MULTIPLE FORMS OF HUMAN TYROSINE HYDROXYLASE

这个项目的长期目标是研究大脑的参与 儿茶酚胺系统与心理健康。多巴胺能系统已经被 与精神分裂症有关，去甲肾上腺素系统也有牵连 在抑郁中。这两个儿茶酚胺能系统的一个共同特征是 它们生物合成中最初的限速酶--酪氨酸羟基酶。 酪氨酸羟化酶(TH)的活性受一定数量的调节。 短期内包括蛋白质在内的过程 磷酸化/去磷酸化，并在长期内，诱导 酶的合成。在人类中，有可能出现额外水平的 监管也是存在的。人TH基因(MRNAHTH)的选择性表达 剪接，以及两种不同的mRNAHTH在 死后的人脑样本。从申请人的工作中 实验室，大脑中存在的两种形式的mRNAHTH的产物是 预测具有不同底物特异性的蛋白激酶。 因此，在人类中，选择性剪接的变化也可能有助于 对TH活性的调节。 到目前为止，发表在多种形式的人类TH上的研究只分析了 该核酸及其重组产物。现在 应用程序建议确定是否存在和区域分布 人死后脑内TH蛋白本身的不同形态 样本。抗体将被提升为代表 由选择性剪接产生的独特的氨基酸序列。有选择性的 对不同形式的TH的识别将针对免疫原进行测试 并对重组载体产生的人TH全酶产生抑制作用。AS 必要时，用异种免疫原预吸附法生产 单特异性抗体。定量的蛋白质印迹分析将是 开发用于分析不同HTH形式的级别和分布 在死后的人脑中。免疫细胞化学将用于分析 HTH在脑内的区域、细胞和亚细胞分布。 预测选择性剪接会改变丝氨酸的磷酸化 31对神经生长因子和佛波酯敏感 作为钙/钙调蛋白依赖性蛋白激酶的底物 重组hTH在AtT-20细胞中的位点特异性磷酸化， 人神经母细胞瘤LA-N细胞中的HTH和人完整尸检中的HTH 组织(纹状体和伏隔核的突触体， 肾上腺髓质中的嗜铬细胞)，以及从 将对人类嗜铬细胞瘤(如有)进行调查。

The long-range goals of this project are to study the involvement of brain catecholamine systems in mental health. Dopaminergic systems have been implicated in schizophrenia, and noradrenergic systems have been implicated in depression. One common feature of both catecholaminergic systems is the initial, rate-limiting enzyme in their biosynthesis-tyrosine hydroxylase. The activity of tyrosine hydroxylase (TH) is regulated by a number processes which include, in the short-term, protein phosphorylation/dephosphorylation and, in the long-term, induction of enzyme synthesis. In humans, the possibility of an additional level of regulation also exists. Human TH mRNA (mRNAHTH) undergoes alternative splicing, and two different species of mRNAHTH have been identified in post-mortem human brain samples. From work on TH in the applicant's laboratory, the products of the two forms of mRNAHTH present in brain are predicted to possess different substrate specificities for protein kinases. Thus, in humans, changes in alternative splicing may also contribute to the regulation of TH activity. To date, studies published on multiple forms of human TH have analyzed only the nucleic acids and recombinant products therefrom. The present application proposes to determine the presence and regional distribution of the different forms of TH protein per se in post-mortem human brain samples. Antibodies will be raised to synthetic peptides representing the unique amino acid sequences created by the alternative splicing. Selective recognition of the different forms of TH will be tested against immunogens and against human TH holoenzymes produced from recombinant vectors. As necessary, preadsorption with the heteroimmunogens will be used to produce monospecific antibodies. Quantitative Western blot analyses will be developed to analyze the levels and distribution of the different HTH forms in post-mortem human brain. Immunocytochemistry will be used to analyze the regional, cellular and subcellular distribution of HTH forms in brain. Alternative splicing is predicted to change the phosphorylation of serine 31 from that of being nerve growth factor and phorbol ester sensitive to that of being a substrate for calcium/calmodulin-dependent protein kinase II. Site-specific phosphorylation of recombinant HTH in AtT-20 cells, of HTH in LA-N human neuroblastoma cells, of HTH in intact post-mortem human tissues (synaptosomes from corpus striatum and nucleus accumbens, chromaffin cells from adrenal medullae), and of HTH in cells isolated from human pheochromocytoma (as available) will be investigated.