Computational and experimental analysis of bioactive peptide linear motifs in the integrin adhesome

Therapeutic modulation of protein interactions is challenging, but short linear motifs (SLiMs) represent potential targets. Focal adhesions play a central role in adhesion by linking cells to the extracellular matrix. Integrins are central to this process, and many other intracellular proteins are components of the integrin adhesome. We applied a peptide network targeting approach to explore the intracellular modulation of integrin function in platelets. Firstly, we computed a platelet-relevant integrin adhesome, inferred via homology of known platelet proteins to adhesome components. We then computationally selected peptides from the set of platelet integrin adhesome cytoplasmic and membrane adjacent protein-protein interfaces. Motifs of interest in the intracellular component of the platelet integrin adhesome were identified using a predictor of SLiMs based on analysis of protein primary amino acid sequences (SLiMPred), a predictor of strongly conserved motifs within disordered protein regions (SLiMPrints), and information from the literature regarding protein interactions in the complex. We then synthesized peptides incorporating these motifs combined with cell penetrating factors (tat peptide and palmitylation for cytoplasmic and membrane proteins respectively). We tested for the platelet activating effects of the peptides, as well as their abilities to inhibit activation. Bioactivity testing revealed a number of peptides that modulated platelet function, including those derived from α-actinin (ACTN1) and syndecan (SDC4), binding to vinculin and syntenin respectively. Both chimeric peptide experiments and peptide combination experiments failed to identify strong effects, perhaps characterizing the adhesome as relatively robust against within-adhesome synergistic perturbation. We investigated in more detail peptides targeting vinculin. Combined experimental and computational evidence suggested a model in which the positively charged tat-derived cell penetrating part of the peptide contributes to bioactivity via stabilizing charge interactions with a region of the ACTN1 negatively charged surface. We conclude that some interactions in the integrin adhesome appear to be capable of modulation by short peptides, and may aid in the identification and characterization of target sites within the complex that may be useful for therapeutic modulation.蛋白质相互作用的治疗性调控颇具挑战，而短线性基序（short linear motifs, SLiMs）是潜在的调控靶点。黏着斑通过将细胞连接至细胞外基质，在细胞黏附过程中发挥核心作用。整合素是该过程的核心分子，众多其他胞内蛋白均为整合素黏着组（integrin adhesome）的组成成分。 我们采用肽网络靶向策略，探究血小板内整合素功能的胞内调控机制。首先，我们基于已知血小板蛋白与整合素黏着组组分的同源性，推导得到血小板相关的整合素黏着组数据集。随后，我们从血小板整合素黏着组的胞质及膜邻近区域的蛋白质-蛋白质互作界面中，通过计算方法筛选得到肽段。 我们依托三种手段鉴定血小板整合素黏着组胞内组分中的目标基序：一是基于蛋白质一级氨基酸序列分析的短线性基序预测工具（SLiMPred），二是用于预测无序蛋白区域内高度保守基序的工具（SLiMPrints），三是来源于文献的复合物内蛋白质相互作用相关信息。 我们随后合成了包含上述目标基序的肽段，并分别结合细胞穿透因子：针对胞内与膜蛋白的Tat肽与棕榈酰化修饰。 我们对这些肽段的血小板激活效应，以及它们抑制血小板激活的能力进行了检测。生物活性测试结果显示，多款肽段可调控血小板功能，其中分别源自α-辅肌动蛋白1（α-actinin, ACTN1）与黏结蛋白聚糖4（syndecan, SDC4）的肽段，可分别结合黏着斑蛋白（vinculin）与黏着蛋白（syntenin）。 嵌合肽实验与肽段组合实验均未发现显著的调控效应，这或许表明整合素黏着组对复合物内部的协同扰动具有较强的鲁棒性。我们针对靶向黏着斑蛋白的肽段开展了更深入的研究。结合实验与计算证据，我们提出如下模型：肽段中源自Tat的带正电荷细胞穿透结构域，通过与α-辅肌动蛋白1表面带负电荷的区域形成稳定的电荷相互作用，从而赋予肽段生物活性。 综上，整合素黏着组内的部分相互作用可被短肽调控，该研究或有助于鉴定并表征该复合物内可用于治疗性调控的靶位点，为相关治疗手段的开发提供支撑。