Elena Tarakhovskaya

Functional Study of the P32T ITPA Variant Associated with Drug Sensitivity in Humans

Sanitization of the cellular nucleotide pools from mutagenic base analogues is necessary for the accuracy of transcription and replication of genetic material and plays a substantial role in cancer prevention. The undesirable mutagenic, recombinogenic, and toxic incorporation of purine base analogues [i.e., ITP, dITP, XTP, dXTP, or 6-hydroxylaminopurine (HAP) deoxynucleoside triphosphate] into nucleic acids is prevented by inosine triphosphate pyrophosphatase (ITPA). The ITPA gene is a highly conserved, moderately expressed gene. Defects in ITPA orthologs in model organisms cause severe sensitivity to HAP and chromosome fragmentation. A human polymorphic allele, 94C-->A, encodes for the enzyme with a P32T amino acid change and leads to accumulation of non-hydrolyzed ITP. ITPase activity is not detected in erythrocytes of these patients. The P32T polymorphism has also been associated with adverse sensitivity to purine base analogue drugs. We have found that the ITPA-P32T mutant is a dimer in solution, as is wild-type ITPA, and has normal ITPA activity in vitro, but the melting point of ITPA-P32T is 5 degrees C lower than that of wild-type. ITPA-P32T is also fully functional in vivo in model organisms as determined by a HAP mutagenesis assay and its complementation of a bacterial ITPA defect. The amount of ITPA protein detected by Western blot is severely diminished in a human fibroblast cell line with the 94C-->A change. We propose that the P32T mutation exerts its effect in certain human tissues by cumulative effects of destabilization of transcripts, protein stability, and availability.

A Snapshot of the Plant Glycated Proteome: STRUCTURAL, FUNCTIONAL, AND MECHANISTIC ASPECTS.

Glycation is the reaction of carbonyl compounds (reducing sugars and α-dicarbonyls) with amino acids, lipids, and proteins, yielding early and advanced glycation end products (AGEs). The AGEs can be formed via degradation of early glycation intermediates (glycoxidation) and by interaction with the products of monosaccharide autoxidation (autoxidative glycosylation). Although formation of these potentially deleterious compounds is well characterized in animal systems and thermally treated foods, only a little information about advanced glycation in plants is available. Thus, the knowledge of the plant AGE patterns and the underlying pathways of their formation are completely missing. To fill this gap, we describe the AGE-modified proteome of Brassica napus and characterize individual sites of advanced glycation by the methods of liquid chromatography-based bottom-up proteomics. The modification patterns were complex but reproducible: 789 AGE-modified peptides in 772 proteins were detected in two independent experiments. In contrast, only 168 polypeptides contained early glycated lysines, which did not resemble the sites of advanced glycation. Similar observations were made with Arabidopsis thaliana. The absence of the early glycated precursors of the AGE-modified protein residues indicated autoxidative glycosylation, but not glycoxidation, as the major pathway of AGE formation. To prove this assumption and to identify the potential modifying agents, we estimated the reactivity and glycative potential of plant-derived sugars using a model peptide approach and liquid chromatography-mass spectrometry-based techniques. Evaluation of these data sets together with the assessed tissue carbohydrate contents revealed dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, ribulose, erythrose, and sucrose as potential precursors of plant AGEs.

Mechanisms of bioadhesion of macrophytic algae

During the evolution the benthic macrophytic algae developed effective mechanisms of bioadhesion enabling their attachment to almost any surface in the aqueous medium. The attachment of algal spores and zygotes includes two successive stages: the primary and the secondary (final) adhesion. Analysis of information on the composition of adhesive materials and attachment mechanisms in brown, green, and red marine macrophytes indicates that synthesis and release of adhesive substances by algal cells can be considered as a temporary intensification of cell wall synthesis. The structure of the primary adhesive material comprises a gel phase (alginate, ulvan, and agar gels) and a structuring component, i.e., a flexible network based on branched chains and/or rings of phenolic compounds, polysaccharides, or glycoproteins. Irreversible hardening of the primary adhesive material arises from phenol polymerization catalyzed by different peroxidases (brown algae) or from polymerization of glycoproteins comprising amino acids with phenolic residues (red algae). In parallel with these processes, covalent cross-links are being formed between the adhesive structural components and the gel phase polysaccharides. This results in the formation of the secondary adhesive and in eventual attachment of the organism to the substrate. The attachment mechanisms of benthic algae appear to have some features in common with the mechanisms of bioadhesion of marine invertebrates.

Osmotic stress is accompanied by protein glycation in Arabidopsis thaliana

Highlight Osmotic stress enhances the rate of protein glycation and monosaccharide autoxidation is the main pathway.

Global Proteomic Analysis of Advanced Glycation End Products in the Arabidopsis Proteome Provides Evidence for Age-related Glycation Hotspots

Glycation is a post-translational modification resulting from the interaction of protein amino and guanidino groups with carbonyl compounds. Initially, amino groups react with reducing carbohydrates, yielding Amadori and Heyns compounds. Their further degradation results in formation of advanced glycation end products (AGEs), also originating from α-dicarbonyl products of monosaccharide autoxidation and primary metabolism. In mammals, AGEs are continuously formed during the life of the organism, accumulate in tissues, are well-known markers of aging, and impact age-related tissue stiffening and atherosclerotic changes. However, the role of AGEs in age-related molecular alterations in plants is still unknown. To fill this gap, we present here a comprehensive study of the age-related changes in the Arabidopsis thaliana glycated proteome, including the proteins affected and specific glycation sites therein. We also consider the qualitative and quantitative changes in glycation patterns in terms of the general metabolic background, pathways of AGE formation, and the status of plant anti-oxidative/anti-glycative defense. Although the patterns of glycated proteins were only minimally influenced by plant age, the abundance of 96 AGE sites in 71 proteins was significantly affected in an age-dependent manner and clearly indicated the existence of age-related glycation hot spots in the plant proteome. Homology modeling revealed glutamyl and aspartyl residues in close proximity (less than 5 Å) to these sites in three aging-specific and eight differentially glycated proteins, four of which were modified in catalytic domains. Thus, the sites of glycation hot spots might be defined by protein structure that indicates, at least partly, site-specific character of glycation.

Defect of Fe-S cluster binding by DNA polymerase δ in yeast suppresses UV-induced mutagenesis, but enhances DNA polymerase ζ – dependent spontaneous mutagenesis

Eukaryotic genomes are duplicated by a complex machinery, utilizing high fidelity replicative B-family DNA polymerases (pols) α, δ and ε. Specialized error-prone pol ζ, the fourth B-family member, is recruited when DNA synthesis by the accurate trio is impeded by replication stress or DNA damage. The damage tolerance mechanism dependent on pol ζ prevents DNA/genome instability and cell death at the expense of increased mutation rates. The pol switches occurring during this specialized replication are not fully understood. The loss of pol ζ results in the absence of induced mutagenesis and suppression of spontaneous mutagenesis. Disruption of the Fe-S cluster motif that abolish the interaction of the C-terminal domain (CTD) of the catalytic subunit of pol ζ with its accessory subunits, which are shared with pol δ, leads to a similar defect in induced mutagenesis. Intriguingly, the pol3-13 mutation that affects the Fe-S cluster in the CTD of the catalytic subunit of pol δ also leads to defective induced mutagenesis, suggesting the possibility that Fe-S clusters are essential for the pol switches during replication of damaged DNA. We confirmed that yeast strains with the pol3-13 mutation are UV-sensitive and defective in UV-induced mutagenesis. However, they have increased spontaneous mutation rates. We found that this increase is dependent on functional pol ζ. In the pol3-13 mutant strain with defective pol δ, there is a sharp increase in transversions and complex mutations, which require functional pol ζ, and an increase in the occurrence of large deletions, whose size is controlled by pol ζ. Therefore, the pol3-13 mutation abrogates pol ζ-dependent induced mutagenesis, but allows for pol ζ recruitment for the generation of spontaneous mutations and prevention of larger deletions. These results reveal differential control of the two major types of pol ζ-dependent mutagenesis by the Fe-S cluster present in replicative pol δ.

Influence of phytohormones on morphology and chlorophyll a fluorescence parameters in embryos of Fucus vesiculosus L. (Phaeophyceae)

While a variety of plant hormones from brown algae were described, there were few studies that examined the combined effects of these hormones on morphogenesis and photosynthetic physiology in developing fucoid embryos. We evaluated the effects of phytohormones to determine the extent, to which responses were similar to those of terrestrial plants. Kinetin, IAA, ABA, GA3, and kinetin + IAA were added to seawater at a physiological concentration (1 mg/L), and embryos of Fucus vesiculosus L. were grown for 10 days. Photosynthetic activity of single embryos or embryo cells were characterized using the following fluorescence parameters: minimum fluorescence yield (F0), maximum quantum yield (Fv/Fm), relative maximum rate of electron transfer to photosystem II under saturation irradiances (rETRmax), photosynthetic efficiency under non-saturating irradiances (αETR) and saturation irradiance (Ek). In addition, embryo length and diameter and apical hair length and number were determined. Morphological changes associated with hormone treatments included an increase in the embryo length in the presence of IAA, an increase in the embryo diameter in the presence of IAA, kinetin, and kinetin + IAA, an increase in the maximum hair length and number in the presence of kinetin + IAA, and a decrease in the hair length and number in the presence of ABA. With respect to fluorescence parameters, significant effects of phytohormones included an increase in the F0 and Fv/Fm at kinetin treatment, a synergistic effect of kinetin + IAA on Fv/Fm, rETRmax, and αETR, a promotion of Fv/Fm by GA, and a decrease of the parameters by ABA. These results are consistent with the data on responses of land plants to the same hormones and suggest that brown algae have evolved regulatory mechanisms for morphogenesis and photosynthetic regulation similar to plants.

Effect of GeO 2 on embryo development and photosynthesis in Fucus vesiculosus (Phaeophyceae)

Germanium dioxide (GeO2) has been used for many years in the cultivation of red and green algae as a means of controlling the growth of diatoms. Brown algae are sensitive to GeO2, however, the basis of this sensitivity has not been characterized. Here we use embryos of Fucus vesiculosus to investigate morphological and physiological impacts of GeO2 toxicity. Morphometric features of embryos were measured microscopically, and physiological features were determined using pulse amplitude modulated (PAM) fluorometry. At 5 mg L -1 GeO2, embryos grew slower than controls and developed growth abnormalities. After 24 h, initial zygote divisions were often oblique rather than transverse. Rhizoids had inflated tips in GeO 2 and were less branched, and apical hairs were deformed, with irregularly aligned, spheroidal cells. Minimum fluorescence ( F0) showed minor differences over the 10 days experiment, and pigment levels (chlorophylls a, c and total carotenoids) showed no difference after 10 days. Optimum quantum yield increased from ca. 0.52 at 24 h to 0.67 at 5 days, and GeO2-treated embryos had higher mean values (significant at 3 and 5 days). Optimum quantum yield of photosystem II (ΦPSII) was stable in control thalli after 5 days, but declined significantly in GeO 2. Addition of silica (as SiO2) did not reverse the effects of GeO2. These results suggest that GeO2 toxicity in brown algae is associated with negative impacts at the cytological level rather than metabolic impacts associated with photosynthesis.

Halocladius variabilis (Diptera: Chironomidae): a marine insect symbiotic with seaweeds from the White Sea, Russia

The commensal (and possibly mutualistic) symbiosis between the marine chironomid, Halocladius variabilis , and brown algal epiphytes of Fucus vesiculosus and Ascophyllum nodosum is described for the first time from Europe, based on field studies from the White Sea, Russia. While the primary host, Elachista fucicola , and the secondary host, A. nodosum , are the same as in eastern Canada where the symbiosis was first described, White Sea populations have a wider range of primary hosts that include Pylaiella littoralis and Dictyosiphon foeniculaceus . About 64% of E. fucicola thalli on A. nodosum in the low intertidal zone were colonized. Significantly lower frequencies were found on E. fucicola when the latter was epiphytic on F. vesiculosus at the same tidal height or on either secondary host when they were in the subtidal zone. For a given tidal height, or secondary host, frequency of H. variabilis was reduced on P. littoralis and further reduced on D. foeniculaceus . We suggest that subtidal populations are colonized by dispersal of larvae from the intertidal zone on to suitable substrata, and that there is limited potential for these individuals to reach the intertidal zone as adults for reproduction.

Hydrogen peroxide content and vanadium-dependent haloperoxidase activity in thalli of six species of Fucales (Phaeophyceae)

Abstract: Patterns of hydrogen peroxide content and vanadium-dependent haloperoxidase (V-HPO) activity were investigated in thalli of six species of Fucales (Fucus vesiculosus, F. serratus, F. edentatus, F. distichus, Ascophyllum nodosum and Pelvetia canaliculata). H2O2 levels in all species were similar to those of photosynthesizing tissues of unstressed terrestrial plants. The highest H2O2 content (up to 0.7 μM g FW−1) was in F. distichus and Pelvetia. H2O2 content in the basal and middle zones of fronds was generally higher than in vegetative apices and receptacles. Similar trends were detected for V-HPO activity with both bromide and iodide. Intrathallus profiles of H2O2 content and V-HPO activity may be attributed to the physiological condition of the respective thallus zones, i.e. photosynthetic activity and stage of cell differentiation. Steady-state kinetics showed significant variation of V-HPO parameters between fucoid species, suggesting that they contain different forms of the enzyme. V-HPO from high-intertidal Pelvetia and F. distichus exhibited the highest Km values for both halide and H2O2. The enzyme form with the maximum kinetic efficiency occurred in the mid-intertidal fucoids. For all investigated species, the iodination activities were two to four times lower than for bromination. Apparently, V-HPOs contribute significantly to the control of reactive oxygen species accumulation in Fucales. The similarity of the longitudinal profiles of H2O2 content and peroxidase activity between Fucales and terrestrial plants suggests a physiological analogy of V-HPO and secretory peroxidases.