賴氨酸乙醯基轉移酶對大腸桿菌細胞分裂和大小的調控

Abstract

乙醯化修飾是細胞中常見且重要的蛋白質轉譯後修飾，可改變蛋白質結構及功能，用以調節細胞生理。細菌中的蛋白質乙醯化修飾可根據機制分為兩類，其中主要的乙醯化修飾來源，是由帶有高能量的乙醯磷酸 (Acetyl-phosphate) 所進行的非酵素催化乙醯化，以及由蛋白質乙醯基轉移酶 (Protein lysine acetyltransferase, Pka) 所催化的專一性乙醯化反應。Pka本身具有高度保留的第一型Gcn5相關乙醯轉移酶 (Gcn5-related N-acetyltransferase, GNAT) 功能性區域，能夠精準的將乙醯基自乙醯輔酶A (Acetyl CoA) 轉移至目標蛋白賴氨酸上的ε胺基上。另一方面，實驗室先前的研究發現，大腸桿菌中的Pka會和細胞分裂相關小細胞蛋白產生直接的交互作用；而Min系統可以協助細菌將分裂的位置放置到中央，使得細菌可以在長軸中心進行分裂，產生兩個等同大小的子細胞。基於蛋白質乙醯化修飾與細胞分裂的重要性，本篇論文主旨在探討大腸桿菌內Pka利用乙醯化修飾影響細胞分裂的機制，以及整體的乙醯化修飾路徑對細菌生長的影響。 在此研究中，我分析了一系列大腸桿菌的乙醯化和去乙醯化的相關酵素基因剔除菌株，發現不同乙醯化突變菌株在生長的對數期與靜止期早期的細胞大小出現明顯差異，因而推論乙醯化修飾可能參與大腸桿菌的細胞大小調節，也加強Pka研究的重要性。為了探討Pka調節細胞分裂的機制，我運用細菌雙雜合系統發現了Pka與細胞分裂蛋白MinC、MinD、MinE、FtsZ、與MreB之間具有直接交互作用關係，代表Pka可能透過與細胞分裂蛋白之間的交互做用調節細胞大小。此外，透過螢光顯微鏡的實驗，觀察到細胞分裂進程上游的關鍵步驟，亦即將FtsZ環定為到細胞中點的能力，會在將pka基因被剔除的菌株中減低產生偏移。而Pka於野生型中會於細胞兩端的形成點狀聚集，而在minCDE基因剔除的背景下Pka均勻散佈於細胞質中。這些細胞內定位的表型，均支持Pka與細胞分裂蛋白之間有密不可分的關聯性。為了進一步了解是否細胞分裂蛋白受Pka產生的乙醯化修飾調節，我以細胞外乙醯化實驗，配合質譜方式鑑定出MinE 蛋白的賴胺酸52為Pka的乙醯化位置。總結目前結果，大腸桿菌的Pka可與細胞分裂蛋白產生交互做用，相互影響並改變Pka與細胞分裂蛋白於細胞內的定位，可能得以調節細胞大小。雖然目前無法確認細胞分裂蛋白乙醯化後對生長分裂的影響，但初步結果已顯示MinE 具有Pka的專一性乙醯化賴氨酸。未來將再進一步進行實驗分析。

Acetylation is a prevalent post-translational modification of proteins in Escherichia coli that regulates cell physiology through modulating protein structure and function. In addition to non-enzymatic acetylation activity involving the high-energy acetyl donor acetyl-phosphate, the protein acetylation can be catalyzed by protein lysine acetyltransferase, Pka. Pka contains a highly conserved, type I Gcn5-related N-acetyltransferases (GNAT) domain at the C-terminus that transfers the acetyl group from acetyl-CoA to the ε-amine of the lysine residue. Interestingly, Pka was identified as an interacting partner of a cell division machinery, the Min system, suggesting that Pka may be involved in regulating cell division. Therefore, this study aims at investigating the interplay between Pka and cell division by asking whether the enzymatic activity of Pka is involved, or Pka possesses moonlighting activity to regulate the process of cell division. To understand the relationships between acetylation and cell division in E. coli, a series of mutants that carried deletions in genes involved in the acetylation and deacetylation cycles was examined for cell growth and morphology. The results showed that there were significant size variations between the mutants and the wild type strain at different growth phases, indicating that the acetylation activity may be involved in the cell size regulation. Among them, the Δpka mutant became smaller proportionally in both width and length at the exponential and late exponential phase when cultured at 30℃. To probe for the mechanism regulating the cell size, the bacterial cytoskeletal proteins FtsZ and MreB, that are involved in coordinating the bacterial growth and division, were found to directly interact with Pka in the bacterial two-hybrid assays. Coincided with the findings, placement of the FtsZ ring at the midcell zone became less robust in the Δpka, and polar localization of GFP-Pka was lost in the absence of minCDE. All evidence suggested that Pka may directly interact with the cell division machineries, including the Min system, FtsZ, and MreB to modulate the cell size. To address whether the acetylation activity of Pka on the cell division proteins could influence the cell division proteins, I used the in vitro acetylation assay in combination with the mass spectrometry analysis to identify lysine 52 of MinE was the specific acetylation site of Pka. Taken together, we conclude that the acetylation network serves as a novel mechanism to regulate cell size, growth and division in E. coli. Whether Pka could directly modulate the functions of the cell division proteins, or regulate cell size via modulating cell metabolism will be investigate.