Lysosomal Proenzyme Sorting: A New Receptor

9604139 Erickson Technical：我们已经鉴定出一种新的细胞内受体，它在酸性pH下将溶酶体半胱氨酸蛋白酶原athepsin L结合到微粒体膜上，而不是将成熟酶结合。与这个43 kda的完整膜蛋白结合是由9个残基前肽序列介导的，该序列与酵母液泡分选序列具有同源性。天冬氨酸蛋白酶原肝组织蛋白酶D经历类似的ph依赖性膜关联。我们的数据表明，溶酶体前酶受体（LPRs）是替代靶向受体，介导溶酶体前酶从反式高尔基体到内体囊泡的不依赖甘露糖6-磷酸的转运。在肿瘤细胞和活化的巨噬细胞中，蛋白酶不能与LPRs结合与酶分泌增加有关。本项目的目的是确定血凝素原L溶酶体前酶受体（LPR）的生理功能。为了实现这一目标，将纯化蛋白酶原L的溶酶体前酶受体。通过兔免疫制备小鼠肝组织蛋白酶原llpr特异性抗血清。通过Edman降解得到原肝蛋白酶LPR的n端序列。如果n端被阻断，通过蛋白酶处理受体制备的肽将被高效液相色谱纯化，并进行n端序列分析。LPR及其配体的细胞内定位将通过超微结构研究来确定，该研究使用针对肝组织蛋白酶原L和纯化的LPR产生的抗体。小鼠原athepsin L将在不产生内源性蛋白酶的小鼠巨噬细胞中表达，并确定蛋白酶的膜关联和溶酶体靶向效率。小鼠原肝蛋白酶L前肽将在小鼠成纤维细胞中表达，以确定该前肽是否具有显性负作用，通过在体内竞争LPR来改变原蛋白酶的靶向性。通过确定为什么ATP添加到完整微粒体的外部会导致原肝蛋白酶L从膜释放到囊泡腔，将研究LPR结合的调节。最初的研究表明，这种释放不会被巴菲霉素阻断，而巴菲霉素会使空泡atp酶失活，这表明这种作用不仅仅是由于调节囊泡内ph。非技术：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着关键作用。组织蛋白酶定位于一个称为溶酶体的离散细胞室，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酶的修饰，将提供寻址信息的糖附着在溶酶体中。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靶向溶酶体，该机制涉及识别部分组织蛋白酶蛋白的特定受体。在资助期间，Erickson博士将鉴定和表征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.