ADP-ribosylation Cycles

ADP-核糖基化循环

基本信息

批准号：
8939756
负责人：
Joel Moss
金额：
$ 220.11万
依托单位：
NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8939756
关键词：
A549 ADP Ribose Transferases ADP ribosylation ADP-Ribosylation Pathway Address Adenosine Diphosphate Ribose Affect Agmatine Amino Acids Anti-Bacterial Agents Antibiotics Apoptotic Arginine Azurophilic Granule Bacteria Bacterial Toxins Biological Biological Assay Biological Preservation Carbamates Carbon Cell Death Cell Differentiation process Cell Line Cell Proliferation Cells Charge Chemotactic Factors Cholera Cholera Toxin Cleaved cell Colony-forming units Complex DNA Repair Data Deacetylation Defensins Disease Disulfides Dithiothreitol Dose Endothelial Cells Epithelial Cells Epithelium Escherichia coli Eukaryotic Cell Event Exhibits Family Fibroblasts Goals Hamman-Rich syndrome Hela Cells High Pressure Liquid Chromatography Host Defense Hour Human Hydrolase IL8 gene Immunoassay In Vitro Incubated Infection Inflammation Inflammatory Response Injury Insecta Interleukin-8 Laboratory Study Laser Microscopy Lung Lysine Mammalian Cell Measures Modification Mono-S Neutrophil Activation Niacinamide O-Acetyl-ADP-Ribose Ornithine Pathway interactions Patients Peptide Antibiotics Peptides Plasma Play Poly Adenosine Diphosphate Ribose Poly(ADP-ribose) Polymerases Positioning Attribute Post-Translational Protein Processing Process Production Protein Family Proteins Pseudomonas aeruginosa Reaction Recruitment Activity Regulation Reporting Role Serum Serum-Free Culture Media Signal Transduction Site Staphylococcus aureus Structure Surface Testing Tetrazolium Time Toxic effect Transforming Growth Factors Water Wound Healing airway epithelium alpha-Defensins analog antimicrobial arginyllysine biological systems carcinogenesis cell motility chemokine cytokine cytotoxic cytotoxicity healthy volunteer human neutrophil peptide 1 injured killings member microorganism neutrophil novel poly ADP-ribose glycohydrolase protein aminoacid sequence protein function repaired response virtual

项目摘要

Explanation The airway epithelium plays a critical role in host defense against infection. The repair process of the injured epithelium involves cell migration and proliferation, processes that increase neutrophil defensins. As reported previously, the proliferative and cytotoxic effects of HNP-1 on human lung fibroblasts and epithelial and endothelial cells are dose dependent. Low concentrations of HNP-1 have a proliferative effect on normal human lung fibroblasts (NHLF) and A549 epithelial cells. However, at concentrations greater than 20g/ml, HNP is cytotoxic for NHLF and A549 cells. To determine the effect of the HNP analogs on eukaryotic cells, lung epithelial cells (A549) or normal human lung fibroblasts (NHLF) were incubated with increasing amounts of HNP or the three HNP-ornithine analogs for 24h in serum-free media. HNP and HNP-(R14,24orn) at 10 ug/ml and HNP-(R14orn) and HNP-(R24orn) at 25 ug/ml increased the number of A549 cells (as measured by a tetrazolium reduction assay). HNP and the three analogs were toxic for NHLF at concentrations less than 10 ug/ml. HNP-(R14orn) and HNP-(R24orn) were toxic for A549 cells at concentrations above 40 ug/ml. In contrast, replacement of arginines with ornithines at amino acids 14 and 24 abolished the cytotoxic effect of HNP on A549 cells at concentrations to 100 ug/ml. The toxicity of HNP and ornithine analogs (50 ug/ml) was also tested on NCI-H441 and SAEC cells and compared to that seen with A549 and NHLF cells. The HNP analogs showed similar or less toxicity than HNP. IL-8, a chemoattractant, modulates the inflammatory response by recruiting neutrophils to the lung. Higher levels of IL-8 in BALF and plasma were found in IPF patients compared to healthy volunteers. HNP-1 has been reported to induce release of IL-8 and other chemokines and cytokines from epithelial cells and fibroblasts. To determine the effects of HNP and the ornithine analogs on IL-8 release by A549 cells, cells were incubated with the ornithine analogs and IL-8 in the medium was measured by immunoassay. HNP with a single ornithine substitution at concentrations up to 20 ug/ml increased IL-8 release as did HNP-(R14,24orn), up to 100 ug/ml, a range of concentrations not cytotoxic for A549 cells. HNP-1 increased TGF-b1 release by fibroblasts but reduced TGF-b1 production by A549 cells. A549 cells were incubated with increasing amounts of HNP analogs and TGF-b1 release was determined by immunoassay. HNP-(R24orn) and HNP-(R14,24orn) inhibited release of less TGF-b1 than did HNP and HNP-(R14orn) at equivalent concentrations. HNP1-4, released from neutrophil azurophilic granules, act as natural antibiotics, killing a variety of microorganisms. Toxicities of the HNP-ornithine analogs for E.coli-TG1 and P.aeruginosa were compared to HNP. After one hour incubation, colony-forming units (CFU) were determined by plate assay. HNP-(R14,24orn) was toxic for P.aeruginosa and E.coli-TG1. HNP-(R24orn) was not toxic for E.coli-TG1 or P.aeruginosa. HNP-(R14orn) was toxic for P.aeruginosa at 3 ug/ml but inactive against E.coli-TG1. Surprisingly, the mono-substituted analogs did not exhibit HNP-1 antibacterial activity but were toxic for epithelial cells; antibacterial activity was observed, however, with ornithine at positions 14 and 24. HNP (3 ug) was not toxic for either E. coli (ATCC 25922) or S. aureus (ATCC 29213). To evaluate how well the ornithine analogs serve as substrates for ART1, nicotinamide release concurrent with ADP-ribose transfer from NAD to the acceptor was measured after incubation of the peptides with ART1 and NAD; HNP was compared to agmatine, a decarboxylated form of arginine which is a known ADP-ribose acceptor used by ART1. HNP and HNP-(R14orn) were better acceptors of ADP-ribose than agmatine. A small amount of nicotinamide release was observed in the presence of ART1, NAD and HNP-(R14,24orn), indicating that under these assay conditions, arginine 5 and arginine 15 are poor ADP-ribose acceptors in the ART1-catalyzed reaction. To identify the products of the reaction of the ornithine analogs, ART1 and NAD, the products were separated by HPLC, purified and analyzed by MS. The early peaks in the HPLC separations were identified as ADP-ribosylated-HNP-(R14orn), or ADP-ribosylated-HNP-(R24orn), whereas the later peaks were identified as the unmodified forms. In contrast, three products were separated from the reaction of HNP-1 with NAD and ART1. Compared to HNP-1, the unmodified analogs were found at an earlier HPLC elution time, suggesting that they are less hydrophobic than HNP. ADP-ribosylated-HNP-(R24orn) eluted at a significantly earlier time, suggesting the product is as hydrophobic as the di-modified HNP-1. ADP-ribosylarginine 5 or 15 HNP were not identified in the reaction products or in HNP-(R14,24orn). Increased ADP-ribosylation of HNP-1 and HNP-(R14,24orn) was observed after reduction of the disulfide bridges with dithiothreitol. These data suggest that the quaternary structure of HNP and the analogs limit the accessibility of arginines for ADP-ribosylation by ART1. As observed by confocal laser microscopy, HNP-1 at high concentrations (10-20 ug/ml) rapidly entered A549 cells, co-localized with the ER and stimulated apoptotic cell death. The reduced cytotoxicity of HNP after di-ornithine substitution could result from changes to HNP folding that may affect its capacity to enter the cell or localize to the ER. Substitution of ornithines for two arginines in oncocin, a 19 amino acid peptide sequence derived from the insect Oncopeltus peptide 4, resulted in a peptide that was active against E.coli, but non-toxic to HeLa cells and stable in serum. Conversion of HNP-1 to the di-ornithine form resulted in reduction of injury to the epithelial cells in the airway and preservation of the proliferative response believed to be involved in wound repair. Arginines or ornithines at positions 14 and 24 in the HNP primary sequence are critical for antibacterial activity. Although lysine, arginine and ornithine have equivalent charge, the replacement of arginine by lysine affected the antimicrobial activity of alpha-defensins. The virtual lethal dose for 90% of bacteria (vLD90) increased when ornithine replaced arginine or lysine at residues 14, 15, and 24. HNP-(R14,24orn) with arginine at residue 15, however, retained significant antibacterial activity against P. aeruginosa and E. coli. suggesting that ornithine 15 had an effect other than charge. Moreover, in contrast to the toxicity of replacing arginine 14 and 24 as in HNP-(R14,24orn), the replacement of arginine 24 with ornithine abolished the antibacterial activity against P. aeruginosa and E. coli, demonstrating that the activity of HNP-1 is affected by the number and position of arginine residues replaced by ornithine. In summary, ADP-ribosylation of arginines at residues 14 and 24 by ART1 regulates HNP-1 biological activities. Site-specific ADP-ribosylation by ART1, abolishes HNP-1 antimicrobial and cytotoxic activities. HNP with ADP-ribosylated arginines 14 and 24 non-enzymatically converted to ornithines is not cytotoxic for epithelial cells but the antibacterial activity is preserved. HNP-(R14,24 orn) is a promising novel candidate; a stable peptide antibiotic that shows diminished toxicity for the epithelial cells that line the airway.

解释气道上皮在宿主防御感染中起着至关重要的作用。受损上皮的修复过程涉及细胞迁移和增殖，这是增加中性粒细胞防御素的过程。如之前报道的，HNP-1 对人肺成纤维细胞以及上皮细胞和内皮细胞的增殖和细胞毒性作用是剂量依赖性的。低浓度的 HNP-1 对正常人肺成纤维细胞 (NHLF) 和 A549 上皮细胞具有增殖作用。然而，当浓度大于 20g/ml 时，HNP 对 NHLF 和 A549 细胞具有细胞毒性。为了确定HNP类似物对真核细胞的影响，将肺上皮细胞(A549)或正常人肺成纤维细胞(NHLF)与逐渐增加量的HNP或三种HNP-鸟氨酸类似物在无血清培养基中孵育24小时。 10 ug/ml 的 HNP 和 HNP-(R14,24orn) 以及 25 ug/ml 的 HNP-(R14orn) 和 HNP-(R24orn) 增加了 A549 细胞的数量（通过四唑还原测定测定）。 HNP 和三种类似物在浓度低于 10 ug/ml 时对 NHLF 有毒。 HNP-(R14orn) 和 HNP-(R24orn) 在浓度高于 40 ug/ml 时对 A549 细胞有毒。相反，在氨基酸14和24处用鸟氨酸替换精氨酸消除了浓度为100ug/ml的HNP对A549细胞的细胞毒性作用。还在 NCI-H441 和 SAEC 细胞上测试了 HNP 和鸟氨酸类似物 (50 ug/ml) 的毒性，并与 A549 和 NHLF 细胞中观察到的毒性进行了比较。 HNP 类似物显示出与 HNP 相似或更低的毒性。 IL-8 是一种化学引诱剂，通过将中性粒细胞募集至肺部来调节炎症反应。与健康志愿者相比，IPF 患者 BALF 和血浆中的 IL-8 水平较高。据报道，HNP-1 可诱导上皮细胞和成纤维细胞释放 IL-8 和其他趋化因子和细胞因子。为了确定HNP和鸟氨酸类似物对A549细胞释放IL-8的影响，将细胞与鸟氨酸类似物一起孵育，并通过免疫测定法测量培养基中的IL-8。浓度高达 20 ug/ml 的具有单一鸟氨酸取代的 HNP 增加了 IL-8 的释放，与浓度高达 100 ug/ml 的 HNP-(R14,24orn) 一样，该浓度范围对 A549 细胞没有细胞毒性。 HNP-1 增加成纤维细胞的 TGF-b1 释放，但减少 A549 细胞的 TGF-b1 产生。 A549 细胞与逐渐增加量的 HNP 类似物一起孵育，并通过免疫测定法测定 TGF-b1 的释放。与同等浓度的HNP和HNP-(R14orn)相比，HNP-(R24orn)和HNP-(R14,24orn)抑制的TGF-b1释放更少。 HNP1-4从中性粒细胞嗜天青颗粒中释放出来，充当天然抗生素，杀死多种微生物。将 HNP-鸟氨酸类似物对大肠杆菌-TG1 和铜绿假单胞菌的毒性与 HNP 进行了比较。孵育一小时后，通过平板测定确定菌落形成单位（CFU）。 HNP-(R14,24orn) 对铜绿假单胞菌和大肠杆菌-TG1 有毒。 HNP-(R24orn) 对大肠杆菌-TG1 或铜绿假单胞菌没有毒性。 HNP-(R14orn) 在 3 ug/ml 时对铜绿假单胞菌有毒性，但对大肠杆菌-TG1 无活性。令人惊讶的是，单取代类似物不表现出 HNP-1 抗菌活性，但对上皮细胞具有毒性；然而，在位置 14 和 24 处观察到鸟氨酸具有抗菌活性。HNP (3 ug) 对大肠杆菌 (ATCC 25922) 或金黄色葡萄球菌 (ATCC 29213) 没有毒性。为了评估鸟氨酸类似物作为 ART1 底物的效果，在将肽与 ART1 和 NAD 一起孵育后，测量与 ADP-核糖从 NAD 转移到受体同时发生的烟酰胺释放；将 HNP 与胍丁胺进行比较，胍丁胺是精氨酸的脱羧形式，是 ART1 使用的已知 ADP-核糖受体。 HNP 和 HNP-(R14orn) 是比胍丁胺更好的 ADP-核糖受体。在 ART1、NAD 和 HNP-(R14,24orn) 存在的情况下观察到少量烟酰胺释放，表明在这些测定条件下，精氨酸 5 和精氨酸 15 在 ART1 催化反应中是较差的 ADP-核糖受体。为了鉴定鸟氨酸类似物、ART1和NAD的反应产物，通过HPLC分离产物，通过MS纯化和分析。 HPLC 分离中的早期峰被鉴定为 ADP-核糖基化-HNP-(R14orn) 或 ADP-核糖基化-HNP-(R24orn)，而后期峰被鉴定为未修饰形式。相比之下，HNP-1 与 NAD 和 ART1 的反应分离出三种产物。与 HNP-1 相比，未修饰的类似物在更早的 HPLC 洗脱时间中被发现，表明它们的疏水性低于 HNP。 ADP-核糖基化-HNP-(R24orn) 在明显更早的时间洗脱，表明该产物与双修饰的 HNP-1 一样疏水。在反应产物或 HNP-(R14,24orn) 中未鉴定出 ADP-核糖精氨酸 5 或 15 HNP。用二硫苏糖醇还原二硫键后，观察到 HNP-1 和 HNP-(R14,24orn) 的 ADP-核糖基化增加。这些数据表明，HNP 及其类似物的四级结构限制了 ART1 对精氨酸进行 ADP-核糖基化的可及性。通过共聚焦激光显微镜观察到，高浓度（10-20 ug/ml）的 HNP-1 迅速进入 A549 细胞，与 ER 共定位并刺激细胞凋亡。二鸟氨酸取代后 HNP 的细胞毒性降低可能是由于 HNP 折叠的变化导致的，这可能会影响其进入细胞或定位到 ER 的能力。 Oncocin 是一种源自昆虫 Oncopeltus 肽 4 的 19 个氨基酸肽序列，用鸟氨酸取代了 Oncopeltus 肽 4 中的两个精氨酸，产生了一种对大肠杆菌有活性、但对 HeLa 细胞无毒且在血清中稳定的肽。 HNP-1 转化为二鸟氨酸形式可减少气道上皮细胞的损伤，并保留据信参与伤口修复的增殖反应。 HNP 一级序列中第 14 和 24 位的精氨酸或鸟氨酸对于抗菌活性至关重要。尽管赖氨酸、精氨酸和鸟氨酸具有相同的电荷，但用赖氨酸替代精氨酸会影响α-防御素的抗菌活性。当鸟氨酸取代残基 14、15 和 24 处的精氨酸或赖氨酸时，90% 细菌的虚拟致死剂量 (vLD90) 增加。然而，HNP-(R14,24orn) 与残基 15 处的精氨酸保留了对疟原虫的显着抗菌活性。铜绿假单胞菌和大肠杆菌。表明鸟氨酸 15 具有电荷以外的作用。此外，与HNP-(R14,24orn)中替换精氨酸14和24的毒性相反，用鸟氨酸替换精氨酸24消除了对铜绿假单胞菌和大肠杆菌的抗菌活性，这表明HNP-的活性1受到鸟氨酸取代的精氨酸残基的数量和位置的影响。总之，ART1 对残基 14 和 24 处的精氨酸进行 ADP 核糖基化可调节 HNP-1 的生物活性。 ART1 的位点特异性 ADP-核糖基化消除了 HNP-1 抗菌和细胞毒性活性。具有 ADP 核糖基化精氨酸 14 和 24 非酶促转化为鸟氨酸的 HNP 对上皮细胞没有细胞毒性，但保留了抗菌活性。 HNP-(R14,24 orn)是一个有前途的新候选者；一种稳定的肽抗生素，对气道上皮细胞的毒性较小。