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

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.

Targeting intrinsically disordered regions facilitates discovery of calcium channels 3.2 inhibitory peptides for adeno-associated virus–mediated peripheral analgesia

“…SLiMPrints specifically identifies the relatively overconstrained proximal groupings of residues within IDRs, indicative of putatively functional IDDs. 19 , 58 The enumerated motifs predicted within Ca V 3.2 IDRs suggest many possible functional peptides as hot-spots of functional IDDs, including proteolytic cleavage sites, ligand binding sites, posttranslational modification sites, and subcellular targeting sites. Notably, Ca V 3.2 IDRs contain several linear polybasic peptide (PBP) sequences composed of approximately 20 aa that are enriched with positively charged arginine (R) and lysine (K), other disordered aa, and phosphorylation sites that are clustered in ICL1-3 and C-terminus and can be considered as potential regulatory IDDs.…”

The Importance of Alpha-Actinin Proteins in Platelet Formation and Function, and Their Causative Role in Congenital Macrothrombocytopenia

“…It would also be nice to see some of our knowledge on actinin biology translated into useful therapeutics. O'Brien et al (2019) reported a step in that direction with the description of an actinin peptide that can potentially modulate platelet activation [97]. In their search for short linear motifs (SLiMs) as potential peptide therapeutics, they identified an actinin-1 peptide that binds vinculin.…”