Tracking a protein following dissociation from a protein–protein complex using a split SNAP-tag system

Protein–protein interactions (PPIs) are important for various biological processes in living cells. Several methods have been developed for the visualization of PPIs in vivo; however, these methods are unsuitable for visualization of post-PPI events such as dissociation and translocation. In this study, we applied a split SNAP-tag system for the visualization of post-PPI events. This method enabled tracking of the protein following dissociation from the protein–protein complex. Thus, the split SNAP-tag system should prove to be a useful tool for visualization of post-PPI events.     

Characterization and optimization of a new immobilized system of κ-carrageenan through interaction with carob bean gum and polyols

The optimum relationships of κ-carrageenan and carob bean gum were investigated in order to obtain an immobilization system with better compression resistance, trapping capacity, and storage stability, and less syneresis phenomenon, when compared to κ-carrageenan itself. With that objective, different concentrations of polyols (glycerol and propylene glycol) were added, because of their water-retention characterization in the containing system. In this way, an improved system with good compression resistance was obtained: 15 × 10⁻⁴ N/m² in modified κ-carrageenan gel without E. coli cells and 11 × 10⁻⁴ N/m² with Escherichia coli cells. In the modified κ-carrageenan gel, the syneresis phenomenon decreased. The enzymatic activity in the system was 18 U/g, which did not present a change over a storage period of six months.     

LEC–BiFC: a new method for rapid assay of protein interaction

Abstract

Protein–protein interactions play fundamental roles in most biological processes. Bimolecular fluorescence complementation (BiFC) is a promising method for its simplicity and direct visualization of protein–protein interactions in cells. This method, however, is limited by background fluorescence that appears without specific interaction between the proteins. We report here a point mutation (V150L) in one Venus BiFC fragment that efficiently decreases background fluorescence of BiFC assay. Furthermore, by combining this modified BiFC and linear expression cassette (LEC), we develop a simple and rapid method (LEC–BiFC) for protein interaction analysis that is demonstrated by a case study of the interaction between Bcl–X_L and Bak BH3 peptide. The total analysis procedure can be completed in two days for screening tens of mutants. LEC–BiFC can be applied easily in any lab equipped with a fluorescence microscope.     

Heterodimerization of the Entamoeba histolytica EhCPADH virulence complex through molecular dynamics and protein–protein docking

EhCPADH is a protein complex involved in the virulence of Entamoeba histolytica, the protozoan responsible for human amebiasis. It is formed by the EhCP112 cysteine protease and the EhADH adhesin. To explore the molecular basis of the complex formation, three-dimensional models were built for both proteins and molecular dynamics simulations (MDS) and docking calculations were performed. Results predicted that the pEhCP112 proenzyme and the mEhCP112 mature enzyme were globular and peripheral membrane proteins. Interestingly, in pEhCP112, the propeptide appeared hiding the catalytic site (C167, H329, N348); while in mEhCP112, this site was exposed and its residues were found structurally closer than in pEhCP112. EhADH emerged as an extended peripheral membrane protein with high fluctuation in Bro1 and V shape domains. 500 ns-long MDS and protein–protein docking predictions evidenced different heterodimeric complexes with the lowest free energy. pEhCP112 interacted with EhADH by the propeptide and C-terminal regions and mEhCP112 by the C-terminal through hydrogen bonds. In contrast, EhADH bound to mEhCP112 by 442–479 residues, adjacent to the target cell-adherence region (480–600 residues), and by the Bro1 domain (9–349 residues). Calculations of the effective binding free energy and per residue free energy decomposition showed that EhADH binds to mEhCP112 with a higher binding energy than to pEhCP112, mainly through van der Waals interactions and the nonpolar part of solvation energy. The EhADH and EhCP112 structural relationship was validated in trophozoites by immunofluorescence, TEM, and immunoprecipitation assays. Experimental findings fair agreed with in silico results.     

Purification and functional analysis of protein kinase G-1α using a bacterial expression system

3',5' Cyclic guanosine monophosphate (cGMP)-dependent protein kinase G-1α (PKG-1α) is an enzyme that is a target of several anti-hypertensive and erectile dysfunction drugs. Binding of cGMP to PKG-1α produces a conformational change that leads to enzyme activation. Activated PKG-1α performs important roles both in blood vessel vasodilation and in maintaining the smooth muscle cell in a differentiated contractile state. Recombinant PKG-1α has been expressed and purified using Sf9-insect cells. However, attempts at purifying full length protein in a soluble and active form in prokaryotes have thus far been unsuccessful. These attempts have been hampered by the lack of proper eukaryotic protein folding machinery in bacteria. In this study, we report the successful expression and purification of PKG-1α using a genetically engineered Escherichia coli strain, Rosetta-gami 2(DE3), transduced with full-length human PKG-1α cDNA containing a C-terminal histidine tag. PKG-1α was purified to homogeneity using sequential nickel affinity chromatography, gel filtration and ion exchange MonoQ columns. Protein identity was confirmed by immunoblot analysis. N-terminal sequencing using Edman degradation demonstrated that the purified protein was full length. Analysis of enzyme kinetics, using a nonlinear regression curve, identified that, at constant cGMP levels (10μM) and varying ATP concentrations, PKG-1α had a maximal velocity (V(max)) of 5.02±0.25pmol/min/μg and a Michaelis-Menten constant (K(m)) of 11.78±2.68μM ATP. Recent studies have suggested that endothelial function can be attenuated by oxidative and/or nitrosative stress but the role of PKG-1α under these conditions is unclear. We found that PKG-1α enzyme activity was attenuated by exposure to the NO donor, spermine NONOate, hydrogen peroxide, and peroxynitrite but not by superoxide, suggesting that the attenuation of PKG-1α activity may be an under-appreciated mechanism underlying the development of endothelial dysfunction in a number of cardiovascular diseases.     

A new potential approach to block HIV-1 replication via protein–protein interaction and strand-transfer inhibition

Therapeutic treatment of AIDS is recently characterized by a crescent effort towards the identification of multiple ligands able to target different steps of HIV-1 life cycle. Taking into consideration our previously obtained SAR information and combining some important chemical structural features we report herein the synthesis of novel benzyl-indole derivatives as anti-HIV agents. Through this work we identified new dual target small molecules able to inhibit both IN-LEDGF/p75 interaction and the IN strand-transfer step considered as two crucial phases of viral life cycle.     

Identification of antisense RNA stem–loops that inhibit RNA–protein interactions using a bacterial reporter system

Many well-characterized examples of antisense RNAs from prokaryotic systems involve hybridization of the looped regions of stem–loop RNAs, presumably due to the high thermodynamic stability of the resulting loop–loop and loop–linear interactions. In this study, the identification of RNA stem–loops that inhibit U1A protein binding to the hpII RNA through RNA–RNA interactions was attempted using a bacterial reporter system based on phage λ N-mediated antitermination. As a result, loop sequences possessing 7–8 base complementarity to the 5′ region of the boxA element important for functional antitermination complex formation, but not the U1 hpII loop, were identified. In vitro and in vivo mutational analysis strongly suggested that the selected loop sequences were binding to the boxA region, and that the structure of the antisense stem–loop was important for optimal inhibitory activity. Next, in an attempt to demonstrate the ability to inhibit the interaction between the U1A protein and the hpII RNA, the rational design of an RNA stem–loop that inhibits U1A-binding to a modified hpII was carried out. Moderate inhibitory activity was observed, showing that it is possible to design and select antisense RNA stem–loops that disrupt various types of RNA–protein interactions.     

Discovery of a small-molecule inhibitor and cellular probe of Keap1–Nrf2 protein–protein interaction

A high-throughput screen (HTS) of the MLPCN library using a homogenous fluorescence polarization assay identified a small molecule as a first-in-class direct inhibitor of Keap1–Nrf2 protein–protein interaction. The HTS hit has three chiral centers; a combination of flash and chiral chromatographic separation demonstrated that Keap1-binding activity resides predominantly in one stereoisomer (SRS)-5 designated as ML334 (LH601A), which is at least 100× more potent than the other stereoisomers. The stereochemistry of the four cis isomers was assigned using X-ray crystallography and confirmed using stereospecific synthesis. (SRS)-5 is functionally active in both an ARE gene reporter assay and an Nrf2 nuclear translocation assay. The stereospecific nature of binding between (SRS)-5 and Keap1 as well as the preliminary but tractable structure–activity relationships support its use as a lead for our ongoing optimization     

Phospho-tyrosine dependent protein–protein interaction network

Post-translational protein modifications, such as tyrosine phosphorylation, regulate protein–protein interactions (PPIs) critical for signal processing and cellular phenotypes. We extended an established yeast two-hybrid system employing human protein kinases for the analyses of phospho-tyrosine (pY)-dependent PPIs in a direct experimental, large-scale approach. We identified 292 mostly novel pY-dependent PPIs which showed high specificity with respect to kinases and interacting proteins and validated a large fraction in co-immunoprecipitation experiments from mammalian cells. About one-sixth of the interactions are mediated by known linear sequence binding motifs while the majority of pY-PPIs are mediated by other linear epitopes or governed by alternative recognition modes. Network analysis revealed that pY-mediated recognition events are tied to a highly connected protein module dedicated to signaling and cell growth pathways related to cancer. Using binding assays, protein complementation and phenotypic readouts to characterize the pY-dependent interactions of TSPAN2 (tetraspanin 2) and GRB2 or PIK3R3 (p55γ), we exemplarily provide evidence that the two pY-dependent PPIs dictate cellular cancer phenotypes.     

ModuleSearch: finding functional modules in a protein–protein interaction network

Many biological processes are performed by a group of proteins rather than by individual proteins. Proteins involved in the same biological process often form a densely connected sub-graph in a protein–protein interaction network. Therefore, finding a dense sub-graph provides useful information to predict the function or protein complex of uncharacterised proteins in the sub-graph. We developed a heuristic algorithm that finds functional modules in a protein–protein interaction network and visualises the modules. The algorithm has been implemented in a platform-independent, standalone program called ModuleSearch. In an interaction network of yeast proteins, ModuleSearch found 366 overlapping modules. Of the modules, 71% have a function shared by more than half the proteins in the module and 58% have a function shared by all proteins in the module. Comparison of ModuleSearch with other programs shows that ModuleSearch finds more sub-graphs than most other programs, yet a higher proportion of the sub-graphs correspond to known functional modules. ModuleSearch and sample data are freely available to academics at http://bclab.inha.ac.kr/ModuleSearch.     

Improved NMR spectra of a protein–DNA complex through rational mutagenesis and the application of a sensitivity optimized isotope-filtered NOESY experiment

The NMR spectra of the complex between the DNA-binding domain of the Dead ringer protein (DRI-DBD, Gly262-Gly398) and its DNA binding site (DRI-DBD:DNA, 26 kDa) have been optimized by biochemical and spectroscopic means. First, we demonstrate the utility of a modified 2D [F1,F2] ¹³C-filtered NOESY experiment that employs a ¹J_HC versus chemical shift optimized adiabatic ¹³C inversion pulse [Zwahlen, C. et al. (1997) J. Am. Chem. Soc., 119, 6711–6721]. The new sequence is shown to be more sensitive than previously published pulse schemes (up to 40% in favorable cases) and its utility is demonstrated using two protein–DNA complexes. Second, we demonstrate that the targeted replacement of an interfacial aromatic residue in the DRI-DBD:DNA complex substantially reduces line broadening within its NMR spectra. The spectral changes are dramatic, salvaging a protein–DNA complex that was originally ill suited for structural analysis by NMR. This biochemical approach is not a general method, but may prove useful in the spectral optimization of other protein complexes that suffer from interfacial line broadening caused by dynamic changes in proximal aromatic rings.     

Construction and analysis of the protein–protein interaction network for the olfactory system of the silkworm Bombyx mori

Olfaction plays an essential role in feeding and information exchange in insects. Previous studies on the olfaction of silkworms have provided a wealth of information about genes and proteins, yet, most studies have only focused on a single gene or protein related to the insect's olfaction. The aim of the current study is to determine key proteins in the olfactory system of the silkworm, and further understand protein–protein interactions (PPIs) in the olfactory system of Lepidoptera. To achieve this goal, we integrated Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and network analyses. Furthermore, we selected 585 olfactory-related proteins and constructed a (PPI) network for the olfactory system of the silkworm. Network analysis led to the identification of several key proteins, including GSTz1, LOC733095, BGIBMGA002169-TA, BGIBMGA010939-TA, GSTs2, GSTd2, Or-2, and BGIBMGA013255-TA. A comprehensive evaluation of the proteins showed that glutathione S-transferases (GSTs) had the highest ranking. GSTs also had the highest enrichment levels in GO and KEGG. In conclusion, our analysis showed that key nodes in the biological network had a significant impact on the network, and the key proteins identified via network analysis could serve as new research targets to determine their functions in olfaction.     

Evolutionarily conserved motifs and modules in mitochondrial protein–protein interaction networks

Advances in organelle interactomics have led to new insights into organelle functions. In this study, we considered the common mitochondrial PIN of four evolutionarily distant eukaryotic species, namely Homo sapiens, Mus musculus, Drosophila melanogaster and Caenorhabditis elegans. By comparative interactomics analysis of mitochondrial PINs in these organisms, five conserved modules were identified. Modules comprise the main mitochondrial tasks, including proteins involved in translation process, mitochondrial import inner membrane proteins, TCA cycle enzymes, mitochondrial electron transport chain, and metabolic enzymes. Furthermore, we reemphasize that subgraphs of network, i.e., motifs and themes, may represent evolutionarily conserved topological units which are biologically significant.     

Probing the amino acids critical for protein oligomerisation and protein–nucleotide interaction in Mycobacterium tuberculosis PII protein through integration of computational and experimental approaches

We investigated the interacting amino acids critical for the stability and ATP binding of Mycobacterium tuberculosis PII protein through a series of site specific mutagenesis experiments. We assessed the effect of mutants using glutaraldehyde crosslinking and size exclusion chromatography and isothermal titration calorimetry. Mutations in the amino acid pair R60–E62 affecting central electrostatic interaction resulted in insoluble proteins. Multiple sequence alignment of PII orthologs displayed a conserved pattern of charged residues at these positions. Mutation of amino acid D97 to a neutral residue was tolerated whereas positive charge was not acceptable. Mutation of R107 alone had no effect on trimer formation. However, the combination of neutral residues both at positions 97 and 107 was not acceptable even with the pair at 60–62 intact. Reversal of charge polarity could partially restore the interaction. The residues including K90, R101 and R103 with potential to form H-bonds to ATP are conserved throughout across numerous orthologs of PII but when mutated to Alanine, they did not show significant differences in the total free energy change of the interaction as examined through isothermal titration calorimetry. The ATP binding pattern showed anti-cooperativity using three-site binding model. We observed compensatory effect in enthalpy and entropy changes and these may represent structural adjustments to accommodate ATP in the cavity even in absence of some interactions to perform the requisite function. In this respect these small differences between the PII orthologs may have evolved to suite species specific physiological niches.     

Co-evolution of quaternary organization and novel RNA tertiary interactions revealed in the crystal structure of a bacterial protein–RNA toxin–antitoxin system

Genes encoding toxin–antitoxin (TA) systems are near ubiquitous in bacterial genomes and they play key roles in important aspects of bacterial physiology, including genomic stability, formation of persister cells under antibiotic stress, and resistance to phage infection. The CptIN locus from Eubacterium rectale is a member of the recently-discovered Type III class of TA systems, defined by a protein toxin suppressed by direct interaction with a structured RNA antitoxin. Here, we present the crystal structure of the CptIN protein–RNA complex to 2.2 Å resolution. The structure reveals a new heterotetrameric quaternary organization for the Type III TA class, and the RNA antitoxin bears a novel structural feature of an extended A-twist motif within the pseudoknot fold. The retention of a conserved ribonuclease active site as well as traits normally associated with TA systems, such as plasmid maintenance, implicates a wider functional role for Type III TA systems. We present evidence for the co-variation of the Type III component pair, highlighting a distinctive evolutionary process in which an enzyme and its substrate co-evolve.     

Inhibition of protein–protein interaction of HER2–EGFR and HER2–HER3 by a rationally designed peptidomimetic

Protein–protein interactions (PPI) play a crucial role in many biological processes and modulation of PPI using small molecules to target hot spots has therapeutic value. As a model system we will use PPI of human epidermal growth factor receptors (EGFRs). Among the four EGFRs, EGFR–HER2 and HER2–HER3 are well known in cancer. We have designed a small molecule that is targeted to modulate HER2-mediated signaling. Our approach is novel because the small molecule designed disrupts dimerization not only of EGFR–HER2, but also of HER2–HER3. In the present study we have shown, using surface plasmon resonance analysis, that a peptidomimetic, compound 5, binds specifically to HER2 protein extracellular domain and disrupts the dimerization of EGFRs. To evaluate the effect of compound 5 on HER2 signaling in vitro, Western blot and PathHunter assays were used. Results indicated that compound 5 inhibits the phosphorylation of HER2 kinase domain and inhibits the heterodimerization in a dose-dependent manner. Molecular modeling methods were used to model the PPI of HER2–HER3 heterodimer.     

The HOPS complex mediates autophagosome–lysosome fusion through interaction with syntaxin 17

Membrane fusion is generally controlled by Rabs, soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs), and tethering complexes. Syntaxin 17 (STX17) was recently identified as the autophagosomal SNARE required for autophagosome–lysosome fusion in mammals and Drosophila. In this study, to better understand the mechanism of autophagosome–lysosome fusion, we searched for STX17-interacting proteins. Immunoprecipitation and mass spectrometry analysis identified vacuolar protein sorting 33A (VPS33A) and VPS16, which are components of the homotypic fusion and protein sorting (HOPS)–tethering complex. We further confirmed that all HOPS components were coprecipitated with STX17. Knockdown of VPS33A, VPS16, or VPS39 blocked autophagic flux and caused accumulation of STX17- and microtubule-associated protein light chain (LC3)–positive autophagosomes. The endocytic pathway was also affected by knockdown of VPS33A, as previously reported, but not by knockdown of STX17. By contrast, ultraviolet irradiation resistance–associated gene (UVRAG), a known HOPS-interacting protein, did not interact with the STX17–HOPS complex and may not be directly involved in autophagosome–lysosome fusion. Collectively these results suggest that, in addition to its well-established function in the endocytic pathway, HOPS promotes autophagosome–lysosome fusion through interaction with STX17.     

Sugar recognition and protein–protein interaction of mammalian lectins conferring diverse functions

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