Role of pathogen's effectors in understanding host-pathogen interaction

Pathogens can pose challenges to plant growth and development at various stages of their life cycle. Two interconnected defense strategies prevent the growth of pathogens in plants, i.e., molecular patterns triggered immunity (PTI) and pathogenic effector-triggered immunity (ETI) that often provides resistance when PTI no longer functions as a result of pathogenic effectors. Plants may trigger an ETI defense response by directly or indirectly detecting pathogen effectors via their resistance proteins. A typical resistance protein is a nucleotide-binding receptor with leucine-rich sequences (NLRs) that undergo structural changes as they recognize their effectors and form associations with other NLRs. As a result of dimerization or oligomerization, downstream components activate “helper” NLRs, resulting in a response to ETI. It was thought that ETI is highly dependent on PTI. However, recent studies have found that ETI and PTI have symbiotic crosstalk, and both work together to create a robust system of plant defense. In this article, we have summarized the recent advances in understanding the plant's early immune response, its components, and how they cooperate in innate defense mechanisms. Moreover, we have provided the current perspective on engineering strategies for crop protection based on up-to-date knowledge.     

Acetylcholinesterase. I. Kinetic aspects of interaction with reversible effectors]

Interaction of an effector M with acetylcholinesterase (EC 3.1.1.7) according to the model of Krupka and Laidler was analysed. Some usual functions of [M] : 1/VM, [(VO/VM)-1]/[M] (where VO and VM are the steady state rates in the absence and in the presence of modifiers, respectively), vertical intercept 1/VM, slope KM/VM and absolute value of reciprocal horizontal intercept KM of Lineweaver-Burk plots are investigated and corresponding plots described. It is particularly shown that if Dixon plots are curves concave downwards, plots of [VO/VM)-1]/[M] and 1/VM against [M] are hyperbolas concave upwards and downwards respectively. If Dixon plots are curves concave upwards, plots of [(VO/VM)-1]/[M] and 1/VM versus [M] are hyperbolas concave downwards and upwards respectively. Moreover plots of KM/VM against [M] are linear. However, this model does not explain some observations, under conditions of high ionic strength (gamma/2 greater than or equal to 0,1), where Dixon plots are curves concave upwards, plots of [VO/VM)-1]/[M] versus [M] strainght lines, the plot of 1/VM against [M] is a straight line or a curve concave upwards of positives slopes and the plot of KM/VM versus [M] a curve of positive slope concave upwards. These experimental data might be interpreted by an extension of the preceding model to a mechanism with two enzymatic binding sites under kinetic conditions that are determined.     

Stopping ras in its tracks

Ras interacts with many downstream effectors that regulate complex cytoplasmic signaling networks. In this issue, Gupta et al. (2007) use mouse models of Ras-mediated tumorigenesis to show that the interaction of Ras with a single isoform of phosphatidylinositol 3-kinase (PI3K), called p110alpha (PIK3CA), is critical for tumor formation. This result will stimulate re-evaluation of pharmacological approaches to target Ras for cancer treatment.     

On modeling two immune effectors two strain antigen interaction

In this paper we consider the fractional order model with two immune effectors interacting with two strain antigen. The systems may explain the recurrence of some diseases e.g. tuberculosis (TB). The stability of equilibrium points are studied. Numerical solutions of this model are given. Using integer order system the system oscillates. Using fractional order system the system converges to a stable internal equilibrium. Ulam-Hyers stability of the system has been studied.     

Spectrophotometric study of cytochrome P-450 (11 beta) interaction with physiological effectors

Using the optical absorbance spectroscopy method, the interaction of a number of biospecific ligands (steroids, adrenodoxin) with homogeneous cytochrome P-450 (11 beta) from bovine adrenal mitochondria was investigated. The parameters of the steroid-protein interaction in a number of substrates and products of the 11 beta- and 18 (19)-hydroxylation with the active site of cytochrome P-450 (11 beta) were determined. A sharp decrease in the cytochrome affinity for steroids upon the insertion of the first hydroxy group was observed, which provides for a predominant formation of monohydroxylated products from the substrate and minimum amounts of dihydroxylated ones, despite the presence of more than one position for the substrate hydroxylation by cytochrome P-450 (11 beta). Some structural elements of the steroid molecule were determined as any alterations in these strongly affect the enzyme affinity for the steroid. These structures are: 1) delta 4-3-oxo structure; 2) either 21-hydroxy group of pregnen steroids or the one fulfilling its functions, 17 beta-hydroxy or 17-oxo group of androsten steroids, and 3) the 11th position of all the substrates under study. It was shown that the binding of various substrates into stoichiometric (1:1) steroid-protein complexes provides a transition to high spin state from 30-40% (cortisol, corticosterone) to 90-95% (11-deoxycorticosterone) of hemoprotein iron. Using the experimental system containing individual cytochrome P-450 (11 beta) and adrenodoxin, as well as the steroid and nonionic detergent Tween 20, it was shown that the parameters of substrate binding and hemoprotein spin equilibrium did not differ from the corresponding parameters of the cytochrome-adrenodoxin dienzyme complex. The peculiarities of the multiligand interactions in the 11 beta-hydroxylase system, involving cytochrome, substrates and ferredoxin demonstrate some analogy with a bacterial camphor hydroxylase system and some differences from the mitochondrial system for the side chain cleavage of cholesterol.     

Identifying type III effectors of plant pathogens and analyzing their interaction with plant cells

Many bacterial pathogens cause disease by injecting virulence proteins (effectors) into host cells via the specialized type III secretion system. Recently, exceptional progress in identifying effectors was made in the phytopathogen Pseudomonas syringae using a novel genetic screen and bioinformatic approach. These studies, along with localization experiments, suggest that most P. syringae effectors function by targeting the plasma membrane, chloroplasts or mitochondria of host cells. The type III secretome of P. syringae is highly variable and dynamic, a lesson gleaned from a comparative genomic analysis. Variation in the effector repertoire is likely to facilitate the adaptation of P. syringae to different hosts.     

Biophysical characterization of the interaction between hepatic glucokinase and its regulatory protein: impact of physiological and pharmacological effectors

Glucokinase (GK) is a key enzyme of glucose metabolism in liver and pancreatic beta-cells, and small molecule activators of GK (GKAs) are under evaluation for the treatment of type 2 diabetes. In liver, GK activity is controlled by the GK regulatory protein (GKRP), which forms an inhibitory complex with the enzyme. Here, we performed isothermal titration calorimetry and surface plasmon resonance experiments to characterize GK-GKRP binding and to study the influence that physiological and pharmacological effectors of GK have on the protein-protein interaction. In the presence of fructose-6-phosphate, GK-GKRP complex formation displayed a strong entropic driving force opposed by a large positive enthalpy; a negative change in heat capacity was observed (Kd = 45 nm, DeltaH = 15.6 kcal/mol, TDeltaS = 25.7 kcal/mol, DeltaCp = -354 cal mol(-1) K(-1)). With k(off) = 1.3 x 10(-2) s(-1), the complex dissociated quickly. The thermodynamic profile suggested a largely hydrophobic interaction. In addition, effects of pH and buffer demonstrated the coupled uptake of one proton and indicated an ionic contribution to binding. Glucose decreased the binding affinity between GK and GKRP. This decrease was potentiated by an ATP analogue. Prototypical GKAs of the amino-heteroaryl-amide type bound to GK in a glucose-dependent manner and impaired the association of GK with GKRP. This mechanism might contribute to the antidiabetic effects of GKAs.     

Mechanism of peroxynitrite interaction with cytochrome c

Kinetics of the reaction of peroxynitrite with ferric cytochrome c in the absence and presence of bicarbonate was studied. It was found that the heme iron in ferric cytochrome c does not react directly with peroxynitrite. The rates of the absorbance changes in the Soret region of cytochrome c spectrum caused by peroxynitrite or peroxynitrite/bicarbonate were the same as the rate of spontaneous isomerization of peroxynitrite or as the rate of the reaction of peroxynitrite with bicarbonate, respectively. This means that intermediate products of peroxynitrite decomposition, (.)OH/(.)NO(2) or, in the presence of bicarbonate, CO(3)(-)(.)/(.)NO(2), are the species responsible for the absorbance changes in the Soret band of cytochrome c. Modifications of the heme center of cytochrome c by radiolytically produced radicals, (.)OH, (.)NO(2) or CO(3)(-)(.), were also studied. The absorbance changes in the Soret band caused by radiolytically produced (.)OH or CO(3)(-)(.) were much more significant that those observed after peroxynitrite treatment, compared under similar concentrations of radicals. (.)NO(2) produced radiolytically did not interact with the heme center of cytochrome c. Cytochrome c exhibited an increased peroxidase-like activity after reaction with peroxynitrite as well as with radiolytically produced (.)OH, (.)NO(2) or CO(3)(-)(.) radicals. This means that modification of protein structure: oxidation of amino acids and/or tyrosine nitration, facilitates reaction of H(2)O(2) with the heme iron of cytochrome c, followed by reaction with the second substrate.     

Mechanism and rate constants of the Cdc42 GTPase binding with intrinsically disordered effectors

Intrinsically disordered proteins (IDPs) are often involved in signaling and regulatory functions, through binding to cellular targets. Many IDPs undergo disorder‐to‐order transitions upon binding. Both the binding mechanisms and the magnitudes of the binding rate constants can have functional importance. Previously we have found that the coupled binding and folding of any IDP generally follows a sequential mechanism that we term dock‐and‐coalesce, whereby one segment of the IDP first docks to its subsite on the target surface and the remaining segments subsequently coalesce around their respective subsites. Here we applied our TransComp method within the framework of the dock‐and‐coalesce mechanism to dissect the binding kinetics of two Rho‐family GTPases, Cdc42 and TC10, with two intrinsically disordered effectors, WASP and Pak1. TransComp calculations identified the basic regions preceding the GTPase binding domains (GBDs) of the effectors as the docking segment. For Cdc42 binding with both WASP and Pak1, the calculated docking rate constants are close to the observed overall binding rate constants, suggesting that basic‐region docking is the rate‐limiting step and subsequent conformational coalescence of the GBDs on the Cdc42 surface is fast. The possibility that conformational coalescence of the WASP GBD on the TC10 surface is slow warrants further experimental investigation. The account for the differences in binding rate constants among the three GTPase‐effector systems and mutational effects therein yields deep physical and mechanistic insight into the binding processes. Our approach may guide the selection of mutations that lead to redesigned binding pathways. Proteins 2016; 84:674–685. © 2016 Wiley Periodicals, Inc.     

Leukocytes on the move with phosphoinositide 3-kinase and its downstream effectors

Cell signalling mediators derived from membrane phospholipids are frequent participants in biological processes. The family of phosphoinositide 3-kinases (PI3Ks) phosphorylate the membrane lipid phosphatidylinositol, generating second messengers that direct diverse responses. These PI3K products are fundamental for leukocyte migration or chemotaxis, a pivotal event during the immune response. This system is therefore of significant biomedical interest. This review focuses on the biochemistry and signalling pathways of PI3K, with particular emphasis on chemokine (chemotactic cytokine)-directed responses. The key objectives of chemotaxis are motility and direction. The latter--direction--requires distinct events at the front and back of a cell. In light of this, the coordinated localisation of signalling factors, an event choreographed by a sharp intracellular gradient of PI3K-derived products, is a common theme.     

Model systems of carcinogenesis in vitro: the interaction of the ras oncogene with immortalized cells

Gene transfer experiments have shown that ras effector functions are sufficient to transform cells from a variety of established lines (e. g., mouse NIH3T3 cells). In contrast, primary cells and early passage rodent cells can be transformed by ras oncogenes only at low frequencies, unless cotransfected with collaborating genes such as adenovirus early region IA (EIA) or myc retroviral oncogene homologue. Primary rat embryo fibroblasts (REF) were chosen as a model for the analysis of multistep cellular transformation. Transfection of REF, immortalized by early region of simian adenovirus SA7 with c-Ha-ras oncogene cannot induce their morphological transformation. This phenomenon is observed only after second transfection with the same oncogene. These different cell lines can be used for further analysis of the mechanisms of carcinogenesis.     

Mechanism of interaction of citrate synthase with citryl-CoA

Arguments are presented which indicate that the low steady-state rates of citrate production governing the catalytic interaction of citrate synthase from pig heart with citryl-CoA reflect the formation of a non-productive enzyme.citryl-CoA complex. The kinetic predictions of such an extended reaction mechanism are examined and are shown to account in satisfactory detail for the complex multiphasic rate behaviour exhibited by the enzyme under a variety of conditions in reactions involving citryl-CoA as a substrate.     

Mechanism for the interaction of thiols with methylcobalamin

The reaction between methylcobalamin and ethane-thiol sulfonic acid (Coenzyme M) has been studied under aerobic conditions. For this reaction evidence is presented for a catalytic cycle which promotes homolytic cleavage of the Cobalt-carbon σ-bond to give Cob(II)alamin (B12-r) and methylcoenzyme M as the products. This reaction is especially pertinent to our understanding of the mechanism of methane-biosynthesis.In addition, we have used 220 MHz ¹H NMR and ¹³C NMR to show that thiols do not react with methylcorrinoids by displacing the base trans-axial to the cobalt-carbon bond. This NMR study is especially important since the coordination of thiols to cobalt has previously been reported to occur by a number of of researh groups including our own.     

Mechanism of interaction of Dictyostelium severin with actin filaments

Severin, a 40,000-dalton protein from Dictyostelium that disassembles actin filaments in a Ca2+ -dependent manner, was purified 500-fold to greater than 99% homogeneity by modifications of the procedure reported by Brown, Yamamoto, and Spudich (1982. J. Cell Biol. 93:205-210). Severin has a Stokes radius of 29 A and consists of a single polypeptide chain. It contains a single methionyl and five cysteinyl residues. We studied the action of severin on actin filaments by electron microscopy, viscometry, sedimentation, nanosecond emission anisotropy, and fluorescence energy transfer spectroscopy. Nanosecond emission anisotropy of fluoresence-labeled severin shows that this protein changes its conformation on binding Ca2+. Actin filaments are rapidly fragmented on addition of severin and Ca2+, but severin does not interact with actin filaments in the absence of Ca2+. Fluorescence energy transfer measurements indicate that fragmentation of actin filaments by severin leads to a partial depolymerization (t1/2 approximately equal to 30 s). Depolymerization is followed by exchange of a limited number of subunits in the filament fragments with the disassembled actin pool (t1/2 approximately equal to 5 min). Disassembly and exchange are probably restricted to the ends of the filament fragments since only a few subunits in each fragment participate in the disassembly or exchange process. Steady state hydrolysis of ATP by actin in the presence of Ca2+-severin is maximal at an actin: severin molar ratio of approximately 10:1, which further supports the inference that subunit exchange is limited to the ends of actin filaments. The observation of sequential depolymerization and subunit exchange following the fragmentation of actin by severin suggests that severin may regulate site-specific disassembly and turnover of actin filament arrays in vivo.