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赖氨酸乙酰化在细菌中被认为很少见，但现在被认为会影响数百种具有不同细胞功能的细菌蛋白。乙酰化最初是作为翻译后修饰发现的（PTM）真核组蛋白的非结构化，高基本的N末端尾巴。组蛋白乙酰化构成了 “组蛋白代码”，并调节染色体压实和各种DNA过程，例如基因表达，复制，维修和重组。在真核生物中，除组蛋白外，乙酰化调节了许多其他蛋白质广泛的重要生物过程。这种观察在细菌中也是正确的，如表征所证明 30多种不同细菌物种的乙酰基粒。但是，绝大多数的身体意义这些修改仍然未知。另外，乙酰化和脱乙酰化的机制，以及涉及的细菌酶尚不完全了解。为了解决知识中的这些差距，我们专注于研究枯草芽孢杆菌中必需组蛋白样蛋白HBSU的乙酰化。在细菌中，核苷为通过核苷相关蛋白（NAP）的作用进行压实和组织。 HBSU是最广泛的成员保守的午睡家族，被认为是真核组蛋白的功能等效。我们发现HBSU包含七个新型的乙酰化位点，这提出了这些修饰代表“类似组蛋白”的令人兴奋的可能性细菌中的代码。到目前为止，我们发现需要在赖氨酸保留处进行HBSU的乙酰化才能保持正常染色体压实。此外，我们在枯草芽孢杆菌中鉴定了第二个蛋白乙酰转移酶。总体目标我们的研究计划是破译该代码。我们最近的进展支持HBSU的乙酰化的假设调节细胞分裂和孢子形成，并在调节乙酰化中涉及其他酶。短 - 这项工作的术语目标是定义调节HBSU乙酰化调节的酶促机理，并确定 HBSU乙酰化在DNA交易，固定相发育和药物耐受性调节中的重要性。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，识别和表征新颖乙酰化酶，并用乙酰化HBSU和新型进行详细的结构和生化分析酶。最终，我们将设计乙酰化酶或乙酰化HBSU的新型抑制剂，并评估它们作为潜在的新型抗菌疗法的功效。总之，这些研究可能证明了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领域。此外，这些研究将提供设计靶向蛋白乙酰化的新型抗菌药物的基础，无论是酶还是关键乙酰化目标。