The infected cell protein 0 of herpes simplex virus 1 dynamically interacts with proteasomes, binds and activates the cdc34 E2 ubiquitin-conjugating enzyme, and possesses in vitro E3 ubiquitin ligase activity

The infected cell protein 0 (ICP0) of herpes simplex virus 1, a promiscuous transactivator shown to enhance the expression of genes introduced into cells by infection or transfection, interacts with numerous cellular proteins and has been linked to the disruption of ND10 and degradation of several proteins. ICP0 contains a RING finger domain characteristic of a class of E3 ubiquitin ligases. We report that: (i) in infected cells, ICP0 interacts dynamically with proteasomes and is bound to proteasomes in the presence of the proteasome inhibitor MG132. Also in infected cells, cdc34, a polyubiquitinated E2 ubiquitin-conjugating enzyme, exhibits increased ICP0-dependent dynamic interaction with proteasomes. (ii) In an in vitro substrate-independent ubiquitination system, the RING finger domain encoded by exon 2 of ICP0 binds cdc34, whereas the carboxyl-terminal domain of ICP0 functions as an E3 ligase independent of the RING finger domain. The results indicate that ICP0 can act as a unimolecular E3 ubiquitin ligase and that it promotes ubiquitin-protein ligation and binds the E2 cdc34. It differs from other unimolecular E3 ligases in that the domain containing the RING finger binds E2, whereas the ligase activity maps to a different domain of the protein. The results also suggest that ICP0 shuttles between nucleus and cytoplasm as a function of its dynamic interactions with proteasomes.

Effect of prolonged intravenous glucose and essential amino acid infusion on nitrogen balance, muscle protein degradation and ubiquitin-conjugating enzyme gene expression in calves

Background: Intravenous infusions of glucose and amino acids increase both nitrogen balance and muscle accretion. We hypothesised that co-infusion of glucose ( to stimulate insulin) and essential amino acids (EAA) would act additively to improve nitrogen balance by decreasing muscle protein degradation in association with alterations in muscle expression of components of the ubiquitin-proteasome proteolytic pathway. Methods: We examined the effect of a 5 day intravenous infusions of saline, glucose, EAA and glucose + EAA, on urinary nitrogen excretion and muscle protein degradation. We carried out the study in 6 restrained calves since ruminants offer the advantage that muscle protein degradation can be assessed by excretion of 3 methyl-histidine and multiple muscle biopsies can be taken from the same animal. On the final day of infusion blood samples were taken for hormone and metabolite measurement and muscle biopsies for expression of ubiquitin, the 14-kDa E2 ubiquitin conjugating enzyme, and proteasome sub-units C2 and C8. Results: On day 5 of glucose infusion, plasma glucose, insulin and IGF-1 concentrations were increased while urea nitrogen excretion and myofibrillar protein degradation was decreased. Co-infusion of glucose + EAA prevented the loss of urinary nitrogen observed with EAA infusions alone and enhanced the increase in plasma IGF-1 concentration but there was no synergistic effect of glucose + EAA on the decrease in myofibrillar protein degradation. Muscle mRNA expression of the ubiquitin conjugating enzyme, 14-kDa E2 and proteasome sub-unit C2 were significantly decreased, after glucose but not amino acid infusions, and there was no further response to the combined infusions of glucose + EAA. Conclusion: Prolonged glucose infusion decreases myofibrillar protein degradation, prevents the excretion of infused EAA, and acts additively with EAA to increase plasma IGF-1 and improve net nitrogen balance. There was no evidence of synergistic effects between glucose + EAA infusion on muscle protein degradation or expression of components of the ubiquitin-proteasome proteolytic pathway.

Provirus integration into a gene encoding a ubiquitin-conjugating enzyme results in a placental defect and embryonic lethality.

Ubiquitin-conjugating enzymes (E2 or Ubc) constitute a family of conserved proteins that play a key role in ubiquitin-dependent degradation of proteins in eukaryotes. We describe here a transgenic mouse strain where retrovirus integration into an Ubc gene, designated UbcM4, results in a recessive-lethal mutation. UbcM4 is the mouse homologue of the previously described human UbcH7 that is involved in the in vitro ubiquitination of several proteins including the tumor suppressor protein p53. The provirus is located in the first intron of the gene. When both alleles are mutated the level of steady-state mRNA is reduced by about 70%. About a third of homozygous mutant embryos die around day 11.5 of gestation. Embryos that survive that stage are growth retarded and die perinatally. The lethal phenotype is most likely caused by impairment of placenta development as this is the only organ that consistently showed pathological defects. The placental labyrinth is drastically reduced in size and vascularization is disturbed. The UbcM4 mouse mutant represents the first example in mammals of a mutation in a gene involved in ubiquitin conjugation. Its recessive-lethal phenotype demonstrates that the ubiquitin system plays an essential role during mouse development.

Small ubiquitin-like modifier conjugating enzyme with active site mutation acts as dominant negative inhibitor of SUMO conjugation in arabidopsis(F).

Small ubiquitin-like modifier (SUMO) conjugation affects a broad range of processes in plants, including growth, flower initiation, pathogen defense, and responses to abiotic stress. Here, we investigate in vivo and in vitro a SUMO conjugating enzyme with a Cys to Ser change in the active site, and show that it has a dominant negative effect. In planta expression significantly perturbs normal development, leading to growth retardation, early flowering and gene expression changes. We suggest that the mutant protein can serve as a probe to investigate sumoylation, also in plants for which poor genetic infrastructure precludes analysis via loss-of-function mutants.

A Ubiquitin-Conjugating Enzyme Is Essential for Developmental Transitions in Dictyostelium

We have identified a developmentally essential gene, UbcB, by insertional mutagenesis. The encoded protein (UBC1) shows very high amino acid sequence identity to ubiquitin-conjugating enzymes from other organisms, suggesting that UBC1 is involved in protein ubiquitination and possibly degradation during Dictyostelium development. Consistent with the homology of the UBC1 protein to UBCs, the developmental pattern of protein ubiquitination is altered in ubcB-null cells. ubcB-null cells are blocked in the ability to properly execute the developmental transition that occurs between the induction of postaggregative gene expression during mound formation and the induction of cell-type differentiation and subsequent morphogenesis. ubcB-null cells plated on agar form mounds with normal kinetics; however, they remain at this stage for ∼10 h before forming multiple tips and fingers that then arrest. Under other conditions, some of the fingers form migrating slugs, but no culmination is observed. In ubcB-null cells, postaggregative gene transcripts accumulate to very high levels and do not decrease significantly with time as they do in wild-type cells. Expression of cell-type-specific genes is very delayed, with the level of prespore-specific gene expression being significantly reduced compared with that in wild-type cells. lacZ reporter studies using developmentally regulated and cell-type-specific promoters suggest that ubcB-null cells show an unusually elevated level of staining of lacZ reporters expressed in anterior-like cells, a regulatory cell population found scattered throughout the aggregate, and reduced staining of a prespore reporter. ubcB-null cells in a chimeric organism containing predominantly wild-type cells are able to undergo terminal differentiation but show altered spatial localization. In contrast, in chimeras containing only a small fraction of wild-type cells, the mature fruiting body is very small and composed almost exclusively of wild-type cells, with the ubcB-null cells being present as a mass of cells located in extreme posterior of the developing organism. The amino acid sequence analysis of the UbcB open reading frame (ORF) and the analysis of the developmental phenotypes suggest that tip formation and subsequent development requires specific protein ubiquitination, and possibly degradation.

Mammalian ubiquitin-conjugating enzyme Ubc9 interacts with Rad51 recombination protein and localizes in synaptonemal complexes.

Hsubc9, a human gene encoding a ubiquitin-conjugating enzyme, has been cloned. The 18-kDa HsUbc9 protein is homologous to the ubiquitin-conjugating enzymes Hus5 of Schizosaccharomyces pombe and Ubc9 of Saccharomyces cerevisiae. The Hsubc9 gene complements a ubc9 mutation of S. cerevisiae. It has been mapped to chromosome 16p13.3 and is expressed in many human tissues, with the highest levels in testis and thymus. According to the Ga14 two-hybrid system analysis, HsUbc9 protein interacts with human recombination protein Rad51. A mouse homolog, Mmubc9, encodes an amino acid sequence that is identical to the human protein. In mouse spermatocytes, MmUbc9 protein, like Rad51 protein, localizes in synaptonemal complexes, which suggests that Ubc9 protein plays a regulatory role in meiosis.

Reconstitution of p53-ubiquitinylation reactions from purified components: the role of human ubiquitin-conjugating enzyme UBC4 and E6-associated protein (E6AP).

The E6 protein of the high-risk human papillomaviruses inactivates the tumor suppressor protein p53 by stimulating its ubiquitinylation and subsequent degradation. Ubiquitinylation is a multistep process involving a ubiquitin-activating enzyme, one of many distinct ubiquitin-conjugating enzymes, and in certain cases, a ubiquitin ligase. In human papillomavirus-infected cells, E6 and the E6-associated protein are thought to act as a ubiquitin-protein ligase in the ubiquitinylation of p53. Here we describe the cloning of a human ubiquitin-conjugating enzyme that specifically ubiquitinylates E6-associated protein. Furthermore, we define the biochemical pathway of p53 ubiquitinylation and demonstrate that in vivo inhibition of various components in the pathway leads to an inhibition of E6-stimulated p53 degradation.

Etudes structurales et fonctionnelles des interactions de SUMO avec des proteines d'echafaudage modeles: TIF1beta, PIAS1 et PML

L’adaptation des cellules à leur environnement externe repose sur la transduction adéquate de signaux régulés par une pléthore d'événements moléculaires. Parmi ces événements moléculaires, les modifications post-traductionnelles (MPT) de protéines aident à intégrer, à traduire et à organiser de façon spatiotemporelle ces signaux pour que les cellules puissent réagir aux stimuli externes. Parmi les modifications post-traductionnelles, les petites protéines de la famille de l’Ubiquitine (Ublps, Ubiquitin-like proteins) jouent un rôle majeur dans presque toutes les voies de signalisation. Cette thèse rapporte des études fonctionnelles et structurales des interactions covalentes et non covalentes entre SUMO (Small Ubiquitin related MOdifier), un membre de la famille des Ublps, et trois protéines d'échafaudage, TIF1beta, le corépresseur universel des protéines KRAB-multidoigt de zinc, PIAS1, une ligase E3 pour SUMO et PML, un suppresseur de tumeur. La première étude rapporte l'identification et la caractérisation biochimique des sites de SUMOylation de TIF1beta. Nous avons déterminé que la modification covalente de six résidus lysine par SUMO est essentielle à l’activité de répression de la transcription induit par TIF1beta. En outre, nous présentons des évidences indiquant que la SUMOylation de TIF1 exige non seulement sa capacité à homo-oligomériser, mais est aussi positivement régulée par son interaction avec le domaine KRAB des protéines à doigts de zinc. Partant de ce constat, nous postulons que les protéines KRAB-multidoigt de zinc recrutent leur corépresseur TIF1betaà des gènes cibles, mais aussi accentuent son activité répressive grâce à l'augmentation de sa SUMOylation. Notre seconde étude révèle qu’en plus de réprimer la transcription en tant que MPT covalente, SUMO joue aussi un rôle important dans la répression en tant que partenaire non covalent d’interactions protéine-protéine. Nous avons montré que SUMO interagit simultanément avec deux enzymes de la machinerie de SUMOylation, l’unique enzyme de conjugaison E2, UBC9, et la ligase E3 PIAS1 au sein d’un complexe ternaire répresseur. En outre, nous révélons que la formation du complexe ternaire PIAS1:SUMO:UBC9 est modulée par le niveau de phosphorylation de résidus sérine juxtaposés à un motif d’interaction avec SUMO (SIM) dans PIAS1. Ainsi, SUMO agit comme un adaptateur spécifique qui stabilise les interactions UBC9 E2: E3 PIAS1. Partant de ce constat, nous proposons que les enzymes E2 et E3 des autres systèmes Ublps exploitent des mécanismes similaires dans le cadre de leur fonction Enfin, notre troisième étude explore la régulation des interactions non covalentes de SUMO par la phosphorylation. En utilisant une combinaison d'études in vivo et in vitro, nous démontrons que l'interaction entre SUMO1 et PML est régi par la phosphorylation dépendant de CK2 sur quatre résidus sérine de PML. Les structures cristallographiques des complexes PML-SIM:SUMO1 révèlent que les phospho-sérines de PML contactent des résidus de la région basique de SUMO1. Sachant que la kinase CK2 peut être induite par des kinases activables par le stress, ces résultats suggèrent que les interactions non-covalentes avec SUMO sont modulées par le stress cellulaire. Sur la base de cette constatation, nous postulons que des événements analogues affectent des protéines contenant des séquences SIM ciblées par CK2. En résumé, cette étude révèle qu’en plus de son rôle de MPT, SUMO peut fonctionner comme un adaptateur permettant des interactions spécifiques entre protéines tel que pour les enzymes E3 et E2.

Regulation of tail-anchored ubiquitin conjugating enzymes that are localised to the endoplasmic reticulum

The vast majority of secreted and membrane proteins are translated and folded at the endoplasmic reticulum (ER), where a sophisticated quality control mechanism ensures that only correctly folded proteins exit the ER and traffic to their final destinations. On the other hand, proteins that persistently misfold are eliminated through a process known as ER associated degradation (ERAD). This involves retrotranslocation of the misfolded protein through the ER membrane, and ubiquitination in advance of degradation by cytosolic proteasomes. The process of ERAD is best described in yeast where ubiquitin conjugating enzymes Ubc6p and Ubc7p function with a limited number of E3 ubiquitin ligases to ubiquitinate misfolded proteins. Interestingly, although the mechanistic principles of ERAD have been conserved through evolution, there is increasing evidence that homologues of the yeast enzymes have gained divergent roles and novel regulatory functions in higher eukaryotes, meaning that the process in humans is more complex and involves a larger repertoire of participating proteins. Two homologues of Ubc6p have been described in humans, and have been named as Ubc6 (UBE2J2) and Ubc6e (UBE2J1). However, little work has been done on these enzymes and thus our main objective of this study was to progress the functional characterisation of these ERAD E2 conjugating enzymes. Our studies included a detailed analysis of conditions whereby these proteins are stabilised and degraded. We’ve also explored the different molecular signalling pathways that induced changes on their steady state protein levels. Furthermore, Ubc6e has a phosphorylatable serine residue at position 184. Thus, our studies also involved delineating the signalling kinases that phosphorylate Ubc6e and examining its function in ERAD. Our studies confirm that the E2 Ubc enzymes are regulated posttranslationally and may have important implications in the regulation of ERAD.

Phosphomimetic Mutation of the N-Terminal Lid of MDM2 Enhances the Polyubiquitination of p53 through Stimulation of E2-Ubiquitin Thioester Hydrolysis.

Mouse double minute 2 (MDM2) has a phosphorylation site within a lid motif at Ser17 whose phosphomimetic mutation to Asp17 stimulates MDM2-mediated polyubiquitination of p53. MDM2 lid deletion, but not Asp17 mutation, induced a blue shift in the λmax of intrinsic fluorescence derived from residues in the central domain including Trp235, Trp303, Trp323, and Trp329. This indicates that the Asp17 mutation does not alter the conformation of MDM2 surrounding the tryptophan residues. In addition, Phe235 mutation enhanced MDM2 binding to p53 but did not stimulate its ubiquitination function, thus uncoupling increases in p53 binding from its E3 ubiquitin ligase function. However, the Asp17mutation inMDM2 stimulated its discharge of the UBCH5a-ubiquitin thioester adduct (UBCH5a is a ubiquitin-conjugating enzyme E2D 1 UBC4/5 homolog yeast). This stimulation of ubiquitin discharge fromE2 was independent of the p53 substrate. There are now four known effects of the Asp17 mutation on MDM2: (i) it alters the conformation of the isolated N-terminus as defined by NMR; (ii) it induces increased thermostability of the isolated N-terminal domain; (iii) it stimulates the allosteric interaction ofMDM2 with the DNA-binding domain of p53; and (iv) it stimulates a novel protein–protein interaction with the E2-ubiquitin complex in the absence of substrate p53 that, in turn, increases hydrolysis of theE2-ubiquitin thioester bond. These data also suggest a new strategy to disrupt MDM2 function by targeting the E2-ubiquitin discharge reaction.

Karyotype variability in KP1(+) and KP1(-) strains of Trypanosoma rangeli isolated in Brazil and Colombia

In the present study, the molecular karyotypes of 12 KP1(+) and KP1(-) Trypanosoma rangeli strains were determined and 10 different molecular markers were hybridized to the chromosomes of the parasite, including seven obtained from T. rangeli [ubiquitin hydrolase (UH), a predicted serine/threonine protein kinase (STK), hexose transporter, hypothetical protein, three anonymous sequences] and three from Trypanosoma cruzi [ubiquitin-conjugating enzyme E2 (UBE2), ribosomal RNA methyltransferase (rRNAmtr), proteasome non-ATPase regulatory subunit 6 (PSMD6)]. Despite intraspecific variation, analysis of the karyotype profiles permitted the division of the T rangeli strains into two groups coinciding with the KP1(+) and KP1(-) genotypes. Southern blot hybridization showed that, except for the hexose transporter probe, all other probes produced distinct patterns able to differentiate the KP1(+) and KP1(-) genotypes. The UH, STK and An-1A04 probes exclusively hybridized to the chromosomes of KP1(+) strains and can be used as markers of this group. In addition, the UBE2, rRNAmtr and PSMD6 markers, which are present in a conserved region in all trypanosomatid species sequenced so far, co-hybridized to the same T. rangeli chromosomal bands, suggesting the occurrence of gene synteny in these species. The finding of distinct molecular karyotypes in KP1(+) and KP1 (-) strains of T rangeli is noteworthy and might be used as a new approach to the study of genetic variability in this parasite. Together with the Southern blot hybridization results, these findings demonstrate that differences at the kDNA level might be associated with variations in nuclear DNA. (c) 2009 Elsevier BY. All rights reserved.

The role of the TRAF-interacting protein in proliferation and differentiation.

Ubiquitination of proteins is a post-translational modification, which decides on the cellular fate of the protein. Addition of ubiquitin moieties to proteins is carried out by the sequential action of three enzymes: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; and E3, ubiquitin ligase. The TRAF-interacting protein (TRAIP, TRIP, RNF206) functions as Really Interesting New Gene (RING)-type E3 ubiquitin ligase, but its physiological substrates are not yet known. TRAIP was reported to interact with TRAF [tumor necrosis factor (TNF) receptor-associated factors] and the two tumor suppressors CYLD and Syk (spleen tyrosine kinase). Ectopically expressed TRAIP was shown to inhibit nuclear factor-kappa B (NF-κB) signalling. However, recent results suggested a role for TRAIP in biological processes other than NF-κB regulation. Knock-down of TRAIP in human epidermal keratinocytes repressed cellular proliferation and induced a block in the G1/S phase of the cell cycle without affecting NF-κB signalling. TRAIP is necessary for embryonal development as mutations affecting the Drosophila homologue of TRAIP are maternal effect-lethal mutants, and TRAIP knock-out mice die in utero because of aberrant regulation of cell proliferation and apoptosis. These findings underline the tight link between TRAIP and cell proliferation. In this review, we summarize the data on TRAIP and put them into a larger perspective regarding the role of TRAIP in the control of tissue homeostasis.

In silico analysis identifies a C3HC4-RING finger domain of a putative E3 ubiquitin-protein ligase located at the C-terminus of a polyglutamine-containing protein

Almost identical polyglutamine-containing proteins with unknown structures have been found in human, mouse and rat genomes (GenBank AJ277365, AF525300, AY879229). We infer that an identical new gene (RING) finger domain of real interest is located in each C-terminal segment. A three-dimensional (3-D) model was generated by remote homology modeling and the functional implications are discussed. The model consists of 65 residues from terminal position 707 to 772 of the human protein with a total length of 796 residues. The 3-D model predicts a ubiquitin-protein ligase (E3) as a binding site for ubiquitin-conjugating enzyme (E2). Both enzymes are part of the ubiquitin pathway to label unwanted proteins for subsequent enzymatic degradation. The molecular contact specificities are suggested for both the substrate recognition and the residues at the possible E2-binding surface. The predicted structure, of a ubiquitin-protein ligase (E3, enzyme class number 6.3.2.19, CATH code 3.30.40.10.4) may contribute to explain the process of ubiquitination. The 3-D model supports the idea of a C3HC4-RING finger with a partially new pattern. The putative E2-binding site is formed by a shallow hydrophobic groove on the surface adjacent to the helix and one zinc finger (L722, C739, P740, P741, R744). Solvent-exposed hydrophobic amino acids lie around both zinc fingers (I717, L722, F738, or P765, L766, V767, V733, P734). The 3-D structure was deposited in the protein databank theoretical model repository (2B9G, RCSB Protein Data Bank, NJ).

Identification du rôle et des modifications post-traductionnelles modulant l’export nucléaire de l’hélicase virale E1 au cours du cycle de réplication du virus du papillome humain

Les virus du papillome humain (VPH) sont de petits virus à ADN double brin infectant les épithéliums de la peau et des muqueuses. La réplication nécessaire au maintien de leur génome dans les cellules infectées dépend des protéines virales E1 et E2. Au cours de la réplication, E1 est recrutée à l’origine de réplication par E2 afin d’être assemblée en doubles hexamères capables de dérouler l’ADN. E1 contient un domaine C-terminal responsable de l’activité ATPase/hélicase, un domaine central de liaison à l’origine et une région N-terminale régulant la réplication in vivo. Cette région contient des signaux de localisation et d’export nucléaire qui modulent le transport intracellulaire de E1. Chez le virus du papillome bovin (VPB), il a été proposé que ce transport est régulé par la sumoylation de E1. Finalement, la région N-terminale de E1 contient un motif de liaison aux cyclines permettant son interaction avec la cycline E/A-Cdk2. La phosphorylation de E1 par cette dernière régule différemment l’export nucléaire des protéines E1 du VPB et du VPH. Dans la première partie de cette étude, nous avons démontré que bien que la protéine E1 des VPH interagit avec Ubc9, l’enzyme de conjugaison de la voie de sumoylation, cette voie n’est pas requise pour son accumulation au noyau. Dans la seconde partie, nous avons déterminé que l’accumulation nucléaire de E1 est plutôt régulée pas sa phosphorylation. En fait, nous avons démontré que l’export nucléaire de E1 est inhibé par la phosphorylation de sérines conservées de la région N-terminale de E1 par Cdk2. Puis, nous avons établi que l’export nucléaire de E1 n’est pas nécessaire à l’amplification du génome dans les kératinocytes différenciés mais qu’il est requis pour le maintien du génome dans les kératinocytes non différenciés. En particulier, nous avons découvert que l’accumulation nucléaire de E1 inhibe la prolifération cellulaire en induisant un arrêt du cycle cellulaire en phase S et que cet effet anti-prolifératif est contrecarrée par l’export de E1 au cytoplasme. Dans la troisième partie de cette étude, nous avons démontré que l’arrêt cellulaire induit par E1 dépend de sa liaison à l’ADN et à l’ATP, et qu’il est accompagné par l’activation de la voie de réponse aux dommages à l’ADN dépendante de ATM (Ataxia Telangiectasia Mutated). Ces deux événements semblent toutefois distincts puisque la formation d’un complexe E1-E2 réduit l’activation de la voie de réponse aux dommages par E1 sans toutefois prévenir l’arrêt de cycle cellulaire. Finalement, nous avons démontré que la réplication transitoire de l’ADN viral peut avoir lieu dans des cellules arrêtées en phase S, indépendamment de l’activation de la voie de réponse aux dommages à l’ADN et de la kinase ATM. Globalement, nos résultats démontrent que l’export nucléaire de E1 est régulé par sa phosphorylation et non par sa sumoylation. Ils démontrent également que l’export nucléaire de E1 est essentiel au maintien du génome dans les kératinocytes, possiblement parce qu’il prévient l’inhibition de la prolifération cellulaire et l’activation de la voie de réponse aux dommages à l’ADN en limitant l’accumulation de E1 au noyau.

A novel mechanism of 3-bromopyruvate-induced cell death through targeting hexokinase and ubiquitination

Increased dependence on aerobic glycolysis for energy (ATP) supply has been observed in various human cancer cells. It is plausible to exploit this metabolic alteration for therapeutic benefits by inhibiting glycolysis to preferentially abolish cancer energy metabolism and kill the malignant cells. 3-Bromopyruvate has been shown to be a potent inhibitor of glycolysis capable of inducing severe ATP reduction and cell death in various cancer cell lines, especially cancer cells with mitochondrial defects or under hypoxic conditions. However, the detailed mechanisms of this novel anticancer agent still remain unclear. My study demonstrated that 3-Bromopyruvate caused a covalent modification of hexokinase II, a key glycolytic enzyme, and disrupted its association with mitochondria. This led to mitochondrial permeability transition and a substantial release of apoptosis-inducing faction (AIF) prior to cytochrome c release. Dissociation of HK II from mitochondria using a cell permeable specific peptide also induced the release of AIF and cytochrome c, and caused substantial cell death. HK II-targeted peptide did not cause significant change in mitochondria respiration and glycolysis activity, suggesting that dissociation of this molecule from mitochondria alone can also cause cell death, and that this may be a novel mechanism by which 3-Bromopyruvate exerts its potent cytotoxic action, in addition to its inhibition of the enzyme activity. Another significant new discovery was that 3-Bromopyruvate induced rapid reduction of protein ubiquitination in vivo, which occurred within several hours of drug incubation and before ATP reduction and cell death. Further mechanistic studies showed that this was due to the inhibition the ubiquitin activating enzyme E1 and the conjugating enzyme E2. Knocking down ubiquitin protein expression by siRNA did not suppress mitochondria respiration and glycolysis, but caused significant cell death. Taken together, this study demonstrated that induction of HK II dissociation from mitochondria and inhibition of glycolysis are two newly discovered mechanisms that contribute to the potent anticancer activity of 3-Bromopyruvate, and identified this compound as a valuable chemical tool for research in protein ubiquitination. ^