Lysine Pyruvylation Mediated by HAT1/p300-SIRT3 Couples Glycolytic Flux to Epigenetic Regulation [CUT&Tag]

Post-translational modifications (PTMs) serve as critical regulatory mechanisms connecting metabolism and protein functions. Following our prior discovery of lysine lactylation (Kla), we herein report the identification and characterization of lysine pyruvylation (Kpy), a previously undescribed PTM driven by the central glycolytic hub metabolite pyruvate. Through integrated biochemical and proteomic strategies, we established the existence and specificity of Kpy. Proteomic profiling revealed Kpy sites across both histones and non-histone proteins, implicating its broad physiological significance. Notably, our investigations demonstrated that Kpy abundance fluctuates in respond to alterations in glycolytic flux and pyruvate concentration, providing a direct mechanism by which metabolic alterations influence protein functions. Additionally, we identified Sirtuin 3 (SIRT3) as the "eraser" enzyme for Kpy removal, while Histone acetyltransferase 1 (HAT1) and p300 (EP300) function as "writer" enzymes responsible for Kpy deposition. Initial mechanistic studies suggested Kpy's regulatory role in transcriptional control. Together, the elucidation of Kpy expands our understanding of metabolite-protein crosstalk through PTMs, providing mechanistic insights into pathophysiological processes and facilitating the development of targeted therapeutic interventions for metabolic disorders. Overall design: RNA-seq profiling of control and 20 mM oxamate-treated HEK293T cells. And CUT&Tag analysis of Kpy in HEK293T cells.翻译后修饰（PTMs）作为连接代谢与蛋白质功能的关键调控机制发挥重要作用。继我们此前发现赖氨酸乳酰化（Kla）之后，本文报道了赖氨酸丙酮酰化（Kpy）的鉴定与特征解析——这是一种此前未被报道的、由糖酵解核心枢纽代谢物丙酮酸驱动的翻译后修饰。通过整合生化与蛋白质组学策略，我们证实了Kpy的存在及其修饰特异性。蛋白质组谱分析显示，组蛋白与非组蛋白中均存在Kpy修饰位点，提示其具有广泛的生理意义。值得注意的是，我们的研究表明Kpy的修饰丰度会随糖酵解通量与丙酮酸浓度的变化而波动，这为代谢改变调控蛋白质功能提供了直接的分子机制。此外，我们鉴定出Sirtuin 3（SIRT3）为Kpy的"擦除酶"（负责移除Kpy修饰），而组蛋白乙酰转移酶1（HAT1）与p300（EP300）则作为"写入酶"介导Kpy的沉积。初步机制研究显示Kpy参与转录调控过程。综上，对Kpy的解析拓展了我们对翻译后修饰介导的代谢物-蛋白质互作的认知，为病理生理过程提供了机制层面的见解，并助力代谢疾病靶向治疗干预手段的开发。实验整体设计：对对照组与20 mM草氨酸处理的HEK293T细胞进行RNA测序（RNA-seq）谱分析，同时对HEK293T细胞开展Kpy的CUT&Tag分析。

搜集汇总

背景与挑战

背景概述

该数据集是一个基于CUT&Tag技术的人类表观遗传学数据集，研究赖氨酸丙酮酰化（Lysine Pyruvylation）通过HAT1/p300-SIRT3介导机制耦合糖酵解通量与表观遗传调控。它包含12个Illumina NovaSeq X Plus测序运行，覆盖不同处理条件（如PBS和Oxamate）和重复，数据为成对外显子组fastq文件，适用于表观遗传修饰和基因表达分析。