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.     

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.     

Structural and mechanistic commonalities of amyloid-β and the prion protein

Amyloid beta (Aβ) is a major causative agent of Alzheimer disease (AD). This neurotoxic peptide is generated as a result of the cleavage of the Amyloid-Precursor-Protein (APP) by the action of β-secretase and γ-secretase. The neurotoxicity was previously thought to be the result of aggregation. However, recent studies suggest that the interaction of Aβ with numerous cell surface receptors such as N-methyl-D-aspartate (NMDA), receptor for advanced glycosylation end products (RAGE), P75 neurotrophin receptor (P75^NTR) as well as cell surface proteins such as the cellular prion protein (PrP^c) and heparan sulfate proteoglycans (HSPG) strongly enhances Aβ induced apoptosis and thereby contributes to neurotoxicity. This review focuses on the molecular mechanism resulting in Aβ-shedding as well as Aβ-induced apoptotic processes, genetic risk factors for familial AD and interactions of Aβ with cell surface receptors and proteins, with particular emphasis on the cellular prion protein. Furthermore, comparisons are drawn between AD and prion disorders and the role of laminin, an extracellular matrix protein, glycosaminoglycans and the 37 kDa/67 kDa laminin receptor (LRP/LR) have been highlighted with regards to both neurodegenerative diseases.Key words: Alzheimer disease, amyloid β, apoptosis, 37 kDa/67 kDa laminin receptor, prion proteinsAlzheimer disease (AD), primarily defined by psychiatrist Alois Alzheimer in 1906, is a neurodegenerative disorder and currently exhibits a prevalence that “doubles approximately every five years from 0.5% at the common age of onset-65 years old.”¹ This disease is the most common form of dementia afflicting the elderly and at present affects in excess of 37 million people globally² and it is predicted that 100 million people will be living with the disease by 2050.³AD has received mounting scientific interest and has stimulated tireless research endeavours not only due to the complex mechanism by which it is caused; the multitude of contributing factors and contradictions which have arisen between hypotheses and acquired results, but also due to the rise in life expectancies⁴ owing to the advent of modern medicine, which has socio-economic implications particularly in terms of strain placed upon national health systems.     

Purification and characterization of the human γ-secretase activating protein

Alzheimer's disease is a fatal neurological disorder that is a leading cause of death, with its prevalence increasing as the average life expectancy increases worldwide. There is an urgent need to develop new therapeutics for this disease. A newly described protein, the γ-secretase activating protein (GSAP), has been proposed to promote elevated levels of amyloid-β production, an activity that seems to be inhibited using the well-establish cancer drug, imatinib (Gleevec). Despite much interest in this protein, there has been little biochemical characterization of GSAP. Here we report protocols for the recombinant bacterial expression and purification of this potentially important protein. GSAP is expressed in inclusion bodies, which can be solubilized using harsh detergents or urea; however, traditional methods of refolding were not successful in generating soluble forms of the protein that contained well-ordered and homogeneous tertiary structure. However, GSAP could be solubilized in detergent micelle solutions, where it was seen to be largely α-helical but to adopt only heterogeneous tertiary structure. Under these same conditions, GSAP did not associate with either imatinib or the 99-residue transmembrane C-terminal domain of the amyloid precursor protein. These results highlight the challenges that will be faced in attempts to manipulate and characterize this protein.     

Intracellular trafficking of the β-secretase and processing of amyloid precursor protein

The main component of the amyloid plaques found in the brains of those with Alzheimer's disease (AD) is a polymerized form of the β-amyloid peptide (Aβ) and is considered to play a central role in the pathogenesis of this neurodegenerative disorder. Aβ is derived from the proteolytic processing of the amyloid precursor protein (APP). Beta site APP-cleaving enzyme, BACE1 (also known as β-secretase) is a membrane-bound aspartyl protease responsible for the initial step in the generation of Aβ peptide and is thus a prime target for therapeutic intervention. Substantive evidence now indicates that the processing of APP by BACE1 is regulated by the intracellular sorting of the enzyme and, moreover, perturbations in these intracellular trafficking pathways have been linked to late-onset AD. In this review, we highlight the recent advances in the understanding of the regulation of the intracellular sorting of BACE1 and APP and illustrate why the trafficking of these cargos represent a key issue for understanding the membrane-mediated events associated with the generation of the neurotoxic Aβ products in AD.     

On the functional and structural characterization of hubs in protein–protein interaction networks

A number of interesting issues have been addressed on biological networks about their global and local properties. The connection between the topological properties of proteins in Protein–Protein Interaction (PPI) networks and their biological relevance has been investigated focusing on hubs, i.e. proteins with a large number of interacting partners. We will survey the literature trying to answer the following questions: Do hub proteins have special biological properties? Do they tend to be more essential than non-hub proteins? Are they more evolutionarily conserved? Do they play a central role in modular organization of the protein interaction network? Are there structural properties that characterize hub proteins?     

Mapping the protein–protein and genetic interactions of cancer to guide precision medicine

Massive efforts to sequence cancer genomes have compiled an impressive catalogue of cancer mutations, revealing the recurrent exploitation of a handful of ‘hallmark cancer pathways’. However, unraveling how sets of mutated proteins in these and other pathways hijack pro-proliferative signaling networks and dictate therapeutic responsiveness remains challenging. Here, we show that cancer driver protein–protein interactions are enriched for additional cancer drivers, highlighting the power of physical interaction maps to explain known, as well as uncover new, disease-promoting pathway interrelationships. We hypothesize that by systematically mapping the protein–protein and genetic interactions in cancer—thereby creating Cancer Cell Maps—we will create resources against which to contextualize a patient’s mutations into perturbed pathways/complexes and thereby specify a matching targeted therapeutic cocktail.     

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.     

Inhibition of dynamin-dependent endocytosis increases shedding of the amyloid precursor protein ectodomain and reduces generation of amyloid β protein

Background  

The amyloid precursor protein (APP) is transported via the secretory pathway to the cell surface, where it may be cleaved within its ectodomain by α-secretase, or internalized within clathrin-coated vesicles. An alternative proteolytic pathway occurs within the endocytic compartment, where the sequential action of β- and γ-secretases generates the amyloid β protein (Aβ). In this study, we investigated the effects of modulators of endocytosis on APP processing.     

Studies on the biosynthesis of protein by lactating guinea-pig mammary gland: Characteristics of the synthesis of α-lactalbumin and total protein by slices and cell-free systems

1. Slices of lactating guinea-pig mammary gland were incubated with radioactive amino acids and the various subcellular fractions separated by centrifugation after disruption of the cells by mincing and homogenization. The most active fraction for protein synthesis appeared to be the `mitochondrial'. 2. When the subcellular fractions were prepared without previous incubation of the cells and were then incubated with radioactive amino acid and an energy-generating system, the `mitochondrial fraction' was at least as active for protein synthesis as the `microsomal fraction'. 3. The ribosomes in the microsomal fraction are mainly unattached to membrane whereas those in the mitochondrial fraction are probably attached to fragments of the rough-surfaced endoplasmic reticulum. This latter fraction contains few mitochondria. 4. The combined mitochondrial and microsomal fractions incorporated radioactive amino acids into α-lactalbumin. 5. The radioactive leucine isolated from tryptic and chymotryptic peptides of α-lactalbumin synthesized in the cell-free system was not of uniform specific radioactivity. This was consistent with the polypeptide being assembled by the sequential addition of amino acids. 6. Evidence is presented for the polypeptide chain of α-lactalbumin being assembled from the N-terminus and for chain initiation in the cell-free system. 7. It is concluded that cell-free extracts of lactating mammary gland synthesize α-lactalbumin.     

Computational design and experimental testing of the fastest-folding β-sheet protein

One of the most important and elusive goals of molecular biology is the formulation of a detailed, atomic-level understanding of the process of protein folding. Fast-folding proteins with low free-energy barriers have proved to be particularly productive objects of investigation in this context, but the design of fast-folding proteins was previously driven largely by experiment. Dramatic advances in the attainable length of molecular dynamics simulations have allowed us to characterize in atomic-level detail the folding mechanism of the fast-folding all-β WW domain FiP35. In the work reported here, we applied the biophysical insights gained from these studies to computationally design an even faster-folding variant of FiP35 containing only naturally occurring amino acids. The increased stability and high folding rate predicted by our simulations were subsequently validated by temperature-jump experiments. The experimentally measured folding time was 4.3 μs at 80 °C—about three times faster than the fastest previously known protein with β-sheet content and in good agreement with our prediction. These results provide a compelling demonstration of the potential utility of very long molecular dynamics simulations in redesigning proteins well beyond their evolved stability and folding speed.     

Conformational flexibility and binding interactions of the G protein βγ heterodimer

Previous NMR experiments on unbound G protein βγ heterodimer suggested that particular residues in the binding interface are mobile on the nanosecond timescale. In this work we performed nanosecond‐timescale molecular dynamics simulations to investigate conformational changes and dynamics of Gβγ in the presence of several binding partners: a high‐affinity peptide (SIGK), phosducin, and the GDP‐bound α subunit. In these simulations, the high mobility of GβW99 was reduced by SIGK, and it appeared that a tyrosine might stabilize GβW99 by hydrophobic or aromatic stacking interactions in addition to hydrogen bonds. Simulations of the phosducin‐Gβγ complex showed that the mobility of GβW99 was restricted, consistent with inferences from NMR. However, large‐scale conformational changes of Gβγ due to binding, which were hypothesized in the NMR study, were not observed in the simulations, most likely due to their short (nanosecond) duration. A pocket consisting of hydrophobic amino acids on Gα appears to restrict GβW99 mobility in the crystal structure of the Gαβγ? heterotrimer. The simulation trajectories are consistent with this idea. However, local conformational changes of residues GβW63, GβW211, GβW297, GβW332, and GβW339 were detected during the MD simulations. As expected, the magnitude of atomic fluctuations observed in simulations was greater for α than for the βγ subunits, suggesting that α has greater flexibility. These observations support the notion that to maintain the high mobility of GβW99 observed by solution NMR requires that the Gβ–α interface must open up on time scale longer than can be observed in nanosecond scale simulations. Proteins 2011. © 2010 Wiley‐Liss, Inc.     

Specificity of protein — Nucleic acid interaction and the biochemical evolution

The water soluble carbodiimide mediated condensation of dipeptides of the general form Gly-X was carried out in the presence of mono- and poly-nucleotides. The observed yield of the tetrapeptide was found to be higher for peptide-nucleotide system of higher interaction specificity following mainly the anticodon-amino acid relationship (Basu, H.S. & Podder, S.K., 1981, Ind. J. Biochem. Biophys.,19, 251–253). The yield of the condensation product of L-peptide was more because of its higher interaction specificity. The extent of the racemization during the condensation of Gly-L-Phe, Gly-L-Tyr and Gly-D-Phe was found to be dependent on the specificity of the interaction —the higher the specificity, the lesser the racemization. The product formed was shown to have a catalytic effect on the condensation reaction. These data thus provide a mechanism showing how the specific interaction between amino acids/dipeptides and nucleic acids could lead to the formation of the primitive translation machinery.     

Acetoxy drug: protein transacetylase of buffalo liver—characterization and mass spectrometry of the acetylated protein product

The purification and characterization of the buffalo liver microsomal transacetylase (TAase) catalyzing the transfer of acetyl groups from a model acetoxy drug: 7,8-diacetoxy-4-methylcoumarin (DAMC) to GST3–3 has been described here. The enzyme was routinely assayed using DAMC and cytosolic GST as the substrates and was partially purified from microsomes of the buffalo liver. The enzyme was found to have approximate molecular of weight 65 kDa. The action of TAase and DAMC on liver cytosolic GST resulted in the formation of monoacetoxymonohydroxy-4-methylcoumarin (MAMHC) and 7,8-dihydroxy-4-methylcoumarin (DHMC), although the former was the major metabolite. The buffalo liver microsomal TAase exhibited hyperbolic kinetics and yielded K_m (1667 μM) and V_max (192 units) when the concentration of DAMC was varied keeping the concentration of GST constant. After having characterized the nature of the substrates and a product of the TAase-catalyzed reaction, we set out to identify the acetylated protein which is another product of the reaction. GST3–3 was used as a model protein substrate for the action of TAase using DAMC as the acetyl donor. The subunit of control and modified GST3–3 were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and digested with trypsin. The tryptic peptides were extracted from the gel pieces and analyzed by matrix assisted laser desorption/ionization–time of flight–mass spectrometry (MALDI-TOFMS). The data search for calibrated and labeled mass peaks of peptides was performed on the Matrix Science Server using the search engine Mascot. The peptide maps so obtained covered 97% of the GST3–3 sequence. On comparison of MALDI peptide maps of modified and control GST, seven new peaks were recognized corresponding to the potentially acetylated peptides in peptide map. The mass value of each of them was 42 Da higher than the theoretical mass of a non-modified GST3–3 tryptic peptide, strongly suggesting acetylation. By examining the fragmentation patterns and by comparing experimental and predicted values for MS/MS daughter ions, the identity of the seven acetylated GST tryptic peptides could be confirmed by the application of LC/MS/MS. In the modified GST, N-terminal proline and six lysines (Lys₅₁, Lys₈₂, Lys₁₂₃, Lsy₁₈₁, Lys₁₉₁ and Lys₂₁₀) were found to be acetylated. The structure of acetylated GST revealed that the lysines that underwent acetylation were peripheral in positions.     

Physicochemical study of the protein–liposome interactions: influence of liposome composition and concentration on protein binding

Abstract

The aim of the present study is to investigate the interactions between liposomes and proteins and to evaluate the role of liposomal lipid composition and concentration in the formation of protein corona. Liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or hydrogenated soybean phosphatidylcholine (HSPC) with 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt) (DPPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000] (DPPE-PEG 3000), cholesterol (CH) or mixtures of these lipids, were prepared at different concentrations by the thin-film hydration method. After liposomes were dispersed in HPLC-grade water and foetal bovine serum (FBS), their physicochemical characteristics, such as size, size distribution, and ζ-potential, were determined using dynamic and electrophoretic light scattering. Aggregation of DPPC, HSPC, DPPC:CH (9:1 molar ratio), and HSPC:CH (9:1 molar ratio) in FBS was observed. On the contrary, liposomes incorporating DPPG lipids and CH both in a molar ratio of 11% were found to be stable over time, while their size did not alter dramatically in biological medium. Liposomes containing CH and PEGylated lipids retain their size in the presence of serum as well as their physical stability. In addition, our results indicate that the protein binding depends on the presence of polyethylene glycol (PEG), CH, concentration and surface charge. In this paper, we introduce a new parameter, fraction of stealthiness (F_s), for investigating the extent of protein binding to liposomes. This parameter depends on the changes in size of liposomes after serum incubation, while liposomes have stealth properties when F_s is close to 1. Thus, we conclude that lipid composition and concentration affect the adsorption of proteins and the liposomal stabilization.     

Identification of a protein–protein interaction between KCNE1 and the activation gate machinery of KCNQ1

KCNQ1 channels assemble with KCNE1 transmembrane (TM) peptides to form voltage-gated K⁺ channel complexes with slow activation gate opening. The cytoplasmic C-terminal domain that abuts the KCNE1 TM segment has been implicated in regulating KCNQ1 gating, yet its interaction with KCNQ1 has not been described. Here, we identified a protein–protein interaction between the KCNE1 C-terminal domain and the KCNQ1 S6 activation gate and S4–S5 linker. Using cysteine cross-linking, we biochemically screened over 300 cysteine pairs in the KCNQ1–KCNE1 complex and identified three residues in KCNQ1 (H363C, P369C, and I257C) that formed disulfide bonds with cysteine residues in the KCNE1 C-terminal domain. Statistical analysis of cross-link efficiency showed that H363C preferentially reacted with KCNE1 residues H73C, S74C, and D76C, whereas P369C showed preference for only D76C. Electrophysiological investigation of the mutant K⁺ channel complexes revealed that the KCNQ1 residue, H363C, formed cross-links not only with KCNE1 subunits, but also with neighboring KCNQ1 subunits in the complex. Cross-link formation involving the H363C residue was state dependent, primarily occurring when the KCNQ1–KCNE1 complex was closed. Based on these biochemical and electrophysiological data, we generated a closed-state model of the KCNQ1–KCNE1 cytoplasmic region where these protein–protein interactions are poised to slow activation gate opening.     

Expression of the Arabidopsis G-protein GPα1: purification and characterisation of the recombinant protein

The Arabidopsis G subunit, GP1, was expressedwithin Escherichia coli by co-transformation with the expressionvector and the dnaY gene which encodes tRNA^Arg _AGA/AGG. Isolation of the recombinant GP1 in a highly pureform could be achieved by a combination of anion exchange and dyeaffinity chromatography or by a single step affinity procedure viachromatography on 4-amino-anilido-GTP agarose. The recombinant proteinyielded by both procedures was highly active and bound GTPS withan apparent K_d in the nM range. GTPS binding wasstimulated two-fold in the presence of Zn²⁺ compared with that inthe presence of Mg²⁺, Mn²⁺ or Ca²⁺.Abbreviations: 4aaGTP, 4-amino-anilido-GTP; GTPS,guanosine- 5-(3-O-thiotriphosphate), PMSF,phenylmethylsulphonyl fluoride; PVDF, polvinylidene fluoride;rGP1, recombinant GP1     

Both the N-terminal fragment and the protein–protein interaction domain (PDZ domain) are required for the pro-apoptotic activity of presenilin-associated protein PSAP

Presenilin-associated protein (PSAP) was originally identified as a PS1-associated, PDZ domain protein. In a subsequent study, PSAP was found to be a mitochondrial apoptotic molecule. In this study, we cloned the PSAP gene and found that it is composed of 12 exons and localizes on chromosome 6. To better understand the structure and function of PSAP, we have generated a series of antibodies that recognize different regions of PSAP. Using these antibodies, we found that PSAP is expressed in four isoforms as a result of differential splicing of exon 8 in addition to the use of either the first or the second ATG codon as the start codon. We also found that all these isoforms are localized in the mitochondria and are pro-apoptotic. Furthermore, our data revealed that the PDZ domain and N-terminal fragment are required for the pro-apoptotic activity of PSAP.