Functional role of transmembrane domain interactions and intramembrane cleavage of microglial innate immunity receptors

这个项目解决了这个研究单位的一个中心问题，即什么定义了要被膜内裂解酶切割的底物。尽管到目前为止已经确定了许多伽马分泌酶的底物，但我们仍然不知道决定1型蛋白跨膜结构域是否可用于伽马分泌酶加工的确切决定因素。这个问题不仅具有学术意义，而且对阿尔茨海默病患者的伽马分泌酶抑制治疗也是最重要的。TREM2的处理与小胶质细胞的生理活性高度相关，我们先前的工作表明，与阿尔茨海默病和许多其他神经退行性疾病有关的突变会影响TREM2的蛋白分解处理和小胶质细胞的吞噬活性。TREM2在体内与TYROBP(DAP12)结合。而未结合的TREM2在脱落后似乎是膜内蛋白分解的良好底物，而DAP12缺乏N-末端结构域，其去除似乎是所有伽马分泌酶底物的绝对要求，可能不被伽马分泌酶切割。已知DAP12形成高度稳定的同源二聚体。这样，我们就有了潜在的底物，它们的相互作用和处理在小胶质细胞吞噬过程中起着至关重要的作用。因此，通过研究生理和病理上高度相关的TREM2/DAP12和DAP12/DAP12相互作用作为体内模型，我们可以解决跨膜结构域的相互作用是否影响膜内蛋白分解以及在生理条件下以二聚体形式出现的跨膜结构域是否可以被伽马分泌酶切割的问题。我们推测，TREM2和DAP12跨膜区的紧密相互作用可能阻止甚至调控随后的膜内蛋白降解。这也可能进一步影响启动小胶质细胞吞噬作用所需的信号传递过程。此外，尽管DAP12显然满足真正的伽马分泌酶底物的所有要求，但我们可能能够将它鉴定为伽马分泌酶非底物。然后，DAP12将被用作支架，以确认研究单位成员确定的伽马分泌酶底物的要求。因此，我们的项目提供了一个自然产生的生物学上高度相关的测试系统来解决这个研究单元的核心问题。我们将共同阐明伽马分泌酶切割的决定因素(目标1)，以及跨膜结构域二聚在体内膜内切割和信号转导中的作用(目标2)。

This project addresses a central question of this research unit, namely what defines a substrate to be cleaved by intramembrane cleaving proteases. Although many substrates of gamma-secretase have been identified until today, we still do not know the exact determinants, which make a transmembrane domain of a type 1 protein accessible for gamma-secretase processing or not. This question is not only of academic interest, but is also of greatest importance for therapeutic gamma-secretase inhibition in Alzheimer's disease patients. TREM2 processing is highly relevant for the physiological activity of microglia cells, and mutations, which have been linked to Alzheimer's disease and many other neurodegenerative disorders, were shown in our previous work to affect proteolytic processing of TREM2 and the phagocytic activity of microglia cells. TREM2 binds in vivo to TYROBP (DAP12). Whereas unbound TREM2 appears to be a good substrate for intramembrane proteolysis after shedding, DAP12, which lacks a N-terminal domain, whose removal appears to be an absolute requirement for all gamma-secretase substrates, may not be cleaved by gamma-secretase. DAP12 is known to form a highly stable homodimer. By this, we have potential substrates available, the interaction and processing of which play an essential role in microglial phagoytosis. Thus, by investigating the physiologically and pathologically highly relevant TREM2/DAP12 and DAP12/DAP12 interactions as an in vivo model we can address the questions if interactions of transmembrane domains influence intramembrane proteolysis and if transmembrane domains, which occur under physiological conditions as dimers, can be cleaved by gamma-secretase. We hypothesize that the tight interaction of the transmembrane domains of TREM2 and DAP12 may prevent or even regulate subsequent intramembrane proteolysis. This may also further affect signaling processes, which are required to initiate microglial phagocytosis. Moreover, we may be able to identify DAP12 as a gamma-secretase non-substrate although it apparently fulfills all requirements of a bona fide gamma-secretase substrate. DAP12 will then be used as a scaffold to confirm requirements of gamma-secretase substrates identified by the members of the research unit. Thus our project provides a naturally occurring biologically highly relevant test system to address central questions of this research unit.Taken together, we will elucidate determinants for gamma-secretase-cleavage (goal 1), as well as the role of transmembrane domain dimerization for intramembrane cleavage and signaling (goal 2) in vivo.