Global characterization of lysine acetylation in cancer by a proteomics approach

描述(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抑制剂被开发出来调节LysAc状态，目前正在进行80多项抗肿瘤治疗的临床试验。分子生物学和遗传学方法已尝试表征LysAc途径和HDAC抑制剂的抗肿瘤作用。然而，这类研究对LysAc位点的功能及其动态提供的洞察力有限。一些底物蛋白之间的LysAc位点已经通过一种候选方法使用质谱学进行了优雅的分析，为下游生物学研究确定其已知功能提供了关键信息。然而，大量的LysAc底物蛋白及其在癌症相关环境中的LysAc动态还没有被全球研究。这一知识空白需要填补，以提高我们对LysAc在癌症和其他疾病中的理解。我们假设LysAc的调节失调在癌症进展中起关键作用。这项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，并利用这一知识更好地了解其生物学功能。我们建议通过一种新的、集成的蛋白质组学方法来研究癌细胞中的全局LysAc动力学，包括(I)高分辨率蛋白质预分离，(Ii)用抗LysAc抗体高效纯化LysAc多肽，以及(Iii)利用SILAC结合1D-HPLC/MS/MS或2D-HPLC/MS/MS来鉴定和定量LysAc多肽。本研究有望鉴定大量非核LysAc蛋白质，并揭示它们在癌症进展中的动力学，从而挑战当前的概念，即调节DNA模板过程是修饰的主要功能。考虑到识别新的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生物学领域的一个关键障碍。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。LysAc将在两组匹配的正常细胞和癌细胞之间进行量化，重点是以尽可能高的灵敏度量化LysAc。(2)确定第一个被批准临床应用的HDAC抑制剂--琥珀酰苯胺异羟肟酸(SAHA)的下游蛋白质靶点。我们将对正常(SAHA耐药细胞)和匹配的癌细胞(SAHA敏感细胞)中的LysAc进行量化，无论有没有SAHA。(3)确定p300乙酰转移酶的赖氨酸乙酰化蛋白靶点。我们将量化转化细胞系中的LysAc蛋白，无论是否表达活性p300，以生成p300底物的图谱。(4)分析并向研究界传播LysAc数据集。这项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中的一种翻译后修饰，在癌症的发生过程中处于失调状态。本研究旨在阐明肿瘤进展过程中赖氨酸乙酰化的动态变化。所获得的知识可用于了解该修饰的生物学功能，辅助开发用于癌症诊断的新生物标记物，以及开发高效、更安全的新型抗肿瘤药物。

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.