Zebrafish TPSTs and Tyrosine-sulfated Proteins

描述（由申请人提供）：酪氨酸磺化翻译后蛋白质修饰在多细胞真核生物的蛋白质中广泛存在。负责蛋白酪氨酸硫酸化的酶已被确定为高尔基膜结合酪氨酸蛋白硫转移酶（TPST）。我们和其他人的研究表明，TPST在人类、小鼠和斑马鱼中存在多种亚型。不同的，但同源的，TPSTs的发现提出了一些关于其功能相关性的基本问题。它们是否表现出不同的底物特异性并作用于不同的靶蛋白亚群？它们是在相同还是不同的细胞类型/组织/器官中表达？它们在胚胎发育到成熟的不同阶段有相同的表达模式吗？在生理学的背景下，它们是单独起作用还是相互补充？为了找到这些未解决问题的答案，我们将使用斑马鱼作为实验模型。提出的研究是由翻译后酪氨酸硫酸化对参与多种生理过程的蛋白质功能至关重要的假设驱动的。在斑马鱼和其他脊椎动物中发现的不同的TPSTs在相同的细胞或不同（尽管重叠）的细胞类型或组织群体中用于硫酸盐不同的蛋白质亚群，并且可能处于不同的发育阶段，具有不同的功能目的。我们通过三个相互关联的具体目标来解决这一假设：1)表达和纯化三种斑马鱼TPSTs，并系统地表征它们的不同酶学性质和底物特异性。各种生化和分子生物学技术，以及RNA干扰策略，将被采用。2)鉴定斑马鱼不同发育阶段的胚胎和幼虫以及成年雄性和雌性斑马鱼中酪氨酸硫酸化的蛋白。将使用先前开发的结合MALDI-TOF分析的方法。3)阐明斑马鱼三种TPST基因的个体发生、细胞类型/组织/器官分布，以及TPST基因敲低的生理后果。在胚胎发育过程中，三种TPSTs的分布和表达将使用分子生物学和显微镜技术进行检查。将采用morpholino反义寡核苷酸方法来研究个体TPST基因敲低的生理后果。本研究旨在为斑马鱼中TPSTs和酪氨酸-硫酸化蛋白的生化特性和功能相关性提供基础信息。鉴定斑马鱼TPSTs的新特征或发现出现在不同发育阶段或不同细胞类型/组织/器官中的新的酪氨酸-硫酸化蛋白可以作为进一步研究人类TPSTs和酪氨酸-硫酸化蛋白的生化和生理相关性的起点。公共卫生相关性：拟议的研究旨在获得斑马鱼中酪氨酸蛋白硫转移酶（TPSTs）和酪氨酸-硫酸化蛋白的生化特性和功能相关性的基本信息。发现斑马鱼TPSTs的新特征或发现出现在不同发育阶段或不同细胞类型/组织/器官中的新的酪氨酸-硫酸化蛋白，可以作为进一步研究人类TPSTs和酪氨酸-硫酸化蛋白的生化和生理相关性的起点。

DESCRIPTION (provided by applicant): Post-translational protein modification by tyrosine sulfation has a widespread occurrence among proteins of multicellular eukaryotic organisms. The enzyme responsible for protein tyrosine sulfation has been identified as the Golgi membrane-bound tyrosylprotein sulfotransferase (TPST). Studies by us and others have revealed the existence of multiple isoforms of TPST in human, mouse, and zebrafish. The discovery of distinct, yet homologous, TPSTs raises some fundamental questions concerning their functional relevance. Do they exhibit differential substrate specificity and act upon different subsets of target proteins? Are they expressed in the same or different cell types/tissues/organs? Do they share the same pattern of expression at different stages during embryonic development onto maturity? Do they function separately or can they complement each other within the context of physiology? To find answers to these unresolved issues, we will use the zebrafish as an experimental model. The proposed research is driven by the hypothesis that post-translational tyrosine sulfation is critical to the functioning of proteins involved in multiple physiological processes. The different TPSTs found in zebrafish and other vertebrates serve to sulfate different subsets of proteins in the same cells or in different, albeit overlapping, populations of cell types or tissues, and possibly at different developmental stages, for distinct functional purposes. We address this hypothesis in three interrelated specific aims: 1) To express and purify the three zebrafish TPSTs and systematically characterize their differential enzymatic properties and substrate specificity. A variety of biochemical and molecular biological techniques, as well as RNA interference strategy, will be employed. 2) To identify the proteins that are subjected to tyrosine sulfation in zebrafish embryos and larvae at different developmental stages, as well as in adult male and female zebrafish. A previously developed methodology in conjunction with MALDI-TOF analysis will be used. 3) To clarify the ontogeny and cell type/tissue/organ distribution of the three zebrafish TPSTs, as well as the physiological consequences of the knockdown of individual TPST genes. The distribution and expression, during embryonic development, of each of the three TPSTs will be examined using molecular biology and microscopy techniques. The morpholino antisense oligonucleotide approach will be taken to investigate the physiological consequences of the knockdown of individual TPST genes. The proposed studies are expected to provide fundamental information concerning the biochemical properties and functional relevance of TPSTs and tyrosine-sulfated proteins in zebrafish. Identification of novel characteristics of the zebrafish TPSTs or discovery of novel tyrosine-sulfated proteins appearing at different developmental stages or in different cell types/tissues/organs may serve as a starting point to investigate further the biochemical and physiological relevance of TPSTs and tyrosine-sulfated proteins in humans. PUBLIC HEALTH RELEVANCE: The proposed studies are designed to obtain fundamental information concerning the biochemical properties and functional relevance of tyrosylprotein sulfotransferases (TPSTs) and tyrosine-sulfated proteins in zebrafish. Identification of novel characteristics of the zebrafish TPSTs or discovery of novel tyrosine-sulfated proteins appearing at different developmental stages or in different cell types/tissues/organs may serve as a starting point to further investigate the biochemical and physiological relevance of TPSTs and tyrosine-sulfated proteins in humans.