Three Brachypodium distachyon Uev1s Promote Ubc13-Mediated Lys63-Linked Polyubiquitination and Confer Different Functions.
ABSTRACT: In this study, we report the identification and functional characterization of three Brachypodium distachyon UEV genes. All three BdUev1s form heterodimers with BdUbc13s, which are capable of catalyzing Lys63-linked polyubiquitination in vitro. The three BdUEV1 genes are also able to functionally complement the budding yeast mms2 mutant defective in DNA-damage tolerance. BdUev1A differs from the other two BdUev1s in that it contains an 18-amino acid tail, which appears to compromise its function in yeast, as deletion of this tail restores full function. BdUev1A is excluded from the nucleus, whereas BdUev1B, BdUev1C and the C-terminally truncated BdUev1A are mainly found in the nucleus. These and the BdUEV1 gene expression analysis allow us to speculate that although all three BdUev1s function by promoting Lys63-linked polyubiquitination, BdUev1B and BdUev1C are involved in DNA-damage response and possibly other nuclear functions, while BdUev1A is required for non-nuclear function(s).
Project description:The error-free branch of the DNA-damage tolerance (DDT) pathway is orchestrated by Lys63-linked polyubiquitination of proliferating cell nuclear antigen (PCNA), and this polyubiquitination is mediated by a Ubc13-Uev complex in yeast. We have previously cloned OsUBC13 from rice, whose product functions as an E2 to promote Lys63-linked ubiquitin chain assembly in the presence of yeast or human Uev.Here we identify four highly conserved UEV1 genes in rice whose products are able to form stable heterodimers with OsUbc13 and mediate Lys63-linked ubiquitin chain assembly. Expression of OsUEV1s is able to rescue the yeast mms2 mutant from death caused by DNA-damaging agents. Interestingly, OsUev1A contains a unique C-terminal tail with a conserved prenylation site not found in the other three OsUev1s, and this post-translational modification appears to be required for its unique subcellular distribution and association with the membrane. The analysis of OsUEV1 expression profiles obtained from the Genevestigator database indicates that these genes are differentially regulated.We speculate that different OsUev1s play distinct roles by serving as a regulatory subunit of the Ubc13-Uev1 complex to respond to diverse cellular, developmental and environmental signals.
Project description:UEV1A encodes a ubiquitin-conjugating enzyme variant (Ubc13), which is required for Ubc13-catalyzed Lys63-linked polyubiquitination of target proteins and nuclear factor ?B (NF-?B) activation. Previous reports have correlated the level of UEV1A expression with tumorigenesis; however, the detailed molecular events leading to tumors particularly breast cancer and metastasis are unclear. This study is to investigate roles of different UEV1 splicing variants, and its close homolog MMS2, in promoting tumorigenesis and metastasis in breast cancer cells.We experimentally manipulated the UEV1 and MMS2 levels in MDA-MB-231 breast cancer cells and monitored their effects on cell invasion and migration, as well as tumor formation and metastasis in xenograft mice. The underlying molecular mechanisms leading to metastasis were also examined.It was found that overexpression of UEV1A alone, but not UEV1C or MMS2, is sufficient to induce cell invasion in vitro and metastasis in vivo. This process is mediated by NF-?B activation and requires functional Ubc13. Our experimental data establish that among NF-?B target genes, UEV1A-regulated matrix metalloproteinase-1 (MMP1) expression plays a critical role in cell invasion and metastasis. Interestingly, experimental depletion of UEV1 in MDA-MB-231 cells reduces MMP1 expression and prevents tumor formation and metastasis in a xenograft mouse model, while overexpression of MMP1 overrides the metastasis effects in UEV1-depleted cells.These results identify UEV1A as a potential therapeutic target in the treatment of metastasic breast cancers.
Project description:Ubc13 is an ubiquitin E2 conjugating enzyme that participates with many different E3 ligases to form lysine 63-linked (Lys63) ubiquitin chains that are critical to signaling in inflammatory and DNA damage response pathways. Recent studies have suggested Ubc13 as a potential therapeutic target for intervention in various human diseases including several different cancers, alleviation of anti-cancer drug resistance, chronic inflammation, and viral infections. Understanding a potential therapeutic target from different angles is important to assess its usefulness and potential pitfalls. Here we present a global review of Ubc13 from its structure, function, and cellular activities, to its natural and chemical inhibition. The aim of this article is to review the literature that directly implicates Ubc13 in a biological function, and to integrate structural and mechanistic insights into the larger role of this critical E2 enzyme. We discuss observations of multiple Ubc13 structures that suggest a novel mechanism for activation of Ubc13 that involves conformational change of the active site loop.
Project description:The cellular response to hypoxia is characterised by a switch in the transcriptional program, mediated predominantly by the hypoxia inducible factor family of transcription factors (HIF). Regulation of HIF1 is primarily controlled by post-translational modification of the HIF1? subunit, which can alter its stability and/or activity. This study identifies an unanticipated role for the X-linked inhibitor of apoptosis (XIAP) protein as a regulator of Lys63-linked polyubiquitination of HIF1?. Lys63-linked ubiquitination of HIF1? by XIAP is dependent on the activity of E2 ubiquitin conjugating enzyme Ubc13. We find that XIAP and Ubc13 dependent Lys63-linked polyubiquitination promotes HIF1? nuclear retention leading to an increase in the expression of HIF1 responsive genes. Inhibition of the Lys63-linked polyubiquitination pathway leads to reduced levels of nuclear HIF1?, promoter occupancy, HIF-dependent gene expression and cell viability. Our data reveals an additional and significant level of control of the HIF1 by XIAP, with important implications in understanding the role of HIF1 and XIAP in human disease.
Project description:The RAF kinase family is essential in mediating signal transduction from RAS to ERK. BRAF constitutively active mutations correlate with human cancer development. However, the precise molecular regulation of BRAF activation is not fully understood. Here we report that BRAF is modified by Lys63-linked polyubiquitination at lysine 578 within its kinase domain once it is activated by gain of constitutively active mutation or epidermal growth factor (EGF) stimulation. Substitution of BRAF lysine 578 with arginine (K578R) inhibited BRAF-mediated ERK activation. Furthermore, ectopic expression of BRAF K578R mutant inhibited anchorage-independent colony formation of MCF7 breast cancer cell line. Our studies have identified a previously unrecognized regulatory role of Lys63-linked polyubiquitination in BRAF-mediated normal and oncogenic signalings.
Project description:Transforming growth factor ? (TGF?) can act either as a tumor promoter or a tumor suppressor in a context-dependent manner. High levels of TGF? are found in prostate cancer tissues and correlate with poor patient prognosis. We recently identified a novel TGF?-regulated signaling cascade in which TGF? type I receptor (T?RI) is activated by the E3 ligase TNF-receptor-associated factor 6 (TRAF6) via the Lys63-linked polyubiquitination of T?RI. TRAF6 also contributes to activation of TNF-?-converting enzyme and presenilin-1, resulting in the proteolytic cleavage of T?RI and releasing the intracellular domain of T?RI, which is translocated to the nucleus to promote tumor invasiveness. In this report, we provide evidence that Lys178 of T?RI is polyubiquitinated by TRAF6. Moreover, our data suggest that TRAF6-mediated Lys63-linked ubiquitination of the T?RI intracellular domain is a prerequisite for TGF? regulation of mRNA for cyclin D1 (CCND1), expression, as well as for the regulation of other genes controlling the cell cycle, differentiation, and invasiveness of prostate cancer cells.
Project description:While plant growth and reproduction is dependent on sunlight, UV irradiation from sunlight is one of the major genotoxic stresses that threaten plant survival and genome stability. In addition, many environmental chemicals can also damage the plant genome. In yeast and mammalian cells protection against the above genome instability is provided by an error-free DNA-damage tolerance (DDT) pathway, which is dependent on Ubc13-mediated K63-linked polyubiquitination of the proliferating cell nuclear antigen (PCNA). In this study, we isolated the UBC13 gene from rice and characterized its functions. Expression of OsUBC13 can protect a yeast ubc13 null mutant against spontaneous and environmental DNA damage. Furthermore, OsUbc13 physically interacts with human Ubc13 partners Mms2 and Uev1A, and catalyzes K63 polyubiquitination in vitro. These observations collectively suggest that the K63 polyubiquitination is conserved in rice, and that OsUBC13 may be involved in DDT and other cellular processes. In addition, OsUBC13 is constitutively expressed at a high level even under various stress conditions, suggesting that it is a housekeeping gene.
Project description:Living organisms are constantly subject to DNA damage from environmental sources. Due to the sessile nature of plants, UV irradiation is a major genotoxic agent and imposes a significant threat on plant survival, genome stability and crop yield. In addition, other environmental chemicals can also influence the stability of the plant genome. Eukaryotic organisms have evolved a mechanism to cope with replication-blocking lesions and stabilize the genome. This mechanism is known as error-free DNA damage tolerance, and is mediated by K63-linked PCNA polyubiquitination. Genes related to K63-linked polyubiquitination have been isolated recently from model plants like Arabidopsis and rice, but we are unaware of such reports on the crop model Brachypodium distachyon. Here, we report the identification and functional characterization of two B. distachyon UBC13 genes. Both Ubc13s form heterodimers with Uevs from other species, which are capable of catalyzing K63 polyubiquitination in vitro. Both genes can functionally rescue the yeast ubc13 null mutant from killing by DNA-damaging agents. These results suggest that Ubc13-Uev-promoted K63-linked polyubiquitination is highly conserved in eukaryotes including B. distachyon. Consistent with recent findings that K63-linked polyubiquitination is involved in several developmental and stress-responsive pathways, the expression of BdUbc13s appears to be constitutive and is regulated by abnormal temperatures.
Project description:Post-replication DNA repair in eukaryotes is regulated by ubiquitination of proliferating cell nuclear antigen (PCNA). Monoubiquitination catalyzed by RAD6-RAD18 (an E2-E3 complex) stimulates translesion DNA synthesis, whereas polyubiquitination, promoted by additional factors such as MMS2-UBC13 (a UEV-E2 complex) and HLTF (an E3 ligase), leads to template switching in humans. Here, using an in vitro ubiquitination reaction system reconstituted with purified human proteins, we demonstrated that PCNA is polyubiquitinated predominantly via en bloc transfer of a pre-formed ubiquitin (Ub) chain rather than by extension of the Ub chain on monoubiquitinated PCNA. Our results support a model in which HLTF forms a thiol-linked Ub chain on UBC13 (UBC13?Ubn) and then transfers the chain to RAD6?Ub, forming RAD6?Ubn+1. The resultant Ub chain is subsequently transferred to PCNA by RAD18. Thus, template switching may be promoted under certain circumstances in which both RAD18 and HLTF are coordinately recruited to sites of stalled replication.
Project description:The data described herein are related to the article entitled "Lys63-linked ubiquitin chain adopts multiple conformational states for specific target recognition" , and to the coordinates for the ensemble structure of Lys63-linked diubiquitin (PDB code 2N2K). A Lys63-linked diubiquitin exists in three conformational states with different orientations for the two subunits, each responsible for binding to a target protein and encoding a specific cell signal. An atomic entry in the ensemble structure file consists multiple lines, representing alternative locations of the atom and recapitulating the dynamics of the protein. Experimental details about obtaining strictly intramolecular paramagnetic restraints and determining the relative occupancies of the conformational states are presented. The experimental design and procedures in this Data article can be useful for characterizing the structure and dynamics of other multi-domain proteins.