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

描述（由申请人提供）：High Mobility Group Box-1 （HMGB1）是一种转录因子样蛋白，最近被定性为一种典型的损伤相关分子模式分子（DAMP）。HMGB1是脓毒症、严重脓毒症和脓毒症休克患者的重要迟发性脓毒症介质。虽然人们对细胞外HMGB1的功能关注较多，但对HMGB1在脓毒症中的释放机制研究甚少。HMGB1缺乏分泌信号肽；因此，它不能通过内质网-高尔基系统分泌。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HMGB1从细胞核主动转运到内体室，并阻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PARPenzyme家族中最丰富的异构体，它的持续激活导致其底物烟酰胺腺嘌呤二核苷酸（NAD+）的耗竭，随之而来的是腺苷-5'-三磷酸（ATP）的耗竭，能量衰竭和细胞死亡。本研究计划将明确PARP-1在脂多糖（LPS）介导的HMGB1转录、翻译后修饰和分泌调控中的作用。这项拨款的中心假设

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.