The Alb3/Oxa1/YidC protein family: membrane-localized chaperones facilitating membrane protein insertion?

The recently identified Alb3/Oxa1/YidC family constitutes a novel class of proteins that function in promoting membrane insertion in chloroplasts, mitochondria and bacteria. These proteins mediate membrane insertion of a diverse group of membrane proteins that range from phage coat proteins in bacteria and respiratory-chain protein subunits in mitochondria to the light-harvesting chlorophyll-binding proteins in chloroplasts. Here, we discuss the Alb3/Oxa1/YidC protein family and their possible function as membrane chaperones, helping newly synthesized proteins to fold into the membrane bilayer.     

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.     

The crystal structure of human protein α1M reveals a chromophore-binding site and two putative protein–protein interfaces

Lipocalin α1-microglobulin (α1M) is a conserved glycoprotein present in plasma and in the interstitial fluids of all tissues. α1M is linked to a heterogeneous yellow–brown chromophore of unknown structure, and interacts with several target proteins, including α1-inhibitor-3, fibronectin, prothrombin and albumin. To date, there is little knowledge about the interaction sites between α1M and its partners. Here, we report the crystal structure of the human α1M. Due to the crystallization occurring in a low ionic strength solution, the unidentified chromophore with heavy electron density is observed at a hydrophobic inner tube of α1M. In addition, two conserved surface regions of α1M are proposed as putative protein–protein interface sites. Further study is needed to unravel the detailed information about the interaction between α1M and its partners.     

Predicting protein–protein interactions from protein sequences using meta predictor

A novel method is proposed for predicting protein–protein interactions (PPIs) based on the meta approach, which predicts PPIs using support vector machine that combines results by six independent state-of-the-art predictors. Significant improvement in prediction performance is observed, when performed on Saccharomyces cerevisiae and Helicobacter pylori datasets. In addition, we used the final prediction model trained on the PPIs dataset of S. cerevisiae to predict interactions in other species. The results reveal that our meta model is also capable of performing cross-species predictions. The source code and the datasets are available at     

Heat shock 70 kDa protein 5/glucose-regulated protein 78 “AMP”ing up autophagy

Breast cancer is one of the most prevalent cancers in women, with more than 240,000 new cases reported in the United States in 2011. Classification of breast cancer based upon hormone and growth factor receptor profiling shows that approximately 70% of all breast cancers express estrogen receptor-α. Thus, drugs that either block estrogen biosynthesis (aromatase inhibitors like Letrozole), or compete with estrogen for estrogen receptor (ER) binding (selective ER modulators including tamoxifen; TAM) and/or cause ER degradation (selective estrogen receptor downregulators such as fulvestrant), are among the most prescribed targeted therapeutics for breast cancer. However, overall clinical benefit from the use of these drugs is often limited by resistance; ER⁺ breast cancers either fail to respond to endocrine therapies initially (de novo resistance), or they respond and then lose sensitivity over time (acquired resistance). While several preclinical studies postulate how antiestrogen resistance occurs, for the most part, the molecular mechanism(s) of resistance is unknown.     

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.     

Scoring functions for protein–protein interactions

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Production of the hybrid protein staphylococcal protein A/Escherichia coli β-galactosidase with E. coli

We have investigated the cultivation of an Escherichia coli strain producing the hybrid protein SpA-βgal. The hybrid protein consists of protein A from Staphylococcus aureus and β-galactosidase from E. coli with retained biological activity of both protein A and β-galactosidase. The expression was controlled by the temperature regulated P_R promoter from phage lambda. By late induction of the product synthesis it was possible to circumvent the problem with plasmid instability. The amount of produced SpA-βgal corresponded to approximately 1256 of the cell dry weight. In shake flask cultures most of the hybrid protein was found in an insoluble form and typical inclusion bodies were observed. However, the major part of the protein could be produced in a soluble and biological active form under controlled conditions in a reactor.     

Inhibitors of the p53/hdm2 protein–protein interaction—path to the clinic

A growing number of the elements identified in intracellular signaling events that affect cell growth and transformation are proteins that physically interact with each other via domains or specifically recognized amino acid sequences. Some of these intracellular protein–protein interactions are attractive targets for anticancer targeted therapy, but progress in this field has been compromised by the paucity of compounds with suitable biological profiles and pharmacological properties. This Letter covers salient achievements in the identification and development of inhibitors of the p53–hdm2 protein–protein interaction, and highlights different screening techniques and structure-based design approaches that may be brought to bear on the discovery and development of inhibitors of other therapeutically relevant intracellular protein–protein interactions.     

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.     

Thermostable single domain antibody–maltose binding protein fusion for Bacillus anthracis spore protein BclA detection

We constructed a genetic fusion of a single domain antibody (sdAb) with the thermal stable maltose binding protein from the thermophile Pyrococcus furiosus (PfuMBP). Produced in the Escherichia coli cytoplasm with high yield, it proved to be a rugged and effective immunoreagent. The sdAb–A5 binds BclA, a Bacillus anthracis spore protein, with high affinity (K_D ∼ 50 pM). MBPs, including the thermostable PfuMBP, have been demonstrated to be excellent folding chaperones, improving production of many recombinant proteins. A three-step purification of E. coli shake flask cultures of PfuMBP–sdAb gave a yield of approximately 100 mg/L highly purified product. The PfuMBP remained stable up to 120 °C, whereas the sdAb–A5 portion unfolded at approximately 68 to 70 °C but could refold to regain activity. This fusion construct was stable to heating at 1 mg/ml for 1 h at 70 °C, retaining nearly 100% of its binding activity; nearly one-quarter (24%) activity remained after 1 h at 90 °C. The PfuMBP–sdAb construct also provides a stable and effective method to coat gold nanoparticles. Most important, the construct was found to provide enhanced detection of B. anthracis Sterne strain (34F2) spores relative to the sdAb–A5 both as a capture reagent and as a detection reagent.     

Molecular glues to stabilise protein–protein interactions

Targeting protein–protein interactions (PPIs) has become a common approach to tackle various diseases whose pathobiology is driven by their mis-regulation in important signalling pathways. Modulating PPIs has tremendous untapped therapeutic potential and different approaches can be used to modulate PPIs. Initially, therapeutic effects were mostly sought by inhibiting PPIs. However, by gaining insight in the mode of action of certain therapeutic compounds, it became clear that stabilising (i.e. enhancing) PPIs can also be useful. The latter strategy is recently gaining a lot of attention, as stabilising physiologic, or even inducing novel interactions of a target protein with E3 ubiquitin ligases forms the basis of the targeted protein degradation (TPD) approach. An emerging additional example for drug discovery based on PPI stabilisation are the 14-3-3 proteins, a family of regulatory proteins, which engages in many protein–protein interactions, some of which might become therapeutical targets.     

PCS-based structure determination of protein–protein complexes

A simple and fast nuclear magnetic resonance method for docking proteins using pseudo-contact shift (PCS) and ¹H^N/¹⁵N chemical shift perturbation is presented. PCS is induced by a paramagnetic lanthanide ion that is attached to a target protein using a lanthanide binding peptide tag anchored at two points. PCS provides long-range (~40 Å) distance and angular restraints between the lanthanide ion and the observed nuclei, while the ¹H^N/¹⁵N chemical shift perturbation data provide loose contact-surface information. The usefulness of this method was demonstrated through the structure determination of the p62 PB1-PB1 complex, which forms a front-to-back 20 kDa homo-oligomer. As p62 PB1 does not intrinsically bind metal ions, the lanthanide binding peptide tag was attached to one subunit of the dimer at two anchoring points. Each monomer was treated as a rigid body and was docked based on the backbone PCS and backbone chemical shift perturbation data. Unlike NOE-based structural determination, this method only requires resonance assignments of the backbone ¹H^N/¹⁵N signals and the PCS data obtained from several sets of two-dimensional ¹⁵N-heteronuclear single quantum coherence spectra, thus facilitating rapid structure determination of the protein–protein complex.     

Structure-based method for analyzing protein–protein interfaces

Hydrogen bond, hydrophobic and vdW interactions are the three major non-covalent interactions at protein–protein interfaces. We have developed a method that uses only these properties to describe interactions between proteins, which can qualitatively estimate the individual contribution of each interfacial residue to the binding and gives the results in a graphic display way. This method has been applied to analyze alanine mutation data at protein–protein interfaces. A dataset containing 13 protein–protein complexes with 250 alanine mutations of interfacial residues has been tested. For the 75 hot-spot residues (G1.5 kcal mol^-1), 66 can be predicted correctly with a success rate of 88%. In order to test the tolerance of this method to conformational changes upon binding, we utilize a set of 26 complexes with one or both of their components available in the unbound form. The difference of key residues exported by the program is 11% between the results using complexed proteins and those from unbound ones. As this method gives the characteristics of the binding partner for a particular protein, in-depth studies on protein–protein recognition can be carried out. Furthermore, this method can be used to compare the difference between protein–protein interactions and look for correlated mutation. Figure Key interaction grids at the interface between barnase and barstar. Key interaction grid for barnase and barstar are presented in one figure according to their coordinates. In order to distinguish the two proteins, different icons were assigned. Crosses represent key grids for barstar and dots represent key grids for barnase. The four residues in ball and stick are Asp40 in barstar and Arg83, Arg87, His102 in barnase.