E3 Ubiquitin Research Articles

Recruitment to the Proteasome Is Necessary but Not Sufficient for Chemically Induced, Ubiquitin-Independent Degradation of Native Proteins.

Targeted protein degradation (TPD) is a promising strategy for drug development. Most degraders function by forcing the association of the target protein (TP) with an E3 Ubiquitin (Ub) ligase, which, in favorable cases, results in the polyubiquitylation of the TP and its subsequent degradation by the 26S proteasome. An alternative strategy would be to create chemical dimerizers that bypass the requirement for polyubiquitylation by recruiting the target protein directly to the proteasome. Direct-to-proteasome degraders (DPDs) may exhibit different characteristics than ubiquitin-dependent degraders, but few studies of this type of TPD have been published, largely due to the dearth of suitable proteasome ligands. To facilitate studies of DPDs, we report here a mammalian cell line in which the HaloTag protein is fused to the proteasome via Rpn13, one of the ubiquitin receptors. In these cells, a chloroalkane serves as a covalent proteasome ligand surrogate. We show that chimeric molecules comprised of a chloroalkane linked to a ligand for the BET family of proteins or the Cdk2/7/9 family of kinases result in ubiquitin-independent degradation of some of these target proteins. We use this system, the first that allows facile degradation of native proteins in a ubiquitin-independent fashion, to probe two issues: the effect of varying the length of the linker connecting the chloroalkane and the target ligand and the selectivity of degradation within the protein families engaged by the target ligand.

Potato E3 Ubiquitin Ligase StXERICO1 Positively Regulates Drought Resistance by Enhancing ABA Accumulation in Potato and Tobacco and Interacts with the miRNA Novel-miR1730-3p and Proteins StUBC and StTLP

Potato (Solanum tuberosum L.) is sensitive to drought, which severely impacts tuber yield and quality. In this study, we characterized a XERICO gene, encoding a RING-H2 type E3 ubiquitin ligase, StXERICO1, from a diploid potato, investigated its role in enhancing drought resistance and ABA accumulation, and identified its interaction with the miRNA novel-miR1730-3p, as well as its protein interactions with StUBC and StTLP. StXERICO1, with a complete Open Reading Frame (ORF) of 459 bp encoding 152 amino acids, was highly responsive to drought, ABA treatment, and abiotic stresses in potato plants. Overexpression of the StXERICO1 significantly enhanced drought resistance and ABA accumulation in transgenic potato and tobacco plants and exhibited greater sensitivity to ABA treatment, which was associated with the upregulation of expression of ABA biosynthetic genes NCED and CYP707A. Furthermore, our results revealed that StXERICO1 and its encoding protein interacted with miRNAs and other proteins. 5′ RLM-RACE (cDNA terminal rapid amplification) experiment showed that the miRNA novel-miR1730-3p targets 5′ UTR region of the StXERICO1 gene. Dual luciferase assay and virus-based miRNA silencing experiment showed that the novel-miR1730-3p negatively regulates StXERICO1 expression. Moreover, yeast two-hybrid assay indicated that StXERICO1 interacts with StUBC (an E2 ubiquitin ligase) and StTLP (a Tubby-like protein), suggesting that StXERICO1 might function on ABA homeostasis at the post-translational level. These findings elucidate the molecular mechanisms by which StXERICO1, a RING-H2 type E3 ubiquitin ligase, enhances drought resistance through increased ABA accumulation, how its expression is regulated by miRNA, and how it exerts its function through interactions with other proteins. The results also provide a potential candidate gene for subsequent precision molecular breeding aimed at improving crop drought resistance.

UBA1 dysfunction in VEXAS and cancer.

UBA1, an X-linked gene, encodes one of the only two ubiquitin E1 enzymes, playing a pivotal role in initiating one of the most essential post-translational modifications. In late 2020, partial loss-of-function mutations in UBA1 within hematopoietic stem and progenitor cells were found to be responsible for VEXAS Syndrome, a previously unidentified hematoinflammatory disorder predominantly affecting older males. The condition is characterized by severe inflammation, cytopenias, and an association to hematologic malignancies. In this research perspective, we comprehensively review the molecular significance of UBA1 loss of function as well as advancements in VEXAS research over the past four years for each of the VEXAS manifestations - inflammation, cytopenias, clonality, and possible oncogenicity. Special attention is given to contrasting the M41 and non-M41 mutations, aiming to elucidate their differential effects and to identify targetable mechanisms responsible for each of the symptoms. Finally, we explore the therapeutic landscape for VEXAS Syndrome, discussing the efficacy and potential of clone-targeting drugs based on the pathobiology of VEXAS. This includes azacitidine, currently approved for myelodysplastic neoplasms (MDS), novel UBA1 inhibitors being developed for a broad spectrum of cancers, Protein Kinase R-like Endoplasmic Reticulum Kinase (PERK) inhibitors, and auranofin, a long-established drug for rheumatoid arthritis. This perspective bridges basic research to clinical symptoms and therapeutics.

A Novel Confocal Scanning Protein-Protein Interaction Assay (PPI-CONA) Reveals Exceptional Selectivity and Specificity of CC0651, a Small Molecule Binding Enhancer of the Weak Interaction between the E2 Ubiquitin-Conjugating Enzyme CDC34A and Ubiquitin.

Protein-protein interactions (PPIs) are some of the most challenging target classes in drug discovery. Highly sensitive detection techniques are required for the identification of chemical modulators of PPIs. Here, we introduce PPI confocal nanoscanning (PPI-CONA), a miniaturized, microbead based high-resolution fluorescence imaging assay. We demonstrate the capabilities of PPI-CONA by detecting low affinity ternary complex formation between the human CDC34A ubiquitin-conjugating (E2) enzyme, ubiquitin, and CC0651, a small molecule enhancer of the CDC34A-ubiquitin interaction. We further exemplify PPI-CONA with an E2 enzyme binding study on CC0651 and a CDC34A binding specificity study of a series of CC0651 analogues. Our results indicate that CC0651 is highly selective toward CDC34A. We further demonstrate how PPI-CONA can be applied to screening very low affinity interactions. PPI-CONA holds potential for high-throughput screening for modulators of PPI targets and characterization of their affinity, specificity, and selectivity.

Ubiquitin-specific protease UBP14 stabilizes HY5 by deubiquitination to promote photomorphogenesis in Arabidopsis thaliana

Transcription factor ELONGATED HYPOCOTYL5 (HY5) is the central hub for seedling photomorphogenesis. E3 ubiquitin (Ub) ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) inhibits HY5 protein accumulation through ubiquitination. However, the process of HY5 deubiquitination, which antagonizes E3 ligase-mediated ubiquitination to maintain HY5 homeostasis has never been studied. Here, we identified that Arabidopsis thaliana deubiquitinating enzyme, Ub-SPECIFIC PROTEASE 14 (UBP14) physically interacts with HY5 and enhances its protein stability by deubiquitination. The da3-1 mutant lacking UBP14 function exhibited a long hypocotyl phenotype, and UBP14 deficiency led to the failure of rapid accumulation of HY5 during dark to light. In addition, UBP14 preferred to stabilize nonphosphorylated form of HY5 which is more readily bound to downstream target genes. HY5 promoted the expression and protein accumulation of UBP14 for positive feedback to facilitate photomorphogenesis. Our findings thus established a mechanism by which UBP14 stabilizes HY5 protein by deubiquitination to promote photomorphogenesis in A. thaliana.

A eukaryotic-like ubiquitination system in bacterial antiviral defence.

Ubiquitination pathways have crucial roles in protein homeostasis, signalling and innate immunity1-3. In these pathways, an enzymatic cascade of E1, E2 and E3 proteins conjugates ubiquitin or a ubiquitin-like protein (Ubl) to target-protein lysine residues4. Bacteria encode ancient relatives of E1 and Ubl proteins involved in sulfur metabolism5,6, but these proteins do not mediate Ubl-target conjugation, leaving open the question of whether bacteria can perform ubiquitination-like protein conjugation. Here we demonstrate that a bacterial operon associated with phage defence islands encodes a complete ubiquitination pathway. Two structures of a bacterial E1-E2-Ubl complex reveal striking architectural parallels with canonical eukaryotic ubiquitination machinery. The bacterial E1 possesses an amino-terminal inactive adenylation domain and a carboxy-terminal active adenylation domain with a mobile α-helical insertion containing the catalytic cysteine (CYS domain). One structure reveals a pre-reaction state with the bacterial Ubl C terminus positioned for adenylation, and a second structure mimics an E1-to-E2 transthioesterification state with the E1 CYS domain adjacent to the bound E2. We show that a deubiquitinase in the same pathway preprocesses the bacterial Ubl, exposing its C-terminal glycine for adenylation. Finally, we show that the bacterial E1 and E2 collaborate to conjugate Ubl to target-protein lysine residues. Together, these data reveal that bacteria possess bona fide ubiquitination systems with strong mechanistic and architectural parallels to canonical eukaryotic ubiquitination pathways, suggesting that these pathways arose first in bacteria.

Phage display identification of high-affinity ligands for human TSG101-UEV: A structural and thermodynamic study of PTAP recognition

The ubiquitin E2 variant domain of TSG101 (TSG101-UEV) plays a pivotal role in protein sorting and virus budding by recognizing PTAP motifs within ubiquitinated proteins. Disrupting TSG101-UEV/PTAP interactions has emerged as a promising strategy for the development of novel host-oriented antivirals with a broad spectrum of action. Nonetheless, finding inhibitors with good properties as therapeutic agents remains a challenge since the key determinants of binding affinity and specificity are still poorly understood. Here we present a detailed thermodynamic, structural, and dynamic characterization viral PTAP Late domain recognition by TSG101-UEV, combining isothermal titration calorimetry, X-ray diffraction structural studies, molecular dynamics simulations, and computational analysis of intramolecular communication pathways. Our analysis highlights key contributions from conserved hydrophobic contacts and water-mediated hydrogen bonds at the PTAP binding interface. We have identified additional electrostatic hotspots adjacent to the core motif that modulate affinity. Using competitive phage display screening we have improved affinity by 1–2 orders of magnitude, producing novel peptides with low micromolar affinities that combine critical elements found in the best natural binders. Molecular dynamics simulations revealed that optimized peptides engage new pockets on the UEV domain surface. This study provides a comprehensive view of the molecular forces directing TSG101-UEV recognition of PTAP motifs, revealing that binding is governed by conserved structural elements yet tuneable through targeted optimization. These insights open new venues to design inhibitors targeting TSG101-dependent pathways with potential application as novel broad-spectrum antivirals.

VEXAS syndrome.

VEXAS syndrome is a recently identified, adult-onset autoinflammatory disease caused by somatic mutations in UBA1. UBA1 is an X-linked gene encoding E1 ubiquitin activating enzyme and its mutation in hematopoietic stem and progenitor cells leads to their clonal expansion and myeloid-skewed differentiation. UBA1 mutations in VEXAS are clustered at the second methionine (p.Met41), eliminating UBA1b isoform translated from p.Met41. Loss of UBA1b impairs ubiquitination and activates innate immune pathways, leading to systemic autoinflammation manifested as recurrent fever, chondritis, pulmonary involvement, vasculitis, or neutrophilic dermatitis. VEXAS syndrome is frequently associated with hematological disorders such as myelodysplastic syndrome (MDS), plasma cell dyscrasia and venous thromboembolism. Macrocytic anemia/macrocytosis and vacuoles in myeloid/erythroid precursors are prominent features of VEXAS syndrome, and their presence in patients with autoinflammatory symptoms prompts physicians to screen for UBA1 variant. Treatment of VEXAS syndrome is challenging and no consistently effective therapies have been established. Anti-inflammation therapies including glucocorticoids and anti-interleukin-6 have shown limited efficacy, while azacytidine and JAK inhibitors such as ruxolitinib were found to induce favorable, mid-term responses. Hematopoietic stem cell transplantation is the only curative option for VEXAS and should be considered for younger, fit patients with poor prognostic factors or recalcitrant symptoms.

Using Förster Resonance Energy Transfer (FRET) to Understand the Ubiquitination Landscape.

Förster resonance energy transfer (FRET) is a fluorescence technique that allows quantitative measurement of protein interactions, kinetics and dynamics. This review covers the use of FRET to study the structures and mechanisms of ubiquitination and related proteins. We survey FRET assays that have been developed where donor and acceptor fluorophores are placed on E1, E2 or E3 enzymes and ubiquitin (Ub) to monitor steady-state and real-time transfer of Ub through the ubiquitination cascade. Specialized FRET probes placed on Ub and Ub-like proteins have been developed to monitor Ub removal by deubiquitinating enzymes (DUBs) that result in a loss of a FRET signal upon cleavage of the FRET probes. FRET has also been used to understand conformational changes in large complexes such as multimeric E3 ligases and the proteasome, frequently using sophisticated single molecule methods. Overall, FRET is a powerful tool to help unravel the intricacies of the complex ubiquitination system.

Proinsulin degradation and presentation of a proinsulin B-chain autoantigen involves ER-associated protein degradation (ERAD)-enzyme UBE2G2.

Type 1 diabetes (T1D) is characterized by HLA class I-mediated presentation of autoantigens on the surface of pancreatic β-cells. Recognition of these autoantigens by CD8+ T cells results in the destruction of pancreatic β-cells and, consequently, insulin deficiency. Most epitopes presented at the surface of β-cells derive from the insulin precursor molecule proinsulin. The intracellular processing pathway(s) involved in the generation of these peptides are poorly defined. In this study, we show that a proinsulin B-chain antigen (PPIB5-14) originates from proinsulin molecules that are processed by ER-associated protein degradation (ERAD) and thus originate from ER-resident proteins. Furthermore, screening genes encoding for E2 ubiquitin conjugating enzymes, we identified UBE2G2 to be involved in proinsulin degradation and subsequent presentation of the PPIB10-18 autoantigen. These insights into the pathway involved in the generation of insulin-derived peptides emphasize the importance of proinsulin processing in the ER to T1D pathogenesis and identify novel targets for future T1D therapies.

Ocular and orbital manifestations in VEXAS syndrome

BackgroundVEXAS (Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic) is a hematoinflammatory disease that typically affects adults. It results from a somatic mutation of the E1 ubiquitin conjugating enzyme encoded by the UBA1 gene. VEXAS is frequently accompanied by myelodysplastic syndrome (MDS). The purpose of this study is to describe the ocular and orbital manifestations of VEXAS patients in a case series in our medical centre.MethodsA retrospective chart review was performed for all patients who were diagnosed with VEXAS syndrome in a tertiary medical centre over two years.ResultsEight patients were identified with VEXAS. In six patients, the diagnosis was confirmed by genomic sequencing. Two patients were identified based on their phenotype. All patients were males. The mean age at diagnosis was 78.7 years. In two patients, the ocular manifestation was the presenting symptom for VEXAS. Seven patients (87.5%) had history of MDS. Systemic inflammation manifestations include: skin rash (n = 5), recurrent fevers (n = 2), relapsing polychondritis (n = 2), pleuritis and pleural effusion (n = 2), poly arteritis nodosa- PAN (n = 1) and thrombophlebitis (n = 1). Seven (87%) patients were presented with periorbital oedema. Three patients showed orbital inflammation. Dacryoadenitis was observed in two patients, and extraocular muscle (EOM) myositis was detected in two patients. Four patients demonstrated ocular inflammation such as: episcleritis, scleritis and anterior uveitis.Conclusionocular manifestations in VEXAS include orbital inflammation, dacryoadenitis, myositis, uveitis, scleritis, episcleritis and periorbital oedema. We recommend that in old male patients, with history of haematological disorder, presenting with ocular symptom, VEXAS investigation should be taken into consideration.

Structure of the human Bre1 complex bound to the nucleosome

Histone H2B monoubiquitination (at Lys120 in humans) regulates transcription elongation and DNA repair. In humans, H2B monoubiquitination is catalyzed by the heterodimeric Bre1 complex composed of Bre1A/RNF20 and Bre1B/RNF40. The Bre1 proteins generally function as tumor suppressors, while in certain cancers, they facilitate cancer cell proliferation. To obtain structural insights of H2BK120 ubiquitination and its regulation, we report the cryo-electron microscopy structure of the human Bre1 complex bound to the nucleosome. The two RING domains of Bre1A and Bre1B recognize the acidic patch and the nucleosomal DNA phosphates around SHL 6.0–6.5, which are ideally located to recruit the E2 enzyme and ubiquitin for H2BK120-specific ubiquitination. Mutational experiments suggest that the two RING domains bind in two orientations and that ubiquitination occurs when Bre1A binds to the acidic patch. Our results provide insights into the H2BK120-specific ubiquitination by the Bre1 proteins and suggest that H2B monoubiquitination can be regulated by nuclesomal DNA flexibility.

Physio-biochemical and DNA methylation analysis of the defense response network of wheat to drought stress

In the present work, physio-biochemical and DNA methylation analysis were conducted in wheat (Triticum aestivum L.) cultivars “Bolani” (drought-tolerant) and “Sistan” (drought-sensitive) during drought treatments: well-watered (at 90% field capacity (FC)), mild stress (at 50% FC, and severe stress (at 25% FC). During severe stress, O2•− and H2O2 content in cultivar Sistan showed significant increase (by 1.3 and 2.5-fold, respectively) relative to cultivar Bolani. In Bolani, the increased levels of radical scavenging activity (by 32%), glycine betaine (GB) (by 11.44%), proline (4-fold), abscisic acid (by 63.76%), and more stability of relative water content (RWC) (2-fold) were observed against drought-induced oxidative stress. Methylation level significantly decreased from 70.26% to 60.64% in Bolani and from 69.06% to 59.85% in Sistan during stress, and higher decreased tendency was related to CG and CHG in Bolani but CG in Sistan under severe stress. Methylation patterns showed that the highest polymorphism in Bolani was mainly as CG. As the intensity of stress increased, the enhanced physio-biochemical responses of Bolani cultivar were accompanied by a more decrease in the number of unchanged bands. According to heat map analysis, the highest difference (84.38%) in methylation patterns was observed between control and severe stress. Multivariate analysis using principal component analysis (PCA) showed a cultivar-specific methylation during stress and that methylation changes between cultivars are much higher than that of within a cultivar. Higher methylation to demethylation in Bolani (30.06 vs. 22.12%) compared to that of cultivar Sistan (23.21 vs. 30.15%) indicated more demethylation did not induce tolerance responses in Sistan. Sequencing differentially methylated fragments along with qRT-PCR analysis showed the efficient role of various DNA fragments, including demethylated fragments such as phosphoenol pyruvate carboxylase (PEPC), beta-glucosidase (BGlu), glycosyltransferase (GT), glutathione S-transferase (GST) and lysine demethylase (LSD) genes and methylated fragments like ubiquitin E2 enzyme genes in the development of drought tolerance. These results suggested the specific roles of DNA methylation in development of drought tolerance in wheat landrace.

Identification of membrane curvature sensing motifs essential for VPS37A phagophore recruitment and autophagosome closure

VPS37A, an ESCRT-I complex component, is required for recruiting a subset of ESCRT proteins to the phagophore for autophagosome closure. However, the mechanism by which VPS37A is targeted to the phagophore remains obscure. Here, we demonstrate that the VPS37A N-terminal domain exhibits selective interactions with highly curved membranes, mediated by two membrane-interacting motifs within the disordered regions surrounding its Ubiquitin E2 variant-like (UEVL) domain. Site-directed mutations of residues in these motifs disrupt ESCRT-I localization to the phagophore and result in defective phagophore closure and compromised autophagic flux in vivo, highlighting their essential role during autophagy. In conjunction with the UEVL domain, we postulate that these motifs guide a functional assembly of the ESCRT machinery at the highly curved tip of the phagophore for autophagosome closure. These results advance the notion that the distinctive membrane architecture of the cup-shaped phagophore spatially regulates autophagosome biogenesis.

Recruitment of ubiquitin E2 enzymes is determined jointly by the U-box domains and substrates of E3 ligases.

Ubiquitination is a cascade reaction involving E1, E2, and E3 enzymes. The orthogonal ubiquitin transfer (OUT) method has been previously established to identify potential substrates of E3 ligases. In this study, we verified the ubiquitination of five substrates mediated by the E3 ligases CHIP and E4B. To further explore the activity of U-box domains of E3 ligases, two mutants with the U-box domains interchanged between CHIP and E4B were generated. They exhibited a significantly reduced ubiquitination ability. Additionally, different E3s recruited similar E2 ubiquitin-conjugating enzymes when ubiquitinating the same substrates, highlighting that U-box domains determined the E2 recruitment, while the substrate determined the E2 selectivity. This study reveals the influence of substrates and U-box domains on E2 recruitment, providing a novel perspective on the function of U-box domains of E3 ligases.

A Survey on the Expression of the Ubiquitin Proteasome System Components HECT- and RBR-E3 Ubiquitin Ligases and E2 Ubiquitin-Conjugating and E1 Ubiquitin-Activating Enzymes during Human Brain Development.

Human brain development involves a tightly regulated sequence of events that starts shortly after conception and continues up to adolescence. Before birth, neurogenesis occurs, implying an extensive differentiation process, sustained by changes in the gene expression profile alongside proteome remodeling, regulated by the ubiquitin proteasome system (UPS) and autophagy. The latter processes rely on the selective tagging with ubiquitin of the proteins that must be disposed of. E3 ubiquitin ligases accomplish the selective recognition of the target proteins. At the late stage of neurogenesis, the brain starts to take shape, and neurons migrate to their designated locations. After birth, neuronal myelination occurs, and, in parallel, neurons form connections among each other throughout the synaptogenesis process. Due to the malfunctioning of UPS components, aberrant brain development at the very early stages leads to neurodevelopmental disorders. Through deep data mining and analysis and by taking advantage of machine learning-based models, we mapped the transcriptomic profile of the genes encoding HECT- and ring-between-ring (RBR)-E3 ubiquitin ligases as well as E2 ubiquitin-conjugating and E1 ubiquitin-activating enzymes during human brain development, from early post-conception to adulthood. The inquiry outcomes unveiled some implications for neurodevelopment-related disorders.

Arabidopsis thaliana ubiquitin-associated protein 2 (AtUAP2) functions as an E4 ubiquitin factor and negatively modulates dehydration stress response.

E4, a ubiquitin (Ub) chain assembly factor and post-translational modification protein, plays a key role in the regulation of multiple cellular functions in plants during biotic or abiotic stress. We have more recently reported that E4 factor AtUAP1 is a negative regulator of the osmotic stress response and enhances the multi-Ub chain assembly of E3 ligase Arabidopsis thaliana RING Zinc Finger 1 (AtRZF1). To further investigate the function of other E4 Ub factors in osmotic stress, we isolated AtUAP2, an AtUAP1 homolog, which interacted with AtRZF1, using pull-down assay and bimolecular fluorescence complementation analysis. AtUAP2, a Ub-associated motif-containing protein, interacts with oligo-Ub5, -Ub6, and -Ub7 chains. The yeast functional complementation experiment revealed that AtUAP2 functions as an E4 Ub factor. In addition, AtUAP2 is localized in the cytoplasm, different from AtUAP1. The activity of AtUAP2 was relatively strongly induced in the leaf tissue of AtUAP2 promoter-β-glucuronidase transgenic plants by abscisic acid, dehydration, and oxidative stress. atuap2 RNAi lines were more insensitive to osmotic stress condition than wild-type during the early growth of seedlings, whereas the AtUAP2-overexpressing line exhibited relatively more sensitive responses. Analyses of molecular and physiological experiments showed that AtUAP2 could negatively mediate the osmotic stress-induced signaling. Genetic studies showed that AtRZF1 mutation could suppress the dehydration-induced sensitive phenotype of the AtUAP2-overexpressing line, suggesting that AtRZF1 acts genetically downstream of AtUAP2 during osmotic stress. Taken together, our findings show that the AtRZF1-AtUAP2 complex may play important roles in the ubiquitination pathway, which controls the osmotic stress response in Arabidopsis.

Rad6, a ubiquitin conjugator required for insect-pathogenic lifestyle, UV damage repair, and genomic expression of Beauveria bassiana

The E2 ubiquitin conjugator Rad6 is required for DNA damage bypass in budding yeast but remain functionally unknown in filamentous fungi. Here, we report pleiotropic effect of Rad6 ortholog in Beauveria bassiana, a wide-spectrum fungal insecticide. Global ubiquitination signal was greatly attenuated in the absence of rad6. The blocked ubiquitination led to severe growth defect, blocked asexual development, and abolished infectivity/insect pathogenicity, which correlated with compromised conidial quality (including viability, hydrophobicity, adherence to insect cuticle, and thermotolerance) and blocked secretion of cuticle-degrading enzymes including Pr1 family proteases. Importantly, Rad6 played much greater role in photoreactivation of UVB-impaired conidia by a 3- or 5-h light plus 9- or 7-h dark incubation than in dark reactivation of those impaired conidia by a 12-h dark incubation. The high activity of Rad6 in photoreactivation in vivo was derived from its link to a protein complex cored by the photolyase regulators WC1 and WC2 via the strong interactions of Rad6 with the E3 partner Rad18 and Rad18 with WC2 revealed in yeast two-hybrid assays. Transcriptomic analysis resulted in identification of 2700 differentially regulated genes involved in various function categories and metabolism pathways, indicating a regulatory role of Rad6-mediated ubiquitination in gene expression networks and genomic stability. Conclusively, Rad6 is required for asexual and insect-pathogenic lifecycles, solar UV damage repair, and genomic expression of B. bassiana. The primary dependence of its strong anti-UV role on photoreactivation in vivo unveils a scenario distinct from the core role of its yeast ortholog in DNA damage bypass.

Topology of ubiquitin chains in the e3 ubiquitin ligase rnf168 chromatosome entourage

Genome stability is critical for normal functioning of cells and depends on accuracy of DNA replication, chromosome segregation, and DNA repair. Cellular defense mechanisms against DNA damage are important for preventing the development of cancer and aging. The E3 ubiquitin ligase RNF168 of the RING superfamily is an essential component of the complex responsible for the ubiquitination of H2A/H2A.X histones near DNA double-strand breaks, which is a key step in attracting repair factors to the injury site. In this study, we unequivocally showed that RNF168 does not have ability to directly distinguish the architecture of polyubiquitin chains, except for tropism of its two ubiquitin-binding domains UDM1/2 to K63 ubiquitin chains. Analysis of the intracellular chromatosomal environment of full-length RNF168 and its domains by ligand-induced bioluminescence resonance energy transfer (BRET) revealed that the C-terminal part of UDM1 is associated with K63 ubiquitin chains; RING and the N-terminal part of UDM2 are sterically close to K63- and K48- ubiquitin chains, while the C-terminal part of UDM1 is colocalized with all possible ubiquitin variants. Our observations together with the available structural data suggest that the C-terminal part of UDM1 binds K63 polyubiquitin chains on linker histone H1; RING and the N-terminal part of UDM2 are located in the central part of the nucleosome and sterically close to H1 and K48-ubiquitinated alternative substrates of RNF168, such as JMJD2A/B demethylases, while the C-terminal part of UDM1 is in the region of an activated ubiquitin residue associated with E2 ubiquitin ligase, engaged by RNF168.

Parkin-mediated ubiquitination inhibits BAK apoptotic activity by blocking its canonical hydrophobic groove

BAK permeabilizes the mitochondrial outer membrane, causing apoptosis. This apoptotic activity of BAK is stimulated by binding prodeath activators within its canonical hydrophobic groove. Parkin, an E3 ubiquitin (Ub) ligase, can ubiquitinate BAK, which inhibits BAK apoptotic activity. However, the molecular mechanism underlying the inhibition of ubiquitination remains structurally uncharacterized. Here, we utilize truncated and soluble BAK to construct a mimetic of K113-ubiquitinated BAK (disulfide-linked UbG76C ~ BAKK113C) and further present its NMR-derived structure model. The classical L8-I44-H68-V70 hydrophobic patch of the conjugated Ub subunit binds within the canonical hydrophobic groove of BAK. This Ub occludes the binding of prodeath BID activators in the groove and impairs BID-triggered BAK activation and membrane permeabilization. Reduced interaction between Ub and BAK subunits allows BID to activate K113-ubiquitinated BAK. These mechanistic insights suggest a nonsignaling function of Ub in that it directly antagonizes stimuli targeting Ub-modified proteins rather than by recruiting downstream partners for cellular messaging.