Lysosomal Proenzyme Sorting: A New Receptor

溶酶体酶原分选：一种新受体

• 批准号: 9604139
• 负责人: Ann Erickson
• 金额: $ 39万
• 依托单位: University of North Carolina at Chapel Hill
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 2002-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9604139&HistoricalAwards=false
• 关键词: Lysosomal; Proenzyme; Sorting; New; Receptor

9604139 Erickson Technical: We have identified a new intracellular receptor that binds the lysosomal cysteine protease procathepsin L, but not the mature enzyme, to microsomal membranes at an acid pH . Binding to this 43-kDa integral membrane protein is mediated by a 9-residue propeptide sequence that showq homology to yeast vacuolar sorting sequences. The aspartic protease procathepsin D undergoes similar pH-dependent membrane association. Our data suggest that lysosomal proenzyme receptors (LPRs) are alternate targeting receptors which mediate mannose 6-phosphate-independent transport of lysosomal proenzymes from the trans Golgi to endosomal vesicles. Failure of the proteases to bind to LPRs in tumor cells and activated macrophages correlates with increased enzyme secretion. The goal of this project is to determine the physiological function(s) of the procathepsin L lysosomal proenzyme receptor (LPR). In order to accomplish this objective the lysosomal proenzyme receptor for procathepsin L will be purified. Antiserum specific for the mouse procathepsin L LPR will be prepared by immunization of rabbits. The N-terminal sequence of the procathepsin L LPR will be obtained by Edman degradation. If the N-terminus is blocked, peptides prepared by protease treatment of the receptor will be purified by HPLC and subjected to N-terminal sequence analysis. The intracellular localization of the LPR and its ligand will be determined by ultrastructural studies using antibodies raised against procathepsin L and the purified LPR. The physiological function of the LPR will be studied using eucaryotic expression systems Mouse procathepsin L will be expressed in mouse macrophages which do not produce endogenous protease and the membrane association and efficiency of lysosomal targeting of the protease will be determined. The mouse procathepsin L propeptide will be expressed in mouse fibroblasts to determine if the propeptide has a dominant-negative effect, altering proprotease targeting by competing fo r the LPR in vivo. Regulation of LPR binding will be studied by determing why ATP added to the exterior of intact microsomes causes procathepsin L to be released from the membranes to the vesicle lumen. Initial studies indicate the release is not blocked by bafilomycin, which inactivates the vacuolar ATPase, suggesting that the effect is not merely due to modulation of intravesicular pH. Non Technical: The protease cathepsin has a critical role in degrading cellular proteins during development and in response to injury. Cathepsin is localized in a discrete cellular compartment termed lysosomes that functions to degrade nonfunctional and nonessential cellular components. Degradative enzymes are targeted for inclusion into lysosome by a modification of the enzymes that attaches a sugar that provides addressing information. The preliminary results of thiq project indicate that the cathepsin protease is targeted to lysosomes by an entirely different mechanism that involves a specific receptor that recognizes part of the cathepsin protein. During the grant award period Dr. Erickson will identify and characterize the cathepsin targeting receptor and demonstrate its function in lysosome assembly.

小行星9604139 技术支持：我们已经确定了一个新的细胞内受体，结合溶酶体半胱氨酸蛋白酶proatepsin L，但不是成熟的酶，在酸性pH值的微粒体膜。结合到这个43 kDa的整合膜蛋白介导的9个残基的前肽序列，showq同源性酵母液泡分选序列。 天冬氨酸蛋白酶前体蛋白酶D经历类似的pH依赖性膜缔合。 我们的数据表明，溶酶体酶原受体（LPRs）是交替的靶向受体介导的甘露糖6-磷酸不依赖运输的溶酶体酶原从反式高尔基体的内体囊泡。 在肿瘤细胞和活化的巨噬细胞中，蛋白酶不能与LPR结合与酶分泌增加相关。本项目的目的是确定前组织蛋白酶L溶酶体酶原受体（LPR）的生理功能。为了实现这一目标，将纯化用于组织蛋白酶原L的溶酶体酶原受体。将通过免疫家兔制备对小鼠组织蛋白原L LPR具有特异性的抗血清。将通过Edman降解获得组织蛋白原L LPR的N-末端序列。 如果N-末端被封闭，则通过蛋白酶处理受体制备的肽将通过HPLC纯化并进行N-末端序列分析。LPR及其配体的细胞内定位将通过使用针对前组织蛋白酶L和纯化的LPR产生的抗体的超微结构研究来确定。将使用真核表达系统研究LPR的生理功能，将在不产生内源性蛋白酶的小鼠巨噬细胞中表达小鼠组织蛋白酶原L，并测定蛋白酶的膜结合和溶酶体靶向效率。小鼠组织蛋白酶原L前肽将在小鼠成纤维细胞中表达，以确定前肽是否具有显性负效应，通过体内竞争LPR来改变蛋白酶原靶向。LPR结合的调节将通过确定为什么ATP添加到完整微粒体的外部导致前组织蛋白酶L从膜释放到囊泡腔来研究。 最初的研究表明，释放不被巴弗洛霉素阻断，巴弗洛霉素使液泡ATP酶失活，这表明这种作用不仅仅是由于囊内pH的调节。 非技术性：蛋白酶组织蛋白酶在发育过程中降解细胞蛋白和对损伤的反应中起关键作用。组织蛋白酶位于称为溶酶体的离散细胞区室中，其功能是降解非功能性和非必需的细胞组分。降解酶通过修饰附着提供寻址信息的糖的酶而被靶向包含到溶酶体中。thiq项目的初步结果表明，组织蛋白酶通过一种完全不同的机制靶向溶酶体，该机制涉及识别组织蛋白酶蛋白的一部分的特异性受体。Erickson博士将鉴定和表征组织蛋白酶靶向受体，并证明其在溶酶体组装中的功能。