Specific Recognition of Linear Ubiquitin Chains by NEMO Is Important for NF-κB Activation

Activation of nuclear factor-κB (NF-κB), a key mediator of inducible transcription in immunity, requires binding of NF-κB essential modulator (NEMO) to ubiquitinated substrates. Here, we report that the UBAN (ubiquitin binding in ABIN and NEMO) motif of NEMO selectively binds linear (head-to-tail) ubiquitin chains. Crystal structures of the UBAN motif revealed a parallel coiled-coil dimer that formed a heterotetrameric complex with two linear diubiquitin molecules. The UBAN dimer contacted all four ubiquitin moieties, and the integrity of each binding site was required for efficient NF-κB activation. Binding occurred via a surface on the proximal ubiquitin moiety and the canonical Ile44 surface on the distal one, thereby providing specificity for linear chain recognition. Residues of NEMO involved in binding linear ubiquitin chains are required for NF-κB activation by TNF-α and other agonists, providing an explanation for the detrimental effect of NEMO mutations in patients suffering from X-linked ectodermal dysplasia and immunodeficiency

OTU deubiquitinases reveal mechanisms of linkage specificity and enable ubiquitin chain restriction analysis

Sixteen ovarian tumor (OTU) family deubiquitinases (DUBs) exist in humans, and most members regulate cell-signaling cascades. Several OTU DUBs were reported to be ubiquitin (Ub) chain linkage specific, but comprehensive analyses are missing, and the underlying mechanisms of linkage specificity are unclear. Using Ub chains of all eight linkage types, we reveal that most human OTU enzymes are linkage specific, preferring one, two, or a defined subset of linkage types, including unstudied atypical Ub chains. Biochemical analysis and five crystal structures of OTU DUBs with or without Ub substrates reveal four mechanisms of linkage specificity. Additional Ub-binding domains, the ubiquitinated sequence in the substrate, and defined S1’ and S2 Ub-binding sites on the OTU domain enable OTU DUBs to distinguish linkage types. We introduce Ub chain restriction analysis, in which OTU DUBs are used as restriction enzymes to reveal linkage type and the relative abundance of Ub chains on substrates

p38-MK2 signaling axis regulates RNA metabolism after UV-light-induced DNA damage

UV-light-induced DNA damage affects RNA metabolism but the underlying signalling pathways are largely unexplored. Here, the authors show that UV light triggers p38-MK2-mediated phosphorylation of the NELF complex, promoting its release from chromatin and concurrent transcriptional elongation

Discovery of Protein-Protein Interaction Inhibitors by Integrating Protein Engineering and Chemical Screening Platforms

Protein-protein interactions (PPIs) govern intracellular life, and identification of PPI inhibitors is challenging. Roadblocks in assay development stemming from weak binding affinities of natural PPIs impede progress in this field. We postulated that enhancing binding affinity of natural PPIs via protein engineering will aid assay development and hit discovery. This proof-of-principle study targets PPI between linear ubiquitin chains and NEMO UBAN domain, which activates NF-κB signaling. Using phage display, we generated ubiquitin variants that bind to the functional UBAN epitope with high affinity, act as competitive inhibitors, and structurally maintain the existing PPI interface. When utilized in assay development, variants enable generation of robust cell-based assays for chemical screening. Top compounds identified using this approach directly bind to UBAN and dampen NF-κB signaling. This study illustrates advantages of integrating protein engineering and chemical screening in hit identification, a development that we anticipate will have wide application in drug discovery

Molecular basis of Lys11-polyubiquitin specificity in the deubiquitinase Cezanne

The post-translational modification of proteins with polyubiquitin regulates virtually all aspects of cell biology. Eight distinct chain linkage types in polyubiquitin co-exist and are independently regulated in cells. This ‘ubiquitin code’ determines the fate of the modified protein1. Deubiquitinating enzymes of the Ovarian Tumour (OTU) family regulate cellular signalling by targeting distinct linkage types within polyubiquitin2, and understanding their mechanisms of linkage specificity gives fundamental insights into the ubiquitin system. We here reveal how the deubiquitinase Cezanne/OTUD7B specifically targets Lys11-linked polyubiquitin. Crystal structures of Cezanne alone and in complex with mono- and Lys11-linked diubiquitin, in combination with hydrogen-deuterium exchange mass spectrometry, enable reconstruction of the enzymatic cycle in exquisite detail. An intricate mechanism of ubiquitin-assisted conformational changes activate the enzyme, and while all chain types interact with the enzymatic S1 site, only Lys11-linked chains can bind productively across the active site and stimulate catalytic turnover. Our work highlights the fascinating plasticity of deubiquitinases, and indicates that new conformational states can occur when a true substrate, such as diubiquitin, is bound at the active site

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目的:獨協医科大学では2010年度より地域枠入学制度が導入された.そこで2013年に,地域枠入学者の意識調査を施行し,「将来地域医療を行うという自覚」と「自分の将来についての不安」に着目してその調査結果を検討した.方法:対象は,地域枠入学者全員を含む地域包括医療実習生70名である.内訳は,第1学年20名,第2学年20名,第3学年18名,第4学年12名である.68名は学習支援システムを利用して記名式のWebアンケート調査に回答し,2名は自記式で同一調査に回答した.結果:「将来地域医療を行うことを大変自覚している・自覚している」ものは,第1学年85.0%,第2学年90.0%,第3学年77.8%,第4学年100%となった.また「将来地域医療を行うことを大変自覚している」ことは,「将来の自分のめざす医師像をもっている」ことと関与することが示唆された.自分の将来に不安があるものは,第1学年は60%,第2学年は55%,第3学年は50.0%,第4学年は50%であり,全体の半数以上が不安を持っていた.「不安がある」ものは有意に「地域包括医療実習以外にもっと地域医療について勉強の機会を与えてほしい」と考えていた.結論:地域医療教育はカリキュラムを越えてさらに発展する必要があることが示唆された.Objective:Four years have passed since Dokkyo Medical University first introduced an admission system for community medicine?oriented students. Therefore, a survey of community-mindedness among these students was held, and based on this survey, we examined the associated factors of willingness toward future engagement and present anxiety.Methods:Participants were 70 students(20 first-year students, 20 second-year students, 18 third-year students, and 12 fourth-year students)who took the comprehensive community medicine practice course. A total of 68 students answered the questionnaire using the learning medical system website and 2 students answered the questionnaire by hand.Results:Willingness to engage in community-oriented medicine was reported by 85.0% of first-years, 90.0% of second-years, 77.8% of third-years, and 100% of fourthyears, and willingness for future engagement was significantly associated with students\u27 possessing an ideal image of the doctor that they want to be. Present anxiety was reported by 60% of first-years, 55% of second-years, 50%of third-years, and 50% of fourth-years;over 50% of all students had some level of anxiety. Additionally, students who wanted to study more about community medicine beyond this practice course were significantly more likely to have present anxiety.Conclusion:Results suggested that education in community medicine was expected further developments

New Hashing Techniques for Three-Dimensional Protein Structures

This paper describes new methods to evaluate the structural similarity of proteins