Alpha2-macroglobulin in peripheral nerve injury

周围神经损伤中的α2-巨球蛋白

• 批准号: 7874555
• 负责人: STEVEN L. GONIAS
• 金额: $ 33.46万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 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/7874555
• 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 Culture Techniques; Cell physiology; Complex; Crush Injury; Development; 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; Tissues; Toxin; Tumor Necrosis Factor-alpha; Tumor Necrosis Factors; Wallerian Degeneration; Work; alpha 2-Glucoproteins; amyloid peptide; base; chronic constriction injury; chronic neuropathic pain; constriction; cytokine; design; extracellular; 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

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）：在周围神经损伤中，炎性细胞因子，例如肿瘤坏死因子-Alpha（TNF-A），有助于沃勒（Wallerian）变性的进展和疼痛的神经病的发展。 Alpha 2-杆球蛋白（A2M）是在血浆和细胞外空间中发现的宽光谱蛋白酶抑制剂，它也调节细胞因子和生长因子的活性。后一种函数反映了A2M亚基结构中两个非共价蛋白相互作用域（PID）的活性。一个不同的序列介导了A2M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A2M构象变化的调节。我们开发了一种稳定A2M构象中间体的方法。所得的制剂（称为MAC）表示对TNF-A和白介素1Beta的结合亲和力增加。 MAC还表现出小鼠的有效抗炎活性。在新的未发表的研究中，我们表明MAC在受伤的周围神经中表达抗炎活性，因此可能是轴突保护性。我们在工作中证明了通过重组蛋白突变破坏各种A2M活性的快速进步。我们假设MAC和其他A2M衍生物可能是周围神经损伤，坐骨神经痛和腰椎椎间盘突出症的有效实验疗法。为了检验这一假设，在AIM 1中，将在小鼠的坐骨神经挤压和慢性收缩损伤实验中比较A2M，甲胺激活的A2M和MAC的活性。这些药物阻止疼痛神经病的发展的能力也将得到评估。为了测试天然发生的A2M的活性，将在A2M/Murinoglobulin基因敲除小鼠中进行神经损伤实验。为了检验以下假设：除中和TNF-A外，MAC通过机制功能，在TNF-A基因删除小鼠中将进行神经损伤研究。在特定的目标2中，将设计全长的人A2M，以独立中和或修改两个PID和LRP-1识别位点的功能。然后，我们将利用巨噬细胞和Schwann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A2M可以调节受伤神经中的细胞生理。在特定的目标3中，将在神经损伤模型系统中测试特定域的功能的突变全长A2M。这些研究的目的是测试MAC和其他A2M衍生物在体内具有保护性的机制。计划在AIM 3中进行其他研究，以利用我们在A2M中阐明结构 - 功能连接的关系，通过设计突变形式的A2M突变形式，具有增强的神经损伤活性。该项目将有助于我们对周围神经损伤中细胞外介体的理解，并为产生新型实验蛋白质疗法提供了潜力。