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.     

Short-term effects of experimental boreal forest &;logging disturbance on bumble bees,bumble &;bee-pollinated flowers and the bee–flower match

This study examines how, over the short term, logging affects the density of bumble bees (Apidae: Bombus), the understory plants commonly visited by bumble bees, and the numerical relationship between bumble bees and flowers. In the summers before and after winter logging, bumble bees and plants were surveyed in 50 deciduous stands (each of 8–10 ha) in the boreal forest of northern Alberta, Canada. Logging was replicated at three different intensities: 0, 10–20, and 50–75% of trees remaining. There were generally more bumble bees, species of bumble bee-visited plants, and flowers in moderately (50–75%) logged sites, but this pattern depended on the time of year. Before logging, bumble bees matched resources according to an ideal free distribution (IFD). Logging affected the distribution of bumble bees across floral resources: the slope of the regression relating bumble bee and flower proportions was less than one for clearcut and control treatments (i.e., undermatching), with too many bumble bees in the flower-poor compartments and too few in the flower-rich ones. Deviations from an IFD were negative in control sites, such that fewer bumble bees occurred here than warranted by flower numbers. Controlling for flower density, bumble bee density was significantly greater in clearcuts than in the other treatments. By disproportionately visiting plants in clearcuts (relative to flower density), and by undermatching, bumble bees in clearcuts should experience higher levels of competition. Conversely, the fewer (and undermatching) bumble bees in control sites (relative to flower abundances there) may cause these plants to obtain diminished pollination service. The proximity of clearcut logging to pristine areas may therefore negatively impact plants and bumble bees in the pristine areas, at least in the season immediately following logging.     

Current trends in endotoxin detection and analysis of endotoxin–protein interactions

Endotoxin is a type of pyrogen that can be found in Gram-negative bacteria. Endotoxin can form a stable interaction with other biomolecules thus making its removal difficult especially during the production of biopharmaceutical drugs. The prevention of endotoxins from contaminating biopharmaceutical products is paramount as endotoxin contamination, even in small quantities, can result in fever, inflammation, sepsis, tissue damage and even lead to death. Highly sensitive and accurate detection of endotoxins are keys in the development of biopharmaceutical products derived from Gram-negative bacteria. It will facilitate the study of the intermolecular interaction of an endotoxin with other biomolecules, hence the selection of appropriate endotoxin removal strategies. Currently, most researchers rely on the conventional LAL-based endotoxin detection method. However, new methods have been and are being developed to overcome the problems associated with the LAL-based method. This review paper highlights the current research trends in endotoxin detection from conventional methods to newly developed biosensors. Additionally, it also provides an overview of the use of electron microscopy, dynamic light scattering (DLS), fluorescence resonance energy transfer (FRET) and docking programs in the endotoxin–protein analysis.     

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.     

Disorderness in Escherichia coli proteome: perception of folding fidelity and protein–protein interactions

Traditionally biased usage of synonymous codons renders selective advantage to proteins expressed at high levels with a few exceptions like in Escherichia coli. Proteome-wide characteristics indicative of trends in highly expressed proteins of E. coli is analyzed in this communication. Implications for the nature of interactions performed by these two groups of highly expressed proteins are discussed here. The group of highly expressed proteins having optimized codon usage through employment of most abundant tRNAs is already shielded from misfolding by their improved error-prone translational machinery. Our data also provide evidence for mechanism by which a significant proportion of highly expressed proteins with high intrinsic disorder evade degradation and successfully carry out their function.     

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.     

Cholesterol transport in steroid biosynthesis: Role of protein–protein interactions and implications in disease states

The transfer of cholesterol from the outer to the inner mitochondrial membrane is the rate-limiting step in hormone-induced steroid formation. To ensure that this step is achieved efficiently, free cholesterol must accumulate in excess at the outer mitochondrial membrane and then be transferred to the inner membrane. This is accomplished through a series of steps that involve various intracellular organelles, including lysosomes and lipid droplets, and proteins such as the translocator protein (18 kDa, TSPO) and steroidogenic acute regulatory (StAR) proteins. TSPO, previously known as the peripheral-type benzodiazepine receptor, is a high-affinity drug- and cholesterol-binding mitochondrial protein. StAR is a hormone-induced mitochondria-targeted protein that has been shown to initiate cholesterol transfer into mitochondria. Through the assistance of proteins such as the cAMP-dependent protein kinase regulatory subunit Iα (PKA-RIα) and the PKA-RIα- and TSPO-associated acyl-coenzyme A binding domain containing 3 (ACBD3) protein, PAP7, cholesterol is transferred to and docked at the outer mitochondrial membrane. The TSPO-dependent import of StAR into mitochondria, and the association of TSPO with the outer/inner mitochondrial membrane contact sites, drives the intramitochondrial cholesterol transfer and subsequent steroid formation. The focus of this review is on (i) the intracellular pathways and protein–protein interactions involved in cholesterol transport and steroid biosynthesis and (ii) the roles and interactions of these proteins in endocrine pathologies and neurological diseases where steroid synthesis plays a critical role.     

Single-spanning transmembrane domains in cell growth and cell-cell interactions: More than meets the eye?

As a whole, integral membrane proteins represent about one third of sequenced genomes, and more than 50% of currently available drugs target membrane proteins, often cell surface receptors. Some membrane protein classes, with a defined number of transmembrane (TM) helices, are receiving much attention because of their great functional and pharmacological importance, such as G protein-coupled receptors possessing 7 TM segments. Although they represent roughly half of all membrane proteins, bitopic proteins (with only 1 TM helix) have so far been less well characterized. Though they include many essential families of receptors, such as adhesion molecules and receptor tyrosine kinases, many of which are excellent targets for biopharmaceuticals (peptides, antibodies, et al.). A growing body of evidence suggests a major role for interactions between TM domains of these receptors in signaling, through homo and heteromeric associations, conformational changes, assembly of signaling platforms, etc. Significantly, mutations within single domains are frequent in human disease, such as cancer or developmental disorders. This review attempts to give an overview of current knowledge about these interactions, from structural data to therapeutic perspectives, focusing on bitopic proteins involved in cell signaling.Key words: bitopic membrane proteins, transmembrane domains, transmembrane signaling, helix-helix interactions, receptors     

Post-translational environmental switch of RadA activity by extein–intein interactions in protein splicing

Post-translational control based on an environmentally sensitive intervening intein sequence is described. Inteins are invasive genetic elements that self-splice at the protein level from the flanking host protein, the exteins. Here we show in Escherichia coli and in vitro that splicing of the RadA intein located in the ATPase domain of the hyperthermophilic archaeon Pyrococcus horikoshii is strongly regulated by the native exteins, which lock the intein in an inactive state. High temperature or solution conditions can unlock the intein for full activity, as can remote extein point mutations. Notably, this splicing trap occurs through interactions between distant residues in the native exteins and the intein, in three-dimensional space. The exteins might thereby serve as an environmental sensor, releasing the intein for full activity only at optimal growth conditions for the native organism, while sparing ATP consumption under conditions of cold-shock. This partnership between the intein and its exteins, which implies coevolution of the parasitic intein and its host protein may provide a novel means of post-translational control.     

Evaluation of temperature and guanidine hydrochloride-induced protein–liposome interactions by using immobilized liposome chromatography

Hydrophobic interactions between nine model proteins and net-neutral lipid bilayer membranes (liposomes) under stress conditions were quantitatively examined by using immobilized liposome chromatography (ILC). Small or large unilamellar liposomes were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and immobilized in a gel matrix by utilizing covalent coupling between amino-containing lipids and activated gel beads or avidin–biotin biospecific binding. Retardation of bovine carbonic anhydrase (CAB) in ILC was pronounced at particular temperatures (50 and 60 °C) where the local hydrophobicity of theses protein molecules becomes sufficiently large. Protein-induced leakage of a hydrophilic dye (calcein) from immobilized liposomes interior was also drastically enhanced at particular temperatures where large retardation was observed. For other proteins examined, similar results were also observed. The specific capacity factor of the proteins characteristic for the ILC and the amount of calcein released from immobilized liposomes were successfully expressed as a function of the product of the local hydrophobicities of proteins and liposomes, regardless of protein species and the type of the stress conditions applied (denaturant and heating). These findings indicate that lipid membranes have an ability to non-specifically recognize local hydrophobicities of proteins to form stress-mediated supramolecular assemblies with proteins, which may have potential applications in bioprocesses such as protein refolding and separation. ILC was thus found to be a very useful method for the quantitative detection of dynamic protein–liposome interactions triggered by stress conditions.     

Emergence of structure through protein–protein interactions and pH changes in dually predicted coiled-coil and disordered regions of centrosomal proteins

Human centrosomal proteins show a significant, 3.5 fold, bias to be both unstructured and coiled-coils with respect to generic human proteins, based on results from state of the art bioinformatics tools. We hypothesize that this bias means that these proteins adopt an ensemble of disordered and partially helical conformations, with the latter becoming stabilized when these proteins form complexes. Characterization of the structural properties of 13 peptides from 10 different centrosomal proteins ranging in size from 20 to 61 residues by biophysical methods led us to confirm our hypothesis in most cases. Interestingly, the secondary structure adopted by most of these peptides becomes stabilized at acidic pH and it is concentration dependent. For two of them, PIK3R1^453–513 and BRCA1^1253–1273, we observed not only the stabilization of helical structure through self-association, but also the presence of β-structures linked to the formation of high molecular weight oligomers. These oligomers are the predominant forms detected by CD, but unobservable by liquid state NMR. BRCA1^1397–1424 and MAP3K11^396–441 populate helical structures that can also self-associate at pH 3 through oligomeric species. Four peptides, derived from three proteins, namely CCNA2^103–123, BRCA1^1253–1273, BRCA1^1397–1424 and PIK3R1^453–513, can form intermolecular associations that are concomitant with alpha or beta structure stabilization. The self-phosphorylation previously described for the kinase NEK2 did not lead to any stabilization in the peptide's structure of NEK2^303–333, NEK2^341–361, and NEK2^410–430. Based on these results, obtained from a series of peptides derived from a significant number of different centrosomal proteins, we propose that conformational polymorphism, modulated by intermolecular interactions is a general property of centrosomal proteins.     

Genotype–environment interactions affect flower and fruit herbivory and plant chemistry of Arabidopsis thaliana in a transplant experiment

Large differences exist in flower and fruit herbivory between dune and inland populations of plants of Arabidopsis thaliana (Brassicaceae). Two specialist weevils Ceutorhynchus atomus and C. contractus (Curculionidae) and their larvae are responsible for this pattern in herbivory. We test, by means of a reciprocal transplant experiment, whether these differences reflect environmental influences or genetic variation in plant defense level. All plants suffered more damage after being transplanted to the dune site than after being transplanted to the inland site. Plants of inland origin suffered more flower and fruit herbivory than plants of dune origin when grown at the dune transplant site, but differences were much smaller at the inland site. Both flower damage by adult weevils and fruit damage by their larvae were subject to significant genotype × environment interactions. The observed pattern in herbivory is a strong indication for local adaption of plant defense to the level of herbivory by Ceutorhynchus. In order to identify the mechanism of defense, a quantitative analysis of glucosinolates was performed on the seeds with HPLC. Highly significant differences were found in glucosinolate types and total concentration. These patterns were mainly determined by the origin of the plants (dune or inland) and by a genotype × environment interaction. Herbivory was not significantly correlated to the concentration of glucosinolates in seeds. We therefore analyzed the total metabolic composition of seeds, using NMR spectroscopy and multivariate data analysis. Major differences in chemical composition were found in the water–methanol fractions: more glucosinolate and sucrose in the dune and more fatty acids, lipids and sinapoylmalate in the inland populations. We discuss which of these chemical factors could explain the marked differences in damage between populations.     

Insights into protein–polysorbate interactions analysed by means of isothermal titration and differential scanning calorimetry

Therapeutic proteins formulated as liquid solutions at high protein concentration are very sensitive to chemical and physical degradation. Especially avoiding the formation of protein aggregates is very crucial for product quality. In order to stabilize the colloidal properties of protein therapeutics various excipient are used. Especially the detergents polysorbate 20 and 80 are common. However, the mechanism upon which the detergents protect the protein from aggregation is not really known. The present study investigates the interaction of polysorbate 20 and 80 with different proteins: lysozyme, bovine serum albumin (BSA) and an immunoglobulin. The interaction and binding of the detergents to the proteins is investigated by isothermal titration calorimetry (ITC). From ITC the thermodynamic parameters (ΔH: change in enthalpy, ΔS: entropy and ΔG: free energy) upon binding are derived as well as the binding constant K _a. The thermal stability of the proteins in the presence of the detergent is assessed by differential scanning calorimetry (DSC). The results show that both detergents bind to BSA with K _a between 8 and 12 × 10³ M⁻¹ with ΔH −50 to −60 kJ/mol (25°C). One to two detergent molecules bind to BSA. The presence of both detergents induces a weak stabilisation of the thermal denaturation properties of BSA. However, the interaction of polysorbate 20 and 80 with lysozyme and the immunoglobulin is quite negligible. The presence of the detergents up to a concentration of 2 mM has no impact on the heat capacity curve neither a destabilisation nor a stabilisation of the native conformation is observed.