Raymond Anne-Aurélie, Gonzalez de Peredo Anne, Stella Alexandre, Ishida-Yamamoto Akemi, Bouyssie David, Serre Guy, Monsarrat Bernard, Simon Michel
UMR5165 CNRS-University of Toulouse III, Institut Fédératif de Recherche Claude de Préval (INSERM-CNRS)-Université Paul Sabatier-Centre Hospitalier Universitaire de Toulouse) 31059 Toulouse, France.
Mol Cell Proteomics. 2008 Nov;7(11):2151-75. doi: 10.1074/mcp.M700334-MCP200. Epub 2008 Jul 12.
Lamellar bodies (LBs) are tubulovesicular secretory organelles of epithelial cells related to lysosomes. In the epidermis, they play a crucial role in permeability barrier homeostasis, secreting their contents, lipids, a variety of hydrolases, protease inhibitors, and antimicrobial peptides, in the upper keratinocyte layers. The identification of proteins transported in epidermal LBs is still far from complete, and the way their secretion is controlled unknown. In this study, we describe the first proteomics characterization by nano-LC-MS/MS of a fraction enriched in epidermal LBs. We identified 984 proteins, including proteins known or thought to be secreted by LBs. Moreover 31 proteins corresponded to lysosomal components further suggesting that LBs are a new class of secretory lysosomes. Many of the newly found proteins could play a role in the epidermal barrier and desquamation (one acid ceramidase-like protein, apolipoproteins, glycosidases, protease inhibitors, and peptidases) and in LB trafficking (e.g. Rab, Arf, and motor complex proteins). We focus here on CLIP-170/restin, a protein that mediates interactions between organelles and microtubules. Western blotting confirmed the presence of CLIP-170 and its known effectors IQGAP1 and Cdc42 in the LB-enriched fraction. We showed, by confocal microscopy analysis of skin cryosections, that CLIP-170 was expressed in differentiated keratinocytes, first at the periphery of the nucleus then with a granular cytoplasmic labeling evocative of LBs. It was preferentially co-localized with Cdc42 and with the known LB protein cathepsin D. CLIP-170 was also largely co-localized with Rab7. This study strongly suggests a new function for CLIP-170, its involvement together with Cdc42 and/or Rab7 in the intracellular trafficking of LBs, and provides evidence that nano-LC-MS/MS combined with monodimensional electrophoresis separation constitutes a powerful method for identifying proteins in a complex mixture such as subcellular structures.
板层小体（LBs）是与溶酶体相关的上皮细胞的管状小泡分泌细胞器。在表皮中，它们在渗透屏障稳态中发挥关键作用，在上层角质形成细胞层中分泌其内容物，包括脂质、多种水解酶、蛋白酶抑制剂和抗菌肽。对表皮板层小体中运输的蛋白质的鉴定仍远未完成，其分泌的控制方式也尚不清楚。在本研究中，我们描述了通过纳升液相色谱-串联质谱法（nano-LC-MS/MS）对富含表皮板层小体的部分进行的首次蛋白质组学表征。我们鉴定出了984种蛋白质，包括已知或被认为由板层小体分泌的蛋白质。此外，31种蛋白质对应于溶酶体成分，进一步表明板层小体是一类新的分泌性溶酶体。许多新发现的蛋白质可能在表皮屏障和脱屑过程中发挥作用（如一种酸性神经酰胺酶样蛋白、载脂蛋白、糖苷酶、蛋白酶抑制剂和肽酶）以及在板层小体运输过程中发挥作用（如Rab、Arf和运动复合体蛋白）。我们在此重点关注CLIP-170/restin，一种介导细胞器与微管之间相互作用的蛋白质。蛋白质免疫印迹法证实了CLIP-170及其已知效应器IQGAP1和Cdc42在富含板层小体的部分中的存在。通过对皮肤冷冻切片的共聚焦显微镜分析，我们发现CLIP-170在分化的角质形成细胞中表达，首先在细胞核周边，然后呈现出颗粒状细胞质标记，让人联想到板层小体。它优先与Cdc42以及已知的板层小体蛋白组织蛋白酶D共定位。CLIP-170也在很大程度上与Rab7共定位。这项研究强烈暗示了CLIP-170的新功能，即它与Cdc42和/或Rab7一起参与板层小体的细胞内运输，并提供了证据表明纳升液相色谱-串联质谱法结合一维电泳分离是鉴定复杂混合物（如亚细胞结构）中蛋白质的一种强大方法。
