Molecular Interactions Modulating Targeting of Procathepsin L

分子相互作用调节组织蛋白酶 L 的靶向

• 批准号: 0235680
• 负责人: Ann Erickson
• 金额: $ 41.5万
• 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: 2003
• 资助国家: 美国
• 起止时间: 2003-04-15 至 2007-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0235680&HistoricalAwards=false
• 关键词: Molecular; Interactions; Modulating; Targeting; Procathepsin

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.

溶酶体蛋白酶通常被认为仅在酸性晚期内体或溶酶体内协同介导内吞和内源性蛋白质的末端降解中起作用。然而，最近的数据表明，这些蛋白酶参与其他不同的过程，如抗原加工，细胞凋亡和细胞外基质重塑，表明蛋白酶可能被靶向和激活不同于溶酶体的细胞位点。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的相互作用有效靶向溶酶体，所述甘露糖磷酸受体识别TGN中的蛋白质并将其转运至晚期内体。当转录调节增加蛋白酶的表达时，无论是在正常发育阶段，如在精子成熟期间在支持细胞中发生的，还是作为细胞转化的结果，合成的额外蛋白酶主要分泌而不是靶向溶酶体，这与该蛋白酶在细胞外过程中的潜在作用一致。这个项目的目标是获得的机制，筛选从溶酶体到细胞外部作为一个单一的蛋白酶的表达增加的结果靶向的理解。 组织蛋白酶L与两个细胞分子的关联将被表征，并且将研究这些分子相互作用如何调节蛋白酶的细胞内靶向。将要检验的假设是，四跨膜蛋白是一种分子促进剂，被认为构成了一个稳定和促进蛋白质相互作用的网络，它作为包装蛋白酶原L的分子伴侣或受体。在多泡内体的致密核心中，前组织蛋白酶L与43-kDa的四跨膜蛋白CD 63共定位，如EM免疫金标记所示。通过酵母双杂交测定，前组织蛋白酶L结合CD 82，其已被证明与CD 63相互作用。 另一个假设是，前组织蛋白酶L也结合到RMR的哺乳动物同源物，RMR是一种含有环指的植物受体，其识别液泡半胱氨酸蛋白酶糊粉蛋白中的C-末端分选信号，以介导该酶靶向拟南芥中的蛋白质储存液泡。初步数据显示，植物受体的抗体识别小鼠成纤维细胞微粒体中正确大小的单一蛋白质，并且在下拉测定中，植物受体的配体结合结构域结合活性双链组织蛋白酶L。通过表位标签的添加和点突变的引入，已经在原组织蛋白酶L中鉴定出调节蛋白酶靶向的两个序列。一个突变导致酶在高尔基体中积累，而另一个突变则诱导在核周囊泡中积累，如免疫荧光显微镜所示。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，四跨膜蛋白促进高尔基体出口的蛋白原L的N-末端位点的相互作用，而植物受体同源物参与后来的靶向事件，通过识别的C-末端序列。我们进一步假设，这些分子间的相互作用介导的生物合成途径，直接组织蛋白酶L的多囊内体，溶酶体和分泌pathway.In动物cells，靶向溶酶体酶的细胞内室发生通过至少两个靶向途径的离散步骤。 其中之一，已被广泛表征，涉及识别溶酶体酶上的甘露糖-6-磷酸（Man-6-P）残基并与其结合以将其携带至溶酶体的特异性受体。 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非依赖性途径显然是重要的。 该项目有望揭示Man-6-P独立运输途径中至少一条的分子机制，这条途径多年来一直困扰着研究人员。拟议项目的更广泛影响：拟议的实验将被用作本科化学和生物学专业以及生物化学研究生的教学工具。动手研究不仅教学生基本技术，但教他们批判性思考。他们不仅要学会利用先例和设计控制，而且要确定除了构成其原始假设基础的变量之外，哪些变量还可能产生所获得的结果。这种批判性思维只能随着时间的推移而出现，不能从教科书上学到。通过参与研究，并被迫批判性地思考他们所产生的数据，学生学习的思维过程中取得成功的研究至关重要。