Investigation of the physiological significance of protein acetylation in Bacillus subtilis

摘要 直到最近，N--赖氨酸乙酰化在细菌中还被认为是罕见的，但现在被认为影响了数以百计的 具有多种细胞功能的细菌蛋白质。乙酰化最初是作为翻译后修饰被发现的。 (PTM)在真核生物组蛋白的无结构、高度碱性的N末端。组蛋白乙酰化是 “组蛋白密码”，调节染色体紧凑和各种DNA过程，如基因表达，复制， 修复和重组。在真核生物中，乙酰化除了调节组蛋白外，还调节许多其他蛋白质，参与一种 广泛的重要生物过程。这一观察结果在细菌中也是正确的，这一特征得到了证明 30多种不同细菌的乙酰体。然而，绝大多数人的生理意义 这些修改中的哪些仍不清楚。此外，还讨论了乙酰化和脱乙酰化的机理，以及 所涉及的细菌酶还不完全清楚。为了解决这些知识差距，我们将重点放在 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，类核是 通过类核相关蛋白(NAP)的作用而紧凑和组织。HBsu是世界上最广泛的 保守的NAP家族，被认为是真核组蛋白的功能等价物。我们发现HBsu含有 7个新的乙酰化位点，这提出了一个令人兴奋的可能性，即这些修饰代表了一个“组蛋白样” 细菌中的密码。到目前为止，我们发现关键赖氨酸残基上HBsu的乙酰化是维持正常所必需的。 染色体紧凑。此外，我们还鉴定了枯草杆菌中的第二个蛋白乙酰转移酶。总目标 我们研究项目的关键就是破译这一密码。我们的最新进展支持HBsu乙酰化的假说 调节细胞分裂和孢子形成，还有其他酶参与调节乙酰化。简而言之- 这项工作的术语目标是定义调节HBsu乙酰化的酶机制，并确定 HBsu乙酰化在DNA交易、稳定期发育和药物耐受性调节中的意义。 此外，我们将开发新的基于生化和质谱学的蛋白质组学技术来研究 细菌中的乙酰化作用。我们的长期目标是表征更多的HBsu PTM，识别和表征新的 用乙酰化的HBsu和NOVE进行了详细的结构和生化分析 酵素。最终，我们将设计新型的细菌乙酰化酶或乙酰化HBsu的抑制剂，并评估它们的 作为潜在的新型抗菌疗法的有效性。总之，这些研究可能会证明组蛋白的存在- 就像细菌中的密码一样，这是生物学中一个意想不到的令人兴奋的新领域。此外，这些研究将提供 设计针对蛋白质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。

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.