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Alpha2-macroglobulin in peripheral nerve injury

α2-巨球蛋白在周围神经损伤中的作用

基本信息

批准号：
7423916
负责人：
STEVEN L. GONIAS
金额：
$ 33.8万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-07-01 至 2012-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7423916
关键词：
Adult Affect Affinity Animal Model Anti-Inflammatory Agents Anti-inflammatory Applications Grants Axon Binding Binding Sites Biological Availability Biological Models Catabolism Cell physiology Complex Constriction procedure Crush Injury Cultured Cells Development Endopeptidases Engineering Equilibrium Event Extracellular Space Generations Genes Genetic Engineering Glycoproteins Goals Growth Factor Health Human In Vitro Inflammation Inflammatory Injury Interleukin-1 beta Interleukin-18 Knockout Mice Knowledge LDL-Receptor Related Protein 1 Length Libraries Ligation Lipoprotein Receptor Macroglobulins Mediating Mediator of activation protein Mental Depression Methods Methylamines Modeling Modification Molecular Molecular Conformation Morbidity - disease rate Mus Mutate Mutation Nerve Nerve Crush Neuraxis Outcome Peptide Hydrolases Peripheral Nerves Peripheral Nervous System Peripheral nerve injury Phenotype Plasma Play Preparation Process Protease Inhibitor Protein Binding Domain Proteins Recombinant Proteins Recombinants Regulation Research Personnel Role Schwann Cells Sciatica Shipping Ships Signal Transduction Site Structure Testing Therapeutic Thinking Tissues Toxin Tumor Necrosis Factor-alpha Tumor Necrosis Factors Wallerian Degeneration Work alpha 2-Glucoproteins amyloid peptide base chronic constriction injury chronic neuropathic pain cytokine design extracellular human TNF protein improved in vivo injured macrophage methylamine mortality mouse model murinoglobulin nerve injury novel novel therapeutics painful neuropathy peptide A prevent programs protein function receptor research study response sciatic nerve therapeutic protein

项目摘要

DESCRIPTION (provided by applicant): In peripheral nerve injury, inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-a), contribute to the progression of Wallerian degeneration and the development of painful neuropathies. Alpha 2-Macroglobulin (a2M) is a broad spectrum protease inhibitor found in the plasma and extracellular spaces, which also regulates the activity of cytokines and growth factors. The latter function reflects the activity of two non-covalent protein interaction domains (PIDs) in the structure of the a2M subunit. A distinct sequence mediates interaction of a2M with the receptor, low density lipoprotein receptor-related protein-1 (LRP-1). Exposure of the PIDs and the LRP-1 recognition site is regulated by a2M conformational change. We developed a method for stabilizing a2M conformational intermediates. The resulting preparation (referred to as MAC) expresses increased binding affinity for TNF-a and interleukin-1beta. MAC also demonstrates potent anti-inflammatory activity in mice. In new unpublished studies, we show that MAC expresses anti-inflammatory activity in injured peripheral nerves and, as a result, may be axonal protective. We demonstrate rapid progress in our work to disrupt various a2M activities by mutation of the recombinant protein. We hypothesize that MAC and other a2M derivatives may be potent experimental therapeutics in peripheral nerve injury, sciatica, and lumbar disc herniation. To test this hypothesis, in Aim 1, the activities of a2M, methylamine-activated a2M, and MAC will be compared in sciatic nerve crush and chronic constriction injury experiments in mice. The ability of these agents to block development of painful neuropathies also will be assessed. To test the activity of naturally occurring a2M, nerve injury experiments will be performed in a2M/murinoglobulin gene knock-out mice. To test the hypothesis that MAC functions by mechanisms in addition to neutralizing TNF-a, nerve injury studies will be performed in TNF-a gene-deleted mice. In Specific Aim 2, full-length human a2M will be engineered to independently neutralize or modify the function of the two PIDs and the LRP-1 recognition site. We will then utilize macrophage and Schwann cell culture model systems to test mechanisms, including cytokine- binding and regulation of LRP-1-dependent cell signaling, by which MAC and other forms of a2M may regulate cell physiology in the injured nerve. In Specific Aim 3, mutated full-length a2M, in which the function of specific domains is disrupted, will be tested in the nerve injury model systems. The goal of these studies is to test the mechanism by which MAC and other a2M derivatives are protective in peripheral nerve injury in vivo. Additional studies are planned in Aim 3 to capitalize on our work elucidating structure-function relation- ships in a2M by designing mutated forms of a2M with enhanced activity in nerve injury. This project will contribute to our understanding of extracellular mediators in peripheral nerve injury and offer the potential for generating novel experimental protein therapeutics.

描述（由申请人提供）：在外周神经损伤中，炎性细胞因子，如肿瘤坏死因子-α（TNF-α），有助于沃勒变性的进展和疼痛性神经病的发展。α 2-巨球蛋白（α 2 M）是在血浆和细胞外空间中发现的广谱蛋白酶抑制剂，其还调节细胞因子和生长因子的活性。后一种功能反映了α 2 M亚基结构中两个非共价蛋白质相互作用结构域（PID）的活性。不同的序列介导α 2 M与受体低密度脂蛋白受体相关蛋白-1（LRP-1）的相互作用。PID和LRP-1识别位点的暴露受a2 M构象变化的调节。我们发展了一种稳定α 2 M构象中间体的方法。所得制剂（称为MAC）表达对TNF-α和白细胞介素-1 β的增加的结合亲和力。MAC还在小鼠中显示出有效的抗炎活性。在新的未发表的研究中，我们表明MAC在受损的外周神经中表达抗炎活性，因此可能具有轴突保护作用。我们在我们的工作中展示了通过重组蛋白的突变来破坏各种α 2 M活性的快速进展。我们假设MAC和其他α 2 M衍生物可能是周围神经损伤、坐骨神经痛和腰椎间盘突出症的有效实验治疗剂。为了检验这一假设，在目的1中，将在小鼠坐骨神经挤压和慢性压迫性损伤实验中比较α 2 M、甲胺活化的α 2 M和MAC的活性。还将评估这些药物阻断疼痛性神经病发展的能力。为了测试天然存在的α 2 M的活性，将在α 2 M/鼠胰岛素基因敲除小鼠中进行神经损伤实验。为了检验MAC通过除中和TNF-α之外的机制起作用的假设，将在TNF-α基因缺失的小鼠中进行神经损伤研究。在特异性目标2中，全长人α 2 M将被工程化以独立地中和或修饰两个PID和LRP-1识别位点的功能。然后，我们将利用巨噬细胞和许旺细胞培养模型系统来测试机制，包括细胞因子结合和LRP-1依赖性细胞信号传导的调节，MAC和其他形式的α 2 M可以通过该机制调节受损神经中的细胞生理学。在特定目标3中，将在神经损伤模型系统中测试突变的全长α 2 M，其中特定结构域的功能被破坏。这些研究的目的是测试MAC和其它α 2 M衍生物在体内外周神经损伤中保护的机制。在目标3中计划了额外的研究，以利用我们的工作，通过设计在神经损伤中具有增强活性的α 2 M的突变形式来阐明α 2 M中的结构-功能关系。该项目将有助于我们了解周围神经损伤中的细胞外介质，并为产生新的实验性蛋白质疗法提供潜力。