Vaithilingam Archana, Lai Nicole Y, Duong Ellen, Boucau Julie, Xu Yang, Shimada Mariko, Gandhi Malini, Le Gall Sylvie
Ragon Institute of MGH, MIT and Harvard, 400 Technology Square, Cambridge, MA 02139, USA.
BMC Cell Biol. 2013 Aug 9;14:35. doi: 10.1186/1471-2121-14-35.
Endolysosomes play a key role in maintaining the homeostasis of the cell. They are made of a complex set of proteins that degrade lipids, proteins and sugars. Studies involving endolysosome contribution to cellular functions such as MHC class I and II epitope production have used recombinant endolysosomal proteins, knockout mice that lack one of the enzymes or purified organelles from human tissue. Each of these approaches has some caveats in analyzing endolysosomal enzyme functions.
In this study, we have developed a simple methodology to assess endolysosomal protease activity. By varying the pH in crude lysate from human peripheral blood mononuclear cells (PBMCs), we documented increased endolysosomal cathepsin activity in acidic conditions. Using this new method, we showed that the degradation of HIV peptides in low pH extracts analyzed by mass spectrometry followed similar kinetics and degradation patterns as those performed with purified endolysosomes.
By using crude lysate in the place of purified organelles this method will be a quick and useful tool to assess endolysosomal protease activities in primary cells of limited availability. This quick method will especially be useful to screen peptide susceptibility to degradation in endolysosomal compartments for antigen processing studies, following which detailed analysis using purified organelles may be used to study specific peptides.
内溶酶体在维持细胞内稳态中起关键作用。它们由一组复杂的蛋白质组成，可降解脂质、蛋白质和糖类。涉及内溶酶体对细胞功能（如MHC I类和II类表位产生）贡献的研究使用了重组内溶酶体蛋白、缺乏其中一种酶的基因敲除小鼠或从人体组织中纯化的细胞器。这些方法中的每一种在分析内溶酶体酶功能时都有一些注意事项。
在本研究中，我们开发了一种简单的方法来评估内溶酶体蛋白酶活性。通过改变来自人外周血单核细胞（PBMC）的粗裂解物的pH值，我们记录了在酸性条件下内溶酶体组织蛋白酶活性的增加。使用这种新方法，我们表明通过质谱分析在低pH提取物中HIV肽的降解遵循与用纯化的内溶酶体进行的降解相似的动力学和降解模式。
通过使用粗裂解物代替纯化的细胞器，该方法将成为评估有限可用性原代细胞中内溶酶体蛋白酶活性的快速且有用的工具。这种快速方法对于筛选内溶酶体区室中肽对降解的敏感性以进行抗原加工研究特别有用，之后可以使用纯化的细胞器进行详细分析以研究特定肽。
