Ubiquitination Of Cellular Proteins Research Articles

Depletion of proteasome subunit PSMD1 induces cancer cell death via protein ubiquitination and DNA damage, irrespective of p53 status

Hepatocellular carcinoma (HCC) is characterized by high incidence and fatality rates worldwide. In our exploration of prognostic factors in HCC, the 26s proteasome subunit, non-ATPase 1 (PSMD1) protein emerged as a significant contributor, demonstrating its potential as a therapeutic target in this aggressive cancer. PSMD1 is a subunit of the 19S regulatory particle in the 26S proteasome complex; the 19S particle controls the deubiquitination of ubiquitinated proteins, which are then degraded by the proteolytic activity of the complex. Proteasome-targeting in cancer therapy has received significant attention because of its practical application as an established anticancer agent. We investigated whether PSMD1 plays a critical role in cancer owing to its prognostic significance. PSMD1 depletion induced cell cycle arrest in G2/M phase, DNA damage and apoptosis of cancer cells, irrespective of the p53 status. PSMD1 depletion-mediated cell death was accompanied by an increase in overall protein ubiquitination. These phenotypes occurred exclusively in cancer cells, with no effects observed in normal cells. These findings indicate that PSMD1 depletion-mediated ubiquitination of cellular proteins induces cell cycle arrest and eventual death in cancer cells, emphasizing PSMD1 as a potential therapeutic target in HCC.

Allantopyrone A interferes with the degradation of hypoxia-inducible factor 1α protein by reducing proteasome activity in human fibrosarcoma HT-1080 cells.

Allantopyrone A is an α-pyrone metabolite that was originally isolated from the endophytic fungus Allantophomopsis lycopodina KS-97. We previously demonstrated that allantopyrone A exhibits anti-cancer, anti-inflammatory, and neuroprotective activities. In the present study, we showed that allantopyrone A up-regulated the protein expression of hypoxia-inducible factor (HIF)-1α in human fibrosarcoma HT-1080 cells. It also up-regulated the mRNA expression of BNIP3 and ENO1, but not other HIF target genes or HIF1A. Allantopyrone A did not inhibit the prolyl hydroxylation of HIF-1α, but enhanced the ubiquitination of cellular proteins. Consistent with this result, chymotrypsin-like and trypsin-like proteasome activities were reduced, but not completely inactivated by allantopyrone A. Allantopyrone A decreased the amount of proteasome catalytic subunits. Therefore, the present results showed that allantopyrone A interfered with the degradation of HIF-1α protein by reducing proteasome activity in human fibrosarcoma HT-1080 cells.

Novel role of HIV-1 Nef in regulating the ubiquitination of cellular proteins.

Our recent data established that HIV-1 Nef is pivotal in determining the fate of cellular proteins by modulating ubiquitination. However, it is unknown which proteins are ubiquitinated in the presence of Nef, a question critical for understanding the proliferation/restriction strategies of HIV-1 in infected cells. To identify cellular proteins ubiquitinated by Nef, we conducted a proteomic analysis of cellular proteins in the presence and absence of Nef. Proteomic analysis in HEK293T cells indicated that 93 proteins were upregulated and 232 were downregulated in their ubiquitination status by Nef. Computational analysis classified these proteins based on molecular function, biological process, subcellular localization, and biological pathway. Of those proteins, we found a majority of molecular functions to be involved in binding and catalytic activity. With respect to biological processes, a significant portion of the proteins identified were related to cellular and metabolic processes. Subcellular localization analysis showed the bulk of proteins to be localized to the cytosol and cytosolic compartments, which is consistent with the known function and location of Nef during HIV-1 infection. As for biological pathways, the wide range of affected proteins was denoted by the multiple modes to fulfill function, as distinguished from a strictly singular means, which was not detected. Among these ubiquitinated proteins, six were found to directly interact with Nef, wherein two were upregulated and four downregulated. We also identified 14 proteins involved in protein stability through directly participating in the Ubiquitin Proteasome System (UPS)-mediated proteasomal degradation pathway. Of those proteins, we found six upregulated and eight downregulated. Taken together, these analyses indicate that HIV-1 Nef is integral to regulating the stability of various cellular proteins via modulating ubiquitination. The molecular mechanisms directing Nef-triggered regulation of cellular protein ubiquitination are currently under investigation.

The UBE2D ubiquitin conjugating enzymes: Potential regulatory hubs in development, disease and evolution.

Ubiquitination of cellular proteins plays critical roles in key signalling pathways and in the regulation of protein turnover in eukaryotic cells. E2 ubiquitin conjugating enzymes function as essential intermediates in ubiquitination reactions by acting as ubiquitin donors for the E3 ubiquitin ligase enzymes that confer substrate specificity. The members of the UBE2D family of E2 enzymes are involved in regulating signalling cascades through ubiquitination of target proteins that include receptor tyrosine kinases (RTKs) and components of the Hedgehog, TGFβ and NFκB pathways. UBE2D enzymes also function in transcriptional control by acting as donors for ubiquitination of histone tails by the Polycomb protein Ring1B and the DNA methylation regulator UHRF1 as well as having roles in DNA repair and regulation of the level of the tumour suppressor p53. Here we review the functional roles and mechanisms of regulation of the UBE2D proteins including recent evidence that regulation of the level of UBE2D3 is critical for controlling ubiquitination of specific targets during development. Cellular levels of UBE2D3 have been shown to be regulated by phosphorylation, which affects folding of the protein, reducing its stability. Specific variations in the otherwise highly conserved UBE2D3 protein sequence in amniotes and in a subgroup of teleost fishes, the Acanthomorpha, suggest that the enzyme has had important roles during vertebrate evolution.

In vitro Auto- and Substrate-Ubiquitination Assays.

The precise regulation of the homeostasis of the cellular proteome is critical for the appropriate growth and development of plants. It also allows the plants to respond to various environmental stresses, by modulating their biochemical and physiological aspects in a timely manner. Ubiquitination of cellular proteins is one of the major protein degradation routes for maintaining cellular protein homeostasis, and ubiquitin E3 ligases, components of ubiquitin ligase complexes, play an important role in the selective degradation of target proteins via substrate-specific interactions. Thus, understanding the role of E3 ligases and their substrate regulation uncovers their specific cellular and physiological functions. Here, we provide protocols for auto- and substrate-ubiquitination analyses that utilize the combination of in vitro purified E3 ubiquitin ligase proteins and immunoprecipitation.

Targeting a helix-in-groove interaction between E1 and E2 blocks ubiquitin transfer.

The ubiquitin-proteasome system (UPS) is a highly-regulated protein disposal process critical to cell survival. Inhibiting the pathway induces proteotoxic stress and can be effective cancer treatment. The therapeutic window observed upon proteosomal blockade has motivated multiple UPS-targeting strategies, including preventing ubiquitination altogether. E1 initiates the cascade by transferring ubiquitin to E2 enzymes. A small molecule that engages the E1 ATP-binding site and derivatizes ubiquitin disrupts enzymatic activity and kills cancer cells. However, binding-site mutations cause resistance, motivating alternative approaches to block this promising target. We identified an interaction between the E2 N-terminal alpha-1 helix and a pocket within the E1 ubiquitin-fold domain as a potentially druggable site. Stapled peptides modeled after the E2 alpha-1 helix bound to the E1 groove, induced a consequential conformational change, and inhibited E1 ubiquitin-thiotransfer, disrupting E2 ubiquitin-charging and ubiquitination of cellular proteins. Thus, we provide a blueprint for a distinct E1-targeting strategy to treat cancer.

HIV-1 Impairment via UBE3A and HIV-1 Nef Interactions Utilizing the Ubiquitin Proteasome System.

Molecular basis of HIV-1 life cycle regulation has thus far focused on viral gene stage-specificity, despite the quintessence of post-function protein elimination processes in the virus life cycle and consequent pathogenesis. Our studies demonstrated that a key pathogenic HIV-1 viral protein, Nef, interacted with ubiquitin (Ub)-protein ligase E3A (UBE3A/E6AP), suggesting that interaction between Nef and UBE3A is integral to regulation of viral and cellular protein decay and thereby the competing HIV-1 and host cell survivals. In fact, Nef and UBE3A degraded reciprocally, and UBE3A-mediated degradation of Nef was significantly more potent than Nef-triggered degradation of UBE3A. Further, UBE3A degraded not only Nef but also HIV-1 structural proteins, Gag, thus significantly inhibiting HIV-1 replication in Jurkat T cells only in the presence of Nef, indicating that interaction between Nef and UBE3Awas pivotal for UBE3A-mediated degradation of the viral proteins. Mechanistic study showed that Nef and UBE3A were specific and antagonistic to each other in regulating proteasome activity and ubiquitination of cellular proteins in general, wherein specific domains of Nef overlapping with the long terminal repeat (LTR) were essential for the observed actions. Further, Nef itself reduced the level of intracellular Gag by degrading a cardinal transcription regulator, Tat, demonstrating a broad role for Nef in the regulation of the HIV-1 life cycle. Taken together, these data demonstrated that the Nef and UBE3A complex plays a crucial role in coordinating viral protein degradation and hence HIV-1 replication, providing insights as to the nature of pathobiologic and defense strategies of HIV-1 and HIV-infected host cells.

Regulation of Phosphoribosyl-Linked Serine Ubiquitination by Deubiquitinases DupA and DupB.

SummaryThe family of bacterial SidE enzymes catalyzes non-canonical phosphoribosyl-linked (PR) serine ubiquitination and promotes infectivity of Legionella pneumophila. Here, we describe identification of two bacterial effectors that reverse PR ubiquitination and are thus named deubiquitinases for PR ubiquitination (DUPs; DupA and DupB). Structural analyses revealed that DupA and SidE ubiquitin ligases harbor a highly homologous catalytic phosphodiesterase (PDE) domain. However, unlike SidE ubiquitin ligases, DupA displays increased affinity to PR-ubiquitinated substrates, which allows DupA to cleave PR ubiquitin from substrates. Interfering with DupA-ubiquitin binding switches its activity toward SidE-type ligase. Given the high affinity of DupA to PR-ubiquitinated substrates, we exploited a catalytically inactive DupA mutant to trap and identify more than 180 PR-ubiquitinated host proteins in Legionella-infected cells. Proteins involved in endoplasmic reticulum (ER) fragmentation and membrane recruitment to Legionella-containing vacuoles (LCV) emerged as major SidE targets. The global map of PR-ubiquitinated substrates provides critical insights into host-pathogen interactions during Legionella infection.

A biosensor study of protein interaction with the 20S proteasome core particle

It becomes increasingly clear that ubiquitination of cellular proteins is not an indispensable prerequisite of their degradation in proteasomes. There are a number of proteins to be eliminated which are not pre-ubiquitinated for their recognition by regulatory subcomplex of 26S proteasome, but which directly interact with the 20S proteasome core particle (20S proteasome). The obligatory precondition for such interaction consists in existence of disordered (hydrophobic) fragments in the target protein. In this study we have investigated the interaction of a number of multifunctional (moonlighting) proteins (glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase, pyruvate kinase) and neurodegeneration-related proteins (a-synuclein, myelin basic protein) with 20S proteasome immobilized on the SPR-biosensor chip and stabilized by means of a bifunctional agent dimethyl pimelimidate (in order to prevent possible dissociation of this subcomplex). Only two of all investigated proteins (aldolase and pyruvate kinase) interacted with the immobilized 20S proteasome (Kd of 8.17´10-7 M and 5.56´10-7 M, respectively). In addition to earlier detected GAPDH ubiquitination, mass spectrometric analysis of the studied proteins revealed the presence of the ubiquitin signature (Lys-e-Gly-Gly) only in aldolase. Oxidation of aldolase and pyruvate kinase, which promotes elimination of proteins via their direct interaction with 20S proteasome, caused a 2-3-fold decrease in their Kd values as comparison with this parameter obtained for the intact proteins. The results of this study provide further evidence for direct interaction of both ubiquitinated proteins (aldolase), and non-ubiquitinated proteins (pyruvate kinase) with the 20S proteasome core particle (20S proteasome). The effectiveness of this interaction is basically equal for the ubiquitinated proteins and non-ubiquitinated proteins.

Global Proteomic Changes Induced by the Epstein-Barr Virus Oncoproteins Latent Membrane Protein 1 and 2A.

ABSTRACTThe Epstein-Barr virus (EBV) oncoproteins latent membrane protein 1 (LMP1) and LMP2A constitutively activate multiple signaling pathways, and both have been shown to interact with cellular ubiquitin ligases and affect cellular ubiquitination. To detect the LMP1- and LMP2A-mediated effects on the global cellular proteome, epithelial cell lines expressing LMP1 or LMP2A were analyzed using label-free quantitative proteomics. To identify proteins whose ubiquitination is affected by the viral proteins, the cells were cultured in the presence and absence of deubiquitinase (DUB) and proteasome inhibitors. More than 7,700 proteins were identified with high confidence and considerably more proteins showed significant differences in expression in the presence of inhibitors. Few of the differentially expressed proteins with or without inhibitors were common between LMP1 and LMP2A, confirming that the viral proteins induce unique changes in cell expression and function. However, ingenuity pathway analysis (IPA) of the data indicated that LMP1 and LMP2A modulate many of the same cellular regulatory pathways, including cell death and survival, cell movement, and actin filament dynamics. In addition, various proteasome subunits, ubiquitin-specific peptidases and conjugating enzymes, vesicle trafficking proteins, and NF-κB and mitogen-activated protein kinase signaling proteins were affected by LMP1 or LMP2A. These findings suggest that LMP1 and LMP2A may commonly target critical cell pathways through effects on distinct genes, with many cellular proteins modified by ubiquitination and/or degradation.

Interfering with Arginine Metabolism As a New Treatment Strategy for Multiple Myeloma

IntroductionStarvation of tumor cells from the amino acid arginine has recently gained particular interest because of the downregulation of the rate-limiting enzyme argininosuccinate synthethase 1 (ASS1) in various cancer entities. ASS1-deficient cells cannot resynthesize arginine from citrulline and are therefore considered arginine auxotrophic. The arginine depleting enzyme arginine deiminase (ADI-PEG20, Polaris Pharmaceuticals) is currently tested in phase I-III clinical trials for different arginine auxotrophic cancers. The natural arginine analogue canavanine can compete with arginine for arginyl-tRNA-binding sites and consequently be incorporated into nascent proteins instead of arginine. Canavanine could therefore potentially further disturb intracellular protein homeostasis, especially under arginine deprivation. The sensitivity of myeloma cells towards arginine depletion strategies has not been analyzed so far.MethodsHuman myeloma cell lines and CD138-sorted primary human myeloma cells from patient bone marrow were screened for ASS1 expression by western blotting (WB). The cells were cultured in arginine free medium and assessed for proliferation and metabolic activity (CFSE/MTT assays), apoptosis (caspase-3 cleavage) and cell death (annexinV/propidium iodide). Canavanine was supplied in both arginine-sufficient and -deficient conditions. The level of intracellular protein stress was determined by WB and/or flow cytometry analysis for ubiquitinated proteins, phosphorylated eukaryotic initiation factor 2α (peIF2α) and the spliced isoform of the X-Box binding protein 1 (Xbp1s). Repetitive ADI-PEG20 ± canavanine application i.p. were tested in vivo in an U266 myeloma xenograft model in NOD/SCID/IL2Rcg-/- (NSG) mice. Arginine and canavanine levels in plasma were determined by HPLC. Tumor growth was measured, mice were assessed for survival, weight and side effects. Tumor tissues were analyzed for caspase-3 cleavage and Ki67 expression by immunohistochemistry.Results5 of 6 myeloma cell lines were negative for ASS1. Also, ASS1 was either not or only weakly expressed in the majority of primary CD138+ myeloma patient samples. Arginine starvation induced an arrest of cell proliferation and/or metabolic activity of primary myeloma cells and myeloma cell lines after 18-24 h. Addition of citrulline could only rescue ASS1 positive myeloma cells due to the intracellular resynthesis of arginine. Arginine starvation alone led to delayed induction of apoptosis (e.g. 35% cell death of NCI-H929 cells after 72 h of treatment). Addition of 100 mM canavanine strongly increased cell death specifically in the context of arginine deficiency (e.g. cell death in NCI-H929 cells: 87% after 24 h, 100 % after 48h) while it was non-toxic and had no effect on cell viability under physiological arginine conditions. Co-application of canavanine induced ubiquitination of cellular proteins and led to the prolongation of a fatal unfolded protein response (UPR) as measured by markedly elevated Xbp1s levels. Prolonged UPR ultimately led to the induction of apoptosis as reflected by annexin V binding and caspase-3 cleavage. In an U266 myeloma NSG xenograft model, systemic arginine depletion by ADI-PEG20 suppressed tumor growth in vivo and significantly prolonged median survival of mice when compared with the control group (22±3 vs. 15±3 days). Canavanine treatment alone had no influence on viability (13±0 days). However, the combination of ADI-PEG20 and canavanine demonstrated the longest median survival (27±7 days). Histological examination of explanted tumors showed the highest rates of caspase-3 cleavage in the ADI-PEG20/canavanine group.ConclusionMyeloma cells are mostly arginine auxotrophic and can be selectively targeted by arginine starvation. Combination of arginine depletion with the arginine analogue canavanine leads to a highly efficient and specific tumor cell eradication and should be further optimized in multiple myeloma preclinical models. DisclosuresBomalaski:Polaris Pharmaceuticals Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

The nonstructural protein 11 of porcine reproductive and respiratory syndrome virus inhibits NF-κB signaling by means of its deubiquitinating activity.

Since its emergence in the late 1980s, porcine reproductive and respiratory syndrome (PRRS) has been devastating the swine industry worldwide. The causative agent is an Arterivirus, referred to as PRRS virus (PRRSV). The pathogenic mechanisms of PRRS are poorly understood, but are believed to correlate with the ability of PRRSV to inhibit immune responses of the host. However, precisely how the virus is capable of doing so remains obscure. In this study, we showed that PRRSV infection led to reduced ubiquitination of cellular proteins. Screening all of the 12 nonstructural proteins (Nsps) encoded by PRRSV revealed that, apart from the Nsp2 which contains the deubiqintinating (DUB) ovarian tumor (OTU) domain, Nsp11, which encodes a unique and conserved endoribonuclease (NendoU) throughout the Nidovirus order, also possesses DUB activity. In vivo assay demonstrated that Nsp11 specifically removed lysine 48 (K48)-linked polyubiquitin chains and the conserved sites C112, H144, D173, K180, and Y219 were critical for its DUB activity. Remarkably, DUB activity was responsible for the capacity of Nsp11 to inhibit nuclear factor κB (NF-κB) activation. Mutations abrogating the DUB activity of Nsp11 toward K48-linked polyubiquitin chains of IκBα nullified the suppressive effect on NF-κB. Our data add Nsp11 to the list of DUBs encoded by PRRSV and uncover a novel mechanism by which PRRSV cripples host innate immune responses.

Exosome poly‐ubiquitin inhibits platelet activation, downregulates CD36 and inhibits pro‐atherothombotic cellular functions

Activated platelets shed microparticles from plasma membranes, but also release smaller exosomes from internal compartments. While microparticles participate in athero-thrombosis, little is known of exosomes in this process. Ex vivo biochemical experiments with human platelets and exosomes, and FeCl3 -induced murine carotid artery thrombosis. Both microparticles and exosomes were abundant in human plasma. Platelet-derived exosomes suppressed ex vivo platelet aggregation and reduced adhesion to collagen-coated microfluidic channels at high shear. Injected exosomes inhibited occlusive thrombosis in FeCl3 -damaged murine carotid arteries. Control platelets infused into irradiated, thrombocytopenic mice reconstituted thrombosis in damaged carotid arteries, but failed to do so after prior ex vivo incubation with exosomes.CD36 promotes platelet activation, and exosomes dramatically reduced platelet CD36.CD36 is also expressed by macrophages, where it binds and internalizes oxidized LDL and microparticles, supplying lipid to promote foam cell formation. Platelet exosomes inhibited oxidized-LDL binding and cholesterol loading into macrophages. Exosomes were not competitive CD36 ligands, but instead sharply reduced total macrophage CD36 content. Exosomal proteins, in contrast to microparticle or cellular proteins, were highly adducted by ubiquitin. Exosomes enhanced ubiquitination of cellular proteins, including CD36, and blockade of proteosome proteolysis with MG-132 rescued CD36 expression. Recombinant unanchored K48 poly-ubiquitin behaved similarly to exosomes, inhibiting platelet function, macrophage CD36 expression and macrophage particle uptake. Platelet-derived exosomes inhibit athero-thrombotic processes by reducing CD36-dependent lipid loading of macrophages and by suppressing platelet thrombosis. Exosomes increase protein ubiquitination and enhance proteasome degradation of CD36.

Enterovirus 71 induces degradation of TRIM38, a potential E3 ubiquitin ligase

BackgroundThe tripartite motif (TRIM) proteins are a family of more than 70 members in human. However, only a few of them have been well studied. The TRIM proteins contain the conserved RING, B-box, coiled-coil, and SPRY domains, most of which are involved in protein ubiquitination. TRIM38 is a member of the TRIM protein family, which we studied in more detail here as its functions are largely unknown.ResultsOur study shows that, similar to other TRIM family members, TRIM38 is localized in the cytoplasm. TRIM38 increases ubiquitination of other cellular proteins and catalyzes self-ubiquitination. TRIM38 also promotes K63- and K48-linked ubiquitination of cellular proteins. An intact RING domain is important for the functions of TRIM38. In addition, enterovirus 71 infection induces TRIM38 degradation.ConclusionsOur observations demonstrate that TRIM38 has E3 ubiquitin ligase activity and can be degraded during virus infection. These findings may provide insight into innate immune signaling pathways.

Nitric Oxide Increases Lysine-48-Linked Ubiquitination Of Cellular Proteins

Introduction: Proteins are targeted for degradation by the 26S proteasome by the addition of a polyubiquitin chain. Ubiquitin molecules can be linked through different lysine residues. The lysine 48 linkage (K48-Ub) is associated with protein degradation while the lysine 63 linkage (K63-Ub) is associated with intracellular signaling. Nitric oxide (NO) has many vasoprotective properties, including inhibition of neointimal hyperplasia, and we have previously shown that NO inhibits the ubiquitin-conjugating enzymes that regulate cell cycle progression. The aim of this current study is to characterize the effect of arterial injury on K48 or K63 ubiquitin bonds, with and without exposure to NO. Our hypothesis is that injury will increase, while NO will decrease, K48-linked ubiquitination of cellular proteins. Methods: The carotid injury model was performed in 11-wk male Sprague Dawley rats. Treatment groups included control, injury, injury+NO (PROLI/NO, 10mg), and NO alone (n=3/group). Arteries were harvested at 2 wks for immunohistochemical analysis and blinded grading of K48-Ub and K63-Ub expression (scale 0-3). Arteries were also harvested after 2, 24 and 72 hrs and homogenized for protein analysis. Vascular smooth muscle cells (VSMC) and adventitial fibroblasts (AF) were exposed to the NO donor DETA/NO (0.125-1 mM). Protein expression was determined by Western blot analysis, or immunoprecipitation (IP) and Western blot analysis. Results: Minimal expression of K48-Ub and K63-Ub was observed in uninjured control arteries (0±0). Injury increased K48-Ub in the neointima (3.0±0.0) and adventitia (2.0±0.2). NO decreased K48-Ub in the neointima (1.0±0.2) following injury, but had no effect on the adventitia (2.2±0.1). Interestingly, NO alone increased K48-Ub in the adventitia (0.7±0.0). Changes in K63-Ub expression were smaller. Injury slightly increased K63-Ub in the neointima (1.1±0.4) and adventitia (0.7±0.2). NO slightly decreased K63-Ub in the neointima (0.3±0.2) following injury, but slightly increased K63-Ub in the adventitia (1.3±0.2). No changes were seen in the media in any treatment group. Analysis of carotid lystates showed that injury increased K48-Ub expression at all time points, while injury+NO further increased K48-Ub. Minimal changes in K63-Ub were seen among all groups. Analysis of VSMC showed an increase in K48-Ub and no change in K63-Ub expression with DETA/NO in vitro. In VSMC and AF, IP revealed that DETA/NO increased expression of K48-Ub-linked cyclin A and B but did not change expression of K63-Ub-linked cyclin A and B. Conclusions: Supporting our hypothesis, we have shown that injury increases K48-linked ubiquitination in vivo. Surprisingly, NO further increases K48-linked ubiquitination in vitro and in vivo. These changes are a remarkable contrast to K63-linked ubiquitination, where minimal changes were seen. These novel findings provide important new insights into the mechanism by which NO prevents neointimal hyperplasia by regulating the ubiquitin-proteasome pathway.

De-Ubiquitinase Inhibition by WP1130 Induces Formation of Aggresomes, Engages Autophagy and Activates Apoptosis in B-Cell Malignancies.

Abstract 3769Poster Board III-705Nearly all protein in mammalian cells is subject to regulation by ubiquitination and it is becoming clear that modification of this process may have therapeutic activity in several diseases, including cancer. Bortezomib and Hsp90 inhibitors affect protein behavior through ubiquitination and have therapeutic activity in specific cancers. These observations suggest that additional molecules that alter protein ubiquitination through a distinct mechanism may have therapeutic value. Previously we described a small molecule, WP1130, that inhibited cytokine and Bcr-Abl mediated signaling through a unique mechanism (Blood. 2007;109(8):3470-8, Cancer Res. 2007;67(8):3912-8). In this study we examined the mechanism of WP1130 action. Like bortezomib, WP1130 induced rapid, time and dose-dependent protein ubiquitination in treated cells. However unlike bortezomib, WP1130 treatment resulted in accumulation of ubiquitinated proteins in the detergent insoluble cell fraction. Sub-cellular fractionation studies revealed deposition of ubiquitinated proteins into the cytoskeletal-enriched fraction of intra-cellular structures called aggresomes. Since proteosome inhibition also results in aggresome formation, confocal microscopic examination of WP1130 treated cells was performed which demonstrated formation of peri-nuclear aggresomes containing ubiquitinated proteins, in complex with heat shock chaperones and other aggresome markers such as HDAC6 and γ-tubulin. However, WP1130 did not directly or indirectly inhibit the 20S proteasome, did not disrupt HSP90 activity or induce oxidative stress, distinguishing its mechanism from other agents. Since aggresomes also acts as disposal grounds for accumulated insoluble protein aggregates through autophagy, we assessed whether autophagy was engaged as a consequence of aggresome formation in WP1130 treated cells. We observed activation of the autophagy pathway, evident by the rapid conversion of LC3-I to LC3-II and decrease in p62/SQSTM1 protein levels. We also compared WP1130 to its parental compound, AG490, and found no changes in ubiquitination or an induction of autophagy with AG490. Since autophagy and aggresome formation are known to be cytoprotective measures initiated by cells to cope with unfavorable growth conditions, inhibition of autophagy may enhance cytotoxicity associated with WP1130. Co-treatment of cells with WP1130 and wortmannin, a known inhibitor of autophagy, resulted in a rapid increase of caspase-3 activity, culminating in enhanced cell death. Biochemical evidence supports a role for de-ubiquitinase (DUB) inhibition in WP1130 treated cells as a mechanism of enhanced resident ubiquitination of cellular proteins. Lysates from WP1130 treated cells displayed time- and concentration-dependent decreased DUB activity. These results suggest that WP1130 initiates rapid and robust protein ubiquitination through suppression of cellular DUB activity to initiate an alternate pathway to control protein behavior and induce apoptosis in specific B-cell malignancies which are highly sensitive to WP1130 and other ubiquitin content modifying agents. Disclosures:No relevant conflicts of interest to declare.

Synthetic fibril peptide promotes clearance of scrapie prion protein by lysosomal degradation

Transmissible spongiform encephalopathies are infectious and neurodegenerative disorders that cause neural deposition of aggregates of the disease-associated form of PrP(Sc). PrP(Sc) reproduces by recruiting and converting the cellular PrP(C), and ScN2a cells support PrP(Sc) propagation. We found that incubation of ScN2a cells with a fibril peptide named P9, which comprises an intrinsic sequence of residues 167-184 of mouse PrP(C), significantly reduced the amount of PrP(Sc) in 24 hr. P9 did not affect the rates of synthesis and degradation of PrP(C). Interestingly, immunofluorescence analysis showed that the incubation of ScN2a cells with P9 induced colocalization of the accumulation of PrP with cathepsin D-positive compartments, whereas the accumulation of PrP in the cells without P9 colocalized mainly with lysosomal associated membrane proteins (LAMP)-1-positive compartments but rarely with cathepsin D-positive compartments in perinuclear regions. Lysosomal enzyme inhibitors attenuated the anti-PrP(Sc) activity; however, a proteasome inhibitor did not impair P9 activity. In addition, P9 neither promoted the ubiquitination of cellular proteins nor caused the accumulation of LC3-II, a biochemical marker of autophagy. These results indicate that P9 promotes PrP(Sc) redistribution from late endosomes to lysosomes, thereby attaining PrP(Sc) degradation.

Pyruvate Dehydrogenase Complex Deficiency Caused by Ubiquitination and Proteasome-mediated Degradation of the E1β Subunit

Congenital deficiencies of the human pyruvate dehydrogenase (PDH) complex are considered to be due to loss of function mutations in one of the component enzymes. Here we describe a case of PDH deficiency associated with the PDH E1beta subunit (PDHB) gene. The clinical phenotype of the patient was consistent with reported cases of PDH deficiency. Cultured skin fibroblasts demonstrated a 55% reduction in PDH activity and markedly decreased immunoreactivity for PDHB protein, compared with healthy controls. Surprisingly, nucleotide sequence analyses of cDNAs corresponding to the patient PDH E1alpha (PDHA1) and PDHB genes revealed no pathological mutations. Moreover, the relative expression level of PDHB mRNA and the rates of transcription and translation of the PDHB gene were normal. However, PDC activity could be restored in cells from this patient following treatment with MG132, a specific proteasome inhibitor, and normal levels of E1beta could be detected in MG132-treated cells. Similar results were obtained following treatment with Tyr-phostin 23 (Tyr23), a specific inhibitor of epidermal growth factor receptor-protein-tyrosine kinase (EGFR-PTK), which also restored E1beta protein levels to those in cells from healthy subjects or from patients with PDHA1 deficiency. The index patient's cells contained a high basal level of EGFR-PTK activity that correlated with the high level of ubiquitination of cellular proteins, although the total EGFR protein levels were similar to those in cells from Elalpha-deficient subjects and healthy subjects. These data indicate that PDH deficiency in our patient involves a post-translational modification in which EGFR-PTK-mediated tyrosine phosphorylation of the E1beta protein leads to enhanced ubiquitination followed by proteasome-mediated degradation. They also provide a novel mechanism accounting for congenital deficiency of the PDH complex and perhaps other inborn errors of metabolism.

7-ketocholesterol induces protein ubiquitination, myelin figure formation, and light chain 3 processing in vascular smooth muscle cells.

Oxysterols such as 7-ketocholesterol (7-KC) are important mediators of cell death in atherosclerosis. Therefore, in vitro studies of human smooth muscle cell (SMC) death in response to 7-KC were undertaken to investigate the potential mechanisms. Human aortic SMCs treated with 7-KC showed enhanced immunoreactivity for the oxidative stress marker 4-hydroxy-2-nonenal and upregulated several stress genes (70-kDa heat shock protein 1, heme oxygenase 1, and growth arrest and DNA damage-inducible protein 153) at the mRNA but not at the protein level. 7-KC-treated SMCs rapidly underwent cell death as determined by neutral red, counting of adherent cells, and depolarization of the mitochondrial inner membrane. Cell death was associated with upregulation of ubiquitin mRNA and ubiquitination of cellular proteins. Inhibition of the proteasome by lactacystin potentiated considerably the toxicity of 7-KC. Transmission electron microscopy revealed formation of myelin figures, extensive vacuolization, and depletion of organelles. Formation of autophagosomes was suggested by labeling cells with LysoTracker and monitoring processing of microtubule-associated protein 1 light chain 3 (LC3). Analogous to our in vitro studies, human atherosclerotic plaques showed signs of ubiquitination in SMCs. 7-KC activates the ubiquitin-proteasome system and induces a complex mode of cell death associated with myelin figure formation and processing of LC3 evocating autophagic processes.

The ubiquitin system: from basic mechanisms to the patient bed.

In the ubiquitin system, a target substrate is modified by ubiquitin or a ubiquitin-like protein. This modification remodels the surface of the target proteins, affecting, among other properties, their stability, interactions with other proteins, activity, and subcellular localization. At least 10 different modifiers have been described in mammalian cells and conjugation of each modifier to its target may result in a different biological effect. In many cases proteins are modified by multiple moieties of ubiquitin that generate a branched polyubiquitin chain. For most proteins, this modification leads to their degradation by the 26S proteasome. Yet, dependent on the character of the internal linkage between the ubiquitin moieties, it can also lead to activation of transcriptional regulators. Modification by a single moiety of ubiquitin can target proteins for degradation in the lysosome/vacuole. Conjugation of ubiquitin or ubiquitin-like proteins can serve a variety of non-proteolytic functions, such as activation of enzymes, modulation of membrane dynamics, or routing of the tagged proteins to their sub-cellular destination. Ubiquitination of cellular proteins is a highly complex, temporally controlled, and tightly regulated process that targets, in a specific manner, thousands of cellular proteins. It is carried out by a modular cascade of enzymes with high specificity towards defined structural motifs in the target proteins. It has emerged as a critically important post-translational modification that plays major roles in regulating a broad array of basic cellular processes, such as cell division, differentiation, signal transduction, trafficking, and quality control. Not surprisingly, aberrations in the system have been implicated in the pathogenesis of many diseases, certain malignancies, neurodegenerative disorders and pathologies of the inflammatory and immune response among them. Understanding of the underlying mechanisms involved is important for the development of novel, mechanism-based drugs.