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Acquistion of multimode plate reader for "Investigation of the physiological significance of protein acetylation in Bacillus subtilis"

购买多模式读板机用于“枯草芽孢杆菌蛋白质乙酰化的生理意义研究”

基本信息

批准号：
10807381
负责人：
Valerie Jean Carabetta
金额：
$ 5.87万
依托单位：
ROWAN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-01 至 2025-06-30
项目状态：
未结题

项目摘要

ABSTRACT Until recently, N-lysine acetylation was thought to be rare in bacteria, but is now appreciated to affect hundreds of bacterial proteins with diverse cellular functions. Acetylation was initially discovered as a post-translational modification (PTM) on the unstructured, highly basic N-terminal tails of eukaryotic histones. Histone acetylation constitutes part of the “histone code,” and regulates chromosome compaction and various DNA processes, such as gene expression, replication, repair and recombination. In eukaryotes, acetylation regulates many other proteins in addition to histones, involved in a wide array of important biological processes. This observation is also true in bacteria, as evidenced by the characterization of the acetylomes of more than 30 different bacterial species. However, the physiological significance of the vast majority of these modifications remains unknown. In addition, the mechanisms of acetylation and deacetylation, and the bacterial enzymes involved are not completely understood. To address these gaps in knowledge, we have focused on studying the acetylation of the essential, histone-like protein HBsu in Bacillus subtilis. In bacteria, the nucleoid is compacted and organized by the action of nucleoid-associated proteins (NAPs). HBsu is a member of the most widely conserved NAP family, and is considered a functional equivalent of eukaryotic histones. We found that HBsu contains seven novel acetylation sites, and this raised the exciting possibility that these modifications represent a “histone-like” code in bacteria. So far, we discovered that acetylation of HBsu at key lysine residues is required to maintain normal chromosome compaction. Additionally, we identified the second protein acetyltransferase in B. subtilis. The overall goal of our research program is to decipher this code. Our recent progress supports the hypotheses that acetylation of HBsu regulates cell division and sporulation, and that there are additional enzymes involved in regulating acetylation. The short- term goals of this work are to define the enzymatic mechanism of regulation of HBsu acetylation and determine the significance of HBsu acetylation in the regulation of DNA transactions, stationary phase development and drug tolerance. Additionally, we will develop new biochemical and mass-spectrometry based proteomics techniques for the study of acetylation in bacteria. Our long-term goals are to characterize additional HBsu PTMs, identify and characterize novel enzymes of acetylation, and perform a detailed structural and biochemical analysis with acetylated HBsu and novel enzymes. Ultimately, we will design novel inhibitors of bacterial acetylation enzymes or acetylated HBsu and assess their efficacy as potential novel antimicrobial therapies. Together, these studies may demonstrate the existence of a histone- like code in bacteria, an unexpected and exciting new field of biology. Furthermore, these studies will provide the foundation for designing novel antimicrobial drugs that target protein acetylation, either the enzymes or key acetylated targets.

抽象的直到最近，N-赖氨酸乙酰化还被认为在细菌中很少见，但现在人们认识到它会影响数百种细菌。具有多种细胞功能的细菌蛋白质。乙酰化最初被发现是一种翻译后修饰 (PTM) 作用于真核组蛋白的非结构化、高碱性 N 末端尾部。组蛋白乙酰化构成了 “组蛋白密码”，并调节染色体压缩和各种 DNA 过程，例如基因表达、复制、修复和重组。在真核生物中，除了组蛋白之外，乙酰化还调节许多其他蛋白质，参与一系列重要的生物过程。这一观察结果在细菌中也成立，正如表征所证明的那样超过 30 种不同细菌物种的乙酰基组。然而，绝大多数的生理意义这些修改的内容仍然未知。此外，乙酰化和脱乙酰化的机制以及所涉及的细菌酶尚未完全了解。为了解决这些知识差距，我们重点关注研究枯草芽孢杆菌中必需的组蛋白样蛋白 HBsu 的乙酰化。在细菌中，核仁是通过类核相关蛋白（NAP）的作用进行压缩和组织。 HBsu 是最广泛的成员保守的 NAP 家族，被认为是真核组蛋白的功能等同物。我们发现 HBsu 含有七个新的乙酰化位点，这提出了令人兴奋的可能性，即这些修饰代表“组蛋白样” 细菌中的密码。到目前为止，我们发现 HBsu 在关键赖氨酸残基上的乙酰化是维持正常的所必需的染色体压缩。此外，我们在枯草芽孢杆菌中鉴定了第二种蛋白质乙酰转移酶。总体目标我们的研究计划就是破译这个密码。我们最近的进展支持了 HBsu 乙酰化的假设调节细胞分裂和孢子形成，并且还有其他酶参与调节乙酰化。短- 这项工作的长期目标是定义 HBsu 乙酰化调节的酶促机制并确定 HBsu 乙酰化在 DNA 交易调节、稳定期发育和药物耐受性中的重要性。此外，我们将开发新的基于生化和质谱的蛋白质组学技术来研究细菌中的乙酰化。我们的长期目标是表征其他 HBsu PTM，识别和表征新颖的乙酰化酶，并使用乙酰化 HBsu 和新型酶进行详细的结构和生化分析酶。最终，我们将设计新型细菌乙酰化酶或乙酰化 HBsu 抑制剂并评估其效果作为潜在的新型抗菌疗法的功效。总之，这些研究可能证明组蛋白的存在- 就像细菌中的代码一样，这是一个意想不到的、令人兴奋的生物学新领域。此外，这些研究将提供为设计针对蛋白质乙酰化（酶或关键乙酰化）的新型抗菌药物奠定了基础目标。