Stabilization of AIMP1/p43 and EMAP II recombinant proteins in the complexes with polysaccharide dextran-70.

BACKGROUND: Protein-based pharmaceuticals are among the fastest growing categories of therapeutic agents in the clinic and as commercial products, and typically target high-impact areas such as various cancers, autoimmune diseases and metabolic disorders. The aim of our work was to explore the possibility of reducing the level of aggregation and improve the stability of the recombinant proteins AIMP1/p43 (aminoacyl-tRNA synthetase complex component of the higher eukaryotes) and antitumor cytokine EMAP II (proteolytic cleavage product of AIMP1/p43) in combination with dextran-70 polysaccharide for structural-functional research and development of new sustainable biomedical products. METHODS: We studied interaction strength between these recombinant proteins with polymer by fluorescence spectroscopy and molecular docking. RESULTS: During experimental studies, optimal concentration ratio of AIMP1/p43 and EMAP II recombinant proteins with dextran-70 in which proteins bind to ligand and form complex was established. As a result of molecular docking investigations, spatial structure of the AIMP1/p43-dextran-70 and EMAP II-dextran-70 complexes was obtained and their binding energy was evaluation. CONCLUSIONS: The effect of temperature increase on the stability of these two complexes was determined by fluorescence spectroscopy method. It was found that dextran-70 specifically connects with recombinant proteins. Binding stoichiometry of dextran-70 with protein is about 1:1, which confirms the formation of a specific complex.

[ENDOTHELIAL MONOCYTEACTIVATING FACTOR II CANCELS OXIDATIVE STRESS, CONSTITUTIVE NOS UNCOUPLING AND INDUCED VIOLATIONS OF CARDIAC HEMODYNAMICS IN HYPERTENSION (PART II)].

The purpose of this study was to investigate the effect of EMAP II on free radical state of the heart and blood vessels, to restore cNOS coupling and cardiac hemodynamics in spontaneously hypertensive rats. It was found that, due to the combined inhibition of oxidative and nitrosative stress, EMAP I quickly restores impaired in hypertension constitutive de novo synthesis of NO by restoring cNOS coupling. Restoration by EMAP II of constitutive de novo synthesis NO abolished cardiac and endothelial dysfunction in spontaneously hypertensive rats. In hypertension, the introduction of EMAP II helped to improve the performance of the pumping function of the heart (stroke volume increased by 18.2 %, cardiac output -22 %), an arterial stiffness decreased by 23.2 %, process of relaxation of the left ventricle improved, due to decreased in 4,7 times myocardial end-diastolic stiffness.

[COMPUTATIONAL MODELING OF MOLECULAR DYNAMICS OF G41R MUTANT FORM OF HUMAN TYROSYL-tRNA SYNTHETASE, ASSOSIATED WITH CHARCOT-MARIE-TOOTH NEUROPATHY].

The computational structural models of human tyrosyl-tRNA synthetase and its mutant form G41R (Charcot-Marie-Tooth associated) were constructed, while their whole structural coordinates are still unknown. Grid-services of MolDynGrid Virtual Laboratory and Ukrainian National Grid-infrastructure were used for molecular dynamics (MD) simulations. The analyses of trajectories of MD simulations have shown the ß-sheet formation in region Lys147 - Glu157 between H9 and H10 helices (CP1 insertion of Rossman fold) for G41R mutant.

Interdomain compactization in human tyrosyl-tRNA synthetase studied by the hierarchical rotations technique.

Aminoacyl-tRNA synthetases are key enzymes of protein biosynthesis which usually possess multidomain structures. Mammalian tyrosyl-tRNA synthetase is composed of two structural modules: N-terminal catalytic core and an EMAPII-like C-terminal domain separated by long flexible linker. The structure of full-length human cytoplasmic tyrosyl-tRNA synthetase is still unknown. The structures of isolated N-terminal and C-terminal domains of the protein are resolved, but their compact packing in a functional enzyme is a subject of debates. In this work we studied putative compactization of the N- and C-terminal modules of human tyrosyl-tRNA synthetase by the coarse-grained hierarchical rotations technique (HIEROT). The large number of distinct types of binding interfaces between N- and C-terminal modules is revealed in the absence of enzyme substrates. The binding propensities of different residues are computed and several binding "hot spots" are observed on the surfaces of N and C modules. These results could be used to govern atomistic molecular dynamics simulations, which will sample preferable binding interfaces effectively.

Cooperative antitumor effect of endothelial-monocyte activating polypeptide II and flutamide on human prostate cancer xenografts.

UNLABELLED: Recombinant cytokine-like endothelial monocyte-activating polypeptide II (EMAP II) and antiandrogen flutamide target different mechanisms of growth of androgen-dependent prostate cancer (PC). The aim of this study was to clarify whether combined treatment with EMAP II and flutamide is more effective than monotherapy with regard to retardation of PC progression. MATERIALS AND METHODS: Antitumor effects of EMAP II (10 µg/kg b.w./d, s.c., 3d), or flutamide (10 mg/kg b.w./d, per os, 3d), or their combination were studied in CBA male mice bearing human androgen-dependent PC xenografts for 7 days. Androgen-dependent phenotype of the tumors was verified in preliminary castrated mice. The xenografts were weighed and underwent a histopathologic examination. The results were compared with those of non-treated mice. RESULTS: EMAP II and flutamide used separately inhibited growth of the xenografts by 74% and 53% respectively. Both drugs caused destructive changes in malignant epithelial cells along with leukocyte infiltration of the tumor. Combined treatment inhibited tumor growth by 85%, and was more effective than monotherapy with regard to morphological changes. CONCLUSIONS: This study demonstrates cooperative inhibitory effect of EMAP II and flutamide on growth and morphology of human PC xenografts that could represent a new modality of palliative treatment of this disease.

Antitumor effect of endothelial monocyte-activating polypeptide-II on human prostate adenocarcinoma in mouse xenograft model.

UNLABELLED: Endothelial monocyte-activating polypeptide-II (EMAP-II) is a novel proinflammatory cytokine with anti-angiogenic properties. The aim of this work was to evaluate in vivo antitumor activity of EMAP-II in growing human prostate adenocarcinoma xenograft mouse model. MATERIALS AND METHODS: Recombinant human EMAP-II was expressed in Escherichia coli and purified after cleavage with enterokinase (EMAP-II e). EMAP-II preparations were injected to CBA mice bearing subrenal capsule xenografts of human prostate adenocarcinoma. After 3-days treatment, the xenografts were isolated and weighed, then the transplants exposed to EMAP II e (100 or 200 microg/kg b. w.) were examined histologically. RESULTS: EMAP-II administered daily at a dose of 100 or 200 microg/kg b. w. caused striking retardation of local tumor progression as compared to the controls. Low dose (10 microg/kg) was effective in some cases. CONCLUSION: EMAP II exhibits significant antitumor activity in vivo in human prostate adenocarcinoma xenografts in mouse model.

The improvement of the algorithm for order parameter calculation (S2) from molecular dynamics simulation using the correlation motion function.

The generalized order parameter, S2, calculated from MD simulation trajectory using time-dependent internal Correlation Motion Function (CMF) agrees well with NMR derived S2 processed with the extended model-free analysis approach. However, the former lies considerably lower comparing to simple model-free derived data from NMR experiments. In the present study we analyze possible reasons of such disagreement. In the general case we propose to use preexponential factors from expression for internal CMF rather than ordinary S2 values. Particularly, in case of the simple model-free S(2) experimental values we suggest comparing them with S2(eff)=1+S2-Sf2 computed from MD simulation data. We show that the S2(eff) values are in a good agreement with NMR derived S2 values obtained using the simple model-free analysis.

Bioinformatic analysis of changes in expression level of tyrosyl-tRNA synthetase during sporulation process in Saccharomyces cerevisiae.

Study of tyrosyl-tRNA synthetases (TyrRS) gene expression during sporulation cycle in Saccharomyces cerevisiae by bioinformatic analysis of microarray data showed high correlation of TyrRS expression to genes that participate in cell wall assembly. Furthermore, the cell wall biogenesis protein KNR4 which physically interacts with TyrRS and cooperates in beta-1,3 glucan biosynthesis falls into a single gene cluster with TyrRS. One third of genes (13 from 42) in TyrRS gene cluster are responsible for the functions directly related to cell wall assembly and maintenance during sporulation. Putative transcription factor binding site on TyrRS upstream sequences was localized with expectation maximization algorithm. The site could be involved in the control of TyrRS expression during sporulation. Absence of correlation in gene expression between KNR4 and TyrRS in S. cerevisiae and their homologues in Schizosaccharomyces pombe--genes SPBC30D10.17 and TYR1 as well as the lack of correlation in the expression level of TyrRS with other sporulation cycle genes indicates that participation of TyrRS in cell wall assembly in S. cerevisiae appeared later in evolution after divergence of S. pombe and S. cerevisiae.

Conformational change of mammalian tyrosyl-tRNA synthetase induced by tyrosyl adenylate formation.

The fluorescent probe 1,5-I-AEDANS was covalently attached to bovine tyrosyl-tRNA synthetase outside of enzyme active site in a nearly stoichiometric amount (2 probe molecules per enzyme dimer). Singlet-singlet resonance energy transfer has been used for the measurement of the apparent distance between tryptophan residues of enzyme and covalently attached 1,5-I-AEDANS. This distance was estimated as 27.4 A in the assumption of the random orientation of the donor and acceptor fluorophores. Tyrosyl adenylate formation catalyzed by bovine tyrosyl-tRNA synthetase resulted in the highly specific enhancement of 1,5-I-AEDANS fluorescence and concomitant decrease of the apparent distance between the probe and tryptophanyls to 22.3-25.7 A. These results are consistent with the conformational change of tyrosyl-tRNA synthetase during tyrosyl adenylate formation which propagates to distant from active site regions of enzyme structure.