The role of the ubiquitin-like protein FAT10 in thymic selection

泛素样修饰剂FAT 10与数百种内源性靶蛋白共价结合，并作为蛋白酶体降解它们的信号。虽然我们最近已经鉴定了FAT 10结合的E1和E2酶，但FAT 10的生物学功能仍然难以捉摸。最近我们发现FAT 10在成熟的树突状细胞和小鼠胸腺上皮细胞中表达最显著，这两种细胞在未成熟胸腺细胞的阴性选择中都起抗原提呈细胞的作用。在这里，我们调查的假设，FAT 10的目标自我蛋白，表达的mTECs在一个Aire依赖的方式，介绍了MHC I类分子的阴性选择的自我反应性CD 8 + T细胞。我们将研究野生型和FAT 10-/-小鼠的mTEC之间的MHC I表面表达和抗原呈递到T细胞杂交瘤是否不同。我们将确定C57 BL/6野生型和FAT 10-/-小鼠之间外周T细胞库和TCR-Vβ使用是否不同。FAT 10在细胞系中的过表达和MHC I结合肽的洗脱将用于评估FAT 10表达是否改变MHC I配体的谱。将从小鼠mTEC细胞系中分离FAT 10偶联物，并通过质谱法鉴定FAT 10偶联的靶蛋白。最后，通过将几只T细胞受体（TCR）转基因小鼠与FAT 10-/-小鼠杂交，我们将研究这些TCR的阴性选择是否在相应同源抗原存在下受到影响。我们希望我们的工作将阐明为什么FAT 10在mTECs中如此特异性地表达，以及它在这些细胞中发挥什么样的生物学功能。

The ubiquitin-like modifier FAT10 is covalently bound to hundreds of endogenous target proteins and serves as a signal for their degradation by the proteasome. While we have recently identified the E1 and E2 enzymes of FAT10 conjugation, the biological function of FAT10 has remained elusive. Recently we have found that FAT10 is most prominently expressed in mature dendritic cells and mouse thymic epithelial cells which both function as antigen presenting cells in negative selection of immature thymocytes. Here we investigate the hypothesis that FAT10 targets self proteins, expressed by mTECs in an Aire dependent manner, for presentation of MHC class I molecules for negative selection of self-reactive CD8+ T cells. We will investigate if MHC I surface expression and antigen presentation to T cell hybridomas differs between mTECs from wildtype and FAT10-/- mice. We will determine if the peripheral T cell repertoire and the TCR-Vβ usage differs between C57BL/6 wildtype and FAT10-/- mice. Overexpression of FAT10 in cell lines and elution of MHC I bound peptides will be used to assess if FAT10 expression changes the spectrum of MHC I ligands. FAT10 conjugates will be isolated from mouse mTEC cell lines and target proteins of FAT10 conjugation will be identified by mass spectrometry. Finally, by crossing several T cell receptor (TCR) transgenic mice with FAT10-/- mice, we will investigate if the negative selection of these TCRs is affected in the presence of the respective cognate antigen. We hope that our work will elucidate why FAT10 is so specifically expressed in mTECs and what biological function it serves in these cells.