利用散彈槍蛋白質體分析法尋找及鑑定與人類Lon蛋白酶結合之蛋白質:以Hsp60與mtHsp70為例

Abstract

人類Lon蛋白酶是一種存在粒線體基質的ATP依賴型多功能蛋白質，參與分解遭氧化損壞的粒腺體蛋白質，重新摺疊損壞蛋白質的錯誤結構以及藉由結合粒線體DNA與粒線體DNA結合蛋白來調節粒線體DNA的基因表達。近年來許多研究指出，人類Lon蛋白酶不正常表現或缺失與許多人類疾病成因有關，如腫瘤生成、老化、糖尿病、腦神經細胞退化以及類中風發作症候群 (MELAS syndrome)。儘管我們知道人類Lon蛋白酶在粒線體生理功能調節中扮演重要角色，但它如何影響粒線體功能的分子機制尚待更進一步的研究。由於人類Lon蛋白酶被發現在癌症組織中過量表現，故在本論文中我們以過量表現人類Lon蛋白酶的細胞為對象利用蛋白質體的策略來探討與人類Lon有交互作用的蛋白質。 散彈槍蛋白質體分析法 (Shotgun proteomics) 是一種目前廣泛用於研究蛋白質與蛋白質間交互作用的研究方法。此方法主要是利用串聯式質譜 (Tandem mass spectrometry, MS/MS) 來決定某蛋白質經酵素分解後所得胜肽的序列。在我們的研究當中，我們設計了一套策略來連結免疫共沉澱法 (Co-immunoprecipitation) 及散彈槍蛋白質體分析法來研究與人類Lon蛋白酶有交互作用的蛋白質。過程中我們利用會穩定表現帶有Myc標記的人類Lon蛋白酶細胞–293M來進行散彈槍蛋白質體分析以及過程中我們利用特殊方法克服了必須使用十二烷基硫酸鈉聚丙烯酰胺凝膠電泳 (SDS-PAGE) 來去除蛋白質溶液中的介面活性劑和抗體蛋白質的限制，解決介面活性劑和抗體蛋白質出現在溶液中將會嚴重影響散彈槍蛋白質體分析法分析結果的問題。 藉由這個方法我們總共發現245個與人類Lon蛋白酶結合的蛋白質，包含參與粒線體伴護子系統 (Mitochondrial chaperone system) 的蛋白質、粒線體能量代謝的蛋白質以及目前已被發現會影響粒線體DNA穩定性的細胞骨架蛋白。為了驗證散彈槍蛋白質體分析法結果的可信度，我們利用西方墨點法 (Western blotting) 及免疫螢光染色 (Immunofluorescence) 做進一步的確認，並證明人類熱休克蛋白60 (Heat shock protein 60, Hsp60) 和粒線體人類熱休克蛋白70 (Mitochondrial heat shock protein 70, mtHsp70) 為人類Lon蛋白酶交互作用的蛋白質。進一步我們發現， 在人類細胞面臨氧化壓力下，過量表現的人類Lon蛋白酶可能藉由其chaperone 活性來影響Hsp60 和mtHsp70的穩定性並藉此作用抑制細胞凋亡 (Apoptosis) 發生。

Human Lon protease (hLon), an ATP-stimulated mitochondrial matrix protein, is a multifunctional protein that catalyzes the degradation of oxidized mitochondrial proteins, chaperons the assembly of matrix proteins and regulates gene expression of mitochondrial DNA (mtDNA) due to its ability to bind mtDNA and interact with mtDNA binding proteins. Recent studies indicated that dys-regulation of hLon relates to tumorigenesis, aging, diabetes, and neurodegeneration. Although hLon appears to play an essential role in regulating physiological functions of mitochondria, its physiological binding-proteins and molecular mechanism are not fully characterized. As hLon is overexpressed in various types of cancer, in this report, we used a shotgun proteomic approach to investigate the interactome of the protein using the cells overexpressing hLon. The shotgun proteomics strategy, based on digesting proteins into peptides and sequencing them using tandem mass spectrometry (MS/MS), has widely used to investigate protein-protein interactions. In the present study, we designed a strategy connecting co-immunoprecipitation (Co-IP) with in-solution digestion and liquid chromatography tandem mass spectrum (LTQ-Orbitrap) to study hLon-associated proteins by using AD293 cells which stably overexpressed hLon with myc tag (called 293M cells). Here we overcame the restriction that Co-IP samples must be separated by SDS-PAGE to remove detergents and to avoid antibodies disturbance, both dramatically interfere with the result of mass spectrometry, before applying to shotgun proteomics analysis. Using this method, we totally identified 245 hLon-associated proteins including proteins participated in mitochondrial chaperone system, cellular metabolism and even cytoskeleton proteins which recently have been indicated that may associate with the stability of mtDNA. To verify the credibility of the result, we further confirmed candidate proteins by Co-IP/western blotting and in vivo immunofluorescence assay. We thus confirmed that Hsp60 and mtHsp70 are hLon-associated proteins. Moreover, we found that hLon may utilize its chaperone activity to influence the stability of Hsp60 and mtHsp70 and suggested that, under oxidative stress, overexpressed hLon inhibits apoptosis due to its ability to stabilize Hsp60 and mtHsp70.