Abstract
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.
Highlights
Protein interactions control many key cellular processes, but are often difficult to target with compounds
To generate a set of proteins that make up the platelet integrin adhesome, we first created a dataset describing the set of proteins present in platelets
Clinical Tissue Alliance dataset, published on GPMDB [15] contains proteomics data which indicates which proteins are frequently found in different tissue types and biological samples. (C) A third dataset containing proteins from an integrin pull-down experiment listing proteins that are likely to associate with integrin αIIbβ3 was obtained ([3]; NM unpublished data)
Summary
Protein interactions control many key cellular processes, but are often difficult to target with compounds. Integrin signaling between the states of the extracellular matrix and intracellular actin filaments[2] relies on direct interactors [3,4,5,6,7], but on large focal adhesion complexes characterized as an ‘adhesome’ comprising over 180 proteins and at least 742 interactions [2,8] This adhesome plays a key role in platelet signaling, as integrin activation is the final common step that leads to platelet activation following stimulation via numerous pathways[9]. Platelet integrins[12] are targeted extracellularly via small molecule, peptidomimetic and antibody therapeutics[13], but given their “inside-out” signaling mechanisms, it is of interest to modulate the multiplicity of different adhesome conformations, targeting various intracellular adhesome interfaces
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