Alpha2-macroglobulin in peripheral nerve injury

描述（由申请人提供）：在周围神经损伤中，炎性细胞因子，如肿瘤坏死因子- α (TNF-a)，有助于沃勒氏变性的进展和疼痛性神经病的发展。α 2-巨球蛋白（α 2-Macroglobulin, a2M）是一种广谱蛋白酶抑制剂，存在于血浆和细胞外间隙，还能调节细胞因子和生长因子的活性。后者的功能反映了a2M亚基结构中两个非共价蛋白相互作用域（PIDs）的活性。一个独特的序列介导a2M与受体低密度脂蛋白受体相关蛋白-1 （LRP-1）的相互作用。PIDs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。由此产生的制剂（称为MAC）表达了对TNF-a和白细胞介素-1 β的增强结合亲和力。MAC在小鼠中也显示出强大的抗炎活性。在新的未发表的研究中，我们发现MAC在受损的周围神经中表达抗炎活性，因此可能具有轴突保护作用。我们在通过重组蛋白的突变来破坏各种a2M活性的工作中取得了快速进展。我们假设MAC和其他a2M衍生物可能是周围神经损伤、坐骨神经痛和腰椎间盘突出症的有效实验治疗药物。为了验证这一假设，在Aim 1中，我们将在小鼠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。这些药物阻断疼痛性神经病变发展的能力也将被评估。为了检测自然产生的a2M的活性，我们将在a2M/小蛋白血红蛋白基因敲除小鼠中进行神经损伤实验。为了验证MAC除中和TNF-a外还通过其他机制起作用的假设，将在TNF-a基因缺失的小鼠中进行神经损伤研究。在Specific Aim 2中，全长人类a2M将被设计成独立中和或修饰这两个pid和LRP-1识别位点的功能。然后，我们将利用巨噬细胞和雪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依赖性细胞信号的调节，MAC和其他形式的a2M可能通过这些机制调节受损神经的细胞生理。在Specific Aim 3中，将在神经损伤模型系统中测试突变的全长a2M，其中特定结构域的功能被破坏。这些研究的目的是测试MAC和其他a2M衍生物在体内保护周围神经损伤的机制。Aim 3计划进一步的研究，利用我们的工作阐明a2M的结构-功能关系，通过设计a2M的突变形式，增强神经损伤的活性。该项目将有助于我们对周围神经损伤中的细胞外介质的理解，并为产生新的实验性蛋白质疗法提供潜力。

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 In new unpublished studies, we show that MAC expresses anti-inflammatory activity in injured peripheral nerves and, as a result, may be axonal protective.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.