic alleles are dominant active mutations. This conclusion is further supported by the observation that TER94-induced neurodegenerative defects require the formation of hexamer complex, a prerequisite for a functional AAA ATPase. Surprisingly, while disruptions of the ubiquitin-proteasome system (UPS) and the ER-associated degradation (ERAD) have been implicated as causes for VCP-induced tissue degeneration, these processes are not significantly affected in our fly model. Instead, the neurodegenerative defect of TER94 mutants seems sensitive to the level of cellular ATP. We show that increasing cellular ATP by independent mechanisms could suppress the phenotypes of TER94 mutants. Conversely, decreasing cellular ATP would enhance the TER94 mutant phenotypes. Taken together, our analyses have defined the nature of IBMPFD-causing VCP mutations and made an unexpected link between cellular ATP level and IBMPFD pathogenesis.     

Specific Inhibition of p97/VCP ATPase and Kinetic Analysis Demonstrate Interaction between D1 and D2 ATPase Domains

The p97 AAA (ATPase associated with diverse cellular activities), also called VCP (valosin-containing protein), is an important therapeutic target for cancer and neurodegenerative diseases. p97 forms a hexamer composed of two AAA domains (D1 and D2) that form two stacked rings and an N-terminal domain that binds numerous cofactor proteins. The interplay between the three domains in p97 is complex, and a deeper biochemical understanding is needed in order to design selective p97 inhibitors as therapeutic agents. It is clear that the D2 ATPase domain hydrolyzes ATP in vitro, but whether D1 contributes to ATPase activity is controversial. Here, we use Walker A and B mutants to demonstrate that D1 is capable of hydrolyzing ATP and show for the first time that nucleotide binding in the D2 domain increases the catalytic efficiency (k_cat/K_m) of D1 ATP hydrolysis 280-fold, by increasing k_cat 7-fold and decreasing K_m about 40-fold. We further show that an ND1 construct lacking D2 but including the linker between D1 and D2 is catalytically active, resolving a conflict in the literature. Applying enzymatic observations to small-molecule inhibitors, we show that four p97 inhibitors (DBeQ, ML240, ML241, and NMS-873) have differential responses to Walker A and B mutations, to disease-causing IBMPFD mutations, and to the presence of the N domain binding cofactor protein p47. These differential effects provide the first evidence that p97 cofactors and disease mutations can alter p97 inhibitor potency and suggest the possibility of developing context-dependent inhibitors of p97.     

EWSR1, a multifunctional protein,regulates cellular function and aging via genetic and epigenetic pathways

Ewing's sarcoma (EWS) is a bone cancer arising predominantly in young children. EWSR1 (Ewing Sarcoma breakpoint region 1/EWS RNA binding protein 1) gene is ubiquitously expressed in most cell types, indicating it has diverse roles in various cellular processes and organ development. Recently, several studies have shown that missense mutations of EWSR1 genes are known to be associated with central nervous system disorders such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Otherwise, EWSR1 plays epigenetic roles in gene expression, RNA processing, and cellular signal transduction. Interestingly, EWSR1 controls micro RNA (miRNA) levels via Drosha, leading to autophagy dysfunction and impaired dermal development. Ewsr1 deficiency also leads to premature senescence of blood cells and gamete cells with a high rate of apoptosis due to the abnormal meiosis. Despite these roles of EWSR1 in various cellular functions, the exact mechanisms are not yet understood. In this context, the current review overviews a large body of evidence and discusses on what EWSR1 genetic mutations are associated with brain diseases and on how EWSR1 modulates cellular function via the epigenetic pathway. This will provide a better understanding of bona fide roles of EWSR1 in aging and its association with brain disorders.     

VPS13D interacts with VCP/p97 and negatively regulates endoplasmic reticulum–mitochondria interactions

Membrane contact sites (MCSs) between the endoplasmic reticulum (ER) and mitochondria are emerging as critical hubs for diverse cellular events, and alterations in the extent of these contacts are linked to neurodegenerative diseases. However, the mechanisms that control ER–mitochondria interactions are so far elusive. Here, we demonstrate a key role of vacuolar protein sorting–associated protein 13D (VPS13D) in the negative regulation of ER–mitochondria MCSs. VPS13D suppression results in extensive ER–mitochondria tethering, a phenotype that can be substantially rescued by suppression of the tethering proteins VAPB and PTPIP51. VPS13D interacts with valosin-containing protein (VCP/p97) to control the level of ER-resident VAPB at contacts. VPS13D is required for the stability of p97. Functionally, VPS13D suppression leads to severe defects in mitochondrial morphology, mitochondrial cellular distribution, and mitochondrial DNA synthesis. Together, our results suggest that VPS13D negatively regulates the ER–mitochondria MCSs, partially through its interactions with VCP/p97.     

The slow Wallerian degeneration protein, WldS, binds directly to VCP/p97 and partially redistributes it within the nucleus

Slow Wallerian degeneration (Wld(S)) mutant mice express a chimeric nuclear protein that protects sick or injured axons from degeneration. The C-terminal region, derived from NAD(+) synthesizing enzyme Nmnat1, is reported to confer neuroprotection in vitro. However, an additional role for the N-terminal 70 amino acids (N70), derived from multiubiquitination factor Ube4b, has not been excluded. In wild-type Ube4b, N70 is part of a sequence essential for ubiquitination activity but its role is not understood. We report direct binding of N70 to valosin-containing protein (VCP; p97/Cdc48), a protein with diverse cellular roles including a pivotal role in the ubiquitin proteasome system. Interaction with Wld(S) targets VCP to discrete intranuclear foci where ubiquitin epitopes can also accumulate. Wld(S) lacking its N-terminal 16 amino acids (N16) neither binds nor redistributes VCP, but continues to accumulate in intranuclear foci, targeting its intrinsic NAD(+) synthesis activity to these same foci. Wild-type Ube4b also requires N16 to bind VCP, despite a more C-terminal binding site in invertebrate orthologues. We conclude that N-terminal sequences of Wld(S) protein influence the intranuclear location of both ubiquitin proteasome and NAD(+) synthesis machinery and that an evolutionary recent sequence mediates binding of mammalian Ube4b to VCP.     

The determination of the relationship between p97/VCP,small VCP-interacting protein,two ERAD proteins and steroidogenesis in Leydig cell lines

Background

In recent studies, it was shown that Endoplasmic reticulum-associated degradation (ERAD) is regulated by androgens and small VCP-interacting protein (SVIP) is an ERAD inhibitor. There is no data available about the interactions of ERAD proteins with proteins involved in steroidogenesis. The aim of the study was to investigate the expressions of SVIP, p97/VCP, StAR, CYP17A1 and 3β-HSD in human and mouse.

Methods and results

HLC, TM3 and MA-10 Leydig cell lines were used to determine roles of ERAD proteins in steroidogenesis based on immunofluorescence, Western blot, qRT-PCR, ELISA. Findings showed that StAR, CYP17A1 and 3β-HSD were colocalized with SVIP and p97/VCP in Leydig cells. A decrease in CYP17A1, 3β-HSD and StAR expressions was observed as a result of suppression of SVIP siRNAs and p97/VCP siRNAs expressions in MA10, TM3 and HLC. When siSVIP transfected cells were compared with siSVIP transfected with hCG-exposed cells, SVIP protein expression was significantly increased as compared to the SVIP transfected group in human Leydig cells.

Conclusion

We suggest that the suppression of protein expressions by p97/VCP and SVIP siRNAs in Leydig cells, the effects of proteins involved in steroidogenesis (StAR, CYP17A1 and 3β-HSD) have proven to be originating from p97/VCP and SVIP which were playing a role in the steroidogenesis process. Additionally, it was demonstrated that testosterone levels decreased after transfection with p97/VCP siRNA and SVIP siRNA, p97/VCP and SVIP created an effect on testosterone synthesis while taking place in the steps of testosterone synthesis. Further, it was determined in the study that the SVIP was affected by hCG stimulations.