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Cellular senescence plays essential roles in tissue homeostasis as well as a host of diseases ranging from cancers to age-related neurodegeneration. Various molecular pathways can induce senescence and these different pathways dictate the phenotypic and metabolic changes that accompany the transition to, and maintenance of, the senescence state. Here, we describe a novel senescence phenotype induced by depletion of UBE2E3, a highly-conserved, metazoan ubiquitin conjugating enzyme. Cells depleted of UBE2E3 become senescent in the absence of overt DNA damage and have a distinct senescence-associated secretory phenotype, increased mitochondrial and lysosomal mass, an increased sensitivity to mitochondrial and lysosomal poisons, and an increased basal autophagic flux. This senescence phenotype can be partially suppressed by co-depletion of either p53 or its cognate target gene, p21 CIP1/WAF1 , or by co-depleting the tumor suppressor p16 INK4a . Together, these data describe a direct link of a ubiquitin conjugating enzyme to cellular senescence and further underscore the consequences of disrupting the integration between the ubiquitin proteolysis system and the autophagy machinery., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

The transcription factor NF-E2 p45-related factor (Nrf2) induces the expression of cytoprotective proteins that maintain and restore redox homeostasis. Nrf2 levels and activity are tightly regulated, and three subcellular populations of the transcription factor have been identified. During homeostasis, the majority of Nrf2 is degraded in the cytoplasm by ubiquitin (Ub)-mediated degradation. A second population is transcriptionally active in the nucleus, and a third population localizes to the outer mitochondrial membrane. Still unresolved are the mechanisms and factors that govern Nrf2 distribution between its subcellular locales. We show here that the Ub-conjugating enzyme UBE2E3 and its nuclear import receptor importin 11 (Imp-11) regulate Nrf2 distribution and activity. Knockdown of UBE2E3 reduces nuclear Nrf2, decreases Nrf2 target gene expression, and relocalizes the transcription factor to a perinuclear cluster of mitochondria. In a complementary manner, Imp-11 functions to restrict KEAP1, the major suppressor of Nrf2, from prematurely extracting the transcription factor off of a subset of target gene promoters. These findings identify a novel pathway of Nrf2 modulation during homeostasis and support a model in which UBE2E3 and Imp-11 promote Nrf2 transcriptional activity by restricting the transcription factor from partitioning to the mitochondria and limiting the repressive activity of nuclear KEAP1., (© 2015 Plafker and Plafker. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Proteins can be modified on lysines (K) with a single ubiquitin (Ub) or with polymers of Ub (polyUb). These different configurations and their respective topologies are primary factors for determining whether substrates are targeted to the proteasome for degradation or directed to nonproteolytic outcomes. We report here on the intrinsic ubiquitylation properties of UbcM2 (UBE2E3/UbcH9), a conserved Ub-conjugating enzyme linked to cell proliferation, development, and the cellular antioxidant defense system. Using a fully recombinant ubiquitylation assay, we show that UbcM2 is severely limited in its ability to synthesize polyUb chains with wild-type Ub. Restriction to monoubiquitylation is governed by multiple residues on the backside of the enzyme, far removed from its active site, and by lysine 48 of Ub. UbcM2 with mutated backside residues can synthesize K63-linked polyUb chains and to a lesser extent K6- and K48-linked chains. Additionally, we identified a single residue on the backside of the enzyme that promotes monoubiquitylation. Together, these findings reveal that a combination of noncatalytic residues within the Ubc catalytic core domain of UbcM2 as well as a lysine(s) within Ub can relegate a Ub-conjugating enzyme to monoubiquitylate its cognate targets despite having the latent capacity to construct polyUb chains. The two-fold mechanism for restricting activity to monoubiquitylation provides added insurance that UbcM2 will not build polyUb chains on its substrates, even under conditions of high local Ub concentrations.

Purpose: Mounting evidence implicates chronic oxidative stress as a significant pathogenic factor in the development and progression of retinopathies, including age-related macular degeneration (AMD). The age-dependent toxic accumulation of oxidatively damaged proteins, lipids, and DNA in susceptible cells of the retina arises, at least in part, from a decreased capacity to eliminate these damaged biomolecules. The goal of this study was to determine the expression patterns and function of class III ubiquitin-conjugating enzymes (UbcM3, UBE2E2, and UbcM2) in the retina. These enzymes have been implicated in the ubiquitin-dependent degradation of oxidatively damaged and misfolded proteins., Methods: Complementary western blotting and immunohistochemistry was performed with specific antibodies to determine the retinal cell expression pattern of each enzyme. Additional analyses using antibodies raised against UbcM2 were performed to determine the relative levels of the enzyme in lysates derived from various mouse organs as compared to the retina. An established light-damage model of oxidative stress-induced retinal degeneration was used to determine alterations in the susceptibility of mice harboring a single intact allele of UbcM2. Ubiquitin charging and auto-ubiquitylation assays were done to assess the catalytic state of UbcM2 following photo-oxidative stress., Results: Expression of the class III ubiquitin-conjugating enzymes in the retina, from highest to lowest, is UbcM2>UbcM3>UBE2E2. In addition to being the most robustly expressed, UbcM2 is further distinguished by its expression in photoreceptors and retinal pigment epithelial cells. UbcM2 is expressed in most mouse tissues analyzed and is most abundant in the retina. Studies using a bright-light-damage model of acute oxidative stress in mice harboring a single disrupted allele of UbcM2 revealed that a 58% reduction in enzyme levels did not increase the susceptibility of photoreceptors to acute photo-oxidative toxicity. This result may be explained by the observation that UbcM2 retained an intact and functional active site following exposure to acute bright light., Conclusions: The class III ubiquitin-conjugating enzymes, and in particular UbcM2, are expressed in the retina and may function to counter the accumulation of oxidatively damaged and misfolded proteins. A 58% reduction in UbcM2 does not increase the susceptibility of photoreceptors to an acute photo-oxidative stress, suggesting the existence of compensating enzymes and/or that the remaining UbcM2 activity is sufficient to target oxidatively damaged proteins for destruction.

The transcription factor nuclear factor E2-related factor 2 (Nrf2) induces the expression of antioxidant gene products that neutralize reactive oxygen species and restore redox homeostasis. Nrf2 is constitutively degraded by the ubiquitin proteolytic system in unperturbed cells, but this turnover is arrested in response to oxidative stress, thereby leading to Nrf2 accumulation. Yet, a mechanistic understanding of how Nrf2 stabilization and transcriptional activation are coupled remains to be determined. We have discovered that the ubiquitin-conjugating enzyme UbcM2 is a novel regulator of Nrf2. Recombinant Nrf2 and UbcM2 form a complex upon alkylation of a non-catalytic cysteine in UbcM2, Cys-136. Substitution of this cysteine with a phenylalanine (C136F) to mimic cysteine oxidation/alkylation results in constitutive binding of UbcM2 to Nrf2 and an increased half-life of the transcription factor in vivo. We provide evidence that UbcM2 and Nrf2 form a nuclear complex utilizing the DNA binding, Neh1 domain, of Nrf2. Finally, we demonstrate that UbcM2 can enhance the transcriptional activity of endogenous Nrf2 and that Cys-136 and the active-site cysteine, Cys-145, jointly contribute to this regulation. Collectively, these data identify UbcM2 as a novel component of the Nrf2 regulatory circuit and position cysteine 136 as a putative redox sensor in this signaling pathway. This work implicates UbcM2 in the restoration of redox homeostasis following oxidative stress.

The class III ubiquitin conjugating enzymes (E2s) are distinguished from other E2s by the presence of unique N-terminal domains, and the utilization of importin-11 for transport into the nucleus in an activation dependent fashion. To begin determining the physiological roles of these enzymes, we carried out a yeast two-hybrid screen with the class III E2, UbcM2. This screen retrieved RCBTB1, a putative substrate adaptor for a cullin3 (CUL3) E3 ligase. We initially established through biochemical studies that RCBTB1 has the properties of a CUL3 substrate adaptor. Further analysis of the UbcM2-RCBTB1 complex led to the discovery and characterization of the following novel interactions: (i) UbcM2 binds an N-terminal domain of CUL3 requiring the first 57 amino acids, the same domain that binds to RCBTB1 and other substrate adaptors; (ii) UbcM2 does not bind mutants of CUL3 that are deficient in substrate adaptor recruitment; (iii) UbcM2 interacts with CUL3 independent of a bridging RING-finger protein; and (iv) can engage the neddylated (i.e., activated) form of CUL3. We also present evidence that UbcM2 can bind to the N-terminal halves of multiple cullins, implying that this E2 is a general cofactor for this class of ligases. Together, these studies represent the first evidence that UbcM2, in concert with substrate adaptors, engages activated CUL3 ligases, thus suggesting that class III E2s are novel regulators of cullin ligases.

Ubiquitin is a small polypeptide that is conjugated to proteins and commonly serves as a degradation signal. The attachment of ubiquitin (Ub) to a substrate proceeds through a multi-enzyme cascade involving an activating enzyme (E1), a conjugating enzyme (E2), and a protein ligase (E3). We previously demonstrated that a murine E2, UbcM2, is imported into nuclei by the transport receptor importin-11. We now show that the import mechanism for UbcM2 and two other human class III E2s (UbcH6 and UBE2E2) uniquely requires the covalent attachment of Ub to the active site cysteine of these enzymes. This coupling of E2 activation and transport arises from the selective interaction of importin-11 with the Ub-loaded forms of these enzymes. Together, these findings reveal that Ub charging can function as a nuclear import trigger, and identify a novel link between E2 regulation and karyopherin-mediated transport.