MECHANISMS OF SULFATE TRANSPORT AND REGULATION

硫酸盐运输和调节机制

• 批准号: 7193533
• 负责人: MICHAEL L JENNINGS
• 金额: $ 22.19万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7193533
• 关键词: Affect; Amino Acids; Anion Transport Proteins; Anions; Area; Bone and Cartilage Funding; Catabolism; Cell Line; Cell membrane; Cell physiology; Cells; Condition; Cysteine; Development; Dysplasia; Erythrocyte Anion Exchange Protein 1; Erythrocytes; Functional disorder; Glycosaminoglycans; Goals; Golgi Apparatus; Human; Inorganic Sulfates; Label; Mammalian Cell; Measures; Mediating; Membrane Transport Proteins; Methionine; Molecular; Mutation; Pathway interactions; Physiologic pulse; Preparation; Process; Proteins; Proteoglycan; Pulse taking; Rate; Rattus; Regulation; Reverse Transcriptase Polymerase Chain Reaction; Role; Skeletal system; System; Time; Tyrosine; Unspecified or Sulfate Ion Sulfates; Work; aggrecan; extracellular; insight; macromolecule; research study; response; sulfate transporter; sulfation; sulfotransferase; transport inhibitor

DESCRIPTION (provided by applicant): The long-term goal of these studies is to understand the regulation of the pathway for sulfation of macromolecules in mammalian cells. The emphasis of the current proposal is on SO4= transport mediated by DTDST (Diastrophic Dysplasia Sulfate Transporter), mutations in which cause a variety of chondrodysplasias. The goal is to provide an integrated understanding of the relationships among several steps in the sulfation pathway: SO4= transport, steady state intracellular [SO4=], synthesis of the universal SO4= donor PAPS (3'-phosphoadenosine-5'-phosphosulfate), PAPS transport into the trans-Golgi, and rate of sulfation of glycosaminoglycans (GAG) and protein tyrosine residues. The cells to be studied are human and rat chondrocytic and osteoblastic cell lines, which express DTDST and secrete sulfated macromolecules at a high rate. There are 6 Specific Aims. Specific Aim 1 is to quantify the role of plasma membrane sulfate transport in regulating the intracellular SO4= pool. This will be done by defining the catalytic mechanism (e.g., SO4= + H+ exchange for CI) of transport by DTDST and possible regulation of DTDST expression or activity. Specific Aim 2 is to evaluate the role of cysteine and methionine catabolism in supplying SO4= under conditions of varying supply of exogenous SO4= and to determine whether cells can up regulate amino acid catabolism in response to SO4= deficiency. Specific Aim 3 is to measure and understand the factors that govern the rate of synthesis of PAPS in intact cells, including the effect of cytoplasmic SO4= concentration and possible regulation of expression of PAPS synthases. Specific Aim 4 is to determine whether there is substrate channeling of SO4= to PAPS synthase, either from the plasma membrane transporter DTDST or from catabolism of amino acids. Specific Aim 5 is to use cell-free preparations to determine whether PAPS transport into the Golgi is rate-limiting for sulfation of macromolecules. Specific Aim 6 is to measure the possible effect of sulfation rate on the Golgi transit time of the major sulfated protein aggrecan. The general approach in these studies is to perform 35S labeling and pulse-chase experiments as well as RT-PCR and immunodetection of expression levels of key proteins in the sulfation pathway. The difference between the approach proposed here and all the preceding work in this area is that we propose to measure the levels of key intermediates (cytoplasmic SO4=, PAPS, labeled protein in the secretory pathway) and the rates of turnover of these intermediates. These experiments will provide new insights into the regulation of a basic pathway (SO4= supply to sulfotransferases) that is of importance not only in the skeletal system but also in many branches of human pathophysiology.

描述（由申请方提供）：这些研究的长期目标是了解哺乳动物细胞中大分子硫酸化途径的调控。目前建议的重点是SO 4 = DTDST（骨发育不良硫酸盐转运蛋白）介导的转运，其中突变导致各种软骨发育不良。目的是提供对硫酸化途径中几个步骤之间关系的综合理解：SO 4 =转运，稳态细胞内[SO 4 =]，通用SO 4 =供体PAPS（3 '-磷酸腺苷-5'-磷酸硫酸盐）的合成，PAPS转运到trans-Golgi中，以及糖胺聚糖（GAG）和蛋白酪氨酸残基的硫酸化速率。待研究的细胞是人类和大鼠软骨细胞和成骨细胞系，其表达DTDST并以高速率分泌硫酸化大分子。有6个具体目标。具体目标1是量化质膜硫酸盐转运在调节细胞内SO 4 =池中的作用。这将通过定义催化机制（例如，SO 4 = + H+交换为CI）的转运和可能的DTDST表达或活性调节。具体目标2是评估在不同的外源性SO 4 =供应条件下半胱氨酸和甲硫氨酸催化剂在供应SO 4 =中的作用，并确定细胞是否可以响应于SO 4 =缺乏而上调氨基酸催化剂。具体目标3是测量和了解在完整细胞中控制PAPS合成速率的因素，包括细胞质SO 4 =浓度的影响和PAPS酶表达的可能调节。具体目标4是确定是否存在SO 4 =至PAPS合酶的底物通道，无论是来自质膜转运蛋白DTDST还是来自氨基酸的催化剂。具体目标5是使用无细胞制剂，以确定是否PAPS运输到高尔基体是速率限制的硫酸化的大分子。具体目标6是测量硫酸化速率对主要硫酸化蛋白聚集蛋白聚糖的高尔基体通过时间的可能影响。在这些研究中的一般方法是进行35 S标记和脉冲追踪实验以及RT-PCR和免疫检测硫酸化途径中的关键蛋白质的表达水平。这里提出的方法和所有以前的工作在这一领域的区别是，我们建议测量的关键中间体（细胞质SO 4 =，PAPS，标记的蛋白质在分泌途径）的水平和这些中间体的周转率。这些实验将提供新的见解调节的基本途径（SO 4 =供应磺基转移酶），这是重要的，不仅在骨骼系统，而且在人类病理生理学的许多分支。