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

描述(申请人提供)：高迁移率组盒-1(HMGB1)是一种转录因子样蛋白，最近被鉴定为典型的损伤相关分子模式分子(DAMP)。HMGB1是脓毒症、严重脓毒症和感染性休克患者脓毒症的重要迟效介质。虽然细胞外HMGB1的功能引起了人们的广泛关注，但在脓毒症中HMGB1的释放机制却鲜有人关注。HMGB1缺乏分泌信号肽，因此不能通过内质网-高尔基体系统分泌。新合成的HMGB1经历了广泛的翻译后修饰，例如赖氨酸残基的乙酰化，促进了HMGB1从细胞核到内体间隙的主动运输，并防止其重新进入细胞核。另一种在类似炎症条件下被激活的核蛋白是聚(ADP-核糖)聚合酶-1(PARP-1)。PARP-1是PARP家族中含量最丰富的亚型，它的持续激活导致其底物烟酰胺腺嘌呤二核苷酸(NAD+)的枯竭，继而导致腺苷-5‘-三磷酸(ATP)的枯竭、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HMGB1转录、翻译后修饰和分泌中的作用。这笔赠款的中心假设是 应用是PARP-1是内毒素介导的HMGB1分泌所必需的。在这项R01拨款申请中，我们提出了一种全面的方法，包括体外和体内研究，以确定PARP-1在调节内毒素介导的HMGB1释放中的作用。在目标1中，我们确定了PARP-1调节内毒素诱导的单核细胞HMGB1分泌的分子机制。我们假设，1)化学和遗传的PARP-1抑制调节丝裂原活化蛋白激酶(MAPK)途径的活性；2)PARP-1抑制调节细胞外信号调节激酶(ERK)1/2介导组蛋白乙酰转移酶(HAT)的活性以及与之密切相关的蛋白cAMP反应元件结合蛋白(CREB)结合蛋白(CBP)；3)PARP-1抑制内毒素介导的HMGB1基因转录。在特定的目标2中，我们将确定PARP-1抑制是否调节HMGB1的核输出和转运到单核细胞的内毒素介导的分泌。在这一目标下，我们将确定：1)内毒素介导的HMGB1转运到溶酶体内是否需要PARP-1活性；2)溶酶体内HMGB1浓度在PARP-1抑制或不抑制的情况下；3)HMGB1乙酰化及其与ADP-核糖化的关系。为了验证我们的体外研究结果，我们将在相关的脓毒症临床模型中测试PARP-1抑制剂；因此，在目标3中，我们将使用盲肠结扎和穿孔(CLP)程序在野生型和PARP-/-小鼠中评估PARP激活在脓毒症中的潜在作用。将进行其他研究，以确定在小鼠脓毒症中使用PARP-1抑制剂的治疗窗口。

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.