Alpha2-macroglobulin in peripheral nerve injury

描述(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，有助于沃勒变性的进展和痛性神经疾病的发展。α2-巨球蛋白(A2M)是一种广泛存在于血浆和细胞外间隙的蛋白水解酶抑制剂，它还可以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质相互作用结构域(PID)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。PID和LRP-1识别位点的暴露受2M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。由此得到的制剂(称为MAC)表达了对肿瘤坏死因子-α和白介素1-β的结合亲和力增加。Mac在小鼠身上也显示出强大的抗炎活性。在新的未发表的研究中，我们表明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。我们展示了我们通过突变重组蛋白来扰乱各种a2M活性的工作的快速进展。我们推测，MAC和其他a2M衍生物可能是治疗周围神经损伤、坐骨神经痛和腰椎间盘突出症的有效实验疗法。为了验证这一假设，在目标1中，将在小鼠坐骨神经挤压和慢性挤压损伤实验中比较a2M、甲胺激活的a2M和MAC的活性。这些药物阻止疼痛神经疾病发展的能力也将得到评估。为了测试自然产生的a2M的活性，将在a2M/鼠球蛋白基因敲除小鼠中进行神经损伤实验。为了验证MAC除了中和肿瘤坏死因子-a外还通过其他机制发挥作用的假设，将在肿瘤坏死因子-a基因缺失的小鼠身上进行神经损伤研究。在具体目标2中，将对全长人类a2M进行工程改造，以独立中和或修改两个ID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调节依赖LRP-1的细胞信号，通过这些机制，MAC和其他形式的a2M可能调节受损神经的细胞生理。在具体目标3中，将在神经损伤模型系统中测试突变的全长a2M，其中特定区域的功能被破坏。这些研究的目的是在体内测试MAC和其他a2M衍生物在周围神经损伤中的保护机制。在目标3中计划进行更多的研究，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突变形式的a2M来阐明a2M的结构-功能关系。该项目将有助于我们理解细胞外介质在周围神经损伤中的作用，并为产生新的实验性蛋白质疗法提供潜在的可能性。

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 In new unpublished studies, we show that MAC expresses anti-inflammatory activity in injured peripheral nerves and, as a result, may be axonal protective.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