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1.
Cell ; 175(6): 1546-1560.e17, 2018 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-30500537

RESUMO

Mammalian folate metabolism is comprised of cytosolic and mitochondrial pathways with nearly identical core reactions, yet the functional advantages of such an organization are not well understood. Using genome-editing and biochemical approaches, we find that ablating folate metabolism in the mitochondria of mammalian cell lines results in folate degradation in the cytosol. Mechanistically, we show that QDPR, an enzyme in tetrahydrobiopterin metabolism, moonlights to repair oxidative damage to tetrahydrofolate (THF). This repair capacity is overwhelmed when cytosolic THF hyperaccumulates in the absence of mitochondrially produced formate, leading to THF degradation. Unexpectedly, we also find that the classic antifolate methotrexate, by inhibiting its well-known target DHFR, causes even more extensive folate degradation in nearly all tested cancer cell lines. These findings shed light on design features of folate metabolism, provide a biochemical basis for clinically observed folate deficiency in QDPR-deficient patients, and reveal a hitherto unknown and unexplored cellular effect of methotrexate.


Assuntos
Carbono/metabolismo , Citosol/metabolismo , Formiatos/metabolismo , Mitocôndrias/metabolismo , Neoplasias/metabolismo , Tetra-Hidrofolatos/metabolismo , Citosol/patologia , Células HCT116 , Células HeLa , Humanos , Células MCF-7 , Metotrexato/farmacocinética , Metotrexato/farmacologia , Mitocôndrias/patologia , Proteínas Mitocondriais/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Tetra-Hidrofolato Desidrogenase/metabolismo
2.
J Proteome Res ; 18(10): 3703-3714, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31398040

RESUMO

Recent advances in genome editing technologies have enabled the insertion of epitope tags at endogenous loci with relative efficiency. We describe an approach for investigation of protein interaction dynamics of the AMP-activated kinase complex AMPK using a catalytic subunit AMPKα2 (PRKAA2 gene) as the bait, based on CRISPR/Cas9-mediated genome editing coupled to stable isotope labeling in cell culture, multidimensional protein identification technology, and computational and statistical analyses. Furthermore, we directly compare this genetic epitope tagging approach to endogenous immunoprecipitations of the same gene under homologous conditions to assess differences in observed interactors. Additionally, we directly compared each enrichment strategy in the genetically modified cell-line with two separate endogenous antibodies. For each approach, we analyzed the interaction profiles of this protein complex under basal and activated states, and after implementing the same analytical, computational, and statistical analyses, we found that high-confidence protein interactors vary greatly with each method and between commercially available endogenous antibodies.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Genômica/métodos , Mapeamento de Interação de Proteínas/métodos , Anticorpos , Células Cultivadas , Cromatografia de Afinidade , Edição de Genes , Células HEK293 , Humanos , Imunoprecipitação , Marcação por Isótopo , Espectrometria de Massas
3.
Nat Methods ; 18(7): 763-767, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34140701
4.
Nat Methods ; 10(1): 54-6, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23160281

RESUMO

Two major challenges in proteomics are the large number of proteins and their broad dynamic range in the cell. We exploited the abundance-dependent Michaelis-Menten kinetics of trypsin digestion to selectively digest and deplete abundant proteins with a method we call DigDeAPr. We validated the depletion mechanism with known yeast protein abundances, and we observed greater than threefold improvement in low-abundance human-protein identification and quantitation metrics. This methodology should be broadly applicable to many organisms, proteases and proteomic pipelines.


Assuntos
Espectrometria de Massas/métodos , Fragmentos de Peptídeos/análise , Proteínas/metabolismo , Proteoma/análise , Proteômica/métodos , Humanos
5.
Trends Biochem Sci ; 36(7): 388-96, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21616670

RESUMO

Cellular membranes are composed of proteins and glyco- and phospholipids and play an indispensible role in maintaining cellular integrity and homeostasis, by physically restricting biochemical processes within cells and providing protection. Membrane proteins perform many essential functions, which include operating as transporters, adhesion-anchors, receptors, and enzymes. Recent advancements in proteomic mass spectrometry have resulted in substantial progress towards the determination of the plasma membrane (PM) proteome, resolution of membrane protein topology, establishment of numerous receptor protein complexes, identification of ligand-receptor pairs, and the elucidation of signaling networks originating at the PM. Here, we discuss the recent accelerated success of discovery-based proteomic pipelines for the establishment of a complete membrane proteome.


Assuntos
Espectrometria de Massas/métodos , Proteínas de Membrana/análise , Proteômica/métodos , Animais , Membrana Celular/química , Membrana Celular/metabolismo , Humanos , Espectrometria de Massas/tendências , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteômica/tendências
6.
J Proteome Res ; 14(11): 4815-22, 2015 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-26445171

RESUMO

Quantification of proteomes by mass spectrometry has proven to be useful to study human pathology recapitulated in cellular or animal models of disease. Enriching and quantifying newly synthesized proteins (NSPs) at set time points by mass spectrometry has the potential to identify important early regulatory or expression changes associated with disease states or perturbations. NSP can be enriched from proteomes by employing pulsed introduction of the noncanonical amino acid, azidohomoalanine (AHA). We demonstrate that pulsed introduction of AHA in the feed of mice can label and identify NSP from multiple tissues. Furthermore, we quantitate differences in new protein expression resulting from CRE-LOX initiated knockout of LKB1 in mouse livers. Overall, the PALM strategy allows for the first time in vivo labeling of mouse tissues to differentiate protein synthesis rates at discrete time points.


Assuntos
Alanina/análogos & derivados , Fígado/metabolismo , Proteínas Serina-Treonina Quinases/deficiência , Proteoma/isolamento & purificação , Proteômica/métodos , Proteínas Quinases Ativadas por AMP , Alanina/administração & dosagem , Alanina/metabolismo , Alcinos/química , Animais , Azidas/química , Biotina/química , Química Click , Alimentos Formulados , Expressão Gênica , Integrases/genética , Integrases/metabolismo , Fígado/química , Fígado/efeitos dos fármacos , Masculino , Metionina/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Anotação de Sequência Molecular , Proteínas Serina-Treonina Quinases/genética , Proteoma/genética , Proteoma/metabolismo
7.
Science ; 383(6683): eadj1415, 2024 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-38330136

RESUMO

Lung adenocarcinoma (LUAD) and small cell lung cancer (SCLC) are thought to originate from different epithelial cell types in the lung. Intriguingly, LUAD can histologically transform into SCLC after treatment with targeted therapies. In this study, we designed models to follow the conversion of LUAD to SCLC and found that the barrier to histological transformation converges on tolerance to Myc, which we implicate as a lineage-specific driver of the pulmonary neuroendocrine cell. Histological transformations are frequently accompanied by activation of the Akt pathway. Manipulating this pathway permitted tolerance to Myc as an oncogenic driver, producing rare, stem-like cells that transcriptionally resemble the pulmonary basal lineage. These findings suggest that histological transformation may require the plasticity inherent to the basal stem cell, enabling tolerance to previously incompatible oncogenic driver programs.


Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-myc , Carcinoma de Pequenas Células do Pulmão , Humanos , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/terapia , Células Epiteliais/patologia , Pulmão/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/patologia , Carcinoma de Pequenas Células do Pulmão/terapia , Oncogenes , Linhagem da Célula , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-akt/genética , Terapia de Alvo Molecular
8.
bioRxiv ; 2023 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-37205525

RESUMO

Mitochondria-rich brown adipocytes dissipate cellular fuel as heat by thermogenic energy expenditure (TEE). Prolonged nutrient excess or cold exposure impair TEE and contribute to the pathogenesis of obesity, but the mechanisms remain incompletely understood. Here we report that stress-induced proton leak into the matrix interface of mitochondrial innermembrane (IM) mobilizes a group of proteins from IM into matrix, which in turn alter mitochondrial bioenergetics. We further determine a smaller subset that correlates with obesity in human subcutaneous adipose tissue. We go on to show that the top factor on this short list, acyl-CoA thioesterase 9 (ACOT9), migrates from the IM into the matrix upon stress where it enzymatically deactivates and prevents the utilization of acetyl-CoA in TEE. The loss of ACOT9 protects mice against the complications of obesity by maintaining unobstructed TEE. Overall, our results introduce aberrant protein translocation as a strategy to identify pathogenic factors. One-Sentence Summary: Thermogenic stress impairs mitochondrial energy utilization by forcing translocation of IM-bound proteins into the matrix.

9.
Cancer Discov ; 13(4): 1002-1025, 2023 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-36715544

RESUMO

KRAS is the most frequently mutated oncogene in human lung adenocarcinomas (hLUAD), and activating mutations frequently co-occur with loss-of-function mutations in TP53 or STK11/LKB1. However, mutation of all three genes is rarely observed in hLUAD, even though engineered comutation is highly aggressive in mouse lung adenocarcinoma (mLUAD). Here, we provide a mechanistic explanation for this difference by uncovering an evolutionary divergence in the regulation of triosephosphate isomerase (TPI1). In hLUAD, TPI1 activity is regulated via phosphorylation at Ser21 by the salt inducible kinases (SIK) in an LKB1-dependent manner, modulating flux between the completion of glycolysis and production of glycerol lipids. In mice, Ser21 of TPI1 is a Cys residue that can be oxidized to alter TPI1 activity without a need for SIKs or LKB1. Our findings suggest this metabolic flexibility is critical in rapidly growing cells with KRAS and TP53 mutations, explaining why the loss of LKB1 creates a liability in these tumors. SIGNIFICANCE: Utilizing phosphoproteomics and metabolomics in genetically engineered human cell lines and genetically engineered mouse models (GEMM), we uncover an evolutionary divergence in metabolic regulation within a clinically relevant genotype of human LUAD with therapeutic implications. Our data provide a cautionary example of the limits of GEMMs as tools to study human diseases such as cancers. This article is highlighted in the In This Issue feature, p. 799.


Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Triose-Fosfato Isomerase , Animais , Humanos , Camundongos , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Mutação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Triose-Fosfato Isomerase/genética , Triose-Fosfato Isomerase/metabolismo
11.
Cancer Discov ; 10(9): 1352-1373, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32571778

RESUMO

A hallmark of metastasis is the adaptation of tumor cells to new environments. Metabolic constraints imposed by the serine and glycine-limited brain environment restrict metastatic tumor growth. How brain metastases overcome these growth-prohibitive conditions is poorly understood. Here, we demonstrate that 3-phosphoglycerate dehydrogenase (PHGDH), which catalyzes the rate-limiting step of glucose-derived serine synthesis, is a major determinant of brain metastasis in multiple human cancer types and preclinical models. Enhanced serine synthesis proved important for nucleotide production and cell proliferation in highly aggressive brain metastatic cells. In vivo, genetic suppression and pharmacologic inhibition of PHGDH attenuated brain metastasis, but not extracranial tumor growth, and improved overall survival in mice. These results reveal that extracellular amino acid availability determines serine synthesis pathway dependence, and suggest that PHGDH inhibitors may be useful in the treatment of brain metastasis. SIGNIFICANCE: Using proteomics, metabolomics, and multiple brain metastasis models, we demonstrate that the nutrient-limited environment of the brain potentiates brain metastasis susceptibility to serine synthesis inhibition. These findings underscore the importance of studying cancer metabolism in physiologically relevant contexts, and provide a rationale for using PHGDH inhibitors to treat brain metastasis.This article is highlighted in the In This Issue feature, p. 1241.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Encéfalo/patologia , Fosfoglicerato Desidrogenase/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Encéfalo/metabolismo , Neoplasias Encefálicas/secundário , Linhagem Celular Tumoral , Conjuntos de Dados como Assunto , Resistencia a Medicamentos Antineoplásicos , Feminino , Técnicas de Silenciamento de Genes , Glicina/análise , Glicina/metabolismo , Humanos , Metabolômica , Camundongos , Fosfoglicerato Desidrogenase/genética , Fosfoglicerato Desidrogenase/metabolismo , Proteômica , RNA-Seq , Serina/análise , Serina/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Cell Rep ; 29(10): 3331-3348.e7, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31801093

RESUMO

Metformin is the front-line treatment for type 2 diabetes worldwide. It acts via effects on glucose and lipid metabolism in metabolic tissues, leading to enhanced insulin sensitivity. Despite significant effort, the molecular basis for metformin response remains poorly understood, with a limited number of specific biochemical pathways studied to date. To broaden our understanding of hepatic metformin response, we combine phospho-protein enrichment in tissue from genetically engineered mice with a quantitative proteomics platform to enable the discovery and quantification of basophilic kinase substrates in vivo. We define proteins whose binding to 14-3-3 are acutely regulated by metformin treatment and/or loss of the serine/threonine kinase, LKB1. Inducible binding of 250 proteins following metformin treatment is observed, 44% of which proteins bind in a manner requiring LKB1. Beyond AMPK, metformin activates protein kinase D and MAPKAPK2 in an LKB1-independent manner, revealing additional kinases that may mediate aspects of metformin response. Deeper analysis uncovered substrates of AMPK in endocytosis and calcium homeostasis.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Metformina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Cálcio/metabolismo , Linhagem Celular , Endocitose/efeitos dos fármacos , Células HEK293 , Homeostase/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Fosforilação , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteômica/métodos
13.
J Med Chem ; 55(7): 2945-59, 2012 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-22364528

RESUMO

The discovery of 1,3,8-triazaspiro[4.5]decane-2,4-diones (spirohydantoins) as a structural class of pan-inhibitors of the prolyl hydroxylase (PHD) family of enzymes for the treatment of anemia is described. The initial hit class, spirooxindoles, was identified through affinity selection mass spectrometry (AS-MS) and optimized for PHD2 inhibition and optimal PK/PD profile (short-acting PHDi inhibitors). 1,3,8-Triazaspiro[4.5]decane-2,4-diones (spirohydantoins) were optimized as an advanced lead class derived from the original spiroindole hit. A new set of general conditions for C-N coupling, developed using a high-throughput experimentation (HTE) technique, enabled a full SAR analysis of the spirohydantoins. This rapid and directed SAR exploration has resulted in the first reported examples of hydantoin derivatives with good PK in preclinical species. Potassium channel off-target activity (hERG) was successfully eliminated through the systematic introduction of acidic functionality to the molecular structure. Undesired upregulation of alanine aminotransferese (ALT) liver enzymes was mitigated and a robust on-/off-target margin was achieved. Spirohydantoins represent a class of highly efficacious, short-acting PHD1-3 inhibitors causing a robust erythropoietin (EPO) upregulation in vivo in multiple preclinical species. This profile deems spirohydantoins as attractive short-acting PHDi inhibitors with the potential for treatment of anemia.


Assuntos
Anemia/tratamento farmacológico , Compostos Aza/síntese química , Hidantoínas/síntese química , Fator 1 Induzível por Hipóxia/metabolismo , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Compostos de Espiro/síntese química , Animais , Compostos Aza/farmacocinética , Compostos Aza/farmacologia , Cães , Canal de Potássio ERG1 , Eritropoetina/biossíntese , Canais de Potássio Éter-A-Go-Go/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Hidantoínas/farmacocinética , Hidantoínas/farmacologia , Prolina Dioxigenases do Fator Induzível por Hipóxia , Indóis/síntese química , Indóis/farmacocinética , Indóis/farmacologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Macaca mulatta , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Ratos , Compostos de Espiro/farmacocinética , Compostos de Espiro/farmacologia , Relação Estrutura-Atividade , Regulação para Cima
14.
J Am Chem Soc ; 130(49): 16704-10, 2008 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-19049464

RESUMO

To discover antifungal treatments that possess the desired characteristics of broad spectrum activity, a strong safety profile, and oral bioavailability, new discovery strategies must be implemented to identify structural classes of molecules capable of combating these microorganisms. One such technique that has been implemented is the Candida albicans Fitness Test, a whole cell screening platform capable of delineating the mechanism of action of compounds that demonstrate activity against the clinically relevant pathogenic fungus, C. albicans. Screening crude natural product extracts with this technology has resulted in the identification of a novel family of antifungal natural products, named the parnafungins, which inhibit the enzyme polyadenosine polymerase (PAP), a key component of the mRNA cleavage and polyadenylation complex. Owing to the rapid interconversion of the structural and stereoisomers of the parnafungins at neutral pH, the determination of the structural isomer with the highest affinity for PAP with standard biochemical assays has not been possible. Herein, we present an application of affinity-selection/mass spectrometry (AS-MS) to determine that the "straight" parnafungin structural isomer (parnafungin A) binds preferentially to PAP compared to the "bent" structural isomer (parnafungin B).


Assuntos
Oxazolidinonas/química , Oxazolidinonas/metabolismo , Polinucleotídeo Adenililtransferase/metabolismo , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Cromatografia Líquida , Fungos/enzimologia , Humanos , Isomerismo , Ligantes , Espectrometria de Massas , Oxazolidinonas/análise
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