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1.
Mol Cell ; 75(4): 711-724.e5, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31278053

RESUMO

The energetic costs of duplicating chromatin are large and therefore likely depend on nutrient sensing checkpoints and metabolic inputs. By studying chromatin modifiers regulated by epithelial growth factor, we identified histone acetyltransferase 1 (HAT1) as an induced gene that enhances proliferation through coordinating histone production, acetylation, and glucose metabolism. In addition to its canonical role as a cytoplasmic histone H4 acetyltransferase, we isolated a HAT1-containing complex bound specifically at promoters of H4 genes. HAT1-dependent transcription of H4 genes required an acetate-sensitive promoter element. HAT1 expression was critical for S-phase progression and maintenance of H3 lysine 9 acetylation at proliferation-associated genes, including histone genes. Therefore, these data describe a feedforward circuit whereby HAT1 captures acetyl groups on nascent histones and drives H4 production by chromatin binding to support chromatin replication and acetylation. These findings have important implications for human disease, since high HAT1 levels associate with poor outcomes across multiple cancer types.


Assuntos
Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Regiões Promotoras Genéticas , Fase S , Transcrição Gênica , Células A549 , Acetilação , Animais , Cromatina/genética , Cromatina/metabolismo , Feminino , Histona Acetiltransferases/genética , Histonas/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Neoplasias/genética , Neoplasias/genética
2.
J Am Chem Soc ; 137(7): 2484-94, 2015 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-25689372

RESUMO

Nonreceptor protein tyrosine kinases (NRTKs) are essential for cellular homeostasis and thus are a major focus of current drug discovery efforts. Peptide substrates that can enhance lanthanide ion luminescence upon tyrosine phosphorylation enable rapid, sensitive screening of kinase activity, however design of suitable substrates that can distinguish between tyrosine kinase families is a huge challenge. Despite their different substrate preferences, many NRTKs are structurally similar even between families. Furthermore, the development of lanthanide-based kinase assays is hampered by incomplete understanding of how to integrate sequence selectivity with metal ion binding, necessitating laborious iterative substrate optimization. We used curated proteomic data from endogenous kinase substrates and known Tb(3+)-binding sequences to build a generalizable in silico pipeline with tools to generate, screen, align, and select potential phosphorylation-dependent Tb(3+)-sensitizing substrates that are most likely to be kinase specific. We demonstrated the approach by developing several substrates that are selective within kinase families and amenable to high-throughput screening (HTS) applications. Overall, this strategy represents a pipeline for developing efficient and specific assays for virtually any tyrosine kinase that use HTS-compatible lanthanide-based detection. The tools provided in the pipeline also have the potential to be adapted to identify peptides for other purposes, including other enzyme assays or protein-binding ligands.


Assuntos
Ensaios Enzimáticos/métodos , Proteínas Tirosina Quinases/metabolismo , Térbio/química , Sequência de Aminoácidos , Técnicas Biossensoriais , Simulação por Computador , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala , Humanos , Células K562 , Medições Luminescentes , Dados de Sequência Molecular , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/farmacologia , Fosforilação , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/química , Bibliotecas de Moléculas Pequenas/farmacologia
3.
Anal Chem ; 87(15): 7555-8, 2015 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-26207839

RESUMO

Kinase signaling is a major mechanism driving many cancers. While many inhibitors have been developed and are employed in the clinic, resistance due to crosstalk and pathway reprogramming is an emerging problem. High-throughput assays to detect multiple pathway kinases simultaneously could better model these complex relationships and enable drug development to combat this type of resistance. We developed a strategy to take advantage of time-resolved luminescence of Tb(3+)-chelated phosphotyrosine-containing peptides, which facilitated efficient energy transfer to small molecule fluorophores conjugated to the peptides to produce orthogonally colored biosensors for two different kinases. This enabled multiplexed detection with high signal-to-noise in a high-throughput-compatible format. This proof-of-concept study provides a platform that could be applied to other lanthanide metal and fluorophore combinations to achieve even greater multiplexing without the need for phosphospecific antibodies.


Assuntos
Técnicas Biossensoriais , Ensaios Enzimáticos/métodos , Proteínas Tirosina Quinases/metabolismo , Térbio/química , Anticorpos/química , Cor , Fluorescência
4.
J Vis Exp ; (203)2024 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-38345235

RESUMO

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome assembly. It is required for tumor growth in various systems, making it a potential target for cancer treatment. To facilitate the identification of compounds that can inhibit HAT1 enzymatic activity, we have devised an acetyl-click assay for rapid screening. In this simple assay, we employ recombinant HAT1/Rbap46, which is purified from activated human cells. The method utilizes the acetyl-CoA analog 4-pentynoyl-CoA (4P) in a click-chemistry approach. This involves the enzymatic transfer of an alkyne handle through a HAT1-dependent acylation reaction to a biotinylated H4 N-terminal peptide. The captured peptide is then immobilized on neutravidin plates, followed by click-chemistry functionalization with biotin-azide. Subsequently, streptavidin-peroxidase recruitment is employed to oxidize amplex red, resulting in a quantitative fluorescent output. By introducing chemical inhibitors during the acylation reaction, we can quantify enzymatic inhibition based on a reduction of the fluorescence signal. Importantly, this reaction is scalable, allowing for high throughput screening of potential inhibitors for HAT1 enzymatic activity.


Assuntos
Química Click , Histonas , Humanos , Histonas/metabolismo , Acetilação , Histona Acetiltransferases/metabolismo , Peptídeos
5.
Anal Chem ; 85(5): 2582-8, 2013 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-23414415

RESUMO

Disruption of regulatory protein phosphorylation can lead to disease and is particularly prevalent in cancers. Inhibitors that target deregulated kinases are therefore a major focus of chemotherapeutic development. Achieving sensitivity and specificity in high-throughput compatible kinase assays is key to successful inhibitor development. Here, we describe the application of time-resolved luminescence detection to the direct sensing of spleen tyrosine kinase (Syk) activity and inhibition using a novel peptide substrate. Chelation and luminescence sensitization of Tb(3+) allowed the direct detection of peptide phosphorylation without any antibodies or other labeling reagents. Characterizing the Tb(3+) coordination properties of the phosphorylated vs unphosphorylated form of the peptide revealed that an inner-sphere water was displaced upon phosphorylation, which likely was responsible for both enhancing the luminescence intensity and also extending the lifetime, which enabled gating of the luminescence signal to improve the dynamic range. Furthermore, a shift in the optimal absorbance maximum for excitation was observed, from 275 nm (for the unphosphorylated tyrosine peptide) to 266 nm (for the phosphorylated tyrosine peptide). Accordingly, time-resolved measurements with excitation at 266 nm via a monochromator enabled a 16-fold improvement in base signal-to-noise for distinguishing phosphopeptide from unphosphorylated peptide. This led to a high degree of sensitivity and quantitative reproducibility, demonstrating the amenability of this method to both research laboratory and high-throughput applications.


Assuntos
Ensaios Enzimáticos/métodos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Medições Luminescentes , Proteínas Tirosina Quinases/metabolismo , Baço/enzimologia , Térbio/química , Sequência de Aminoácidos , Quelantes/química , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Razão Sinal-Ruído , Quinase Syk , Fatores de Tempo
6.
Anal Chem ; 85(10): 5071-7, 2013 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-23611696

RESUMO

Characterization of ligand-protein binding is of crucial importance in drug discovery. Classical competition binding assays measure the binding of a labeled ligand in the presence of various concentrations of unlabeled ligand and typically use single purified proteins. Here, we introduce a high-throughput approach to study ligand-protein interactions by coupling competition binding assays with mass spectrometry-based quantitative proteomics. With the use of a phosphorylated immunoreceptor tyrosine-based activation motif (pITAM) peptide as a model, we characterized pITAM-interacting partners in human lymphocytes. The shapes of competition binding curves of various interacting partners constructed in a single set of quantitative proteomics experiments reflect relative affinities for the pITAM peptide. This strategy can provide an efficient approach to distinguish specific interacting partners, including two signaling kinases possessing tandem SH2 domains, SYK and ZAP-70, as well as other SH2 domain-containing proteins such as CSK and PI3K, from contaminants and to measure relative binding affinities of multiple proteins in a single experiment.


Assuntos
Ligação Competitiva , Motivo de Ativação do Imunorreceptor Baseado em Tirosina , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Proteômica , Sequência de Aminoácidos , Linhagem Celular Tumoral , Humanos , Ligantes , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Especificidade por Substrato
7.
J Med Chem ; 66(8): 5774-5801, 2023 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-37027002

RESUMO

HAT1 is a central regulator of chromatin synthesis that acetylates nascent histone H4. To ascertain whether targeting HAT1 is a viable anticancer treatment strategy, we sought to identify small-molecule inhibitors of HAT1 by developing a high-throughput HAT1 acetyl-click assay. Screening of small-molecule libraries led to the discovery of multiple riboflavin analogs that inhibited HAT1 enzymatic activity. Compounds were refined by synthesis and testing of over 70 analogs, which yielded structure-activity relationships. The isoalloxazine core was required for enzymatic inhibition, whereas modifications of the ribityl side chain improved enzymatic potency and cellular growth suppression. One compound (JG-2016 [24a]) showed relative specificity toward HAT1 compared to other acetyltransferases, suppressed the growth of human cancer cell lines, impaired enzymatic activity in cellulo, and interfered with tumor growth. This is the first report of a small-molecule inhibitor of the HAT1 enzyme complex and represents a step toward targeting this pathway for cancer therapy.


Assuntos
Histonas , Neoplasias , Humanos , Histonas/metabolismo , Histona Acetiltransferases/metabolismo , Cromatina , Linhagem Celular , Acetilação
8.
Biochemistry ; 51(38): 7515-24, 2012 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-22920457

RESUMO

Spleen tyrosine kinase (Syk) has been implicated in a number of pathologies including cancer and rheumatoid arthritis and thus has been pursued as a novel therapeutic target. Because of the complex relationship between Syk's auto- and other internal phosphorylation sites, scaffolding proteins, enzymatic activation state and sites of phosphorylation on its known substrates, the role of Syk's activity in these diseases has not been completely clear. To approach such analyses, we developed a Syk-specific artificial peptide biosensor (SAStide) to use in a cell-based assay for direct detection of intracellular Syk activity and inhibition in response to physiologically relevant stimuli in both laboratory cell lines and primary splenic B cells. This peptide contains a sequence derived from known Syk substrate preference motifs linked to a cell permeable peptide, resulting in a biosensor that is phosphorylated in live cells in a Syk-dependent manner, thus serving as a reporter of Syk catalytic activity in intact cells. Because the assay is compatible with live, primary cells and can report pharmacodynamics for drug action on an intended target, this methodology could be used to facilitate a better understanding of Syk's function and the effect of its inhibition in disease.


Assuntos
Técnicas Biossensoriais , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Animais , Linfócitos B/enzimologia , Linhagem Celular , Ativação Enzimática , Ensaio de Imunoadsorção Enzimática , Camundongos , Fosforilação , Espectrometria de Fluorescência , Quinase Syk
9.
Nat Commun ; 13(1): 3007, 2022 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-35637192

RESUMO

RNA polymerase III (Pol III) includes two alternate isoforms, defined by mutually exclusive incorporation of subunit POLR3G (RPC7α) or POLR3GL (RPC7ß), in mammals. The contributions of POLR3G and POLR3GL to transcription potential has remained poorly defined. Here, we discover that loss of subunit POLR3G is accompanied by a restricted repertoire of genes transcribed by Pol III. Particularly sensitive is snaR-A, a small noncoding RNA implicated in cancer proliferation and metastasis. Analysis of Pol III isoform biases and downstream chromatin features identifies loss of POLR3G and snaR-A during differentiation, and conversely, re-establishment of POLR3G gene expression and SNAR-A gene features in cancer contexts. Our results support a model in which Pol III identity functions as an important transcriptional regulatory mechanism. Upregulation of POLR3G, which is driven by MYC, identifies a subgroup of patients with unfavorable survival outcomes in specific cancers, further implicating the POLR3G-enhanced transcription repertoire as a potential disease factor.


Assuntos
Neoplasias , Pequeno RNA não Traduzido , Animais , Cromatina , Humanos , Mamíferos/genética , Neoplasias/genética , Isoformas de Proteínas/genética , RNA Polimerase III/genética , RNA Polimerase III/metabolismo
10.
Anal Biochem ; 397(1): 73-8, 2010 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-19818327

RESUMO

Many cancers are characterized by changes in protein phosphorylation as a result of kinase dysregulation. Disruption of Abl kinase signaling through the Philadelphia chromosome (causing the Bcr-Abl mutation) in chronic myeloid leukemia (CML) has provided a paradigm for development of kinase inhibitor drugs such as the specific inhibitor imatinib (also known as STI571 or Gleevec). However, because patients are treated indefinitely with this drug to maintain remission, resistance is increasingly becoming an issue. Although there are many ways to detect kinase activity, most lack the ability to "multiplex" the analysis (i.e., to detect more than one substrate simultaneously). Here we report a novel biosensor for detecting Abl kinase activity and sensitivity to inhibitor in live intact cells overexpressing a CML model Abl kinase construct. This straightforward methodology could eventually provide a new tool for detecting kinase activity and inhibitor drug response in cancer cells that overexpress oncogenic kinases.


Assuntos
Técnicas Biossensoriais/métodos , Peptídeos/análise , Proteínas Proto-Oncogênicas c-abl/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sequência de Aminoácidos , Linhagem Celular , Corantes Fluorescentes/química , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Dados de Sequência Molecular , Peptídeos/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-abl/genética , Raios Ultravioleta
11.
Protein Sci ; 29(2): 350-359, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31697410

RESUMO

Most signal transduction pathways in humans are regulated by protein kinases through phosphorylation of their protein substrates. Typical eukaryotic protein kinases are of two major types: those that phosphorylate-specific sequences containing tyrosine (~90 kinases) and those that phosphorylate either serine or threonine (~395 kinases). The highly conserved catalytic domain of protein kinases comprises a smaller N lobe and a larger C lobe separated by a cleft region lined by the activation loop. Prior studies find that protein tyrosine kinases recognize peptide substrates by binding the polypeptide chain along the C-lobe on one side of the activation loop, while serine/threonine kinases bind their substrates in the cleft and on the side of the activation loop opposite to that of the tyrosine kinases. Substrate binding structural studies have been limited to four families of the tyrosine kinase group, and did not include Src tyrosine kinases. We examined peptide-substrate binding to Src using paramagnetic-relaxation-enhancement NMR combined with molecular dynamics simulations. The results suggest Src tyrosine kinase can bind substrate positioning residues C-terminal to the phosphoacceptor residue in an orientation similar to serine/threonine kinases, and unlike other tyrosine kinases. Mutagenesis corroborates this new perspective on tyrosine kinase substrate recognition. Rather than an evolutionary split between tyrosine and serine/threonine kinases, a change in substrate recognition may have occurred within the TK group of the human kinome. Protein tyrosine kinases have long been therapeutic targets, but many marketed drugs have deleterious off-target effects. More accurate knowledge of substrate interactions of tyrosine kinases has the potential for improving drug selectivity.


Assuntos
Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Quinases da Família src/química , Humanos , Peptídeos/metabolismo , Ligação Proteica , Especificidade por Substrato , Quinases da Família src/metabolismo
12.
Cell Rep ; 28(8): 2182-2193.e6, 2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31433991

RESUMO

Individuals with a single functional copy of the BRCA2 tumor suppressor have elevated risks for breast, ovarian, and other solid tumor malignancies. The exact mechanisms of carcinogenesis due to BRCA2 haploinsufficiency remain unclear, but one possibility is that at-risk cells are subject to acute periods of decreased BRCA2 availability and function ("BRCA2-crisis"), which may contribute to disease. Here, we establish an in vitro model for BRCA2-crisis that demonstrates chromatin remodeling and activation of an NF-κB survival pathway in response to transient BRCA2 depletion. Mechanistically, we identify BRCA2 chromatin binding, histone acetylation, and associated transcriptional activity as critical determinants of the epigenetic response to BRCA2-crisis. These chromatin alterations are reflected in transcriptional profiles of pre-malignant tissues from BRCA2 carriers and, therefore, may reflect natural steps in human disease. By modeling BRCA2-crisis in vitro, we have derived insights into pre-neoplastic molecular alterations that may enhance the development of preventative therapies.


Assuntos
Proteína BRCA2/metabolismo , Montagem e Desmontagem da Cromatina , Animais , Proteína BRCA2/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular , Proliferação de Células , Cromatina/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Feminino , Deleção de Genes , Regulação da Expressão Gênica , Humanos , Camundongos , Mutação/genética , NF-kappa B/metabolismo , Transdução de Sinais , Transcrição Gênica , Transcriptoma/genética
13.
Curr Biol ; 24(5): 473-83, 2014 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-24530065

RESUMO

BACKGROUND: The venoms of predators have been an excellent source of diverse highly specific peptides targeting ion channels. Here we describe the first known peptide antagonist of the nociceptor ion channel transient receptor potential ankyrin 1 (TRPA1). RESULTS: We constructed a recombinant cDNA library encoding ∼100 diverse GPI-anchored peptide toxins (t-toxins) derived from spider venoms and screened this library by coexpression in Xenopus oocytes with TRPA1. This screen resulted in identification of protoxin-I (ProTx-I), a 35-residue peptide from the venom of the Peruvian green-velvet tarantula, Thrixopelma pruriens, as the first known high-affinity peptide TRPA1 antagonist. ProTx-I was previously identified as an antagonist of voltage-gated sodium (NaV) channels. We constructed a t-toxin library of ProTx-I alanine-scanning mutants and screened this library against NaV1.2 and TRPA1. This revealed distinct partially overlapping surfaces of ProTx-I by which it binds to these two ion channels. Importantly, this mutagenesis yielded two novel ProTx-I variants that are only active against either TRPA1or NaV1.2. By testing its activity against chimeric channels, we identified the extracellular loops of the TRPA1 S1-S4 gating domain as the ProTx-I binding site. CONCLUSIONS: These studies establish our approach, which we term "toxineering," as a generally applicable method for isolation of novel ion channel modifiers and design of ion channel modifiers with altered specificity. They also suggest that ProTx-I will be a valuable pharmacological reagent for addressing biophysical mechanisms of TRPA1 gating and the physiology of TRPA1 function in nociceptors, as well as for potential clinical application in the context of pain and inflammation.


Assuntos
Peptídeos/farmacologia , Venenos de Aranha/química , Canais de Potencial de Receptor Transitório/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Sítios de Ligação , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Biblioteca Gênica , Humanos , Dados de Sequência Molecular , Canal de Sódio Disparado por Voltagem NAV1.2/metabolismo , Oócitos , Peptídeos/genética , Estrutura Terciária de Proteína , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Proteínas de Xenopus/antagonistas & inibidores , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo
14.
Cell Signal ; 24(5): 1064-73, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22261254

RESUMO

The engagement of antigen receptors on lymphocytes leads to the activation of phospholipase C-γ, the mobilization of intracellular calcium and the activation of the NFAT transcription factor. The coupling of antigen receptors to the activation of NFAT is modulated by numerous cellular effectors including phospho-inositide 3-kinase (PI3K), which is activated following receptor cross-linking. The activation of PI3K has both positive and negative effects on the receptor-mediated activation of NFAT. An increase in the level and activity of Akt2, a target of activated PI3K, potently inhibits the subsequent activation of NFAT. In contrast, an elevation in Akt1 has no effect on signaling. Signaling pathways operating both upstream and downstream of inositol 1,4,5-trisphosphate (IP3)-stimulated calcium release from intracellular stores are unaffected by Akt2. An increase in the level of Akt2 has no significant effect on the initial amplitude, but substantially reduces the duration of calcium mobilization. The ability of Akt2 to inhibit prolonged calcium mobilization is abrogated by the administration of a cell permeable peptide that blocks the interaction between Bcl-2 and the IP3 receptor. Thus, Akt2 is a negative regulator of NFAT activation through its ability to inhibit calcium mobilization from the ER.


Assuntos
Sinalização do Cálcio , Retículo Endoplasmático/metabolismo , Fatores de Transcrição NFATC/metabolismo , Proteínas Proto-Oncogênicas c-akt/fisiologia , Androstadienos/farmacologia , Animais , Linhagem Celular , Genes Reporter , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Luciferases/biossíntese , Luciferases/genética , Linfócitos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Regiões Promotoras Genéticas , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Antígenos de Linfócitos B , Quinase Syk , Transcrição Gênica , Wortmanina
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