Interactions between subunits of protein kinase CK2 and their protein substrates influences its sensitivity to specific inhibitors

Five isoforms of CK2 may exist simultaneously in yeast cells: free catalytic subunits CK2α', CK2α and three holoenzymatic structures composed of αα'ββ', α(2)ββ' and α'(2)ββ'. Each isolated and purified form exhibits properties typical for CK2, but they differ in substrate specificity as well as in sensitivity to specific modulators. All five isoforms of protein kinase CK2 from Saccharomyces cerevisiae were examined for their binding capacity with ATP/GTP and two commonly used ATP-competitive inhibitors TBB and TBI. Enzymes were tested with protein substrates differently interacting with CK2 subunits: Elf1, Fip1, Svf1, P2B and synthetic peptide. Obtained results show that K(m) for ATP varies from 2.4-53 μM for Elf1/CK2α' and Svf1/CK2α, respectively. Similar differences can be seen in case when GTP was used as phosphate donor. The inhibitory effect depends on composition of CK2/substrate complexes. Highest sensitivity to TBB shows all complexes containing αα'ββ' isoform with K (i) values between 0.2 and 1.1 μM. The prospect that TBB and TBI could be utilized to discriminate between different molecular forms of CK2 in yeast cells was examined. Both inhibitors, TBB as well as TBI, decreases cell growth to extents devoting interactions with different CK2 isoforms present in the cell but the presence of β/β'-dimer has a high importance towards sensitivity. Conceivably, a given inhibitor concentration can inhibit only selected CK2-mediated processes in the cell.     

Structural studies of hydroxycitrates and their relevance to certain enzymatic mechanisms.

The crystal structures of the ethylenediamine salts of two diastereoisomeric hydroxycitratesy are described, and their conformations in the solid state are analyzed. In both structures, the HOCCOH torsion angle is approximately 60 ° as found for many tartrates and mesotartrates. The presence of three carboxyl groups and two hydroxyl groups in hydroxycitrates leads to 10 possible types of tridentate metal chelates. Since bacterial citrate lyase and ATP citrate lyase require metal ions, the possible geometries of hydroxycitrate chelation have been compared with those of citrate, and as a result, some information on the geometry of each enzymic active site has been obtained. If the hydroxycitrate binds in the same manner as citrate, the C(3)&;z.sbnd;C(4) bond will be in the correct position to be cleaved. Other modes of binding of hydroxycitrate, if they can be accommodated in the active site of the enzyme, are nonproductive and compete with the citrate-like mode causing inhibition. It is possible, in these alternate modes of binding of hydroxycitrates, for additional binding to side chains in the active site of the enzyme to occur, resulting in extremely potent inhibition.     

Interactions of different inhibitors with active-site aspartyl residues of HIV-1 protease and possible relevance to pepsin

The importance of the active site region aspartyl residues 25 and 29 of the mature HIV-1 protease (PR) for the binding of five clinical and three experimental protease inhibitors [symmetric cyclic urea inhibitor DMP323, nonhydrolyzable substrate analog (RPB) and the generic aspartic protease inhibitor acetyl-pepstatin (Ac-PEP)] was assessed by differential scanning calorimetry. DeltaT(m) values, defined as the difference in T(m) for a given protein in the presence and absence of inhibitor, for PR with DRV, ATV, SQV, RTV, APV, DMP323, RPB, and Ac-PEP are 22.4, 20.8, 19.3, 15.6, 14.3, 14.7, 8.7, and 6.5 degrees C, respectively. Binding of APV and Ac-PEP is most sensitive to the D25N mutation, as shown by DeltaT(m) ratios [DeltaT(m)(PR)/DeltaT(m)(PR(D25N))] of 35.8 and 16.3, respectively, whereas binding of DMP323 and RPB (DeltaT(m) ratios of 1-2) is least affected. Binding of the substrate-like inhibitors RPB and Ac-PEP is nearly abolished (DeltaT(m)(PR)/DeltaT(m)(PR(D29N)) > or = 44) by the D29N mutation, whereas this mutation only moderately affects binding of the smaller inhibitors (DeltaT(m) ratios of 1.4-2.2). Of the nine FDA-approved clinical HIV-1 protease inhibitors screened, APV, RTV, and DRV competitively inhibit porcine pepsin with K(i) values of 0.3, 0.6, and 2.14 microM, respectively. DSC results were consistent with this relatively weak binding of APV (DeltaT(m) 2.7 degrees C) compared with the tight binding of Ac-PEP (DeltaT(m) > or = 17 degrees C). Comparison of superimposed structures of the PR/APV complex with those of PR/Ac-PEP and pepsin/pepstatin A complexes suggests a role for Asp215, Asp32, and Ser219 in pepsin, equivalent to Asp25, Asp25', and Asp29 in PR in the binding and stabilization of the pepsin/APV complex.     

Interactions between the amyloid and cholinergic mechanisms in Alzheimer's disease

Alzheimer's disease (AD) is a progressive, neurodegenerative disease characterized by memory and cognitive loss, the formation of senile plaques containing amyloid-beta (Abeta) peptide, degeneration of the cholinergic neurons and the development of neurofibrillary tangles. The build-up of Abeta is considered to be a central feature in the pathogenesis of AD. However, other critical molecular and neurochemical alterations too occur, such as a cholinergic dysfunction. As concerns the pathomechanism of the disease, both the amyloid cascade hypothesis and the cholinergic hypothesis of AD are widely accepted. This review surveys recent in vitro and in vivo experimental evidence relating to these two hypotheses. Bidirectional pathways linking them as regards the cholinergic neurotoxicity of Abeta and the regulatory mechanisms of cholinergic receptor activation or enzyme inhibition in the processing of the amyloid precursor protein are also discussed. Further work is warranted to elucidate the exact effects in the interactions between the cholinergic and amyloid hypotheses of the candidate drugs used in AD therapy.     

Interactions between inhibitors of dihydrofolate reductase.

The binding of substrates and inhibitors to dihydrofolate reductase was studied by steady-state kinetics and high-field 1H-n.m.r. spectroscopy. A series of 5-substituted 2,4-diaminopyrimidines were examined and were found to be 'tightly binding' inhibitors of the enzyme (Ki less than 10(-9) M). Studies on the binding of 4-substituted benzenesulphonamides and benzenesulphonic acids also established the existence of a 'sulphonamide-binding site' on the enzyme. Subsequent n.m.r. experiments showed that there are two binding sites for the sulphonamides on the enzyme, one of which overlaps the coenzyme (NADPH) adenine-ring-binding site. An examination of the pH-dependence of the binding of sulphonamides to the enzyme indicated the influence of an ionizable group on the enzyme that was not directly involved in the sulphonamide binding. The change in pKa value from 6.7 to 7.2 observed on sulphonamide binding suggests the involvement of a histidine residue, which could be histidine-28.     

Interactions between ticks and their hosts

Tick populations on hosts are limited by varied mechanisms expressed through natural host-parasite relationships. Tickborne disease losses in nature are minimal because of enzootic stability.Study of these natural interactions between ticks and their hosts provides guidelines for managing interactions with domestic livestock.     

Interrelationship between various aging methods,and their relevance to palaeodemography

Age estimations for 64 adult individuals from the Byzantine city of Rehovot-in-the-Negev were carried out using seven different aging methods. Correlation coefficients were calculated between five of the methods, which employed the following anatomical structures: teeth attrition rate, cranial sutures (closure stages), sternal end of ribs, symphysis pubis, and sacroiliac joints. Age estimations were checked against ‘summary age’ (Lovejoy et al., 1985a). Life tables were reconstructed based on summary ages and revised ages separately. It was found that ages obtained from the innominate bones correlated best with the summary age and showed less bias and inaccuracy, while ages obtained from the cranial sutures, showed the opposite. Reconstructing the ‘revised-age’ has no advantage over other aging methods when calculating demographic parameters.     

Lipid-apolipoprotein interactions in amyloid fibril formation and relevance to atherosclerosis

The apolipoprotein family is a set of highly conserved proteins characterized by the presence of amphipathic α-helical sequences that mediate lipid binding. Paradoxically, this family of proteins is also prominent among the proteins known to form amyloid fibrils, characterized by extensive cross-β structure. Several apolipoproteins including apolipoprotein (apo) A-I, apoA-II and apoC-II accumulate in amyloid deposits of atherosclerotic lesions. This review illustrates the role of lipid-apolipoprotein interactions in apolipoprotein folding and aggregation with a specific focus on human apoC-II, a well-studied member of the family. In the presence of high concentrations of micellar lipid mimetics apoC-II adopts a stable and predominantly α-helical structure, similar to other members of the family and presumed to be the structure of apoC-II in circulating plasma lipoproteins. In contrast, lipid-free apoC-II aggregates to form long amyloid fibrils with a twisted ribbon-like morphology. Detailed structural analyses identify a letter G-like conformation as the basic building block within these fibrils. Phospholipids at submicellar concentrations accelerate apoC-II fibril formation by promoting the formation of a discrete tetrameric intermediate. Conversely, several small molecule lipid-mimetics inhibit apoC-II fibril formation at submicellar concentrations, inducing well-defined dimers unable to further aggregate. Finally, low concentrations of phospholipid micelles and bilayers induce the slow formation of amyloid fibrils with distinct rod-like fibril morphology. These studies highlight the diversity of lipid effects on apolipoprotein amyloid formation and reveal a conformational adaptability that could underlie the widespread occurrence of apolipoproteins in amyloid deposits and atheroma.     

Interactions between branched DNAs and peptide inhibitors of DNA repair

The RecG helicase of Escherichia coli unwinds both Holliday junction (HJ) and replication fork DNA substrates. Our lab previously identified and characterized peptides (WRWYCR and KWWCRW) that block the activity of RecG on these substrates. We determined that the peptides bind HJ DNA and prevent the binding of RecG. Herein, we present further evidence that the peptides are competitive inhibitors of RecG binding to its substrates. We have generated structural models of interactions between WRWYCR and a junction substrate. Using the fluorescent probe 2-aminopurine, we show that inhibitors interact with highest affinity with HJs (K_d = 14 nM) and ~4- to 9-fold more weakly with replication fork substrates. The fluorescence assay results agree with the structural model, and predict the molecular basis for interactions between HJ-trapping peptides and branched DNA molecules. Specifically, aromatic amino acids in the peptides stack with bases at the center of the DNA substrates. These interactions are stabilized by hydrogen bonds to the DNA and by intrapeptide interactions. These peptides inhibit several proteins involved in DNA repair in addition to RecG, have been useful as tools to dissect recombination, and possess antibiotic activity. Greater understanding of the peptides’ mechanism of action will further increase their utility.     

Interactions between pairs of DNA-binding dyes: results and implications of chromosome analysis

A number of DNA-binding dyes, with spectral properties making them suitable as components of energy donor-acceptor pairs, are described. If such pairs are used to stain metaphase chromosomes, and if the energy acceptor (e.g., actinomycin D or methyl green) has a binding specificity opposite to the binding or fluorescence specificity of the donor (e.g., 33258 Hoechst, quinacrine or chromomycin A3), contrast in donor fluorescence can be enhanced, leading to patterns selectively highlighting standard or reverse chromosome bands or particular polymorphic regions. Such results presumably reflect chromosomal regions enriched in 10-20 base pair clusters to which the donor binds and fluoresces but to which the acceptor cannot bind. For other pairs, involving counterstains such as netropsin or echinomycin, which are not suitable as energy acceptors, specific changes observed in polymorphic region fluorescence are most likely due to binding competition between dyes. Dye pairs producing contrast by either method can be used to differentiate between homologous chromosomes or to facilitate detection of specific chromosomal rearrangements. Preliminary data indicate that contrast enhancement generated in fixed metaphase chromosomes spread on microscopic slides can also be observed in suspensions of unfixed metaphase chromosomes, reinforcing the expectation that the methodology described will be of use in flow cytometry.     

The reaction between some dyes and synthetic hydroxyapatite I. The uptake of dyes in relation to their structures

Synopsis The uptake of dyes from dilute solutions by synthetic hydroxyapatite and other sparingly soluble calcium compounds has been determined. About 30 dyes, mostly azo-, dis-azo and anthraquinonoid types were used in 95% ethanol or 0.1 M tris buffer. Many had closely related configurations. Chemical groupings possibly responsible for the adsorption of particular dyes by hydroxyapatite have been deduced from an analysis of the results. The uptake of most dyes from alcoholic solutions was, linearly related to the surface area of hydroxyapatite. Calcium carbonate and secondary calcium phosphate took up less stain than hydroxyapatite of similar surface area. With the simpler anthraquinonoid dyes, the uptake was higher from aqueous than alcoholic solutions, but specificity for hydroxyapatite was much less. The increased uptake of dye by powdered bone or dentine when rendered anorganic was proportional to the increased surface area. It was found that several dyes in common use as stains for bone and calcified tissue were only poorly adsorbed by synthetic hydroxyapatite under the particular conditions of these experiments.The experimental data presented could be used as a basis for the development of histochemical reactions for calcified tissue or inclusions. By suitable choice of dyes, solvent and rinsing solution it ought to be possible to differentiate various forms of calcified material.     

Interactions between monogenean parasites and their fish hosts

Parasite factors associated with recognition and selection of the host and the mechanisms in the host responsible for acceptance or rejection of the invading organism were evaluated. Sensory structures in parasites are able to detect differences between different fish species and this ability to discern between fishes may be based on both chemical and mechanical stimuli on the host surface. Complex glycoproteins, proteins, carbohydrates and simple molecules attract parasites or modify their behaviour. Furthermore, attachment of the monogenean parasite to a host is dependent on both mechanical structures and chemical factors in the parasite. These systems comprise anterior pads, posterior haptors, gland secretions, and muscular elements. The parasite needs access to appropriate nutrients which can be absorbed and used for reproduction and in this context signals from the host are needed for an optimal physiological response of the parasite. The innate and adaptive immune systems of the host are important elements in this question. Investigations have indicated that innate host factors (complement, lectins, acute phase reactants, macrophages) can bind to monogeneans and elicit severe damage to the parasites. The targets for these hostile products are not only the monogenean tegument, but may involve the gastrodermis and glands. However, the parasite's ability to avoid and even exploit the wide array of immunological elements of the host may be an important player in the dynamic interactions between host and monogenean determining host specificity. Even fish hosts susceptible to a certain parasite show an ability to mount a protective response at post-infection periods. Elevation of the host's production of adaptive and non-adaptive factors following monogenean infections of a certain duration may explain the acquired response.     

Interactions between phytophagous insects and their Opuntia hosts

Abstract.

• 1 The cactophagous insect community on opuntias is analysed to show the number of insect species in different taxa. An extension of this analysis gives the average species complement on large and small opuntias.
• 2 A highly significant positive correlation is found between the total number of phytophagous insect species on individual Opuntia species and a measure of the overall ‘architecture’ of their host plants.
• 3 The specificity of the phytophagous insects on opuntias is briefly considered and the community as a whole analysed by guilds. The co-evolution of the Opuntia-feeding insects and their hosts has culminated in a community of specialist insects to the exclusion of nearly all generalist phytophages.
• 4 The life history strategies of the Opuntia-feeding insects are reviewed. Common to all developmental stages are morphological and behavioural adaptations that reduce the risk of attack by natural enemies. This is clearly the consequence of living on structurally simple host plants where there is little place to hide.
• 5 The possible influence of insect herbivores on Opuntia evolution is discussed.
• 6 An understanding of the interactions between the phytophagous insect community and opuntias has clear implications for the biological control of alien Opuntia weeds.

     

Structural studies of cysteine proteases and their inhibitors.

Cysteine proteases (CPs) are responsible for many biochemical processes occurring in living organisms and they have been implicated in the development and progression of several diseases that involve abnormal protein turnover. The activity of CPs is regulated among others by their specific inhibitors: cystatins. The main aim of this review is to discuss the structure-activity relationships of cysteine proteases and cystatins, as well as of some synthetic inhibitors of cysteine proteases structurally based on the binding fragments of cystatins.     

Interactions of Alzheimer amyloid peptides with cultured cells and brain tissue, and their biological consequences

The Alzheimer amyloid peptides are the main constituent of the diagnostic hallmark of Alzheimer disease, the senile plaque. A halo of neurodegeneration surrounds the senile plaques observed in the brains of Alzheimer patients. Significant efforts are under way to determine whether the Alzheimer peptides are the causal agents of this neurodegeneration. We review the developments in identifying the putative interaction sites of Alzheimer amyloid peptides on cells and intact brain tissue. We focus on the specificity of this interaction and on the molecular nature of potential receptors. These studies form the bases for developing therapeutics that target potential interaction sites and inhibit Alzheimer amyloid peptide deposition.