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1.
Nat Commun ; 10(1): 231, 2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30651545

RESUMO

USP7 is a highly abundant deubiquitinating enzyme (DUB), involved in cellular processes including DNA damage response and apoptosis. USP7 has an unusual catalytic mechanism, where the low intrinsic activity of the catalytic domain (CD) increases when the C-terminal Ubl domains (Ubl45) fold onto the CD, allowing binding of the activating C-terminal tail near the catalytic site. Here we delineate how the target protein promotes the activation of USP7. Using NMR analysis and biochemistry we describe the order of activation steps, showing that ubiquitin binding is an instrumental step in USP7 activation. Using chemically synthesised p53-peptides we also demonstrate how the correct ubiquitinated substrate increases catalytic activity. We then used transient reaction kinetic modelling to define how the USP7 multistep mechanism is driven by target recognition. Our data show how this pleiotropic DUB can gain specificity for its cellular targets.


Assuntos
Processamento de Proteína Pós-Traducional , Peptidase 7 Específica de Ubiquitina/metabolismo , Ubiquitina/metabolismo , Isótopos de Carbono/química , Domínio Catalítico/genética , Ensaios Enzimáticos/métodos , Cinética , Modelos Químicos , Mutagênese Sítio-Dirigida , Isótopos de Nitrogênio/química , Ressonância Magnética Nuclear Biomolecular/métodos , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Ressonância de Plasmônio de Superfície , Proteína Supressora de Tumor p53/química , Ubiquitina/química , Peptidase 7 Específica de Ubiquitina/química , Peptidase 7 Específica de Ubiquitina/genética , Peptidase 7 Específica de Ubiquitina/isolamento & purificação
2.
Nucleic Acids Res ; 46(21): 11251-11261, 2018 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-30203048

RESUMO

The histone methyltransferase Dot1 is conserved from yeast to human and methylates lysine 79 of histone H3 (H3K79) on the core of the nucleosome. H3K79 methylation by Dot1 affects gene expression and the response to DNA damage, and is enhanced by monoubiquitination of the C-terminus of histone H2B (H2Bub1). To gain more insight into the functions of Dot1, we generated genetic interaction maps of increased-dosage alleles of DOT1. We identified a functional relationship between increased Dot1 dosage and loss of the DUB module of the SAGA co-activator complex, which deubiquitinates H2Bub1 and thereby negatively regulates H3K79 methylation. Increased Dot1 dosage was found to promote H2Bub1 in a dose-dependent manner and this was exacerbated by the loss of SAGA-DUB activity, which also caused a negative genetic interaction. The stimulatory effect on H2B ubiquitination was mediated by the N-terminus of Dot1, independent of methyltransferase activity. Our findings show that Dot1 and H2Bub1 are subject to bi-directional crosstalk and that Dot1 possesses chromatin regulatory functions that are independent of its methyltransferase activity.


Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitinação , Cromatina/genética , Cromatina/metabolismo , Histona-Lisina N-Metiltransferase/genética , Proteínas Nucleares/genética , Ligação Proteica , Mapas de Interação de Proteínas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
3.
Mol Cell ; 62(4): 572-85, 2016 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-27203180

RESUMO

Deubiquitinating enzymes (DUBs) recognize and cleave linkage-specific polyubiquitin (polyUb) chains, but mechanisms underlying specificity remain elusive in many cases. The severe acute respiratory syndrome (SARS) coronavirus papain-like protease (PLpro) is a DUB that cleaves ISG15, a two-domain Ub-like protein, and Lys48-linked polyUb chains, releasing diUb(Lys48) products. To elucidate this specificity, we report the 2.85 Å crystal structure of SARS PLpro bound to a diUb(Lys48) activity-based probe. SARS PLpro binds diUb(Lys48) in an extended conformation via two contact sites, S1 and S2, which are proximal and distal to the active site, respectively. We show that specificity for polyUb(Lys48) chains is predicated on contacts in the S2 site and enhanced by an S1-S1' preference for a Lys48 linkage across the active site. In contrast, ISG15 specificity is dominated by contacts in the S1 site. Determinants revealed for polyUb(Lys48) specificity should prove useful in understanding PLpro deubiquitinating activities in coronavirus infections.


Assuntos
Cisteína Endopeptidases/metabolismo , Citocinas/metabolismo , Enzimas Desubiquitinantes/metabolismo , Poliubiquitina/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologia , Ubiquitinas/metabolismo , Proteínas Virais/metabolismo , Sítios de Ligação , Proteases 3C de Coronavírus , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Citocinas/química , Enzimas Desubiquitinantes/química , Células HeLa , Humanos , Lisina , Modelos Moleculares , Mutação , Poliubiquitina/química , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Relação Estrutura-Atividade , Ubiquitinação , Ubiquitinas/química , Proteínas Virais/química , Proteínas Virais/genética
4.
Cell Chem Biol ; 23(4): 472-82, 2016 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-27066941

RESUMO

Ubiquitin chains are important post-translational modifications that control a large number of cellular processes. Chains can be formed via different linkages, which determines the type of signal they convey. Deubiquitylating enzymes (DUBs) regulate ubiquitylation status by trimming or removing chains from attached proteins. DUBs can contain several ubiquitin-binding pockets, which confer specificity toward differently linked chains. Most tools for monitoring DUB specificity target binding pockets on opposing sides of the active site; however, some DUBs contain additional pockets. Therefore, reagents targeting additional pockets are essential to fully understand linkage specificity. We report the development of active site-directed probes and fluorogenic substrates, based on non-hydrolyzable diubiquitin, that are equipped with a C-terminal warhead or a fluorogenic activity reporter moiety. We demonstrate that various DUBs in lysates display differential reactivity toward differently linked diubiquitin probes, as exemplified by the proteasome-associated DUB USP14. In addition, OTUD2 and OTUD3 show remarkable linkage-specific reactivity with our diubiquitin-based reagents.


Assuntos
Corantes Fluorescentes/química , Linfoma/metabolismo , Peptídeo Hidrolases/metabolismo , Ubiquitinas/química , Animais , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Linfoma/patologia , Camundongos , Ubiquitinação , Ubiquitinas/síntese química , Ubiquitinas/metabolismo
5.
Nat Commun ; 7: 10292, 2016 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-26739236

RESUMO

The deubiquitinating enzyme BAP1 is an important tumor suppressor that has drawn attention in the clinic since its loss leads to a variety of cancers. BAP1 is activated by ASXL1 to deubiquitinate mono-ubiquitinated H2A at K119 in Polycomb gene repression, but the mechanism of this reaction remains poorly defined. Here we show that the BAP1 C-terminal extension is important for H2A deubiquitination by auto-recruiting BAP1 to nucleosomes in a process that does not require the nucleosome acidic patch. This initial encounter-like complex is unproductive and needs to be activated by the DEUBAD domains of ASXL1, ASXL2 or ASXL3 to increase BAP1's affinity for ubiquitin on H2A, to drive the deubiquitination reaction. The reaction is specific for Polycomb modifications of H2A as the complex cannot deubiquitinate the DNA damage-dependent ubiquitination at H2A K13/15. Our results contribute to the molecular understanding of this important tumor suppressor.


Assuntos
Proteínas Repressoras/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Clonagem Molecular , Escherichia coli , Regulação da Expressão Gênica/fisiologia , Histonas/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Proteínas Repressoras/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética
6.
Mol Cell ; 57(5): 887-900, 2015 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-25702870

RESUMO

Deubiquitinating enzymes (DUBs) control vital processes in eukaryotes by hydrolyzing ubiquitin adducts. Their activities are tightly regulated, but the mechanisms remain elusive. In particular, the DUB UCH-L5 can be either activated or inhibited by conserved regulatory proteins RPN13 and INO80G, respectively. Here we show how the DEUBAD domain in RPN13 activates UCH-L5 by positioning its C-terminal ULD domain and crossover loop to promote substrate binding and catalysis. The related DEUBAD domain in INO80G inhibits UCH-L5 by exploiting similar structural elements in UCH-L5 to promote a radically different conformation, and employs molecular mimicry to block ubiquitin docking. In this process, large conformational changes create small but highly specific interfaces that mediate activity modulation of UCH-L5 by altering the affinity for substrates. Our results establish how related domains can exploit enzyme conformational plasticity to allosterically regulate DUB activity. These allosteric sites may present novel insights for pharmaceutical intervention in DUB activity.


Assuntos
Proteínas de Ligação a DNA/química , Glicoproteínas de Membrana/química , Estrutura Terciária de Proteína , Ubiquitina Tiolesterase/química , Sequência de Aminoácidos , Cristalografia por Raios X , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Ubiquitina/química , Ubiquitina/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo
7.
Curr Opin Chem Biol ; 23: 63-70, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25461387

RESUMO

Protein ubiquitylation is an important regulator of protein function, localization and half-life. It plays a key role in most cellular processes including immune signaling. Deregulation of this process is a major causative factor for many diseases. A major advancement in the identification and characterization of the enzymes that remove ubiquitin, deubiquitylases (DUBs) was made by the development of activity-based probes (ABPs). Recent advances in chemical protein synthesis and ligation methodology has yielded novel reagents for use in ubiquitylation research. We describe recent advances and discuss future directions in reagent development for studying DUBs.


Assuntos
Proteases Específicas de Ubiquitina/metabolismo , Ubiquitina/metabolismo , Domínio Catalítico , Humanos , Sondas Moleculares , Ubiquitina/química , Proteases Específicas de Ubiquitina/química
8.
J Immunol ; 193(10): 4803-13, 2014 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-25311806

RESUMO

Virus or tumor Ag-derived peptides that are displayed by MHC class I molecules are attractive starting points for vaccine development because they induce strong protective and therapeutic cytotoxic T cell responses. In thus study, we show that the MHC binding and consequent T cell reactivity against several HLA-A*02 restricted epitopes can be further improved through the incorporation of nonproteogenic amino acids at primary and secondary anchor positions. We screened more than 90 nonproteogenic, synthetic amino acids through a range of epitopes and tested more than 3000 chemically enhanced altered peptide ligands (CPLs) for binding affinity to HLA-A*0201. With this approach, we designed CPLs of viral epitopes, of melanoma-associated Ags, and of the minor histocompatibility Ag UTA2-1, which is currently being evaluated for its antileukemic activity in clinical dendritic cell vaccination trials. The crystal structure of one of the CPLs in complex with HLA-A*0201 revealed the molecular interactions likely responsible for improved binding. The best CPLs displayed enhanced affinity for MHC, increasing MHC stability and prolonging recognition by Ag-specific T cells and, most importantly, they induced accelerated expansion of antitumor T cell frequencies in vitro and in vivo as compared with the native epitope. Eventually, we were able to construct a toolbox of preferred nonproteogenic residues with which practically any given HLA-A*02 restricted epitope can be readily optimized. These CPLs could improve the therapeutic outcome of vaccination strategies or can be used for ex vivo enrichment and faster expansion of Ag-specific T cells for transfer into patients.


Assuntos
Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Antígeno HLA-A2/imunologia , Neoplasias/prevenção & controle , Peptídeos/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Linfócitos B , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/química , Cristalografia por Raios X , Epitopos , Expressão Gênica , Antígeno HLA-A2/química , Antígeno HLA-A2/genética , Humanos , Imunização , Camundongos , Camundongos Transgênicos , Antígenos de Histocompatibilidade Menor/química , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/imunologia , Modelos Moleculares , Dados de Sequência Molecular , Neoplasias/imunologia , Peptídeos/administração & dosagem , Peptídeos/química , Peptídeos/genética , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo
9.
Cell ; 154(1): 169-84, 2013 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-23827681

RESUMO

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.


Assuntos
Endopeptidases/química , Endopeptidases/metabolismo , Neoplasias Ovarianas/enzimologia , Ubiquitinação , Catálise , Domínio Catalítico , Cristalografia por Raios X , Endopeptidases/genética , Feminino , Humanos , Modelos Moleculares , Neoplasias Ovarianas/metabolismo , Estrutura Terciária de Proteína , Tioléster Hidrolases/química , Tioléster Hidrolases/metabolismo , Ubiquitinas/metabolismo
10.
J Am Chem Soc ; 135(8): 2867-70, 2013 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-23387960

RESUMO

Active-site directed probes are powerful in studies of enzymatic function. We report an active-site directed probe based on a warhead so far considered unreactive. By replacing the C-terminal carboxylate of ubiquitin (Ub) with an alkyne functionality, a selective reaction with the active-site cysteine residue of de-ubiquitinating enzymes was observed. The resulting product was shown to be a quaternary vinyl thioether, as determined by X-ray crystallography. Proteomic analysis of proteins bound to an immobilized Ub alkyne probe confirmed the selectivity toward de-ubiquitinating enzymes. The observed reactivity is not just restricted to propargylated Ub, as highlighted by the selective reaction between caspase-1 (interleukin converting enzyme) and a propargylated peptide derived from IL-1ß, a caspase-1 substrate.


Assuntos
Alcinos/química , Cisteína/química , Peptídeo Hidrolases/metabolismo , Domínio Catalítico
12.
J Proteomics ; 73(10): 1945-53, 2010 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-20546957

RESUMO

T cell epitopes are peptides, for instance derived from foreign, mutated or overexpressed proteins, that are displayed by MHC molecules on the cell surface and that are recognized by T lymphocytes. Knowledge of the identity of epitopes displayed by MHC molecules is of high value for diagnostic purposes and for the development of prophylactic and therapeutic immunotherapy regimens. Here we review key techniques in MHC class I epitope definition and we discuss recent developments in epitope discovery and their implications. Developments in epitope discovery strategies should ultimately lead to the definition of the MHC-associated peptidome.


Assuntos
Proteômica/métodos , Algoritmos , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Genes MHC Classe I , Humanos , Imunoterapia , Marcação por Isótopo , Espectrometria de Massas/métodos , Linfócitos T/imunologia
13.
Anal Chem ; 80(23): 9042-51, 2008 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-19551932

RESUMO

In LC-MS, derivatization is primarily used to improve ionization characteristics, especially for analytes that are not (efficiently) ionized by ESI or APCI such as aldehydes, sugars, and steroids. Derivatization strategies are then directed at the incorporation of a group with a permanent charge. A compound class that typically requires derivatization prior to LC-MS is the group of small aliphatic aldehydes that are, for instance, analyzed as the key biomarkers for lipid peroxidation in organisms. Here we report the development of a new tailor-made, highly sensitive, and selective derivatization agent 4-(2-(trimethylammonio)ethoxy)benzenaminium halide (4-APC) for the quantification of aldehydes in biological matrixes with positive ESI-MS/ MS without additional extraction procedures. 4-APC possesses an aniline moiety for a fast selective reaction with aliphatic aldehydes as well as a quaternary ammonium group for improved MS sensitivity. The derivatization reaction is a convenient one-pot reaction at a mild pH (5.7) and temperature (10 degrees C). As a result, an in-vial derivatization can be performed before analysis with an LC-MS/MS system. All aldehydes are derivatized within 30 min to a plateau, except malondialdehyde, which requires 300 min to reach a plateau. All derivatized aldehydes are stable for at least 35 h. Linearity was established between 10 and 500 nM and the limits of detection were in the 3-33 nM range for the aldehyde derivatives. Furthermore, the chosen design of these structures allows tandem MS to be used to monitor the typical losses of 59 and 87 from aldehyde derivatives, thereby enabling screening for aldehydes. Finally, of all aldehydes, pentanal and hexanal were detected at elevated levels in pooled healthy human urine samples.


Assuntos
Aldeídos/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Aldeídos/química , Aldeídos/urina , Humanos , Hidrocarbonetos Halogenados/síntese química , Hidrocarbonetos Halogenados/química , Cetonas/química , Malondialdeído/química , Compostos de Amônio Quaternário/síntese química , Compostos de Amônio Quaternário/química , Sensibilidade e Especificidade
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