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1.
Sci Signal ; 17(819): eabn1101, 2024 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-38227684

RESUMO

Ubiquitination is a posttranslational modification that is crucial for the dynamic regulation of diverse signaling pathways. To enhance our understanding of ubiquitination-mediated signaling, we generated a new class of bispecific antibodies that combine recognition of ubiquitination substrates and specific polyubiquitin linkages. RIP1-K63 and RIP1-linear (Lin) linkage polyubiquitin bispecific antibodies detected linkage-specific ubiquitination of the proinflammatory kinase RIP1 in cells and in tissues and revealed RIP1 ubiquitination by immunofluorescence. Similarly, ubiquitination of the RIP1-related kinase RIP2 with K63 or linear linkages was specifically detected with the RIP2-K63 and RIP2-Lin bispecific antibodies, respectively. Furthermore, using the RIP2-K63 and RIP2-Lin bispecific antibodies, we found prominent K63-linked and linear RIP2 ubiquitination in samples from patients with ulcerative colitis and Crohn's disease. We also developed a bispecific antibody (K63-Lin) that simultaneously recognizes K63-linked and linear ubiquitination of components of various signaling pathways. Together, these bispecific antibodies represent a new class of reagents with the potential to be developed for the detection of inflammatory biomarkers.


Assuntos
Anticorpos Biespecíficos , Ubiquitina , Humanos , Anticorpos Biespecíficos/metabolismo , Poliubiquitina/metabolismo , Transdução de Sinais/fisiologia , Ubiquitina/metabolismo , Ubiquitinação
2.
J Proteome Res ; 22(7): 2218-2231, 2023 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-37285454

RESUMO

Recent advances in targeted covalent inhibitors have aroused significant interest for their potential in drug development for difficult therapeutic targets. Proteome-wide profiling of functional residues is an integral step of covalent drug discovery aimed at defining actionable sites and evaluating compound selectivity in cells. A classical workflow for this purpose is called IsoTOP-ABPP, which employs an activity-based probe and two isotopically labeled azide-TEV-biotin tags to mark, enrich, and quantify proteome from two samples. Here we report a novel isobaric 11plex-AzidoTMT reagent and a new workflow, named AT-MAPP, that significantly expands multiplexing power as compared to the original isoTOP-ABPP. We demonstrate its application in identifying cysteine on- and off-targets using a KRAS G12C covalent inhibitor ARS-1620. However, changes in some of these hits can be explained by modulation at the protein and post-translational levels. Thus, it would be crucial to interrogate site-level bona fide changes in concurrence to proteome-level changes for corroboration. In addition, we perform a multiplexed covalent fragment screening using four acrylamide-based compounds as a proof-of-concept. This study identifies a diverse set of liganded cysteine residues in a compound-dependent manner with an average hit rate of 0.07% in intact cell. Lastly, we screened 20 sulfonyl fluoride-based compounds to demonstrate that the AT-MAPP assay is flexible for noncysteine functional residues such as tyrosine and lysine. Overall, we envision that 11plex-AzidoTMT will be a useful addition to the current toolbox for activity-based protein profiling and covalent drug development.


Assuntos
Cisteína , Proteoma , Proteoma/metabolismo , Cisteína/metabolismo , Proteômica , Processamento de Proteína Pós-Traducional , Descoberta de Drogas
3.
bioRxiv ; 2023 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-37162991

RESUMO

5-fluorouracil (5-FU) is a successful and broadly used anti-cancer therapeutic. A major mechanism of action of 5-FU is thought to be through thymidylate synthase (TYMS) inhibition resulting in dTTP depletion and activation of the DNA damage response. This suggests that 5-FU should synergize with other DNA damaging agents. However, we found that combinations of 5-FU and oxaliplatin or irinotecan failed to display any evidence of synergy in clinical trials, and resulted in sub-additive killing in a panel of colorectal cancer (CRC) cell lines. In seeking to understand this antagonism, we unexpectedly found that an RNA damage response during ribosome biogenesis dominates the drug's efficacy in tumor types for which 5-FU shows clinical benefit. 5-FU has an inherent bias for RNA incorporation, and blocking this greatly reduced drug-induced lethality, indicating that accumulation of damaged RNA is more deleterious than the lack of new RNA synthesis. Using 5-FU metabolites that specifically incorporate into either RNA or DNA revealed that CRC cell lines and patient-derived colorectal cancer organoids are inherently more sensitive to RNA damage. This difference held true in cell lines from other tissues in which 5-FU has shown clinical utility, whereas cell lines from tumor tissues that lack clinical 5-FU responsiveness typically showed greater sensitivity to the drug's DNA damage effects. Analysis of changes in the phosphoproteome and ubiquitinome shows RNA damage triggers the selective ubiquitination of multiple ribosomal proteins leading to autophagy-dependent rRNA catabolism and proteasome-dependent degradation of ubiquitinated ribosome proteins. Further, RNA damage response to 5-FU is selectively enhanced by compounds that promote ribosome biogenesis, such as KDM2A inhibitors. These results demonstrate the presence of a strong RNA damage response linked to apoptotic cell death, with clear utility of combinatorially targeting this response in cancer therapy.

4.
Anal Chem ; 95(11): 4834-4839, 2023 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-36876898

RESUMO

The growing opportunities recognized for covalent drug inhibitors, like KRAS G12C inhibitors, are driving the need for mass spectrometry methods that can quickly and robustly measure therapeutic drug activity in vivo for drug discovery research and development. Effective front-end sample preparation is critical for proteins extracted from tumors but is generally labor intensive and impractical for large sample numbers typical in pharmacodynamic (PD) studies. Herein, we describe an automated and integrated sample preparation method for the measurement of activity levels of KRAS G12C drug inhibitor alkylation from complex tumor samples involving high throughput detergent removal and preconcentration followed by quantitation using mass spectrometry. We introduce a robust assay with an average intra-assay coefficient of variation (CV) of 4% and an interassay CV of 6% obtained from seven studies, enabling us to understand the relationship between KRAS G12C target occupancy and the therapeutic PD effect from mouse tumor samples. Further, the data demonstrated that the drug candidate GDC-6036, a KRAS G12C covalent inhibitor, shows dose-dependent target inhibition (KRAS G12C alkylation) and MAPK pathway inhibition, which correlate with high antitumor potency in the MIA PaCa-2 pancreatic xenograft model.


Assuntos
Antineoplásicos , Proteínas Proto-Oncogênicas p21(ras) , Humanos , Animais , Camundongos , Proteínas Proto-Oncogênicas p21(ras)/genética , Linhagem Celular Tumoral , Mutação , Antineoplásicos/farmacologia , Modelos Animais de Doenças
5.
J Biol Chem ; 299(1): 102729, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36410439

RESUMO

Fibroblast growth factor receptor 1 (FGFR1) is a receptor tyrosine kinase that plays a major role in developmental processes and metabolism. The dysregulation of FGFR1 through genetic aberrations leads to skeletal and metabolic diseases as well as cancer. For this reason, FGFR1 is a promising therapeutic target, yet a very challenging one due to potential on-target toxicity. More puzzling is that both agonistic and antagonistic FGFR1 antibodies are reported to exhibit similar toxicity profiles in vivo, namely weight loss. In this study, we aimed to assess and compare the mechanism of action of these molecules to better understand this apparent contradiction. By systematically comparing the binding of these antibodies and the activation or the inhibition of the major FGFR1 signaling events, we demonstrated that the molecules displayed similar properties and can behave either as an agonist or antagonist depending on the presence or the absence of the endogenous ligand. We further demonstrated that these findings translated in xenografts mice models. In addition, using time-resolved FRET and mass spectrometry analysis, we showed a functionally distinct FGFR1 active conformation in the presence of an antibody that preferentially activates the FGFR substrate 2 (FRS2)-dependent signaling pathway, demonstrating that modulating the geometry of a FGFR1 dimer can effectively change the signaling outputs and ultimately the activity of the molecule in preclinical studies. Altogether, our results highlighted how bivalent antibodies can exhibit both agonistic and antagonistic activities and have implications for targeting other receptor tyrosine kinases with antibodies.


Assuntos
Anticorpos Monoclonais , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Transdução de Sinais , Animais , Humanos , Camundongos , Neoplasias , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/agonistas , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Anticorpos Monoclonais/farmacologia
6.
J Am Chem Soc ; 143(28): 10571-10575, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34236858

RESUMO

We hypothesized that the proximity-driven ubiquitylation of E3-interacting small molecules could affect the degradation of E3 ubiquitin ligases. A series of XIAP BIR2 domain-binding small molecules was modified to append a nucleophilic primary amine. This modification transforms XIAP binders into inducers of XIAP degradation. The degradation of XIAP is E1- and proteasome-dependent, dependent on the ligase function of XIAP, and is rescued by subtle modifications of the small molecule that would obviate ubiquitylation. We demonstrate in vitro ubiquitylation of the small molecule that is dependent on its interaction with XIAP. Taken together, these results demonstrate the designed ubiquitylation of an engineered small molecule and a novel approach for the degradation of E3 ubiquitin ligases.


Assuntos
Aminas/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Aminas/química , Cristalografia por Raios X , Humanos , Modelos Moleculares , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/química , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo
7.
Cell ; 184(17): 4480-4494.e15, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34320407

RESUMO

In neutrophils, nicotinamide adenine dinucleotide phosphate (NADPH) generated via the pentose phosphate pathway fuels NADPH oxidase NOX2 to produce reactive oxygen species for killing invading pathogens. However, excessive NOX2 activity can exacerbate inflammation, as in acute respiratory distress syndrome (ARDS). Here, we use two unbiased chemical proteomic strategies to show that small-molecule LDC7559, or a more potent designed analog NA-11, inhibits the NOX2-dependent oxidative burst in neutrophils by activating the glycolytic enzyme phosphofructokinase-1 liver type (PFKL) and dampening flux through the pentose phosphate pathway. Accordingly, neutrophils treated with NA-11 had reduced NOX2-dependent outputs, including neutrophil cell death (NETosis) and tissue damage. A high-resolution structure of PFKL confirmed binding of NA-11 to the AMP/ADP allosteric activation site and explained why NA-11 failed to agonize phosphofructokinase-1 platelet type (PFKP) or muscle type (PFKM). Thus, NA-11 represents a tool for selective activation of PFKL, the main phosphofructokinase-1 isoform expressed in immune cells.


Assuntos
Fagocitose , Fosfofrutoquinase-1 Hepática/metabolismo , Explosão Respiratória , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Regulação Alostérica/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Glicólise/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cinética , Viabilidade Microbiana/efeitos dos fármacos , Modelos Moleculares , NADPH Oxidases/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Fagocitose/efeitos dos fármacos , Proteínas de Ligação a Fosfato/metabolismo , Fosfofrutoquinase-1 Hepática/antagonistas & inibidores , Fosfofrutoquinase-1 Hepática/ultraestrutura , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Recombinantes/isolamento & purificação , Explosão Respiratória/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia
8.
Nat Commun ; 11(1): 465, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31974362

RESUMO

The ability to quantitatively measure a small molecule's interactions with its protein target(s) is crucial for both mechanistic studies of signaling pathways and in drug discovery. However, current methods to achieve this have specific requirements that can limit their application or interpretation. Here we describe a complementary target-engagement method, HIPStA (Heat Shock Protein Inhibition Protein Stability Assay), a high-throughput method to assess small molecule binding to endogenous, unmodified target protein(s) in cells. The methodology relies on the change in protein turnover when chaperones, such as HSP90, are inhibited and the stabilization effect that drug-target binding has on this change. We use HIPStA to measure drug binding to three different classes of drug targets (receptor tyrosine kinases, nuclear hormone receptors, and cytoplasmic protein kinases), via quantitative fluorescence imaging. We further demonstrate its utility by pairing the method with quantitative mass spectrometry to identify previously unknown targets of a receptor tyrosine kinase inhibitor.


Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Chaperonas Moleculares/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Benzoquinonas/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/metabolismo , Imunofluorescência , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Humanos , Hidroxibutiratos/metabolismo , Hidroxibutiratos/farmacologia , Lactamas Macrocíclicas/farmacologia , Espectrometria de Massas , Chaperonas Moleculares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Proteoma/análise , Proteínas Proto-Oncogênicas c-raf/metabolismo , Receptor ErbB-2/metabolismo
9.
Nature ; 574(7777): 249-253, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31578523

RESUMO

The integrity of the mammalian epidermis depends on a balance of proliferation and differentiation in the resident population of stem cells1. The kinase RIPK4 and the transcription factor IRF6 are mutated in severe developmental syndromes in humans, and mice lacking these genes display epidermal hyperproliferation and soft-tissue fusions that result in neonatal lethality2-5. Our understanding of how these genes control epidermal differentiation is incomplete. Here we show that the role of RIPK4 in mouse development requires its kinase activity; that RIPK4 and IRF6 expressed in the epidermis regulate the same biological processes; and that the phosphorylation of IRF6 at Ser413 and Ser424 primes IRF6 for activation. Using RNA sequencing (RNA-seq), histone chromatin immunoprecipitation followed by sequencing (ChIP-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) of skin in wild-type and IRF6-deficient mouse embryos, we define the transcriptional programs that are regulated by IRF6 during epidermal differentiation. IRF6 was enriched at bivalent promoters, and IRF6 deficiency caused defective expression of genes that are involved in the metabolism of lipids and the formation of tight junctions. Accordingly, the lipid composition of the stratum corneum of Irf6-/- skin was abnormal, culminating in a severe defect in the function of the epidermal barrier. Collectively, our results explain how RIPK4 and IRF6 function to ensure the integrity of the epidermis and provide mechanistic insights into why developmental syndromes that are characterized by orofacial, skin and genital abnormalities result when this axis goes awry.


Assuntos
Diferenciação Celular , Células Epidérmicas/citologia , Epiderme/fisiologia , Fatores Reguladores de Interferon/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Anormalidades Múltiplas/genética , Animais , Fenda Labial/genética , Fissura Palatina/genética , Cistos/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Células Epidérmicas/metabolismo , Epiderme/embriologia , Anormalidades do Olho/genética , Feminino , Dedos/anormalidades , Regulação da Expressão Gênica , Fatores Reguladores de Interferon/deficiência , Fatores Reguladores de Interferon/genética , Joelho/anormalidades , Articulação do Joelho/anormalidades , Lábio/anormalidades , Metabolismo dos Lipídeos/genética , Deformidades Congênitas das Extremidades Inferiores/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Fosfosserina/metabolismo , Proteínas Serina-Treonina Quinases/genética , Sindactilia/genética , Anormalidades Urogenitais/genética
10.
J Proteome Res ; 18(3): 1264-1277, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30525646

RESUMO

Lupus nephritis (LN) is a severe clinical manifestation of systemic lupus erythematosus (SLE) associated with significant morbidity and mortality. Assessment of severity and activity of renal involvement in SLE requires a kidney biopsy, an invasive procedure with limited prognostic value. Noninvasive biomarkers are needed to inform treatment decisions and to monitor disease activity. Proteinuria is associated with disease progression in LN; however, the composition of the LN urinary proteome remains incompletely characterized. To address this, we profiled LN urine samples using complementary mass spectrometry-based methods:  protein gel fractionation, chemical labeling using tandem mass tags, and data-independent acquisition. Combining results from these approaches yielded quantitative information on 2573 unique proteins in urine from LN patients. A multiple-reaction monitoring (MRM) method was established to confirm eight proteins in an independent cohort of LN patients, and seven proteins (transferrin, α-2-macroglobulin, haptoglobin, afamin, α-1-antitrypsin, vimentin, and ceruloplasmin) were confirmed to be elevated in LN urine compared to healthy controls. In this study, we demonstrate that deep mass spectrometry profiling of a small number of patient samples can identify high-quality biomarkers that replicate in an independent LN disease cohort. These biomarkers are being used to inform clinical biomarker strategies to support longitudinal and interventional studies focused on evaluating disease progression and treatment efficacy of novel LN therapeutics.


Assuntos
Biomarcadores/urina , Lúpus Eritematoso Sistêmico/urina , Nefrite Lúpica/urina , Proteoma/genética , Adolescente , Adulto , Idoso , Biópsia , Proteínas de Transporte/urina , Ceruloplasmina/urina , Feminino , Glicoproteínas/urina , Haptoglobinas/urina , Humanos , Rim/metabolismo , Rim/patologia , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/patologia , Nefrite Lúpica/genética , Nefrite Lúpica/patologia , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Prognóstico , Albumina Sérica Humana/urina , Transferrina/urina , Vimentina/urina , Adulto Jovem , alfa 1-Antitripsina/urina , alfa-Macroglobulinas/urina
11.
Nat Commun ; 9(1): 1162, 2018 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-29563501

RESUMO

Activity-based probes (ABPs) are widely used to monitor the activity of enzyme families in biological systems. Inferring enzyme activity from probe reactivity requires that the probe reacts with the enzyme at its active site; however, probe-labeling sites are rarely verified. Here we present an enhanced chemoproteomic approach to evaluate the activity and probe reactivity of deubiquitinase enzymes, using bioorthogonally tagged ABPs and a sequential on-bead digestion protocol to enhance the identification of probe-labeling sites. We confirm probe labeling of deubiquitinase catalytic Cys residues and reveal unexpected labeling of deubiquitinases on non-catalytic Cys residues and of non-deubiquitinase proteins. In doing so, we identify ZUFSP (ZUP1) as a previously unannotated deubiquitinase with high selectivity toward cleaving K63-linked chains. ZUFSP interacts with and modulates ubiquitination of the replication protein A (RPA) complex. Our reactive-site-centric chemoproteomics method is broadly applicable for identifying the reaction sites of covalent molecules, which may expand our understanding of enzymatic mechanisms.


Assuntos
Enzimas Desubiquitinantes/química , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Proteína de Replicação A/metabolismo , Coloração e Rotulagem/métodos , Biocatálise , Domínio Catalítico , Cisteína/química , Cisteína/metabolismo , Enzimas Desubiquitinantes/classificação , Enzimas Desubiquitinantes/genética , Enzimas Desubiquitinantes/metabolismo , Células HEK293 , Células HeLa , Humanos , Lisina/química , Lisina/metabolismo , Sondas Moleculares , Proteína de Replicação A/genética , Sumoilação , Ubiquitinação
12.
Mol Cell ; 69(4): 551-565.e7, 2018 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-29452636

RESUMO

Inflammatory responses mediated by NOD2 rely on RIP2 kinase and ubiquitin ligase XIAP for the activation of nuclear factor κB (NF-κB), mitogen-activated protein kinases (MAPKs), and cytokine production. Herein, we demonstrate that selective XIAP antagonism blocks NOD2-mediated inflammatory signaling and cytokine production by interfering with XIAP-RIP2 binding, which removes XIAP from its ubiquitination substrate RIP2. We also establish that the kinase activity of RIP2 is dispensable for NOD2 signaling. Rather, the conformation of the RIP2 kinase domain functions to regulate binding to the XIAP-BIR2 domain. Effective RIP2 kinase inhibitors block NOD2 signaling by disrupting RIP2-XIAP interaction. Finally, we identify NOD2 signaling and XIAP-dependent ubiquitination sites on RIP2 and show that mutating these lysine residues adversely affects NOD2 pathway signaling. Overall, these results reveal a critical role for the XIAP-RIP2 interaction in NOD2 inflammatory signaling and provide a molecular basis for the design of innovative therapeutic strategies based on XIAP antagonists and RIP2 kinase inhibitors.


Assuntos
Aminoquinolinas/farmacologia , Inflamação/prevenção & controle , Proteína Adaptadora de Sinalização NOD2/antagonistas & inibidores , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Sulfonas/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Animais , Células Cultivadas , Humanos , Inflamação/metabolismo , Inflamação/patologia , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Fosforilação , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/antagonistas & inibidores , Transdução de Sinais , Ubiquitina/metabolismo , Ubiquitinação , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores
13.
Nature ; 550(7677): 534-538, 2017 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-29045385

RESUMO

The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.


Assuntos
Aminopiridinas/química , Aminopiridinas/farmacologia , Indazóis/química , Indazóis/farmacologia , Fenóis/química , Fenóis/farmacologia , Piridinas/química , Piridinas/farmacologia , Peptidase 7 Específica de Ubiquitina/antagonistas & inibidores , Ubiquitina/metabolismo , Animais , Ligação Competitiva , Linhagem Celular Tumoral , Sinergismo Farmacológico , Feminino , Humanos , Camundongos , Camundongos SCID , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/patologia , Ligação Proteica , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Especificidade por Substrato , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina/química , Peptidase 7 Específica de Ubiquitina/química , Peptidase 7 Específica de Ubiquitina/deficiência , Peptidase 7 Específica de Ubiquitina/metabolismo
14.
Cell Death Differ ; 24(1): 26-37, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27518435

RESUMO

Proper regulation of cell death signaling is crucial for the maintenance of homeostasis and prevention of disease. A caspase-independent regulated form of cell death called necroptosis is rapidly emerging as an important mediator of a number of human pathologies including inflammatory bowel disease and ischemia-reperfusion organ injury. Activation of necroptotic signaling through TNF signaling or organ injury leads to the activation of kinases receptor-interacting protein kinases 1 and 3 (RIP1 and RIP3) and culminates in inflammatory cell death. We found that, in addition to phosphorylation, necroptotic cell death is regulated by ubiquitination of RIP1 in the necrosome. Necroptotic RIP1 ubiquitination requires RIP1 kinase activity, but not necroptotic mediators RIP3 and MLKL (mixed lineage kinase-like). Using immunoaffinity enrichment and mass spectrometry, we profiled numerous ubiquitination events on RIP1 that are triggered during necroptotic signaling. Mutation of a necroptosis-related ubiquitination site on RIP1 reduced necroptotic cell death and RIP1 ubiquitination and phosphorylation, and disrupted the assembly of RIP1 and RIP3 in the necrosome, suggesting that necroptotic RIP1 ubiquitination is important for maintaining RIP1 kinase activity in the necrosome complex. We also observed RIP1 ubiquitination in injured kidneys consistent with a physiological role of RIP1 ubiquitination in ischemia-reperfusion disease. Taken together, these data reveal that coordinated and interdependent RIP1 phosphorylation and ubiquitination within the necroptotic complex regulate necroptotic signaling and cell death.


Assuntos
Apoptose , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Apoptose/efeitos dos fármacos , Sistemas CRISPR-Cas/genética , Linhagem Celular , Creatinina/sangue , Células HEK293 , Células HT29 , Humanos , Nefropatias/etiologia , Nefropatias/metabolismo , Camundongos , Complexo de Proteínas Formadoras de Poros Nucleares/deficiência , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Oligopeptídeos/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/patologia , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Ubiquitinação/efeitos dos fármacos
15.
Mol Cell Proteomics ; 15(7): 2293-307, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27125827

RESUMO

Many diseases are associated with endoplasmic reticulum (ER) stress, which results from an accumulation of misfolded proteins. This triggers an adaptive response called the "unfolded protein response" (UPR), and prolonged exposure to ER stress leads to cell death. Caspases are reported to play a critical role in ER stress-induced cell death but the underlying mechanisms by which they exert their effect continue to remain elusive. To understand the role caspases play during ER stress, a systems level approach integrating analysis of the transcriptome, proteome, and proteolytic substrate profile was employed. This quantitative analysis revealed transcriptional profiles for most human genes, provided information on protein abundance for 4476 proteins, and identified 445 caspase substrates. Based on these data sets many caspase substrates were shown to be downregulated at the protein level during ER stress suggesting caspase activity inhibits their cellular function. Additionally, RNA sequencing revealed a role for caspases in regulation of ER stress-induced transcriptional pathways and gene set enrichment analysis showed expression of multiple gene targets of essential transcription factors to be upregulated during ER stress upon inhibition of caspases. Furthermore, these transcription factors were degraded in a caspase-dependent manner during ER stress. These results indicate that caspases play a dual role in regulating the cellular response to ER stress through both post-translational and transcriptional regulatory mechanisms. Moreover, this study provides unique insight into progression of the unfolded protein response into cell death, which may help identify therapeutic strategies to treat ER stress-related diseases.


Assuntos
Caspases/metabolismo , Estresse do Retículo Endoplasmático , Perfilação da Expressão Gênica/métodos , Proteômica/métodos , Resposta a Proteínas não Dobradas , Apoptose , Morte Celular , Regulação para Baixo , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica , Células HCT116 , Células HeLa , Humanos , Proteólise , Análise de Sequência de RNA , Fatores de Transcrição/genética
17.
Mol Cell Proteomics ; 15(4): 1220-31, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26711467

RESUMO

The KEAP1/Nrf2 pathway senses and responds to changes in intracellular oxidative stress. Mutations that result in constitutive activation of Nrf2 are present in several human tumors, especially non-small cell lung cancer. Therefore, compounds that inhibit Nrf2 activity might be beneficial in treating patients whose tumors show activation of this pathway. Recent reports suggest that the natural product brusatol can potently and selectively inhibit Nrf2 activity, resulting in cell cytotoxicity, and can be effectively combined with chemotherapeutic agents. Here, we analyzed the effects of brusatol on the cellular proteome in the KEAP1 mutant non-small cell lung cancer cell line A549. Brusatol was found to rapidly and potently decrease the expression of the majority of detected proteins, including Nrf2. The most dramatically decreased proteins are those that display a short half-life, like Nrf2. This effect was confirmed by restricting the analysis to newly synthesized proteins using a labeled methionine analogue. Moreover, brusatol increased the expression of multiple components of the ribosome, suggesting that it regulates the function of this macromolecular complex. Finally, we show that brusatol induces its potent cellular cytotoxicity effects on multiple cancer cell lines in a manner independent of KEAP1/Nrf2 activity and with a profile similar to the protein translation inhibitor silvestrol. In conclusion, our data show that the activity of brusatol is not restricted to Nrf2 but, rather, functions as a global protein synthesis inhibitor.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Espectrometria de Massas/métodos , Fator 2 Relacionado a NF-E2/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Quassinas/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Neoplasias Pulmonares/tratamento farmacológico , Mutação , Proteômica/métodos
18.
Nature ; 528(7582): 370-5, 2015 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-26649818

RESUMO

Inactivation of the TNFAIP3 gene, encoding the A20 protein, is associated with critical inflammatory diseases including multiple sclerosis, rheumatoid arthritis and Crohn's disease. However, the role of A20 in attenuating inflammatory signalling is unclear owing to paradoxical in vitro and in vivo findings. Here we utilize genetically engineered mice bearing mutations in the A20 ovarian tumour (OTU)-type deubiquitinase domain or in the zinc finger-4 (ZnF4) ubiquitin-binding motif to investigate these discrepancies. We find that phosphorylation of A20 promotes cleavage of Lys63-linked polyubiquitin chains by the OTU domain and enhances ZnF4-mediated substrate ubiquitination. Additionally, levels of linear ubiquitination dictate whether A20-deficient cells die in response to tumour necrosis factor. Mechanistically, linear ubiquitin chains preserve the architecture of the TNFR1 signalling complex by blocking A20-mediated disassembly of Lys63-linked polyubiquitin scaffolds. Collectively, our studies reveal molecular mechanisms whereby A20 deubiquitinase activity and ubiquitin binding, linear ubiquitination, and cellular kinases cooperate to regulate inflammation and cell death.


Assuntos
Cisteína Endopeptidases/metabolismo , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Animais , Morte Celular , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Feminino , Inflamação/genética , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lisina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Fosforilação , Poliubiquitina/química , Poliubiquitina/metabolismo , Ligação Proteica , Proteínas Quinases/metabolismo , Transdução de Sinais , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitinação
19.
PLoS One ; 10(6): e0130635, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26086088

RESUMO

Perturbation of endoplasmic reticulum (ER) homeostasis triggers the ER stress response (also known as Unfolded Protein Response), a hallmark of many pathological disorders. However the connection between ER stress and inflammation remains largely unexplored. Recent data suggest that ER stress controls the activity of inflammasomes, key signaling platforms that mediate innate immune responses. Here we report that expression of NLRP1, a core inflammasome component, is specifically up-regulated during severe ER stress conditions in human cell lines. Both IRE1α and PERK, but not the ATF6 pathway, modulate NLRP1 gene expression. Furthermore, using mutagenesis, chromatin immunoprecipitation and CRISPR-Cas9-mediated genome editing technology, we demonstrate that ATF4 transcription factor directly binds to NLRP1 promoter during ER stress. Although involved in different types of inflammatory responses, XBP-1 splicing was not required for NLRP1 induction. This study provides further evidence that links ER stress with innate.


Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Estresse do Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Inflamassomos/metabolismo , Fator 4 Ativador da Transcrição/química , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/genética , Sistemas CRISPR-Cas/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endorribonucleases/metabolismo , Células HCT116 , Células HeLa , Humanos , Células Jurkat , Células K562 , Mutagênese , Proteínas NLR , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Splicing de RNA , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição de Fator Regulador X , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para Cima , Proteína 1 de Ligação a X-Box , eIF-2 Quinase/metabolismo
20.
J Mol Biol ; 427(11): 2121-34, 2015 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-25861760

RESUMO

Ubiquitination is one of the most prevalent posttranslational modifications in eukaryotic cells, with functional importance in protein degradation, subcellular localization and signal transduction pathways. Immunoaffinity enrichment coupled with quantitative mass spectrometry enables the in-depth characterization of protein ubiquitination events at the site-specific level. We have applied this strategy to investigate cellular response triggered by two distinct type agents: small molecule inhibitors of the tumor-associated kinases MEK and PI3K or the pro-inflammatory cytokine IL-17. Temporal profiling of protein ubiquitination events across a series of time points covering the biological response permits interrogation of signaling through thousands of quantified proteins, of which only a subset display significant and physiologically meaningful regulation. Distinctive clusters of residues within proteins can display distinct temporal patterns attributable to diverse molecular functions, although the majority of differential ubiquitination appears as a coordinated response across the modifiable residues present within an individual substrate. In cells treated with a combination of MEK and PI3K inhibitors, we found differential ubiquitination of MEK within the first hour after treatment and a series of mitochondria proteins at later time points. In the IL-17 signaling pathway, ubiquitination events on several signaling proteins including HOIL-1 and Tollip were observed. The functional relevance of these putative IL-17 mediators was subsequently validated by knockdown of HOIL-1, HOIP and TOLIP, each of which decreased IL-17-stimulated cytokine production. Together, these data validate proteomic profiling of protein ubiquitination as a viable approach for identifying dynamic signaling components in response to intracellular and extracellular perturbations.


Assuntos
Proteínas Mitocondriais/metabolismo , Proteínas/imunologia , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Ubiquitina/metabolismo , Apoptose/efeitos dos fármacos , Azetidinas/farmacologia , Linhagem Celular Tumoral/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Indazóis/farmacologia , Interleucina-17/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Piperidinas/farmacologia , Proteínas/metabolismo , Transdução de Sinais , Sulfonamidas/farmacologia , Fatores de Transcrição , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
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