ALS-related p97 R155H mutation disrupts lysophagy in iPSC-derived motor neurons.

Mutations in the AAA+ ATPase p97 cause multisystem proteinopathy 1, which includes amyotrophic lateral sclerosis; however, the pathogenic mechanisms that contribute to motor neuron loss remain obscure. Here, we use two induced pluripotent stem cell models differentiated into spinal motor neurons to investigate how p97 mutations perturb the motor neuron proteome. Using quantitative proteomics, we find that motor neurons harboring the p97 R155H mutation have deficits in the selective autophagy of lysosomes (lysophagy). p97 R155H motor neurons are unable to clear damaged lysosomes and have reduced viability. Lysosomes in mutant motor neurons have increased pH compared with wild-type cells. The clearance of damaged lysosomes involves UBXD1-p97 interaction, which is disrupted in mutant motor neurons. Finally, inhibition of the ATPase activity of p97 using the inhibitor CB-5083 rescues lysophagy defects in mutant motor neurons. These results add to the evidence that endo-lysosomal dysfunction is a key aspect of disease pathogenesis in p97-related disorders.

UBXN1 maintains ER proteostasis and represses UPR activation by modulating translation.

ER protein homeostasis (proteostasis) is essential for proper folding and maturation of proteins in the secretory pathway. Loss of ER proteostasis can lead to the accumulation of misfolded or aberrant proteins in the ER and triggers the unfolded protein response (UPR). In this study, we find that the p97 adaptor UBXN1 is an important negative regulator of the UPR. Loss of UBXN1 sensitizes cells to ER stress and activates the UPR. This leads to widespread upregulation of the ER stress transcriptional program. Using comparative, quantitative proteomics we show that deletion of UBXN1 results in a significant enrichment of proteins involved in ER-quality control processes including those involved in protein folding and import. Notably, we find that loss of UBXN1 does not perturb p97-dependent ER-associated degradation (ERAD). Our studies indicate that loss of UBXN1 increases translation in both resting and ER-stressed cells. Surprisingly, this process is independent of p97 function. Taken together, our studies have identified a new role for UBXN1 in repressing translation and maintaining ER proteostasis in a p97 independent manner.

The p97-UBXD8 complex regulates ER-Mitochondria contact sites by altering membrane lipid saturation and composition.

The intimate association between the endoplasmic reticulum (ER) and mitochondrial membranes at ER-Mitochondria contact sites (ERMCS) is a platform for critical cellular processes, particularly lipid synthesis. How contacts are remodeled and the impact of altered contacts on lipid metabolism remains poorly understood. We show that the p97 AAA-ATPase and its adaptor ubiquitin-X domain adaptor 8 (UBXD8) regulate ERMCS. The p97-UBXD8 complex localizes to contacts and its loss increases contacts in a manner that is dependent on p97 catalytic activity. Quantitative proteomics and lipidomics of ERMCS demonstrates alterations in proteins regulating lipid metabolism and a significant change in membrane lipid saturation upon UBXD8 deletion. Loss of p97-UBXD8 increased membrane lipid saturation via SREBP1 and the lipid desaturase SCD1. Aberrant contacts can be rescued by unsaturated fatty acids or overexpression of SCD1. We find that the SREBP1-SCD1 pathway is negatively impacted in the brains of mice with p97 mutations that cause neurodegeneration. We propose that contacts are exquisitely sensitive to alterations to membrane lipid composition and saturation.

Cooperative assembly of p97 complexes involved in replication termination.

The p97 ATPase extracts polyubiquitylated proteins from diverse cellular structures in preparation for destruction by the proteasome. p97 functions with Ufd1-Npl4 and a variety of UBA-UBX co-factors, but how p97 complexes assemble on ubiquitylated substrates is unclear. To address this, we investigated how p97 disassembles the CMG helicase after it is ubiquitylated during replication termination. We show that p97Ufd1-Npl4 recruitment to CMG requires the UBA-UBX protein Ubxn7, and conversely, stable Ubxn7 binding to CMG requires p97Ufd1-Npl4. This cooperative assembly involves interactions between Ubxn7, p97, Ufd1-Npl4, and ubiquitin. Another p97 co-factor, Faf1, partially compensates for the loss of Ubxn7. Surprisingly, p97Ufd1-Npl4-Ubxn7 and p97Ufd1-Npl4-Faf1 also assemble cooperatively on unanchored ubiquitin chains. We propose that cooperative and substrate-independent recognition of ubiquitin chains allows p97 to recognize an unlimited number of polyubiquitylated proteins while avoiding the formation of partial, inactive complexes.

The functional importance of VCP to maintaining cellular protein homeostasis.

The AAA-ATPase (ATPases associated with diverse cellular activities) valosin-containing protein (VCP), is essential for many cellular pathways including but not limited to endoplasmic reticulum-associated degradation (ERAD), DNA damage responses, and cell cycle regulation. VCP primarily identifies ubiquitylated proteins in these pathways and mediates their unfolding and degradation by the 26S proteasome. This review summarizes recent research on VCP that has uncovered surprising new ways that this ATPase is regulated, new aspects of recognition of substrates and novel pathways and substrates that utilize its activity.

The Cure VCP Scientific Conference 2021: Molecular and clinical insights into neurodegeneration and myopathy linked to multisystem proteinopathy-1 (MSP-1).

The 2021 VCP Scientific Conference took place virtually from September 9-10, 2021. This conference, planned and organized by the nonprofit patient advocacy group Cure VCP Disease, Inc. (https://www.curevcp.org), was the first VCP focused meeting since the 215th ENMC International Workshop VCP-related multi-system proteinopathy in 2016 (Evangelista et al., 2016). Mutations in VCP cause a complex and heterogenous disease termed inclusion body myopathy (IBM) with Paget's disease of the bone (PDB) and frontotemporal dementia (FTD) (IBMPFD), or multisystem proteinopathy 1 (MSP-1) Kimonis (n.d.), Kovach et al. (2001), Kimonis et al. (2000). In addition, VCP mutations also cause other age-related neurodegenerative disorders including amyptrophic lateral sclerosis (ALS), Parkinsonism, Charcot-Marie type II-B, vacuolar tauopathy among others (Korb et al., 2022). The objectives of this conference were as follows: (1) to provide a forum that facilitates sharing of published and unpublished information on physiological roles of p97/VCP, and on how mutations of VCP lead to diseases; (2) to bolster understanding of mechanisms involved in p97/VCP-relevant diseases and to enable identification of therapeutics to treat these conditions; (3) to identify gaps and barriers of further discoveries and translational research in the p97/VCP field; (4) to set a concrete basic and translational research agenda for future studies including crucial discussions on biomarker discoveries and patient longitudinal studies to facilitate near-term clinical trials; (5) to accelerate cross-disciplinary research collaborations among p97/VCP researchers; (6) to enable attendees to learn about new tools and reagents with the potential to facilitate p97/VCP research; (7) to assist trainees in propelling their research and to foster mentorship from leaders in the field; and (8) to promote diversity and inclusion of under-represented minorities in p97/VCP research as diversity is critically important for strong scientific research. Given the range of topics, the VCP Scientific Conference brought together over one hundred and forty individuals representing a diverse group of research scientists, trainees, medical practitioners, industry representatives, and patient advocates. Twenty-five institutions with individuals from thirteen countries attended this virtual meeting. In this report, we summarize the major topics presented at this conference by a range of experts.

The p97-UBXN1 complex regulates aggresome formation.

The recognition and disposal of misfolded proteins is essential for the maintenance of cellular homeostasis. Perturbations in the pathways that promote degradation of aberrant proteins contribute to a variety of protein aggregation disorders broadly termed proteinopathies. The AAA-ATPase p97 (also known as VCP), in combination with adaptor proteins, functions to identify ubiquitylated proteins and target them for degradation by the proteasome or through autophagy. Mutations in p97 cause multi-system proteinopathies; however, the precise defects underlying these disorders are unclear. Here, we systematically investigate the role of p97 and its adaptors in the process of formation of aggresomes, membrane-less structures containing ubiquitylated proteins that arise upon proteasome inhibition. We demonstrate that p97 mediates aggresome formation and clearance, and identify a novel role for the adaptor UBXN1 in the process of aggresome formation. UBXN1 is recruited to aggresomes, and UBXN1-knockout cells are unable to form aggresomes. Loss of p97-UBXN1 results in increased Huntingtin polyQ inclusion bodies both in mammalian cells and in a C. elegans model of Huntington's disease. Together, our results identify evolutionarily conserved roles for p97-UBXN1 in the disposal of protein aggregates.

The WD40-repeat protein WDR-48 promotes the stability of the deubiquitinating enzyme USP-46 by inhibiting its ubiquitination and degradation.

Ubiquitination is a reversible post-translational modification that has emerged as a critical regulator of synapse development and function. However, the mechanisms that regulate the deubiquitinating enzymes (DUBs) responsible for the removal of ubiquitin from target proteins are poorly understood. We have previously shown that the DUB ubiquitin-specific protease 46 (USP-46) removes ubiquitin from the glutamate receptor GLR-1 and regulates its trafficking and degradation in Caenorhabditis elegans We found that the WD40-repeat proteins WDR-20 and WDR-48 bind and stimulate the catalytic activity of USP-46. Here, we identified another mechanism by which WDR-48 regulates USP-46. We found that increased expression of WDR-48, but not WDR-20, promotes USP-46 abundance in mammalian cells in culture and in C. elegans neurons in vivo Inhibition of the proteasome increased USP-46 abundance, and this effect was nonadditive with increased WDR-48 expression. We found that USP-46 is ubiquitinated and that expression of WDR-48 reduces the levels of ubiquitin-USP-46 conjugates and increases the t1/2 of USP-46. A point-mutated WDR-48 variant that disrupts binding to USP-46 was unable to promote USP-46 abundance in vivo Finally, siRNA-mediated knockdown of wdr48 destabilizes USP46 in mammalian cells. Together, these results support a model in which WDR-48 binds and stabilizes USP-46 protein levels by preventing the ubiquitination and degradation of USP-46 in the proteasome. Given that a large number of USPs interact with WDR proteins, we propose that stabilization of DUBs by their interacting WDR proteins may be a conserved and widely used mechanism that controls DUB availability and function.

AggreCount: an unbiased image analysis tool for identifying and quantifying cellular aggregates in a spatially defined manner.

Protein quality control is maintained by a number of integrated cellular pathways that monitor the folding and functionality of the cellular proteome. Defects in these pathways lead to the accumulation of misfolded or faulty proteins that may become insoluble and aggregate over time. Protein aggregates significantly contribute to the development of a number of human diseases such as amyotrophic lateral sclerosis, Huntington's disease, and Alzheimer's disease. In vitro, imaging-based, cellular studies have defined key biomolecular components that recognize and clear aggregates; however, no unifying method is available to quantify cellular aggregates, limiting our ability to reproducibly and accurately quantify these structures. Here we describe an ImageJ macro called AggreCount to identify and measure protein aggregates in cells. AggreCount is designed to be intuitive, easy to use, and customizable for different types of aggregates observed in cells. Minimal experience in coding is required to utilize the script. Based on a user-defined image, AggreCount will report a number of metrics: (i) total number of cellular aggregates, (ii) percentage of cells with aggregates, (iii) aggregates per cell, (iv) area of aggregates, and (v) localization of aggregates (cytosol, perinuclear, or nuclear). A data table of aggregate information on a per cell basis, as well as a summary table, is provided for further data analysis. We demonstrate the versatility of AggreCount by analyzing a number of different cellular aggregates including aggresomes, stress granules, and inclusion bodies caused by huntingtin polyglutamine expansion.

The VCP-UBXN1 Complex Mediates Triage of Ubiquitylated Cytosolic Proteins Bound to the BAG6 Complex.

A balance between protein synthesis and degradation is necessary to maintain cellular homeostasis. Failure to triage aberrant proteins may result in their accumulation and aggregation in the cytosol. The valosin-containing protein (VCP)-BCL2-associated athanogene 6 (BAG6) complex facilitates a wide variety of ubiquitin-mediated quality control events at the endoplasmic reticulum (ER), both prior to ER translocation and during ER-associated degradation (ERAD). However, how ubiquitylated clients associated with BAG6 are recognized by VCP for proteasomal degradation is presently unknown. We have identified UBXN1 as the VCP adaptor in BAG6-dependent processes occurring prior to ER insertion but not during ERAD. The loss of VCP-UBXN1 results in the inappropriate stabilization of ubiquitylated BAG6 clients and their accumulation in insoluble aggregates and sensitizes cells to proteotoxic stress. Our results identify how VCP is specifically targeted to ubiquitylated substrates in the BAG6 triage pathway and suggest that the degradation of ubiquitylated clients by the proteasome is reliant on the association of UBXN1 with ubiquitylated substrates and the catalytic activity of VCP.

Systematic proteomics of the VCP-UBXD adaptor network identifies a role for UBXN10 in regulating ciliogenesis.

The AAA-ATPase VCP (also known as p97 or CDC48) uses ATP hydrolysis to 'segregate' ubiquitylated proteins from their binding partners. VCP acts through UBX-domain-containing adaptors that provide target specificity, but the targets and functions of UBXD proteins remain poorly understood. Through systematic proteomic analysis of UBXD proteins in human cells, we reveal a network of over 195 interacting proteins, implicating VCP in diverse cellular pathways. We have explored one such complex between an unstudied adaptor UBXN10 and the intraflagellar transport B (IFT-B) complex, which regulates anterograde transport into cilia. UBXN10 localizes to cilia in a VCP-dependent manner and both VCP and UBXN10 are required for ciliogenesis. Pharmacological inhibition of VCP destabilized the IFT-B complex and increased trafficking rates. Depletion of UBXN10 in zebrafish embryos causes defects in left-right asymmetry, which depends on functional cilia. This study provides a resource for exploring the landscape of UBXD proteins in biology and identifies an unexpected requirement for VCP-UBXN10 in ciliogenesis.

Landscape of the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization.

The PARKIN ubiquitin ligase (also known as PARK2) and its regulatory kinase PINK1 (also known as PARK6), often mutated in familial early-onset Parkinson's disease, have central roles in mitochondrial homeostasis and mitophagy. Whereas PARKIN is recruited to the mitochondrial outer membrane (MOM) upon depolarization via PINK1 action and can ubiquitylate porin, mitofusin and Miro proteins on the MOM, the full repertoire of PARKIN substrates--the PARKIN-dependent ubiquitylome--remains poorly defined. Here we use quantitative diGly capture proteomics (diGly) to elucidate the ubiquitylation site specificity and topology of PARKIN-dependent target modification in response to mitochondrial depolarization. Hundreds of dynamically regulated ubiquitylation sites in dozens of proteins were identified, with strong enrichment for MOM proteins, indicating that PARKIN dramatically alters the ubiquitylation status of the mitochondrial proteome. Using complementary interaction proteomics, we found depolarization-dependent PARKIN association with numerous MOM targets, autophagy receptors, and the proteasome. Mutation of the PARKIN active site residue C431, which has been found mutated in Parkinson's disease patients, largely disrupts these associations. Structural and topological analysis revealed extensive conservation of PARKIN-dependent ubiquitylation sites on cytoplasmic domains in vertebrate and Drosophila melanogaster MOM proteins. These studies provide a resource for understanding how the PINK1-PARKIN pathway re-sculpts the proteome to support mitochondrial homeostasis.

A genome-wide screen identifies p97 as an essential regulator of DNA damage-dependent CDT1 destruction.

Several proteins, including the replication licensing factor CDT1 and the histone methyltransferase SET8, are targeted for proteolysis during DNA replication and repair by the E3 ubiquitin ligase CRL4(CDT2). CRL4(CDT2) function is coupled to replication and repair because it only ubiquitinates substrates that associate with chromatin-bound PCNA. Here, we report a genome-wide siRNA screen that identifies multiple factors necessary for CDT1 destruction after UV irradiation. Among these, nucleotide excision repair factors promote CDT1 destruction due to a role in recruiting PCNA to damaged DNA. The COP9/Signalosome regulates CDT2 stability through CUL4 deneddylation. Finally, the p97 AAA(+)-ATPase and its cofactor UFD1 are required for proteasome-dependent removal of ubiquitinated CDT1 and SET8 from chromatin and their subsequent degradation both in vivo and in a Xenopus egg extract system in vitro. This study provides insight into and a resource for the further exploration of pathways that promote timely degradation of chromatin-associated CRL4(CDT2) substrates.

TAO kinases mediate activation of p38 in response to DNA damage.

Thousand and one amino acid (TAO) kinases are Ste20p-related MAP kinase kinase kinases (MAP3Ks) that activate p38 MAPK. Here we show that the TAO kinases mediate the activation of p38 in response to various genotoxic stimuli. TAO kinases are activated acutely by ionizing radiation, ultraviolet radiation, and hydroxyurea. Full-length and truncated fragments of dominant negative TAOs inhibit the activation of p38 by DNA damage. Inhibition of TAO expression by siRNA also decreases p38 activation by these agents. Cells in which TAO kinases have been knocked down are less capable of engaging the DNA damage-induced G2/M checkpoint and display increased sensitivity to IR. The DNA damage kinase ataxia telangiectasia mutated (ATM) phosphorylates TAOs in vitro; radiation induces phosphorylation of TAO on a consensus site for phosphorylation by the ATM protein kinase in cells; and TAO and p38 activation is compromised in cells from a patient with ataxia telangiectasia that lack ATM. These findings indicate that TAO kinases are regulators of p38-mediated responses to DNA damage and are intermediates in the activation of p38 by ATM.

TGF-beta regulation by Emilin1: new links in the etiology of hypertension.

Hypertension is a complex disease influenced by multiple genetic and environmental factors. The TGF-beta signaling pathway has a long recognized role in blood pressure homeostasis. In this issue of Cell, Zacchigna et al. (2006) report that the secreted protein Emilin1 is a negative regulator of TGF-beta signaling. Emilin1 knockout mice display elevated blood pressure due to increased TGF-beta signaling in the vasculature.

Crystal structure of the TAO2 kinase domain: activation and specificity of a Ste20p MAP3K.

TAO2 is a mitogen-activated protein kinase kinase kinase (MAP3K) that doubly phosphorylates and activates the MAP kinase kinases (MAP2Ks) MEK3 and MEK6. The structure of the kinase domain of TAO2 (1-320) has been solved in its phosphorylated active conformation. The structure, together with structure-based mutagenic analysis, reveals that positively charged residues in the substrate binding groove mediate the first step in the dual phosphorylation of MEK6, on the threonine residue in the motif DS*VAKT*I (*denotes phosphorylation site) of MEK6. TAO2 is a Ste20p homolog, and the structure of active TAO2, in comparison with that of low-activity p21-activated protein kinase (PAK1), a Ste20p-related MAP4K, reveals how this group of kinases is activated by phosphorylation. Finally, active TAO2 displays unusual interactions with ATP, involving, in part, a subgroup-specific C-terminal extension of TAO2. The observed interactions may be useful in making specific inhibitors of TAO kinases.

TAO (thousand-and-one amino acid) protein kinases mediate signaling from carbachol to p38 mitogen-activated protein kinase and ternary complex factors.

The TAO (for thousand-and-one amino acids) protein kinases activate p38 mitogen-activated protein (MAP) kinase cascades in vitro and in cells by phosphorylating the MAP/ERK kinases (MEKs) 3 and 6. We found that TAO2 activity was increased by carbachol and that carbachol and the heterotrimeric G protein Galphao could activate p38 in 293 cells. Using dominant interfering kinase mutants, we found that MEKs 3 and 6 and TAOs were required for p38 activation by carbachol or the constitutively active mutant GalphaoQ205L. To explore events downstream of TAOs, the effects of TAO2 on ternary complex factors (TCFs) were investigated. Transfection studies demonstrated that TAO2 stimulates phosphorylation of the TCF Elk1 on the major activating site, Ser383, and that TAO2 stimulates transactivation of Elk1 and the related TCF, Sap1. Reporter activity was reduced by the p38-selective inhibitor SB203580. Taken together, these studies suggest that TAO protein kinases relay signals from carbachol through heterotrimeric G proteins to the p38 MAP kinase, which then activates TCFs in the nucleus.