Uncovering the role of the small intracellular loop of tetraspanins by using CD9 as example

四联蛋白是一种小的膜蛋白，在单细胞向多细胞转变的过程中起着进化作用，在所有后生动物中都有表达。它们通过与免疫球蛋白超家族蛋白、蛋白酶、整合素和其他几种受体形成复合物，在四球蛋白富集微结构域（TEMs）中组织，参与多种细胞过程和疾病。这些复合物如何调节功能尚不清楚。可能，它们稳定四联蛋白及其相互作用伙伴，协助它们折叠，引导它们到达目标细胞器，调节它们在tem中的功能和排列，或介导膜曲率。了解它们是如何工作的是很复杂的，因为有许多类型的参与，并且观察到四蛋白蛋白在不同的细胞类型中会表现出相反的作用。一幅连贯图像的先决条件可能是一些尚待发现的新特性。该提案是关于发现迄今为止几乎没有研究过的蛋白质片段的功能，即小细胞内环。一项研究表明，它介导相互作用，可能是通过与细胞膜内小叶的功能相互作用。我们在环中发现了包含谷氨酸的五个氨基酸的保守序列，在CD9的晶体结构中，谷氨酸与n端赖氨酸形成盐桥。没有盐桥，CD9及其相互作用伙伴EWI-2的tem关联增加。盐桥在其他四联蛋白中也起作用。我们的目标是解决两个基本问题：1。为什么没有盐桥的突变体与EWI-2的关联更强？因为对EWI-2有更高的亲和力，四蛋白间的交联更强，或者棕榈酰化？通过生化方法检测到的更强的cd9 - ewi -2关联是如何与蛋白质的横向组织相关的？tem实际上是什么样的，哪些参数定义了它们的纳米结构？2. CD9的n端有三个正电荷，其中一个形成盐桥，注定与磷酸肌苷信号脂质相互作用。对于CD9的相互作用伙伴EWI-2，这种相互作用已经被证明。因此，tem中CD9和EWI-2之间的关联可能涉及磷酸肌苷，甚至可能受到盐桥和Ca2+的调节。四联蛋白和第二信使蛋白之间的直接联系将是非常令人兴奋的，并有助于了解该蛋白家族的一般功能。由于这个环不仅决定CD9的行为，我们期望我们的发现将为这个神秘的蛋白质家族提供普遍重要的见解。

Tetraspanins are small membrane proteins playing an evolutionary role in the unicell-to-multicell transition, and are expressed in all metazoans. They are involved in a large variety of cellular processes and diseases, through forming complexes with immunoglobulin superfamily proteins, proteases, integrins, and several other receptors, that they organize in tetraspanin enriched microdomains (TEMs). How the complexes regulate function is unclear. Possibly, they stabilize tetraspanins and their interaction partners, assist in their folding, direct them to their destination organelle, regulate their function and arrangement in TEMs, or mediate membrane curvature. Obtaining an understanding of how they work in general is complicated, because of the many types of involvements, and the observation that tetraspanins can exhibit opposite effects in different cell types. A prerequisite for a coherent picture could be novel, yet to be discovered properties. The proposal is about the uncovering of the function of a so far barely studied protein segment, the small intracellular loop. One study suggests that it mediates interactions, possibly via functional interactions with the intracellular membrane leaflet. We identified in the loop a conserved sequence of five amino acids harboring a glutamate that in the crystal structure of the tetraspanin CD9 forms a salt-bridge with a lysine of the N-terminus. Without salt-bridge, the TEM-association of CD9 and its interaction partner EWI-2 increases. The salt-bridge plays a role in other tetraspanins as well. We aim for addressing two basic questions: 1. Why does the mutant without salt-bridge associate stronger with EWI-2? Because of a higher affinity to EWI-2, a stronger cross-linking between tetraspanins, or palmitoylation? How does the stronger CD9-EWI-2-association, detected by biochemical methods, relate to the lateral organization of the proteins? How do TEMs actually look like, what parameters define their nano-architecture? 2. Three positive charges at the N-terminus of CD9, from which one forms the salt-bridge, are predestined for an interaction with phosphoinositide signaling lipids. For the CD9 interaction partner EWI-2 such an interaction has been already demonstrated. Therefore, the association between CD9 and EWI-2 in TEMs possibly involves phosphoinositides, and perhaps is even regulated by the salt-bridge and Ca2+. A direct link between tetraspanins and second messengers would be very exciting and strongly contribute to the understanding of the general functionality of this protein family. As the loop does not only determine the behavior of CD9, we expect that our findings will provide generally important insights into this yet enigmatic protein family.