Abstract：
Filaggrin (FLG) is a well-known biomarker of atopic dermatitis and skin dryness. Its full proteolysis (or filaggrinolysis) produces the major constituents of the natural moisturizing factor. Some proteases/peptidases remain to be identified in this multistep process. Mining 16 omics analyses, we identified prolyl endopeptidase (PREP) as a candidate peptidase. Indirect immunofluorescence and confocal analysis demonstrated its localization in the granular and deep cornified layers, where it co-localized with FLG. Tandem mass spectroscopy and fluorescent quenching activity assays showed that PREP cleaved several synthetic peptides derived from the FLG sequence, at the carboxyl side of an internal proline. Deimination of these peptides increased PREP enzymatic efficiency. Specific inhibition of PREP in reconstructed human epidermis (RHEs) using benzyloxycarbonyl-Pro-Prolinal (ZPP) induced the accumulation of FLG monomers. Down-regulation of PREP expression in RHEs using RNA interference confirmed the impact of PREP on FLG metabolism, and highlighted a more general role of PREP in keratinocyte differentiation. Indeed, quantitative global proteomic, Western blotting and RT-qPCR analyses showed a strong reduction in the expression of bleomycin hydrolase, known to be involved in filaggrinolysis, and of several other actors of cornification like loricrin. Consequently, at the functional level, the trans-epidermal electric resistance was drastically reduced.
摘要：
Filaggrin（FLG）是特应性皮炎和皮肤干燥的众所周知的生物标志物。其完全的蛋白水解(或丝团分解)产生天然保湿因子的主要成分。一些蛋白酶/肽酶仍有待在该多步骤过程中鉴定。挖掘16个组学分析，我们确定了脯氨酸内肽酶（PREP）为候选肽酶。间接免疫荧光和共聚焦分析表明其定位在颗粒状和深角化层中，它与FLG共同本地化。串联质谱和荧光猝灭活性测定表明，PREP裂解了几种来自FLG序列的合成肽，在内部脯氨酸的羧基侧。这些肽的脱亚胺增加了PREP酶效率。使用苄氧羰基-Pro-Prolinal（ZPP）对重建的人表皮（RHE）中的PREP的特异性抑制诱导了FLG单体的积累。使用RNA干扰下调RHE中的PREP表达证实了PREP对FLG代谢的影响，并强调了PREP在角质形成细胞分化中的更普遍作用。的确,定量全球蛋白质组学，Western印迹和RT-qPCR分析显示博来霉素水解酶的表达强烈减少，已知参与丝草分解，以及其他几个角化演员，如洛林。因此,在功能层面，经表皮电阻急剧下降。
