Lysosomal Proenzyme Sorting: A New Receptor

9604139埃里克森技术公司：我们发现了一种新的细胞内受体，它能在酸性pH下将溶酶体半胱氨酸蛋白酶原L结合到微粒体膜上，而不是结合到成熟的酶上。与这个43 kDa的完整膜蛋白的结合是由一个9个残基的前肽序列介导的，该序列与酵母空泡分选序列同源。天冬氨酸蛋白酶Proathepsin D经历类似的pH依赖的膜结合。我们的数据表明，溶酶体酶原受体(LPR)是一种交替靶向受体，它介导6-磷酸甘露糖不依赖于甘露糖将溶酶体酶原从反式高尔基体转运到内体小泡。在肿瘤细胞和激活的巨噬细胞中，蛋白酶不能与LPR结合与酶分泌增加有关。本项目的目的是确定L溶酶体酶原受体的生理功能(S)。为了达到这一目的，我们将纯化L原的溶酶体酶原受体。用免疫兔的方法制备针对小鼠前组织蛋白酶L LPR的抗血清。L LPR原蛋白的N端序列将通过Edman降解获得。如果N-末端被封堵，则通过对受体进行蛋白酶处理而制备的多肽将被高效液相色谱纯化，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抗L原蛋白抗体的超微结构研究和纯化的LPR来确定。将利用真核表达系统研究LPR的生理功能，在不产生内源性蛋白酶的小鼠巨噬细胞中表达小鼠前组织蛋白酶L，并测定该酶的膜结合和溶酶体靶向效率。将小鼠血管紧张素原L前肽在小鼠成纤维细胞中表达，以确定该前肽是否具有显性-负性作用，在体内通过竞争LPR来改变蛋白水解酶的靶向性。LPR结合的调节将通过确定为什么添加到完整微粒体外部的三磷酸腺苷导致L原蛋白从膜释放到囊泡腔来研究。初步研究表明，巴非霉素不会阻止这种释放，它会使液泡中的ATPase失活，这表明这种影响不仅仅是由于囊泡内pH的调节。非技术：在发育过程中和对损伤的反应中，组织蛋白酶在降解细胞蛋白方面起着关键作用。组织蛋白酶定位于一个离散的细胞隔间，称为溶酶体，其功能是降解非功能性和非必需的细胞成分。降解酶的目标是通过修饰结合提供寻址信息的糖的酶来包含在溶酶体中。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定位于溶酶体，该机制涉及识别部分组织蛋白酶蛋白的特定受体。在授予期间，埃里克森博士将鉴定和鉴定组织蛋白酶靶向受体，并展示其在溶酶体组装中的功能。

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.