Discovery of binding proteins for a protein target using protein–protein docking‐based virtual screening

Target structure‐based virtual screening, which employs protein‐small molecule docking to identify potential ligands, has been widely used in small‐molecule drug discovery. In the present study, we used a protein–protein docking program to identify proteins that bind to a specific target protein. In the testing phase, an all‐to‐all protein–protein docking run on a large dataset was performed. The three‐dimensional rigid docking program SDOCK was used to examine protein–protein docking on all protein pairs in the dataset. Both the binding affinity and features of the binding energy landscape were considered in the scoring function in order to distinguish positive binding pairs from negative binding pairs. Thus, the lowest docking score, the average Z‐score, and convergency of the low‐score solutions were incorporated in the analysis. The hybrid scoring function was optimized in the all‐to‐all docking test. The docking method and the hybrid scoring function were then used to screen for proteins that bind to tumor necrosis factor‐α (TNFα), which is a well‐known therapeutic target for rheumatoid arthritis and other autoimmune diseases. A protein library containing 677 proteins was used for the screen. Proteins with scores among the top 20% were further examined. Sixteen proteins from the top‐ranking 67 proteins were selected for experimental study. Two of these proteins showed significant binding to TNFα in an in vitro binding study. The results of the present study demonstrate the power and potential application of protein–protein docking for the discovery of novel binding proteins for specific protein targets. Proteins 2014; 82:2472–2482. © 2014 Wiley Periodicals, Inc.     

Activated α2 macroglobulin induces matrix metalloproteinase 9 expression by low‐density lipoprotein receptor‐related protein 1 through MAPK‐ERK1/2 and NF‐κB activation in macrophage‐derived cell lines

Macrophages under certain stimuli induce matrix metalloproteinase 9 (MMP‐9) expression and protein secretion through the activation of MAPK‐ERK and NF‐κB signaling pathways. Previously, we demonstrated that activated α₂‐macroglulin (α₂M*) through the interaction with its receptor low‐density lipoprotein receptor‐related protein 1 (LRP1) induces macrophage proliferation mediated by the activation of MAPK‐ERK1/2. In the present work, we examined whether α₂M*/LRP1interaction could induce the MMP‐9 production in J774 and Raw264.7 macrophage‐derived cell lines. It was shown that α₂M* promoted MMP‐9 expression and protein secretion by LRP1 in both macrophage‐derived cell lines, which was mediated by the activation of MAPK‐ERK1/2 and NF‐κB. Both intracellular signaling pathways activated by α₂M* were effectively blocked by calphostin‐C, suggesting involvement of PKC. In addition, we demonstrate that α₂M* produced extracellular calcium influx via LRP1. However, when the intracellular calcium mobilization was inhibited by BAPTA‐AM, the α₂M*‐induced MAPK‐ER1/2 activation was fully blocked in both macrophage cell lines. Finally, using specific pharmacological inhibitors for PKC, Mek1, and NF‐κB, it was shown that the α₂M*‐induced MMP‐9 protein secretion was inhibited, indicating that the MMP production promoted by the α₂M*/LRP1 interaction required the activation of both signaling pathways. These findings may prove useful in the understanding of the macrophage LRP1 role in the vascular wall during atherogenic plaque progression. J. Cell. Biochem. 111: 607–617, 2010. © 2010 Wiley‐Liss, Inc.     

Computational analysis of the CB1 carboxyl‐terminus in the receptor‐G protein complex

Despite the important role of the carboxyl‐terminus (Ct) of the activated brain cannabinoid receptor one (CB1) in the regulation of G protein signaling, a structural understanding of interactions with G proteins is lacking. This is largely due to the highly flexible nature of the CB1 Ct that dynamically adapts its conformation to the presence of G proteins. In the present study, we explored how the CB1 Ct can interact with the G protein by building on our prior modeling of the CB1‐Gi complex (Shim, Ahn, and Kendall, The Journal of Biological Chemistry 2013;288:32449–32465) to incorporate a complete CB1 Ct (Glu416^Ct–Leu472^Ct). Based on the structural constraints from NMR studies, we employed ROSETTA to predict tertiary folds, ZDOCK to predict docking orientation, and molecular dynamics (MD) simulations to obtain two distinct plausible models of CB1 Ct in the CB1‐Gi complex. The resulting models were consistent with the NMR‐determined helical structure (H9) in the middle region of the CB1 Ct. The CB1 Ct directly interacted with both Gα and Gβ and stabilized the receptor at the Gi interface. The results of site‐directed mutagenesis studies of Glu416^Ct, Asp423^Ct, Asp428^Ct, and Arg444^Ct of CB1 Ct suggested that the CB1 Ct can influence receptor‐G protein coupling by stabilizing the receptor at the Gi interface. This research provided, for the first time, models of the CB1 Ct in contact with the G protein. Proteins 2016; 84:532–543. © 2016 Wiley Periodicals, Inc.     

Cellular prion protein in human plasma–derived extracellular vesicles promotes neurite outgrowth via the NMDA receptor–LRP1 receptor system

Exosomes and other extracellular vesicles (EVs) participate in cell–cell communication. Herein, we isolated EVs from human plasma and demonstrated that these EVs activate cell signaling and promote neurite outgrowth in PC-12 cells. Analysis of human plasma EVs purified by sequential ultracentrifugation using tandem mass spectrometry indicated the presence of multiple plasma proteins, including α₂-macroglobulin, which is reported to regulate PC-12 cell physiology. We therefore further purified EVs by molecular exclusion or phosphatidylserine affinity chromatography, which reduced plasma protein contamination. EVs subjected to these additional purification methods exhibited unchanged activity in PC-12 cells, even though α₂-macroglobulin was reduced to undetectable levels. Nonpathogenic cellular prion protein (PrP^C) was carried by human plasma EVs and essential for the effects of EVs on PC-12 cells, as EV-induced cell signaling and neurite outgrowth were blocked by the PrP^C-specific antibody, POM2. In addition, inhibitors of the N-methyl-d-aspartate (NMDA) receptor (NMDA-R) and low-density lipoprotein receptor–related protein-1 (LRP1) blocked the effects of plasma EVs on PC-12 cells, as did silencing of Lrp1 or the gene encoding the GluN1 NMDA-R subunit (Grin1). These results implicate the NMDA-R–LRP1 complex as the receptor system responsible for mediating the effects of EV-associated PrP^C. Finally, EVs harvested from rat astrocytes carried PrP^C and replicated the effects of human plasma EVs on PC-12 cell signaling. We conclude that interaction of EV-associated PrP^C with the NMDA-R–LRP1 complex in target cells represents a novel mechanism by which EVs may participate in intercellular communication in the nervous system.     

The postsynaptic density protein PSD-95 differentially regulates insulin- and Src-mediated current modulation of mouse NMDA receptors expressed in Xenopus oocytes

The NMDA subtype of glutamate receptor is physically associated with the postsynaptic density protein PSD-95 at glutamatergic synapses. The channel activity of NMDA receptors is regulated by different signaling molecules, including protein tyrosine kinases. Because previous results have suggested a role for protein kinase C (PKC) in insulin potentiation of NMDA currents in oocytes, the effects of coexpression of PSD-95 on insulin and PKC potentiation of NMDA currents from these receptors were compared. Another primary objective was to determine if PSD-95 could enable Src to potentiate currents from NR2A/NR1 and NR2B/NR1 receptors expressed in XENOPUS: oocytes. The results show opposite effects of PSD-95 coexpression on Src and insulin modulation of NR2A/NR1 receptor currents. Src potentiation of mouse NR2A/NR1 currents required PSD-95 coexpression. In contrast, PSD-95 coexpression eliminated insulin-mediated potentiation of NR2A/NR1 receptor currents. PSD-95 coexpression also eliminated PKC potentiation of NR2A/NR1 receptor currents. PSD-95 may therefore play a key role in controlling kinase modulation of NR2A/NR1 receptor currents at glutamatergic synapses.     

Low‐density lipoprotein receptor‐related protein 6 regulates alternative pre‐mRNA splicing

Low‐density lipoprotein receptor‐related protein 6 (LRP6) serves as a Wnt coreceptor. Although Wnt/LRP6 signalling is best known for the β‐catenin‐dependent regulation of target genes in tissue development and homeostasis, emerging evidence demonstrates the biological aspects of LRP6 beyond a Wnt coreceptor. Whether LRP6 modulates tissue development in a Wnt/β‐catenin signalling‐independent manner remains unknown. Using a model of striated muscle development, we observed that LRP6 was almost undetectable in proliferating myoblasts, whereas its expression gradually increased in the nucleus of myodifferentiating cells. During myodifferentiation, LRP6 modulated the muscle‐specific splicing of integrin‐β1D and consequent myotube maturation independently of the β‐catenin‐dependent Wnt signalling. Furthermore, we identified that the carboxy‐terminal serine‐rich region in LRP6 bond to the adenine‐rich sequence within alternative exon D (AED) of integrin‐β1 pre‐mRNA, and therefore, elicited AED inclusion when the spliceosome was recruited to the splice site. The interaction of LRP6 with the adenine‐rich sequence was sufficient to overcome AED exclusion by a splicing repressor, polypyrimidine tract binding protein‐1. Besides the integrin‐β1, deep RNA sequencing in different types of cells revealed that the LRP6‐mediated splicing regulation was widespread. Thus, our findings implicate LRP6 as a potential regulator for alternative pre‐mRNA splicing.     

Computational design of small molecular modulators of protein–protein interactions with a novel thermodynamic cycle: Allosteric inhibitors of HIV‐1 integrase

Targeting protein–protein interactions for therapeutic development involves designing small molecules to either disrupt or enhance a known PPI. For this purpose, it is necessary to compute reliably the effect of chemical modifications of small molecules on the protein–protein association free energy. Here we present results obtained using a novel thermodynamic free energy cycle, for the rational design of allosteric inhibitors of HIV‐1 integrase (ALLINI) that act specifically in the early stage of the infection cycle. The new compounds can serve as molecular probes to dissect the multifunctional mechanisms of ALLINIs to inform the discovery of new allosteric inhibitors. The free energy protocol developed here can be more broadly applied to study quantitatively the effects of small molecules on modulating the strengths of protein–protein interactions.     

Molecular dynamics simulations of the auxin‐binding protein 1 in complex with indole‐3‐acetic acid and naphthalen‐1‐acetic acid

Auxin‐binding protein 1 (ABP1) is suggested to be an auxin receptor which plays an important role in several processes in green plants. Maize ABP1 was simulated with the natural auxin indole‐3‐acetic acid (IAA) and the synthetic analog naphthalen‐1‐acetic acid (NAA), to elucidate the role of the KDEL sequence and the helix at the C‐terminus. The KDEL sequence weakens the intermolecular interactions between the monomers but stabilizes the C‐terminal helix. Conformational changes at the C‐terminus occur within the KDEL sequence and are influenced by the binding of the simulated ligands. This observation helps to explain experimental findings on ABP1 interactions with antibodies that are modulated by the presence of auxin, and supports the hypothesis that ABP1 acts as an auxin receptor. Stable hydrogen bonds between the monomers are formed between Glu40 and Glu62, Arg10 and Thr97, Lys39, and Glu62 in all simulations. The amino acids Ile22, Leu25, Trp44, Pro55, Ile130, and Phe149 are located in the binding pocket and are involved in hydrophobic interactions with the ring system of the ligand. Trp151 is stably involved in a face to end interaction with the ligand. The calculated free energy of binding using the linear interaction energy approach showed a higher binding affinity for NAA as compared to IAA. Our simulations confirm the asymmetric behavior of the two monomers, the stronger interaction of NAA than IAA and offers insight into the possible mechanism of ABP1 as an auxin receptor. Proteins 2014; 82:2744–2755. © 2014 Wiley Periodicals, Inc.     

Exploring new strategies for grafting binding peptides onto protein loops using a consensus‐designed tetratricopeptide repeat scaffold

Peptide display approaches, in which peptide epitopes of known binding activities are grafted onto stable protein scaffolds, have been developed to constrain the peptide in its bioactive conformation and to enhance its stability. However, peptide grafting can be a lengthy process requiring extensive computational modeling and/or optimisation by directed evolution techniques. In this study, we show that ultra‐stable consensus‐designed tetratricopeptide repeat (CTPR) proteins are amenable to the grafting of peptides that bind the Kelch‐like ECH‐associated protein 1 (Keap1) onto the loop between adjacent repeats. We explore simple strategies to optimize the grafting process and show that modest improvements in Keap1‐binding affinity can be obtained by changing the composition of the linker sequence flanking either side of the binding peptide.     

Examining post‐translational modification‐mediated protein–protein interactions using a chemical proteomics approach

Post‐translational modifications (PTM) of proteins can control complex and dynamic cellular processes via regulating interactions between key proteins. To understand these regulatory mechanisms, it is critical that we can profile the PTM‐dependent protein–protein interactions. However, identifying these interactions can be very difficult using available approaches, as PTMs can be dynamic and often mediate relatively weak protein–protein interactions. We have recently developed CLASPI (cross‐linking‐assisted and stable isotope labeling in cell culture‐based protein identification), a chemical proteomics approach to examine protein–protein interactions mediated by methylation in human cell lysates. Here, we report three extensions of the CLASPI approach. First, we show that CLASPI can be used to analyze methylation‐dependent protein–protein interactions in lysates of fission yeast, a genetically tractable model organism. For these studies, we examined trimethylated histone H3 lysine‐9 (H3K9Me₃)‐dependent protein–protein interactions. Second, we demonstrate that CLASPI can be used to examine phosphorylation‐dependent protein–protein interactions. In particular, we profile proteins recognizing phosphorylated histone H3 threonine‐3 (H3T3‐Phos), a mitotic histone “mark” appearing exclusively during cell division. Our approach identified survivin, the only known H3T3‐Phos‐binding protein, as well as other proteins, such as MCAK and KIF2A, that are likely to be involved in weak but selective interactions with this histone phosphorylation “mark”. Finally, we demonstrate that the CLASPI approach can be used to study the interplay between histone H3T3‐Phos and trimethylation on the adjacent residue lysine 4 (H3K4Me₃). Together, our findings indicate the CLASPI approach can be broadly applied to profile protein–protein interactions mediated by PTMs.     

The nuclear architectural protein HMGA1a triggers receptor‐mediated endocytosis

High mobility group proteins A (HMGA), nuclear architectural factors, locate in the cell nuclei and mostly execute gene‐regulation function. However, our results reveal that a HMGA member (HMGA1a) has a unique plasma membrane receptor; this receptor specifically binds to HMGA‐decorated species, effectively mediates endocytosis, and internalizes extracellular HMGA‐functionalized cargoes. Indeed, dyes or nanoparticles labeled with HMGA1a protein readily enter Hela cells. Using a stratagem chemical cross‐linker, we covalently bonded the HMGA receptor to the HMGA1a‐GFP fusion protein, thus capturing the plasma membrane receptor. Subsequent Western blots and SDS–PAGE gel revealed that the HMGA receptor is a 26‐kDa protein. Confocal live‐cell microscopic imaging was used to monitor the whole endocytic process, in which the internalized HMGA1a‐decorated species are transported by motor proteins on microtubules and eventually arrive at the late endosomes/lysosomes. Cell viability assays also suggested that extracellular HMGA1a protein directly influences the survival ability of Hela cells in a dose‐dependent manner, implying versatility of HMGA1a protein and its potent role to suppress cancer cell survivability and to regulate growth. J. Cell. Biochem. 108: 791–801, 2009. © 2009 Wiley‐Liss, Inc.