Global characterization of lysine acetylation in cancer by a proteomics approach

描述（由申请人提供）：赖氨酸乙酰化（LysAc）及其调节酶与癌症广泛相关。因此，HDAC抑制剂已被开发用于调节LysAc状态，并且目前正在进行80多项临床试验作为抗肿瘤治疗剂。分子生物学和遗传学方法已被尝试表征LysAc途径和HDAC抑制剂的抗肿瘤作用。然而，这样的研究提供了有限的洞察LysAc网站的功能和它们的动态。一些底物蛋白中的LysAc位点已经通过使用质谱的候选方法进行了优雅的分析，为下游生物学研究提供了关键信息，以确定其已知的功能。然而，大量的LysAc底物蛋白及其LysAc动力学尚未在癌症相关环境中进行全面研究。需要填补这一知识空白，以提高我们对LysAc在癌症和其他疾病中的理解。我们假设LysAc的失调在癌症进展中起关键作用。该研究计划的长期目标是全面阐明癌症中赖氨酸乙酰化的动态变化，并利用这些知识更好地了解其生物学功能。我们提出通过一种新的、整合的蛋白质组学方法来研究癌细胞中的整体LysAc动力学，该方法包括（i）高分辨率蛋白质预分级分离，（ii）用抗LysAc抗体有效纯化LysAc肽，和（iii）通过SILAC结合1D-HPLC/MS/MS或2D-HPLC/MS/MS鉴定和定量LysAc肽。核LysAc蛋白，并揭示其在癌症进展中的动力学，因此挑战了目前的概念，即DNA模板化过程的调节是修饰的主要功能。鉴于识别新的LysAc底物的困难，本研究中建立的新颖、全面的LysAc数据集将解决LysAc生物学领域的关键障碍。本研究的具体目的是：（1）明确癌细胞中赖氨酸乙酰化的异常变化。将在两组匹配的正常细胞和癌细胞之间定量LysAc，重点是以尽可能高的灵敏度定量LysAc。(2)鉴定首个批准用于临床应用的HDAC抑制剂辛二酰苯胺异羟肟酸（SAHA）的下游蛋白靶点。我们将在正常（SAHA抗性细胞）和匹配的癌细胞（SAHA敏感细胞）中定量LysAc（有或没有SAHA）。(3)确定p300乙酰转移酶作用的赖氨酸乙酰化蛋白靶点。我们将量化转化的细胞系中的LysAc蛋白，有或没有活性p300的表达，以产生p300底物的图谱。（4）分析和传播LysAc数据集到研究社区。本研究的新信息将通过生物信息学工具进行分析，并用于构建LysAc数据库，为刺激药物开发和LysAc生物学研究提供基础设施。 赖氨酸乙酰化是蛋白质的翻译后修饰，在癌症发展中失调。该研究旨在阐明赖氨酸乙酰化在癌症进展中的动态变化。由此产生的知识可用于理解修饰的生物学功能，以帮助开发用于诊断癌症的新型生物标志物和开发具有高效力和更好安全性的新型抗肿瘤药物。

DESCRIPTION (provided by applicant): Lysine-acetylation (LysAc) and its regulatory enzymes are broadly associated with cancers. Consequently, HDAC inhibitors have been developed to modulate the LysAc status, and are currently undergoing more than 80 clinical trials as anti-tumor therapeutics. Molecular biology and genetics approaches have been attempted to characterize LysAc pathway and the anti-tumor effects of HDAC inhibitors. However, such research provides limited insight into the functions of LysAc sites and their dynamics. LysAc sites among some substrate proteins have been elegantly analyzed by a candidate approach using mass spectrometry, generating key information for downstream biological studies to establish its known functions. Nevertheless, a large number of LysAc substrate proteins and their LysAc dynamics have not been globally studied in cancer-related environments before. This knowledge gap needs to be filled to improve our understanding of LysAc in cancer and other diseases. We hypothesize that dysregulation of LysAc plays a key role in cancer progression. The long-term objective of this research plan is to comprehensively elucidate the dynamic changes of lysine acetylation in cancer, and to use this knowledge to gain a better understanding of its biological functions. We propose to study global LysAc dynamics in cancer cells by a novel, integrated proteomics approach, consisting of (i) high- resolution protein pre-fractionation, (ii) efficient purification of LysAc peptides with anti- LysAc antibodies, and (iii) identification and quantification of the LysAc peptides by SILAC combined with 1D- HPLC/MS/MS or 2D-HPLC/MS/MS. This study is anticipated to identify a large number of non-nuclear LysAc proteins and to reveal their dynamics in cancer progression, therefore challenging the current concept that regulation of DNA-templated process is the major function of the modification. Given the difficulty in identifying new LysAc substrates, the novel, comprehensive LysAc datasets established in this study will address a critical barrier in the LysAc biology field. Specific aims of this research are: (1) To define the aberrant changes in lysine acetylation in cancer cells. LysAc will be quantified between two sets of matched normal and cancer cells, with an emphasis on quantifying LysAc with the highest possible sensitivity. (2) To identify downstream protein targets of suberoylanilide hydroxamic acid (SAHA), the first HDAC inhibitor approved for clinical application. We will quantify LysAc, with or without SAHA, in both normal (SAHA-resistant cells) and matched cancer cells (SAHA-sensitive cells). (3) To identify the lysine-acetylation protein targets for p300 acetyltransferase. We will quantify LysAc proteins among transformed cell lines, with or without expression of the active p300 to generate an atlas of p300 substrates. And (4) to analyze and disseminate LysAc datasets to research community. The novel information from this study will be analyzed by bioinformatics tools and used to construct a LysAc database, providing an infrastructure to stimulate drug development and LysAc-biology research. Lysine acetylation, a post-translational modification in proteins, is dysregulated in cancer development. The proposed study aims to elucidate the dynamic changes of lysine acetylation in cancer progression. The resulting knowledge can be used to understand the biological functions of the modification, to assist development of novel biomarkers for diagnosis of cancer and development of novel anti-tumor drug with high potency and better safety profile.