MAP1A AND MAP1B--SYNTHESIS, STRUCTURE, AND FUNCTION

MAP1A 和 MAP1B——合成、结构和功能

• 批准号: 2268950
• 负责人: JAMES A HAMMARBACK
• 金额: $ 11.49万
• 依托单位: WAKE FOREST UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-04-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2268950
• 关键词: endopeptidases; enzyme activity; epitope mapping; immunocytochemistry; laboratory mouse; laboratory rabbit; laboratory rat; microtubule associated protein; molecular cloning; nucleic acid sequence; protein metabolism; protein sequence; protein structure function

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.

神经退行性疾病每年影响数百万人， 在情感上和经济上都很严重 神经恢复 系统功能将需要神经元结构再生，包括 轴突和树突状突起。 微管形成细胞骨架系统 已知有助于轴突的结构完整性， 参与轴突内物质的运输。 在本研究中， 微管蛋白的分子结构和相互作用 与轴突生长相关的研究。 其中一种的基因 蛋白质-微管相关蛋白1B -很可能是 致命的人类疾病脊髓性肌萎缩症的缺陷基因。 一种不寻常的加工机制，作用于微管相关的 蛋白1B（MAP 1B）是最近发现的一种蛋白质。 该机制产生两个 MAP 1B亚基来自单个多聚蛋白前体。 如果多聚蛋白 未被加工，微管组织的显著改变 可能是由于微管交联。 MAP 1B的处理 将进一步通过鉴定处理蛋白酶来表征 MAP 1B。 体外表达的多聚蛋白将用作底物， 测定MAP 1B特异性蛋白酶活性。 新的证据表明， MAP 1A在结构上与MAP 1B相关，并且它也来源于一种 多蛋白前体含有两个亚基，MAP 1A重链 链和轻链2.识别抗原表位的抗体 推定的MAP 1A多蛋白的羧基末端将用于 肽图谱实验，以确定MAP 1A多蛋白是否 如最近提出的在体内可检测的（Langkopf等，待印）。 轻链3（一种低分子量亚基）的一级序列 MAP 1A和MAP 1B的共同点，将通过克隆和测序来确定 来自λ gt 11表达文库的编码该蛋白质的cDNA。 的 用于选择这些克隆的抗体将被生成为合成的 肽，其序列是通过Edman降解 轻链3的N-末端。抗轻链3抗体也将是 用于确定轻链3是否是微管结合的一部分 MAP 1A和MAP 1B。 最后，轻链1的分布， 脑中的轻链2和轻链3将通过 免疫组织化学方法。 低分子量的国产化 MAP 1A和MAP 1B亚基将与MAP 1A和MAP 1B亚基的定位进行比较。 沉重的锁链 亚基相对于 它们的重链可能表明亚基组成调节重链， 链式函数