RESUMO
Histone H2B monoubiquitylation plays essential roles in chromatin-based transcriptional processes. A RING-type E3 ligase (yeast Bre1 or human RNF20/RNF40) and an E2 ubiquitin-conjugating enzyme (yeast Rad6 or human hRAD6A), together, precisely deposit ubiquitin on H2B K123 in yeast or K120 in humans. Here, we developed a chemical trapping strategy and successfully captured the transient structures of Bre1- or RNF20/RNF40-mediated ubiquitin transfer from Rad6 or hRAD6A to nucleosomal H2B. Our structures show that Bre1 and RNF40 directly bind nucleosomal DNA, exhibiting a conserved E3/E2/nucleosome interaction pattern from yeast to humans for H2B monoubiquitylation. We also find an uncanonical non-hydrophobic contact in the Bre1 RING-Rad6 interface, which positions Rad6 directly above the target H2B lysine residue. Our study provides mechanistic insights into the site-specific monoubiquitylation of H2B, reveals a critical role of nucleosomal DNA in mediating E3 ligase recognition, and provides a framework for understanding the cancer-driving mutations of RNF20/RNF40.
Assuntos
Nucleossomos , Proteínas de Saccharomyces cerevisiae , Humanos , Nucleossomos/genética , Histonas/genética , Saccharomyces cerevisiae/genética , Ubiquitina , Ubiquitina-Proteína Ligases/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The N-degron pathway targets proteins that bear a destabilizing residue at the N terminus for proteasome-dependent degradation1. In yeast, Ubr1-a single-subunit E3 ligase-is responsible for the Arg/N-degron pathway2. How Ubr1 mediates the initiation of ubiquitination and the elongation of the ubiquitin chain in a linkage-specific manner through a single E2 ubiquitin-conjugating enzyme (Ubc2) remains unknown. Here we developed chemical strategies to mimic the reaction intermediates of the first and second ubiquitin transfer steps, and determined the cryo-electron microscopy structures of Ubr1 in complex with Ubc2, ubiquitin and two N-degron peptides, representing the initiation and elongation steps of ubiquitination. Key structural elements, including a Ubc2-binding region and an acceptor ubiquitin-binding loop on Ubr1, were identified and characterized. These structures provide mechanistic insights into the initiation and elongation of ubiquitination catalysed by Ubr1.
Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Sítios de Ligação , Biocatálise , Microscopia Crioeletrônica , Lisina/metabolismo , Modelos Moleculares , Proteólise , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/ultraestruturaRESUMO
Protein ubiquitination is a multifaceted post-translational modification that controls almost every process in eukaryotic cells. Recently, the Legionella effector SdeA was reported to mediate a unique phosphoribosyl-linked ubiquitination through successive modifications of the Arg42 of ubiquitin (Ub) by its mono-ADP-ribosyltransferase (mART) and phosphodiesterase (PDE) domains. However, the mechanisms of SdeA-mediated Ub modification and phosphoribosyl-linked ubiquitination remain unknown. Here we report the structures of SdeA in its ligand-free, Ub-bound and Ub-NADH-bound states. The structures reveal that the mART and PDE domains of SdeA form a catalytic domain over its C-terminal region. Upon Ub binding, the canonical ADP-ribosyltransferase toxin turn-turn (ARTT) and phosphate-nicotinamide (PN) loops in the mART domain of SdeA undergo marked conformational changes. The Ub Arg72 might act as a 'probe' that interacts with the mART domain first, and then movements may occur in the side chains of Arg72 and Arg42 during the ADP-ribosylation of Ub. Our study reveals the mechanism of SdeA-mediated Ub modification and provides a framework for further investigations into the phosphoribosyl-linked ubiquitination process.
Assuntos
Legionella pneumophila/enzimologia , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , ADP Ribose Transferases/química , ADP Ribose Transferases/metabolismo , Arginina/metabolismo , Proteínas de Bactérias , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Chaperonas Moleculares/metabolismo , NAD/metabolismo , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/metabolismo , Processamento de Proteína Pós-Traducional , Especificidade por Substrato , Ubiquitina/químicaRESUMO
Protein ubiquitination shows remarkable topological and functional diversity through the polymerization of ubiquitin via different linkages. Deciphering the cellular ubiquitin code is of central importance to understand the physiology of the cell. However, our understanding of its function is rather limited due to the lack of specific binders as tools to detect K29-linked polyubiquitin. In this study, we screened and characterized a synthetic antigen-binding fragment, termed sAB-K29, that can specifically recognize K29-linked polyubiquitin using chemically synthesized K29-linked diubiquitin. We further determined the crystal structure of this fragment bound to the K29-linked diubiquitin, which revealed the molecular basis of specificity. Using sAB-K29 as a tool, we uncovered that K29-linked ubiquitination is involved in different kinds of cellular proteotoxic stress response as well as cell cycle regulation. In particular, we showed that K29-linked ubiquitination is enriched in the midbody and downregulation of the K29-linked ubiquitination signal arrests cells in G1/S phase.
Assuntos
Ubiquitina-Proteína Ligases/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , Humanos , Modelos Moleculares , Transdução de Sinais , Estresse Fisiológico , Ubiquitina-Proteína Ligases/química , UbiquitinaçãoRESUMO
Ag2Te is one of the most promising semiconductors with a narrow band gap and low toxicity; however, it remains a challenge to tune the emission of Ag2Te quantum dots (QDs) precisely and continuously in a wide range. Herein, Ag2Te QDs emitting from 950 to 2100 nm have been synthesized via trialkylphosphine-controlled growth. Trialkylphosphine has been found to induce the dissolution of small-sized Ag2Te QDs due to its stronger ability to coordinate to the Ag ion than that of 1-octanethiol, predicated by the density functional theory. By controlling this dissolution effect, the monomer supply kinetics can be regulated, achieving precise size control of Ag2Te QDs. This synthetic strategy results in state-of-the-art silver-based QDs with emission tunability. Only by taking advantage of such an ultrawide emission has the sizing curve of Ag2Te been obtained. Moreover, the absolute photoluminescence quantum yield of Ag2Te QDs can reach 12.0% due to their well-passivated Ag-enriched surface with a density of 5.0 ligands/nm2, facilitating noninvasive in vivo fluorescence imaging. The high brightness in the long-wavelength near-infrared (NIR) region makes the cerebral vasculature and the tiny vessel with a width of only 60 µm clearly discriminable. This work reveals a nonclassical growth mechanism of Ag2Te QDs, providing new insight into precisely controlling the size and corresponding photoluminescence properties of semiconductor nanocrystals. The ultrasmall, low-toxicity, emission-tunable, and bright NIR-II Ag2Te QDs synthesized in this work offer a tremendous promise for multicolor and deep-tissue in vivo fluorescence imaging.
RESUMO
Triazole-based deubiquitylase (DUB)-resistant ubiquitin (Ub) probes have recently emerged as effective tools for the discovery of Ub chain-specific interactors in proteomic studies, but their structural diversity is limited. A new family of DUB-resistant Ub probes is reported based on isopeptide-N-ethylated dimeric or polymeric Ub chains, which can be efficiently prepared by a one-pot, ubiquitin-activating enzyme (E1)-catalyzed condensation reaction of recombinant Ub precursors to give various homotypic and even branched Ub probes at multi-milligram scale. Proteomic studies using label-free quantitative (LFQ) MS indicated that the isopeptide-N-ethylated Ub probes may complement the triazole-based probes in the study of Ub interactome. Our study highlights the utility of modern protein synthetic chemistry to develop structurally and new families of tool molecules needed for proteomic studies.
Assuntos
Sondas Moleculares/química , Poliubiquitina/química , Enzimas Ativadoras de Ubiquitina/química , Ciclina B1/química , Ciclina B1/genética , Células HEK293 , Células HeLa , Histonas/química , Histonas/genética , Humanos , Sondas Moleculares/síntese química , Mutação , Poliubiquitina/síntese química , ProteômicaRESUMO
Histone ubiquitination affects the structure and function of nucleosomes through tightly regulated dynamic reversible processes. The efficient preparation of ubiquitinated histones and their analogs is important for biochemical and biophysical studies on histone ubiquitination. Here, we report the CAACU (cysteine-aminoethylation assisted chemical ubiquitination) strategy for the efficient synthesis of ubiquitinated histone analogs. The key step in the CAACU strategy is the installation of an N-alkylated 2-bromoethylamine derivative into a recombinant histone through cysteine aminoethylation, followed by native chemical ligation assisted by Seitz's auxiliary to produce mono- and diubiquitin (Ub) and small ubiquitin-like modifier (SUMO) modified histone analogs. This approach enables the rapid production of modified histones from recombinant proteins at about 1.5-6 mg/L expression. The thioether-containing isopeptide bonds in the products are chemically stable and bear only one atomic substitution in the structure, compared to their native counterparts. The ubiquitinated histone analogs prepared by CAACU can be readily reconstituted into nucleosomes and selectively recognized by relevant interacting proteins. The thioether-containing isopeptide bonds can also be recognized and hydrolyzed by deubiquitinases (DUBs). Cryo-electron microscopy (cryo-EM) of the nucleosome containing H2BKC34Ub indicated that the obtained CAACU histones were of good quality for structural studies. Collectively, this work exemplifies the utility of the CAACU strategy for the simple and efficient production of homogeneous ubiquitinated and SUMOylated histones for biochemical and biophysical studies.
Assuntos
Cisteína/química , Etilaminas/química , Histonas/química , Ubiquitinação , Modelos Moleculares , Estrutura Molecular , Proteínas Recombinantes/químicaRESUMO
New synthetic strategies that exploited the strengths of both chemoselective ligation and recombinant protein expression were developed to prepare K27 di-ubiquitins (diUb), which enabled mechanistic studies on the molecular recognition of K27-linked Ubs by single-molecule Förster resonance energy transfer (smFRET) and X-ray crystallography. The results revealed that free K27 diUb adopted a compact conformation, whereas upon binding to UCHL3, K27 diUb was remodeled to an open conformation. The K27 isopeptide bond remained rigidly buried inside the diUb moiety during binding, an interesting unique structural feature that may explain the distinctive biological function of K27 Ub chains.
Assuntos
Ubiquitina/síntese química , Cristalografia por Raios X , Transferência Ressonante de Energia de Fluorescência , Modelos Moleculares , Conformação Proteica , Processamento de Proteína Pós-Traducional , Ubiquitina/químicaRESUMO
Quasi-racemic crystallography has been used to determine the X-ray structures of K27-linked ubiquitin (Ub) chains prepared through total chemical synthesis. Crystal structures of K27-linked di- and tri-ubiquitins reveal that the isopeptide linkages are confined in a unique buried conformation, which provides the molecular basis for the distinctive function of K27 linkage compared to the other seven Ub chains. K27-linked di- and triUb were found to adopt different structural conformations in the crystals, one being symmetric whereas the other triangular. Furthermore, bioactivity experiments showed that the ovarian tumor family de-ubiquitinase 2 significantly favors K27-linked triUb than K27-linked diUb. K27-linked triUb represents the so-far largest chemically synthesized protein (228 amino acids) that has been crystallized to afford a high-resolution X-ray structure.
Assuntos
Lisina/química , Poliubiquitina/química , Poliubiquitina/síntese química , Sítios de Ligação , Cristalografia por Raios X , Endopeptidases/metabolismo , Lisina/metabolismo , Modelos Moleculares , Poliubiquitina/metabolismo , Conformação Proteica , Tioléster Hidrolases/metabolismo , UbiquitinaçãoRESUMO
Racemic or quasi-racemic crystallography recently emerges as a useful technology for solution of the crystal structures of biomacromolecules. It remains unclear to what extent the biomacromolecules of opposite handedness can differ from each other in racemic or quasi-racemic crystallography. Here we report a finding that monomeric d-ubiquitin (Ub) has propensity to cocrystallize with different dimers, trimers, and even a tetramer of l-Ub. In these cocrystals the unconnected monomeric d-Ubs can self-assemble to form pseudomirror images of different oligomers of l-Ub. This monomer/oligomer cocrystallization phenomenon expands the concept of racemic crystallography. Using the monomer/oligomer cocrystallization technology we obtained, for the first time the X-ray structures of linear M1-linked tri- and tetra-Ubs and a K11/K63-branched tri-Ub.
Assuntos
Multimerização Proteica , Ubiquitina/química , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Estrutura Quaternária de Proteína , EstereoisomerismoRESUMO
A new thiol protecting group Hmb(off/on) is described, which has a switchable activity that may be useful in the chemical synthesis of proteins. When placed on the side chain of Cys, Cys(Hmb(off)) is stable to trifluoroacetic acid (TFA) in the process of solid-phase peptide synthesis. When Cys(Hmb(off)) is treated with neutral aqueous buffers, it is cleanly converted to acid-labile Cys(Hmb(on)), which can later be fully deprotected by TFA to generate free Cys. The utility of Cys(Hmb(off/on)) is demonstrated by the chemical synthesis of an erythropoietin segment, EPO[Cys(98)-Arg(166)]-OH through native chemical ligation.
Assuntos
Peptídeos/química , Peptídeos/síntese química , Técnicas de Síntese em Fase Sólida , Compostos de Sulfidrila/químicaRESUMO
Mambalgins are a class of 57-residue polypeptide toxins isolated from the venom of the African mamba. They exhibit potent analgesic effects by inhibiting the acid-sensing ion channels. Classified as members of the family of three-finger toxins, mambalgins contain four pairs of disulfide bridges that help to stabilize the three-finger scaffold. Here, we report the chemical synthesis of functional mambalgin-1/2/3 by using one-step two-segment hydrazide-based native chemical ligation. The two-segment ligation approach reported here may enable efficient production of mambalgin toxins. These synthetic mambalgins are useful compounds for development of diagnostic or therapeutic reagents. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.
Assuntos
Venenos Elapídicos/síntese química , Peptídeos/síntese química , Azidas/química , Dissulfetos/química , Venenos Elapídicos/química , Modelos Moleculares , Estrutura Molecular , Peptídeos/químicaRESUMO
As a unique and unappreciated protein posttranslational modification, arginine N-glycosylation was recently discovered to play an important role in the process that bacteria counteract host defenses. To provide chemical tools for further proteomic and biochemical studies on arginine N-glycosylation, we report the first general strategy for a rapid and cost-effective synthesis of glycopeptides carrying single or multiple arginine N-GlcNAcyl groups. These glycopeptides were successfully utilized to generate the first antibodies that can specifically recognize arginine N-GlcNAcylated peptides or proteins in a sequence-independent manner.
Assuntos
Anticorpos/química , Arginina/química , Glucosamina/química , Glicopeptídeos/química , Anticorpos/metabolismo , Proteínas de Escherichia coli/metabolismo , Glicosilação , Prata/química , Técnicas de Síntese em Fase Sólida , Fatores de Virulência/metabolismoRESUMO
Sortase-mediated hydrazinolysis of proteins with hydrazine or its derivatives was developed for the production of recombinant protein hydrazides. This process provides an alternative approach for protein semisynthesis through the use of recombinant protein hydrazides as thioester surrogates. It also provides an alternative method for C-terminal modification of proteins with functional units as well as for the preparation of C-to-C fusion proteins.
Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Hidrazinas/química , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Fluoresceína/química , Hidrólise , Peptídeos/química , Peptídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologiaRESUMO
Limited understanding of human proteoforms with complex posttranslational modifications and the underlying mechanisms poses a major obstacle to research on human health and disease. This Outlook discusses opportunities and challenges of de novo chemical protein synthesis in human proteoform studies. Our analysis suggests that to develop a comprehensive, robust, and cost-effective methodology for chemical synthesis of various human proteoforms, new chemistries of the following types need to be developed: (1) easy-to-use peptide ligation chemistries allowing more efficient de novo synthesis of protein structural domains, (2) robust temporary structural support strategies for ligation and folding of challenging targets, and (3) efficient transpeptidative protein domain-domain ligation methods for multidomain proteins. Our analysis also indicates that accurate chemical synthesis of human proteoforms can be applied to the following aspects of biomedical research: (1) dissection and reconstitution of the proteoform interaction networks, (2) structural mechanism elucidation and functional analysis of human proteoform complexes, and (3) development and evaluation of drugs targeting human proteoforms. Overall, we suggest that through integrating chemical protein synthesis with in vivo functional analysis, mechanistic biochemistry, and drug development, synthetic chemistry would play a pivotal role in human proteoform research and facilitate the development of precision diagnostics and therapeutics.
RESUMO
Epigenetic regulators have a crucial effect on gene expression based on their manipulation of histone modifications. Histone H2AK119 monoubiquitination (H2AK119Ub), a well-established hallmark in transcription repression, is dynamically regulated by the opposing activities of Polycomb repressive complex 1 (PRC1) and nucleosome deubiquitinases including the primary human USP16 and Polycomb repressive deubiquitinase (PR-DUB) complex. Recently, the catalytic mechanism for the multi-subunit PR-DUB complex has been described, but how the single-subunit USP16 recognizes the H2AK119Ub nucleosome and cleaves the ubiquitin (Ub) remains unknown. Here we report the cryo-EM structure of USP16-H2AK119Ub nucleosome complex, which unveils a fundamentally distinct mode of H2AK119Ub deubiquitination compared to PR-DUB, encompassing the nucleosome recognition pattern independent of the H2A-H2B acidic patch and the conformational heterogeneity in the Ub motif and the histone H2A C-terminal tail. Our work highlights the mechanism diversity of H2AK119Ub deubiquitination and provides a structural framework for understanding the disease-causing mutations of USP16.
RESUMO
Ubiquitination, catalyzed usually by a three-enzyme cascade (E1, E2, E3), regulates various eukaryotic cellular processes. E3 ligases are the most critical components of this catalytic cascade, determining both substrate specificity and polyubiquitination linkage specificity. Here, we reveal the mechanism of a naturally occurring E3-independent ubiquitination reaction of a unique human E2 enzyme UBE2E1 by solving the structure of UBE2E1 in complex with substrate SETDB1-derived peptide. Guided by this peptide sequence-dependent ubiquitination mechanism, we developed an E3-free enzymatic strategy SUE1 (sequence-dependent ubiquitination using UBE2E1) to efficiently generate ubiquitinated proteins with customized ubiquitinated sites, ubiquitin chain linkages and lengths. Notably, this strategy can also be used to generate site-specific branched ubiquitin chains or even NEDD8-modified proteins. Our work not only deepens the understanding of how an E3-free substrate ubiquitination reaction occurs in human cells, but also provides a practical approach for obtaining ubiquitinated proteins to dissect the biochemical functions of ubiquitination.
Assuntos
Enzimas de Conjugação de Ubiquitina , Ubiquitina-Proteína Ligases , Humanos , Peptídeos/metabolismo , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Ubiquitinadas/metabolismo , Ubiquitinação , Engenharia de ProteínasRESUMO
The cancer-specific fusion oncoprotein SS18-SSX1 disturbs chromatin accessibility by hijacking the BAF complex from the promoters and enhancers to the Polycomb-repressed chromatin regions. This process relies on the selective recognition of H2AK119Ub nucleosomes by synovial sarcoma X breakpoint 1 (SSX1). However, the mechanism underlying the selective recognition of H2AK119Ub nucleosomes by SSX1 in the absence of ubiquitin (Ub)-binding capacity remains unknown. Here we report the cryo-EM structure of SSX1 bound to H2AK119Ub nucleosomes at 3.1-Å resolution. Combined in vitro biochemical and cellular assays revealed that the Ub recognition by SSX1 is unique and depends on a cryptic basic groove formed by H3 and the Ub motif on the H2AK119 site. Moreover, this unorthodox binding mode of SSX1 induces DNA unwrapping at the entry/exit sites. Together, our results describe a unique mode of site-specific ubiquitinated nucleosome recognition that underlies the specific hijacking of the BAF complex to Polycomb regions by SS18-SSX1 in synovial sarcoma.
Assuntos
Nucleossomos , Sarcoma Sinovial , Humanos , Sarcoma Sinovial/metabolismo , Cromatina , Membrana Celular/metabolismo , Proteínas de Fusão Oncogênica/genéticaRESUMO
Three alkyne-containing pyrrolysine derivatives were synthesized and genetically encoded into proteins by a mutant PylRS-tRNA pair with high efficiencies. With these alkyne handles, site-specific dual labeling of proteins can be achieved via a bioorthogonal thiol-yne ligation reaction.
Assuntos
Alcinos/química , Aminoacil-tRNA Sintetases/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Lisina/análogos & derivados , Compostos de Sulfidrila/química , Escherichia coli/química , Proteínas de Escherichia coli/análise , Lisina/química , Lisina/genética , Modelos Moleculares , Mutação , Coloração e Rotulagem/métodosRESUMO
Ubiquitination regulates almost every life process of eukaryotes. The study of the ubiquitin (Ub) coupling or decoupling process and the interaction study of Ub-Ub binding protein have always been the central focus. However, such studies are challenging, owing to the transient nature of Ub-coupling enzymes and deubiquitinases in the reactions, as well as the difficulty in preparing large quantities of polyubiquitinated samples. Here we describe a recently developed strategy for the efficient preparation of analogs of Ub chains and analogs for Ub coupling and uncoupling intermediates, which facilitate the study of the ubiquitination process. The strategy includes mainly the following steps: (i) the bifunctional molecule conjugation on the only cysteine (Cys) residue of a target protein (usually a Ub or Ub-conjugating enzyme), exposing an orthogonal reactive site for native chemical ligation; (ii) covalent ligation with a Ub-derived thioester, exposing a free sulfhydryl; and (iii) (optional) a disulfide bond formation with a substrate protein (mainly with only one Cys protein) through nonactivity-based cross-linking or with a deubiquitinase (mainly with several Cys residues) through activity-based cross-linking. When the bifunctional molecule and target proteins are obtained, the final products can be prepared in milligram quantities within 2 weeks.