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.     

Zinc transporter‐1: a novel NMDA receptor‐binding protein at the postsynaptic density

Zinc (Zn²⁺) is believed to play a relevant role in the physiology and pathophysiology of the brain. Hence, Zn²⁺ homeostasis is critical and involves different classes of molecules, including Zn²⁺ transporters. The ubiquitous Zn²⁺ transporter‐1 (ZNT‐1) is a transmembrane protein that pumps cytosolic Zn²⁺ to the extracellular space, but its function in the central nervous system is not fully understood. Here, we show that ZNT‐1 interacts with GluN2A‐containing NMDA receptors, suggesting a role for this transporter at the excitatory glutamatergic synapse. First, we found that ZNT‐1 is highly expressed at the hippocampal postsynaptic density (PSD) where NMDA receptors are enriched. Two‐hybrid screening, coimmunoprecipitation experiments and clustering assay in COS‐7 cells demonstrated that ZNT‐1 specifically binds the GluN2A subunit of the NMDA receptor. GluN2A deletion mutants and pull‐down assays indicated GluN2A(1390–1464) domain as necessary for the binding to ZNT‐1. Most importantly, ZNT‐1/GluN2A complex was proved to be dynamic, since it was regulated by induction of synaptic plasticity. Finally, modulation of ZNT‐1 expression in hippocampal neurons determined a significant change in dendritic spine morphology, PSD‐95 clusters and GluN2A surface levels, supporting the involvement of ZNT‐1 in the dynamics of excitatory PSD.

     

Sua5p a single‐stranded telomeric DNA‐binding protein facilitates telomere replication

In budding yeast Saccharomyces cerevisiae, telomere length maintenance involves a complicated network as more than 280 telomere maintenance genes have been identified in the nonessential gene deletion mutant set. As a supplement, we identified additional 29 telomere maintenance genes, which were previously taken as essential genes. In this study, we report a novel function of Sua5p in telomere replication. Epistasis analysis and telomere sequencing show that sua5Δ cells display progressively shortened telomeres at early passages, and Sua5 functions downstream telomerase recruitment. Further, biochemical, structural and genetic studies show that Sua5p specifically binds single‐stranded telomeric (ssTG) DNA in vitro through a distinct DNA‐binding region on its surface, and the DNA‐binding ability is essential for its telomere function. Thus, Sua5p represents a novel ssTG DNA‐binding protein and positively regulates the telomere length in vivo.     

Stn1 is critical for telomere maintenance and long‐term viability of somatic human cells

Disruption of telomere maintenance pathways leads to accelerated entry into cellular senescence, a stable proliferative arrest that promotes aging‐associated disorders in some mammals. The budding yeast CST complex, comprising Cdc13, Stn1, and Ctc1, is critical for telomere replication, length regulation, and end protection. Although mammalian homologues of CST have been identified recently, their role and function for telomere maintenance in normal somatic human cells are still incompletely understood. Here, we characterize the function of human Stn1 in cultured human fibroblasts and demonstrate its critical role in telomere replication, length regulation, and function. In the absence of high telomerase activity, shRNA‐mediated knockdown of hStn1 resulted in aberrant and fragile telomeric structures, stochastic telomere attrition, increased telomere erosion rates, telomere dysfunction, and consequently accelerated entry into cellular senescence. Oxidative stress augmented the defects caused by Stn1 knockdown leading to almost immediate cessation of cell proliferation. In contrast, overexpression of hTERT suppressed some of the defects caused by hStn1 knockdown suggesting that telomerase can partially compensate for hStn1 loss. Our findings reveal a critical role for human Stn1 in telomere length maintenance and function, supporting the model that efficient replication of telomeric repeats is critical for long‐term viability of normal somatic mammalian cells.     

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.     

LuTHy: a double‐readout bioluminescence‐based two‐hybrid technology for quantitative mapping of protein–protein interactions in mammalian cells

Information on protein–protein interactions (PPIs) is of critical importance for studying complex biological systems and developing therapeutic strategies. Here, we present a double‐readout bioluminescence‐based two‐hybrid technology, termed LuTHy, which provides two quantitative scores in one experimental procedure when testing binary interactions. PPIs are first monitored in cells by quantification of bioluminescence resonance energy transfer (BRET) and, following cell lysis, are again quantitatively assessed by luminescence‐based co‐precipitation (LuC). The double‐readout procedure detects interactions with higher sensitivity than traditional single‐readout methods and is broadly applicable, for example, for detecting the effects of small molecules or disease‐causing mutations on PPIs. Applying LuTHy in a focused screen, we identified 42 interactions for the presynaptic chaperone CSPα, causative to adult‐onset neuronal ceroid lipofuscinosis (ANCL), a progressive neurodegenerative disease. Nearly 50% of PPIs were found to be affected when studying the effect of the disease‐causing missense mutations L115R and ?L116 in CSPα with LuTHy. Our study presents a robust, sensitive research tool with high utility for investigating the molecular mechanisms by which disease‐associated mutations impair protein activity in biological systems.     

Interaction of serum amyloid A with human cystatin C—identification of binding sites

Serum amyloid A (SAA) is a multifunctional acute‐phase protein whose natural role seems to be participation in many physiologic and pathological processes. Prolonged increased SAA level in a number of chronic inflammatory and neoplastic diseases gives rise to reactive systemic amyloid A amyloidosis, where the N‐terminal 76‐amino acid residue‐long segment of SAA is deposited as amyloid fibrils. Recently, a specific interaction between SAA and the ubiquitous inhibitor of cysteine proteases—human cystatin C (hCC)—has been described. Here, we report further evidence corroborating this interaction, and the identification of the SAA and hCC binding sites in the SAA–hCC complex, using a combination of selective proteolytic excision and high‐resolution mass spectrometry. The shortest binding site in the SAA sequence was determined as SAA(86–104), whereas the binding site in hCC sequence was identified as hCC(96–102). Binding specificities of both interacting sequences were ascertained by affinity experiments (ELISA) and by registration of mass spectrum of SAA–hCC complex. Copyright © 2012 John Wiley & Sons, Ltd.     

Mitotic spindle association of TACC3 requires Aurora‐A‐dependent stabilization of a cryptic α‐helix

Aurora‐A regulates the recruitment of TACC3 to the mitotic spindle through a phospho‐dependent interaction with clathrin heavy chain (CHC). Here, we describe the structural basis of these interactions, mediated by three motifs in a disordered region of TACC3. A hydrophobic docking motif binds to a previously uncharacterized pocket on Aurora‐A that is blocked in most kinases. Abrogation of the docking motif causes a delay in late mitosis, consistent with the cellular distribution of Aurora‐A complexes. Phosphorylation of Ser558 engages a conformational switch in a second motif from a disordered state, needed to bind the kinase active site, into a helical conformation. The helix extends into a third, adjacent motif that is recognized by a helical‐repeat region of CHC, not a recognized phospho‐reader domain. This potentially widespread mechanism of phospho‐recognition provides greater flexibility to tune the molecular details of the interaction than canonical recognition motifs that are dominated by phosphate binding.     

Exon‐independent recruitment of SRSF1 is mediated by U1 snRNP stem‐loop 3

SRSF1 protein and U1 snRNPs are closely connected splicing factors. They both stimulate exon inclusion, SRSF1 by binding to exonic splicing enhancer sequences (ESEs) and U1 snRNPs by binding to the downstream 5′ splice site (SS), and both factors affect 5′ SS selection. The binding of U1 snRNPs initiates spliceosome assembly, but SR proteins such as SRSF1 can in some cases substitute for it. The mechanistic basis of this relationship is poorly understood. We show here by single‐molecule methods that a single molecule of SRSF1 can be recruited by a U1 snRNP. This reaction is independent of exon sequences and separate from the U1‐independent process of binding to an ESE. Structural analysis and cross‐linking data show that SRSF1 contacts U1 snRNA stem‐loop 3, which is required for splicing. We suggest that the recruitment of SRSF1 to a U1 snRNP at a 5′SS is the basis for exon definition by U1 snRNP and might be one of the principal functions of U1 snRNPs in the core reactions of splicing in mammals.     

Hot‐spot analysis to dissect the functional protein–protein interface of a tRNA‐modifying enzyme

Interference with protein–protein interactions of interfaces larger than 1500 Ų by small drug‐like molecules is notoriously difficult, particularly if targeting homodimers. The tRNA modifying enzyme Tgt is only functionally active as a homodimer. Thus, blocking Tgt dimerization is a promising strategy for drug therapy as this protein is key to the development of Shigellosis. Our goal was to identify hot‐spot residues which, upon mutation, result in a predominantly monomeric state of Tgt. The detailed understanding of the spatial location and stability contribution of the individual interaction hot‐spot residues and the plasticity of motifs involved in the interface formation is a crucial prerequisite for the rational identification of drug‐like inhibitors addressing the respective dimerization interface. Using computational analyses, we identified hot‐spot residues that contribute particularly to dimer stability: a cluster of hydrophobic and aromatic residues as well as several salt bridges. This in silico prediction led to the identification of a promising double mutant, which was validated experimentally. Native nano‐ESI mass spectrometry showed that the dimerization of the suggested mutant is largely prevented resulting in a predominantly monomeric state. Crystal structure analysis and enzyme kinetics of the mutant variant further support the evidence for enhanced monomerization and provide first insights into the structural consequences of the dimer destabilization. Proteins 2014; 82:2713–2732. © 2014 Wiley Periodicals, Inc.     

MicroRNA-340-5p increases telomere length by targeting telomere protein POT1 to improve Alzheimer's disease in mice

Alzheimer′s disease (AD) is a chronic neurodegenerative disorder which is the primary cause of dementia in the elderly. Telomere attrition has been proposed as a hallmark of aging. Our study aimed to explore the mechanism of the protection of telomere 1 (POT1) in regulating telomere length and affecting cellular senescence in AD. The AD mouse model was established by d -galactose and aluminum chloride, and the water maze test and dark avoidance test were used to detect the behaviors of mice and confirm the success of AD mouse model. AD cell model was established with HT22 cells induced by Aβ₄₂ oligomers. POT1 expression in the AD model was detected by quantitative real-time polymerase chain reaction. Cellular telomere length in hippocampal tissue was analyzed by telomere restriction fragment. Localization of intracellular POT1, telomerase, and telomeres was analyzed by immunofluorescence and fluorescence in situ hybridization. Dual-luciferase assay was used to validate the targeted binding relationship between microRNA-340-5p (miR-340-5p) and POT1. After inhibiting POT1 expression, the symptoms of AD in mice were improved. Aβ_1–42 deposition was reduced, whereas telomere length and telomerase activity was increased. Dual-luciferase assay verified the binding relationship between miR-340-5p and POT1. An increase in miR-340-5p expression could alleviate cellular senescence and AD symptoms. miR-340-5p increased cellular telomere length and delayed cell senescence by inhibiting POT1 expression to improve AD symptoms. This study made a conclusion that miR-340-5p increased cellular telomere length and delayed cell senescence by inhibiting POT1 expression to improve AD symptoms in mice.