Substrate recognition and binding by Signal Peptide Peptidase-like 2 (SPPL2) family

信号肽肽酶（SPP）和同源的SPP样（SPPL）蛋白酶SPPL2a、SPPL2b、SPPL2c和SPPL3属于GxGD蛋白酶家族，因此代表了一种膜内切割酶的原型。在第一个资助期，我们应用了蛋白质组宽筛选，并确定了SPPL2a， SPPL2b以及SPPL2c的各种新的候选底物，SPPL2c是迄今为止的孤儿蛋白酶。此外，我们通过候选方法鉴定了三个新的SPPL2a/b底物，并确定了这些底物中c端SPPL2的切割位点。基于对已建立的SPPL2底物TNFalpha和非底物Bri3以及TNF突变底物嵌合结构的分析，我们假设SPPL2b处理底物的多步骤切割模型，其中每一步都需要单独的决定因素。虽然全长底物与酶的结合似乎是非特异性的，但活性中心的底物识别有利于短腔底物结构域。底物TM结构域c末端的初始裂解由底物管腔近膜结构域的氨基酸8-14决定。底物是否随后在其TM结构域内经历连续的解理是由TM结构域的螺旋稳定性决定的。最后，裂解产物的稳定性以及它们可能的信号传导能力取决于底物胞质TM边界的s -棕榈酰化。目标1：在新的资助期内，我们将在我们建立的细胞培养模型以及我们新建立的SPPL2a和SPPL2b敲除细胞中验证新确定的SPPL2候选底物。目标2：由于我们的SPPL2切割模型主要基于对TNFalpha的分析和对Bri2的肤浅了解，我们将对其他底物进行针对性的改变，以确认我们结果的普遍性。最后，我们的目标是通过应用最小突变集将非底物Bri3转化为真正的SPPL2底物。目标3：阐明了SPPL2蛋白酶有效处理底物所需的内在决定因素后，我们现在将研究酶内底物识别的特定先决条件。SPPL2b优先处理具有短腔内近膜结构域的底物，而SPPL3接受与外膜结构域长度无关的底物。在此基础上，我们将建立SPPL2/SPPL3嵌合蛋白，以确定SPPL2中负责选择具有短外结构域的底物的结构域。总的来说，P2项目将提供对允许SPPL2蛋白酶识别和切割的底物决定因子的更详细的了解，并将有助于了解蛋白酶和底物如何相互作用。因此，P2项目有助于实现课题组的两大目标，并有助于进一步揭示膜内切割的分子机制。

Signal peptide peptidase (SPP) and the homologous SPP-like (SPPL) proteases, SPPL2a, SPPL2b, SPPL2c and SPPL3, belong to the family of GxGD proteases and thus represent one prototype of intramembrane cleaving enzymes. In the first funding period we applied a proteome wide screen and identified a variety of new candidate substrates for SPPL2a, SPPL2b and, in addition, also for SPPL2c, a so far orphan protease. Moreover, we identified three new SPPL2a/b substrates by candidate approach and determined the C-terminal SPPL2 cleavage sites in these substrates. Based on the analysis of chimeric constructs from the established SPPL2 substrate TNFalpha and the non-substrate Bri3 as well as of TNF mutant substrates we postulate a multistep cleavage model for substrate processing by SPPL2b, in which every step demands individual determinants. While binding of full length substrates to the enzyme seems to occur rather non-specifically, substrate recognition in the active centre is favoured by a short luminal substrate domain. The initial cleavages at the C-terminal end of the substrates TM domain are determined by amino acids 8-14 of the substrates luminal juxtamembrane domain. Whether the substrate subsequently undergoes consecutive cleavage within its TM domain is determined by the helical stability of the TM domain. And finally, the stability of the cleavage products and thus most likely also their putative signaling capacity depends on an S-palmitoylation at the cytosolic TM boundary of the substrate.Goal 1: In the new funding period we will validate the newly identified SPPL2 candidate substrates in our established cell culture models as well as in our newly established SPPL2a and SPPL2b knock out cells. Goal 2: Since our model of SPPL2 cleavage is mainly based on the analysis of TNFalpha and superficial knowledge on Bri2, we will apply targeted changes to other substrates to confirm the generality of our results. Finally, we aim to convert the non-substrate Bri3 by applying a minimal set of mutations into a bonafide SPPL2 substrate.Goal 3: Having elucidated the substrate intrinsic determinants required for efficient processing by SPPL2 proteases, we will now investigate the specific prerequisites for substrate recognition within the enzyme. While SPPL2b preferentially process substrates with a short luminal juxtamembrane domain, SPPL3 accepts substrates independent of their ectodomain length. Based on this, we will establish SPPL2/SPPL3 chimeric proteins to identify the domains within SPPL2 that are responsible for selection of substrates with short ectodomains. Altogether, project P2 will provide a more detailed insight into the substrate determinates that allow recognition and cleavage by SPPL2 proteases and will help to understand how protease and substrate interact. Thus, project P2 contributes to the two major aims of the research group and helps to further unravel the molecular mechanism of intramembrane cleavage.