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1.
Cell Rep ; 22(6): 1522-1530, 2018 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-29425507

RESUMO

The age-related effects of GDF11 have been a subject of controversy. Here, we find that elevated GDF11 causes signs of cachexia in mice: reduced food intake, body weight, and muscle mass. GDF11 also elicited a significant elevation in plasma Activin A, previously shown to contribute to the loss of skeletal muscle. The effects of GDF11 on skeletal muscle could be reversed by administration of antibodies to the Activin type II receptors. In addition to the effects on muscle, GDF11 increased plasma GDF15, an anorectic agent. The anorexia, but not the muscle loss, could be reversed with a GDF15-neutralizing antibody. GDF15 upregulation is due to GDF11-induced recruitment of SMAD2/3 to the GDF15 promoter. Inhibition of GDF15 can restore appetite but cannot restore the GDF11-induced loss of muscle mass, which requires blockade of ActRII signaling. These findings are relevant for treatment of cachexia.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Caquexia , Fator 15 de Diferenciação de Crescimento/biossíntese , Fatores de Diferenciação de Crescimento/metabolismo , Ativinas/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/farmacologia , Fatores de Diferenciação de Crescimento/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regulação para Cima
2.
Cancer Res ; 74(12): 3317-31, 2014 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-24755473

RESUMO

Oncogenic mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in several types of cancer, but the metabolic consequences of these genetic changes are not fully understood. In this study, we performed (13)C metabolic flux analysis on a panel of isogenic cell lines containing heterozygous IDH1/2 mutations. We observed that under hypoxic conditions, IDH1-mutant cells exhibited increased oxidative tricarboxylic acid metabolism along with decreased reductive glutamine metabolism, but not IDH2-mutant cells. However, selective inhibition of mutant IDH1 enzyme function could not reverse the defect in reductive carboxylation activity. Furthermore, this metabolic reprogramming increased the sensitivity of IDH1-mutant cells to hypoxia or electron transport chain inhibition in vitro. Lastly, IDH1-mutant cells also grew poorly as subcutaneous xenografts within a hypoxic in vivo microenvironment. Together, our results suggest therapeutic opportunities to exploit the metabolic vulnerabilities specific to IDH1 mutation.


Assuntos
Ciclo do Ácido Cítrico , Isocitrato Desidrogenase/genética , Mitocôndrias/metabolismo , Mutação de Sentido Incorreto , Animais , Antineoplásicos/farmacologia , Hipóxia Celular , Inibidores Enzimáticos/farmacologia , Glutamina/metabolismo , Células HCT116 , Humanos , Isocitrato Desidrogenase/antagonistas & inibidores , Isocitrato Desidrogenase/metabolismo , Camundongos , Oxirredução , Estresse Fisiológico , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Mol Cell Biol ; 33(2): 194-212, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23109432

RESUMO

Molecular mechanisms underlying sarcopenia, the age-related loss of skeletal muscle mass and function, remain unclear. To identify molecular changes that correlated best with sarcopenia and might contribute to its pathogenesis, we determined global gene expression profiles in muscles of rats aged 6, 12, 18, 21, 24, and 27 months. These rats exhibit sarcopenia beginning at 21 months. Correlation of the gene expression versus muscle mass or age changes, and functional annotation analysis identified gene signatures of sarcopenia distinct from gene signatures of aging. Specifically, mitochondrial energy metabolism (e.g., tricarboxylic acid cycle and oxidative phosphorylation) pathway genes were the most downregulated and most significantly correlated with sarcopenia. Also, perturbed were genes/pathways associated with neuromuscular junction patency (providing molecular evidence of sarcopenia-related functional denervation and neuromuscular junction remodeling), protein degradation, and inflammation. Proteomic analysis of samples at 6, 18, and 27 months confirmed the depletion of mitochondrial energy metabolism proteins and neuromuscular junction proteins. Together, these findings suggest that therapeutic approaches that simultaneously stimulate mitochondrogenesis and reduce muscle proteolysis and inflammation have potential for treating sarcopenia.


Assuntos
Envelhecimento/genética , Mitocôndrias/metabolismo , Junção Neuromuscular/patologia , Proteoma/análise , Sarcopenia/patologia , Transcriptoma , Envelhecimento/metabolismo , Animais , DNA Mitocondrial/genética , Metabolismo Energético , Perfilação da Expressão Gênica , Imuno-Histoquímica , Modelos Lineares , Masculino , Análise em Microsséries , Mitocôndrias/genética , Mitocôndrias/patologia , Força Muscular/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Junção Neuromuscular/genética , Junção Neuromuscular/metabolismo , Mudanças Depois da Morte , Proteômica , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Regulação para Cima
5.
J Biomol Screen ; 15(6): 695-702, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20484097

RESUMO

Many attractive targets for therapeutic intervention are enzymes that catalyze biological reactions involving small molecules such as lipids, fatty acids, amino acid derivatives, nucleic acid derivatives, and cofactors. Some of the reactions are difficult to detect by methods commonly used in high-throughput screening (HTS) without specific radioactive or fluorescent labeling of substrates. In addition, there are instances when labeling has a detrimental effect on the biological response. Generally, applicable assay methodologies for detection of such reactions are thus required. Mass spectrometry (MS), being a label-free detection tool, has been actively pursued for assay detection in HTS in the past several years. The authors have explored the use of multiparallel liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) for high-throughput detection of biochemical reactions. In this report, we describe in detail the assay development and screening with a LC/MS-based system for inhibitors of human diacylglycerol acyltransferase (DGAT1) with a chemical library of approximately 800,000 compounds. Several strategies and process improvements have been investigated to overcome technical challenges such as data variation and throughput. Results indicated that, through these innovative approaches, the LC/MS-based screening method is both feasible and suitable for high-throughput primary screening.


Assuntos
Diacilglicerol O-Aciltransferase/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios Enzimáticos/métodos , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/farmacologia , Espectrometria de Massas/métodos , Cromatografia Líquida , Diacilglicerol O-Aciltransferase/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Padrões de Referência , Reprodutibilidade dos Testes , Solventes/química , Fatores de Tempo , Titulometria
6.
Virology ; 397(1): 43-55, 2010 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-19932913

RESUMO

Three cyclophilin inhibitors (DEBIO-025, SCY635, and NIM811) are currently in clinical trials for hepatitis C therapy. The mechanism of action of these, however, is not completely understood. There are at least 16 cyclophilins expressed in human cells which are involved in a diverse set of cellular processes. Large-scale siRNA experiments, chemoproteomic assays with cyclophilin binding compounds, and mRNA profiling of HCV replicon containing cells were used to identify the cyclophilins that are instrumental to HCV replication. The previously reported cyclophilin A was confirmed and additional cyclophilin containing pathways were identified. Together, the experiments provide strong evidence that NIM811 reduces viral replication by inhibition of multiple cyclophilins and pathways with protein trafficking as the most strongly and persistently affected pathway.


Assuntos
Ciclofilinas/metabolismo , Hepacivirus/fisiologia , Interações Hospedeiro-Patógeno , Replicação Viral , Antivirais/química , Antivirais/farmacologia , Linhagem Celular , Ciclosporina/química , Ciclosporina/farmacologia , Perfilação da Expressão Gênica , Inativação Gênica , Humanos , Modelos Biológicos , Estrutura Molecular , Proteoma/análise , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
7.
Anal Chem ; 79(21): 8207-13, 2007 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-17902631

RESUMO

High-throughput screening (HTS) is an important tool for finding active compounds to initiate medicinal chemistry programs in pharmaceutical discovery research. Traditional HTS methods rely on fluorescent or radiolabeled reagents and/or coupling assays to permit quantitation of enzymatic target inhibition or activation. Mass spectrometry-based high-throughput screening (MS-HTS) is an alternative that is not susceptible to the limitations imposed by labeling and coupling enzymes. MS-HTS offers a selective and sensitive analytical method for unlabeled substrates and products. Furthermore, method development times are reduced without the need to incorporate labels or coupling assays. MS-HTS also permits screening of targets that are difficult or impossible to screen by other techniques. For example, enzymes that are challenging to purify can lead to the nonspecific detection of structurally similar components of the impure enzyme or matrix of membraneous enzymes. The high selectivity of tandem mass spectrometry (MS/MS) enables these screens to proceed with low levels of background noise to sensitively discover interesting hits even with relatively weak activity. In this article, we describe three techniques that we have adapted for large-scale (approximately 175,000 sample) compound library screening, including four-way parallel multiplexed electrospray liquid chromatography tandem mass spectrometry (MUX-LC/MS/MS), four-way parallel staggered gradient liquid chromatography tandem mass spectrometry (LC/MS/MS), and eight-way staggered flow injection MS/MS following 384-well plate solid-phase extraction (SPE). These methods are capable of analyzing a 384-well plate in 37 min, with typical analysis times of less than 2 h. The quality of the MS-HTS approach is demonstrated herein with screening data from two large-scale screens.


Assuntos
Técnicas de Química Combinatória , Preparações Farmacêuticas/análise , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Avaliação Pré-Clínica de Medicamentos/instrumentação , Avaliação Pré-Clínica de Medicamentos/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Extração em Fase Sólida/instrumentação , Extração em Fase Sólida/métodos , Espectrometria de Massas em Tandem/instrumentação
8.
J Biol Chem ; 277(17): 14838-43, 2002 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-11815627

RESUMO

Human angiotensin-converting enzyme-related carboxypeptidase (ACE2) is a zinc metalloprotease whose closest homolog is angiotensin I-converting enzyme. To begin to elucidate the physiological role of ACE2, ACE2 was purified, and its catalytic activity was characterized. ACE2 proteolytic activity has a pH optimum of 6.5 and is enhanced by monovalent anions, which is consistent with the activity of ACE. ACE2 activity is increased approximately 10-fold by Cl(-) and F(-) but is unaffected by Br(-). ACE2 was screened for hydrolytic activity against a panel of 126 biological peptides, using liquid chromatography-mass spectrometry detection. Eleven of the peptides were hydrolyzed by ACE2, and in each case, the proteolytic activity resulted in removal of the C-terminal residue only. ACE2 hydrolyzes three of the peptides with high catalytic efficiency: angiotensin II () (k(cat)/K(m) = 1.9 x 10(6) m(-1) s(-1)), apelin-13 (k(cat)/K(m) = 2.1 x 10(6) m(-1) s(-1)), and dynorphin A 1-13 (k(cat)/K(m) = 3.1 x 10(6) m(-1) s(-1)). The ACE2 catalytic efficiency is 400-fold higher with angiotensin II () as a substrate than with angiotensin I (). ACE2 also efficiently hydrolyzes des-Arg(9)-bradykinin (k(cat)/K(m) = 1.3 x 10(5) m(-1) s(-1)), but it does not hydrolyze bradykinin. An alignment of the ACE2 peptide substrates reveals a consensus sequence of: Pro-X((1-3 residues))-Pro-Hydrophobic, where hydrolysis occurs between proline and the hydrophobic amino acid.


Assuntos
Carboxipeptidases/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Enzima de Conversão de Angiotensina 2 , Catálise , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Peptidil Dipeptidase A , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrofotometria Ultravioleta
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