DISRUPTION OF CALMODULIN/GAP 43 BINDING BY LEAD

铅破坏钙调蛋白/GAP 43 的结合

• 批准号: 6134155
• 负责人: PAUL M STEMMER
• 金额: $ 10万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6134155
• 关键词: PC12 cells; atomic absorption spectrometry; binding sites; biological signal transduction; calcium flux; calmodulin; immunocytochemistry; laboratory rat; lead; lead poisoning; microinjections; neural growth associated protein; neurons; neurotoxins; phosphorylation; protein binding; protein kinase C; protein purification; protein transport; tissue /cell culture; western blottings

Work to elucidate mechanisms underlying Pb2+ neurotoxicity now focuses on determining what biochemical or anatomical changes occur at the pM Pb2+ concentrations associated with Pb2+-induced behavioral changes and functional deficits. Protein Kinase C (PKC) is the only enzyme important for signal transduction which is currently known to be affected by Pb2+ at such low concentrations. One other pb2+-induced cellular change that is important for calmodulin-dependent signal transduction is a two-fold increase in the resting intracellular Ca2+ concentration from approximately 100 to 200 nM. At this time, there are no known biochemical or anatomical sequelae of either the PKC stimulation or the increase in resting Ca2+ concentration that can be linked to Pb2+ toxicities. This project is designed to take that next step and demonstrate that Pb2+ disrupts the balance of calmodulin-dependent signal transduction in neurons by causing a redistribution of calmodulin (CaM) away from PKC-sensitive and Ca2+-sensitive CaM binding sites, including CaM-reservoir proteins. The CaM reservoirs in question are proteins such as GAP-43 (neuromodulin) which bind CaM at IQ-domains in a Cat+-inhibitable manner. CaM binding to several of the IQ-domain proteins, including neuromodulin, is blocked when they are phosphorylated by PKC. The CaM-reservoir proteins are most abundant in neurons and neuromodulin is of particular importance to Pb toxicity as it is located predominantly in the nerve growth cone and presynaptic terminals and is essential for normal nerve growth and synaptic transmission. CaM and the interaction of CaM with neuromodulin are also essential for proper nerve growth cone development and for synaptic transmission. The hypothesis to be tested is that low level lead exposure causes redistribution of CaM from membrane associated neuromodulin binding sites to cytosol in neuronal cells. It is further hypothesized that both a Pb2+-dependent stimulation of PKC with a subsequent phosphorylation of neuromodulin and a Pb2+-dependent increase in resting intracellular Ca2+ concentration are causally important in the redistribution of CaM away from neuromodulin and the membrane. The long term goal of this research program is to determine the importance of IQ-domain CaM-binding proteins as targets for Pb2+ toxicity and to elucidate the mechanistic importance of PKC stimulation and resting intracellular Ca2+ fluctuations as underlying the developmental deficits seen in Pb2+ exposed populations.

现在，阐明PB2+神经毒性为基础的机制的工作重点是确定在与PB2+诱导的行为变化和功能缺陷相关的PM PB2+浓度下发生的生化或解剖变化。 蛋白激酶C（PKC）是对信号转导重要的唯一酶，目前已知在这种低浓度下受PB2+的影响。 对于钙调蛋白依赖性信号转导至关重要的另一种PB2+诱导的细胞变化是静止的细胞内Ca2+浓度从大约100到200 nm增加了两倍。 目前，尚无PKC刺激或静息Ca2+浓度的增加的生化或解剖后遗症，可以与PB2+毒性有关。 该项目旨在采取下一步，并证明PB2+通过导致从PKC敏感和CA2+敏感的CAM cAM结合位点（包括CAM-Reservoir蛋白）的重新分布来破坏神经元中钙调蛋白依赖性信号转导的平衡。 所讨论的CAM储存剂是蛋白质，例如GAP-43（NeuroModulin），它们以CAT+可约束的方式在IQ-domain上结合CAM。 当PKC磷酸化时，CAM与几种智商域蛋白（包括神经统计蛋白）的结合被阻断。 CAM-腌制蛋白在神经元中最丰富，神经统治蛋白对PB毒性特别重要，因为它主要位于神经生长锥和突触前终末中，对于正常的神经生长和突触传播至关重要。 CAM和CAM与神经统治的相互作用对于适当的神经生长锥发育和突触传播也是必不可少的。 要测试的假设是，低水平的铅暴露会导致膜相关的神经统计素结合位点与神经元细胞中的胞质溶胶重新分布。 进一步假设，PB2+依赖性刺激PKC具有随后的磷酸化神经统治蛋白的磷酸化和PB2+依赖性的增加，而静止的细胞内Ca2+浓度对于CAM远离神经元素和膜的重新分布都非常重要。 该研究计划的长期目标是确定智商结合蛋白作为PB2+毒性的目标的重要性，并阐明PKC刺激的机理重要性和静止的细胞内CA2+波动，因为它是PB2+暴露群体中所见的发育缺陷的基本。