MECHANISMS OF SULFATE TRANSPORT AND REGULATION

描述（由申请人提供）：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。当前提案的重点是由DTDST（硫酸异型发育不良转运蛋白）介导的SO4=转运，其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：SO4=运输，细胞内稳态[SO4=]，通用SO4=供体PAPS（3'-磷酸腺苷-5'-硫酸磷酸）的合成，PAPS转运到反式高尔基体，以及糖胺聚糖（GAG）和蛋白酪氨酸残基的硫酸化速率。所研究的细胞是人类和大鼠的软骨细胞和成骨细胞系，它们表达DTDST并以高速率分泌硫酸大分子。有6个具体目标。具体目的1是量化质膜硫酸盐运输在调节细胞内SO4=池中的作用。这将通过定义DTDST运输的催化机制（例如，SO4= + H+交换CI）和DTDST表达或活性的可能调节来完成。具体目的2是评估在不同外源SO4=供应条件下，半胱氨酸和蛋氨酸分解代谢在供应SO4=中的作用，并确定细胞在SO4=缺乏时是否可以上调氨基酸分解代谢。具体目的3是测量和了解控制完整细胞中PAPS合成速率的因素，包括细胞质SO4=浓度的影响和可能对PAPS合成酶表达的调节。具体目的4是确定SO4=是否通过质膜转运体DTDST或氨基酸分解代谢向PAPS合成酶传递底物通道。具体目标5是使用无细胞制剂来确定进入高尔基体的PAPS转运是否限制了大分子硫酸化的速率。具体目的6是测量硫酸化速率对主要硫酸化蛋白聚集蛋白的高尔基传递时间的可能影响。这些研究的一般方法是进行35S标记和脉冲追踪实验，以及RT-PCR和免疫检测硫酸化途径关键蛋白的表达水平。本文提出的方法与该领域所有先前工作的不同之处在于，我们建议测量关键中间体（细胞质SO4=， PAPS，分泌途径中的标记蛋白）的水平和这些中间体的周转率。这些实验将为基本途径（SO4=硫转移酶供应）的调控提供新的见解，这不仅在骨骼系统中很重要，而且在人类病理生理学的许多分支中也很重要。

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.