Mechanism of Lys6 poly-ubiquitin specificity by the L. pneumophila deubiquitinase LotA.

The versatility of ubiquitination to control vast domains of eukaryotic biology is due, in part, to diversification through differently linked poly-ubiquitin chains. Deciphering signaling roles for some chain types, including those linked via K6, has been stymied by a lack of specificity among the implicated regulatory proteins. Forged through strong evolutionary pressures, pathogenic bacteria have evolved intricate mechanisms to regulate host ubiquitin during infection. Herein, we identify and characterize a deubiquitinase domain of the secreted effector LotA from Legionella pneumophila that specifically regulates K6-linked poly-ubiquitin. We demonstrate the utility of LotA for studying K6 poly-ubiquitin signals. We identify the structural basis of LotA activation and poly-ubiquitin specificity and describe an essential "adaptive" ubiquitin-binding domain. Without LotA activity during infection, the Legionella-containing vacuole becomes decorated with K6 poly-ubiquitin as well as the AAA ATPase VCP/p97/Cdc48. We propose that LotA's deubiquitinase activity guards Legionella-containing vacuole components from ubiquitin-dependent extraction.

Ubiquitin profiling of lysophagy identifies actin stabilizer CNN2 as a target of VCP/p97 and uncovers a link to HSPB1.

Lysosomal membrane permeabilization (LMP) is an underlying feature of diverse conditions including neurodegeneration. Cells respond by extensive ubiquitylation of membrane-associated proteins for clearance of the organelle through lysophagy that is facilitated by the ubiquitin-directed AAA-ATPase VCP/p97. Here, we assessed the ubiquitylated proteome upon acute LMP and uncovered a large diversity of targets and lysophagy regulators. They include calponin-2 (CNN2) that, along with the Arp2/3 complex, translocates to damaged lysosomes and regulates actin filaments to drive phagophore formation. Importantly, CNN2 needs to be ubiquitylated during the process and removed by VCP/p97 for efficient lysophagy. Moreover, we identified the small heat shock protein HSPB1 that assists VCP/p97 in the extraction of CNN2 and show that other membrane regulators including SNAREs, PICALM, AGFG1, and ARL8B are ubiquitylated during lysophagy. Our data reveal a framework of how ubiquitylation and two effectors, VCP/p97 and HSPB1, cooperate to protect cells from the deleterious effects of LMP.

Ubiquitin-Independent Disassembly by a p97 AAA-ATPase Complex Drives PP1 Holoenzyme Formation.

The functional diversity of protein phosphatase-1 (PP1), with its countless substrates, relies on the ordered assembly of alternative PP1 holoenzymes. Here, we show that newly synthesized PP1 is first held by its partners SDS22 and inhibitor-3 (I3) in an inactive complex, which needs to be disassembled by the p97 AAA-ATPase to promote exchange to substrate specifiers. Unlike p97-mediated degradative processes that require the Ufd1-Npl4 ubiquitin adapters, p97 is targeted to PP1 by p37 and related adapter proteins. Reconstitution with purified components revealed direct interaction of the p37 SEP domain with I3 without the need for ubiquitination, and ATP-driven pulling of I3 into the central channel of the p97 hexamer, which triggers dissociation of I3 and SDS22. Thus, we establish regulatory ubiquitin-independent protein complex disassembly as part of the functional arsenal of p97 and define an unanticipated essential step in PP1 biogenesis that illustrates the molecular challenges of ordered subunit exchange.

Wnt-Dependent Inactivation of the Groucho/TLE Co-repressor by the HECT E3 Ubiquitin Ligase Hyd/UBR5.

Extracellular signals are transduced to the cell nucleus by effectors that bind to enhancer complexes to operate transcriptional switches. For example, the Wnt enhanceosome is a multiprotein complex associated with Wnt-responsive enhancers through T cell factors (TCF) and kept silent by Groucho/TLE co-repressors. Wnt-activated ß-catenin binds to TCF to overcome this repression, but how it achieves this is unknown. Here, we discover that this process depends on the HECT E3 ubiquitin ligase Hyd/UBR5, which is required for Wnt signal responses in Drosophila and human cell lines downstream of activated Armadillo/ß-catenin. We identify Groucho/TLE as a functionally relevant substrate, whose ubiquitylation by UBR5 is induced by Wnt signaling and conferred by ß-catenin. Inactivation of TLE by UBR5-dependent ubiquitylation also involves VCP/p97, an AAA ATPase regulating the folding of various cellular substrates including ubiquitylated chromatin proteins. Thus, Groucho/TLE ubiquitylation by Hyd/UBR5 is a key prerequisite that enables Armadillo/ß-catenin to activate transcription.

Valosin-Containing Protein (VCP)/p97 Oligomerization.

Valosin-containing protein (VCP), also known as p97, is an evolutionarily conserved AAA+ ATPase essential for cellular homeostasis. Cooperating with different sets of cofactors, VCP is involved in multiple cellular processes through either the ubiquitin-proteasome system (UPS) or the autophagy/lysosomal route. Pathogenic mutations frequently found at the interface between the NTD domain and D1 ATPase domain have been shown to cause malfunction of VCP, leading to degenerative disorders including the inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD), amyotrophic lateral sclerosis (ALS), and cancers. Therefore, VCP has been considered as a potential therapeutic target for neurodegeneration and cancer. Most of previous studies found VCP predominantly exists and functions as a hexamer, which unfolds and extracts ubiquitinated substrates from protein complexes for degradation. However, recent studies have characterized a new VCP dodecameric state and revealed a controlling mechanism of VCP oligomeric states mediated by the D2 domain nucleotide occupancy. Here, we summarize our recent knowledge on VCP oligomerization, regulation, and potential implications of VCP in cellular function and pathogenic progression.

Ubiquitin and SUMO as timers during DNA replication.

Every time a cell copies its DNA the genetic material is exposed to the acquisition of mutations and genomic alterations that corrupt the information passed on to daughter cells. A tight temporal regulation of DNA replication is necessary to ensure the full copy of the DNA while preventing the appearance of genomic instability. Protein modification by ubiquitin and SUMO constitutes a very complex and versatile system that allows the coordinated control of protein stability, activity and interactome. In chromatin, their action is complemented by the AAA+ ATPase VCP/p97 that recognizes and removes ubiquitylated and SUMOylated factors from specific cellular compartments. The concerted action of the ubiquitin/SUMO system and VCP/p97 determines every step of DNA replication enforcing the ordered activation/inactivation, loading/unloading and stabilization/destabilization of replication factors. Here we analyze the mechanisms used by ubiquitin/SUMO and VCP/p97 to establish molecular timers throughout DNA replication and their relevance in maintaining genome stability. We propose that these PTMs are the main molecular watch of DNA replication from origin recognition to replisome disassembly.

Tau accumulation is cleared by the induced expression of VCP via autophagy.

Tauopathy, including frontotemporal lobar dementia and Alzheimer's disease, describes a class of neurodegenerative diseases characterized by the aberrant accumulation of Tau protein due to defects in proteostasis. Upon generating and characterizing a stable transgenic zebrafish that expresses the human TAUP301L mutant in a neuron-specific manner, we found that accumulating Tau protein was efficiently cleared via an enhanced autophagy activity despite constant Tau mRNA expression; apparent tauopathy-like phenotypes were revealed only when the autophagy was genetically or chemically inhibited. We performed RNA-seq analysis, genetic knockdown, and rescue experiments with clinically relevant point mutations of valosin-containing protein (VCP), and showed that induced expression of VCP, an essential cytosolic chaperone for the protein quality system, was a key factor for Tau degradation via its facilitation of the autophagy flux. This novel function of VCP in Tau clearance was further confirmed in a tauopathy mouse model where VCP overexpression significantly decreased the level of phosphorylated and oligomeric/aggregate Tau and rescued Tau-induced cognitive behavioral phenotypes, which were reversed when the autophagy was blocked. Importantly, VCP expression in the brains of human Alzheimer's disease patients was severely downregulated, consistent with its proposed role in Tau clearance. Taken together, these results suggest that enhancing the expression and activity of VCP in a spatiotemporal manner to facilitate the autophagy pathway is a potential therapeutic approach for treating tauopathy.

Cross-sectional study of patients with VCP multisystem proteinopathy 1 using dual-energy x-ray absorptiometry.

INTRODUCTION/AIMS: VCP multisystem proteinopathy 1 (MSP1), encompassing inclusion body myopathy (IBM), Paget's disease of bone (PDB) and frontotemporal dementia (FTD) (IBMPFD), features progressive muscle weakness, fatty infiltration, and disorganized bone structure in Pagetic bones. The aim of this study is to utilize dual-energy x-ray absorptiometry (DXA) parameters to examine it as a biomarker of muscle and bone disease in MSP1. METHODS: DXA scans were obtained in 28 patients to assess body composition parameters (bone mineral density [BMD], T-score, total fat, and lean mass) across different groups: total VCP disease (n = 19), including myopathy without Paget's ("myopathy"; n = 12) and myopathy with Paget's ("Paget"; n = 7), and unaffected first-degree relatives serving as controls (n = 6). RESULTS: In the VCP disease group, significant declines in left hip BMD and Z-scores were noted versus the control group (p ≤ .03). The VCP disease group showed decreased whole body lean mass % (p = .04), and increased total body fat % (p = .04) compared to controls. Subgroup comparisons indicated osteopenia in 33.3% and osteoporosis in 8.3% of the myopathy group, with 14.3% exhibiting osteopenia in the Paget group. Moreover, the Paget group displayed higher lumbar L1-L4 T-score values than the myopathy group. DISCUSSION: In MSP1, DXA revealed reduced bone and lean mass, and increased fat mass. These DXA insights could aid in monitoring disease progression of muscle loss and secondary osteopenia/osteoporosis in MSP1, providing value both clinically and in clinical research.

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.

New Insights into Cardiovascular Diseases Treatment Based on Molecular Targets.

Cardiovascular diseases (CVDs) which consist of ischemic heart disease, stroke, heart failure, peripheral arterial disease, and several other cardiac and vascular conditions are one of the most common causes of death worldwide and often co-occur with diabetes mellitus and lipid disorders which worsens the prognosis and becomes a therapeutic challenge. Due to the increasing number of patients with CVDs, we need to search for new risk factors and pathophysiological changes to create new strategies for preventing, diagnosing, and treating not only CVDs but also comorbidities like diabetes mellitus and lipid disorders. As increasing amount of patients suffering from CVDs, there are many therapies which focus on new molecular targets like proprotein convertase subtilisin/kexin type 9 (PCSK9), angiopoietin-like protein 3, ATP-citrate lyase, or new technologies such as siRNA in treatment of dyslipidemia or sodium-glucose co-transporter-2 and glucagon-like peptide-1 in treatment of diabetes mellitus. Both SGLT-2 inhibitors and GLP-1 receptor agonists are used in the treatment of diabetes, however, they proved to have a beneficial effect in CVDs as well. Moreover, a significant amount of evidence has shown that exosomes seem to be associated with myocardial ischaemia and that exosome levels correlate with the severity of myocardial injury. In our work, we would like to focus on the above mechanisms. The knowledge of them allows for the appearance of new strategies of treatment among patients with CVDs.

VCP relocalization limits mitochondrial activity, GSH depletion and ferroptosis during starvation in PC3 prostate cancer cells.

During periods of crisis, cells must compensate to survive. To this end, cells may need to alter the subcellular localization of crucial proteins. Here, we show that during starvation, VCP, the most abundant soluble ATPase, relocalizes and forms aggregate-like structures at perinuclear regions in PC3 prostate cancer cells. This movement is associated with a lowered metabolic state, in which mitochondrial activity and ROS production are reduced. VCP appears to explicitly sense glutamine levels, as removal of glutamine from complete medium triggered VCP relocalization and its addition to starvation media blunted VCP relocalization. Cells cultured in Gln(+) starvation media exhibited uniformly distributed VCP in the cytoplasm (free VCP) and underwent ferroptotic cell death, which was associated with a decrease in GSH levels. Moreover, the addition of a VCP inhibitor, CB-5083, in starvation media prevented VCP relocalization and triggered ferroptotic cell death. Likewise, expression of GFP-fused VCP proteins, irrespective of ATPase activities, displayed free VCP and triggered cell death during starvation. These results indicate that free VCP is essential for the maintenance of mitochondrial function and that PC3 cells employ a strategy of VCP self-aggregation to suppress mitochondrial activity in order to escape cell death during starvation, a novel VCP-mediated survival mechanism.

Methyltransferase-like 21C (METTL21C) methylates alanine tRNA synthetase at Lys-943 in muscle tissue.

Protein-lysine methylation is a common posttranslational modification (PTM) throughout the human proteome that plays important roles in diverse biological processes. In humans, there are >100 known and candidate protein lysine methyltransferases (PKMTs), many of which are linked to human diseases. Methyltransferase-like protein 21C (METTL21C) is a PKMT implicated in muscle biology that has been reported to methylate valosin-containing protein/p97 (VCP) and heat shock 70-kDa protein 8 (HSPA8). However, a clear in vitro methyltransferase activity for METTL21C remains yet to be demonstrated, and whether it is an active enzyme that directly methylates substrate(s) in vivo is unclear. Here, we used an unbiased biochemistry-based screening assay coupled to MS, which identified alanine tRNA synthetase 1 (AARS1) as a direct substrate of METTL21C. We found that METTL21C catalyzes methylation of Lys-943 of AARS1 (AARS1-K943me) both in vitro and in vivoIn vitro METTL21C-mediated AARS1 methylation was independent of ATP or tRNA molecules. Unlike for AARS1, and in conflict with previous reports, we did not detect METTL21C methylation of VCP and HSPA8. AARS1-K943 methylation in HEK293T cells depends upon METTL21C levels. Finally, METTL2C was almost exclusively expressed in muscle tissue, and, accordingly, we detected METTL21C-catalyzed methylation of AARS1 in mouse skeletal muscle tissue. These results reveal that AARS1 is a bona fide in vitro substrate of METTL21C and suggest a role for the METTL21C-AARS1 axis in the regulation of protein synthesis in muscle tissue. Moreover, our study describes a straightforward protocol for elucidating the physiological substrates of poorly characterized or uncharacterized PKMTs.

Targeting the VCP-binding motif of ataxin-3 improves phenotypes in Drosophila models of Spinocerebellar Ataxia Type 3.

Of the family of polyglutamine (polyQ) neurodegenerative diseases, Spinocerebellar Ataxia Type 3 (SCA3) is the most common. Like other polyQ diseases, SCA3 stems from abnormal expansions in the CAG triplet repeat of its disease gene resulting in elongated polyQ repeats within its protein, ataxin-3. Various ataxin-3 protein domains contribute to its toxicity, including the valosin-containing protein (VCP)-binding motif (VBM). We previously reported that VCP, a homo-hexameric protein, enhances pathogenic ataxin-3 aggregation and exacerbates its toxicity. These findings led us to explore the impact of targeting the SCA3 protein by utilizing a decoy protein comprising the N-terminus of VCP (N-VCP) that binds ataxin-3's VBM. The notion was that N-VCP would reduce binding of ataxin-3 to VCP, decreasing its aggregation and toxicity. We found that expression of N-VCP in Drosophila melanogaster models of SCA3 ameliorated various phenotypes, coincident with reduced ataxin-3 aggregation. This protective effect was specific to pathogenic ataxin-3 and depended on its VBM. Increasing the amount of N-VCP resulted in further phenotype improvement. Our work highlights the protective potential of targeting the VCP-ataxin-3 interaction in SCA3, a key finding in the search for therapeutic opportunities for this incurable disorder.

The Human Cytomegalovirus Transmembrane Protein pUL50 Induces Loss of VCP/p97 and Is Regulated by a Small Isoform of pUL50.

The human cytomegalovirus (HCMV) UL50 gene encodes a transmembrane protein, pUL50, which acts as a core component of the nuclear egress complex (NEC) for nucleocapsids. Recently, pUL50 has been shown to have NEC-independent activities: downregulation of IRE1 to repress the unfolded protein response and degradation of UBE1L to inhibit the protein ISG15 modification pathway. Here, we demonstrate that a 26-kDa N-terminal truncated isoform of pUL50 (UL50-p26) is expressed from an internal methionine at amino acid position 199 and regulates the activity of pUL50 to induce the loss of valosin-containing protein (VCP/p97). A UL50(M199V) mutant virus expressing pUL50(M199V) but not UL50-p26 showed delayed growth at a low multiplicity of infection. There was also delayed accumulation of the viral immediate early 2 (IE2) protein in the mutant virus, and this correlated with the reduced expression of VCP/p97, which promotes IE2 expression. Infection with mutant virus did not significantly alter ISGylation levels. In transient expression assays, pUL50 induced VCP/p97 loss posttranscriptionally, and this was dependent on the presence of its transmembrane domain. In contrast, UL50-p26 did not destabilize VCP/p97 but, rather, inhibited pUL50-mediated VCP/p97 loss and the associated major IE gene suppression. Both pUL50 and UL50-p26 interacted with VCP/p97, although UL50-p26 did so more weakly than pUL50. UL50-p26 interacted with pUL50, and this interaction was much stronger than the pUL50 self-interaction. Furthermore, UL50-p26 was able to interfere with the pUL50-VCP/p97 interaction. Our study newly identifies UL50-p26 expression during HCMV infection and suggests a regulatory role for UL50-p26 in blocking pUL50-mediated VCP/p97 loss by associating with pUL50.IMPORTANCE Targeting the endoplasmic reticulum (ER) by viral proteins may affect ER-associated protein homeostasis. During human cytomegalovirus (HCMV) infection, pUL50 targets the ER through its transmembrane domain and moves to the inner nuclear membrane (INM) to form the nuclear egress complex (NEC), which facilitates capsid transport from the nucleus to the cytoplasm. Here, we demonstrate that pUL50 induces the loss of valosin-containing protein (VCP/p97), which promotes the expression of viral major immediate early gene products, in a manner dependent on its membrane targeting but that a small isoform of pUL50 is expressed to negatively regulate this pUL50 activity. This study reports a new NEC-independent function of pUL50 and highlights the fine regulation of pUL50 activity by a smaller isoform for efficient viral growth.

Emerging role of VCP/p97 in cardiovascular diseases: novel insights and therapeutic opportunities.

Valosin-containing protein (VCP/p97) is a member of the conserved type II AAA+ (ATPases associated with diverse cellular activities) family of proteins with multiple biological functions, especially in protein homeostasis. Mutations in VCP/p97 are reportedly related to unique autosomal dominant diseases, which may worsen cardiac function. Although the structure of VCP/p97 has been clearly characterized, with reports of high abundance in the heart, research focusing on the molecular mechanisms underpinning the roles of VCP/p97 in the cardiovascular system has been recently undertaken over the past decades. Recent studies have shown that VCP/p97 deficiency affects myocardial fibers and induces heart failure, while overexpression of VCP/p97 eliminates ischemia/reperfusion injury and relieves pathological cardiac hypertrophy caused by cardiac pressure overload, which is related to changes in the mitochondria and calcium overload. However, certain studies have drawn opposing conclusions, including the mitigation of ischemia/reperfusion injury via inhibition of VCP/p97 ATPase activity. Nevertheless, these emerging studies shed light on the role of VCP/p97 and its therapeutic potential in cardiovascular diseases. In other words, VCP/p97 may be involved in the development of cardiovascular disease, and is anticipated to be a new therapeutic target. This review summarizes current findings regarding VCP/p97 in the cardiovascular system for the first time, and discusses the role of VCP/p97 in cardiovascular disease.

VCP/p97 targets the nuclear export and degradation of p27Kip1 during G1 to S phase transition.

One of the critical regulatory mechanisms for cell cycle progression is the timely degradation of CDK inhibitors, including p21Cip1 and p27Kip1 . VCP/p97, an AAA-ATPase, is reported to be overexpressed in many types of cancers. Here, we found that treatment of MCF-7 human breast cancer cells with DBeQ, a VCP inhibitor, or VCP knockdown in MCF-7 cells arrested cells at G1 phase, accompanied with the blockage of both p21 and p27 degradation. Whereas, double knockdown of p21 and p27 in MCF-7 cells rendered cells refractory to DBeQ-induced G1 arrest. Moreover, inhibition or knockdown of VCP or UFD1, one of VCP's co-factors, in MCF-7, NIH3T3, or HEK293T cells blocked the nuclear export of p27 during earlier G1 phase after mitogen stimulation. We also identified the nuclear localization sequence (NLS) of VCP, and found that adding back wild-type VCP, not the NLS-deleted VCP mutant, restored the nuclear export and degradation of p27 in VCP knockout MCF-7 cells. Importantly, we found that VCP inhibition sensitized breast cancer cells to the treatment of several anticancer therapeutics both in vitro and in vivo. Taken together, our study not only uncovers the mechanisms underlying VCP-mediated cell proliferation control but also provides potential therapeutic option for cancer treatment.

Spatial proteomics reveal that the protein phosphatase PTP1B interacts with and may modify tyrosine phosphorylation of the rhomboid protease RHBDL4.

Rhomboid-like proteins are evolutionarily conserved, ubiquitous polytopic membrane proteins, including the canonical rhomboid intramembrane serine proteases and also others that have lost protease activity during evolution. We still have much to learn about their cellular roles, and evidence suggests that some may have more than one function. For example, RHBDL4 (rhomboid-like protein 4) is an endoplasmic reticulum (ER)-resident protease that forms a ternary complex with ubiquitinated substrates and p97/VCP (valosin-containing protein), a major driver of ER-associated degradation (ERAD). RHBDL4 is required for ERAD of some substrates, such as the pre-T-cell receptor α chain (pTα) and has also been shown to cleave amyloid precursor protein to trigger its secretion. In another case, RHBDL4 enables the release of full-length transforming growth factor α in exosomes. Using the proximity proteomic method BioID, here we screened for proteins that interact with or are in close proximity to RHBDL4. Bioinformatics analyses revealed that BioID hits of RHBDL4 overlap with factors related to protein stress at the ER, including proteins that interact with p97/VCP. PTP1B (protein-tyrosine phosphatase nonreceptor type 1, also called PTPN1) was also identified as a potential proximity factor and interactor of RHBDL4. Analysis of RHBDL4 peptides highlighted the presence of tyrosine phosphorylation at the cytoplasmic RHBDL4 C terminus. Site-directed mutagenesis targeting these tyrosine residues revealed that their phosphorylation modifies binding of RHBDL4 to p97/VCP and Lys63-linked ubiquitinated proteins. Our work lays a critical foundation for future mechanistic studies of the roles of RHBDL4 in ERAD and other important cellular pathways.

Methyltransferase-like 21e inhibits 26S proteasome activity to facilitate hypertrophy of type IIb myofibers.

Skeletal muscles contain heterogeneous myofibers that are different in size and contractile speed, with type IIb myofiber being the largest and fastest. Here, we identify methyltransferase-like 21e (Mettl21e), a member of newly classified nonhistone methyltransferases, as a gene enriched in type IIb myofibers. The expression of Mettl21e was strikingly up-regulated in hypertrophic muscles and during myogenic differentiation in vitro and in vivo. Knockdown (KD) of Mettl21e led to atrophy of cultured myotubes, and targeted mutation of Mettl21e in mice reduced the size of IIb myofibers without affecting the composition of myofiber types. Mass spectrometry and methyltransferase assay revealed that Mettl21e methylated valosin-containing protein (Vcp/p97), a key component of the ubiquitin-proteasome system. KD or knockout of Mettl21e resulted in elevated 26S proteasome activity, and inhibition of proteasome activity prevented atrophy of Mettl21e KD myotubes. These results demonstrate that Mettl21e functions to maintain myofiber size through inhibiting proteasome-mediated protein degradation.-Wang, C., Zhang, B., Ratliff, A. C., Arrington, J., Chen, J., Xiong, Y., Yue, F., Nie, Y., Hu, K., Jin, W., Tao, W. A., Hrycyna, C. A., Sun, X., Kuang, S. Methyltransferase-like 21e inhibits 26S proteasome activity to facilitate hypertrophy of type IIb myofibers.

p97/VCP promotes degradation of CRBN substrate glutamine synthetase and neosubstrates.

Glutamine synthetase (GS) plays an essential role in metabolism by catalyzing the synthesis of glutamine from glutamate and ammonia. Our recent study showed that CRBN, a direct protein target for the teratogenic and antitumor activities of immunomodulatory drugs such as thalidomide, lenalidomide, and pomalidomide, recognizes an acetyl degron of GS, resulting in ubiquitylation and degradation of GS in response to glutamine. Here, we report that valosin-containing protein (VCP)/p97 promotes the degradation of ubiquitylated GS, resulting in its accumulation in cells with compromised p97 function. Notably, p97 is also required for the degradation of all four known CRBN neo-substrates [Ikaros family zinc finger proteins 1 (IKZF1) and 3 (IKZF3), casein kinase 1α (CK1α), and the translation termination factor GSPT1] whose ubiquitylation is induced by immunomodulatory drugs. Together, these data point to an unexpectedly intimate relationship between the E3 ubiquitin ligase CRL4CRBN and p97 pathways.

Valosin-containing protein VCP/p97 is essential for the intracellular development of Leishmania and its survival under heat stress.

Valosin-containing protein (VCP)/p97/Cdc48 is one of the best-characterised type II cytosolic AAA+ ATPases most known for their role in ubiquitin-dependent protein quality control. Here, we provide functional insights into the role of the Leishmania VCP/p97 homologue (LiVCP) in the parasite intracellular development. We demonstrate that although LiVCP is an essential gene, Leishmania infantum promastigotes can grow with less VCP. In contrast, growth of axenic and intracellular amastigotes is dramatically affected upon decreased LiVCP levels in heterozygous and temperature sensitive (ts) LiVCP mutants or the expression of dominant negative mutants known to specifically target the second conserved VCP ATPase domain, a major contributor of the VCP overall ATPase activity. Interestingly, these VCP mutants are also unable to survive heat stress, and a ts VCP mutant is defective in amastigote growth. Consistent with LiVCP's essential function in amastigotes, LiVCP messenger ribonucleic acid undergoes 3'Untranslated Region (UTR)-mediated developmental regulation, resulting in higher VCP expression in amastigotes. Furthermore, we show that parasite mutant lines expressing lower VCP levels or dominant negative VCP forms exhibit high accumulation of polyubiquitinated proteins and increased sensitivity to proteotoxic stress, supporting the ubiquitin-selective chaperone function of LiVCP. Together, these results emphasise the crucial role LiVCP plays under heat stress and during the parasite intracellular development.