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1.
ACS Cent Sci ; 10(6): 1295-1303, 2024 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-38947213

RESUMO

Chemical protein synthesis provides a powerful means to prepare novel modified proteins with precision down to the atomic level, enabling an unprecedented opportunity to understand fundamental biological processes. Of particular interest is the process of gene expression, orchestrated through the interactions between transcription factors (TFs) and DNA. Here, we combined chemical protein synthesis and high-throughput screening technology to decipher the role of post-translational modifications (PTMs), e.g., Lys-acetylation on the DNA binding activity of Max TF. We synthesized a focused library of singly, doubly, and triply modified Max variants including site-specifically acetylated and fluorescently tagged analogs. The resulting synthetic analogs were employed to decipher the molecular role of Lys-acetylation on the DNA binding activity and sequence specificity of Max. We provide evidence that the acetylation sites at Lys-31 and Lys-57 significantly inhibit the DNA binding activity of Max. Furthermore, by utilizing high-throughput binding measurements, we assessed the binding activities of the modified Max variants across diverse DNA sequences. Our results indicate that acetylation marks can alter the binding specificities of Max toward certain sequences flanking its consensus binding sites. Our work provides insight into the hidden molecular code of PTM-TFs and DNA interactions, paving the way to interpret gene expression regulation programs.

2.
Angew Chem Int Ed Engl ; 62(47): e202310913, 2023 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-37642402

RESUMO

The chemical synthesis of site-specifically modified transcription factors (TFs) is a powerful method to investigate how post-translational modifications (PTMs) influence TF-DNA interactions and impact gene expression. Among these TFs, Max plays a pivotal role in controlling the expression of 15 % of the genome. The activity of Max is regulated by PTMs; Ser-phosphorylation at the N-terminus is considered one of the key regulatory mechanisms. In this study, we developed a practical synthetic strategy to prepare homogeneous full-length Max for the first time, to explore the impact of Max phosphorylation. We prepared a focused library of eight Max variants, with distinct modification patterns, including mono-phosphorylated, and doubly phosphorylated analogues at Ser2/Ser11 as well as fluorescently labeled variants through native chemical ligation. Through comprehensive DNA binding analyses, we discovered that the phosphorylation position plays a crucial role in the DNA-binding activity of Max. Furthermore, in vitro high-throughput analysis using DNA microarrays revealed that the N-terminus phosphorylation pattern does not interfere with the DNA sequence specificity of Max. Our work provides insights into the regulatory role of Max's phosphorylation on the DNA interactions and sequence specificity, shedding light on how PTMs influence TF function.


Assuntos
Regulação da Expressão Gênica , Fatores de Transcrição , Fatores de Transcrição/metabolismo , Fosforilação , Sequência de Aminoácidos , Processamento de Proteína Pós-Traducional , DNA/metabolismo
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