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.

为了阐明Pb2+神经毒性的潜在机制，目前的工作集中在确定PmPb2+浓度下发生的与Pb2+诱导的行为变化和功能障碍相关的生化或解剖变化。蛋白激酶C(PKC)是目前已知的唯一重要的信号转导酶，在如此低的浓度下会受到Pb2+的影响。Pb2+引起的另一个对钙调蛋白依赖的信号转导重要的细胞变化是细胞内静息钙离子浓度从大约100 nM增加到200 nM，增加了两倍。目前，还没有已知的PKC刺激或静息钙浓度升高的生化或解剖学后遗症与Pb2+毒性有关。本项目旨在进行下一步的研究，并证明Pb2+通过引起钙调蛋白(CaM)从PKC敏感和钙敏感的CaM结合部位(包括CaM储藏蛋白)重新分布，从而扰乱神经元中钙调蛋白依赖的信号转导的平衡。所讨论的CaM储存库是像GAP-43(神经调制素)这样的蛋白质，它以Cat+抑制的方式将CaM结合在IQ结构域上。当它们被PKC磷酸化时，CaM与包括神经调制素在内的几个IQ结构域蛋白的结合被阻止。CaM储存库蛋白在神经元中含量最丰富，神经调制素对铅中毒特别重要，因为它主要位于神经生长锥和突触前终末，对于正常的神经生长和突触传递是必不可少的。CaM及其与神经调制素的相互作用对于正常的神经生长锥发育和突触传递也是必不可少的。需要检验的假设是，低水平的铅暴露导致CaM在神经细胞中从膜相关的神经调制素结合部位重新分布到胞浆。进一步推测，Pb2+依赖的PKC激活和随后的神经调制素的磷酸化以及Pb2+依赖的细胞内静息钙离子浓度的增加都是CaM从神经调制素和细胞膜重新分布的重要原因。这项研究计划的长期目标是确定IQ结构域CaM结合蛋白作为Pb2+毒性靶标的重要性，并阐明PKC刺激和静息细胞内钙波动作为导致Pb2+暴露人群发育缺陷的机制重要性。