VCP/p97 mediates nuclear targeting of non-ER-imported prion protein to maintain proteostasis.

Mistargeting of secretory proteins in the cytosol can trigger their aggregation and subsequent proteostasis decline. We have identified a VCP/p97-dependent pathway that directs non-ER-imported prion protein (PrP) into the nucleus to prevent the formation of toxic aggregates in the cytosol. Upon impaired translocation into the ER, PrP interacts with VCP/p97, which facilitates nuclear import mediated by importin-ß. Notably, the cytosolic interaction of PrP with VCP/p97 and its nuclear import are independent of ubiquitination. In vitro experiments revealed that VCP/p97 binds non-ubiquitinated PrP and prevents its aggregation. Inhibiting binding of PrP to VCP/p97, or transient proteotoxic stress, promotes the formation of self-perpetuating and partially proteinase resistant PrP aggregates in the cytosol, which compromised cellular proteostasis and disrupted further nuclear targeting of PrP. In the nucleus, RNAs keep PrP in a soluble and non-toxic conformation. Our study revealed a novel ubiquitin-independent role of VCP/p97 in the nuclear targeting of non-imported secretory proteins and highlights the impact of the chemical milieu in triggering protein misfolding.

Bone scan findings of Paget's disease of bone in patients with VCP Multisystem Proteinopathy 1.

Multisystem Proteinopathy 1 (MSP1) disease is a rare genetic disorder caused by mutations in the Valosin-Containing Protein (VCP) gene with clinical features of inclusion body myopathy (IBM), frontotemporal dementia (FTD), and Paget's disease of bone (PDB). We performed bone scan imaging in twelve patients (6 females, 6 males) with confirmed VCP gene mutation six (50%) of which has myopathy alone, four (33%) with both PDB and myopathy, and two (15%) were presymptomatic carriers. We aim to characterize the PDB in diagnosed individuals, and potentially identify PDB in the myopathy and presymptomatic groups. Interestingly, two patients with previously undiagnosed PDB had positive diagnostic findings on the bone scan and subsequent radiograph imaging. Among the individuals with PDB, increased radiotracer uptake of the affected bones were of typical distribution as seen in conventional PDB and those reported in other MSP1 cohorts which are the thoracic spine and ribs (75%), pelvis (75%), shoulder (75%) and calvarium (15%). Overall, we show that technetium-99m bone scans done at regular intervals are a sensitive screening tool in patients with MSP1 associated VCP variants at risk for PDB. However, diagnostic confirmation should be coupled with clinical history, biochemical analysis, and skeletal radiographs to facilitate early treatment and prevention complications, acknowledging its limited specificity.

VCF1 is a p97/VCP cofactor promoting recognition of ubiquitylated p97-UFD1-NPL4 substrates.

The hexameric AAA+ ATPase p97/VCP functions as an essential mediator of ubiquitin-dependent cellular processes, extracting ubiquitylated proteins from macromolecular complexes or membranes by catalyzing their unfolding. p97 is directed to ubiquitylated client proteins via multiple cofactors, most of which interact with the p97 N-domain. Here, we discover that FAM104A, a protein of unknown function also named VCF1 (VCP/p97 nuclear Cofactor Family member 1), acts as a p97 cofactor in human cells. Detailed structure-function studies reveal that VCF1 directly binds p97 via a conserved α-helical motif that recognizes the p97 N-domain with unusually high affinity, exceeding that of other cofactors. We show that VCF1 engages in joint p97 complex formation with the heterodimeric primary p97 cofactor UFD1-NPL4 and promotes p97-UFD1-NPL4-dependent proteasomal degradation of ubiquitylated substrates in cells. Mechanistically, VCF1 indirectly stimulates UFD1-NPL4 interactions with ubiquitin conjugates via its binding to p97 but has no intrinsic affinity for ubiquitin. Collectively, our findings establish VCF1 as an unconventional p97 cofactor that promotes p97-dependent protein turnover by facilitating p97-UFD1-NPL4 recruitment to ubiquitylated targets.

ASPSCR1-TFE3 reprograms transcription by organizing enhancer loops around hexameric VCP/p97.

The t(X,17) chromosomal translocation, generating the ASPSCR1::TFE3 fusion oncoprotein, is the singular genetic driver of alveolar soft part sarcoma (ASPS) and some Xp11-rearranged renal cell carcinomas (RCCs), frustrating efforts to identify therapeutic targets for these rare cancers. Here, proteomic analysis identifies VCP/p97, an AAA+ ATPase with known segregase function, as strongly enriched in co-immunoprecipitated nuclear complexes with ASPSCR1::TFE3. We demonstrate that VCP is a likely obligate co-factor of ASPSCR1::TFE3, one of the only such fusion oncoprotein co-factors identified in cancer biology. Specifically, VCP co-distributes with ASPSCR1::TFE3 across chromatin in association with enhancers genome-wide. VCP presence, its hexameric assembly, and its enzymatic function orchestrate the oncogenic transcriptional signature of ASPSCR1::TFE3, by facilitating assembly of higher-order chromatin conformation structures demonstrated by HiChIP. Finally, ASPSCR1::TFE3 and VCP demonstrate co-dependence for cancer cell proliferation and tumorigenesis in vitro and in ASPS and RCC mouse models, underscoring VCP's potential as a novel therapeutic target.

Characterizing ATP processing by the AAA+ protein p97 at the atomic level.

The human enzyme p97 regulates various cellular pathways by unfolding hundreds of protein substrates in an ATP-dependent manner, making it an essential component of protein homeostasis and an impactful pharmacological target. The hexameric complex undergoes substantial conformational changes throughout its catalytic cycle. Here we elucidate the molecular motions that occur at the active site in the temporal window immediately before and after ATP hydrolysis by merging cryo-EM, NMR spectroscopy and molecular dynamics simulations. p97 populates a metastable reaction intermediate, the ADP·Pi state, which is poised between hydrolysis and product release. Detailed snapshots reveal that the active site is finely tuned to trap and eventually discharge the cleaved phosphate. Signalling pathways originating at the active site coordinate the action of the hexamer subunits and couple hydrolysis with allosteric conformational changes. Our multidisciplinary approach enables a glimpse into the sophisticated spatial and temporal orchestration of ATP handling by a prototype AAA+ protein.

Bidirectional substrate shuttling between the 26S proteasome and the Cdc48 ATPase promotes protein degradation.

Most eukaryotic proteins are degraded by the 26S proteasome after modification with a polyubiquitin chain. Substrates lacking unstructured segments cannot be degraded directly and require prior unfolding by the Cdc48 ATPase (p97 or VCP in mammals) in complex with its ubiquitin-binding partner Ufd1-Npl4 (UN). Here, we use purified yeast components to reconstitute Cdc48-dependent degradation of well-folded model substrates by the proteasome. We show that a minimal system consists of the 26S proteasome, the Cdc48-UN ATPase complex, the proteasome cofactor Rad23, and the Cdc48 cofactors Ubx5 and Shp1. Rad23 and Ubx5 stimulate polyubiquitin binding to the 26S proteasome and the Cdc48-UN complex, respectively, allowing these machines to compete for substrates before and after their unfolding. Shp1 stimulates protein unfolding by the Cdc48-UN complex rather than substrate recruitment. Experiments in yeast cells confirm that many proteins undergo bidirectional substrate shuttling between the 26S proteasome and Cdc48 ATPase before being degraded.

Akt enhances the vulnerability of cancer cells to VCP/p97 inhibition-mediated paraptosis.

Valosin-containing protein (VCP)/p97, an AAA+ ATPase critical for maintaining proteostasis, emerges as a promising target for cancer therapy. This study reveals that targeting VCP selectively eliminates breast cancer cells while sparing non-transformed cells by inducing paraptosis, a non-apoptotic cell death mechanism characterized by endoplasmic reticulum and mitochondria dilation. Intriguingly, oncogenic HRas sensitizes non-transformed cells to VCP inhibition-mediated paraptosis. The susceptibility of cancer cells to VCP inhibition is attributed to the non-attenuation and recovery of protein synthesis under proteotoxic stress. Mechanistically, mTORC2/Akt activation and eIF3d-dependent translation contribute to translational rebound and amplification of proteotoxic stress. Furthermore, the ATF4/DDIT4 axis augments VCP inhibition-mediated paraptosis by activating Akt. Given that hyperactive Akt counteracts chemotherapeutic-induced apoptosis, VCP inhibition presents a promising therapeutic avenue to exploit Akt-associated vulnerabilities in cancer cells by triggering paraptosis while safeguarding normal cells.

Nationwide survey of patients with multisystem proteinopathy in Japan.

OBJECTIVE: Multisystem proteinopathy (MSP) is an inherited disorder in which protein aggregates with TAR DNA-binding protein of 43 kDa form in multiple organs. Mutations in VCP, HNRNPA2B1, HNRNPA1, SQSTM1, MATR3, and ANXA11 are causative for MSP. This study aimed to conduct a nationwide epidemiological survey based on the diagnostic criteria established by the Japan MSP study group. METHODS: We conducted a nationwide epidemiological survey by administering primary and secondary questionnaires among 6235 specialists of the Japanese Society of Neurology. RESULTS: In the primary survey, 47 patients with MSP were identified. In the secondary survey of 27 patients, inclusion body myopathy was the most common initial symptom (74.1%), followed by motor neuron disease (11.1%), frontotemporal dementia (FTD, 7.4%), and Paget's disease of bone (PDB, 7.4%), with no cases of parkinsonism. Inclusion body myopathy occurred most frequently during the entire course of the disease (81.5%), followed by motor neuron disease (25.9%), PDB (18.5%), FTD (14.8%), and parkinsonism (3.7%). Laboratory findings showed a high frequency of elevated serum creatine kinase levels and abnormalities on needle electromyography, muscle histology, brain magnetic resonance imaging, and perfusion single-photon emission computed tomography. INTERPRETATION: The low frequency of FTD and PDB may suggest that FTD and PDB may be widely underdiagnosed and undertreated in clinical practice.

Elevated 4R tau contributes to endolysosomal dysfunction and neurodegeneration in VCP-related frontotemporal dementia.

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are two incurable neurodegenerative diseases that exist on a clinical, genetic and pathological spectrum. The VCP gene is highly relevant, being directly implicated in both FTD and ALS. Here, we investigate the effects of VCP mutations on the cellular homoeostasis of human induced pluripotent stem cell-derived cortical neurons, focusing on endolysosomal biology and tau pathology. We found that VCP mutations cause abnormal accumulation of enlarged endolysosomes accompanied by impaired interaction between two nuclear RNA binding proteins: fused in sarcoma (FUS) and splicing factor, proline- and glutamine-rich (SFPQ) in human cortical neurons. The spatial dissociation of intranuclear FUS and SFPQ correlates with alternative splicing of the MAPT pre-mRNA and increased tau phosphorylation. Importantly, we show that inducing 4R tau expression using antisense oligonucleotide technology is sufficient to drive neurodegeneration in control human neurons, which phenocopies VCP-mutant neurons. In summary, our findings demonstrate that tau hyperphosphorylation, endolysosomal dysfunction, lysosomal membrane rupture, endoplasmic reticulum stress and apoptosis are driven by a pathogenic increase in 4R tau.

KUS121, a VCP modulator, has an ameliorating effect on acute and chronic heart failure without calcium loading via maintenance of intracellular ATP levels.

AIMS: As heart failure (HF) progresses, ATP levels in myocardial cells decrease, and myocardial contractility also decreases. Inotropic drugs improve myocardial contractility but increase ATP consumption, leading to poor prognosis. Kyoto University Substance 121 (KUS121) is known to selectively inhibit the ATPase activity of valosin-containing protein, maintain cellular ATP levels, and manifest cytoprotective effects in several pathological conditions. The aim of this study is to determine the therapeutic effect of KUS121 on HF models. METHODS AND RESULTS: Cultured cell, mouse, and canine models of HF were used to examine the therapeutic effects of KUS121. The mechanism of action of KUS121 was also examined. Administration of KUS121 to a transverse aortic constriction (TAC)-induced mouse model of HF rapidly improved the left ventricular ejection fraction and improved the creatine phosphate/ATP ratio. In a canine model of high frequency-paced HF, administration of KUS121 also improved left ventricular contractility and decreased left ventricular end-diastolic pressure without increasing the heart rate. Long-term administration of KUS121 to a TAC-induced mouse model of HF suppressed cardiac hypertrophy and fibrosis. In H9C2 cells, KUS121 reduced ER stress. Finally, in experiments using primary cultured cardiomyocytes, KUS121 improved contractility and diastolic capacity without changing peak Ca2+ levels or contraction time. These effects were not accompanied by an increase in cyclic adenosine monophosphate or phosphorylation of phospholamban and ryanodine receptors. CONCLUSIONS: KUS121 ameliorated HF by a mechanism totally different from that of conventional catecholamines. We propose that KUS121 is a promising new option for the treatment of HF.

VCIP135 associates with both the N- and C-terminal regions of p97 ATPase.

Two distinct p97ATPase-mediated membrane fusion pathways are required for Golgi and endoplasmic reticulum (ER) biogenesis, namely, the p97/p47 pathway and the p97/p37 pathway. p97 (VCP)/p47 complex-interacting protein p135 (VCIP135) is necessary for both of these pathways. Although VCIP135 is known to form a complex with p97 in the cytosol, the role of this complex in Golgi and ER biogenesis has remained unclear. In this study, we demonstrated that VCIP135 has two distinct p97-binding sites at its N- and C-terminal regions. In particular, the C-terminal binding site includes the SHP motif, which is also found in other p97-binding proteins, such as p47, p37, and Ufd1. We also clarified that VCIP135 binds to both the N- and C-terminal regions of p97; that is, the N- and C-terminal binding sites in VCIP135 interact with the C- and N-terminal regions of p97, respectively. These two interactions within the complex are synchronously controlled by the nucleotide state of p97. We next generated VCIP135 mutants lacking each of the p97-binding sites to investigate their functions in living cells and clarified that VCIP135 is involved in Golgi and ER biogenesis through its two distinct interactions with p97. VCIP135 is hence a unique p97-binding protein that functions by interacting with both the N-and C-terminal regions of p97, which strongly suggests that it plays crucial roles in p97-mediated events.

Proteomics analysis of serum and urine identifies VCP and CTSA as potential biomarkers associated with multiple myeloma.

AIMS: We analyzed the differentially expressed proteins (DEPs) in serum and urine in order to provide new potential biomarkers for MM. METHODS: Data-Independent Acquisition-based proteomics of serum and urine was performed to identify potential biomarkers for MM patients. Then we performed Western Blotting (WB), ELISA along with their ROC curve analysis to confirm DEPs. RESULTS: A total of 1653 proteins in serum and 4519 proteins in urine were identified using Data-Dependent Acquisition method. VCP was the only protein that showed significant differences in different comparison groups in both serum and urine. Pathway analysis revealed that protein processing in the endoplasmic reticulum was the most relevant pathway associated with MM. Furthermore, the increased expression of HSP90B1, VCP, CTSA, HYOU1, PDIA4, and RAB7A was detected by WB. The results of ELISA indicated that a combination of VCP and CTSA provided a high area under curve (AUC) value of 0.883 (95 % CI, 0.769-0.997, p < 0.001) to diagnose NDMM. The combination of VCP, CTSA, ALB, and HGB exhibited better performance (AUC = 0.981), with 100 % specificity and 86.7 % sensitivity. CONCLUSION: These findings suggest VCP and CTSA exhibit potential as biomarkers for MM, which may be helpful in the molecular mechanisms and pathogenesis upon further investigation.

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.

Adolescent-onset multisystem proteinopathy due to a novel VCP variant.

Valosin-containing protein (VCP) pathogenic variants are the most common cause of multisystem proteinopathy presenting with inclusion body myopathy, amyotrophic lateral sclerosis/frontotemporal dementia, and Paget disease of bone in isolation or in combination. We report a patient manifesting with adolescent-onset myopathy caused by a novel heterozygous VCP variant (c.467G > T, p.Gly156Val). The myopathy manifested asymmetrically in lower limbs and extended to proximal, axial, and upper limb muscles, with loss of ambulation at age 35. Creatine kinase value was normal. Alkaline phosphatase was elevated. Electromyography detected mixed low amplitude, short duration and high amplitude, long duration motor unit potentials. Muscle biopsy showed features of inclusion body myopathy, which in combination with newly diagnosed Paget disease of bone, supported the VCP variant pathogenicity. In conclusion, VCP-multisystem proteinopathy is not only a disease of adulthood but can have a pediatric onset and should be considered in differential diagnosis of neuromuscular weakness in the pediatric population.

XAF1 promotes colorectal cancer metastasis via VCP-RNF114-JUP axis.

Metastasis is the main cause of colorectal cancer (CRC)-related death, and the 5-year relative survival rate for CRC patients with distant metastasis is only 14%. X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1) is a zinc-rich protein belonging to the interferon (IFN)-induced gene family. Here, we report a metastasis-promoting role of XAF1 in CRC by acting as a novel adaptor of valosin-containing protein (VCP). XAF1 facilitates VCP-mediated deubiquitination of the E3 ligase RING finger protein 114 (RNF114), which promotes K48-linked ubiquitination and subsequent degradation of junction plakoglobin (JUP). The XAF1-VCP-RNF114-JUP axis is critical for the migration and metastasis of CRC cells. Moreover, we observe correlations between the protein levels of XAF1, RNF114, and JUP in clinical samples. Collectively, our findings reveal an oncogenic function of XAF1 in mCRC and suggest that the XAF1-VCP-RNF114-JUP axis is a potential therapeutic target for CRC treatment.

Betaine attenuates age-related suppression in autophagy via Mettl21c/p97/VCP axis to delay muscle loss.

Age-related impairment of autophagy accelerates muscle loss and lead to sarcopenia. Betaine can delay muscle loss as a dietary methyl donor via increasing S-adenosyl-L-methionine (SAM, a crucial metabolite for autophagy regulation) in methionion cycle. However, whether betaine can regulate autophagy level to attenuate degeneration in aging muscle remains unclear. Herein, male C57BL/6J young mice (YOU, 2-month-old), old mice (OLD, 15-month-old), and 2%-betaine-treated old mice (BET, 15-month-old) were employed and raised for 12 weeks. All mice underwent body composition examination and grip strength test before being sacrificed. Betaine alleviated age-related decline in muscle mass and strength. Meanwhile, betaine preserved the expression autophagy markers (Atg5, Atg7, LC3-II, and Beclin1) both at transcriptional and translational level during the aging process. RNA-sequencing results generated from mice gastrocnemius muscle found Mettl21c, a SAM-dependent autophagy-regulating methyltransferase, was significantly higher expressed in BET and YOU group. Results were further validated by qPCR and western bloting. In vitro, C2C12 cells with or without Mettl21c RNA interference were treated different concentration of betaine (0 mM, 10 mM) under methionine-starved condition. Compared with control group, betaine upregulated autophagy markers expression and autophagy flux. By increasing the SAM level, betaine facilitated trimethylation of p97 (Mettl21c downstream effector) into valosin-containing protein (VCP). Increased VCP promoted autophagic turnover of cellular components, ATP production, and cell differentiation. Knock-down of Metthl21c dismissed improvements mentioned above. Collectively, betaine could enhance aged skeletal muscle autophagy level via Mettl21c/p97/VCP axis to delay muscle loss.

Atypical K6-ubiquitin chains mobilize p97/VCP and the proteasome to resolve formaldehyde-induced RNA-protein crosslinks.

In this issue of Molecular Cell, Rahmanto et al.1 and Zhao et al.2 demonstrate that RNA-protein crosslinks contribute to formaldehyde toxicity by blocking protein synthesis. Furthermore, they identify a ubiquitin-mediated degradation system for RNA-protein crosslink resolution in eukaryotes.

Keratinocyte FABP5-VCP complex mediates recruitment of neutrophils in psoriasis.

One of the hallmarks of intractable psoriasis is neutrophil infiltration in skin lesions. However, detailed molecular mechanisms of neutrophil chemotaxis and activation remain unclear. Here, we demonstrate a significant upregulation of epidermal fatty acid binding protein (E-FABP, FABP5) in the skin of human psoriasis and psoriatic mouse models. Genetic deletion of FABP5 in mice by global knockout and keratinocyte conditional (Krt6a-Cre) knockout, but not myeloid cell conditional (LysM-Cre) knockout, attenuates psoriatic symptoms. Immunophenotypic analysis shows that FABP5 deficiency specifically reduces skin recruitment of Ly6G+ neutrophils. Mechanistically, activated keratinocytes produce chemokines and cytokines that trigger neutrophil chemotaxis and activation in an FABP5-dependent manner. Proteomic analysis further identifies that FABP5 interacts with valosin-containing protein (VCP), a key player in NF-κB signaling activation. Silencing of FABP5, VCP, or both inhibits NF-κB/neutrophil chemotaxis signaling. Collectively, these data demonstrate dysregulated FABP5 as a molecular mechanism promoting NF-κB signaling and neutrophil infiltration in psoriasis pathogenesis.

p97/VCP targets Toxoplasma gondii vacuoles for parasite restriction in interferon-stimulated human cells.

IMPORTANCE: Toxoplasma gondii (Tg) is a ubiquitous parasitic pathogen, infecting about one-third of the global population. Tg is controlled in immunocompetent people by mechanisms that are not fully understood. Tg infection drives the production of the inflammatory cytokine interferon gamma (IFNγ), which upregulates intracellular anti-pathogen defense pathways. In this study, we describe host proteins p97/VCP, UBXD1, and ANKRD13A that control Tg at the parasitophorous vacuole (PV) in IFNγ-stimulated endothelial cells. p97/VCP is an ATPase that interacts with a network of cofactors and is active in a wide range of ubiquitin-dependent cellular processes. We demonstrate that PV ubiquitination is a pre-requisite for recruitment of these host defense proteins, and their deposition directs Tg PVs to acidification in endothelial cells. We show that p97/VCP universally targets PVs in human cells and restricts Tg in different human cell types. Overall, these findings reveal new players of intracellular host defense of a vacuolated pathogen.