Yang Chun-Yao, Bonelli Simone, Calligaris Matteo, Carreca Anna Paola, Müller Stephan A, Lichtenthaler Stefan F, Troeberg Linda, Scilabra Simone D
Centre for Osteoarthritis Pathogenesis Versus Arthritis, Kennedy Institute of Rheumatology, University of Oxford, Roosevelt Drive, Oxford OX3 7FY, UK.
Proteomics Group of Fondazione Ri.MED, Research Department IRCCS ISMETT (Istituto Mediterraneo per i Trapianti e Terapie ad Alta Specializzazione), Via E. Tricomi 5, 90145 Palermo, Italy.
Membranes (Basel). 2022 May 31;12(6):578. doi: 10.3390/membranes12060578.
A disintegrin and metalloproteinase 15 (ADAM15) is a member of the ADAM family of sheddases. Its genetic ablation in mice suggests that ADAM15 plays an important role in a wide variety of biological functions, including cartilage homeostasis. Nevertheless, while the substrate repertoire of other members of the ADAM family, including ADAM10 and ADAM17, is largely established, little is known about the substrates of ADAM15 and how it exerts its biological functions. Herein, we used unbiased proteomics to identify ADAM15 substrates and proteins regulated by the proteinase in chondrocyte-like HTB94 cells. ADAM15 silencing did not induce major changes in the secretome composition of HTB94 cells, as revealed by two different proteomic approaches. Conversely, overexpression of ADAM15 remodeled the secretome, with levels of several secreted proteins being altered compared to GFP-overexpressing controls. However, the analysis did not identify potential substrates of the sheddase, i.e., transmembrane proteins released by ADAM15 in the extracellular milieu. Intriguingly, secretome analysis and immunoblotting demonstrated that ADAM15 overexpression increased secreted levels of tissue inhibitor of metalloproteinases 3 (TIMP-3), a major regulator of extracellular matrix turnover. An inactive form of ADAM15 led to a similar increase in the inhibitor, indicating that ADAM15 regulates TIMP-3 secretion by an unknown mechanism independent of its catalytic activity. In conclusion, high-resolution quantitative proteomics of HTB94 cells manipulated to have increased or decreased ADAM15 expression did not identify canonical substrates of the proteinase in the steady state, but it revealed that ADAM15 can modulate the secretome in a catalytically-independent manner.
解整合素金属蛋白酶15（ADAM15）是解聚酶ADAM家族的成员。在小鼠中的基因敲除表明，ADAM15在包括软骨内环境稳定在内的多种生物学功能中发挥重要作用。然而，虽然ADAM家族其他成员（包括ADAM10和ADAM17）的底物谱在很大程度上已确定，但对于ADAM15的底物以及它如何发挥生物学功能却知之甚少。在此，我们使用非靶向蛋白质组学来鉴定软骨样HTB94细胞中ADAM15的底物和受该蛋白酶调控的蛋白质。两种不同的蛋白质组学方法显示，ADAM15沉默并未引起HTB94细胞分泌蛋白质组组成的重大变化。相反，ADAM15的过表达重塑了分泌蛋白质组，与过表达绿色荧光蛋白的对照相比，几种分泌蛋白的水平发生了改变。然而，该分析未鉴定出解聚酶的潜在底物，即ADAM15在细胞外环境中释放的跨膜蛋白。有趣的是，分泌蛋白质组分析和免疫印迹表明，ADAM15过表达增加了金属蛋白酶组织抑制剂3（TIMP - 3）的分泌水平，TIMP - 3是细胞外基质周转的主要调节因子。ADAM15的无活性形式导致该抑制剂有类似的增加，表明ADAM15通过一种独立于其催化活性的未知机制调节TIMP - 3的分泌。总之，对经操作使ADAM15表达增加或减少的HTB94细胞进行的高分辨率定量蛋白质组学分析，在稳态下未鉴定出该蛋白酶的典型底物，但揭示了ADAM15可以以催化非依赖的方式调节分泌蛋白质组。
