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

聚 (ADP-核糖) 聚合酶 1 调节 LPS 介导的 HMGB1 释放

• 批准号: 8293488
• 负责人: RAJESH K. ANEJA
• 金额: $ 28.24万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8293488
• 关键词: ADP ribosylation; Acetylation; Active Biological Transport; Adenosine; Adenosine Diphosphate Ribose; Applications Grants; Attention; Binding; Binding Proteins; Bone Marrow; Cell Death; Cell Line; Cell Nucleus; Cell Surface Receptors; Cells; Chemicals; Cleaved cell; Clinical; Cyclic AMP-Responsive DNA-Binding Protein; DNA; Data; Disease model; Dose; EP300 gene; Endoplasmic Reticulum; Endothelial Cells; Enterocytes; Failure; Family; Genetic Transcription; Golgi Apparatus; HMGB1 Protein; HMGB1 gene; Histology; Histones; Immune; In Vitro; Inflammatory; Inflammatory Response; Ligation; Lipopolysaccharides; Location; Lysine; Lysosomes; MAPK3 gene; Measures; Mediating; Mediator of activation protein; Mitogen-Activated Protein Kinases; Modeling; Molecular; Morbidity - disease rate; Mus; Niacinamide; Nicotinamide adenine dinucleotide; Nuclear; Nuclear Export; Nuclear Protein; Nuclear Proteins; Organ; Outcome; Pathway interactions; Patients; Pattern; Peptide Signal Sequences; Plasma; Play; Poly(ADP-ribose) Polymerases; Post-Translational Protein Processing; Protein Binding; Protein Isoforms; Proteins; Public Health; Puncture procedure; Regulation; Research; Role; Sepsis; Septic Shock; Serum; Signal Pathway; Stimulus; System; Testing; Tetanus Helper Peptide; Therapeutic; Therapeutic Agents; Transferase; chemical genetics; extracellular; improved; in vivo; inhibitor/antagonist; macrophage; monocyte; mortality; prevent; trafficking; transcription factor; tripolyphosphate

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.

描述（由申请人提供）：高迁移率组盒-1（HMGB1）是一种转录因子样蛋白，最近被表征为原型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脓毒症的重要晚效介质。虽然细胞外 HMGB1 的功能受到了很多关注，但脓毒症中 HMGB1 释放的机制却很少受到关注。 HMGB1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 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 从细胞核主动转运至内体区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 PARP 酶家族中最丰富的亚型，其持续激活会导致其底物烟酰胺腺嘌呤二核苷酸 (NAD+) 耗尽，从而导致 5'-三磷酸腺苷 (ATP) 耗尽、能量衰竭和细胞死亡。拟议的研究计划将明确 PARP-1 在脂多糖 (LPS) 介导的 HMGB1 转录、翻译后修饰和分泌调节中的作用。本次资助的中心假设 应用的一个事实是，PARP-1 对于 LPS 介导的 HMGB1 分泌至关重要。在此 R01 拨款申请中，我们提出了一种包括体外和体内研究在内的综合方法，该方法将定义 PARP-1 在调节 LPS 介导的 HMGB1 释放中的作用。在目标 1 中，我们确定了 PARP-1 调节单核细胞中 LPS 诱导的 HMGB1 分泌的分子机制。我们假设 1) 化学和基因 PARP-1 抑制调节丝裂原激活蛋白激酶 (MAPK) 通路活性； 2) PARP-1 抑制调节 p300 的细胞外信号调节激酶 (ERK) 1/2 介导的组蛋白乙酰转移酶 (HAT) 活性以及密切相关的蛋白 cAMP 反应元件结合蛋白 (CREB) 结合蛋白 (CBP)； 3) PARP-1抑制LPS介导的HMGB1基因转录。在具体目标 2 中，我们将确定 PARP-1 抑制是否调节 HMGB1 的核输出和向内体区室的递送，以实现单核细胞中 LPS 介导的分泌。在此目标下，我们将确定：1）LPS介导的HMGB1向溶酶体的运输是否需要PARP-1活性； 2）有或没有PARP-1抑制的溶酶体中HMGB1浓度3）HMGB1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，我们将在脓毒症相关临床模型中测试 PARP-1 抑制剂；因此，在目标 3 中，我们将使用野生型和 PARP-/- 小鼠的盲肠结扎穿刺 (CLP) 程序来评估 PARP 激活在脓毒症中的潜在作用。将进行其他研究以确定 PARP-1 抑制剂在小鼠脓毒症中使用的治疗窗。 公共卫生相关性：脓毒症仍然是一个全球公共卫生问题，导致显着的死亡率和发病率。尽管对脓毒症控制机制的理解取得了进展，但新的有效治疗药物的发现仍然落后。在本提案中，我们将探讨高迁移率组 Box-1（败血症的关键晚期介质）释放所涉及的机制。