MAP1A AND MAP1B--SYNTHESIS, STRUCTURE, AND FUNCTION

神经退行性疾病每年影响数以百万计的人 在情感上和经济上都是残缺的。神经质的恢复 系统功能将需要再生神经元结构，包括 轴突和树突。微管形成细胞骨架系统 已知有助于轴突的结构完整性和 参与轴突内物质的运输。在这项研究中， 微管蛋白的分子结构及其相互作用 与生长中的轴突相关的将被研究。其中一个的基因 蛋白质-微管相关蛋白1B-可能是 人类致命疾病脊髓性肌萎缩症中的基因缺陷。 一种作用于微管相关的不寻常的处理机制 蛋白1B(MAP1B)是最近发现的一种蛋白。这个机制产生了两个 来自单一多蛋白前体的MAP1B亚基。如果这种多聚蛋白 未处理，微管组织发生显著变化 可能是由于微管交联所致。MAP1B的加工 将通过鉴定处理的蛋白水解酶来进一步表征 MAP1B。体外表达的多聚蛋白将作为底物用于 测定MAP1B特异性蛋白水解酶活性。新的证据表明 MAP1A在结构上与MAP1B相关，它也是从一个 含有两个亚基的多蛋白前体，MAP1AHeavy 链和轻链2.识别附近表位的抗体 推测的MAP1A多聚蛋白的羧基末端将用于 多肽图实验确定MAP1A多蛋白是否为 如最近提出的那样，可在体内检测到(Langkopf等人，在出版中)。 轻链3的一级序列是一种低分子量亚单位 MAP1A和MAP1B的共同点将通过克隆和测序来确定 从lambda GT 11表达文库中获得该蛋白的编码基因。这个 用于选择这些克隆的抗体将生成合成的 其序列是通过Edman降解得到的 轻链3的N末端。抗轻链3的抗体也将 用于确定轻链3是否为微管结合的一部分 MAP1A和MAP1B的结构域。最后，轻链1的分布， 大脑中的轻链2和轻链3将由 免疫组织化学方法。低相对分子质量的定位 MAP1A和MAP1B亚基将与MAP1B的本地化进行比较 沉重的锁链。亚基相对亚基的差异定位 它们的重链可能表明亚基组成调节重 链函数。

Neurodegenerative diseases affect millions of people each year in ways that are emotionally and financially crippling. Restoration of nervous system function will require regeneration of neuron structure including axonal and dendritic processes. Microtubules form a cytoskeletal system known to contribute to the structural integrity of axons and to participate in the transport of materials within axons. In this study, the molecular structure and interactions of microtubule proteins associated with growing axons will be studied. The gene for one of these proteins - microtubule associated protein 1B - is likely to be the defective gene in the fatal human disease spinal muscular atrophy. An unusual processing mechanism that acts on microtubule-associated protein 1B (MAP1B) was recently identified. This mechanism produces two MAP1B subunits from a single polyprotein precursor. If the polyprotein is not processed, significant alterations in microtubule organization might occur due to microtubule cross-linking. The processing of MAP1B will be further characterized by identifying the protease that processes MAP1B. Polyprotein expressed in vitro will be used as the substrate to assay MAP1B specific protease activity. New evidence indicates that MAP1A is structurally related to MAP1B and that it is also derived from a polyprotein precursor containing two of its subunits, the MAP1A heavy chain and light chain 2. Antibodies that recognize epitopes near the carboxyl-terminus of the putative MAP1A polyprotein will be used in peptide-mapping experiments to determine if the MAP1A polyprotein is detectable in vivo as was recently suggested (Langkopf et al., In press). The primary sequence of light chain 3, a low molecular weight subunit common to MAP1A and MAP1B, will be determined by cloning and sequencing cDNA encoding this protein from a lambda gt 11 expression library. The antibody used to select these clones will be generated to a synthetic peptide whose sequence was obtained by Edman degradation of the N-terminus of light chain 3. The anti-light chain 3 antibody will also be used to determine if light chain 3 is part of the microtubule-binding domains of MAP1A and MAP1B. Finally, the distribution of light chain 1, light chain 2, and light chain 3 in brain will be determined by immunohistochemical methods. The localization of low molecular weight MAP1A and MAP1B subunits will be compared to the localization of the heavy chains. Differential localization of the subunits relative to their heavy chains may indicate that subunit composition regulates heavy chain function.