RESUMO
Deubiquitinating enzymes (DUBs) are important regulators of ubiquitin signaling. Here, we report the discovery of deubiquitinating activity in ZUFSP/C6orf113. High-resolution crystal structures of ZUFSP in complex with ubiquitin reveal several distinctive features of ubiquitin recognition and catalysis. Our analyses reveal that ZUFSP is a novel DUB with no homology to any known DUBs, leading us to classify ZUFSP as the seventh DUB family. Intriguingly, the minimal catalytic domain does not cleave polyubiquitin. We identify two ubiquitin binding domains in ZUFSP: a ZHA (ZUFSP helical arm) that binds to the distal ubiquitin and an atypical UBZ domain in ZUFSP that binds to polyubiquitin. Importantly, both domains are essential for ZUFSP to selectively cleave K63-linked polyubiquitin. We show that ZUFSP localizes to DNA lesions, where it plays an important role in genome stability pathways, functioning to prevent spontaneous DNA damage and also promote cellular survival in response to exogenous DNA damage.
Assuntos
Núcleo Celular/enzimologia , Dano ao DNA , Enzimas Desubiquitinantes/metabolismo , Instabilidade Genômica , Poliubiquitina/metabolismo , Sítios de Ligação , Sobrevivência Celular , Enzimas Desubiquitinantes/química , Enzimas Desubiquitinantes/genética , Células HEK293 , Células HeLa , Humanos , Células Jurkat , Lisina , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-Atividade , Especificidade por Substrato , UbiquitinaçãoRESUMO
The ubiquitin system has become synonymous with the modification of lysine residues. However, the substrate scope and diversity of the conjugation machinery have been underappreciated, bringing us to an epoch in ubiquitin system research. The striking discoveries of metazoan enzymes dedicated toward serine and threonine ubiquitination have revealed the important role of nonlysine ubiquitination in endoplasmic reticulum-associated degradation, immune signaling and neuronal processes, while reports of nonproteinaceous substrates have extended ubiquitination beyond the proteome. Bacterial effectors that bypass the canonical ubiquitination machinery and form unprecedented linkage chemistry further redefine long-standing dogma. While chemical biology approaches have advanced our understanding of the canonical ubiquitin system, further study of noncanonical ubiquitination has been hampered by a lack of suitable tools. This Perspective aims to consolidate and contextualize recent discoveries and to propose potential applications of chemical biology, which will be instrumental in unraveling this new frontier of ubiquitin research.
Assuntos
Degradação Associada com o Retículo Endoplasmático , Lisina , Animais , Lisina/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Ubiquitination is initiated by transfer of ubiquitin (Ub) from a ubiquitin-activating enzyme (E1) to a ubiquitin-conjugating enzyme (E2), producing a covalently linked intermediate (E2-Ub) 1 . Ubiquitin ligases (E3s) of the 'really interesting new gene' (RING) class recruit E2-Ub via their RING domain and then mediate direct transfer of ubiquitin to substrates 2 . By contrast, 'homologous to E6-AP carboxy terminus' (HECT) E3 ligases undergo a catalytic cysteine-dependent transthiolation reaction with E2-Ub, forming a covalent E3-Ub intermediate3,4. Additionally, RING-between-RING (RBR) E3 ligases have a canonical RING domain that is linked to an ancillary domain. This ancillary domain contains a catalytic cysteine that enables a hybrid RING-HECT mechanism 5 . Ubiquitination is typically considered a post-translational modification of lysine residues, as there are no known human E3 ligases with non-lysine activity. Here we perform activity-based protein profiling of HECT or RBR-like E3 ligases and identify the neuron-associated E3 ligase MYCBP2 (also known as PHR1) as the apparent single member of a class of RING-linked E3 ligase with esterification activity and intrinsic selectivity for threonine over serine. MYCBP2 contains two essential catalytic cysteine residues that relay ubiquitin to its substrate via thioester intermediates. Crystallographic characterization of this class of E3 ligase, which we designate RING-Cys-relay (RCR), provides insights into its mechanism and threonine selectivity. These findings implicate non-lysine ubiquitination in cellular regulation of higher eukaryotes and suggest that E3 enzymes have an unappreciated mechanistic diversity.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Biocatálise , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular Tumoral , Cristalografia por Raios X , Cisteína/metabolismo , Esterificação , Células HEK293 , Humanos , Lisina/metabolismo , Modelos Moleculares , Domínios Proteicos , Proteômica , Serina/metabolismo , Especificidade por Substrato , Treonina/metabolismo , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
The reversibility of ubiquitination by the action of deubiquitinating enzymes (DUBs) serves as an important regulatory layer within the ubiquitin system. Approximately 100 DUBs are encoded by the human genome, and many have been implicated with pathologies, including neurodegeneration and cancer. Non-lysine ubiquitination is chemically distinct, and its physiological importance is emerging. Here, we couple chemically and chemoenzymatically synthesized ubiquitinated lysine and threonine model substrates to a mass spectrometry-based DUB assay. Using this platform, we profile two-thirds of known catalytically active DUBs for threonine esterase and lysine isopeptidase activity and find that most DUBs demonstrate dual selectivity. However, with two anomalous exceptions, the ovarian tumor domain DUB class demonstrates specific (iso)peptidase activity. Strikingly, we find the Machado-Joseph disease (MJD) class to be unappreciated non-lysine DUBs with highly specific ubiquitin esterase activity rivaling the efficiency of the most active isopeptidases. Esterase activity is dependent on the canonical catalytic triad, but proximal hydrophobic residues appear to be general determinants of non-lysine activity. Our findings also suggest that ubiquitin esters have appreciable cellular stability and that non-lysine ubiquitination is an integral component of the ubiquitin system. Its regulatory sophistication is likely to rival that of canonical ubiquitination.
Assuntos
Enzimas Desubiquitinantes/genética , Esterases/genética , Doença de Machado-Joseph/genética , Ubiquitina/genética , Aminoácidos/genética , Enzimas Desubiquitinantes/isolamento & purificação , Humanos , Lisina/genética , Doença de Machado-Joseph/enzimologia , Doença de Machado-Joseph/patologia , Espectrometria de Massas , Processamento de Proteína Pós-Traducional/genética , Ubiquitinação/genéticaRESUMO
MYCBP2 is a ubiquitin (Ub) E3 ligase (E3) that is essential for neurodevelopment and regulates axon maintenance. MYCBP2 transfers Ub to nonlysine substrates via a newly discovered RING-Cys-Relay (RCR) mechanism, where Ub is relayed from an upstream cysteine to a downstream substrate esterification site. The molecular bases for E2-E3 Ub transfer and Ub relay are unknown. Whether these activities are linked to the neural phenotypes is also unclear. We describe the crystal structure of a covalently trapped E2~Ub:MYCBP2 transfer intermediate revealing key structural rearrangements upon E2-E3 Ub transfer and Ub relay. Our data suggest that transfer to the dynamic upstream cysteine, whilst mitigating lysine activity, requires a closed-like E2~Ub conjugate with tempered reactivity, and Ub relay is facilitated by a helix-coil transition. Furthermore, neurodevelopmental defects and delayed injury-induced degeneration in RCR-defective knock-in mice suggest its requirement, and that of substrate esterification activity, for normal neural development and programmed axon degeneration.
Assuntos
Axônios/metabolismo , Cisteína/metabolismo , Domínios RING Finger , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Sítios de Ligação , Feminino , Técnicas de Introdução de Genes , Humanos , Lisina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL/embriologia , Camundongos Transgênicos , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Conformação Proteica , Transdução de Sinais , Relação Estrutura-Atividade , UbiquitinaçãoRESUMO
E3 ligases represent an important class of enzymes, yet there are currently no chemical probes for profiling their activity. We develop a new class of activity-based probe by re-engineering a ubiquitin-charged E2 conjugating enzyme and demonstrate the utility of these probes by profiling the transthiolation activity of the RING-in-between-RING (RBR) E3 ligase parkin in vitro and in cellular extracts. Our study provides valuable insight into the roles, and cellular hierarchy, of distinct phosphorylation events in parkin activation. We also profile parkin mutations associated with patients with Parkinson's disease and demonstrate that they mediate their effect largely by altering transthiolation activity. Furthermore, our probes enable direct and quantitative measurement of endogenous parkin activity, revealing that endogenous parkin is activated in neuronal cell lines (≥75%) in response to mitochondrial depolarization. This new technology also holds promise as a novel biomarker of PINK1-parkin signaling, as demonstrated by its compatibility with samples derived from individuals with Parkinson's disease.
Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Fibroblastos/metabolismo , Células HeLa , Humanos , Mutação , Ubiquitina-Proteína Ligases/genéticaRESUMO
The full breadth of the field: The 2016 EMBO Chemical Biology Conference, covering topics from tool development to biological applications and from computational drug design to synthetic chemistry, took place in Heidelberg from 31st August to 3rd September.
Assuntos
Descoberta de Drogas , Produtos Biológicos/química , Epigenômica , Engenharia de Proteínas , Pesquisa , Sistemas do Segundo MensageiroRESUMO
The modification of proteins with ubiquitin (Ub) is an important regulator of eukaryotic biology and deleterious perturbation of this process is widely linked to the onset of various diseases. The regulatory capacity of the Ub signal is high and, in part, arises from the capability of Ub to be enzymatically polymerised to form polyubiquitin (polyUb) chains of eight different linkage types. These distinct polyUb topologies can then be site-specifically conjugated to substrate proteins to elicit a number of cellular outcomes. Therefore, to further elucidate the biological significance of substrate ubiquitination, methodologies that allow the production of defined polyUb species, and substrate proteins that are site-specifically modified with them, are essential to progress our understanding. Many chemically inspired methods have recently emerged which fulfil many of the criteria necessary for achieving deeper insight into Ub biology. With a view to providing immediate impact in traditional biology research labs, the aim of this review is to provide an overview of the techniques that are available for preparing Ub conjugates and polyUb chains with focus on approaches that use recombinant protein building blocks. These approaches either produce a native isopeptide, or analogue thereof, that can be hydrolysable or non-hydrolysable by deubiquitinases. The most significant biological insights that have already been garnered using such approaches will also be summarized.
Assuntos
Ubiquitina/química , Ubiquitina/metabolismo , Animais , Humanos , Modelos Biológicos , Conformação Proteica , UbiquitinaçãoRESUMO
We describe the genetically directed incorporation of aminooxy functionality into recombinant proteins by using a mutant Methanosarcina barkeri pyrrolysyl-tRNA synthetase/tRNACUA pair. This allows the general production of nonhydrolysable ubiquitin conjugates of recombinant origin by bioorthogonal oxime ligation. This was exemplified by the preparation of nonhydrolysable versions of diubiquitin, polymeric ubiquitin chains and ubiquitylated SUMO. The conjugates exhibited unrivalled isostery with the native isopeptide bond, as inferred from structural and biophysical characterisation. Furthermore, the conjugates functioned as nanomolar inhibitors of deubiquitylating enzymes and were recognised by linkage-specific antibodies. This technology should provide a versatile platform for the development of powerful tools for studying deubiquitylating enzymes and for elucidating the cellular roles of diverse polyubiquitin linkages.
Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Methanosarcina barkeri/enzimologia , Ubiquitinas/química , Bioengenharia/métodos , Enzimas Desubiquitinantes , Methanosarcina barkeri/genética , Methanosarcina barkeri/metabolismo , Oximas/química , PolimerizaçãoRESUMO
Modification of proteins with ubiquitin (Ub) occurs through a variety of topologically distinct Ub linkages, including Ube2W-mediated monoubiquitylation of N-terminal alpha amines to generate peptide-linked linear mono-Ub fusions. Protein ubiquitylation can be reversed by the action of deubiquitylating enzymes (DUBs), many of which show striking preference for particular Ub linkage types. Here, we have screened for DUBs that preferentially cleave N-terminal Ub from protein substrates but do not act on Ub homopolymers. We show that members of the Ub C-terminal hydrolase (UCH) family of DUBs demonstrate this preference for N-terminal deubiquitylating activity as they are capable of cleaving N-terminal Ub from SUMO2 and Ube2W, while displaying no activity against any of the eight Ub linkage types. Surprisingly, this ability to cleave Ub from SUMO2 was 100 times more efficient for UCH-L3 when we deleted the unstructured N-terminus of SUMO2, demonstrating that UCH enzymes can cleave Ub from structured proteins. However, UCH-L3 could also cleave chemically synthesized isopeptide-linked Ub from lysine 11 (K11) of SUMO2 with similar efficiency, demonstrating that UCH DUB activity is not limited to peptide-linked Ub. These findings advance our understanding of the specificity of the UCH family of DUBs, which are strongly implicated in cancer and neurodegeneration but whose substrate preference has remained unclear. In addition, our findings suggest that the reversal of Ube2W-mediated N-terminal ubiquitylation may be one physiological role of UCH DUBs in vivo.
Assuntos
Proteínas de Escherichia coli/metabolismo , Polímeros/metabolismo , Ubiquitina Tiolesterase/metabolismo , Proteínas de Escherichia coli/química , Polímeros/química , Estrutura Terciária de Proteína , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Ubiquitina Tiolesterase/química , Ubiquitinação/fisiologiaRESUMO
Ubiquitin phosphorylation is emerging as an important regulatory layer in the ubiquitin system. This is exemplified by the phosphorylation of ubiquitin on Ser65 by the Parkinson's disease-associated kinase PINK1, which mediates the activation of the E3 ligase Parkin. Additional phosphorylation sites on ubiquitin might also have important cellular roles. Here we report a versatile strategy for preparing phosphorylated ubiquitin. We biochemically and structurally characterise semisynthetic phospho-Ser65-ubiquitin. Unexpectedly, we observed disulfide bond formation between ubiquitin molecules, and hence a novel crystal form. The method outlined provides a direct approach to study the combinatorial effects of phosphorylation on ubiquitin function. Our analysis also suggests that disulfide engineering of ubiquitin could be a useful strategy for obtaining alternative crystal forms of ubiquitin species thereby facilitating structural validation.
Assuntos
Serina/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Dissulfetos/química , Ativação Enzimática , Modelos Moleculares , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosforilação , Conformação Proteica , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Crimean Congo hemorrhagic fever virus (CCHFV) is a deadly human pathogen that evades innate immune responses by efficiently interfering with antiviral signaling pathways mediated by NF-κB, IRF3, and IFNα/ß. These pathways rely on protein ubiquitination for their activation, and one outcome is the modification of proteins with the ubiquitin (Ub)-like modifier interferon-stimulated gene (ISG)15. CCHFV and related viruses encode a deubiquitinase (DUB) of the ovarian tumor (OTU) family, which unlike eukaryotic OTU DUBs also targets ISG15 modifications. Here we characterized the viral OTU domain of CCHFV (vOTU) biochemically and structurally, revealing that it hydrolyzes four out of six tested Ub linkages, but lacks activity against linear and K29-linked Ub chains. vOTU cleaved Ub and ISG15 with similar kinetics, and we were able to understand vOTU cross-reactivity at the molecular level from crystal structures of vOTU in complex with Ub and ISG15. An N-terminal extension in vOTU not present in eukaryotic OTU binds to the hydrophobic Ile44 patch of Ub, which results in a dramatically different Ub orientation compared to a eukaryotic OTU-Ub complex. The C-terminal Ub-like fold of ISG15 (ISG15-C) adopts an equivalent binding orientation. Interestingly, ISG15-C contains an additional second hydrophobic surface that is specifically contacted by vOTU. These subtle differences in Ub/ISG15 binding allowed the design of vOTU variants specific for either Ub or ISG15, which will be useful tools to understand the relative contribution of ubiquitination vs. ISGylation in viral infection. Furthermore, the crystal structures will allow structure-based design of antiviral agents targeting this enzyme.
Assuntos
Citocinas/imunologia , Vírus da Febre Hemorrágica da Crimeia-Congo/imunologia , Neoplasias Ovarianas , Peptídeo Hidrolases/imunologia , Ubiquitina/imunologia , Ubiquitinação/imunologia , Ubiquitinas/imunologia , Proteínas Virais/imunologia , Citocinas/genética , Citocinas/metabolismo , Feminino , Vírus da Febre Hemorrágica da Crimeia-Congo/enzimologia , Vírus da Febre Hemorrágica da Crimeia-Congo/genética , Humanos , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
UBR4 is a 574 kDa E3 ligase (E3) of the N-degron pathway with roles in neurodevelopment, age-associated muscular atrophy and cancer. The catalytic module that carries out ubiquitin (Ub) transfer remains unknown. Here we identify and characterize a distinct E3 module within human UBR4 consisting of a 'hemiRING' zinc finger, a helical-rich UBR zinc-finger interacting (UZI) subdomain, and an N-terminal region that can serve as an affinity factor for the E2 conjugating enzyme (E2). The structure of an E2-E3 complex provides atomic-level insight into the specificity determinants of the hemiRING toward the cognate E2s UBE2A/UBE2B. Via an allosteric mechanism, the UZI subdomain modestly activates the Ub-loaded E2 (E2â¼Ub). We propose attenuated activation is complemented by the intrinsically high lysine reactivity of UBE2A, and their cooperation imparts a reactivity profile important for substrate specificity and optimal degradation kinetics. These findings reveal the mechanistic underpinnings of a neuronal N-degron E3, its specific recruitment of UBE2A, and highlight the underappreciated architectural diversity of cross-brace domains with Ub E3 activity.
Assuntos
Enzimas de Conjugação de Ubiquitina , Ubiquitina-Proteína Ligases , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo , Catálise , Ubiquitinação , Proteínas de Ligação a Calmodulina/metabolismoRESUMO
Ubiquitination is a reversible post-translational modification that regulates a myriad of eukaryotic functions. Our ability to study the effects of ubiquitination is often limited by the inaccessibility of homogeneously ubiquitinated proteins. In particular, elucidating the roles of the so-called 'atypical' ubiquitin chains (chains other than Lys48- or Lys63-linked ubiquitin), which account for a large fraction of ubiquitin polymers, is challenging because the enzymes for their biosynthesis are unknown. Here we combine genetic code expansion, intein chemistry and chemoselective ligations to synthesize 'atypical' ubiquitin chains. We solve the crystal structure of Lys6-linked diubiquitin, which is distinct from that of structurally characterized ubiquitin chains, providing a molecular basis for the different biological functions this linkage may regulate. Moreover, we profile a panel containing 10% of the known human deubiquitinases on Lys6- and Lys29-linked ubiquitin and discover that TRABID cleaves the Lys29 linkage 40-fold more efficiently than the Lys63 linkage.
Assuntos
Endopeptidases/metabolismo , Lisina/metabolismo , Neoplasias Ovarianas/enzimologia , Peptídeos/metabolismo , Engenharia de Proteínas , Ubiquitinas/síntese química , Ubiquitinas/metabolismo , Endopeptidases/química , Feminino , Humanos , Lisina/química , Peptídeos/química , Especificidade por Substrato , Ubiquitinas/química , Ubiquitinas/genéticaRESUMO
The degeneration of nerve fibres following injury was first described by Augustus Waller over 170 years ago. Initially assumed to be a passive process, it is now evident that axons respond to insult via regulated cellular signaling events resulting in their programmed degeneration. Pro-survival and pro-degenerative factors have been identified and their regulatory mechanisms are beginning to emerge. The ubiquitin system has been implicated in the pro-degenerative process and a key component is the ubiquitin E3 ligase MYCBP2 (also known as PHR1). Ubiquitin E3 ligases are tasked with the transfer of the small protein modifier ubiquitin to substrates and consist of hundreds of members. They can be classified as single subunit systems or as multi-subunit complexes. Their catalytic domains can also be assigned to three general architectures. Hints that MYCBP2 might not conform to these established formats came to light and it is now clear from biochemical and structural studies that MYCBP2 is indeed an outlier in terms of its modus operandi. Furthermore, the unconventional way in which MYCBP2 transfers ubiquitin to substrates has been linked to neurodevelopmental and pro-degenerative function. Herein, we will summarize these research developments relating to the unusual features of MYCBP2 and postulate therapeutic strategies that prevent Wallerian degeneration. These have exciting potential for providing relief from pathological neuropathies and neurodegenerative diseases.
RESUMO
A general approach for the rapid and selective inhibition of enzymes in cells using a common tool compound would be of great value for research and therapeutic development. We previously reported a chemogenetic strategy that addresses this challenge for kinases, relying on bioorthogonal tethering of a pan inhibitor to a target kinase through a genetically encoded non-canonical amino acid. However, pan inhibitors are not available for many enzyme classes. Here, we expand the scope of the chemogenetic strategy to cysteine-dependent enzymes by bioorthogonal tethering of electrophilic warheads. For proof of concept, selective inhibition of two E2 ubiquitin-conjugating enzymes, UBE2L3 and UBE2D1, was demonstrated in biochemical assays. Further development and optimization of this approach should enable its use in cells as well as with other cysteine-dependent enzymes, facilitating the investigation of their cellular function and validation as therapeutic targets.
Assuntos
Cisteína , Enzimas de Conjugação de Ubiquitina , Cisteína/química , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Enzimas de Conjugação de Ubiquitina/genéticaRESUMO
Ubiquitylation enzymes are involved in all aspects of eukaryotic biology and are frequently disrupted in disease. One example is the E3 ubiquitin ligase RNF12/RLIM, which is mutated in the developmental disorder Tønne-Kalscheuer syndrome (TOKAS). RNF12 TOKAS variants largely disrupt catalytic E3 ubiquitin ligase activity, which presents a pressing need to develop approaches to assess the impact of variants on RNF12 activity in patients. Here, we use photocrosslinking activity-based probes (photoABPs) to monitor RNF12 RING E3 ubiquitin ligase activity in normal and pathogenic contexts. We demonstrate that photoABPs undergo UV-induced labelling of RNF12 that is consistent with its RING E3 ligase activity. Furthermore, photoABPs robustly report the impact of RNF12 TOKAS variants on E3 activity, including variants within the RING domain and distal non-RING regulatory elements. Finally, we show that this technology can be rapidly deployed in human pluripotent stem cells. In summary, photoABPs are versatile tools that can directly identify disruptions to RING E3 ubiquitin ligase activity in human disease, thereby providing new insight into pathogenic mechanisms.
Assuntos
Ubiquitina-Proteína Ligases , Humanos , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Protein ubiquitination is a post-translational modification that regulates almost all aspects of eukaryotic biology. Here we discover the first routes for the efficient site-specific incorporation of δ-thiol-L-lysine (7) and δ-hydroxy-L-lysine (8) into recombinant proteins, via evolution of a pyrrolysyl-tRNA synthetase/tRNA(CUA) pair. We combine the genetically directed incorporation of 7 with native chemical ligation and desulfurization to yield an entirely native isopeptide bond between substrate proteins and ubiquitin. We exemplify this approach by demonstrating the synthesis of a ubiquitin dimer and the first synthesis of ubiquitinated SUMO.
Assuntos
Engenharia de Proteínas/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ubiquitinação , Aminoacil-tRNA Sintetases/metabolismo , Sítios de Ligação , Lisina/análogos & derivados , Lisina/metabolismo , Methanosarcina barkeri/enzimologia , Proteínas Recombinantes/genética , Especificidade por SubstratoRESUMO
Activity-based protein profiling is an invaluable technique for studying enzyme biology and facilitating the development of therapeutics. Ubiquitin E3 ligases (E3s) are one of the largest enzyme families and regulate a host of (patho)physiological processes. The largest subtype are the RING E3s of which there are >600 members. RING E3s have adaptor-like activity that can be subject to diverse regulatory mechanisms and have become attractive drug targets. Activity-based probes (ABPs) for measuring RING E3 activity do not exist. Here we re-engineer ubiquitin-charged E2 conjugating enzymes to produce photocrosslinking ABPs. We demonstrate activity-dependent profiling of two divergent cancer-associated RING E3s, RNF4 and c-Cbl, in response to their native activation signals. We also demonstrate profiling of endogenous RING E3 ligase activation in response to epidermal growth factor (EGF) stimulation. These photocrosslinking ABPs should advance E3 ligase research and the development of selective modulators against this important class of enzymes.