2011-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/687775摘要：細胞生長與營養、生長因子以及能量狀態息息相關。整合這三條訊息傳遞路徑很重要的兩個磷酸激酶為AMP-activated protein kinase (AMPK)以及target of rapamycin (TOR)。AMPK在細胞能量缺乏狀態下，AMP/ATP比例上升時，會被活化；其活化導致TOR的抑制，進而抑制蛋白轉譯及細胞生長。此一路徑不僅調控細胞生長，在哺乳動物與癌症、神經退化及糖尿病的致病機轉也有密切關聯。在最簡單的真核生物—酵母菌，過去發現AMPK的調節次單元Sip2p，會正向調控複製存活壽命 (Replicative life span，RLS)；TOR則會負向調控複製及時間存活壽命 (chronological life span，CLS)，顯示AMPK及TOR對於酵母菌老化的重要性。吾人過去在酵母菌的乙醯化蛋白微陣列實驗，利用純化之NuA4 complex (酵母菌唯一對其生長必不可缺的乙醯化轉移酶)在含有5,800個酵母菌蛋白的晶片做乙醯化實驗。發現Sip2p以及Tap42p (TOR的下游重要的受動器(effector))會被NuA4乙醯化；使Sip2p活化的myristoylation酶—Nmt1p，也會被NuA4乙醯化。而Sip2p的N-端以共價鍵結加上myristate對於Sip2p延長RLS是必不可缺的，顯示存在著重要的feed forward loop的調控關係。本計畫將利用質譜儀找出被乙醯化的lysine，作位點突變，研究其對於磷酸激酶活性以及下游功能的影響。吾人也將研究乙醯化對於RLS及CLS的調控，並在哺乳動物的細胞株，研究AMPK及TOR的乙醯化，是否在演化上被保留下來。此外吾人將利用酵母菌蛋白晶片，尋找AMPK及TOR的磷酸化受質。AMPK有三個不同的β次元，將純化含有不同β次元的AMPK複合體，分別尋找其下游的受質。TOR分為TOR1與TOR2。TOR1之受質，目前僅知與蛋白轉錄及核糖體生成有關；TOR2之受質則與細胞壁及細胞骨骼形成有關。透過大規模蛋白晶片實驗，尋找AMPK及TOR磷酸化受質及其功能，將有助於了解生命體因應能量狀態如何調控新陳代謝及存活壽命。這些結果也將促進將來對於癌症、神經退化及糖尿病的藥物開發。<br> Abstract: Previous studies revealed that cell growth is tightly coupled to nutrient availability, growth factors, and energy status. These three signal inputs are integrated by two crucial kinases, AMP-activated protein kinase (AMPK) and target of rapamycin (TOR), which regulate cell growth, and hence play critical roles in the pathogenesis of a wide variety of diseases in mammals, such as cancer, neurodegenerative and metabolic disorders. AMPK is activated in response to high intracellular AMP/ATP ratio, an indicator of low cellular energy status; and further inhibits the TOR activity. In the budding yeast, Saccharomyces cerevisiae, a primitive eukaryotic organism, one of the three β-subunits of AMPK, Sip2p, positively regulates the replicative life span (RLS), while the TOR pathway negatively regulates the replicative as well as chronological life span (CLS), indicating a crucial role of AMPK and TOR in yeast ageing.In our previous study, we have identified many nonhistone substrates of the NuA4 complex, the only histone acetyltransferase essential for yeast viability, by performing in vitro acetylation experiments using purified NuA4 complex on a yeast proteome microarray containing ~5,800 yeast proteins. One prominent substrate is Pck1p, the rate–limiting enzyme of the gluconeogenesis pathway, which also serves as the critical effector downstream to NuA4 and Sir2p (an NAD+–dependent histone deacetylase) in regulating calorie restriction–mediated extension of yeast chronological life span. We also found and validated that Sip2p and Tap42p (an essential downstream effector of TOR) were in vivo substrates of the NuA4 complex, indicating an important role of protein acetylation in regulating AMPK and TOR activity. Nmt1p – an important N-myristoyl transferase responsible for the N-terminal myristoylation and hence activation of Sip2, was also found to be another in vivo substrate of the NuA4 complex. Defect of the covalent attachment of myristate to the N-terminal glycine of Sip2p shortens the replicative life span. The NuA4 regulation of Nmt1p and Sip2p potentially forms a “feed-forward loop”, which filters out spurious pulses of signals and ensures a full activation of AMPK only when cells need it. In this study, we will identify the acetylation sites of Sip2p, Tap42p and Nmt1p by using mass spectrometry; perform site-directed mutagenesis on these acetylable lysine residues to examine the effects of acetylation on the kinase activities of AMPK and TOR, and assess whether acetylation will affect their downstream functions. We will further explore the role of NuA4 acetylation of these substrates in regulating yeast replicative and chronological life span. Further studies will be carried on to examine if the acetylation control mechanism is conserved in mammalian systems.Furthermore, we will perform in vitro kinase assay on yeast proteome microarrays to identify the substrates of AMPK and TOR by using purified kinase complexes. AMPK containing three different β-subunits and the two different types of TOR complexes (TORC1 and TORC2) will be purified for the experiments. A systematic search for the different repertoire of downstream substrates of distinct type of AMPK and TOR complexes might lead us to the new therapeutic targets of cancer, neurodegenerative and metabolic disorders.NuA4AMPKTOR乙醯化複製存活壽命時間存活壽命蛋白質體微陣列磷酸化受質NuA4AMPKTORacetylationreplicative life spanchronologic life spanproteome microarrayphosphorylation substrate