Lon蛋白酶與調節因子的生物物理特性之分析

Abstract

Lon是高度保留存在於各個生物體中的蛋白酶，協助蛋白質的代謝及調控、維持DNA穩定正常表現，屬於AAA+ 蛋白酶家族。此蛋白酶家族共有的特徵是需要依賴ATP結合與水解，產生的能量讓蛋白酶構型改變並將蛋白質去折疊，構型改變會產生拉力將蛋白質運送至蛋白酶區域進行降解，此區域在有ATP結合情況下，構型上呈現封閉的腔體 (chamber)，避免進行非特異性結合。之前在研究LonA特殊生化學模式，MtaLonA K361A長晶時無添加ATP，但加入鎂離子時，結構中在ATP結合位發現丙二酸 (malonate)。丙二酸為長晶條件下的添加物，MtaLonA此時的結構也呈現封閉的腔體。丙二酸結構與檸檬酸循環代謝中間物琥珀酸 (succinate) 相似，與琥珀酸競爭琥珀酸去氫酶，進一步抑制其活性。因此，研究假設檸檬酸循環中代謝中間物會調節LonA的活性，LonA形成無活性的封閉狀態，研究也進一步發現部分檸檬酸循環代謝中間物會抑制LonA降解蛋白質的活性，接著進行高通量熱穩定測試 (ThermoFluor) 實驗，以確認結合強度。已有研究指出T4噬菌體利用蛋白酶抑制劑，比如PinA抑制大腸桿菌中EcLonA降解蛋白質的活性，但保留胜肽酶的活性。當LonA截去N端時，蛋白質水解活性降低，推測PinA結合LonA的N端從而抑制LonA的活性。蛋白質體外結合實驗 (protein pull-down assay) 及微量熱泳動技術同時證實PinA結合LonA的N端 (N210及N245)。DNA也被證實具調控LonA的活性，並且促進LonA降解DNA結合蛋白的活性，這些蛋白會結合在LonA的其他相鄰區域。LonA的N端已被證實會參與受質的辨識及結合，微量熱泳動技術證實DNA結合在Lon的核心區域 (3HAAAP)。綜合上述，我們從生物物理的角度去探討這些LonA調節因子的結合位，從體外實驗去推測細胞在生理狀況下如何調控LonA的活性。

Lon protease is a member of AAA+ protein family. Lon protease is an ATP-dependent homo-oligomeric protease which carries both ATPase and protease activities and is well known for its role in cellular functions like protein quality control and metabolic regulation. Besides, DNA binding of Lon is critical for DNA damage tolerance. In E. coli, proteolysis of abnormal proteins is inhibited when the bacteria are infected with bacteriophage T4. Previous studies have shown that the bacteriophage T4 protein PinA (Proteolysis inhibition A) inhibits the Lon protease activity without affecting the peptidase activities, but the mechanism remains unclear. PinA causes similar reduction of protease activity to N-terminal truncation of Lon, indicating that PinA may inhibit Lon via interaction with the N-terminal region. In my study, PinA and E. coli Lon protease (EcLonA) were purified with or without the N-terminal region for several protein-protein interaction assays. Pull-down and microscale thermophoresis (MST) assays were used to confirm the interaction between PinA and two N-terminal constructs of EcLonA (N245 and N210). In vitro, DNA can directly regulate Lon activity and control degradation of DNA bound proteins by adjacent bound Lon. The N-terminal domain is proposed to be involved in binding and recognition of substrate proteins. MST assays revealed the interaction between the core region (3HAAAP) of EcLonA and DNA, as it turned out. In previous study, we discovered that malonate bound with Lon at ATP binding site in the structure of MtaLonA K361A. Malonate is a competitive inhibitor of succinate dehydrogenase which has similar shape to a TCA cycle metabolic intermediate succinate. We further found that some intermediates inhibited degradation activity of Lon protease. The binding affinities between EcLonA (3HAAA) and these intermediates were determined in ThermoFluor.