RESUMEN
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.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Ubiquitinación , Sitios de Unión , Biocatálisis , Microscopía por Crioelectrón , Lisina/metabolismo , Modelos Moleculares , Proteolisis , Reproducibilidad de los Resultados , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/ultraestructura , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/ultraestructuraRESUMEN
Ubiquitination-dependent histone crosstalk plays critical roles in chromatin-associated processes and is highly associated with human diseases. Mechanism studies of the crosstalk have been of the central focus. Here our study on the crosstalk between H2BK34ub and Dot1L-catalyzed H3K79me suggests a novel mechanism of ubiquitination-induced nucleosome distortion to stimulate the activity of an enzyme. We determined the cryo-electron microscopy structures of Dot1L-H2BK34ub nucleosome complex and the H2BK34ub nucleosome alone. The structures reveal that H2BK34ub induces an almost identical orientation and binding pattern of Dot1L on nucleosome as H2BK120ub, which positions Dot1L for the productive conformation through direct ubiquitin-enzyme contacts. However, H2BK34-anchored ubiquitin does not directly interact with Dot1L as occurs in the case of H2BK120ub, but rather induces DNA and histone distortion around the modified site. Our findings establish the structural framework for understanding the H2BK34ub-H3K79me trans-crosstalk and highlight the diversity of mechanisms for histone ubiquitination to activate chromatin-modifying enzymes.
Asunto(s)
Histonas , Nucleosomas , Cromatina , Microscopía por Crioelectrón , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Humanos , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
Ubiquitin (Ub)-like protein ISG15 (interferon-stimulated gene 15) regulates innate immunity and links with the evasion of host response by viruses such as SARS-CoV-2. Dissecting ISGylation pathways recently received increasing attention which can inform related disease interventions, but such studies necessitate the preparation and development of various ISG15 protein tools. Here, we find that the leader protease (Lbpro ) encoded by foot-and-mouth disease virus can promote ligation reactions between recombinant ISG15 and synthetic glycyl compounds, generating protein tools such as ISG15-propargylamide and ISG15-rhodamine110, which are needed for cellular proteomic studies of deISGylases, and the screening and evaluation of inhibitors against SARS-CoV-2 papain-like protease (PLpro). Furthermore, this strategy can be also used to load ISG15 onto the lysine of a synthetic peptide through an isopeptide bond, and prepare Ub and NEDD8 (ubiquitin-like protein Nedd8) protein tools.
Asunto(s)
COVID-19 , Péptido Hidrolasas , Animales , Catálisis , Citocinas/metabolismo , Interferones , Lisina , Proteína NEDD8 , Péptido Hidrolasas/metabolismo , Proteómica , SARS-CoV-2 , Ubiquitinas/químicaRESUMEN
Sortase A (SrtA)-mediated ligation, a popular method for protein labeling and semi-synthesis, is limited by its reversibility and dependence on the LPxTG motif, where "x" is any amino acid. Here, we report that SrtA can mediate the efficient and irreversible ligation of a protein/peptide containing a C-terminal thioester with another protein/peptide bearing an N-terminal Gly, with broad tolerance for a wide variety of LPxT-derived sequences. This strategy, the thioester-assisted SrtA-mediated ligation, enabled the expedient preparation of proteins bearing various N- or C-terminal labels, including post-translationally modified proteins such as the Ser139-phosphorylated histone H2AX and Lys9-methylated histone H3, with less dependence on the LPxTG motif. Our study validates the chemical modification of substrates as an effective means of augmenting the synthetic capability of existing enzymatic methods.
Asunto(s)
Aminoaciltransferasas , Aminoaciltransferasas/química , Proteínas Bacterianas/metabolismo , Cisteína Endopeptidasas/química , Péptidos/química , Compuestos de AzufreRESUMEN
Activity-based E2 conjugating enzyme (E2)-ubiquitin (Ub) probes have recently emerged as effective tools for studying the molecular mechanism of E3 ligase (E3)-catalyzed ubiquitination. However, the preparation of existing activity-based E2-Ub probes depends on recombination technology and bioconjugation chemistry, limiting their structural diversity. Herein we describe an expedient total chemical synthesis of an E2 enzyme variant through a hydrazide-based native chemical ligation, which enabled the construction of a structurally new activity-based E2-Ub probe to covalently capture the catalytic site of Cys-dependent E3s. Chemical cross-linking coupled with mass spectrometry (CXMS) demonstrated the utility of this new probe in structural analysis of the intermediates formed during Nedd4 and Parkin-mediated transthiolation. This study exemplifies the utility of chemical protein synthesis for the development of protein probes for biological studies.
Asunto(s)
Compuestos de Sulfhidrilo/metabolismo , Ubiquitina-Proteína Ligasas/análisis , Ubiquitina/química , Biocatálisis , Humanos , Estructura Molecular , Compuestos de Sulfhidrilo/química , Ubiquitina/síntesis química , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
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.
Asunto(s)
Ubiquitina/síntesis química , Cristalografía por Rayos X , Transferencia Resonante de Energía de Fluorescencia , Modelos Moleculares , Conformación Proteica , Procesamiento Proteico-Postraduccional , Ubiquitina/químicaRESUMEN
Chemical ubiquitination is an effective approach for accessing structurally defined, atypical ubiquitin (Ub) chains that are difficult to prepare by other techniques. Herein, we describe a strategy that uses a readily accessible premade isopeptide-linked 76-mer (isoUb), which has an N-terminal Cys and a C-terminal hydrazide, as the key building block to assemble atypical Ub chains in a modular fashion. This method avoids the use of auxiliary-modified Lys and instead employs the canonical and therefore more robust Cys-based native chemical ligation technique. The efficiency and capacity of this isoUb-based strategy is exemplified by the cost-effective synthesis of several linkage- and length-defined atypical Ub chains, including K27-linked tetra-Ub and K11/K48-branched tri-, tetra-, penta-, and hexa-Ubs.
RESUMEN
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.
Asunto(s)
Enzimas Ubiquitina-Conjugadoras , Ubiquitina-Proteína Ligasas , Humanos , Péptidos/metabolismo , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Ubiquitinadas/metabolismo , Ubiquitinación , Ingeniería de ProteínasRESUMEN
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.
Asunto(s)
Nucleosomas , Sarcoma Sinovial , Humanos , Sarcoma Sinovial/metabolismo , Cromatina , Membrana Celular/metabolismo , Proteínas de Fusión Oncogénica/genéticaRESUMEN
ISG15 is a ubiquitin-like (Ubl) protein attached to substrate proteins by ISG15 conjugating enzymes whose dysregulation is implicated in a multitude of disease processes, but the probing of these enzymes remains to be accomplished. Here, we describe the development of a new activity-based probe ISG15-Dha (dehydroalanine) through protein semi-synthesis. In vitro cross-linking and cell lysate proteomic profiling experiments showed that this probe can sequentially capture ISG15 conjugating enzymes including E1 enzyme UBA7, E2 enzyme UBE2L6, E3 enzyme HERC5, the previously known ISG15 deconjugating enzyme (USP18), as well as some other enzymes (USP5 and USP14) which we additionally confirmed to impart deISGylation activity. Collectively, ISG15-Dha provides a new tool that can simultaneously capture ISG15 conjugating and deconjugating enzymes for biochemical or pharmacological studies. Electronic Supplementary Material: Supplementary material is available for this article at 10.1007/s11426-022-1455-x and is accessible for authorized users.
RESUMEN
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.
Asunto(s)
Enzimas Ubiquitina-Conjugadoras , Ubiquitina , Ubiquitinación , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas/metabolismo , Cisteína/metabolismoRESUMEN
Dynamic monitoring of intracellular ubiquitin (Ub) conjugates is instrumental to understanding the Ub regulatory machinery. Although many biochemical approaches have been developed to characterize protein ubiquitination, chemical tools capable of temporal resolution probing of ubiquitination events remain to be developed. Here, we report the development of the first cell-permeable and stimuli-responsive Ub probe and its application for the temporal resolution profiling of ubiquitinated substrates in live cells. The probe carrying the photolabile group N-(2-nitrobenzyl)-Gly (Nbg) on the amide bond between Ub Gly75 and Gly76 is readily prepared through chemical synthesis and can be delivered to live cells by conjugation via a disulfide bond with the cyclic cell-penetrating peptide cR10D (i.e., 4-((4-(dimethylamino)phenyl)-azo)-benzoic acid-modified cyclic deca-arginine). Both in vitro and in vivo experiments showed that Ub-modifying enzymes (E1, E2s, and E3s) could not install the Ub probe onto substrate proteins prior to removal of the nitrobenzyl group, which was easily accomplished via photoirradiation. The utility and practicality of this probe were exemplified by the time-resolved biochemical and proteomic investigation of ubiquitination events in live cells during a H2O2-mediated oxidative stress response. This work shows a conceptually new family of chemical Ub tools for the time-resolved studies on dynamic protein ubiquitination in different biological processes and highlights the utility of modern chemical protein synthesis in obtaining custom-designed tools for biological studies.
RESUMEN
Linear (Met1-linked) ubiquitination is involved inflammatory and innate immune signaling. Previous studies have characterized enzymes regulating the addition and removal of this modification in mammalian systems. However, only a few plant-derived deubiquitinases targeting Met1-linked ubiquitin chains have been reported and their mechanism of action remains elusive. Here, using a dehydroalanine-bearing Met1-diubiquitin suicide probe, we discover OTUB1 from Oryza sativa (OsOTUB1) as a Met1-linked ubiquitin chain-targeting deubiquitinase. By solving crystal structures of apo OsOTUB1 and an OsOTUB1/Met1-diubiquitin complex, we find that Met1 activity is conferred by Met1-specific motifs in the S1' pocket of OsOTUB1. Large-scale sequence alignments and hydrolysis experiments provide evidence that these motifs are a general determinant of Met1 activity in the OTUB subfamily across species. Analysis of the species distribution of OTUBs capable of hydrolysing Met1-linked ubiquitin chains shows that this activity is conserved in green plants (Viridiplantae) and does not exist in metazoans, providing insights into the evolutionary differentiation between primitive plants and animals.
Asunto(s)
Enzimas Desubicuitinizantes/metabolismo , Oryza , Transducción de Señal , Animales , Humanos , Mamíferos/metabolismo , Oryza/genética , Oryza/metabolismo , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
Protein ubiquitination regulates almost every process in eukaryotic cells. The study of the many enzymes involved in the ubiquitination system and the development of ubiquitination-associated therapeutics are important areas of current research. Synthetic tools such as ubiquitin-based chemical probes have been making an increasing contribution to deciphering various biochemical components involved in ubiquitin conjugation, recruitment, signaling, and deconjugation. In the present minireview, we summarize the progress of ubiquitin-based chemical probes with an emphasis on their various structures and chemical synthesis. We discuss the utility of the ubiquitin-based chemical probes for discovering and profiling ubiquitin-dependent signaling systems, as well as the monitoring and visualization of ubiquitin-related enzymatic machinery. We also show how the probes can serve to elucidate the molecular mechanism of recognition and catalysis. Collectively, the development and application of ubiquitin-based chemical probes emphasizes the importance and utility of chemical protein synthesis in modern chemical biology.
RESUMEN
Phosphorylation of S403 or S407 of the autophagic receptor protein p62 has recently been discovered to enhance the binding of p62 with ubiquitinated protein substrates to upregulate selective autophagy. To elucidate the molecular mechanism of how phosphorylation regulates the recruitment of ubiquitinated proteins, we report the first chemical synthesis of homogeneously phosphorylated p62, which enables the setting up of accurate in vitro systems for biochemical studies. Our synthesis employs the technology of sortase A-mediated protein hydrazide ligation, which successfully affords three types of phosphorylated p62 at the multi-milligram scale. Quantitative biochemical measurements show that the binding affinity of S403/S407-bisphosphorylated p62 to K63 diubiquitin is significantly higher than that of mono-phosphorylated p62. This finding suggests that phosphorylated S403 and S407 sites should bind to different epitopes on the ubiquitin chain. Furthermore, glutamate mutation is found to give a significantly impaired binding affinity, implying the necessity of using chemically synthesized phosphorylated p62 for the biochemical study of selective autophagy.
RESUMEN
The first copper-catalyzed/promoted sp(3)-C Suzuki-Miyaura coupling reaction of gem-diborylalkanes with nonactivated electrophilic reagents is reported. Not only 1, 1-diborylalkanes but also some other gem-diborylalkanes can be coupled with nonactivated primary alkyl halides, offering a new method for sp(3)C-sp(3)C bond formation and, simultaneously, providing a new strategy for the synthesis of alkylboronic esters.