2016-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/659226摘要：Poly(ADP-ribose) polymerase-1 (PARP-1)是真核細胞中一個會與核中染色質結合的多功能酵素。它能催化 ADP核糖的聚合反應，並將 ADP核醣的複合物鍵結在標的蛋白上，這種重要的轉譯後修飾稱為 PARylation。PARylation在細胞生理功能中扮演重要的調控角色，包括偵測和修復去氧核醣核酸損傷、染色質修飾、調控基因轉譯以及細胞死亡路徑等等。在調控細胞死亡路徑的機轉中，PARP-1的過度活化會造成細胞內 NAD+和其前驅物 ATP的大量耗盡，而產生細胞壞死。從我們實驗室之前的研究發現 PARP-1會經由ROS而活化的嶄新路徑，並且在 PARP-1過度活化時，細胞中的粒線體會產生短暫性的生質合成反應增加現象，來適應細胞中能量不足的壓力，以造成過渡性的能量修復。 近來越來越多研究顯示 PARP-1在發炎反應中也扮演了重要的角色，它會在發炎過程中同時被活化，並且在諸多疾病模式中，包括敗血症、糖尿病、心血管疾病、中風、創傷、關節炎和肥胖等等，扮演調控病理現象的角色。目前大多數的研究都著重在PARP-1調控發炎基因的轉錄層次，但對於其是否會調控發炎小體的活化尚未被研究。除了參與發炎反應之外，PARP-1在先天免疫反應和宿主對抗病原菌感染的機制中的角色並不清楚。發炎小體的活化在微生物感染時所誘導的 IL-1產生過程中非常重要，它能促使 caspase-1活化進而切割原型的 IL-1成具活性的 IL-1並釋放到胞外。在我們的前驅研究中，將 Parp-1-/-巨噬細胞以細胞內毒素 LPS誘發前處理並用不同的 NLRP3活化劑做刺激後，發現 PARP-1的缺失會造成 IL-1釋放的下降，且 caspase-1活化以及NLRP3發炎小體的聚合皆降低。當我們使用 PARP-1的活性抑制劑去模擬 PARP-1的缺失情形，發現也會造成 IL-1釋放的降低，而使用 PARP-1的活化劑時會更促進 NLRP3造成的 IL-1釋放，所以 PARP-1的酵素活性在調控發炎小體的活化是必須的。值得注意的是 PARP-1並不影響細胞內毒素誘發的原型 IL-1或是 NLRP3蛋白的表現。總而言之，我們目前的實驗發現 PARP-1會正向調控 NLRP3發炎小體的活化，但是詳細的分子機制並不清楚。除此之外，我們也發現 PARP-1會經由不同的機轉來調控 Toll-like receptor (TLR) 活化所誘發的 iNOS和 COX-2表現。在本計劃中，我們預計使用基因、藥理和生化等不同方式探討 PARP-1在先天免疫上的角色，尤其著重在 NLRP3發炎小體的活化和病原菌感染上的機制。 三個主要的研究目標包括： 目標一：探討 PARP-1參與調控 NLRP3發炎小體活化的角色 目標二：探討 PARP-1在 TLR訊息傳遞路徑和誘導發炎反應中扮演的角色 目標三：探討 PARP-1在病原菌感染時參與宿主防禦功能的角色 <br> Abstract: Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear chromatin-associated multifunctional enzyme found in most eukaryotes. It can catalyze an important post-translational modification named PARylation by polymerizing the ADP-ribose units and conjugating the poly(ADP-ribose) (PAR) complex to the target proteins. PARylation plays diverse roles in many molecular and cellular processes, including DNA damage detection and repair, chromatin modification, transcription and cell death pathways. For inducing cell death, overactivation of PARP-1 leads to depletion of cellular NAD+ and its precursor ATP, and subsequently cell necrosis. In our previous work, we note a novel activating pathway of PARP-1 via ROS, and a transient energy recovery response through mitochondrial biogenesis upon PARP-1 hyperactivation. Recently, the emerging role of PARP-1 in regulating inflammation has been revealed, and several studies have shown that PARP-1 can be simultaneously activated under inflammatory responses and contributes to the pathogenesis in various disease models, including sepsis, diabetes, myocardial dysfunction, stroke, trauma, arthritis, and obesity. Most studies have focused on the transcriptional regulation by PARP-1 in inflammatory genes. However, whether PARP-1 plays roles in NLRP3 inflammasome regulation has not been investigated. Moreover, although the deleterious action of PARP-1 in inflammatory responses is suggested, its involvement in innate immune response and host defense against pathogen infection has not yet been well understood. Inflammsome activation is critical for IL-1 production during microbial infection via generating active caspase-1 for cleavage of pro-IL-1. In our preliminary study using PARP-1-/- primary murine macrophages under lipopolysaccharide (LPS) priming, we found that PARP-1 deficiency impaired IL-1production, caspase-1 maturation and NLRP3 complex assembly caused by various NLRP3 activators, and these actions depend on PARP-1 enzymatic activity, as PARP-1 inhibitor can mimic the responses of PARP-1 deficiency, while PARP-1 activator can enhance the IL-1production via NLRP3 inflammasome activation. Notably PARP-1 did not get involved in pro-IL-1nor NLRP3 induction caused by LPS. Overall, our preliminary data suggest the predominantly nuclear enzyme PARP-1 as a positive regulator of NLRP3 inflammasome, but the detailed molecular events are not yet clarified. Moreover, our preliminary data also suggest PARP-1 might regulate TLRs-induced iNOS and COX-2 expression through different and multiple action mechanisms. Thus in this project we attempt to use genetic, pharmacological and biochemical approaches to explore the roles played by PARP-1 in innate immunity, especially focusing on NLRP3 inflammasome activation and pathogen infection. Three specific aims of this study include Aim 1: Determine the roles of PARP-1 in regulating NLRP3 inflammasome activation; Aim 2: Determine the roles of PARP-1 in TLR signaling pathways and inflammatory responses; Aim 3: Determine the roles of PARP-1 in host defense against pathogens.PARP-1NLRP3發炎小體TLRs發炎先天免疫巨噬細胞PARP-1NLRP3 inflammasomeTLRsinflammationinnate immunitymacrophages