Cambridge Institute for Medical Research, Department of Clinical Neurosciences, University of Cambridge, Addenbrooke's Hospital, Hills Road, England, Cambridge, CB2 0XY, United Kingdom.
Proteome Sci. 2012 May 18;10(1):33. doi: 10.1186/1477-5956-10-33.
Signal peptide peptidase (SPP), a member of the presenilin-like intra-membrane cleaving aspartyl protease family, migrates on Blue Native (BN) gels as 100 kDa, 200 kDa and 450 kDa species. SPP has recently been implicated in other non-proteolytic functions such as retro-translocation of MHC Class I molecules and binding of misfolded proteins in the endoplasmic reticulum (ER). These high molecular weight SPP complexes might contain additional proteins that regulate the proteolytic activity of SPP or support its non-catalytic functions.
In this study, an unbiased iTRAQ-labeling mass spectrometry approach was used to identify SPP-interacting proteins. We found that vigilin, a ubiquitous multi-KH domain containing cytoplasmic protein involved in RNA binding and protein translation control, selectively enriched with SPP. Vigilin interacted with SPP and both proteins co-localized in restricted intracellular domains near the ER, biochemically co-fractionated and were part of the same 450 kDa complex on BN gels. However, vigilin does not alter the protease activity of SPP, suggesting that the SPP-vigilin interaction might be involved in the non-proteolytic functions of SPP.
We have identified and validated vigilin as a novel interacting partner of SPP that could play an important role in the non-proteolytic functions of SPP. This data adds further weight to the idea that intramembrane-cleaving aspartyl proteases, such as presenilin and SPPs, could have other functions besides the proteolysis of short membrane stubs.
信号肽肽酶（SPP）是早老素样跨膜切割天冬氨酸蛋白酶家族的成员，在 Blue Native（BN）凝胶上迁移为 100 kDa、200 kDa 和 450 kDa 物种。SPP 最近被牵连到其他非蛋白水解功能，如 MHC Ⅰ类分子的逆向转位和内质网（ER）中错误折叠蛋白的结合。这些高分子量的 SPP 复合物可能含有额外的蛋白质，调节 SPP 的蛋白水解活性或支持其非催化功能。
在这项研究中，我们采用了一种无偏的 iTRAQ 标记质谱方法来鉴定 SPP 的相互作用蛋白。我们发现，vigilin 是一种普遍存在的多 KH 结构域细胞质蛋白，参与 RNA 结合和蛋白质翻译调控，与 SPP 选择性富集。vigilin 与 SPP 相互作用，并且两种蛋白质在靠近 ER 的限制的细胞内区域共定位，生物化学上共分离，并且是 BN 凝胶上 450 kDa 复合物的一部分。然而，vigilin 不改变 SPP 的蛋白酶活性，这表明 SPP-vigilin 相互作用可能参与 SPP 的非蛋白水解功能。
我们已经鉴定并验证了 vigilin 是 SPP 的一种新的相互作用伙伴，它可能在 SPP 的非蛋白水解功能中发挥重要作用。这一数据进一步支持了这样一种观点，即跨膜切割天冬氨酸蛋白酶，如早老素和 SPPs，除了切割短膜片之外，还可能具有其他功能。
