Assessment and significance of protein–protein interactions during development of protein biopharmaceuticals

Early development of protein biotherapeutics using recombinant DNA technology involved progress in the areas of cloning, screening, expression and recovery/purification. As the biotechnology industry matured, resulting in marketed products, a greater emphasis was placed on development of formulations and delivery systems requiring a better understanding of the chemical and physical properties of newly developed protein drugs. Biophysical techniques such as analytical ultracentrifugation, dynamic and static light scattering, and circular dichroism were used to study protein–protein interactions during various stages of development of protein therapeutics. These studies included investigation of protein self-association in many of the early development projects including analysis of highly glycosylated proteins expressed in mammalian CHO cell cultures. Assessment of protein–protein interactions during development of an IgG1 monoclonal antibody that binds to IgE were important in understanding the pharmacokinetics and dosing for this important biotherapeutic used to treat severe allergic IgE-mediated asthma. These studies were extended to the investigation of monoclonal antibody–antigen interactions in human serum using the fluorescent detection system of the analytical ultracentrifuge. Analysis by sedimentation velocity analytical ultracentrifugation was also used to investigate competitive binding to monoclonal antibody targets. Recent development of high concentration protein formulations for subcutaneous administration of therapeutics posed challenges, which resulted in the use of dynamic and static light scattering, and preparative analytical ultracentrifugation to understand the self-association and rheological properties of concentrated monoclonal antibody solutions.     

Peptides and peptidomimetics as regulators of protein–protein interactions

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SH3 domains: modules of protein–protein interactions

Src homology 3 (SH3) domains are involved in the regulation of important cellular pathways, such as cell proliferation, migration and cytoskeletal modifications. Recognition of polyproline and a number of noncanonical sequences by SH3 domains has been extensively studied by crystallography, nuclear magnetic resonance and other methods. High-affinity peptides that bind SH3 domains are used in drug development as candidates for anticancer treatment. This review summarizes the latest achievements in deciphering structural determinants of SH3 function.     

Alternative modulation of protein–protein interactions by small molecules

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Automated feature engineering improves prediction of protein–protein interactions

Amino Acids - Over the last decade, various machine learning (ML) and statistical approaches for protein–protein interaction (PPI) predictions have been developed to help annotating...     

Comparative mapping of host–pathogen protein–protein interactions

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Hot spot-based design of small-molecule inhibitors for protein–protein interactions

Protein–protein interactions (PPIs) are important targets for the development of chemical probes and therapeutic agents. From the initial discovery of the existence of hot spots at PPI interfaces, it has been proposed that hot spots might provide the key for developing small-molecule PPI inhibitors. However, there has been no review on the ways in which the knowledge of hot spots can be used to achieve inhibitor design, nor critical examination of successful examples. This Digest discusses the characteristics of hot spots and the identification of druggable hot spot pockets. An analysis of four examples of hot spot-based design reveals the importance of this strategy in discovering potent and selective PPI inhibitors. A general procedure for hot spot-based design of PPI inhibitors is outlined.     

Simulation of protein diffusion: a sensitive probe of protein–solvent interactions

Aqueous solutions of Candida antarctica lipase B (CALB) were simulated considering three different water models (SPC/E, TIP3P, TIP4P) by a series of molecular dynamics (MD) simulations of three different box sizes (L = 9, 14, and 19 nm) to determine the diffusion coefficient, the water viscosity and the protein density. The protein–water systems were equilibrated for 500 ns, followed by 100 ns production runs which were analysed. The diffusional properties of CALB were characterized by the Stokes radius (R_S), which was derived from the diffusion coefficient and the viscosity. R_S was compared to the geometric radius (R_G) of CALB, which was derived from the protein density. R_S and R_G differed by 0.27 nm for SPC/E and by 0.40 and 0.39 nm for TIP3P and TIP4P, respectively, which characterizes the thickness of the diffusive hydration layer on the protein surface. The simulated hydration layer of CALB resulted in agreement with those experimentally determined for other seven different proteins of comparable size. By avoiding the most common pitfalls, protein diffusion can be reliably simulated: simulating different box sizes to account for the finite size effect, equilibrating the protein–water system sufficiently, and using the complete production run for the determination of the diffusion coefficient.     

Features of protein−protein interactions in the cyanobacterial photoprotection mechanism

Photoprotective mechanisms of cyanobacteria are characterized by several features associated with the structure of their water-soluble antenna complexes–the phycobilisomes (PBs). During energy transfer from PBs to chlorophyll of photosystem reaction centers, the “energy funnel” principle is realized, which regulates energy flux due to the specialized interaction of the PBs core with a quenching molecule capable of effectively dissipating electron excitation energy into heat. The role of the quencher is performed by ketocarotenoid within the photoactive orange carotenoid protein (OCP), which is also a sensor for light flux. At a high level of insolation, OCP is reversibly photoactivated, and this is accompanied by a sig- nificant change in its structure and spectral characteristics. Such conformational changes open the possibility for pro- tein–protein interactions between OCP and the PBs core (i.e., activation of photoprotection mechanisms) or the fluores- cence recovery protein. Even though OCP was discovered in 1981, little was known about the conformation of its active form until recently, as well as about the properties of homologs of its N and C domains. Studies carried out during recent years have made a breakthrough in understanding of the structural-functional organization of OCP and have enabled discovery of new aspects of the regulation of photoprotection processes in cyanobacteria. This review focuses on aspects of protein–pro- tein interactions between the main participants of photoprotection reactions and on certain properties of representatives of newly discovered families of OCP homologs.     

Systems for the detection and analysis of protein–protein interactions

The analysis of protein–protein interactions is important for developing a better understanding of the functional annotations of proteins that are involved in various biochemical reactions in vivo. The discovery that a protein with an unknown function binds to a protein with a known function could provide a significant clue to the cellular pathway concerning the unknown protein. Therefore, information on protein–protein interactions obtained by the comprehensive analysis of all gene products is available for the construction of interactive networks consisting of individual protein–protein interactions, which, in turn, permit elaborate biological phenomena to be understood. Systems for detecting protein–protein interactions in vitro and in vivo have been developed, and have been modified to compensate for limitations. Using these novel approaches, comprehensive and reliable information on protein–protein interactions can be determined. Systems that permit this to be achieved are described in this review.K. Kuroda, M. Kato and J. Mima contributed equally to this work.     

High-throughput single-molecule studies of protein–DNA interactions

Fluorescence and force-based single-molecule studies of protein–nucleic acid interactions continue to shed critical insights into many aspects of DNA and RNA processing. As single-molecule assays are inherently low-throughput, obtaining statistically relevant datasets remains a major challenge. Additionally, most fluorescence-based single-molecule particle-tracking assays are limited to observing fluorescent proteins that are in the low-nanomolar range, as spurious background signals predominate at higher fluorophore concentrations. These technical limitations have traditionally limited the types of questions that could be addressed via single-molecule methods. In this review, we describe new approaches for high-throughput and high-concentration single-molecule biochemical studies. We conclude with a discussion of outstanding challenges for the single-molecule biologist and how these challenges can be tackled to further approach the biochemical complexity of the cell.     

Discovery of novel inhibitors of LEDGF/p75-IN protein–protein interactions

Human lens epithelium-derived growth factor (LEDGF)/p75 plays an important role in the HIV life cycle by stimulating integrase (IN)-led viral DNA integration into cellular chromosomes. Mechanistic studies show the majority of IN inhibitors chelate magnesium ions in the catalytic active site, a region topologically distant from the LEDGF/p75 binding site. Compounds disrupting the formation of LEDGF/p75 and IN complexes serve as a novel mechanistic approach different from current antiretroviral therapies. We previously built pharmacophore models mimicking LEDGF/p75 residues and identified four classes of LEDGF/p75-IN inhibitors. Substructure and similarity searches yielded additional LEDGF/p75-IN inhibitors containing an acylhydrazone moiety. The most potent of the acylhydrazones inhibited LEDGF/p75-IN interaction with an IC₅₀ value of 400 nM. We explored structure–activity relationships (SAR) and identified new acylhydrazones, hydrazines, and diazenes as lead molecules for further optimization. Two lead LEDGF/p75-IN inhibitors showed antiviral activity.     

Two-hybrid-based analysis of protein–protein interactions of the yeast multidrug resistance protein, Pdr5p

The ATP-binding cassette (ABC) transporters are a large family of proteins responsible for the translocation of a variety of compounds across the membranes of both prokaryotes and eukaryotes. The inter-protein and intra-protein interactions in these traffic ATPases are still only poorly understood. In the present study we describe, for the first time, an extensive yeast two-hybrid (Y2H)-based analysis of the interactions of the cytoplasmic loops of the yeast pleiotropic drug resistance (Pdr) protein, Pdr5p, an ABC transporter of Saccharomyces cerevisiae. Four of the major cytosolic loops that have been predicted for this protein [including the two nucleotide-binding domain (NBD)-containing loops and the cytosolic C-terminal region] were subjected to an extensive inter-domain interaction study in addition to being used as baits to identify potential interacting proteins within the cell using the Y2H system. Results of these studies have revealed that the first cytosolic loop (CL1) – containing the first NBD domain – and also the C-terminal region of Pdr5p interact with several candidate proteins. The possibility of an interaction between the CL1 loops of two neighboring Pdr5p molecules was also indicated, which could possibly have implications for dimerization of this protein. Electronic Publication     

Characterization of protein–protein interactions between rice viruses and vector insects

Planthoppers are the most notorious rice pests, because they transmit various rice viruses in a persistent-propagative manner. Protein–protein interactions (PPIs) between virus and vector are crucial for virus transmission by vector insects. However, the number of known PPIs for pairs of rice viruses and planthoppers is restricted by low throughput research methods. In this study, we applied DeNovo, a virus-host sequence-based PPI predictor, to predict potential PPIs at a genome-wide scale between three planthoppers and five rice viruses. PPIs were identified at two different confidence thresholds, referred to as low and high modes. The number of PPIs for the five planthopper-virus pairs ranged from 506 to 1985 in the low mode and from 1254 to 4286 in the high mode. After eliminating the “one-too-many” redundant interacting information, the PPIs with unique planthopper proteins were reduced to 343–724 in the low mode and 758–1671 in the high mode. Homologous analysis showed that 11 sets and 31 sets of homologous planthopper proteins were shared by all planthopper-virus interactions in the two modes, indicating that they are potential conserved vector factors essential for transmission of rice viruses. Ten PPIs between small brown planthopper and rice stripe virus (RSV) were verified using glutathione-S-transferase (GST)/His-pull down or co-immunoprecipitation assay. Five of the ten PPIs were proven positive, and three of the five SBPH proteins were confirmed to interact with RSV. The predicted PPIs provide new clues for further studies of the complicated relationship between rice viruses and their vector insects.