Ghanem Esther, Al-Balushi Mohammed
Department of Biology, Faculty of Natural and Applied Sciences, Notre Dame University, 72, Zouk Mosbeh, Keserwan district, Lebanon.
Department of Microbiology and Immunology, Sultan Qaboos University, Muscat, Oman.
BMC Cell Biol. 2015 Dec 29;16:30. doi: 10.1186/s12860-015-0077-1.
In mammalian cells, the quality control (QC) of properly folded proteins is monitored in the early secretory pathway, particularly in the endoplasmic reticulum (ER). Several proteins, including our protein of interest, major histocompatibility complex class I (MHC class I), can bypass the first line of ER-QC and reside in post-ER compartments in an unfolded form. Such forms entail both monomeric and dimeric structures that are devoid of peptides and thus cannot fulfill the immunological function of antigen presentation at the cell surface. MHC class I structures become mature and properly folded once loaded with the appropriate peptides in the framework of the peptide loading complex (PLC). Despite the flood of information on the diverse trafficking behavior of different MHC class I alleles, there is still controversy on the actual trajectory followed by improperly folded murine MHC class I alleles, namely H-2Kb. In this study, we employ an in vitro rapamycin trapping assay, live cell imaging, and a biochemical COPII budding approach to further investigate the trafficking of H-2Kb beyond the level of the ER.
We confirm the egress of H-2Kb in an unfolded form to a post-ER compartment from where they can cycle back to the ER. Deciphering the exact identity of the post-ER compartment by laser scanning microscopy did not only point to the existence of the ERGIC and cis-Golgi compartments as residency areas for unfolded proteins, but also to the involvement of an addional compartment, that lies in close proximity and possesses high resemblance to the aforementioned compartments. Interestingly, we were capable of showing using the same rapamycin trapping assay that H-2Kb can undergo a potential maturation event during their cycling; this is attained upon addition of peptides and trapping of accumulated post-ER molecules at the cell surface.
Our findings deepen the understanding of H-2Kb trafficking outside the ER and pave the way to decipher the role and the trafficking of certain PLC chaperones, such as tapasin, throughout H-2K(b) post-ER QC. Finally, we demonstrate the plausible usage of the rapamycin assay to assess the trafficking of defected proteins especially in diseases and under therapeutic studies.
在哺乳动物细胞中，正确折叠蛋白质的质量控制（QC）在早期分泌途径中进行监测，特别是在内质网（ER）中。包括我们感兴趣的蛋白质主要组织相容性复合体I类（MHC I类）在内的几种蛋白质可以绕过内质网质量控制的第一道防线，以未折叠的形式存在于内质网后的区室中。这些形式包括单体和二聚体结构，它们没有肽，因此不能在细胞表面发挥抗原呈递的免疫功能。一旦在肽装载复合体（PLC）的框架内装载了适当的肽，MHC I类结构就会成熟并正确折叠。尽管有大量关于不同MHC I类等位基因多样运输行为的信息，但关于未正确折叠的小鼠MHC I类等位基因，即H-2Kb，实际遵循的轨迹仍存在争议。在本研究中，我们采用体外雷帕霉素捕获试验、活细胞成像和生化COPII出芽方法，进一步研究H-2Kb在内质网水平之外的运输。
我们证实H-2Kb以未折叠的形式输出到内质网后的区室，从那里它们可以循环回到内质网。通过激光扫描显微镜确定内质网后区室的确切身份，不仅指出了内质网-高尔基体中间膜囊（ERGIC）和顺式高尔基体区室作为未折叠蛋白质的驻留区域的存在，还指出了另一个区室的参与，该区室与上述区室紧邻且高度相似。有趣的是，我们能够使用相同的雷帕霉素捕获试验表明，H-2Kb在其循环过程中可以经历潜在的成熟事件；这是在添加肽并在细胞表面捕获积累的内质网后分子时实现的。
我们的发现加深了对H-2Kb在内质网外运输的理解，并为破译某些PLC伴侣蛋白，如塔帕辛，在H-2K(b)内质网后质量控制中的作用和运输铺平了道路。最后，我们证明了雷帕霉素试验在评估缺陷蛋白质运输方面的合理用途，特别是在疾病和治疗研究中。
