Dietary protection against the visual and motor deficits induced by experimental autoimmune encephalomyelitis.

Introduction: Five to eight percent of the world population currently suffers from at least one autoimmune disorder. Despite multiple immune modulatory therapies for autoimmune demyelinating diseases of the central nervous system, these treatments can be limiting for subsets of patients due to adverse effects and expense. To circumvent these barriers, we investigated a nutritional intervention in mice undergoing experimental autoimmune encephalomyelitis (EAE), a model of autoimmune-mediated demyelination that induces visual and motor pathologies similar to those experienced by people with multiple sclerosis (MS). Methods: EAE was induced in female and male mice and the impact of limiting dietary carbohydrates by feeding a ketogenic diet (KD) enriched in medium chain triglycerides (MCTs), alpha-linolenic acid (an omega-3 fatty acid), and fiber was evaluated in both a preventive regimen (prior to immunization with MOG antigen) and an interventional regimen (following the onset of symptoms). Motor scores were assigned daily and visual acuity was measured using optokinetic tracking. Immunohistochemical analyses of optic nerves were done to assess inflammatory infiltrates and myelination status. Fatty acid and cytokine profiling from blood were performed to evaluate systemic inflammatory status. Results: The KD was efficacious when fed as a preventive regimen as well as when initiated as an interventional regimen following symptom onset. The KD minimally impacted body weight during the experimental time course, increased circulating ketones, prevented motor and ocular deficits, preserved myelination of the optic nerve, and reduced infiltration of immune cells to optic nerves. The KD also increased anti-inflammatory-associated omega-3 fatty acids in the plasma and reduced select cytokines in the circulation associated with EAE-mediated pathological inflammation. Discussion: In light of ongoing clinical trials using dietary strategies to treat people with MS, these findings support that a KD enriched in MCTs, omega-3 fatty acids, and fiber promotes a systemic anti-inflammatory milieu and ameliorates autoimmune-induced demyelinating visual and motor deficits.

Sulforaphane recovers cone function in an Nrf2-dependent manner in middle-aged mice undergoing RPE oxidative stress.

Purpose: Sulforaphane (SFN) is an isothiocyanate derived from cruciferous vegetables that has therapeutic efficacy in numerous animal models of human disease, including mouse models of retinal degeneration. However, despite dozens of clinical trials, the compound remains to be tested as a clinical treatment for ocular disease. Numerous cellular activities of SFN have been identified, including the activation of Nrf2, a transcription factor that induces a battery of target gene products to neutralize oxidative and xenobiotic stresses. As Nrf2 expression and function reportedly decrease with aging, we tested whether the loss of the transcription factor limits the therapeutic efficacy of SFN against retinal degeneration. Methods: Six- to 8-month-old wild-type and Nrf2 knockout mice were treated with SFN beginning 1 month after ribozyme-mediated knockdown of superoxide dismutase 2 (SOD2) mRNA in the RPE. The impacts of MnSOD (the protein product of SOD2) knockdown and the efficacy of SFN were evaluated using a combination of electroretinography (ERG), spectral domain optical coherence tomography (SD-OCT), and postmortem histology. Results: SFN restored the ERG photopic b-wave suppressed by MnSOD loss in wild-type mice, but not in the Nrf2 knockout mice. In contrast, ERG scotopic a- and b-wave loss was not restored for either genotype. SFN significantly improved retinal thickness in the Nrf2 knockout mice with MnSOD knockdown, but this was not observed in the wild-type mice. In both genotypes, SFN treatment reduced morphological markers of RPE atrophy and degeneration, although these improvements did not correlate proportionally with functional recovery. Conclusions: These findings highlight the capacity of SFN to preserve cone function, as well as the potential challenges of using the compound as a standalone treatment for age-related retinal degeneration under conditions associated with reduced Nrf2 function.

Loss of the ubiquitin conjugating enzyme UBE2E3 induces cellular senescence.

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, p21CIP1/WAF1, or by co-depleting the tumor suppressor p16INK4a. 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.

A PGAM5-KEAP1-Nrf2 complex is required for stress-induced mitochondrial retrograde trafficking.

The Nrf2 transcription factor is a master regulator of the cellular anti-stress response. A population of the transcription factor associates with the mitochondria through a complex with KEAP1 and the mitochondrial outer membrane histidine phosphatase, PGAM5. To determine the function of this mitochondrial complex, we knocked down each component and assessed mitochondrial morphology and distribution. We discovered that depletion of Nrf2 or PGAM5, but not KEAP1, inhibits mitochondrial retrograde trafficking induced by proteasome inhibition. Mechanistically, this disrupted motility results from aberrant degradation of Miro2, a mitochondrial GTPase that links mitochondria to microtubules. Rescue experiments demonstrate that this Miro2 degradation involves the KEAP1-cullin-3 E3 ubiquitin ligase and the proteasome. These data are consistent with a model in which an intact complex of PGAM5-KEAP1-Nrf2 preserves mitochondrial motility by suppressing dominant-negative KEAP1 activity. These data further provide a mechanistic explanation for how age-dependent declines in Nrf2 expression impact mitochondrial motility and induce functional deficits commonly linked to neurodegeneration.

The nuclear transport receptor Importin-11 is a tumor suppressor that maintains PTEN protein.

Phosphatase and tensin homologue (PTEN) protein levels are critical for tumor suppression. However, the search for a recurrent cancer-associated gene alteration that causes PTEN degradation has remained futile. In this study, we show that Importin-11 (Ipo11) is a transport receptor for PTEN that is required to physically separate PTEN from elements of the PTEN degradation machinery. Mechanistically, we find that the E2 ubiquitin-conjugating enzyme and IPO11 cargo, UBE2E1, is a limiting factor for PTEN degradation. Using in vitro and in vivo gene-targeting methods, we show that Ipo11 loss results in degradation of Pten, lung adenocarcinoma, and neoplasia in mouse prostate with aberrantly high levels of Ube2e1 in the cytoplasm. These findings explain the correlation between loss of IPO11 and PTEN protein in human lung tumors. Furthermore, we find that IPO11 status predicts disease recurrence and progression to metastasis in patients choosing radical prostatectomy. Thus, our data introduce the IPO11 gene as a tumor-suppressor locus, which is of special importance in cancers that still retain at least one intact PTEN allele.

The ubiquitin-conjugating enzyme UBE2E3 and its import receptor importin-11 regulate the localization and activity of the antioxidant transcription factor NRF2.

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.

The ubiquitin-conjugating enzyme, UbcM2, is restricted to monoubiquitylation by a two-fold mechanism that involves backside residues of E2 and Lys48 of ubiquitin.

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.

Expression and distribution of the class III ubiquitin-conjugating enzymes in the retina.

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 ubiquitin-conjugating enzyme UbcM2 can regulate the stability and activity of the antioxidant transcription factor Nrf2.

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 ubiquitin conjugating enzyme, UbcM2, engages in novel interactions with components of cullin-3 based E3 ligases.

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.

The human ubiquitin conjugating enzyme, UBE2E3, is required for proliferation of retinal pigment epithelial cells.

PURPOSE: Cell cycle progression is governed by the coordinated activities of kinases, phosphatases, and the ubiquitin system. The entire complement of ubiquitin pathway components that mediate this process in retinal pigment epithelial (RPE) cells remains to be identified. This study was undertaken to determine whether the human ubiquitin-conjugating enzyme, UBE2E3, is essential for RPE cell proliferation. METHODS: UBE2E3 expression and localization in telomerase-immortalized, human RPE cells was determined with a UBE2E3-specific antibody. The necessity for UBE2E3 in RPE proliferation was determined using small interfering (si)RNA to target the expression of the enzyme. Cell counts and immunolabeling for the proliferation marker Ki-67 and the cyclin-dependent kinase inhibitor p27(Kip1) were performed to assess the consequences of UBE2E3 depletion. A mouse strain harboring a disrupted allele of UbcM2 (the mouse counterpart of UBE2E3) with the coding sequence for beta-galactosidase was used to track the developmental expression of the enzyme in murine RPE cells. RESULTS: UBE2E3 localized in the nucleus of the immortalized RPE cells. Depletion of the enzyme by siRNA resulted in a cell-cycle exit accompanied by a loss of Ki-67, an increase in p27(Kip1), and a doubling in cell area. Rescue experiments confirmed the specificity of the RNA interference. In vivo, UbcM2 was transcriptionally downregulated during RPE development in the mouse. CONCLUSIONS: UBE2E3 is essential for the proliferation of RPE-1 cells and is downregulated during RPE layer maturation in the developing mouse eye. These findings indicate that UBE2E3 is a major enzyme in modulating the balance between RPE cell proliferation and differentiation.

Ubiquitin charging of human class III ubiquitin-conjugating enzymes triggers their nuclear import.

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.