■缺陷核糖体产物(DRiP)是在翻译过程中或翻译后快速降解的非功能性蛋白质,是MHCI类配体的必需来源。DRiP的特征是源自新生基因产物的大量子集,这些产物的降解速度比其相应的天然退休人员池更快。到目前为止,质谱分析显示大量的I类HLA肽来源于DRiPs。然而,没有描述蛋白水平的特异性病毒DRiP.在这项研究中,我们旨在表征和鉴定衍生自病毒蛋白的DRiPs。
■使用淋巴细胞脉络膜脑膜炎病毒(LCMV)的核蛋白(NP),其N端与泛素结合,或泛素样修饰剂FAT10或ISG15研究了DRiPs的发生。借助于FLAG标签通过蛋白质印迹监测DRiP的形成和降解。流式细胞术和细胞毒性T细胞用于研究抗原呈递。
■我们确定了几个来自LCMV-NP的短寿命DRiP。值得注意的是,只有当LCMV-NP被泛素或泛素样修饰剂修饰时,才能观察到这些DRiP,但不是野生型形式。使用蛋白酶体抑制剂,我们可以证明LCMV-NP衍生的DRiPs的降解是蛋白酶体依赖性的。有趣的是,当细胞在FCS饥饿的帮助下受到压力时,可以增强DRiPs的合成。当LCMV-NP被泛素或泛素样修饰剂修饰时,观察到NP118-126表达增强。或在FCS饥饿下。
■放在一起,我们首次可视化来自病毒蛋白的DRiPs。此外,DRiPs形成,因此MHC-I呈递,在细胞应激条件下增强。我们对MHCI类抗原呈递中DRiP的研究为开发疫苗接种策略开辟了新方法。
UNASSIGNED: Defective ribosomal products (DRiPs) are non-functional proteins rapidly degraded during or after translation being an essential source for
MHC class I ligands. DRiPs are characterized to derive from a substantial subset of nascent gene products that degrade more rapidly than their corresponding native retiree pool. So far, mass spectrometry analysis revealed that a large number of HLA class I peptides derive from DRiPs. However, a specific viral DRiP on protein level was not described. In this study, we aimed to characterize and identify DRiPs derived from a viral protein.
UNASSIGNED: Using the nucleoprotein (NP) of the lymphocytic choriomeningitis virus (LCMV) which is conjugated N-terminally to ubiquitin, or the ubiquitin-like modifiers FAT10 or ISG15 the occurrence of DRiPs was studied. The formation and degradation of DRiPs was monitored by western blot with the help of a FLAG tag. Flow cytometry and cytotoxic T cells were used to study antigen presentation.
UNASSIGNED: We identified several short lived DRiPs derived from LCMV-NP. Of note, these DRiPs could only be observed when the LCMV-NP was modified with ubiquitin or ubiquitin-like modifiers, but not in the wild type form. Using proteasome inhibitors, we could show that degradation of LCMV-NP derived DRiPs were proteasome dependent. Interestingly, the synthesis of DRiPs could be enhanced when cells were stressed with the help of FCS starvation. An enhanced NP118-126 presentation was observed when the LCMV-NP was modified with ubiquitin or ubiquitin-like modifiers, or under FCS starvation.
UNASSIGNED: Taken together, we visualize for the first time DRiPs derived from a viral protein. Furthermore, DRiPs formation, and therefore
MHC-I presentation, is enhanced under cellular stress conditions. Our investigations on DRiPs in
MHC class I antigen presentation open up new approaches for the development of vaccination strategies.