Zebrafish TPSTs and Tyrosine-sulfated Proteins

描述(申请人提供)：酪氨酸硫化的翻译后蛋白质修饰在多细胞真核生物的蛋白质中广泛发生。负责蛋白质酪氨酸硫化的酶被确定为高尔基膜结合的酪氨酸蛋白磺基转移酶(TPST)。我们和其他人的研究表明，在人类、小鼠和斑马鱼中存在多种TPST同工酶。不同但同源的TPSTs的发现提出了一些关于它们功能相关性的基本问题。它们是否表现出不同的底物专一性，并作用于不同的靶蛋白亚群？它们在相同或不同的细胞类型/组织/器官中表达吗？它们在胚胎发育到成熟的不同阶段有相同的表达模式吗？它们是各自发挥作用，还是在生理学背景下相辅相成？为了找到这些悬而未决的问题的答案，我们将使用斑马鱼作为实验模型。这项拟议的研究是由一种假设推动的，即翻译后酪氨酸硫化对参与多种生理过程的蛋白质的功能至关重要。在斑马鱼和其他脊椎动物中发现的不同的TPST用于在同一细胞中或不同的尽管重叠的细胞类型或组织中，以及可能处于不同发育阶段的不同功能目的的不同蛋白质亚组中硫酸盐。我们在三个相互关联的特定目标中提出了这一假说：1)表达和纯化三种斑马鱼TPST，并系统地表征它们的差异酶特性和底物专一性。将采用各种生化和分子生物学技术，以及RNA干扰策略。2)鉴定不同发育阶段斑马鱼胚胎和仔鱼以及成年雄性斑马鱼和雌性斑马鱼中受酪氨酸硫化影响的蛋白质。将使用先前开发的与MALDI-TOF分析相结合的方法。3)阐明三种斑马鱼TPST的个体发育和细胞类型/组织/器官分布，以及单个TPST基因被敲除的生理后果。在胚胎发育期间，将使用分子生物学和显微技术来检测三个TPST的分布和表达。将采用吗啡反义寡核苷酸的方法来研究单个TPST基因被敲除的生理后果。这项拟议的研究有望提供有关斑马鱼中TPSTs和酪氨酸硫化蛋白的生化特性和功能相关性的基本信息。斑马鱼TPSTs的新特性的鉴定或在不同发育阶段或不同细胞类型/组织/器官中出现的新的酪氨酸硫化蛋白的发现可能是进一步研究TPSTs与人类酪氨酸硫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有关斑马鱼中酪氨酸蛋白硫化转移酶(TPSTs)和酪氨酸硫化蛋白的生化特性和功能相关性的基本信息。斑马鱼TPSTs的新特性的鉴定或在不同发育阶段或不同细胞类型/组织/器官中出现的新的酪氨酸硫化蛋白的发现可能是进一步研究TPSTs与人类酪氨酸硫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.