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.     

Structural insight into protein–protein interactions between intestinal microbiome and host

Protein–protein interactions between the microbiome and host organism play an important role in shaping host health. These host-modulating proteins have therapeutic potential in treating microbiome-linked disorders such as inflammatory bowel disease and obesity. Structural analysis of interacting proteins provides highly mechanistic insight into the domains driving these interactions and the resulting influence on host cell processes. Here, we briefly review recent publication of microbiome protein structures involved in host binding interactions, the effects of these interactions on host physiology, and the need for further study to increase the ability to detect proteins with therapeutic potential.     

Hypoxia activates tumor suppressor p53 by inducing ATR-Chk1 kinase cascade-mediated phosphorylation and consequent 14-3-3γ inactivation of MDMX protein

It has been known that p53 can be induced and activated by hypoxia, an abnormal condition that often occurs in rapidly growing solid tumors or when normal tissues undergo ischemia. Although the ATR-Chk1 kinase cascade was associated with hypoxia-induced p53 activation, molecules that directly link this hypoxia-ATR-Chk1 pathway to p53 activation have been elusive. Here, we showed that hypoxia could induce phosphorylation of MDMX at Ser-367 and enhance the binding of this phosphorylated MDMX to 14-3-3γ, consequently leading to p53 activation. A Chk1 inhibitor or knockdown of ATR and Chk1 inhibited the phosphorylation of MDMX at Ser-367 and impaired the binding of MDMX to 14-3-3γ in addition to p53 activation in response to hypoxia. In primary mouse embryonic fibroblast cells that harbor a mutant MDMX, including the S367A mutation, hypoxia also failed to induce the binding of this mutant MDMX to 14-3-3γ and to activate p53 and its direct targets. These results demonstrate that hypoxia can activate p53 through inactivation of MDMX by the ATR-Chk1-MDMX-14-3-3γ pathway.     

Structural basis of protein–protein interaction studied by NMR

This paper describes efforts of the structural genomics project in the nuclear magnetic resonance (NMR) laboratory at the University of Science and Technology of China. This structural genomics project is biological-functional driven. Targets are mainly selected from two systems: proteins related with regulation of gene expression in humans and other eukaryotes, and proteins existing in the cell junction in humans. The majority of proteins selected from these two systems are related with human health and diseases, and some are potential drug targets. Twenty-five protein structures from Homo sapiens and other eukaryotes have been determined during last 5 years in this laboratory. Nuclear magnetic resonance (NMR) spectroscopy is highly suited to investigate molecular interactions at a close physiological condition and is particularly suited for the study of low-affinity, transient complexes. It can provide information on protein surface interaction, their complex structure, and their dynamic properties during protein recognition. Several examples are given in this paper.     

Structural analysis of protein–protein interactions in type I polyketide synthases

Abstract

Polyketide synthases (PKSs) are responsible for synthesizing a myriad of natural products with agricultural, medicinal relevance. The PKSs consist of multiple functional domains of which each can catalyze a specified chemical reaction leading to the synthesis of polyketides. Biochemical studies showed that protein–substrate and protein–protein interactions play crucial roles in these complex regio-/stereo-selective biochemical processes. Recent developments on X-ray crystallography and protein NMR techniques have allowed us to understand the biosynthetic mechanism of these enzymes from their structures. These structural studies have facilitated the elucidation of the sequence–function relationship of PKSs and will ultimately contribute to the prediction of product structure. This review will focus on the current knowledge of type I PKS structures and the protein–protein interactions in this system.     

Alternative modulation of protein–protein interactions by small molecules

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Evolution of acyl-ACP thioesterases and β-ketoacyl-ACP synthases revealed by protein–protein interactions

The fatty acid synthase (FAS) is a conserved primary metabolic enzyme complex capable of tolerating cross-species engineering of domains for the development of modified and overproduced fatty acids. In eukaryotes, acyl-acyl carrier protein thioesterases (TEs) off-load mature cargo from the acyl carrier protein (ACP), and plants have developed TEs for short/medium-chain fatty acids. We showed that engineering plant TEs into the green microalga Chlamydomonas reinhardtii does not result in the predicted shift in fatty acid profile. Since fatty acid biosynthesis relies on substrate recognition and protein–protein interactions between the ACP and its partner enzymes, we hypothesized that plant TEs and algal ACP do not functionally interact. Phylogenetic analysis revealed major evolutionary differences between FAS enzymes, including TEs and ketoacyl synthases (KSs), in which the former is present only in some species, whereas the latter is present in all, and has a common ancestor. In line with these results, TEs appeared to be selective towards their ACP partners, whereas KSs showed promiscuous behavior across bacterial, plant, and algal species. Based on phylogenetic analyses, in silico docking, in vitro mechanistic cross-linking, and in vivo algal engineering, we propose that phylogeny can predict effective interactions between ACPs and partner enzymes.     

Landscape of π–π and sugar–π contacts in DNA–protein interactions

There were 1765 contacts identified between DNA nucleobases or deoxyribose and cyclic (W, H, F, Y) or acyclic (R, E, D) amino acids in 672 X-ray structures of DNA–protein complexes. In this first study to compare π-interactions between the cyclic and acyclic amino acids, visual inspection was used to categorize amino acid interactions as nucleobase π–π (according to biological edge) or deoxyribose sugar–π (according to sugar edge). Overall, 54% of contacts are nucleobase π–π interactions, which involve all amino acids, but are more common for Y, F, and R, and involve all DNA nucleobases with similar frequencies. Among binding arrangements, cyclic amino acids prefer more planar (stacked) π-systems than the acyclic counterparts. Although sugar–π interactions were only previously identified with the cyclic amino acids and were found to be less common (38%) than nucleobase–cyclic amino acid contacts, sugar–π interactions are more common than nucleobase π–π contacts for the acyclic series (61% of contacts). Similar to DNA–protein π–π interactions, sugar–π contacts most frequently involve Y and R, although all amino acids adopt many binding orientations relative to deoxyribose. These DNA–protein π-interactions stabilize biological systems, by up to approximately ?40 kJ mol^?1 for neutral nucleobase or sugar–amino acid interactions, but up to approximately ?95 kJ mol^?1 for positively or negatively charged contacts. The high frequency and strength, despite variation in structure and composition, of these π-interactions point to an important function in biological systems.     

Peptides and peptidomimetics as regulators of protein–protein interactions

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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.     

Direct targeting of β-catenin: Inhibition of protein–protein interactions for the inactivation of Wnt signaling

The activation of developmental signaling pathways such as Notch, Hedgehog and Wnt has implications in the onset and progression of numerous types of cancer. Consequently, targeting of such pathways is considered an attractive therapeutic approach. Inhibition of the Wnt signaling cascade proves to be complicated, in part, due to the lack of druggable pathway components. The central hub in Wnt signaling is the protein β-catenin, which is involved in numerous protein–protein interactions. In general, the inhibition of protein–protein interactions is challenging in particular with binding interfaces lacking pronounced hydrophobic pockets. Herein, we give an overview of β-catenin–protein interactions, and we review active agents that were reported to inhibit canonical Wnt signaling via direct targeting of β-catenin.