Molecular Interactions Modulating Targeting of Procathepsin L

溶酶体蛋白水解酶通常被认为仅在酸性晚期内切体或溶酶体中内吞和内源性蛋白的最终降解的协同中介中起作用。然而，最近的数据表明，这些酶参与了其他不同的过程，如抗原处理、细胞凋亡和细胞外基质重塑，这表明这些酶可能靶向并在不同于溶酶体的细胞位置被激活。在正常细胞中，溶酶体半胱氨酸蛋白酶组织蛋白酶L通过其磷酸化的甘露糖残基与甘露糖磷酸受体相互作用有效地靶向溶酶体，甘露糖磷酸受体识别TGN中的蛋白质并将其运输到晚期内体。当转录调控增加该酶的表达时，无论是在正常发育阶段，如精子成熟期间的支持细胞，还是作为细胞转化的结果，所合成的额外的蛋白酶主要是分泌的，而不是针对溶酶体的，这与该酶在细胞外过程中的潜在作用是一致的。这个项目的目标是了解由于表达增加而将单一蛋白酶的靶向从溶酶体筛选到细胞外部的机制。组织蛋白酶L与两个细胞分子的结合将被表征，以及这些分子相互作用如何调节蛋白水解酶的细胞内靶向。将被检验的假说是Tetraspanins，被认为是组成稳定和促进蛋白质相互作用的网络的分子促进剂，作为Proathepsin L的包装伴侣或受体。在多囊内体的致密核心，Proathepsin L与43 kDa的TetraspaninCD63共存，EM免疫金标记法证明。通过酵母双杂交实验，L原蛋白与CD82结合，后者被证明与CD63相互作用。另一个假设是，原蛋白L也与RMR的哺乳动物同源物结合，RMR是一种含有无名指的植物受体，它识别液泡半胱氨酸蛋白酶Aleurain中的C-末端分选信号，介导该酶靶向拟南芥蛋白质储存液泡。初步数据表明，抗植物受体的抗体可以识别小鼠成纤维细胞微粒体中大小正确的单个蛋白质，并且在下拉实验中，植物受体的配体结合域通过表位标签添加和引入点突变，与活性双链组织蛋白L结合，已经在原组织蛋白L中鉴定出两个调节蛋白酶靶向的序列。根据免疫荧光显微镜的分析，一个突变导致酶在高尔基体中积累，而另一个突变导致在核周小泡中积累。这表明分子的两个表面与介导靶向的蛋白质相互作用。我们推测，Tetraspanins通过与N-末端相互作用促进原L高尔基体的输出，而植物受体同源物通过识别C-末端序列参与后来的靶向事件。我们进一步假设，这些分子相互作用在将组织蛋白酶L定向到多囊内小体、溶酶体和分泌途径的生物合成途径中介导了离散的步骤。在动物细胞中，溶酶体酶靶向细胞内隔室至少通过两条靶向途径发生。其中一种已被广泛描述，涉及一种特定的受体，它识别溶酶体酶上的甘露糖-6-磷酸(Man-6-P)残基，并与其结合，将其携带到溶酶体内。其他靶向过程独立于Man-6-P识别，并且在很大程度上仍然未知。然而，在植物和酵母中，Man-6-P途径甚至不存在，因此Man-6-P非依赖途径(S)显然是重要的。这个项目有望揭示至少一个多年来一直未被研究人员发现的Man-6-P独立转运途径的分子机制。BROADER项目影响：拟议的实验将作为本科化学和生物专业以及生物化学研究生的教学工具。实践研究不仅教会了学生基本技术，还教会了他们批判性思维。他们不仅必须学会利用先例和设计控制，而且还必须确定除了构成其原始假设基础的变量之外，哪些变量可能会产生所获得的结果。这种批判性思维只能随着时间的推移而来，不能从教科书上学到。通过参与研究，并被迫对他们产生的数据进行批判性思考，学生们学习了对研究成功至关重要的思维过程。

Lysosomal proteases have classically been assumed to function exclusively in the cooperative mediation of terminal degradation of endocytosed and endogenous proteins within acidic late endosomes or lysosomes. Recent data, however, implicate these proteases in additional diverse processes such as antigen processing, apoptosis and extracellular matrix remodeling, suggesting the proteases may be targeted to and activated in cellular sites distinct from the lysosome. In normal cells, the lysosomal cysteine protease cathepsin L is efficiently targeted to lysosomes through interaction of its phosphorylated mannose residues with mannose phosphate receptors that recognize the protein in the TGN and transport it to late endosomes. When transcriptional regulation increases expression of the protease, whether during a normal developmental stage, as occurs in Sertoli cells during sperm maturation, or as a consequence of cell transformation, the additional protease synthesized is predominately secreted rather than targeted to lysosomes, consistent with a potential role for this protease in extracellular processes. The goal of this project is to gain understanding of the mechanism that sifts targeting of a single protease from the lysosome to the cell exterior as a consequence of increased expression. The association of cathepsin L with two cellular molecules will be characterized, and how these molecular interactions modulate intracellular targeting of the protease will be studied. The hypothesis that will be tested is that tetraspanins, which are molecular facilitators thought to comprise a web that stabilizes and facilitates protein interaction, serve as packaging chaperones or receptors for procathepsin L. In the dense cores of multivesicular endosomes, procathepsin L colocalizes with the 43-kDa tetraspanin CD63, as demonstrated by EM immunogold labeling. By yeast two-hybrid assay, procathepsin L binds CD82, which has been shown to interact with CD63. An additional hypothesis is that procathepsin L also binds to a mammalian homologue of RMR, a RING-finger containing plant receptor that recognizes a C-terminal sorting signal in the vacuolar cysteine protease aleurain to mediate targeting of this enzyme to protein storage vacuoles in Arabidopsis thaliana. Preliminary data show that antibodies to the plant receptor recognize a single protein of correct size in mouse fibroblast microsomes, and that in a pull-down assay, the ligand-binding domain of the plant receptor binds active two-chain cathepsin L. By epitope tag addition and introduction of point mutations, two sequences have beeen identified within procathepsin L that modulate protease targeting. One mutation causes the enzyme to accumulate in the Golgi, while the other induces accumulation in perinuclear vesicles, as assayed by immunofluorescence microscopy. This suggests that two surfaces of the molecule interact with proteins which mediate targeting. We hypothesize that the tetraspanins facilitate Golgi export of procathepsin L by interacting with the N-terminal site, while the plant receptor homologue participates in later targeting events through recognition of C-terminal sequences. We further hypothesize that these molecular interactions mediate discrete steps in the biosynthetic pathway that direct cathepsin L to multivesicular endosomes, lysosomes and the secretory pathway.In animal cells, targeting of lysosomal enzymes to intracellular compartments occurs via at least two targeting pathways. One of these, which has been very extensively characterized, involves a specific receptor that recognizes mannose-6-phosphate (Man-6-P) residues on lysosomal enzymes and binds to them to carry them to lysosomes. The other targeting process(es) are independent of Man-6-P recognition and remain largely unknown. In plants and yeast, however, the Man-6-P pathway does not even exist, so the Man-6-P independent pathway(s) is obviously important. This project holds the promise of revealing the molecular mechanism at least one of the Man-6-P independent trafficking pathways that has eluded investigators for so many years.BROADER IMPACT OF PROPOSED PROJECT: The proposed experiments will be utilized as teaching vehicles for both undergraduate chemistry and biology majors and biochemistry graduate students. Hands-on research not only teaches students basic technology but teaches them to think critically. They must not only learn to utilize precedent and design controls but also to determine which variables could have generated the results obtained in addition to the one which formed the basis for their original hypothesis. This critical thinking only comes with time and can't be learned from a textbook. By participating in research, and being forced to think critically about data that they have generated, students learn the thinking processes critical for success in research.