E3 Ub Ligase Research Articles

Ubiquitin protease Ubp8 is necessary for S. cerevisiae respiration

Healthy mitochondria are required in cell metabolism and deregulation of underlying mechanisms is often involved in human diseases and neurological disorders. Post-translational modifications of mitochondrial proteins regulate their function and activity, accordingly, impairment of ubiquitin proteasome system affects mitochondria homeostasis and organelle dynamics. In the present study we have investigated the role of the ubiquitin protease Ubp8 in S. cerevisiae respiration. We show that Ubp8 is necessary for respiration and its expression is upregulated in glycerol respiratory medium. In addition, we show that the respiratory defects in absence of Ubp8 are efficiently rescued by disruption of the E3 Ub-ligase Psh1, suggesting their epistatic link. Interestingly, we found also that Ubp8 is localized into mitochondria as single protein independently of SAGA complex assembly, thus suggesting an independent function from the nuclear one. We also show evidences on the importance of HAT Gcn5 in sustaining Ubp8 expression and affecting the amount of protein in mitochondria. Collectively, our results have investigated the role of Ubp8 in respiratory metabolism and highlight the role of ubiquitin related pathways in the mitochondrial functions of S. cerevisiae.

Age- and disease-dependent increase of the mitophagy marker phospho-ubiquitin in normal aging and Lewy body disease

ABSTRACTAlthough exact causes of Parkinson disease (PD) remain enigmatic, mitochondrial dysfunction is increasingly appreciated as a key determinant of dopaminergic neuron susceptibility in both familial and sporadic PD. Two genes associated with recessive, early-onset PD encode the ubiquitin (Ub) kinase PINK1 and the E3 Ub ligase PRKN/PARK2/Parkin, which together orchestrate a protective mitochondrial quality control (mitoQC) pathway. Upon stress, both enzymes cooperatively identify and decorate damaged mitochondria with phosphorylated poly-Ub (p-S65-Ub) chains. This specific label is subsequently recognized by autophagy receptors that further facilitate mitochondrial degradation in lysosomes (mitophagy). Here, we analyzed human post-mortem brain specimens and identified distinct pools of p-S65-Ub-positive structures that partially colocalized with markers of mitochondria, autophagy, lysosomes and/or granulovacuolar degeneration bodies. We further quantified levels and distribution of the ‘mitophagy tag’ in 2 large cohorts of brain samples from normal aging and Lewy body disease (LBD) cases using unbiased digital pathology. Somatic p-S65-Ub structures independently increased with age and disease in distinct brain regions and enhanced levels in LBD brain were age- and Braak tangle stage-dependent. Additionally, we observed significant correlations of p-S65-Ub with LBs and neurofibrillary tangle levels in disease. The degree of co-existing p-S65-Ub signals and pathological PD hallmarks increased in the pre-mature stage, but decreased in the late stage of LB or tangle aggregation. Altogether, our study provides further evidence for a potential pathogenic overlap among different forms of PD and suggests that p-S65-Ub can serve as a biomarker for mitochondrial damage in aging and disease.Abbreviations: BLBD: brainstem predominant Lewy body disease; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; DLB: dementia with Lewy bodies; DLBD: diffuse neocortical Lewy body disease; EOPD: early-onset Parkinson disease; GVB: granulovacuolar degeneration body; LB: Lewy body; LBD: Lewy body disease; mitoQC: mitochondrial quality control; nbM: nucleus basalis of Meynert; PD: Parkinson disease; PDD: Parkinson disease with dementia; p-S65-Ub: PINK1-phosphorylated serine 65 ubiquitin; SN: substantia nigra; TLBD: transitional Lewy body disease; Ub: ubiquitin

Insights into the ubiquitin transfer cascade catalyzed by the Legionella effector SidC.

The causative agent of Legionnaires' disease, Legionella pneumophila, delivers more than 330 virulent effectors to its host to establish an intracellular membrane-bound organelle called the Legionella containing vacuole. Among the army of Legionella effectors, SidC and its paralog SdcA have been identified as novel bacterial ubiquitin (Ub) E3 ligases. To gain insight into the molecular mechanism of SidC/SdcA as Ub ligases, we determined the crystal structures of a binary complex of the N-terminal catalytic SNL domain of SdcA with its cognate E2 UbcH5C and a ternary complex consisting of the SNL domain of SidC with the Ub-linked E2 UbcH7. These two structures reveal the molecular determinants governing the Ub transfer cascade catalyzed by SidC. Together, our data support a common mechanism in the Ub transfer cascade in which the donor Ub is immobilized with its C-terminal tail locked in an extended conformation, priming the donor Ub for catalysis.

Hierarchical core decomposition of RING structure as a method to capture novel functional residues within RING-type E3 ligases: a structural systems biology approach

BackgroundIncreasing amounts of evidence strongly suggest that mutations located in RING (Really Interesting New Gene) domains of E3 ligases are involved in cancer development. Despite the existence of many experimentally defined E3 RING structures, there are still many RING mutations in the Catalog of Somatic Mutations in Cancer (COSMIC), with an unknown structural or functional significance, which are usually substitutions of amino acids with no conservation at the corresponding position. The core decomposition of networks has long been used in systems biology but never utilized in protein structure networks to identify a tolerant “core” to peripheral structure changes or failures, a region that is usually not easy to be determined with high accuracy through classical 3D protein structure analyses. MethodA new structure decomposition method that utilizes network analysis and computational thermodynamic measures of fold stability changes upon amino acids alterations is proposed. ResultsIn particular, by successively pruning the entire RING structure network of three cancer-associated E3s (BRCA1, MDM2, and cIAP2), a ‘‘core’’ for each RING domain is left. Interestingly, substitutions of ‘‘core’’ residues are associated with cancers according to COSMIC catalog. Unexpectedly, 80% of the residues located in the ‘‘core’’ RING subnetworks are non-conserved within E3 RING domains. To validate the predictions, the function of the identified RING ‘‘core’’ amino acids as well as the effects of their substitutions on E3 Ub ligase activity were mined from the literature and investigated by computational tools and in vitro Ub ligase assays. ConclusionsThis method could be widely applicable as a source of novel functional RING residues, non-conserved in E3s, for which substitutions could be deleterious.

The host ubiquitin system in innate immunity and virus replication: proviral and antiviral functions of the host E3-ubiquitin ligase TRIM family

Abstract Type-I Interferons (IFN-I), which are innate immune cytokines with broad-spectrum antiviral effects, are regulated by the host Ubiquitin (Ub) system. Evidence has shown that new forms of poly-Ub chains, which are not covalently attached to any protein (termed unanchored poly-Ub), are important activators of kinase activity in vitro. We recently reported that unanchored K48-linked poly-Ub synthesized by the E3-Ub ligase TRIM6 promote IFN-mediated antiviral innate immunity in vivo by activating the IKKɛ kinase. Intriguingly, we found that two different highly pathogenic viruses, Nipah (NiV; Paramyxoviridae family) and Ebola (EBOV; Filoviridae family), hijack TRIM6 to promote virus replication through mechanisms that involve both covalent and unanchored Ub. This indicates that host antiviral factors can have direct pro-viral functions. We also found that Zika virus (ZIKV; Flaviviridae family), which can cause congenital microcephaly in newborns, requires ubiquitination of viral proteins for efficient replication. Importantly, ZIKV ubiquitination occurs in vivo in a tissue-specific manner. Recombinant infectious ZIKV mutants that lack ubiquitination on specific sites are attenuated in mice and appear to differentially replicate in tissues. As compared to other flaviviruses, ZIKV acquired an additional ubiquitination site, which may be responsible for providing ZIKV an advantage to replicate in brain and reproductive tissue. This would be the first example of a flavivirus utilizing the Ub system for tissue tropism. Taken together, our work suggests that divergent viruses hijack similar innate immune host factors to enhance their replication. This information could help design novel broad-spectrum antiviral strategies

Roles of E3 Ubiquitin-Ligases in Nuclear Protein Homeostasis during Plant Stress Responses.

Ubiquitination, the reversible protein conjugation with ubiquitin (Ub), is a post-translational modification that enables rapid and specific cellular responses to stimuli without requirement of de novo protein synthesis. Although ubiquitination also displays non-proteolytic functions, it often acts as a signal for selective protein degradation through the ubiquitin-proteasome system (UPS). In plants, it has become increasingly apparent that the UPS is a central regulator of many key cellular and physiological processes, including responses to biotic and abiotic stresses. In the nucleus, protein regulation via the UPS orchestrates gene expression, genome maintenance, and signal transduction. Here, we focus on E3 Ub-ligase proteins as major components of the ubiquitination cascade that confer specificity of substrate recognition. We provide an overview on how they contribute to nuclear proteome plasticity during plant responses to environmental stress signals.

MRNA Processing Factor CstF-50 and Ubiquitin Escort Factor p97 Are BRCA1/BARD1 Cofactors Involved in Chromatin Remodeling during the DNA Damage Response.

ABSTRACTThe cellular response to DNA damage is an intricate mechanism that involves the interplay among several pathways. In this study, we provide evidence of the roles of the polyadenylation factor cleavage stimulation factor 50 (CstF-50) and the ubiquitin (Ub) escort factor p97 as cofactors of BRCA1/BARD1 E3 Ub ligase, facilitating chromatin remodeling during the DNA damage response (DDR). CstF-50 and p97 formed complexes with BRCA1/BARD1, Ub, and some BRCA1/BARD1 substrates, such as RNA polymerase (RNAP) II and histones. Furthermore, CstF-50 and p97 had an additive effect on the activation of the ubiquitination of these BRCA1/BARD1 substrates during DDR. Importantly, as a result of these functional interactions, BRCA1/BARD1/CstF-50/p97 had a specific effect on the chromatin structure of genes that were differentially expressed. This study provides new insights into the roles of RNA processing, BRCA1/BARD1, the Ub pathway, and chromatin structure during DDR.

The E3 Ligase RING1 Targets p53 for Degradation and Promotes Cancer Cell Proliferation and Survival.

As a component of the transcriptional repression complex 1 (PRC1), the ring finger protein RING1 participates in the epigenetic regulation in cancer. However, the contributions of RING1 to cancer etiology or development are unknown. In this study, we report that RING1 is a critical negative regulator of p53 homeostasis in human hepatocellular and colorectal carcinomas. RING1 acts as an E3 ubiquitin (Ub) ligase to directly interact with and ubiquitinate p53, resulting in its proteasome-dependent degradation. The RING domain of RING1 was required for its E3 Ub ligase activity. RING1 depletion inhibited the proliferation and survival of the p53 wild-type cancer cells by inducing cell-cycle arrest, apoptosis, and senescence, with only modest effects on p53-deficient cells. Its growth inhibitory effect was partially rescued by p53 silencing, suggesting an important role for the RING1-p53 complex in human cancer. In clinical specimens of hepatocellular carcinoma, RING1 upregulation was evident in association with poor clinical outcomes. Collectively, our results elucidate a novel PRC1-independent function of RING1 and provide a mechanistic rationale for its candidacy as a new prognostic marker and/or therapeutic target in human cancer.Significance: These results elucidate a novel PRC1-independent function of RING1 and provide a mechanistic rationale for its candidacy as a new prognostic marker and/or therapeutic target in human cancer. Cancer Res; 78(2); 359-71. ©2017 AACR.

Enzymatic Assembly of Ubiquitin Chains.

The availability of different polyubiquitin chains of specific linkage types has changed the appreciation of the specificity in the ubiquitin (Ub) system. Numerous E2 Ub-conjugating enzymes and E3 Ub ligases, Ub-binding domains (UBDs), and deubiquitinases (DUBs) are now known to assemble, bind, or hydrolyze individual linkage types, respectively. Biochemical and structural studies of these processes require milligram quantities of pure polyUb. Here we describe protocols that allow the enzymatic synthesis and purification of six of the eight homotypic polyUb chains through the use of chain-specific Ub ligases and DUBs.

Oryza sativa salt-induced RING E3 ligase 2 (OsSIRP2) acts as a positive regulator of transketolase in plant response to salinity and osmotic stress.

A rice gene (OsSIRP2) encoding the RING Ub E3 ligase was highly induced under salinity stress and physically interacted with a transketolase (OsTKL1). Overexpression of OsSIRP2 conferred salinity and osmotic stress tolerance in plants. The RING E3 ligases play a vital role in post transitional modification through ubiquitination-mediated protein degradation that mediate plants responses during abiotic stresses and signal transduction. In this study, we report an Oryza sativa salt induced Really Interesting New Gene (RING) finger protein 2 gene (OsSIRP2) and elucidate its role under salinity and osmotic stress. The transcript levels of OsSIRP2 in rice leaves were induced in response to different abiotic stresses, such as salt, drought, heat, and abscisic acid (ABA) exposure. In vitro ubiquitination revealed that the OsSIRP2 protein formed poly-ubiquitin products, whereas a single amino acid substitution in OsSIRP2 (OsSIRP2C149A) in the RING domain did not form ubiquitinated substrates, supporting the hypothesis that E3 ligase activity requires the functional RING domain. Using the yeast two-hybrid (Y2H) assay, O. sativa transketolase 1 (OsTKL1) was identified as an interacting partner. OsSIRP2 was localized in the nucleus, whereas its interacting partner (OsTKL1) was localized in the cytosol and plastids in the rice protoplasts. Fluorescence signals between OsSIRP2 and OsTKL1 were observed in the cytosol. The pull-down assay confirmed the physical interaction between OsSIRP2 and OsTKL1. In vitro ubiquitination assay and in vitro protein degradation assay revealed that OsSIRP2 ubiquitinates OsTKL1 and enhances the degradation of OsTKL1 through the 26S proteasomal pathway. Heterogeneous overexpression of OsSIRP2 resulted in conferring tolerance against salinity and osmotic stress. Overall, our findings suggest that OsSIRP2 may be associated with plant responses to abiotic stresses and act as a positive regulator of salt and osmotic stress tolerance.

Structural insights into the mechanism and E2 specificity of the RBR E3 ubiquitin ligase HHARI

RING-in-between-RING (RBR) ubiquitin (Ub) E3 ligases function with Ub E2s through a RING/HECT hybrid mechanism to conjugate Ub to target proteins. Here, we report the crystal structure of the RBR E3, HHARI, in complex with a UbcH7 ~ Ub thioester mimetic which reveals the molecular basis for the specificity of this cognate E2/RBR E3 pair. The structure also reveals mechanistically important conformational changes in the RING1 and UBA-like domains of HHARI that accompany UbcH7 ~ Ub binding and provides a molecular basis by which HHARI recruits E2 ~ Ub in an ‘open’ conformation. In addition to optimally functioning with an E2 that solely performs transthiolation, our data suggests that HHARI prevents spurious discharge of Ub from E2 to lysine residues by: (1) harboring structural elements that block E2 ~ Ub from adopting a ‘closed’ conformation and (2) participating in contacts to ubiquitin that promote an open E2 ~ Ub conformation.

Expanding the host cell ubiquitylation machinery targeting cytosolic Salmonella.

Ubiquitylation is one of the cardinal post-translational modifications in the cell, balancing several distinct biological processes and acting as a pathogen recognition receptor during bacterial pathogen invasion. A dense layer of polyubiquitin chains marks invading bacteria that gain access to the host cytosol for their selective clearance via xenophagy. However, the enzymes that mediate recognition of cytosolic bacteria and generate this ubiquitin (Ub) coat remain largely elusive. To address this, we employed an image-based RNAi screening approach to monitor the loss of Ub on Salmonella upon depletion of human Ub E3 ligases in cells. Using this approach, we identified ARIH1 as one of the ligases involved in the formation of Ub coat on cytosolic bacteria. In addition, we provide evidence that the RING-between-RING ligase ARIH1, together with LRSAM1 and HOIP, forms part of a network of ligases that orchestrates recognition of intracellular Salmonella and participates in the activation of the host cell immune response.

Two functionally distinct E2/E3 pairs coordinate sequential ubiquitination of a common substrate in Caenorhabditis elegans development

Ubiquitination, the crucial posttranslational modification that regulates the eukaryotic proteome, is carried out by a trio of enzymes, known as E1 [ubiquitin (Ub)-activating enzyme], E2 (Ub-conjugating enzyme), and E3 (Ub ligase). Although most E2s can work with any of the three mechanistically distinct classes of E3s, the E2 UBCH7 is unable to function with really interesting new gene (RING)-type E3s, thereby restricting it to homologous to E6AP C-terminus (HECT) and RING-in-between-RING (RBR) E3s. The Caenorhabditis elegans UBCH7 homolog, UBC-18, plays a critical role in developmental processes through its cooperation with the RBR E3 ARI-1 (HHARI in humans). We discovered that another E2, ubc-3, interacts genetically with ubc-18 in an unbiased genome-wide RNAi screen in C. elegans These two E2s have nonoverlapping biochemical activities, and each is dedicated to distinct classes of E3s. UBC-3 is the ortholog of CDC34 that functions specifically with Cullin-RING E3 ligases, such as SCF (Skp1-Cullin-F-box). Our genetic and biochemical studies show that UBCH7 (UBC-18) and the RBR E3 HHARI (ARI-1) coordinate with CDC34 (UBC-3) and an SCF E3 complex to ubiquitinate a common substrate, a SKP1-related protein. We show that UBCH7/HHARI primes the substrate with a single Ub in the presence of CUL-1, and that CDC34 is required to build chains onto the Ub-primed substrate. Our study reveals that the association and coordination of two distinct E2/E3 pairs play essential roles in a developmental pathway and suggests that cooperative action among E3s is a conserved feature from worms to humans.

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.

Ubiquitin phosphorylated at Ser57 hyper-activates parkin

Malfunction of the ubiquitin (Ub) E3 ligase, parkin, leads to defects in mitophagy and protein quality control linked to Parkinson's disease. Parkin activity is stimulated by phosphorylation of Ub at Ser65 (pUbS65). Since the upstream kinase is only known for Ser65 (PINK1), the biochemical function of other phosphorylation sites on Ub remain largely unknown. We used fluorescently labelled and site-specifically phosphorylated Ub substrates to quantitatively relate the position and stoichiometry of Ub phosphorylation to parkin activation. Fluorescence measurements show that pUbS65-stimulated parkin is 5-fold more active than auto-inhibited and un-stimulated parkin, which catalyzes a basal level of auto-ubiquitination. We consistently observed a low but detectable level of parkin activity with pUbS12. Strikingly, pUbS57 hyper-activates parkin, and our data demonstrate that parkin is able to selectively synthesize poly-pUbS57 chains, even when 90% of the Ub in the reaction is un-phosphorylated. We further found that parkin ubiquitinates its physiological substrate Miro-1 with chains solely composed of pUbS65 and more efficiently with pUbS57 chains. Parkin hyper-activation by pUbS57 demonstrates the first PINK1-independent route to active parkin, revealing the roles of multiple ubiquitin phosphorylation sites in governing parkin stimulation and catalytic activity. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O’Donoghue.

Evidence that phosphorylated ubiquitin signaling is involved in the etiology of Parkinson's disease.

The ubiquitin (Ub) kinase PINK1 and the E3 Ub ligase Parkin, two gene products associated with young-onset Parkinson's disease (PD), participate in mitochondrial quality control. The phosphorylation of mitochondrial polyUb by PINK1, which is activated in a mitochondrial membrane potential (ΔΨm)-dependent manner, facilitates the mitochondrial translocation and concomitant enzymatic activation of Parkin, leading to the clearance of phospho-polyUb-tagged mitochondria via mitophagy. Thus, Ub phosphorylation is a key event in PINK1-Parkin-mediated mitophagy. Here, we examined the role of phospho-Ub signaling in the pathogenesis of PD using fly PD models, human brain tissue and dopaminergic neurons derived from induced pluripotent stem cells (iPSCs) containing Parkin or PINK1 mutations, as well as normal controls. We report that phospho-Ub signaling is highly conserved between humans and Drosophila, and that phospho-Ub signaling and the relocation of axonal mitochondria upon ΔΨm reduction are indeed compromised in human dopaminergic neurons containing Parkin or PINK1 mutations. Moreover, phospho-Ub signaling is prominent in tyrosine hydroxylase-positive neurons compared with tyrosine hydroxylase-negative neurons, suggesting that PINK1-Parkin signaling is more required for dopaminergic neurons. These results shed light on the particular vulnerability of dopaminergic neurons to mitochondrial stress.

The PINK1 p.I368N mutation affects protein stability and ubiquitin kinase activity

BackgroundMutations in PINK1 and PARKIN are the most common causes of recessive early-onset Parkinson’s disease (EOPD). Together, the mitochondrial ubiquitin (Ub) kinase PINK1 and the cytosolic E3 Ub ligase PARKIN direct a complex regulated, sequential mitochondrial quality control. Thereby, damaged mitochondria are identified and targeted to degradation in order to prevent their accumulation and eventually cell death. Homozygous or compound heterozygous loss of either gene function disrupts this protective pathway, though at different steps and by distinct mechanisms. While structure and function of PARKIN variants have been well studied, PINK1 mutations remain poorly characterized, in particular under endogenous conditions. A better understanding of the exact molecular pathogenic mechanisms underlying the pathogenicity is crucial for rational drug design in the future.MethodsHere, we characterized the pathogenicity of the PINK1 p.I368N mutation on the clinical and genetic as well as on the structural and functional level in patients’ fibroblasts and in cell-based, biochemical assays.ResultsUnder endogenous conditions, PINK1 p.I368N is expressed, imported, and N-terminally processed in healthy mitochondria similar to PINK1 wild type (WT). Upon mitochondrial damage, however, full-length PINK1 p.I368N is not sufficiently stabilized on the outer mitochondrial membrane (OMM) resulting in loss of mitochondrial quality control. We found that binding of PINK1 p.I368N to the co-chaperone complex HSP90/CDC37 is reduced and stress-induced interaction with TOM40 of the mitochondrial protein import machinery is abolished. Analysis of a structural PINK1 p.I368N model additionally suggested impairments of Ub kinase activity as the ATP-binding pocket was found deformed and the substrate Ub was slightly misaligned within the active site of the kinase. Functional assays confirmed the lack of Ub kinase activity.ConclusionsHere we demonstrated that mutant PINK1 p.I368N can not be stabilized on the OMM upon mitochondrial stress and due to conformational changes in the active site does not exert kinase activity towards Ub. In patients’ fibroblasts, biochemical assays and by structural analyses, we unraveled two pathomechanisms that lead to loss of function upon mutation of p.I368N and highlight potential strategies for future drug development.

Homologous U-box E3 Ubiquitin Ligases OsPUB2 and OsPUB3 Are Involved in the Positive Regulation of Low Temperature Stress Response in Rice (Oryza sativa L.).

Rice U-box E3 Ub ligases (OsPUBs) are implicated in biotic stress responses. However, their cellular roles in response to abiotic stress are poorly understood. In this study, we performed functional analyses of two homologous OsPUB2 and OsPUB3 in response to cold stress (4°C). OsPUB2 was up-regulated by high salinity, drought, and cold, whereas OsPUB3 was constitutively expressed. A subcellular localization assay revealed that OsPUB2 and OsPUB3 were localized to the exocyst positive organelle (EXPO)-like punctate structures. OsPUB2 was also localized to the nuclei. OsPUB2 and OsPUB3 formed a hetero-dimeric complex as well as homo-dimers in yeast cells and in vitro. OsPUB2/OsPUB3 exhibited self-ubiquitination activities in vitro and were rapidly degraded in the cell-free extracts with apparent half-lives of 150–160 min. This rapid degradation of OsPUB2/OsPUB3 was delayed in the presence of the crude extracts of cold-treated seedlings (apparent half-lives of 200–280 min). Moreover, a hetero-dimeric form of OsPUB2/OsPUB3 was more stable than the homo-dimers. These results suggested that OsPUB2 and OsPUB3 function coordinately in response to cold stress. OsPUB2- and OsPUB3-overexpressing transgenic rice plants showed markedly better tolerance to cold stress than did the wild-type plants in terms of survival rates, chlorophyll content, ion leakage, and expression levels of cold stress-inducible marker genes. Taken together, these results suggested that the two homologous rice U-box E3 Ub ligases OsPUB2 and OsPUB3 are positive regulators of the response to cold stress.

CHFR negatively regulates SIRT1 activity upon oxidative stress.

SIRT1, the NAD+-dependent protein deacetylase, controls cell-cycle progression and apoptosis by suppressing p53 tumour suppressor. Although SIRT1 is known to be phosphorylated by JNK1 upon oxidative stress and subsequently down-regulated, it still remains elusive how SIRT1 stability and activity are controlled. Here, we have unveiled that CHFR functions as an E3 Ub-ligase of SIRT1, responsible for its proteasomal degradation under oxidative stress conditions. CHFR interacts with and destabilizes SIRT1 by ubiquitylation and subsequent proteolysis. Such CHFR-mediated SIRT1 inhibition leads to the increase of p53 acetylation and its target gene transcription. Notably, CHFR facilitates SIRT1 destabilization when SIRT1 is phosphorylated by JNK1 upon oxidative stress, followed by prominent apoptotic cell death. Meanwhile, JNK inhibitor prevents SIRT1 phosphorylation, leading to elevated SIRT1 protein levels even in the presence of H2O2. Taken together, our results indicate that CHFR plays a crucial role in the cellular stress response pathway by controlling the stability and function of SIRT1.

Ubiquitin-Dependent Modification of Skeletal Muscle by the Parasitic Nematode, Trichinella spiralis.

Trichinella spiralis is a muscle-specific parasitic worm that is uniquely intracellular. T. spiralis reprograms terminally differentiated skeletal muscle cells causing them to de-differentiate and re-enter the cell cycle, a process that cannot occur naturally in mammalian skeletal muscle cells, but one that holds great therapeutic potential. Although the host ubiquitin pathway is a common target for viruses and bacteria during infection, its role in parasite pathogenesis has been largely overlooked. Here we demonstrate that the secreted proteins of T. spiralis contain E2 Ub-conjugating and E3 Ub-ligase activity. The E2 activity is attributed to TsUBE2L3, a novel and conserved T. spiralis enzyme located in the secretory organ of the parasite during the muscle stages of infection. TsUBE2L3 cannot function with any T.spiralis secreted E3, but specifically binds to a panel of human RING E3 ligases, including the RBR E3 ARIH2 with which it interacts with a higher affinity than the mammalian ortholog UbcH7/UBE2L3. Expression of TsUBE2L3 in skeletal muscle cells causes a global downregulation in protein ubiquitination, most predominantly affecting motor, sarcomeric and extracellular matrix proteins, thus mediating their stabilization with regards to proteasomal degradation. This effect is not observed in the presence of the mammalian ortholog, suggesting functional divergence in the evolution of the parasite protein. These findings demonstrate the first example of host-parasite interactions via a parasite-derived Ub conjugating enzyme; an E2 that demonstrates a novel muscle protein stabilization function.