Ub Proteasome System Research Articles

Ubiquitin-proteasome dependent regulation of Profilin2 (Pfn2) by a cellular inhibitor of apoptotic protein 1 (cIAP1)

The two major isoforms of the profilin (Pfn) family of proteins in mammals are Pfn1 and Pfn2. Pfn1 is a universal actin cytoskeletal regulator, while Pfn2 is an actin binding protein and mediator of synapse architecture, specific to neural tissues. However, it has recently been suggested that Pfn2 is also widely distributed in various tissues and involved in numerous cellular events as well as cytoskeletal regulation. In our previous study, we showed that Pfn1 is regulated by carboxyl terminus of Hsc70-Interacting Protein (CHIP) via an ubiquitin (Ub) proteasome system; although, the mechanism of regulation of Pfn2 is unknown. In this report, we demonstrate that Pfn2 is heavily ubiquitinated via differential Ub-linkages for degradation or as a regulatory signal. We also show that cellular inhibitor of apoptosis 1 (cIAP1) rather than CHIP, functions as an E3 ligase that targets Pfn2 for proteasomal degradation. Finally, we observed that Pfn2 levels, regulated by cIAP1, affected intracellular levels of reactive oxygen species. These results may provide a regulatory mechanism for cellular function of Pfn2 in various tissues.

The N-recognin UBR4 of the N-end rule pathway is targeted to and required for the biogenesis of the early endosome.

The N-end rule pathway is a proteolytic system in which single N-terminal residues of proteins act as N-degrons. These degrons are recognized by N-recognins, facilitating substrate degradation via the ubiquitin (Ub) proteasome system (UPS) or autophagy. We have previously identified a set of N-recognins [UBR1, UBR2, UBR4 (also known as p600) and UBR5 (also known as EDD)] that bind N-degrons through their UBR boxes to promote proteolysis by the proteasome. Here, we show that the 570 kDa N-recognin UBR4 is associated with maturing endosomes through an interaction with Ca2+-bound calmodulin. The endosomal recruitment of UBR4 is essential for the biogenesis of early endosomes (EEs) and endosome-related processes, such as the trafficking of endocytosed protein cargos and degradation of extracellular cargos by endosomal hydrolases. In mouse embryos, UBR4 marks and plays a role in the endosome-lysosome pathway that mediates the heterophagic proteolysis of endocytosed maternal proteins into amino acids. By screening 9591 drugs through the DrugBank database, we identify picolinic acid as a putative ligand for UBR4 that inhibits the biogenesis of EEs. Our results suggest that UBR4 is an essential modulator in the endosome-lysosome system.This article has an associated First Person interview with the first author of the paper.

Pulmonary inflammation-induced loss and subsequent recovery of skeletal muscle mass require functional poly-ubiquitin conjugation

BackgroundPulmonary inflammation in response to respiratory infections can evoke muscle wasting. Increased activity of the ubiquitin (Ub)-proteasome system (UPS) and the autophagy lysosome pathway (ALP) have been implicated in inflammation-induced muscle atrophy. Since poly-Ub conjugation is required for UPS-mediated proteolysis and has been implicated in the ALP, we assessed the effect of impaired ubiquitin conjugation on muscle atrophy and recovery following pulmonary inflammation, and compared activation and suppression of these proteolytic systems to protein synthesis regulation.MethodsPulmonary inflammation was induced in mice by an intratracheal instillation of LPS. Proteolysis (UPS and ALP) and synthesis signaling were examined in gastrocnemius muscle homogenates. Ub-conjugation-dependency of muscle atrophy and recovery was addressed using Ub-K48R (K48R) mice with attenuated poly-ubiquitin conjugation, and compared to UBWT control mice.ResultsPulmonary inflammation caused a decrease in skeletal muscle mass which was accompanied by a rapid increase in expression of UPS and ALP constituents and reduction in protein synthesis signaling acutely after LPS. Muscle atrophy was attenuated in K48R mice, while ALP and protein synthesis signaling were not affected. Muscle mass recovery starting 72 h post LPS, correlated with reduced expression of UPS and ALP constituents and restoration of protein synthesis signaling. K48R mice however displayed impaired recovery of muscle mass.ConclusionPulmonary inflammation-induced muscle atrophy is in part attributable to UPS-mediated proteolysis, as activation of ALP- and suppression of protein synthesis signaling occur independently of poly-Ub conjugation during muscle atrophy. Recovery of muscle mass following pulmonary inflammation involves inverse regulation of proteolysis and protein synthesis signaling, and requires a functional poly-Ub conjugation.

N-terminal arginylation generates a bimodal degron that modulates autophagic proteolysis

The conjugation of amino acids to the protein N termini is universally observed in eukaryotes and prokaryotes, yet its functions remain poorly understood. In eukaryotes, the amino acid l-arginine (l-Arg) is conjugated to N-terminal Asp (Nt-Asp), Glu, Gln, Asn, and Cys, directly or associated with posttranslational modifications. Following Nt-arginylation, the Nt-Arg is recognized by UBR boxes of N-recognins such as UBR1, UBR2, UBR4/p600, and UBR5/EDD, leading to substrate ubiquitination and proteasomal degradation via the N-end rule pathway. It has been a mystery, however, why studies for the past five decades identified only a handful of Nt-arginylated substrates in mammals, although five of 20 principal amino acids are eligible for arginylation. Here, we show that the Nt-Arg functions as a bimodal degron that directs substrates to either the ubiquitin (Ub)-proteasome system (UPS) or macroautophagy depending on physiological states. In normal conditions, the arginylated forms of proteolytic cleavage products, D101-CDC6 and D1156-BRCA1, are targeted to UBR box-containing N-recognins and degraded by the proteasome. However, when proteostasis by the UPS is perturbed, their Nt-Arg redirects these otherwise cellular wastes to macroautophagy through its binding to the ZZ domain of the autophagic adaptor p62/STQSM/Sequestosome-1. Upon binding to the Nt-Arg, p62 acts as an autophagic N-recognin that undergoes self-polymerization, facilitating cargo collection and lysosomal degradation of p62-cargo complexes. A chemical mimic of Nt-Arg redirects Ub-conjugated substrates from the UPS to macroautophagy and promotes their lysosomal degradation. Our results suggest that the Nt-Arg proteome of arginylated proteins contributes to reprogramming global proteolytic flux under stresses.

Pseudo-DUBs as allosteric activators and molecular scaffolds of protein complexes.

The ubiquitin (Ub) proteasome system and Ub signalling networks are crucial to cell biology and disease development. Deubiquitylases (DUBs) control cell signalling by removing mono-Ub and polyubiquitin chains from substrates. DUBs take part in almost all processes that regulate cellular life and are frequently dysregulated in disease. We have catalogued 99 currently known DUBs in the human genome and sequence conservation analyses of catalytic residues suggest that 11 lack enzyme activity and are classed as pseudo-DUBs. These pseudoenzymes play important biological roles by allosterically activating catalytically competent DUBs as well as other active enzymes. Additionally, pseudoenzymes act as assembly scaffolds of macromolecular complexes. We discuss how pseudo-DUBs have lost their catalytic activity, their diverse mechanisms of action and their potential as therapeutic targets. Many known pseudo-DUBs play crucial roles in cell biology and it is likely that unstudied and overlooked pseudo-DUB genes will have equally important functions.

Repair or destruction-an intimate liaison between ubiquitin ligases and molecular chaperones in proteostasis.

Cellular differentiation, developmental processes, and environmental factors challenge the integrity of the proteome in every eukaryotic cell. The maintenance of protein homeostasis, or proteostasis, involves folding and degradation of damaged proteins, and is essential for cellular function, organismal growth, and viability 1, 2. Misfolded proteins that cannot be refolded by chaperone machineries are degraded by specialized proteolytic systems. A major degradation pathway regulating cellular proteostasis is the ubiquitin (Ub)/proteasome system (UPS), which regulates turnover of damaged proteins that accumulate upon stress and during aging. Despite a large number of structurally unrelated substrates, Ub conjugation is remarkably selective. Substrate selectivity is mainly provided by the group of E3 enzymes. Several observations indicate that numerous E3 Ub ligases intimately collaborate with molecular chaperones to maintain the cellular proteome. In this review, we provide an overview of specialized quality control E3 ligases playing a critical role in the degradation of damaged proteins. The process of substrate recognition and turnover, the type of chaperones they team up with, and the potential pathogeneses associated with their malfunction will be further discussed.

Hydrophobic Patch of Ubiquitin is Important for its Optimal Activation by Ubiquitin Activating Enzyme E1.

Protein ubiquitination plays a role in essentially every process in eukaryotic cells. The attachment of ubiquitin (Ub) or Ub-like (UBL) proteins to target proteins is achieved by parallel but distinct cascades of enzymatic reactions involving three enzymes: E1, E2, and E3. The E1 enzyme functions at the apex of this pathway and plays a critical role in activating the C-terminus of ubiquitin or UBL, which is an essential step that triggers subsequent downstream transfer to their cognate E2s resulting in the fidelity of the Ub/UBL conjugation machinery. Despite the central role of the E1 enzyme in protein modification, a quantitative method to measure Ub/UBL activation by E1 is lacking. Here, we present a mass spectrometry-based assay to accurately measure the activation of Ub/UBL by E1 independent of the E2/E3 enzymes. Our method does not require radiolabeling of any components and therefore can be used in any biochemical laboratory having access to a mass spectrometer. This method allowed us to dissect the concerted process of E1-E2-catalyzed Ub conjugation in order to separately characterize the process of Ub activation and how it is affected by select mutations and other factors. We found that the hydrophobic patch of Ub is important for the optimal activation of Ub by E1. We further show that the blockers of the Ub-proteasome system such as ubistatin and fullerenol inhibit Ub activation by E1. Interestingly, our data indicate that the phosphorylation of Ub at the S65 position augments its activation by the E1 enzyme.

Cul3 neddylation is crucial for gradual lipid droplet formation during adipogenesis

Cullin 3 (Cul3) belongs to the family of cullins (Cul1–7) providing the scaffold for cullin-RING ubiquitin (Ub) ligases (CRLs), which are activated by neddylation and represent essential E3 ligases of the Ub proteasome system. During adipogenic differentiation neddylated Cul3 accumulates in LiSa-2 preadipocytes. Downregulation of Cul3 and inhibition of neddylation by MLN4924 blocks the formation of lipid droplets (LDs), the lipid storage organelles and markers of adipogenesis. Neddylation of Cul3 coincides with an increase of Rab18, a GTPase associated with LDs. Immunoprecipitation and confocal fluorescence microscopy revealed physical association of Cul3 and Rab18 at the membrane of LDs. RhoA, a suppressor of adipogenesis decreased during differentiation. Our results in LiSa-2 cells, but also mouse embryonic fibroblasts revealed a connection between Cul3, Rab18 and RhoA. Downregulation of Cul3 led to a marked increase in RhoA protein expression after 6days of LiSa-2 cell differentiation, suggesting that Cul3 is involved in the regulation of RhoA stability.

Protein Quality Control by Molecular Chaperones in Neurodegeneration.

Protein homeostasis (proteostasis) requires the timely degradation of misfolded proteins and their aggregates by protein quality control (PQC), of which molecular chaperones are an essential component. Compared with other cell types, PQC in neurons is particularly challenging because they have a unique cellular structure with long extensions. Making it worse, neurons are postmitotic, i.e., cannot dilute toxic substances by division, and, thus, are highly sensitive to misfolded proteins, especially as they age. Failure in PQC is often associated with neurodegenerative diseases, such as Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), and prion disease. In fact, many neurodegenerative diseases are considered to be protein misfolding disorders. To prevent the accumulation of disease-causing aggregates, neurons utilize a repertoire of chaperones that recognize misfolded proteins through exposed hydrophobic surfaces and assist their refolding. If such an effort fails, chaperones can facilitate the degradation of terminally misfolded proteins through either the ubiquitin (Ub)-proteasome system (UPS) or the autophagy-lysosome system (hereafter autophagy). If soluble, the substrates associated with chaperones, such as Hsp70, are ubiquitinated by Ub ligases and degraded through the proteasome complex. Some misfolded proteins carrying the KFERQ motif are recognized by the chaperone Hsc70 and delivered to the lysosomal lumen through a process called, chaperone-mediated autophagy (CMA). Aggregation-prone misfolded proteins that remain unprocessed are directed to macroautophagy in which cargoes are collected by adaptors, such as p62/SQSTM-1/Sequestosome-1, and delivered to the autophagosome for lysosomal degradation. The aggregates that have survived all these refolding/degradative processes can still be directly dissolved, i.e., disaggregated by chaperones. Studies have shown that molecular chaperones alleviate the pathogenic symptoms by neurodegeneration-causing protein aggregates. Chaperone-inducing drugs and anti-aggregation drugs are actively exploited for beneficial effects on symptoms of disease. Here, we discuss how chaperones protect misfolded proteins from aggregation and mediate the degradation of terminally misfolded proteins in collaboration with cellular degradative machinery. The topics also include therapeutic approaches to improve the expression and turnover of molecular chaperones and to develop anti-aggregation drugs.

Crosstalk and Interplay between the Ubiquitin-Proteasome System and Autophagy.

Proteolysis in eukaryotic cells is mainly mediated by the ubiquitin (Ub)-proteasome system (UPS) and the autophagylysosome system (hereafter autophagy). The UPS is a selective proteolytic system in which substrates are recognized and tagged with ubiquitin for processive degradation by the proteasome. Autophagy is a bulk degradative system that uses lysosomal hydrolases to degrade proteins as well as various other cellular constituents. Since the inception of their discoveries, the UPS and autophagy were thought to be independent of each other in components, action mechanisms, and substrate selectivity. Recent studies suggest that cells operate a single proteolytic network comprising of the UPS and autophagy that share notable similarity in many aspects and functionally cooperate with each other to maintain proteostasis. In this review, we discuss the mechanisms underlying the crosstalk and interplay between the UPS and autophagy, with an emphasis on substrate selectivity and compensatory regulation under cellular stresses.

Acute intermittent hypoxia in rats activates muscle proteolytic pathways through a gluccorticoid-dependent mechanism.

Although it is well known that chronic hypoxia induces muscle wasting, the effects of intermittent hypoxia on skeletal muscle protein metabolism remain unclear. We hypothesized that acute intermittent hypoxia (AIH), a challenge that activates the hypothalamic-pituitary-adrenal axis, would alter muscle protein homeostasis through a glucocorticoid-dependent mechanism. Three-week-old rats were submitted to adrenalectomy (ADX) and exposed to 8 h of AIH (6% O2 for 40 s at 9-min intervals). Animals were euthanized, and the soleus and extensor digitorum longus (EDL) muscles were harvested and incubated in vitro for measurements of protein turnover. AIH increased plasma levels of corticosterone and induced insulin resistance as estimated by the insulin tolerance test and lower rates of muscle glucose oxidation and the HOMA index. In both soleus and EDL muscles, rates of overall proteolysis increased after AIH. This rise was accompanied by an increased proteolytic activities of the ubiquitin(Ub)-proteasome system (UPS) and lysosomal and Ca2+-dependent pathways. Furthermore, AIH increased Ub-protein conjugates and gene expression of atrogin-1 and MuRF-1, two key Ub-protein ligases involved in muscle atrophy. In parallel, AIH increased the mRNA expression of the autophagy-related genes LC3b and GABARAPl1. In vitro rates of protein synthesis in skeletal muscles did not differ between AIH and control rats. ADX completely blocked the insulin resistance in hypoxic rats and the AIH-induced activation of proteolytic pathways and atrogene expression in both soleus and EDL muscles. These results demonstrate that AIH induces insulin resistance in association with activation of the UPS, the autophagic-lysosomal process, and Ca2+-dependent proteolysis through a glucocorticoid-dependent mechanism.NEW & NOTEWORTHY Since hypoxia is a condition in which the body is deprived of adequate oxygen supply and muscle wasting is induced, the present work provides evidence linking hypoxia to proteolysis through a glucocorticoid-dependent mechanism. We show that the activation of proteolytic pathways, atrophy-related genes, and insulin resistance in rats exposed to acute intermittent hypoxia was abolished by surgical removal of adrenal gland. This finding will be helpful for understanding of the muscle wasting in hypoxemic conditions.

Ubiquitin Associates with the N‐Terminal Domain of Nerve Growth Factor: The Role of Copper(II) Ions

Many biochemical pathways involving nerve growth factor (NGF), a neurotrophin with copper(II) binding abilities, are regulated by the ubiquitin (Ub) proteasome system. However, whether NGF binds Ub and the role played by copper(II) ions in modulating their interactions have not yet been investigated. Herein NMR spectroscopy, circular dichroism, ESI-MS, and titration calorimetry are employed to characterize the interactions of NGF with Ub. NGF1-14 , which is a short model peptide encompassing the first 14 N-terminal residues of NGF, binds the copper-binding regions of Ub (KD =8.6 10-5 m). Moreover, the peptide undergoes a random coil-polyproline type II helix structural conversion upon binding to Ub. Notably, copper(II) ions inhibit NGF1-14 /Ub interactions. Further experiments performed with the full-length NGF confirmed the existence of a copper(II)-dependent association between Ub and NGF and indicated that the N-terminal domain of NGF was a valuable paradigm that recapitulated many traits of the full-length protein.

Overexpression of COP9 signalosome subunits, CSN7A and CSN7B, exerts different effects on adipogenic differentiation.

The COP9 signalosome (CSN) is an essential regulator of cullin‐RING‐ubiquitin (Ub) ligases (CRLs), which ubiquitinate important cellular regulators and target them for degradation by the Ub proteasome system (UPS). The CSN exhibits deneddylating activity localized on subunit CSN5, which removes the ubiquitin‐like protein Nedd8 from the cullins of CRLs. CSN‐mediated deneddylation is an important step in the process of CRL remodeling, in which new substrate recognition units are incorporated into Ub ligases to meet changed requirements for proteolysis in cells. For instance, extensive CRL remodeling occurs during adipogenic differentiation when new CRL3s are formed. Diversification of CSN complexes during evolution is most likely another adaptation to meet different cellular requirements. Best known CSN variants are formed by different CSN subunit isoforms. For instance, in plant cells, isoforms have been identified for the MPN‐domain subunits CSN5 (CSN5A and CSN5B) and CSN6 (CSN6A and CSN6B) which form four distinct CSN variants. In mammalian cells CSNCSN7A and CSNCSN7B variants are generated by CSN7 isoforms. We demonstrate that the two variants coexist in human LiSa‐2 cells and in mouse embryonic fibroblasts. During adipogenic differentiation of LiSa‐2 cells CSN7B increases in parallel with an elevation of the total CSN complex. Permanent overexpression of Flag‐CSN7B but not of Flag‐CSN7A accelerates adipogenesis in LiSa‐2 cells indicating a specific function of the CSNCSN7B variant in stimulating adipogenesis. Silencing of CSN7A as well as of CSN7B in LiSa‐2 cells and in mouse embryonic fibroblasts (MEFs) reduces adipogenic differentiation demonstrating that both CSNCSN7A and CSNCSN7B variants are involved in the process.

Using the Ubiquitin-modified Proteome to Monitor Distinct and Spatially Restricted Protein Homeostasis Dysfunction

Protein homeostasis dysfunction has been implicated in the development and progression of aging related human pathologies. There is a need for the establishment of quantitative methods to evaluate global protein homoeostasis function. As the ubiquitin (ub) proteasome system plays a key role in regulating protein homeostasis, we applied quantitative proteomic methods to evaluate the sensitivity of site-specific ubiquitylation events as markers for protein homeostasis dysfunction. Here, we demonstrate that the ub-modified proteome can exceed the sensitivity of engineered fluorescent reporters as a marker for proteasome dysfunction and can provide unique signatures for distinct proteome challenges which is not possible with engineered reporters. We demonstrate that combining ub-proteomics with subcellular fractionation can effectively separate degradative and regulatory ubiquitylation events on distinct protein populations. Using a recently developed potent inhibitor of the critical protein homeostasis factor p97/VCP, we demonstrate that distinct insults to protein homeostasis function can elicit robust and largely unique alterations to the ub-modified proteome. Taken together, we demonstrate that proteomic approaches to monitor the ub-modified proteome can be used to evaluate global protein homeostasis and can be used to monitor distinct functional outcomes for spatially separated protein populations.

Saturation scanning of ubiquitin variants reveals a common hot spot for binding to USP2 and USP21

A detailed understanding of the molecular mechanisms whereby ubiquitin (Ub) recognizes enzymes in the Ub proteasome system is crucial for understanding the biological function of Ub. Many structures of Ub complexes have been solved and, in most cases, reveal a large structural epitope on a common face of the Ub molecule. However, owing to the generally weak nature of these interactions, it has been difficult to map in detail the functional contributions of individual Ub side chains to affinity and specificity. Here we took advantage of Ub variants (Ubvs) that bind tightly to particular Ub-specific proteases (USPs) and used phage display and saturation scanning mutagenesis to comprehensively map functional epitopes within the structural epitopes. We found that Ubvs that bind to USP2 or USP21 contain a remarkably similar core functional epitope, or "hot spot," consisting mainly of positions that are conserved as the wild type sequence, but also some positions that prefer mutant sequences. The Ubv core functional epitope contacts residues that are conserved in the human USP family, and thus it is likely important for the interactions of Ub across many family members.

Abstract 2770: DERL3 hypermethylation alters rhabdomyosarcoma cell metabolism

Abstract Physiological cellular activity requires a precise control of the proteome. However, cancer cells have a distorted protein profile. It is likely that intrinsic defects in protein homeostasis participate in human tumorigenesis, such as alterations in de novo protein synthesis, or defects in protein degradation. In this last case, the ubiquitin (Ub) proteasome system (UPS) targets a variety of proteins, including functional proteins that are no longer needed. Without appropriate protein homeostasis maintained by the UPS, healthy cells can undergo malignant transformation, and this observation has been therapeutically exploited by the development of proteasome inhibitors as anticancer agents. Many of the cytoplasm UPS-degraded proteins are retrotranslocated from the endoplasmic reticulum (ER). In this regard, ER possesses a quality control mechanism, termed the ER-associated degradation mechanism (ERAD), that is induced in response to ER stress by a transcriptional program, known as the unfolded protein response (UPR), which leads to the accelerated degradation of unfolded proteins. Within the ERAD pathway, the Derlin proteins play a critical role. It has been proposed that Derlins form an export channel in the membrane of the ER through which the ERAD substrates pass to reach the proteasome. A previous study in our lab has shown that DERL3 silencing by DNA methylation deregulate SLC2A1 (GLUT1) degradation, promoting the Warburg effect. SLC2A1 is a key glucose receptor up-regulated in most solid tumors, allowing cancer cells to get the glucose they need for tumor progression. In this study we use an embryonic rhabdomyosarcoma cell line as a model to find out the role of DERL3 in these tumors, as the percentage of promoter hypermethylation is these tumors is approximately 60%. Rhabdomyosarcoma (RMS) is one of the leading causes of cancer related deaths among children, being the histological variant of embryonic rhabdomyosarcoma the most common soft tissue sarcoma of childhood and adolescence, with 350 cases per year in USA. The restoration of DERL3 expression in vitro promotes GLUT1 degradation and GLUT4 up-regulation, becoming cells sensitive to insulin. Moreover, it has been shown that DERL3 confers sensitivity against an ERAD related drug, Eeyarestatin I, opening a new approach for RMS treatment. All together, these initial findings highlight the role of DERL3 in RMS metabolism. Citation Format: Pere Llinas, Paula Lopez, Manel Esteller. DERL3 hypermethylation alters rhabdomyosarcoma cell metabolism. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2770.

UBE2D3 gene overexpression increases radiosensitivity of EC109 esophageal cancer cells in vitro and in vivo.

Ubiquitin-conjugating enzyme E2D3 (UBE2D3), a key component in ubiquitin (Ub) proteasome system, plays a crucial role in tumorigenesis. We previously found that it is bound to hTERT, and UBE2D3 could attenuate radiosensitivity of human breast cancer cells. Here we investigated a contributing role of UBE2D3 in radiosensitivity of esophageal squamous carcinoma. We demonstrated that the overexpression of UBE2D3 in esophageal squamous carcinoma cells (EC109) resulted in prolonged G1 phase and shortened G2/M phase after irradiation. UBE2D3 overexpression also decreased length of telomere and activity of telomerase. In addition, the overexpression of UBE2D3 increased mRNA expression but decreased protein levels of hTERT in both vitro and vivo systems. Compared with untreated cells, the treatment of UBE2D3 overexpressing cells with the specific proteasome inhibitor (MG132) could up-regulate hTERT. MG132 treatment of UBE2D3 overexpressed cells caused a clear and dramatic increase in the amount of ubiquitinated hTERT species. These findings indicate that UBE2D3 enhances radiosensitivity of EC109 cells by degradating hTERT through the ubiquitin proteolysis pathway.

Pharmacogenomics of Bortezomib in Multiple Myeloma Patients Reveals That the Ubiquitin Ligase SCF-Skp2 Promotes Drug Resistance

The ability to couple cancer patients with genetically-targeted therapeutic strategies remains a major challenge due to the heterogeneous and evolvable nature of individual tumors. This is particularly true of the molecularly diverse and complex disease multiple myeloma (MM). Clinical efficacy of the proteasome inhibitor (PI) Bortezomib (VelcadeTM) established the Ub+proteasome system (UPS) as a key target in MM. While the therapeutic benefit of PIs in MM remains unchallenged, many patients do not respond to therapy and drug resistance develops nearly uniformly. Despite marked therapeutic advances for most patients with MM, outcomes remain poor for 15% to 20% of patients presenting with high-risk disease defined by host-related and tumor-related factors and expert guidelines recommend enrollment in a clinical trial for the majority of patients who will ultimately fail approved therapies.The Skp1-Cul1-F-box protein (SCF) ubiquitin ligase SCFSkp2 promotes ubiquitination and degradation of the cell-cycle inhibitor p27. Increased SKP2 expression and reduced p27 levels are frequent in human cancers and are associated with therapeutic resistance and poor prognosis. SCFSkp2 ubiquitination activity is increased by Commd1, a copper metabolism MURR1 domain-containing 1 protein that binds Cul1 and by Cks1B that increases binding of phosphorylated p27 to Skp2. Bortezomib is a proteasome inhibitor that has remarkably improved multiple myeloma (MM) patient outcome, but dose-limiting toxicities and development of resistance limit its long-term utility. Here, we observed higher CUL1 and COMMD1 mRNA levels in Bortezomib-resistant patient MM cells and correlated higher CUL1, COMMD1, SKP2 and CKS1B levels with decreased survival. Genetic knockdown of CUL1 or COMMD1 disrupted the SCFSkp2 complex, stabilized p27, synergistically increased the generation of annexin-positive cells by bortezomib and enhanced the anti-myeloma effect of PIs against resistant cells. Chemical library screening identified a novel compound, DT204, that reduced Skp2 association with Cul1 and Commd1, inhibited p27 degradation and induced synergistic anti-MM activity combined with Bortezomib in patient tumor cells and chemoresistant stem cell-like cells to overcame Bortezomib resistance. Bortezomib treatment with DT204 also reduced the growth of xenotransplanted myeloma cells in mouse models and prolongs host survival. Taken together, our results demonstrate that the SCFSkp2 -p27 axis represents a major determinant in the ability of Bortezomib to induce apoptosis in MM and provide proof-of-concept for the combination of SCFSkp2 inhibitors with Bortezomib as a strategy to overcome Bortezomib resistance. DisclosuresNo relevant conflicts of interest to declare.

Pharmacogenomics of Bortezomib in Multiple Myeloma Patients Reveals that the Ubiquitin Ligase SCF-Skp2 Promotes Drug Resistance

Abstract The ability to couple cancer patients with genetically-targeted therapeutic strategies remains a major challenge due to the heterogeneous and evolvable nature of individual tumors. This is particularly true of the molecularly diverse and complex disease multiple myeloma (MM). Clinical efficacy of the proteasome inhibitor (PI) Bortezomib (VelcadeTM) established the Ub+proteasome system (UPS) as a key target in MM. While the therapeutic benefit of PIs in MM remains unchallenged, many patients do not respond to therapy and drug resistance develops nearly uniformly. Despite marked therapeutic advances for most patients with MM, outcomes remain poor for 15% to 20% of patients presenting with high-risk disease defined by host-related and tumor-related factors and expert guidelines recommend enrollment in a clinical trial for the majority of patients who will ultimately fail approved therapies.The Skp1-Cul1-F-box protein (SCF) ubiquitin ligase SCFSkp2 promotes ubiquitination and degradation of the cell-cycle inhibitor p27. Increased SKP2 expression and reduced p27 levels are frequent in human cancers and are associated with therapeutic resistance and poor prognosis. SCFSkp2 ubiquitination activity is increased by Commd1, a copper metabolism MURR1 domain-containing 1 protein that binds Cul1 and by Cks1B that increases binding of phosphorylated p27 to Skp2. Bortezomib is a proteasome inhibitor that has remarkably improved multiple myeloma (MM) patient outcome, but dose-limiting toxicities and development of resistance limit its long-term utility. Here, we observed higher CUL1 and COMMD1 mRNA levels in Bortezomib-resistant patient MM cells and correlated higher CUL1, COMMD1, SKP2 and CKS1B levels with decreased survival. Genetic knockdown of CUL1 or COMMD1 disrupted the SCFSkp2 complex, stabilized p27, synergistically increased the generation of annexin-positive cells by bortezomib and enhanced the anti-myeloma effect of PIs against resistant cells. Chemical library screening identified a novel compound, DT204, that reduced Skp2 association with Cul1 and Commd1, inhibited p27 degradation and induced synergistic anti-MM activity combined with Bortezomib in patient tumor cells and chemoresistant stem cell-like cells to overcame Bortezomib resistance. Bortezomib treatment with DT204 also reduced the growth of xenotransplanted myeloma cells in mouse models and prolongs host survival. Taken together, our results demonstrate that the SCFSkp2 -p27 axis represents a major determinant in the ability of Bortezomib to induce apoptosis in MM and provide proof-of-concept for the combination of SCFSkp2 inhibitors with Bortezomib as a strategy to overcome Bortezomib resistance. Disclosures No relevant conflicts of interest to declare.

MJD and OTU deubiquitinating enzymes in Schistosoma mansoni.

The ubiquitination and deubiquitination of proteins can alter diverse cellular processes, such as proteolysis, trafficking, subcellular localisation, DNA repair, apoptosis and signal transduction. Deubiquitinating enzymes (DUBs) are responsible for removing ubiquitin from their target proteins. Previous reports have shown the presence of two subfamilies of DUBs in Schistosoma mansoni: Ub carboxyl-terminal hydrolase (UCH) and Ub-specific protease (USP). In this study, we analysed the ovarian tumour (OTU) and Machado-Joseph disease protein domain (MJD) proteases found in the Schistosoma mansoni genome database. An in silico analysis identified two different MJD subfamily members, SmAtaxin-3 and SmJosephin, and five distinct OTU proteases, SmOTU1, SmOTU3, SmOTU5a, SmOTU6b and SmOtubain. The phylogenetic analysis showed the evolutionary conservation of these proteins. Furthermore, the 3D structures confirmed the similarity of these proteins with human proteins. In addition, we performed quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and observed distinct expression profiles for all of the investigated transcripts between the cercariae, schistosomula and adult worm stages. Taken together, our data suggest that MJD and OTU subfamily members contribute to regulating the activity of the Ub-proteasome system during the life cycle of this parasite.