Modification Of Amino Acid Residues Research Articles

Direct Molecular Fishing of Zinc-Dependent Protein Partners of Amyloid-beta 1-16 with the Taiwan (D7H) Mutation and Phosphorylated Ser8 Residue

We previously showed that the metal-binding domain 1-16 of intact amyloid-beta (Aβ) is involved in interactions with a number of proteins from the cytosolic fraction of SK-N-SH human neuroblastoma cells in a zinc-dependent manner only. It is known that hereditary mutations in the Aβ metal-binding domain (Aβ(1-16)), which accelerate the development of Alzheimer's disease and post-translational modifications of amino acid residues, can significantly affect the domain's structure in the presence of zinc ions. In this work, using the molecular fishing methodology for Aβ(l-16) isoforms with the Taiwanese mutation (D7H) and a phosphorylated Ser8 residue, proteins from the cytosol of SK-N-SH cells were found that are able to form zinc-dependent non-covalent complexes with these domains. The partner proteins identified for these isoforms differed from those for intact Aβ(1-16). In contrast, the Aβ(1-16) isoform with the English mutation (H6R) and the Aβ(1-16) isoform containing both an isomerized Asp7 residue and phosphorylated Ser8 residue did not interact with cytosolic proteins. The results are useful for developing methods for rational modulation of protein-protein interactions involving natural isoforms of beta-amyloid, and also indicate the possible role of beta-amyloid with phosphorylated Ser8 as a molecule involved in normal physiological processes.

Molecular Modification of Fluoroquinolone-Biodegrading Enzymes Based on Molecular Docking and Homology Modelling.

To improve the biodegradation efficiency of fluoroquinolone antibiotics during sewage treatment, fluoroquinolone aerobic, anaerobic and facultative degrading enzymes for fluoroquinolone degradation were modified by molecular docking and homology modelling. First, amino acid residues of the binding sites of degrading enzymes for the target fluoroquinolones ciprofloxacin (CIP), norfloxacin (NOR) and ofloxacin (OFL) were analysed by the molecular docking method. The hydrophobic amino acid residues within 5 Å of the target fluoroquinolone molecules were selected as the modification sites. The hydrophobic amino acid residues at the modified sites were replaced by the hydrophilic amino acid residues, and 150 amino acid sequence modification schemes of the degrading enzymes were designed. Subsequently, a reconstruction scheme of the degrading enzyme amino acid sequence reconstruction scheme was submitted to the SWISS-MODEL server and a selected homology modelling method was used to build a new structure of the degrading enzyme. At the same time, the binding affinities between the novel degrading enzymes and the target fluoroquinolones (represented by the docking scoring function) were evaluated by the molecular docking method. It was found that the novel enzymes can simultaneously improve the binding affinities for the three target fluoroquinolones, and the degradation ability of the six modification schemes was increased by more than 50% at the same time. Among the novel enzymes, the affinity effect of the novel anaerobic enzyme (6-1) with CIP, NOR and OFL was significantly increased, with increases of 129.24%, 165.06% and 169.59%, respectively, followed by the facultative enzyme and aerobic enzyme. In addition, the designed degrading enzymes had certain selectivity for the degradation of the target quinolone. Among the novel enzymes, the binding affinities of the novel anaerobic enzyme (6-3) and CIP, the novel aerobic enzyme (3-6) and NOR, and the novel facultative enzyme (13-6) and OFL were increased by 149.71%, 178.57% and 297.12% respectively. Calculations using the Gaussian09 software revealed that the degradation reaction barrier of the novel degrading enzyme (7-1) and CIP NOR and OFL decreased by 37.65 kcal·mol−1, 6.28 kcal·mol−1 and 6.28 kcal·mol−1, respectively, which would result in efficient degradation of the target fluoroquinolone molecules. By analysing the binding affinity of the degrading enzymes before and after the modification with methanol, it was further speculated that the degradation effect of the modified aerobic degrading enzymes on organic matter was lower than that before the modification, and the increase or decrease in the degradation effect was less than 10%. The mechanism analysis found that the interaction between the modified amino acid residues of the degrading enzymes and the fluoroquinolone molecules increased. The average distance between the amino acid residues and the fluoroquinolone molecules represented a comprehensive affinity effect, and its value was positively correlated with the degradation effect of the novel degrading enzymes.

Chemical Modification of 1-Aminocyclopropane Carboxylic Acid (ACC) Oxidase: Cysteine Mutational Analysis, Characterization, and Bioconjugation with a Nitroxide Spin Label.

1-Aminocyclopropane carboxylic acid oxidase (ACCO) catalyzes the last step of ethylene biosynthesis in plants. Although some sets of structures have been described, there are remaining questions on the active conformation of ACCO and in particular, on the conformation and potential flexibility of the C-terminal part of the enzyme. Several techniques based on the introduction of a probe through chemical modification of amino acid residues have been developed for determining the conformation and dynamics of proteins. Cysteine residues are recognized as convenient targets for selective chemical modification of proteins, thanks to their relatively low abundance in protein sequences and to their well-mastered chemical reactivity. ACCOs have generally 3 or 4 cysteine residues in their sequences. By a combination of approaches including directed mutagenesis, activity screening on cell extracts, biophysical and biochemical characterization of purified enzymes, we evaluated the effect of native cysteine replacement and that of insertion of cysteines on the C-terminal part in tomato ACCO. Moreover, we have chosen to use paramagnetic labels targeting cysteine residues to monitor potential conformational changes by electron paramagnetic resonance (EPR). Given the level of conservation of the cysteines in ACCO from different plants, this work provides an essential basis for the use of cysteine as probe-anchoring residues.

Effects of Protein-Derived Amino Acid Modification Products Present in Infant Formula on Metabolic Function, Oxidative Stress, and Intestinal Permeability in Cell Models.

Proteins present in infant formulas are modified by oxidation and glycation during processing. Modified amino acid residues released from proteins may be absorbed in the gastrointestinal tract, and pose a health risk to infants. In this study, the markers of glycation furosine (1.7-3.5 μg per milligram of protein) and Nε-(carboxymethyl)lysine (28-81 ng per milligram of protein) were quantitated in infant formulas. The effects of these species, and other amino acid modifications, at the levels detected in infant formulas, on 3T3-L1 (murine preadipocyte) and Caco-2 (human intestinal epithelial) cells were assessed. Incubation of 3T3-L1 cells for 48 h with amino acid side chain oxidation and glycation products (1 and 10 μM) resulted in a loss (up to 40%, p < 0.05) of cell thiols and decreased metabolic activity compared with those of the controls. In contrast, Caco-2 cells showed a stimulation (10-50%, p < 0.05) of cellular metabolism on exposure to these products for 24 or 48 h. A 28% ( p < 0.05) increase in protein carbonyls was detected upon incubation with 200 μM modified amino acids for 48 h, although no alteration in transepithelial electrical resistance was detected. Oxidation products were detected in the basolateral compartments of Caco-2 monolayers when modified amino acids were applied to the apical side, consistent with limited permeability (up to 3.4%) across the monolayer. These data indicate that modified amino acids present in infant formulas can induce effects on different cell types, with evidence of bioavailability and induction of cellular stress. This may lead to potential health risks for infants consistently exposed to high levels of infant formulas.

Possible Mechanisms of Nonenzymatic Formation of Dehydroalanine Residue Catalyzed by Dihydrogen Phosphate Ion.

Uncommon crosslinked amino acids have been identified in several aging tissues and are suspected to trigger various age-related diseases. Several uncommon residues are formed when the dehydroalanine (Dha) residue undergoes a nucleophilic attack by surrounding residues. Dha residues are considered to be formed by posttranslational modification of serine (Ser) and cysteine residues. In the present study, we investigated the Dha residue formation mechanism catalyzed by dihydrogen phosphate ion (H2PO4-) using quantum chemical calculations. We obtained optimized geometries using the B3LYP density functional method and carried out single-point energy calculations using the second-order Møller-Plesset perturbation method. All calculations were performed using Ace-Ser-Nme (Ace = acetyl, Nme = methylamino) as a model compound. Results of the computational analysis suggest that the mechanism underlying the Dha residue formation from Ser consists of two steps: enolization and 1,3-elimination. The H2PO4- catalyzed both reactions as a proton-relay mediator. The calculated activation barrier for Dha residue formation was estimated as 30.4 kcal mol-1. In this pathway, the catalytic H2PO4- interacts with the Ser residue α-proton, carbonyl oxygen of Ser, and C-terminal side adjacent residues, and the calculated activation energy produced was the same as the experimentally reported value for nonenzymatic modifications of amino acid residues. Therefore, our calculation suggests that H2PO4--catalyzed Ser residue dehydration can proceed nonenzymatically.

Generation of Reactive Oxygen Species by Photosensitizers and their Modes of Action on Proteins.

In this review, we first survey the mechanisms underlying the chemical modification of amino acid residues in proteins by singlet oxygen elicited by photosensitizers. Singlet oxygen has the capacity to cause widespread chemical damage to cellular proteins. Its use in photodynamic therapy of tumors thus requires the development of methodologies for specific addressing of the photosensitizer to malignant cells while sparing normal tissue. We describe three targeting paradigms for achieving this objective. The first involves the use of a photosensitizer with a high affinity for its target protein; in this case, the photosensitizer is methylene blue for acetylcholinesterase. The second paradigm involves the use of the hydrophobic photosensitizer hypericin, which has the capacity to interact selectively with partially unfolded forms of proteins, including nascent species in rapidly dividing or virus-infected and cancer cells, acting preferentially at membrane interfaces. In this case, partially unfolded molten globule species of acetylcholinesterase serve as the model system. In the third paradigm, the photodynamic approach takes advantage of a general approach in 'state-of-the-art' chemotherapy, by coupling the photosensitizer emodin to a specific peptide hormone, GnRH, which recognizes malignant cells via specific GnRH receptors on their surface.

Genetically engineered hydrogenases promote biophotocatalysis-mediated H2 production in the green alga Chlorella sp. DT

Green algal hydrogenase (HydA) can produce hydrogen (H2) biophotocatalytically. Nevertheless, HydA is easily inhibited via its gas tunnel by O2, which is a byproduct of photosynthesis. To promote H2 production, the green alga Chlorella sp. HydA from the DT strain (CsHydAc.DT, encoded by CshydAc.DT) was genetically engineered to modify amino acid residues A105I, V265W, G113I, or V273I around the gas tunnel to prevent O2 accessing the enzyme active site. The mutated-CshydAc.DT genes were generated and homologously transformed into DT. Then the mutated-CsHydAc.DT transgenics with single or double mutations were selected. Using an in vitro assay, it was found that the DT transgenics produced up to 7-fold more H2 than the wild type (WT) in the presence of 5% O2 during biophotocatalysis. Furthermore, using an in vivo assay for several days, one transgenic with double mutations was capable of producing H2 up to 30-fold more than the WT in the presence of arbitrary O2 during biophotocatalysis.

Oxidative Modified Fibrinogen in Cardiovascular Diseases

BackgroundCardiovascular diseases are linked with oxidative stress which is the source of reactive oxidative and nitrative species, contributors of post-translational modification. Fibrinogen due to its high concentration in blood is considered as one of the most sought of targets of oxidative stress substances. Post-translational modifications of fibrinogen might influence its physiological function, thus affect hemostasis in the terms of fibrin nets forming and architecture or interaction with platelets. The aim of this study was to observe influence of in vivo fibrinogen modifications on formation of fibrin net and to identify amino acid residues prone to changes related to oxidative stress.MethodsPlasma samples were collected from patients of The Military University Hospital Prague in the agreement with ethical committees of participating institutions and with informed consents from all subjects. Samples were divided into 4 groups: patients with acute coronary syndrome (A), patients with stroke (B), patients with thrombus localized in carotid vein (C) and control group (patients without coronary atherosclerosis; D). Fibrin net architecture was studied by scanning electron microscopy (Mira 3 LMH, Tescan Orsay Holding, a.s., Brno, Czech Republic). For identification of modified amino acids residue mass spectroscopy was used (Triple TOF 6600, Sciex). Molecular dynamics simulations of hydrated protein were performed in Gromacs software with Gromos force fields. Crystal structure 3GHG was used as a reference structure to which post-translational modifications were introduced manually in Yasara View.ResultsWe found extensive both qualitative and quantitative changes in the structure of fibrinogen molecule in all groups of patients. Oxidative stress level differed among patient groups and between the control group. Different oxidative changes caused by in vivo modifications of fibrinogen affected quite distinctly the architecture of fibrin net. Modified amino acids were detected in all three fibrinogen chains. In gamma chain the localisation of modified amino acid residues correlated with the part of fibrinogen important for fibrin polymerisation. The impact of the most pronounced post-translational modifications on the secondary structure of fibrinogen was described by molecular dynamics simulations.ConclusionsThe results show that the degree of impairment of fibrinogen functions in the cardiovascular diseases is related to the level of oxidative stress. Characterization of oxidative fibrinogen modification and its precise meaning to the function of fibrinogen in hemostasis appears to be extremely helpful to better understanding of thrombotic/bleeding complications linked with various cardiovascular diseases.AcknowledgmentsThis work was supported by the Ministry of Health, Czech Republic, no. 00023736, by the Academy of Sciences, Czech Republic no. P205/12/G118 and NV18-08-00149, by ERDF OPPK CZ.2.16/3.1.00/24001 and by the European Regional Development Fund and the state budget of the Czech Republic (project AIIHHP: CZ.02.1.01/0.0/0.0/16_025/0007428, OP RDE, Ministry of Education, Youth and Sports). DisclosuresNo relevant conflicts of interest to declare.

Validation of Peptide Identification Results in Proteomics Using Amino Acid Counting.

The efficiency of proteome analysis depends strongly on the configuration parameters of the search engine. One of the murkiest and nontrivial among them is the list of amino acid modifications included for the search. Here, an approach called AA_stat is presented for uncovering the unexpected modifications of amino acid residues in the protein sequences, as well as possible artifacts of data acquisition or processing, in the results of proteome analyses. The approach is based on comparing the amino acid frequencies of different mass shifts observed using the open search method introduced recently. In this work, the proposed approach is applied to publicly available proteomic data is applied and its feasibility for discovering unaccounted modifications or possible pitfalls of the identification workflow is demonstrated. The algorithm is implemented in Python as an open-source command-line tool available at https://bitbucket.org/J_Bale/aa_stat/.

Molecular identification of histone acetyltransferases and deacetylases in lower plant Marchantia polymorpha

Histone is the core component of nucleosome and modification of amino acid residues on histone tails is one of the most pivotal epigenetic regulatory mechanisms. Histone acetylation or deacetylation is carried out by two groups of proteins: histone acetyltransferases (HATs) or histone deacetylases (HDACs), and has been proven to be tightly linked to regulation of gene expression in animals and vascular plants. The biological functions of HATs and HDACs in non-flowering plants remain largely unknown. We found that there are seven MpHAT genes and twelve MpHDAC genes present in the Marchantia genome, and the comprehensive protein sequence analysis of the HAT and HDAC families was introduced to investigate their potential functions. On the basis of the functional domain analysis, eight MpHATs and twelve MpHDACs contain the conserved functional domains as the defining feature of each family. Phylogenetic trees of all families of MpHATs and MpHDACs along with their homologs from different plant and green algae species were constructed to illustrate evolutionary relationship of HAT and HDAC proteins. We found both SIR2 family and RPD3/HDA1 superfamily possess lower plant-specific proteins indicating the potential unknown functions of HATs and HDACs in Marchantia and other lower plant or algae species. Subcellular localization prediction suggests that MpHATs and MpHDACs are likely functioning in various organelles. Expression analysis shows that all MpHAT and MpHDAC genes are expressed in all tissues with differences at the transcriptional level. In addition, their expression patterns were altered in response to various treatments with plant hormones and environmental stress. We concluded that all MpHATs and MpHDACs are functional proteins in Marchantia and involved in various signaling pathways. Marchantia could have developed a complex histone acetylation epigenetic mechanism to regulate growth and development, as well as responses to environment.

Molecular discrimination of Panax ginseng cultivar K-1 using pathogenesis-related protein 5 gene

BackgroundThe mixed-cultivation of different Panax ginseng cultivars can cause adverse effects on stability of yield and quality. K-1 is a superior cultivar with good root shape and stronger disease resistance. DNA markers mined from functional genes are clearly desirable for K-1, as they may associate with major traits and can be used for marker-assisted selection to maintain the high quality of Korean ginseng. MethodsFive genes encoding pathogenesis-related (PR) proteins of P. ginseng were amplified and compared for polymorphism mining. Primary, secondary, and tertiary structures of PR5 protein were analyzed by ExPASy-ProtParam, PSSpred, and I-TASSER methods, respectively. A coding single nucleotide polymorphism (SNP)–based specific primer was designed for K-1 by introducing a destabilizing mismatch within the 3′ end. Allele-specific polymerase chain reaction (PCR) and real-time allele-specific PCR assays were conducted for molecular discrimination of K-1 from other cultivars and landraces. ResultsA coding SNP leading to the modification of amino acid residue from aspartic acid to asparagine was exploited in PR5 gene of K-1 cultivar. Bioinformatics analysis showed that the modification of amino acid residue changed the secondary and tertiary structures of the PR5 protein. Primer KSR was designed for specific discrimination of K-1 from other ginseng cultivars and landraces. The developed real-time allele-specific PCR assay enabled easier automation and accurate genotyping of K-1 from a large number of ginseng samples. ConclusionThe SNP marker and the developed real-time allele-specific PCR assay will be useful not only for marker-assisted selection of K-1 cultivar but also for quality control in breeding and seed programs of P. ginseng.

Structural changes of fibrinogen as a consequence of cirrhosis

Cirrhosis is a disease which may develop as a consequence of various conditions. In advanced liver disease, blood coagulation can be seriously affected. Portal hypertension, vascular abnormalities and/or a dysbalance in coagulation factors may result in bleeding disorders or in the development of thrombosis. Fibrinogen is the main protein involved in clot formation and wound healing. The aim of this work was to analyse the glycosylation pattern of the isolated fibrinogen molecules by lectin-based protein microarray, together with the carbonylation pattern of the individual fibrinogen chains, possible changes in the molecular secondary and tertiary structure and reactivity with the insulin-like growth factor-binding protein 1 (IGFBP-1) in patients with cirrhosis. The results pointed to an increase in several carbohydrate moieties: tri/tetra-antennary structures, Gal β-1,4 GlcNAc, terminal α-2,3 Sia and α-1,3 Man, and a decrease in core α-1,6 Fuc and bi-antennary galactosylated N-glycans with bisecting GlcNAc. Fibrinogen Aα chain was the most susceptible to carbonylation, followed by the Bβ chain. Cirrhosis induced additional protein carbonylation, mostly on the α chain. Spectrofluorimetry and CD spectrometry detected reduction in the α-helix content, protein unfolding and/or appearance of modified amino acid residues in cirrhosis. The amount of complexes which fibrinogen forms with IGFBP-1, another factor involved in wound healing was significantly greater in patients with cirrhosis than in healthy individuals. A more detailed knowledge of individual molecules in coagulation process may contribute to deeper understanding of coagulopathies and the results of this study offer additional information on the possible mechanisms involved in impaired coagulation due to cirrhosis.

Development of Broad‐Based Labeling Reagents for Peptide and Protein Footprinting

Interactions between macromolecules in living organisms are governed by fundamental biophysical principles. Different methods to study these biophysical properties exist; including footprinting. Footprinting refers to the structural probing of interaction between macromolecules using chemical reactions ranging from HDX to reactive species such as free radicals and carbenes. Modern footprinting methods allows for a fast, specific, and precise determination of proteins' structure, interaction sites, conformational changes, folding, misfolding, and binding affinity. The complete understanding of these, in‐vitro and in the cell, is crucial for drug development and antibody synthesis for therapeutics and diagnostics. These advances contribute to the understanding of the human proteome. Methods for determining a protein footprint include Mass Spectrometry (MS), which is advantageous given its sensitivity. Covalent modification of amino acid residues coupled with MS has been useful in characterizing non‐membrane and membrane proteins; a field where current biophysical techniques are limited. Covalent modification can be achieved via Fast Photochemical Oxidation of Proteins (FPOP) using radical chemistry. Residue labeling using current radical species lacks generality or broad reactivity among amino acids. Thus, the development of novel chemical labeling methods that increases amino acid labeling are needed. To implement this, we propose the use of carbene species (double radicals), trifluoro‐methyl radicals, and chloride cations. Carbene compounds will be generated via FPOP from diazirine precursors, which were synthesized from ketone precursors. The Togni's Reagent will be used as a precursor for covalent modification through trifluoromethyl radicals and Palau's reagent will be used for modification through chloride cations. If a robust footprinting method is developed to further understand protein biophysics it would lead to further advances in the study of protein folding, protein‐protein interactions, and drug development.Support or Funding InformationAmgen Foundation. Michael Gross's Laboratory. NIH/NIGMS Biomedical Mass Spectrometry Resource.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The structural diversity and the biological meaning of antibody

Along with the development of antibody drugs and catalytic antibodies, the structural diversity issue has attracted much attention. Structural diversity(or structural heterogeneity) is generated caused by the modification of different charges, different molecular sizes, and/or modified amino acid residues. Namely, an antibody takes multi‐molecular forms in the solution. It is desirable that the antibody and/or the subunits must have a defined structure for practical use. Regarding with the difficult issue on the structural diversity of antibody, we found the interesting phenomena in which copper ion can convert the multi‐molecular forms of antibodies to mono‐molecular form (1). The ion hugely contributed to the enrichment of the dimer‐form and the homogenation of the differently charged full‐length of the light chain. The role of copper ion must be significant for preparing a single, defined, not multiple, isoform structure.In addition, we found that the constant region domain (CL) of the antibody light chain also plays an important role in the structural heterogeneity of the light chain (2), which was clarified by expressing the constant domain. Considering the chemical features of the full length light chain (VL+CL) and CL, note that the CL domain can influence to generation of the structural diversity of the VL domain. This finding is important, because, even at the present, the function of CL is still unknown. It is considered that the generation of the structural diversity of a protein may influence on the binding ability as well as the specificity.In the presentation, the authors will discuss not only the reason why antibodies take such structural diversity but also their biological meanings.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pentaminomycins A and B, Hydroxyarginine-Containing Cyclic Pentapeptides from Streptomyces sp. RK88-1441.

Two new cyclic peptides, pentaminomycins A (1) and B (2), were isolated from cultures of Streptomyces sp. RK88-1441. Based on the interpretation of the NMR, UV, IR, and MS data, the planar structures of 1 and 2 were elucidated as cyclic pentapeptides with a modified amino acid residue, N5-hydroxyarginine (N5-OH-Arg). The absolute configurations of the constituent amino acid residues were determined by the advanced Marfey's method. Localization of l- and d-amino acids in the sequence was ascertained by chiral analysis of the fragment peptide obtained from a partial hydrolysate; amino acids were identified by LC-MS. Pentaminomycin A (1) reduced α-MSH-stimulated melanin synthesis by suppressing the expression of melanogenic enzymes including tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2).

Protein-Derived Cofactors Revisited: Empowering Amino Acid Residues with New Functions.

A protein-derived cofactor is a catalytic or redox-active site in a protein that is formed by post-translational modification of one or more amino acid residues. These post-translational modifications are irreversible and endow the modified amino acid residues with new functional properties. This Perspective focuses on the following advances in this area that have occurred during recent years. The biosynthesis of the tryptophan tryptophylquinone cofactor is catalyzed by a diheme enzyme, MauG. A bis-FeIV redox state of the hemes performs three two-electron oxidations of specific Trp residues via long-range electron transfer. In contrast, a flavoenzyme catalyzes the biosynthesis of the cysteine tryptophylquinone (CTQ) cofactor present in a newly discovered family of CTQ-dependent oxidases. Another carbonyl cofactor, the pyruvoyl cofactor found in classes of decarboxylases and reductases, is formed during an apparently autocatalytic cleavage of a precursor protein at the N-terminus of the cleavage product. It has been shown that in at least some cases, the cleavage is facilitated by binding to an accessory protein. Tyrosylquinonine cofactors, topaquinone and lysine tyrosylquinone, are found in copper-containing amine oxidases and lysyl oxidases, respectively. The physiological roles of different families of these enzymes in humans have been more clearly defined and shown to have significant implications with respect to human health. There has also been continued characterization of the roles of covalently cross-linked amino acid side chains that influence the reactivity of redox-active metal centers in proteins. These include Cys-Tyr species in galactose oxidase and cysteine dioxygenase and the Met-Tyr-Trp species in the catalase-peroxidase KatG.

3D-modeling of carboxyl-terminal phosphorylation of plant αβ-tubulin and its role in kinesin-8/microtubule interaction.

The results of computer modeling of plant kinesin-8/αβ-tubulin complexes with such αβ-tubulins' modified amino acid residues as phosphorylated Tyr262 and Tyr107 are reported in this paper. The molecular dynamics of these modified complexes in comparison with the dynamics of non-modified ones suggests that the phosphorylation of both α- and β-tubulins reveals stabilizing effect on the protein structure around the modified residue. It was found also that the phosphorylation of Tyr107 in β-tubulin molecule favors to more advantageous kinesin-8 binding with the phosphorylated microtubule surface in terms of energy.

Metal coordination in kinases and pseudokinases.

Protein phosphorylation, mediated by protein kinases, is a key event in the regulation of eukaryotic signal transduction. The majority of eukaryotic protein kinases perform phosphoryl transfer, assisted by two divalent metal ions. About 10% of all human protein kinases are, however, thought to be catalytically inactive. These kinases lack conserved residues of the kinase core and are classified as pseudokinases. Yet, it has been demonstrated that pseudokinases are critically involved in biological functions. Here, we show how pseudokinases have developed strategies by modifying amino acid residues in order to achieve stable, active-like conformations. This includes binding of the co-substrate ATP in a two metal-, one metal- or even no metal-binding mode. Examples of the respective pseudokinases are provided on a structural basis and compared with a canonical protein kinase, Protein Kinase A. Moreover, the functional roles of both independent metal-binding sites, Me1 and Me2, are discussed. Lack of phosphotransferase activity does not implicate a loss of function and can easily point to alternative roles of pseudokinases, i.e. acting as switches or scaffolds, and having evolved as components crucial for cellular cross-talk and signaling. Interestingly, pseudokinases are present in all kingdoms of life and their specific roles remain enigmatic. More studies are needed to unravel the crucial functions of those interesting proteins.

Mazes of Nrf2 Regulation.

Nrf2 transcription factor plays a key role in maintaining cellular redox balance under stress and is a perspective target for oxidative stress-associated diseases. Under normal conditions, Nrf2 transcriptional activity is low due to its rapid ubiquitination and degradation in the 26S proteasome, as well as through various modifications of amino acid residues of this transcription factor that regulate its transport to the nucleus and binding to DNA. Continuous activation of Nrf2 is possible due to autophagy and epigenetic regulation that may underlie the increased resistance of tumor cells to radiotherapy and chemotherapy. This review deals with the mechanisms of regulation of Nrf2 transcriptional activity and its main elements, and pharmacological approaches to activation of the Keap1/Nrf2/ARE system.

112 - ROS-Mediated Modification of RhCC10 Protein for Potential Gain of Function

The Club cell 10kDa protein is one of the most abundant proteins in the respiratory mucosa. Lowered pulmonary or systemic CC10 levels correlate with poorer outcomes in several respiratory conditions such as neonatal bronchopulmonary dysplasia (BPD), asthma, COPD, and post-lung transplant bronchiolitis obliterans syndrome (BOS). CC10 suppresses inflammation by at least two distinct mechanisms, including inhibition of phospholipase A 2 enzymes and the arachidonic acid cascade and inhibition of NF-κB signaling in respiratory epithelial cells, but CC10 is also chemically modified by inflammatory processes. Lindahl et al (1999) first reported on at least 3 distinct isoforms of CC10 in respiratory mucosal fluids in adults with significant respiratory sequelae to inhaled industrial chemical exposures. Ramsay et al (2001) reported on a CC10 isoform in TAF from premature infants who went on to develop BPD. Arias-Martinez et al (2012) extended that observation to identify up to 7 CC10 isoforms in premature infants experiencing severe RDS who developed BPD. We investigated the possibility that the CC10 isoforms resulted from chemical modification of amino acid residues mediated by reactive oxygen and/or nitrogen species (ROS or RNS) in a series of in vitro reactions using recombinant human CC10 protein (rhCC10). At least a dozen new isoforms of CC10 were generated by reaction with ROS using three different oxidants, including NaOCl, mCPBA, and neutrophil myeloperoxidase (MPO) plus H 2 O 2 . Reaction products were characterized by HPLC, isoelectric point, Western blot, and peptide mapping. Comparison of mCPBA reaction products with NaOCl and MPO-H 2 O 2 , using different reaction times and amounts of oxidizing equivalents, revealed that methionines were initially oxidized prior to other amino acids. Each human CC10 monomer contains 4 methionines, 3 of which are readily oxidized by ROS, suggesting that each CC10 homodimer can absorb at least 6 oxygen radicals, thereby protecting other proteins, cells, and tissues and augmenting its other anti-inflammatory activities. Preliminary data also suggest that ROS-modification of CC10 enhances its anti-neutrophil activity and anti-viral activity.