Discovery of the First Irreversible HDAC6 Isoform Selective Inhibitor with Potent Anti-Multiple Myeloma Activity.

In our previous research, a series of phenylsulfonylfuroxan-based hydroxamates were developed, among which compound 1 exhibited remarkable in vitro and in vivo antitumor potency due to its histone deacetylase (HDAC) inhibitory and nitric oxide (NO)-donating activities. Herein, the in-depth study of compound 1 revealed that this HDAC inhibitor-NO donor hybrid could enduringly increase the intracellular levels of acetyl histones and acetyl α-tubulin, which could be ascribed to its irreversible inhibition toward class I HDACs and HDAC6. Structural modification of compound 1 led to a novel phenylsulfonylfuroxan-based hydroxamate 4, which exhibited considerable HDAC6 inhibitory activity and selectivity. Furthermore, compound 4 could inhibit intracellular HDAC6 both selectively and irreversibly. To the best of our knowledge, this is the first research reporting the irreversible inhibition of HDAC6. It was also demonstrated that compared with ACY-241 (a reversible HDAC6 inhibitor in clinical trials), the irreversible HDAC6 selective inhibitor 4 exhibited not only superior anti-multiple myeloma activity but also improved therapeutic index.

Parkin regulates neuronal lipid homeostasis through SREBP2-lipoprotein lipase pathway-implications for Parkinson's disease.

Abnormal lipid homeostasis has been observed in the brain of Parkinson's disease (PD) patients and experimental models, although the mechanism underlying this phenomenon is unclear. Notably, previous studies have reported that the PD-linked protein Parkin functionally interacts with important lipid regulators, including Sterol Regulatory Element-Binding Proteins (SREBPs) and cluster of differentiation 36 (CD36). Here, we demonstrate a functional relationship between Parkin and lipoprotein lipase (LPL), a triglyceride lipase that is widely expressed in the brain. Using a human neuroblastoma cell line and a Parkin knockout mouse model, we demonstrate that Parkin expression level positively correlates with neuronal LPL protein level and activity. Importantly, our study identified SREBP2, a major regulator of sterol and fatty acid synthesis, as a potential mediator between Parkin and LPL. Supporting this, SREBP2 genetic ablation abolished Parkin effect on LPL expression. We further demonstrate that Parkin-LPL pathway regulates the formation of intracellular lipid droplets, and that this pathway is upregulated upon exposure to PD-linked oxidative stress induced by rotenone. Finally, we show that inhibition of either LPL or SREBP2 exacerbates rotenone-induced cell death. Taken together, our findings reveal a novel pathway linking Parkin, SREBP2 and LPL in neuronal lipid homeostasis that may be relevant to the pathogenesis of PD.

Cathelicidin-related antimicrobial peptide mediates skeletal muscle degeneration caused by injury and Duchenne muscular dystrophy in mice.

BACKGROUND: Cathelicidin, an antimicrobial peptide, plays a key role in regulating bacterial killing and innate immunity; however, its role in skeletal muscle function is unknown. We investigated the potential role of cathelicidin in skeletal muscle pathology resulting from acute injury and Duchenne muscular dystrophy (DMD) in mice. METHODS: Expression changes and muscular localization of mouse cathelicidin-related antimicrobial peptide (Cramp) were examined in the skeletal muscle of normal mice treated with chemicals (cardiotoxin and BaCl2 ) or in dystrophic muscle of DMD mouse models (mdx, mdx/Utrn+/- and mdx/Utrn-/- ). Cramp penetration into myofibres and effects on muscle damage were studied by treating synthetic peptides to mouse skeletal muscles or C2C12 myotubes. Cramp knockout (KO) mice and mdx/Utrn/Cramp KO lines were used to determine whether Cramp mediates muscle degeneration. Muscle pathophysiology was assessed by histological methods, serum analysis, grip strength and lifespan. Molecular factors targeted by Cramp were identified by the pull-down assay and proteomic analysis. RESULTS: In response to acute muscle injury, Cramp was activated in muscle-infiltrating neutrophils and internalized into myofibres. Cramp treatments of mouse skeletal muscles or C2C12 myotubes resulted in muscle degeneration and myotube damage, respectively. Genetic ablation of Cramp reduced neutrophil infiltration and ameliorated muscle pathology, such as fibre size (P < 0.001; n = 6) and fibrofatty infiltration (P < 0.05). Genetic reduction of Cramp in mdx/Utrn+/- mice not only attenuated muscle damage (35%, P < 0.05; n = 9-10), myonecrosis (53%, P < 0.05), inflammation (37-65%, P < 0.01) and fibrosis (14%, P < 0.05) but also restored muscle fibre size (14%, P < 0.05) and muscle force (18%, P < 0.05). Reducing Cramp levels led to a 63% (male, P < 0.05; n = 10-14) and a 124% (female, P < 0.001; n = 20) increase in the lifespan of mdx/Utrn-/- mice. Proteomic and mechanistic studies revealed that Cramp cross-talks with Ca2+ signalling in skeletal muscle through sarcoplasmic/endoplasmic reticulum Ca2+ -ATPase1 (SERCA1). Cramp binds and inactivates SERCA1, leading to the activation of Ca2+ -dependent calpain proteases that exacerbate DMD progression. CONCLUSIONS: These findings identify Cramp as an immune cell-derived regulator of skeletal muscle degeneration and provide a potential therapeutic target for DMD.

RIPK3 upregulation confers robust proliferation and collateral cystine-dependence on breast cancer recurrence.

The molecular and genetic basis of tumor recurrence is complex and poorly understood. RIPK3 is a key effector in programmed necrotic cell death and, therefore, its expression is frequently suppressed in primary tumors. In a transcriptome profiling between primary and recurrent breast tumor cells from a murine model of breast cancer recurrence, we found that RIPK3, while absent in primary tumor cells, is dramatically reexpressed in recurrent breast tumor cells by an epigenetic mechanism. Unexpectedly, we found that RIPK3 knockdown in recurrent tumor cells reduced clonogenic growth, causing cytokinesis failure, p53 stabilization, and repressed the activities of YAP/TAZ. These data uncover a surprising role of the pro-necroptotic RIPK3 kinase in enabling productive cell cycle during tumor recurrence. Remarkably, high RIPK3 expression also rendered recurrent tumor cells exquisitely dependent on extracellular cystine and undergo necroptosis upon cystine deprivation. The induction of RIPK3 in recurrent tumors unravels an unexpected mechanism that paradoxically confers on tumors both growth advantage and necrotic vulnerability, providing potential strategies to eradicate recurrent tumors.

Targeting Histone Deacetylase 6 Reprograms Interleukin-17-Producing Helper T Cell Pathogenicity and Facilitates Immunotherapies for Hepatocellular Carcinoma.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) is often accompanied by resistance to immunotherapies despite the presence of tumor-infiltrating lymphocytes. We report that histone deacetylase 6 (HDAC6) represses interleukin-17 (IL-17)-producing helper T (TH 17) cell pathogenicity and the antitumor immune response, dependent on its deacetylase activity. APPROACH AND RESULTS: Adoptive transfer of HDAC6-deficient TH 17 cells impedes HCC growth, dependent on elevated IL-17A, by enhancing the production of antitumor cytokine and cluster of differentiation 8-positive (CD8+) T cell-mediated antitumor responses. Intriguingly, HDAC6-depleted T cells trigger programmed cell death protein 1 (PD-1)-PD-1 ligand 1 expression to achieve a strong synergistic effect to sensitize advanced HCC to an immune checkpoint blocker, while blockade of IL-17A partially suppresses it. Mechanistically, HDAC6 limits TH 17 pathogenicity and the antitumor effect through regulating forkhead box protein O1 (FoxO1). HDAC6 binds and deacetylates cytosolic FoxO1 at K242, which is required for its nuclear translocation and stabilization to repress retinoic acid-related orphan receptor gamma (RoRγt), the transcription factor of TH 17 cell. This regulation of HDAC6 for murine and human TH 17 cell is highly conserved. CONCLUSIONS: These results demonstrate that targeting the cytosolic HDAC6-FoxO1 axis reprograms the pathogenicity and antitumor response of TH 17 cells in HCC, with a pathogenicity-driven responsiveness to facilitate immunotherapies.

PTHrP targets HDAC4 and HDAC5 to repress chondrocyte hypertrophy.

During endochondral bone formation, chondrocyte hypertrophy represents a crucial turning point from chondrocyte differentiation to bone formation. Both parathyroid hormone-related protein (PTHrP) and histone deacetylase 4 (HDAC4) inhibit chondrocyte hypertrophy. Using multiple mouse genetics models, we demonstrate in vivo that HDAC4 is required for the effects of PTHrP on chondrocyte differentiation. We further show in vivo that PTHrP leads to reduced HDAC4 phosphorylation at the 14-3-3-binding sites and subsequent HDAC4 nuclear translocation. The Hdac4-KO mouse shares a similar but milder phenotype with the Pthrp-KO mouse, indicating the possible existence of other mediators of PTHrP action. We identify HDAC5 as an additional mediator of PTHrP signaling. While the Hdac5-KO mouse has no growth plate phenotype at birth, the KO of Hdac5 in addition to the KO of Hdac4 is required to block fully PTHrP action on chondrocyte differentiation at birth in vivo. Finally, we show that PTHrP suppresses myocyte enhancer factor 2 (Mef2) action that allows runt-related transcription factor 2 (Runx2) mRNA expression needed for chondrocyte hypertrophy. Our results demonstrate that PTHrP inhibits chondrocyte hypertrophy and subsequent bone formation in vivo by allowing HDAC4 and HDAC5 to block the Mef2/Runx2 signaling cascade. These results explain the phenotypes of several genetic abnormalities in humans.

CoA synthase regulates mitotic fidelity via CBP-mediated acetylation.

The temporal activation of kinases and timely ubiquitin-mediated degradation is central to faithful mitosis. Here we present evidence that acetylation controlled by Coenzyme A synthase (COASY) and acetyltransferase CBP constitutes a novel mechanism that ensures faithful mitosis. We found that COASY knockdown triggers prolonged mitosis and multinucleation. Acetylome analysis reveals that COASY inactivation leads to hyper-acetylation of proteins associated with mitosis, including CBP and an Aurora A kinase activator, TPX2. During early mitosis, a transient CBP-mediated TPX2 acetylation is associated with TPX2 accumulation and Aurora A activation. The recruitment of COASY inhibits CBP-mediated TPX2 acetylation, promoting TPX2 degradation for mitotic exit. Consistently, we detected a stage-specific COASY-CBP-TPX2 association during mitosis. Remarkably, pharmacological and genetic inactivation of CBP effectively rescued the mitotic defects caused by COASY knockdown. Together, our findings uncover a novel mitotic regulation wherein COASY and CBP coordinate an acetylation network to enforce productive mitosis.

Discovery of a fluorescent probe with HDAC6 selective inhibition.

There is increasing interest in discovering HDAC6 selective inhibitors as chemical probes to elucidate the biological functions of HDAC6 and ultimately as new therapeutic agents. Small-molecular fluorescent probes are widely used to detect target protein location and function, identify protein complex composition in biological processes of interest. In the present study, structural modification of the previously reported compound 4MS leads to two novel fluorescent HDAC inhibitors, 6a and 6b. Determination of IC50 values against the panel of Zn2+ dependent HDACs (HDAC1-11) reveals that 6b is a HDAC6 selective inhibitor, which can induce hyperacetylation of tubulin but not histone H4. Importantly, fluorescent and immunofluorescent analyses of cells treated with the proteasome inhibitor MG132 demonstrates that 6b can selectively target and image HDAC6 within the inclusion body, the aggresome. These results identify 6b not only as a HDAC6 selective inhibitor but also as a fluorescent probe for imaging HDAC6 and investigating the roles of HDAC6 in various physiological and pathological contexts.

HDAC6 regulates the dynamics of lytic granules in cytotoxic T lymphocytes.

HDAC6 is a tubulin deacetylase involved in many cellular functions related to cytoskeleton dynamics, including cell migration and autophagy. In addition, HDAC6 affects antigen-dependent CD4(+)T cell activation. In this study, we show that HDAC6 contributes to the cytotoxic function of CD8(+)T cells. Immunization studies revealed defective cytotoxic activity in vivo in the absence of HDAC6. Adoptive transfer of wild-type or Hdac6(-/-)CD8(+)T cells to Rag1(-/-)mice demonstrated specific impairment in CD8(+)T cell responses against vaccinia infection. Mechanistically, HDAC6-deficient cytotoxic T lymphocytes (CTLs) showed defective in vitro cytolytic activity related to altered dynamics of lytic granules, inhibited kinesin-1-dynactin-mediated terminal transport of lytic granules to the immune synapse and deficient exocytosis, but not to target cell recognition, T cell receptor (TCR) activation or interferon (IFN)γ production. Our results establish HDAC6 as an effector of the immune cytotoxic response that acts by affecting the dynamics, transport and secretion of lytic granules by CTLs.

HDAC6 regulates cellular viral RNA sensing by deacetylation of RIG-I.

RIG-I is a key cytosolic sensor that detects RNA viruses through its C-terminal region and activates the production of antiviral interferons (IFNs) and proinflammatory cytokines. While posttranslational modification has been demonstrated to regulate RIG-I signaling activity, its significance for the sensing of viral RNAs remains unclear. Here, we first show that the RIG-I C-terminal region undergoes deacetylation to regulate its viral RNA-sensing activity and that the HDAC6-mediated deacetylation of RIG-I is critical for viral RNA detection. HDAC6 transiently bound to RIG-I and removed the lysine 909 acetylation in the presence of viral RNAs, promoting RIG-I sensing of viral RNAs. Depletion of HDAC6 expression led to impaired antiviral responses against RNA viruses, but not against DNA viruses. Consequently, HDAC6 knockout mice were highly susceptible to RNA virus infections compared to wild-type mice. These findings underscore the critical role of HDAC6 in the modulation of the RIG-I-mediated antiviral sensing pathway.

Loss of α-Tubulin Acetylation Is Associated with TGF-ß-induced Epithelial-Mesenchymal Transition.

The epithelial-to-mesenchymal transition (EMT) is a process by which differentiated epithelial cells reprogram gene expression, lose their junctions and polarity, reorganize their cytoskeleton, increase cell motility and assume a mesenchymal morphology. Despite the critical functions of the microtubule (MT) in cytoskeletal organization, how it participates in EMT induction and maintenance remains poorly understood. Here we report that acetylated α-tubulin, which plays an important role in microtubule (MT) stabilization and cell morphology, can serve as a novel regulator and marker of EMT. A high level of acetylated α-tubulin was correlated with epithelial morphology and it profoundly decreased during TGF-ß-induced EMT. We found that TGF-ß increased the activity of HDAC6, a major deacetylase of α-tubulin, without affecting its expression levels. Treatment with HDAC6 inhibitor tubacin or TGF-ß type I receptor inhibitor SB431542 restored the level of acetylated α-tubulin and consequently blocked EMT. Our results demonstrate that acetylated α-tubulin can serve as a marker of EMT and that HDAC6 represents an important regulator during EMT process.

Chaperone-mediated 26S proteasome remodeling facilitates free K63 ubiquitin chain production and aggresome clearance.

Efficient elimination of misfolded proteins by the proteasome system is critical for proteostasis. Inadequate proteasome capacity can lead to aberrant aggregation of misfolded proteins and inclusion body formation, a hallmark of neurodegenerative disease. The proteasome system cannot degrade aggregated proteins; however, it stimulates autophagy-dependent aggregate clearance by producing unanchored lysine (K)63-linked ubiquitin chains via the proteasomal deubiquitinating enzyme Poh1. The canonical function of Poh1, which removes ubiquitin chains en bloc from proteasomal substrates prior to their degradation, requires intact 26S proteasomes. Here we present evidence that during aggresome clearance, 20S proteasomes dissociate from protein aggregates, while Poh1 and selective subunits of 19S proteasomes are retained. The dissociation of 20S proteasome components requires the molecular chaperone Hsp90. Hsp90 inhibition suppresses 26S proteasome remodeling, unanchored ubiquitin chain production, and aggresome clearance. Our results suggest that 26S proteasomes undergo active remodeling to generate a Poh1-dependent K63-deubiquitinating enzyme to facilitate protein aggregate clearance.

Glycolysis-dependent histone deacetylase 4 degradation regulates inflammatory cytokine production.

Activation of the inflammatory response is accompanied by a metabolic shift to aerobic glycolysis. Here we identify histone deacetylase 4 (HDAC4) as a new component of the immunometabolic program. We show that HDAC4 is required for efficient inflammatory cytokine production activated by lipopolysaccharide (LPS). Surprisingly, prolonged LPS treatment leads to HDAC4 degradation. LPS-induced HDAC4 degradation requires active glycolysis controlled by GSK3ß and inducible nitric oxide synthase (iNOS). Inhibition of GSK3ß or iNOS suppresses nitric oxide (NO) production, glycolysis, and HDAC4 degradation. We present evidence that sustained glycolysis induced by LPS treatment activates caspase-3, which cleaves HDAC4 and triggers its degradation. Of importance, a caspase-3-resistant mutant HDAC4 escapes LPS-induced degradation and prolongs inflammatory cytokine production. Our findings identify the GSK3ß-iNOS-NO axis as a critical signaling cascade that couples inflammation to metabolic reprogramming and a glycolysis-driven negative feedback mechanism that limits inflammatory response by triggering HDAC4 degradation.

Microtubule acetylation amplifies p38 kinase signalling and anti-inflammatory IL-10 production.

Reversible acetylation of α-tubulin is an evolutionarily conserved modification in microtubule networks. Despite its prevalence, the physiological function and regulation of microtubule acetylation remain poorly understood. Here we report that macrophages challenged by bacterial lipopolysaccharides (LPS) undergo extensive microtubule acetylation. Suppression of LPS-induced microtubule acetylation by inactivating the tubulin acetyltransferase, MEC17, profoundly inhibits the induction of anti-inflammatory interleukin-10 (IL-10), a phenotype effectively reversed by an acetylation-mimicking α-tubulin mutant. Conversely, elevating microtubule acetylation by inhibiting the tubulin deacetylase, HDAC6, or stabilizing microtubules via Taxol stimulates IL-10 hyper-induction. Supporting the anti-inflammatory function of microtubule acetylation, HDAC6 inhibition significantly protects mice from LPS toxicity. In HDAC6-deficient macrophages challenged by LPS, p38 kinase signalling becomes selectively amplified, leading to SP1-dependent IL-10 transcription. Remarkably, the augmented p38 signalling is suppressed by MEC17 inactivation. Our findings identify reversible microtubule acetylation as a kinase signalling modulator and a key component in the inflammatory response.

VCP is essential for mitochondrial quality control by PINK1/Parkin and this function is impaired by VCP mutations.

Mutations in VCP cause multisystem degeneration impacting the nervous system, muscle, and/or bone. Patients may present with ALS, Parkinsonism, frontotemporal dementia, myopathy, Paget's disease, or a combination of these. The disease mechanism is unknown. We developed a Drosophila model of VCP mutation-dependent degeneration. The phenotype is reminiscent of PINK1 and parkin mutants, including a pronounced mitochondrial defect. Indeed, VCP interacts genetically with the PINK1/parkin pathway in vivo. Paradoxically, VCP complements PINK1 deficiency but not parkin deficiency. The basis of this paradox is resolved by mechanistic studies in vitro showing that VCP recruitment to damaged mitochondria requires Parkin-mediated ubiquitination of mitochondrial targets. VCP recruitment coincides temporally with mitochondrial fission, and VCP is required for proteasome-dependent degradation of Mitofusins in vitro and in vivo. Further, VCP and its adaptor Npl4/Ufd1 are required for clearance of damaged mitochondria via the PINK1/Parkin pathway, and this is impaired by pathogenic mutations in VCP.

Lysine modifications and autophagy.

Nutrient deprivation or cellular stress leads to the activation of a catabolic pathway that is conserved across species, known as autophagy. This process is considered to be adaptive and plays an important role in a number of cellular processes, including metabolism, immunity and development. Autophagy has also been linked to diseases, such as cancer and neurodegeneration, highlighting the importance of a better insight into its regulation. In the present chapter, we discuss how PTMs (post-translational modifications) of lysine residues by acetylation and ubiquitination alter the function of key proteins involved in the activation, maturation and substrate selectivity of autophagy. We also discuss the clinical potential of targeting these modifications to modulate autophagic activities.

Class IIb HDAC6 regulates endothelial cell migration and angiogenesis by deacetylation of cortactin.

Histone deacetylases (HDACs) deacetylate histones and non-histone proteins, thereby affecting protein activity and gene expression. The regulation and function of the cytoplasmic class IIb HDAC6 in endothelial cells (ECs) is largely unexplored. Here, we demonstrate that HDAC6 is upregulated by hypoxia and is essential for angiogenesis. Silencing of HDAC6 in ECs decreases sprouting and migration in vitro and formation of functional vascular networks in matrigel plugs in vivo. HDAC6 regulates zebrafish vessel formation, and HDAC6-deficient mice showed a reduced formation of perfused vessels in matrigel plugs. Consistently, overexpression of wild-type HDAC6 increases sprouting from spheroids. HDAC6 function requires the catalytic activity but is independent of ubiquitin binding and deacetylation of α-tubulin. Instead, we found that HDAC6 interacts with and deacetylates the actin-remodelling protein cortactin in ECs, which is essential for zebrafish vessel formation and which mediates the angiogenic effect of HDAC6. In summary, we show that HDAC6 is necessary for angiogenesis in vivo and in vitro, involving the interaction and deacetylation of cortactin that regulates EC migration and sprouting.

The role of ubiquitin in autophagy-dependent protein aggregate processing.

The efficient management of misfolded protein aggregates is essential for cell viability and requires three interconnected pathways: the molecular chaperone machinery that assists protein folding, the proteasome pathway that degrades misfolded proteins, and the aggresomal pathway that sequesters and delivers toxic proteins aggregates to autophagy for clearance. Although autophagy is generally considered as non-selective degradative machinery, growing evidence supports the existence of a selective autophagy that specifically targets protein aggregates for clearance. This so-called "quality control autophagy" is established by specific ubiquitin E3 ligases, autophagic substrate ubiquitination, and specific ubiquitin binding proteins p62 and HDAC6. In this context, quality control autophagy is similar to the proteasome system and utilizes ubiquitin tags for substrate recognition and processing. Here I will discuss the recent progress towards understanding the molecular basis of this unique form of ubiquitin-dependent autophagy in protein aggregate clearance and its relevance to disease.

The microtubule-associated histone deacetylase 6 (HDAC6) regulates epidermal growth factor receptor (EGFR) endocytic trafficking and degradation.

Histone deacetylase 6 (HDAC6) is a microtubule-associated deacetylase with tubulin deacetylase activity, and it binds dynein motors. Recent studies revealed that microtubule acetylation affects the affinity and processivity of microtubule motors. These unique properties implicate a role for HDAC6 in intracellular organelle transport. Here, we show that HDAC6 associates with the endosomal compartments and controls epidermal growth factor receptor (EGFR) trafficking and degradation. We found that loss of HDAC6 promoted EGFR degradation. Mechanistically, HDAC6 deficiency did not cause aberrant EGFR internalization and recycling. Rather, it resulted in accelerated segregation of EGFR from early endosomes and premature delivery of EGFR to the late endosomal and lysosomal compartments. The deregulated EGFR endocytic trafficking was accompanied by an increase in microtubule-dependent movement of EGFR-bearing vesicles, revealing a novel regulation of EGFR vesicular trafficking and degradation by the microtubule deacetylase HDAC6.

VCP/p97 is essential for maturation of ubiquitin-containing autophagosomes and this function is impaired by mutations that cause IBMPFD.

VCP (VCP/p97) is a ubiquitously expressed member of the AAA(+)-ATPase family of chaperone-like proteins that regulates numerous cellular processes including chromatin decondensation, homotypic membrane fusion and ubiquitin-dependent protein degradation by the proteasome. Mutations in VCP cause a multisystem degenerative disease consisting of inclusion body myopathy, Paget disease of bone, and frontotemporal dementia (IBMPFD). Here we show that VCP is essential for autophagosome maturation. We generated cells stably expressing dual-tagged LC3 (mCherry-EGFP-LC3) which permit monitoring of autophagosome maturation. We determined that VCP deficiency by RNAi-mediated knockdown or overexpression of dominant-negative VCP results in significant accumulation of immature autophagic vesicles, some of which are abnormally large, acidified and exhibit cathepsin B activity. Furthermore, expression of disease-associated VCP mutants (R155H and A232E) also causes this autophagy defect. VCP was found to be essential to autophagosome maturation under basal conditions and in cells challenged by proteasome inhibition, but not in cells challenged by starvation, suggesting that VCP might be selectively required for autophagic degradation of ubiquitinated substrates. Indeed, a high percentage of the accumulated autophagic vesicles contain ubiquitin-positive contents, a feature that is not observed in autophagic vesicles that accumulate following starvation or treatment with Bafilomycin A. Finally, we show accumulation of numerous, large LAMP-1 and LAMP-2-positive vacuoles and accumulation of LC3-II in myoblasts derived from patients with IBMPFD. We conclude that VCP is essential for maturation of ubiquitin-containing autophagosomes and that defect in this function may contribute to IBMPFD pathogenesis.