Identification of Immunoproteasome dependent factors involved in cytokine release and T cell differentiation

造血细胞，特别是淋巴细胞和单核细胞，表达免疫蛋白酶体低分子质量多肽（LMP）2 （beta1i）、LMP7 （beta5i）和多催化内肽酶复合体亚基-1 (MECL-1) （beta2i）蛋白的催化亚基。这三种亚单位也可以在暴露于炎症细胞因子后诱导非造血细胞。免疫蛋白酶体以其在MHC I类途径的抗原加工中的作用而闻名。近年来，免疫蛋白酶体在T细胞增殖、细胞因子产生、T细胞分化和自身免疫性疾病中的新功能被发现，但其潜在的分子机制尚不明确。为了研究免疫蛋白酶体如何在机制上参与控制T细胞活化和细胞因子的产生，将采用几种实验方法。已知参与T细胞活化和干扰素刺激的转录因子和信号分子将在LMP7抑制剂存在和不存在的情况下研究其磷酸化状态和表达水平。为了鉴定参与细胞因子产生的lmp7依赖因子，我们将利用两种新开发的方法。我们将定量评估在lmp7抑制存在或不存在的情况下泛素修饰的蛋白质组。此外，我们打算通过lmp7抑制细胞中底物的末端胺同位素标记来鉴定和量化蛋白质水解事件。通过这些实验，我们打算研究免疫蛋白酶体可能选择性地加工或降解参与细胞因子产生、T细胞分化和T细胞活化的因子的中心假设。

Cells of hematopoietic origin, particularly lymphocytes and monocytes, express the catalytic subunits of the immunoproteasome low-molecular mass polypeptide (LMP)2 (beta1i), LMP7 (beta5i), and multicatalytic endopeptidase complex subunit-1 (MECL-1) (beta2i) proteins. These three subunits can also be induced in non-hematopoietic cells after exposure to inflammatory cytokines. The immunoproteasome is known for its role in antigen processing along the MHC class I pathway. Recently, novel functions of immunoproteasomes in T cell proliferation, cytokine production, T cell differentiation, and autoimmune diseases have been discovered, but the underlying molecular mechanisms have remained elusive. In order to investigate how the immunoproteasome may be mechanistically involved in the control of T cell activation and cytokine production, several experimental approaches will be pursued. Transcription factors and signalling molecules known to be involved in T cell activation and interferon-gamma stimulation will be investigated with respect to their phosphorylation status and expression level in the presence and absence of an LMP7 inhibitor. In order to identify LMP7-dependent factors involved in cytokine production we will take advantage of two newly developed methods. We will quantitatively assess the ubiquitin-modified proteome in the presence or absence of LMP7-inhbition. Additionally, we intend to identify and quantify proteolytic events by terminal amine isotopic labelling of substrates in cells subjected to LMP7-inhibition. With these experiments we intend to investigate the central hypothesis that the immunoproteasome may selectively process or degrade a factor involved in cytokine production, T cell differentiation, and T cell activation.