The molecular role of the invariant chain/CD74 and its proteolytic degradation in dendritic cell function and anti-mycobacterial immune recognition

不变链(CD74)在抗原提呈细胞中MHC-II类(MHCII)复合体的组装和靶向中起着核心作用。在抗原处理隔间，CD74的管腔结构域通过顺序的蛋白分解而降解，从而释放MHCII。最终的CD74膜结合N末端片段(NTF)经过信号肽类2a(SPPL2a)的膜内蛋白降解，正如我们之前所证明的那样。SPPL2a-/-小鼠表现出脾B细胞成熟停滞和树突状细胞(DC)丢失。最近，三名因某些DC亚群缺失而导致孟德尔分枝杆菌病(MSMD)易感性的SPPL2a缺陷型人类患者被鉴定出来。在这两种情况下，积累的未裂解的CD74 NTF诱导了所描述的表型。然而，该片段所触发的病理生理序列仍不完全清楚。在小鼠SPPL2a-/-B细胞中，我们发现中枢生存通路，特别是B细胞抗原受体下游的通路受到干扰。虽然其他影响可能起作用，但我们的发现表明，CD74 NTF诱导的相关受体的错误运输是一个中心机制。在拟议的项目中，我们计划更详细地分析SPPL2a缺乏对树突状细胞功能的影响。初步结果表明，SPPL2a-/-DC在细胞因子应答方面表现出明显的变化，特别是在抗分枝杆菌免疫识别方面。通过对这种功能缺陷的描述，我们想要阐明除了DC减少之外，这是如何对SPPL2a缺陷患者和小鼠的MSMD表型起作用的。与我们在B细胞中的发现类似，我们注意到某些质膜蛋白的细胞分布，以C型凝集素受体Dectin-1为例，在SPPL2a-/-DC中发生变化，导致细胞表面水平降低。我们假设，参与病原体识别或DC分化的更多受体可能经历了CD74 NTF诱导的错误转运。我们希望采用假设驱动和无偏见相结合的方法对此进行系统分析，并将确定这在多大程度上解释了SPPL2a-/-DC的功能缺陷。以Dectin-1为模型，我们将调查哪些贩运路线主要受CD74 NTF的调节。为了在分子水平上了解NTF的影响，我们进行了IP-MS筛选，并确定了该片段的几个潜在的相互作用伙伴，证明了它们在膜运输中的作用。在多步验证策略中，我们的目标是确认这些假定的相互作用，并分析它们与SPPL2a-/-DC细胞表型的功能相关性。由于SPPL2a代表了在自身免疫中耗尽B细胞和/或树突状细胞的一个有希望的治疗靶点，因此从翻译的角度更好地理解这项研究提供的分子机制也是相关的。

The invariant chain (CD74) plays a central role in assembly and targeting of MHC class II (MHCII) complexes in antigen-presenting cells. In antigen-processing compartments, the luminal domain of CD74 is degraded by sequential proteolysis thereby releasing MHCII. The final CD74 membrane-bound N-terminal fragment (NTF) undergoes intramembrane proteolysis by Signal peptide peptidase-like 2a (SPPL2a) as we demonstrated before. SPPL2a-/- mice exhibit an arrest of splenic B cell maturation and a loss of dendritic cells (DCs). Recently, three SPPL2a-deficient human patients with Mendelian Susceptibility to Mycobacterial Disease (MSMD) based on the depletion of certain DC subsets were identified. In both contexts, the accumulating uncleaved CD74 NTF induces the described phenotypes. However, the pathophysiological sequence triggered by this fragment remains incompletely understood. In murine SPPL2a-/- B cells, we found that central survival pathways, in particular downstream of the B cell antigen receptor, were disturbed. Though additional effects may contribute, our findings indicate that a CD74 NTF-induced mis-trafficking of the involved receptors represents a central mechanism. In the proposed project, we plan to analyse the impact of SPPL2a-deficiency on dendritic cell function in more detail. Prelimininary results indicate that SPPL2a-/- DCs exhibit distinct changes in cytokine responses, in particular with regard to anti-mycobacterial immune recognition. By characterising this functional deficit, we want to elucidate how this contributes to the MSMD phenotype of SPPL2a-deficient patients and mice in addition to the DC reduction. In analogy with our findings in B cells, we noticed that the cellular distribution of certain plasma membrane proteins, exemplified by the C-type lectin receptor Dectin-1, is altered in SPPL2a-/- DCs leading to reduced levels at the cell surface. We hypothesise that further receptors involved in pathogen recognition or DC differentiation may undergo mis-trafficking induced by the CD74 NTF. We want to analyse this systematically using a combination of hypothesis-driven and unbiased approaches and will determine to what extent this accounts for the functional deficits of SPPL2a-/- DCs. Using Dectin-1 as a model, we will investigate which trafficking routes are primarily modulated by the CD74 NTF. To understand effects by the NTF at the molecular level, we have performed an IP-MS screen and identified several potential interaction partners of this fragment with a documented role in membrane trafficking. In a multistep validation strategy, we aim to confirm these putative interactions and to analyse their functional relevance with regard to the cellular phenotypes of SPPL2a-/- DCs. Since SPPL2a represents a promising therapeutic target to deplete B cells and/or dendritic cells in autoimmunity, a better understanding of the molecular mechanism as provided by this study will be relevant also from a translational point of view.