Regulation of LPS-mediated HMGB1 Release by Poly (ADP-ribose) Polymerase-1

描述（由申请人提供）：高迁移率族蛋白-1（HMGB 1）是一种转录因子样蛋白，最近被表征为原型损伤相关分子模式分子（DAMP）。HMGB 1是脓毒症、严重脓毒症和脓毒性休克患者中重要的迟发性脓毒症介质。虽然细胞外HMGB 1的功能已受到广泛关注，但脓毒症中HMGB 1释放的机制却很少受到关注。HMGB 1缺乏分泌信号肽;因此，它不能通过内质网-高尔基体系统分泌。新合成的HMGB 1经历了广泛的翻译后修饰，例如，赖氨酸残基的乙酰化，其促进HMGB 1从细胞核主动转运至内体区室并防止其再进入细胞核。另一种在类似炎症条件下活化的核蛋白是聚（ADP-核糖）聚合酶-1（PARP-1）。PARP-1是PARPenzyme家族中最丰富的同种型，其持续活化导致其底物烟酰胺腺嘌呤二核苷酸（NAD+）耗尽，从而导致腺苷-5 '-三磷酸（ATP）耗尽、能量衰竭和细胞死亡。拟议的研究计划将确定PARP-1在脂多糖（LPS）介导的HMGB 1转录，翻译后修饰和分泌的调节中的作用。这项资助的核心假设是 PARP-1是LPS介导的HMGB 1分泌所必需的。在这个R 01基金申请中，我们提出了一个全面的方法，包括体外和体内研究，将确定PARP-1在调节LPS介导的HMGB 1释放中的作用。在目的1中，我们确定PARP-1调节LPS诱导的单核细胞HMGB 1分泌的分子机制。我们假设：1）化学和遗传性PARP-1抑制调节丝裂原活化蛋白激酶（MAPK）通路活性：2）PARP-1抑制调节细胞外信号调节激酶（ERK）1/2介导的p300和一种密切相关的蛋白质，cAMP反应元件结合蛋白（CREB）结合蛋白（CBP）的组蛋白乙酰转移酶（HAT）活性; 3）PARP-1抑制LPS介导的HMGB 1基因转录。在特定目标2中，我们将确定PARP-1抑制是否调节核输出和HMGB 1向内体隔室的递送，以用于单核细胞中LPS介导的分泌。在此目标下，我们将确定：1）LPS介导的HMGB 1运输至溶酶体是否需要PARP-1活性; 2）在有或无PARP-1抑制的情况下溶酶体中的HMGB 1浓度; 3）HMGB 1乙酰化及其与ADP-核糖基化的相关性。为了验证我们的体外研究结果，我们将在脓毒症的相关临床模型中泰特PARP-1抑制剂;因此，在目标3中，我们将在野生型和PARP-/-小鼠中使用盲肠结扎穿孔（CLP）程序评估PARP激活在脓毒症中的潜在作用。将进行其他研究，以确定在鼠败血症中使用PARP-1抑制剂的治疗窗。 公共卫生相关性：脓毒症仍然是一个全球性的公共卫生问题，并导致显著的死亡率和发病率。尽管在对支配脓毒症的机制的理解方面取得了进展，但新的有效治疗剂的发现仍然落后。在本研究中，我们将探讨高迁移率族蛋白-1（一种重要的脓毒症晚期介导因子）的释放机制。

DESCRIPTION (provided by applicant): High Mobility Group Box-1 (HMGB1) is a transcription factor-like protein that has recently been characterized as a prototypical Damage -Associated Molecular Pattern molecule (DAMP). HMGB1 is a crucial late-acting mediator of sepsis in patients with sepsis, severe sepsis and septic shock. While much attention has been focused on the function of extracellular HMGB1, the mechanisms of HMGB1 release in sepsis have received little consideration. HMGB1 lacks a secretory signal peptide; therefore, it cannot be secreted via the endoplasmic reticulum-Golgi system. The newly synthesized HMGB1 undergoes extensive post-translational modifications, e.g., acetylation of lysine residues that promote active transport of HMGB1 from the nucleus to the endosomal compartment and prevent its re-entry into the nucleus. Another nuclear protein, which is activated in similar inflammatory conditions, is Poly (ADP-ribose) Polymerase-1(PARP-1). PARP-1 is the most abundant isoform of the PARPenzyme family and its continued activation leads to depletion of its substrate, nicotinamide adenine dinucleotide (NAD+), with consequent depletion of adenosine-5'-triphosphate (ATP), energy failure and cell death. The proposed research plan will define the role of PARP-1 in the modulation of lipopolysaccharide (LPS)-mediated HMGB1 transcription, post-translational modification and secretion. The central hypothesis for this grant application is that PARP-1 is essential for LPS-mediated HMGB1 secretion. In this R01 grant application, we propose a comprehensive approach including in vitro and in vivo studies that will define the role of PARP-1 in modulating LPS-mediated HMGB1 release. In Aim 1, we determine the molecular mechanisms whereby PARP-1 regulates LPS-induced HMGB1 secretion in monocytes. We hypothesize that 1) chemical and genetic PARP-1 inhibition modulates mitogen-activated protein kinase (MAPK) pathway activity; 2) PARP-1 inhibition modulates extracellular signal-regulated kinase (ERK) 1/2-mediated histone acetyl-transferase (HAT) activity of p300 and a closely related protein, cAMP response element-binding protein (CREB)-binding protein (CBP); 3) PARP-1 inhibits LPS-mediated HMGB1 gene transcription. In Specific Aim 2, we will determine if PARP-1 inhibition modulates the nuclear export and delivery of HMGB1 to the endosomal compartment for LPS-mediated secretion in monocytes. Under this aim, we will determine: 1) if LPS-mediated HMGB1 trafficking to the lysosomes requires PARP-1 activity; 2) HMGB1 concentration in the lysosomes with or without PARP-1 inhibition 3) HMGB1 acetylation and its correlation with ADP-ribosylation. To verify the results of our in vitro studies we will tet PARP-1 inhibitors in relevant clinical models of sepsis; therefore, in Aim 3 we will assess the potential role of PARP activation in sepsis using cecal ligation and puncture (CLP) procedure in wild-type and PARP-/- mice. Additional studies will be performed to determine the therapeutic window for use of PARP-1 inhibitors in murine sepsis. PUBLIC HEALTH RELEVANCE: Sepsis continues to be a global public health problem and results in significant mortality and morbidity. Despite advances in the understanding of the mechanisms that govern sepsis, the discovery of new and efficacious therapeutic agents has lagged behind. In this proposal, we will explore the mechanisms involved in the release of High Mobility Group Box-1, a crucial late - mediator of sepsis.