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1.
Nat Chem Biol ; 16(5): 577-586, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32094923

RESUMO

Receptor tyrosine kinases (RTKs) are transmembrane receptors of great clinical interest due to their role in disease. Historically, therapeutics targeting RTKs have been identified using in vitro kinase assays. Due to frequent development of drug resistance, however, there is a need to identify more diverse compounds that inhibit mutated but not wild-type RTKs. Here, we describe MaMTH-DS (mammalian membrane two-hybrid drug screening), a live-cell platform for high-throughput identification of small molecules targeting functional protein-protein interactions of RTKs. We applied MaMTH-DS to an oncogenic epidermal growth factor receptor (EGFR) mutant resistant to the latest generation of clinically approved tyrosine kinase inhibitors (TKIs). We identified four mutant-specific compounds, including two that would not have been detected by conventional in vitro kinase assays. One of these targets mutant EGFR via a new mechanism of action, distinct from classical TKI inhibition. Our results demonstrate how MaMTH-DS is a powerful complement to traditional drug screening approaches.

2.
J Proteome Res ; 19(3): 1029-1036, 2020 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-32009416

RESUMO

The sequence database searching method is widely used in proteomics for peptide identification. To control the false discovery rate (FDR) of the searching results, the target-decoy method generates and searches a decoy database together with the target database. A known problem is that the target protein sequence database may contain numerous repeated peptides. The structures of these repeats are not preserved by most existing decoy generation algorithms. Previous studies suggest that such discrepancy between the target and decoy databases may lead to an inaccurate FDR estimation. Based on the de Bruijn graph model, we propose a new repeat-preserving algorithm to generate decoy databases. We prove that this algorithm preserves the structures of the repeats in the target database to a great extent. The de Bruijn method has been compared with a few other commonly used methods and demonstrated superior FDR estimation accuracy and an improved number of peptide identification.

3.
J Biol Chem ; 295(7): 2084-2096, 2020 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-31822558

RESUMO

The Maf proteins, including c-Maf, MafA, and MafB, are critical transcription factors in myelomagenesis. Previous studies demonstrated that Maf proteins are processed by the ubiquitin-proteasome pathway, but the mechanisms remain elusive. This study applied MS to identify MafB ubiquitination-associated proteins and found that the ubiquitin-specific protease USP7 was present in the MafB interactome. Moreover, USP7 also interacted with c-Maf and MafA and blocked their polyubiquitination and degradation. Consistently, knockdown of USP7 resulted in Maf protein degradation along with increased polyubiquitination levels. The action of USP7 thus promoted Maf transcriptional activity as evidenced by luciferase assays and by the up-regulation of the expression of Maf-modulated genes. Furthermore, USP7 was up-regulated in myeloma cells, and it was negatively associated with the survival of myeloma patients. USP7 promoted myeloma cell survival, and when it was inhibited by its specific inhibitor P5091, myeloma cell lines underwent apoptosis. These results therefore demonstrated that USP7 is a deubiquitinase of Maf proteins and promotes MM cell survival in association with Maf stability. Given the significance of USP7 and Maf proteins in myeloma genesis, targeting the USP7/Maf axle is a potential strategy to the precision therapy of MM.

4.
Cancer Res ; 79(16): 4057-4071, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31292163

RESUMO

Glioblastoma is the most common primary brain tumor in adults. While the introduction of temozolomide chemotherapy has increased long-term survivorship, treatment failure and rapid tumor recurrence remains universal. The transcriptional regulatory protein, inhibitor of DNA-binding-1 (ID1), is a key regulator of cell phenotype in cancer. We show that CRISPR-mediated knockout of ID1 in glioblastoma cells, breast adenocarcinoma cells, and melanoma cells dramatically reduced tumor progression in all three cancer systems through transcriptional downregulation of EGF, which resulted in decreased EGFR phosphorylation. Moreover, ID1-positive cells were enriched by chemotherapy and drove tumor recurrence in glioblastoma. Addition of the neuroleptic drug pimozide to inhibit ID1 expression enhanced the cytotoxic effects of temozolomide therapy on glioma cells and significantly prolonged time to tumor recurrence. Conclusively, these data suggest ID1 could be a promising therapeutic target in patients with glioblastoma. SIGNIFICANCE: These findings show that the transcriptional regulator ID1 is critical for glioblastoma initiation and chemoresistance and that inhibition of ID1 enhances the effect of temozolomide, delays tumor recurrence, and prolongs survival.

5.
Cancers (Basel) ; 11(7)2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31330989

RESUMO

The burden of somatic mutations and neoantigens has been associated with improved survival in cancer treated with immunotherapies, especially non-small cell lung cancer (NSCLC). However, there is uncertainty about their effect on outcome in early-stage untreated cases. We posited that the burden of mutations in a specific set of genes may also contribute to the prognosis of early NSCLC patients. From a small cohort of 36 NSCLC cases, we were able to identify somatic mutations and copy number alterations in 865 genes that contributed to patient overall survival. Simply, the number of altered genes (NAG) among these 865 genes was associated with longer disease-free survival (hazard ratio (HR) = 0.153, p = 1.48 × 10-4). The gene expression signature distinguishing patients with high/low NAG was also prognostic in three independent datasets. Patients with a high NAG could be further stratified based on the presence of immunogenic mutations, revealing a further subgroup of stage I NSCLC with even better prognosis (85% with >5 years survival), and associated with cytotoxic T-cell expression. Importantly, 95% of the highly-altered genes lacked direct relation to cancer, but were implicated in pathways regulating cell proliferation, motility and immune response.

6.
Mol Cell Proteomics ; 18(10): 2099-2107, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31249099

RESUMO

Deep learning models for prediction of three key LC-MS/MS properties from peptide sequences were developed. The LC-MS/MS properties or behaviors are indexed retention times (iRT), MS1 or survey scan charge state distributions, and sequence ion intensities of HCD spectra. A common core deep supervised learning architecture, bidirectional long-short term memory (LSTM) recurrent neural networks was used to construct the three prediction models. Two featurization schemes were proposed and demonstrated to allow for efficient encoding of modifications. The iRT and charge state distribution models were trained with on order of 105 data points each. An HCD sequence ion prediction model was trained with 2 × 106 experimental spectra. The iRT prediction model and HCD sequence ion prediction model provide improved accuracies over the start-of-the-art models available in literature. The MS1 charge state distribution prediction model offers excellent performance. The prediction models can be used to enhance peptide identification and quantification in data-dependent acquisition and data-independent acquisition (DIA) experiments as well as to assist MRM (multiple reaction monitoring) and PRM (parallel reaction monitoring) experiment design.

7.
J Proteome Res ; 18(5): 2346-2353, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-30938160

RESUMO

The balance between comprehensively analyzing the proteome and using valuable mass spectrometry time is a genuine challenge in the field of proteomics. Multidimensional fractionation strategies have significantly increased proteome coverage, but often at the cost of increased mass analysis time, despite advances in mass spectrometer acquisition rates. Recently, the Evosep One liquid chromatography system was shown to analyze peptide samples in a high-throughput manner without sacrificing in-depth proteomics coverage. We demonstrate the incorporation of Evosep One technology into our multiplexing workflow for analysis of tandem mass tag (TMT)-labeled nonsmall cell lung carcinoma (NSCLC) patient-derived xenografts (PDXs). By the use of a 30 samples per day Evosep workflow, >12 000 proteins were identified in 48 h of mass spectrometry time, which is comparable to the number of proteins identified by our conventional concatenated EASY-nLC workflow in 60 h. Shorter Evosep gradient lengths reduced the number of protein identifications by 10% while decreasing the mass analysis time by 50%. This Evosep workflow will enable quantitative analysis of multiplexed samples in less time without conceding depth of proteome coverage.

8.
Structure ; 27(4): 590-605.e5, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30713027

RESUMO

The multi-domain deubiquitinase USP15 regulates diverse eukaryotic processes and has been implicated in numerous diseases. We developed ubiquitin variants (UbVs) that targeted either the catalytic domain or each of three adaptor domains in USP15, including the N-terminal DUSP domain. We also designed a linear dimer (diUbV), which targeted the DUSP and catalytic domains, and exhibited enhanced specificity and more potent inhibition of catalytic activity than either UbV alone. In cells, the UbVs inhibited the deubiquitination of two USP15 substrates, SMURF2 and TRIM25, and the diUbV inhibited the effects of USP15 on the transforming growth factor ß pathway. Structural analyses revealed that three distinct UbVs bound to the catalytic domain and locked the active site in a closed, inactive conformation, and one UbV formed an unusual strand-swapped dimer and bound two DUSP domains simultaneously. These inhibitors will enable the study of USP15 function in oncology, neurology, immunology, and inflammation.


Assuntos
Fatores de Transcrição/química , Fator de Crescimento Transformador beta1/química , Proteínas com Motivo Tripartido/química , Ubiquitina-Proteína Ligases/química , Proteases Específicas de Ubiquitina/química , Ubiquitina/química , Sequência de Aminoácidos , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteases Específicas de Ubiquitina/antagonistas & inibidores , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismo , Ubiquitinação
9.
Protein Sci ; 28(2): 403-413, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30431205

RESUMO

Protein phosphorylation is the most abundant post-translational modification in cells. Src homology 2 (SH2) domains specifically recognize phosphorylated tyrosine (pTyr) residues to mediate signaling cascades. A conserved pocket in the SH2 domain binds the pTyr side chain and the EF and BG loops determine binding specificity. By using large phage-displayed libraries, we engineered the EF and BG loops of the Fyn SH2 domain to alter specificity. Engineered SH2 variants exhibited distinct specificity profiles and were able to bind pTyr sites on the epidermal growth factor receptor, which were not recognized by the wild-type Fyn SH2 domain. Furthermore, mass spectrometry showed that SH2 variants with additional mutations in the pTyr-binding pocket that enhanced affinity were highly effective for enrichment of diverse pTyr peptides within the human proteome. These results showed that engineering of the EF and BG loops could be used to tailor SH2 domain specificity, and SH2 variants with diverse specificities and high affinities for pTyr residues enabled more comprehensive analysis of the human phosphoproteome. STATEMENT: Src Homology 2 (SH2) domains are modular domains that recognize phosphorylated tyrosine embedded in proteins, transducing these post-translational modifications into cellular responses. Here we used phage display to engineer hundreds of SH2 domain variants with altered binding specificities and enhanced affinities, which enabled efficient and differential enrichment of the human phosphoproteome for analysis by mass spectrometry. These engineered SH2 domain variants will be useful tools for elucidating the molecular determinants governing SH2 domains binding specificity and for enhancing analysis and understanding of the human phosphoproteome.


Assuntos
Fosfoproteínas/análise , Engenharia de Proteínas , Proteoma/análise , Domínios de Homologia de src , Células HeLa , Humanos , Espectrometria de Massas , Fosfotirosina/análise , Fosfotirosina/química , Estrutura Secundária de Proteína
10.
Sci Adv ; 4(11): eaar7653, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30417091

RESUMO

Memory B cells and plasma cells are antigen-experienced cells tasked with the maintenance of humoral protection. Despite these prominent functions, definitive cell surface markers have not been identified for these cells. We report here the isolation and characterization of the monoclonal variable lymphocyte receptor B (VLRB) N8 antibody from the evolutionarily distant sea lamprey that specifically recognizes memory B cells and plasma cells in humans. Unexpectedly, we determined that VLRB N8 recognizes the human leukocyte antigen-I (HLA-I) antigen in a tyrosine sulfation-dependent manner. Furthermore, we observed increased binding of VLRB N8 to memory B cells in individuals with autoimmune disorders multiple sclerosis and systemic lupus erythematosus. Our study indicates that lamprey VLR antibodies uniquely recognize a memory B cell- and plasma cell-specific posttranslational modification of HLA-I, the expression of which is up-regulated during B cell activation.


Assuntos
Anticorpos Monoclonais/imunologia , Linfócitos B/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Memória Imunológica/imunologia , Plasmócitos/imunologia , Receptores de Antígenos/imunologia , Tirosina/análogos & derivados , Animais , Anticorpos Monoclonais/sangue , Linfócitos B/metabolismo , Células Cultivadas , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Região Variável de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/metabolismo , Lampreias/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/metabolismo , Esclerose Múltipla/imunologia , Esclerose Múltipla/metabolismo , Plasmócitos/metabolismo , Receptores de Antígenos/metabolismo , Tirosina/química
11.
Cancer Res ; 78(23): 6539-6548, 2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30297534

RESUMO

: Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is an inherited cancer syndrome associated with a highly aggressive form of type 2 papillary renal cell carcinoma (PRCC). Germline inactivating alterations in fumarate hydratase (FH) cause HLRCC and result in elevated levels of reactive oxygen species (ROS). Recent work indicates that FH-/- PRCC cells have increased activation of ABL1, which promotes tumor growth, but how ABL1 is activated remains unclear. Given that oxidation can regulate protein-tyrosine phosphatase (PTP) catalytic activity, inactivation of an ABL-directed PTP by ROS might account for ABL1 activation in this malignancy. Our group previously developed "q-oxPTPome," a method that globally monitors the oxidation of classical PTPs. In this study, we present a refined q-oxPTPome, increasing its sensitivity by >10×. Applying q-oxPTPome to FH-deficient cell models showed that multiple PTPs were either highly oxidized (including PTPN12) or overexpressed. Highly oxidized PTPs were those with relatively high sensitivity to exogenous H2O2. Most PTP oxidation in FH-deficient cells was reversible, although nearly 40% of PTPN13 was irreversibly oxidized to the sulfonic acid state. Using substrate-trapping mutants, we mapped PTPs to their putative substrates and found that only PTPN12 could target ABL1. Furthermore, knockdown experiments identified PTPN12 as the major ABL1 phosphatase, and overexpression of PTPN12 inhibited ABL1 phosphorylation and HLRCC cell growth. These results show that ROS-induced oxidation of PTPN12 accounts for ABL1 phosphorylation in HLRCC-associated PRCC, revealing a novel mechanism for inactivating a tumor suppressor gene product and establishing a direct link between pathologic PTP oxidation and neoplastic disease. SIGNIFICANCE: This work identifies a novel mechanism of activation of the oncogenic kinase ABL1 via ROS-induced, oxidation-mediated inactivation of cognate protein tyrosine phosphatases.


Assuntos
Leiomiomatose/etiologia , Leiomiomatose/metabolismo , Síndromes Neoplásicas Hereditárias/etiologia , Síndromes Neoplásicas Hereditárias/metabolismo , Oxirredução , Proteína Tirosina Fosfatase não Receptora Tipo 12/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/metabolismo , Neoplasias Uterinas/etiologia , Neoplasias Uterinas/metabolismo , Biomarcadores , Linhagem Celular Tumoral , Fumarato Hidratase/genética , Fumarato Hidratase/metabolismo , Mutação em Linhagem Germinativa , Humanos , Leiomiomatose/diagnóstico , Metaboloma , Metabolômica/métodos , Modelos Biológicos , Síndromes Neoplásicas Hereditárias/diagnóstico , Fosforilação , Ligação Proteica , Espécies Reativas de Oxigênio , Neoplasias Cutâneas/diagnóstico , Neoplasias Uterinas/diagnóstico
12.
PLoS Biol ; 16(9): e2006092, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30212448

RESUMO

N6-Methyladenosine (m6A) accounts for approximately 0.2% to 0.6% of all adenosine in mammalian mRNA, representing the most abundant internal mRNA modifications. m6A RNA immunoprecipitation followed by high-throughput sequencing (MeRIP-seq) is a powerful technique to map the m6A location transcriptome-wide. However, this method typically requires 300 µg of total RNA, which limits its application to patient tumors. In this study, we present a refined m6A MeRIP-seq protocol and analysis pipeline that can be applied to profile low-input RNA samples from patient tumors. We optimized the key parameters of m6A MeRIP-seq, including the starting amount of RNA, RNA fragmentation, antibody selection, MeRIP washing/elution conditions, methods for RNA library construction, and the bioinformatics analysis pipeline. With the optimized immunoprecipitation (IP) conditions and a postamplification rRNA depletion strategy, we were able to profile the m6A epitranscriptome using 500 ng of total RNA. We identified approximately 12,000 m6A peaks with a high signal-to-noise (S/N) ratio from 2 lung adenocarcinoma (ADC) patient tumors. Through integrative analysis of the transcriptome, m6A epitranscriptome, and proteome data in the same patient tumors, we identified dynamics at the m6A level that account for the discordance between mRNA and protein levels in these tumors. The refined m6A MeRIP-seq method is suitable for m6A epitranscriptome profiling in a limited amount of patient tumors, setting the ground for unraveling the dynamics of the m6A epitranscriptome and the underlying mechanisms in clinical settings.


Assuntos
Perfilação da Expressão Gênica , Imunoprecipitação/métodos , RNA/metabolismo , Células A549 , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Adenosina/análogos & derivados , Adenosina/metabolismo , Anticorpos/metabolismo , Sequência de Bases , Humanos , RNA/genética
13.
Nat Commun ; 9(1): 3510, 2018 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-30158528

RESUMO

In most solid tumors, the Hippo pathway is inactivated through poorly understood mechanisms that result in the activation of the transcriptional regulators, YAP and TAZ. Here, we identify NUAK2 as a YAP/TAZ activator that directly inhibits LATS-mediated phosphorylation of YAP/TAZ and show that NUAK2 induction by YAP/TAZ and AP-1 is required for robust YAP/TAZ signaling. Pharmacological inhibition or loss of NUAK2 reduces the growth of cultured cancer cells and mammary tumors in mice. Moreover, in human patient samples, we show that NUAK2 expression is elevated in aggressive, high-grade bladder cancer and strongly correlates with a YAP/TAZ gene signature. These findings identify a positive feed forward loop in the Hippo pathway that establishes a key role for NUAK2 in enforcing the tumor-promoting activities of YAP/TAZ. Our results thus introduce a new opportunity for cancer therapeutics by delineating NUAK2 as a potential target for re-engaging the Hippo pathway.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Carcinogênese/metabolismo , Fosfoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Carcinogênese/genética , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Feminino , Células HEK293 , Humanos , Immunoblotting , Imunoprecipitação , Microscopia de Fluorescência , Fosfoproteínas/genética , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
14.
Nat Commun ; 8(1): 499, 2017 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-28894103

RESUMO

The capacity to coordinate environmental sensing with initiation of cellular responses underpins microbial survival and is crucial for virulence and stress responses in microbial pathogens. Here we define circuitry that enables the fungal pathogen Candida albicans to couple cell cycle dynamics with responses to cell wall stress induced by echinocandins, a front-line class of antifungal drugs. We discover that the C. albicans transcription factor Cas5 is crucial for proper cell cycle dynamics and responses to echinocandins, which inhibit ß-1,3-glucan synthesis. Cas5 has distinct transcriptional targets under basal and stress conditions, is activated by the phosphatase Glc7, and can regulate the expression of target genes in concert with the transcriptional regulators Swi4 and Swi6. Thus, we illuminate a mechanism of transcriptional control that couples cell wall integrity with cell cycle regulation, and uncover circuitry governing antifungal drug resistance.Cas5 is a transcriptional regulator of responses to cell wall stress in the fungal pathogen Candida albicans. Here, Xie et al. show that Cas5 also modulates cell cycle dynamics and responses to antifungal drugs.


Assuntos
Candida albicans/genética , Pontos de Checagem do Ciclo Celular/genética , Farmacorresistência Fúngica/genética , Regulação Fúngica da Expressão Gênica/genética , Fatores de Transcrição/genética , Antifúngicos/farmacologia , Western Blotting , Candida albicans/efeitos dos fármacos , Candida albicans/metabolismo , Parede Celular/genética , Parede Celular/metabolismo , Equinocandinas/farmacologia , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Mutação , Fosforilação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/metabolismo , beta-Glucanas/metabolismo
15.
J Cell Biol ; 216(11): 3655-3675, 2017 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-28877995

RESUMO

Axon degeneration is an early event and pathological in neurodegenerative conditions and nerve injuries. To discover agents that suppress neuronal death and axonal degeneration, we performed drug screens on primary rodent neurons and identified the pan-kinase inhibitor foretinib, which potently rescued sympathetic, sensory, and motor wt and SOD1 mutant neurons from trophic factor withdrawal-induced degeneration. By using primary sympathetic neurons grown in mass cultures and Campenot chambers, we show that foretinib protected neurons by suppressing both known degenerative pathways and a new pathway involving unliganded TrkA and transcriptional regulation of the proapoptotic BH3 family members BimEL, Harakiri,and Puma, culminating in preservation of mitochondria in the degenerative setting. Foretinib delayed chemotherapy-induced and Wallerian axonal degeneration in culture by preventing axotomy-induced local energy deficit and preserving mitochondria, and peripheral Wallerian degeneration in vivo. These findings identify a new axon degeneration pathway and a potentially clinically useful therapeutic drug.


Assuntos
Anilidas/farmacologia , Lesões por Esmagamento/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Quinolinas/farmacologia , Receptor trkA/antagonistas & inibidores , Nervo Isquiático/efeitos dos fármacos , Neuropatia Ciática/tratamento farmacológico , Degeneração Walleriana , Fibras Adrenérgicas/efeitos dos fármacos , Fibras Adrenérgicas/enzimologia , Fibras Adrenérgicas/patologia , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Axônios/efeitos dos fármacos , Axônios/enzimologia , Axônios/patologia , Células Cultivadas , Lesões por Esmagamento/enzimologia , Lesões por Esmagamento/genética , Lesões por Esmagamento/patologia , Citoproteção , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Genótipo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/enzimologia , Neurônios Motores/patologia , Mutação , Neurônios/enzimologia , Neurônios/patologia , Fenótipo , Fosforilação , Ratos Sprague-Dawley , Receptor trkA/genética , Receptor trkA/metabolismo , Nervo Isquiático/enzimologia , Nervo Isquiático/lesões , Nervo Isquiático/patologia , Neuropatia Ciática/enzimologia , Neuropatia Ciática/genética , Neuropatia Ciática/patologia , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/enzimologia , Células Receptoras Sensoriais/patologia , Transdução de Sinais , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Fatores de Tempo , Transcrição Genética
16.
Cell Death Dis ; 8(9): e3058, 2017 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-28933784

RESUMO

The deubiquitinase USP5 stabilizes c-Maf, a key transcription factor in multiple myeloma (MM), but the mechanisms and significance are unclear. In the present study, USP5 was found to interact with c-Maf and prevented it from degradation by decreasing its polyubiquitination level. Specifically, the 308th and 347th lysine residues in c-Maf were critical for USP5-mediated deubiquitination and stability. There are five key domains in the USP5 protein and subsequent studies revealed that the cryptic ZnF domain and the C-box domain interacted with c-Maf but the UBA1/UBA2 domain partly increased its stability. Notably, MafA and MafB are also members of the c-Maf family, however, USP5 failed to deubiquitinate MafA, suggesting its substrate specificity. In the functional studies, USP5 was found to promoted the transcriptional activity of c-Maf. Consistent with the high level of c-Maf protein in MM cells, USP5 was also highly expressed. When USP5 was knocked down, c-Maf underwent degradation. Interestingly, USP5 silence led to apoptosis of MM cells expressing c-Maf but not MM cells lacking c-Maf, indicating c-Maf is a key factor in USP5-mediated MM cell proliferation and survival. Consistent with this finding, WP1130, an inhibitor of several Dubs including USP5, suppressed the transcriptional activity of c-Maf and induced MM cell apoptosis. When c-Maf was overexpressed, WP1130-induced MM cell apoptosis was abolished. Taken together, these findings suggest that USP5 regulates c-Maf stability and MM cell survival. Targeting the USP5/c-Maf axis could be a potential strategy for MM treatment.


Assuntos
Apoptose , Endopeptidases/metabolismo , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Proteólise , Proteínas Proto-Oncogênicas c-maf/metabolismo , Apoptose/genética , Endopeptidases/química , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Lisina/metabolismo , Terapia de Alvo Molecular , Mieloma Múltiplo/genética , Poliubiquitina/metabolismo , Ligação Proteica , Domínios Proteicos , Estabilidade Proteica , Relação Estrutura-Atividade , Transcrição Genética , Ubiquitinação
17.
J Hematol Oncol ; 10(1): 132, 2017 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-28673317

RESUMO

BACKGROUND: UBE2O is proposed as a ubiquitin-conjugating enzyme, but its function was largely unknown. METHODS: Mass spectrometry was applied to identify c-Maf ubiquitination-associated proteins. Immunoprecipitation was applied for c-Maf and UBE2O interaction. Immunoblotting was used for Maf protein stability. Luciferase assay was used for c-Maf transcriptional activity. Lentiviral infections were applied for UBE2O function in multiple myeloma (MM) cells. Flow cytometry and nude mice xenografts were applied for MM cell apoptosis and tumor growth assay, respectively. RESULTS: UBE2O was found to interact with c-Maf, a critical transcription factor in MM, by the affinity purification/tandem mass spectrometry assay and co-immunoprecipitation assays. Subsequent studies showed that UBE2O mediated c-Maf polyubiquitination and degradation. Moreover, UBE2O downregulated the transcriptional activity of c-Maf and the expression of cyclin D2, a typical gene modulated by c-Maf. DNA microarray revealed that UBE2O was expressed in normal bone marrow cells but downregulated in MGUS, smoldering MM and MM cells, which was confirmed by RT-PCR in primary MM cells, suggesting its potential role in myeloma pathophysiology. When UBE2O was restored, c-Maf protein in MM cells was significantly decreased and MM cells underwent apoptosis. Furthermore, the human MM xenograft in nude mice showed that re-expression of UBE2O delayed the growth of myeloma xenografts in nude mice in association with c-Maf downregulation and activation of the apoptotic pathway. CONCLUSIONS: UBE2O mediates c-Maf polyubiquitination and degradation, induces MM cell apoptosis, and suppresses myeloma tumor growth, which provides a novel insight in understanding myelomagenesis and UBE2O biology.


Assuntos
Apoptose , Mieloma Múltiplo/metabolismo , Proteínas Proto-Oncogênicas c-maf/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação , Animais , Linhagem Celular Tumoral , Células Cultivadas , Células HEK293 , Humanos , Camundongos Nus , Mieloma Múltiplo/patologia , Mapas de Interação de Proteínas , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-maf/análise , Enzimas de Conjugação de Ubiquitina/análise
18.
Curr Biol ; 27(6): 868-876, 2017 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-28285997

RESUMO

Cerebral cavernous malformations (CCMs) are vascular defects of the CNS that arise from loss of integrity of the endothelial cells lining blood capillaries, causing leakage of blood into the brain [1]. This results in headaches, seizures, and/or hemorrhagic stroke, depending on the location of the lesion. CCM affects 0.5% of the population and follows an autosomal dominant inheritance pattern caused by mutations in one of the three genes: CCM1 (gene name KRIT1), CCM2 (also known as malcavernin or OSM), and CCM3 (gene name PDCD10) [2, 3], with the earliest onset and most severe prognosis occurring in CCM3 patients [4]. The three CCM genes encode structurally distinct scaffold proteins that function in multiple complexes [5-9]. Using the C. elegans germline as a model of multicellular tube development, we show here that CCM-3 is enriched at the luminal membrane of the germline and the contractile ring of dividing cells in the embryo. Loss of ccm-3 results in defective RAB-11-mediated endocytic recycling, which in turn is necessary for gonadal lumen (rachis) formation, completion of cytokinesis, and localization of cell-surface receptors. CCM-3-mediated localization of anillin and non-muscle myosin to the lateral surfaces of germ cells is required for proper cytoskeletal organization, subsequent oocyte growth, and localization of polarity proteins. Biochemical analysis reveals conservation of the STRIPAK complex and distinct roles for GCK-1 (germinal center kinase III family protein) and striatin/CASH-1 in controlling the localization and function of CCM-3. Taken together, our data establish CCM-3 as a novel regulator of rachis lumenization and polarity establishment during embryogenesis.


Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/embriologia , Polaridade Celular/fisiologia , Citocinese/fisiologia , Desenvolvimento Embrionário/genética , Células Germinativas/metabolismo , Proteínas de Membrana/fisiologia , Animais , Proteínas Reguladoras de Apoptose/genética , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Polaridade Celular/genética , Citocinese/genética , Proteínas de Membrana/genética , Transporte Proteico
19.
Proteomics ; 17(6)2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28176486

RESUMO

Reversible protein-tyrosine phosphorylation is catalyzed by the antagonistic actions of protein-tyrosine kinases (PTKs) and phosphatases (PTPs), and represents a major form of cell regulation. Acute myeloid leukemia (AML) is an aggressive hematological malignancy that results from the acquisition of multiple genetic alterations, which in some instances are associated with deregulated protein-phosphotyrosine (pY) mediated signaling networks. However, although individual PTKs and PTPs have been linked to AML and other malignancies, analysis of protein-pY networks as a function of activated PTKs and PTPs has not been done. In this study, MS was used to characterize AML proteomes, and phospho-proteome-subsets including pY proteins, PTKs, and PTPs. AML proteomes resolved into two groups related to high or low degrees of maturation according to French-American-British classification, and reflecting differential expression of cell surface antigens. AML pY proteomes reflect canonical, spatially organized signaling networks, unrelated to maturation, with heterogeneous expression of activated receptor and nonreceptor PTKs. We present the first integrated analysis of the pY-proteome, activated PTKs, and PTPs. Every PTP and most PTKs have both positive and negative associations with the pY-proteome. pY proteins resolve into groups with shared PTK and PTP correlations. These findings highlight the importance of pY turnover and the PTP phosphatome in shaping the pY-proteome in AML.


Assuntos
Leucemia Mieloide Aguda/metabolismo , Fosfotirosina/metabolismo , Proteínas Quinases/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Humanos , Leucemia Mieloide Aguda/enzimologia , Fosforilação , Transdução de Sinais
20.
Int J Cancer ; 140(3): 662-673, 2017 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-27750381

RESUMO

Availability of lung cancer models that closely mimic human tumors remains a significant gap in cancer research, as tumor cell lines and mouse models may not recapitulate the spectrum of lung cancer heterogeneity seen in patients. We aimed to establish a patient-derived tumor xenograft (PDX) resource from surgically resected non-small cell lung cancer (NSCLC). Fresh tumor tissue from surgical resection was implanted and grown in the subcutaneous pocket of non-obese severe combined immune deficient (NOD SCID) gamma mice. Subsequent passages were in NOD SCID mice. A subset of matched patient and PDX tumors and non-neoplastic lung tissues were profiled by whole exome sequencing, single nucleotide polymorphism (SNP) and methylation arrays, and phosphotyrosine (pY)-proteome by mass spectrometry. The data were compared to published NSCLC datasets of NSCLC primary and cell lines. 127 stable PDXs were established from 441 lung carcinomas representing all major histological subtypes: 52 adenocarcinomas, 62 squamous cell carcinomas, one adeno-squamous carcinoma, five sarcomatoid carcinomas, five large cell neuroendocrine carcinomas, and two small cell lung cancers. Somatic mutations, gene copy number and expression profiles, and pY-proteome landscape of 36 PDXs showed greater similarity with patient tumors than with established cell lines. Novel somatic mutations on cancer associated genes were identified but only in PDXs, likely due to selective clonal growth in the PDXs that allows detection of these low allelic frequency mutations. The results provide the strongest evidence yet that PDXs established from lung cancers closely mimic the characteristics of patient primary tumors.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Xenoenxertos/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Adulto , Idoso , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Mutação/genética , Polimorfismo de Nucleotídeo Único/genética , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
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