Computer simulation of the spatial structure of MUC 1 peptides capable of inhibiting apoptosis

Identification of new effective inhibitors of apoptosis is an important task for drug development for treatment of a number diseases including neurogenerative diseases. Initiation of apoptosis occurs via the formationof macromolecular protein complexes. In these complexes, activation of key enzymes in apoptosis, caspases, takes place. One of those macromolecular complexes is DISC (death- inducing signaling complex) playing a central role in the induction of the extrinsic apoptosis pathway. The adaptor protein FA DD has a major role in the formation of the DISC. Therefore, inhibitors of FA DD, preventing its function in the DISC, can act as potential drugs inhibiting apoptosis. Furthermore, the study of the mechanisms of action of these inhibitors is of great interest for understanding the mechanisms of the signal transduction pathways of apoptosis. It has been reported that a natural protein inhibitor of FA DD is mucin-type 1 glycoprotein (MUC1). In particular, two fragments of the primary structure of the cytoplasmic domain of MUC1 (MUC1- CD) are capable of inhibiting the binding of caspase-8 to FA DD. However, the three-dimensional structure of MUC1 has not been obtained yet. It complicates significantly the rational design of potential drugs on the basis of these peptides. In this context, the aim of the present study was in silico prediction ofthree-dimensional structures of MUC1-CD peptides corresponding to protein fragments (1-20 and 46-72), as well as analysis of their conformational properties. The main focus of the work was given to the peptide MUC1-CD (46-72), which is capable of binding to FA DD. Using the methods of molecular dynamics in the implicit water it was shown that the peptide MUC1-CD (46-72) can take conformations similar to the conformations of a number of fragments of the caspase-8 DED domain. It was found that  the structure of the peptide MUC1-CD (46-72) is similar to the spatial structure of at least four fragments of caspase-8. These results indicate that the molecular mechanism of the inhibitory activity of the peptide can be explained by competitive binding with FA DD due to the structural and conformational similarity with the fragments of the caspase-8 DED domain

Design and experimental validation of the action of small molecule-based inhibitors of the FADD protein

CD95 is one of the best studied members of the death receptor family. Activation of CD95 leads to the induction of the cell death programme, apoptosis, via formation of the death-inducing signaling complex (DISC). FA DD is a key adaptor protein for the formation of the C D95 DISC and activation of caspase-8 in the receptor complex. FA DD comprises the death domain and the death effector domain (DED). The death domain is essential for the interactions of FA DD with CD95, while DED is necessary for the recruitment of procaspase-8, -10 and the protein c-FLIP into the DISC. The search for the inhibitors that would block the interactions of FA DD with the other core proteins of the DISC is essential for the studies of the structure and function of this complex, investigation of the apoptosis mechanisms and development of new treatments for neurodegenerative diseases. In the course of this work, the screening for small inhibitors in silico that selectively interact with DED has been performed. For this purpose, the molecular modeling of the protein complexes and virtual screening of the potential inhibitors of FA DD has been performed. In addition, a new technology to test the activity of these inhibitors has been developed. The computational and experimental analysis performed allowed us to characterize the optimal conformation of the FA DD protein for the design of the small molecules that can bind in the region of amino acid residue Y25. We presume that further optimization of the structures of chemical compounds that can bind with the hydrophobic pocket next to the residue Y25 of FA DD will allow for the creation of the new perspective inhibitors of the programmed cell death

Interaction of PARP2 with DNA structures mimicking DNA repair intermediates

Poly(ADP-ribosyl)ation is a posttranslational protein modification significant for the genomic stability and cell survival in response to DNA damage. Poly(ADP-ribosyl)ation is catalyzed by poly(ADP-ribose)polymerases (PARPs). Whereas the role of PARP1 in response to DNA damage has been widely illustrated, the contribution of another DNA-dependent PARP, PARP2, has not been studied so far. Aim. To find out specific DNA targets of PARP2. Methods. The EMSA and the PARP activity tests were used. Results. We evaluated Kd values of PARP2-DNA complexes for several DNA structures mimicking intermediates of different DNA metabolizing processes and tested these DNA as «activators» of PARP1 and PARP2 in poly(ADP-ribose) synthesis. Conclusions. Like PARP1, PARP2 does not show correlation between the activation efficiency and Kd values for DNA. PARP2 was activated most effectively in the presence of over5DNA

Glycyrrhetinic acid and its derivatives as inhibitors of poly(ADP-ribose)polymerases 1 and 2, apurinic/apyrimidinic endonuclease 1 and DNA polymerase β

Aim. For strengthening the efficiency of monofunctional alkylating antineoplastic drugs it is important to lower the capacity of base excision repair (BER) system which corrects the majority of DNA damages caused by these reagents. The objective was to create inhibitors of the key BER enzymes (PARP1, PARP2, DNA polymerase β, and APE1) by the directed modification of glycyrrhetinic acid (GA). Methods. Amides of GA were produced from the GA acetate by formation of the corresponding acyl chloride, amidation with the appropriate amine and subsequent deacylation. Small library of 2-cyano substituted derivatives of GA methyl esters was obtained by the structural modification of GA framework and carboxylic acid group. The inhibitory capacity of the compounds was estimated by comparison of the enzyme activities in specific tests in the presence of compounds versus their absence. Results. None of tested compounds inhibits PARP1 significantly. Unmodified GA and its morpholinic derivative were shown to be weak inhibitors of PARP2. The derivatives of GA containing keto-group in 11 triterpene framework were shown to be moderate inhibitors of pol β. Compound 3, containing 12-oxo-9(11)-en moiety in the ring C, was shown to be a single inhibitor of APE1 among all compounds studied. Conclusions. The class of GA derivatives, selective pol β inhibitors, was found out. The selective inhibitor of APE1 and weak selective inhibitor of PARP2 were also revealed

Human base excision repair enzymes apurinic/apyrimidinic endonuclease1 (APE1), DNA polymerase β and poly(ADP-ribose) polymerase 1: interplay between strand-displacement DNA synthesis and proofreading exonuclease activity

We examined interactions between base excision repair (BER) DNA intermediates and purified human BER enzymes, DNA polymerase β (pol β), apurinic/apyrimidinic endonuclease (APE1) and poly(ADP-ribose) polymerase-1 (PARP-1). Studies under steady-state conditions with purified BER enzymes and BER substrates have already demonstrated interplay between these BER enzymes that is sensitive to the respective concentrations of each enzyme. Therefore, in this study, using conditions of enzyme excess over substrate DNA, we further examine the question of interplay between BER enzymes on BER intermediates. The results reveal several important differences compared with data obtained using steady-state assays. Excess PARP-1 antagonizes the action of pol β, producing a complete block of long patch BER strand-displacement DNA synthesis. Surprisingly, an excess of APE1 stimulates strand-displacement DNA synthesis by pol β, but this effect is blocked by PARP-1. The APE1 exonuclease function appears to be modulated by the other BER proteins. Excess APE1 over pol β may allow APE1 to perform both exonuclease function and stimulation of strand-displacement DNA synthesis by pol β. This enables pol β to mediate long patch sub-pathway. These results indicate that differences in the stoichiometry of BER enzymes may regulate BER

Neutron spin echo is a "quantum tale of two paths''

We describe an experiment that strongly supports a two-path interferometric model in which the spin-up and spin-down components of each neutron propagate coherently along spatially separated parallel paths in a typical neutron spin echo small angle scattering (SESANS) experiment. Specifically, we show that the usual semi-classical, single-path treatment of the Larmor precession of a polarized neutron in an external magnetic field predicts a damping as a function of the spin-echo length of the SESANS signal obtained with a periodic phase grating when the transverse width of the neutron wave packet is finite. However, no such damping is observed experimentally, implying either that the Larmor model is incorrect or that the transverse extent of the wave packet is very large. In contrast, we demonstrate theoretically that a quantum-mechanical interferometric model in which the two mode-entangled (i.e. intraparticle entangled) spin states of a single neutron are separated in space when they interact with the grating accurately predicts the measured SESANS signal, which is independent of the wave packet width

Molecular-genetic mechanisms of the interaction between processes of cell response to mechanical stress and neuronal apoptosis in primary open-angle glaucoma

Glaucoma is a chronic and progressive disease, which affects more than 60 million people worldwide. Primary open-angle glaucoma (POAG) is one of the most common forms of glaucoma. For example, about 2.71 million people in the USA had primary open-angle glaucoma in 2011. Currently POAG is a major cause of irreversible vision loss. In patients with treated open-angle glaucoma the risk of blindness reached to be about 27 %. It is known that the death of optic nerve cells can be triggered by mechanical stress caused by increased intraocular pressure, which induces neuronal apoptosis and is observed in patients with POAG. Currently, there is a large number of scientific publications describing proteins and genes involved in the pathogenesis of POAG, including neuronal apoptosis and the cell response to mechanical stress. However, the molecular- genetic mechanisms underlying the pathophysiology of POAG are still poorly understood. Reconstruction of associative networks describing the functional interactions between these genes/proteins, including biochemical reactions, regulatory interactions, transport, etc., requires the use of methods of automated knowledge extraction from texts of scientific publications. The aim of the work was the analysis of associative networks, describing the molecular-genetic interactions between proteins and genes involved in cell response to mechanical stress (CRMS), neuronal apoptosis and pathogenesis of POAG using ANDSystem, our previous development for automated text analysis. It was shown that genes associated with POAG are statistically significantly more often represented among the genes involved in the interactions between CRMS and neuronal apoptosis than it was expected by random reasons, which can be an explanation for the effect of POAG leading to the retinal ganglion cell death

ANALYSIS OF THE INTERACTIONS OF NEURONAL APOPTOSIS GENES IN THE ASSOCIATIVE GENE NETWORK OF PARKINSON’S DISEASE

Parkinson’s disease (PD) affects an estimated 7–10 million people worldwide and 210000 people in Russia. PD is accompanied by degeneration of dopaminergic neurons and because of that neuronal apoptosis is an important factor in this disease. Analysis of gene networks is one of the key approaches in systems biology. We previously developed the ANDSystem tool, designed to automatically extract knowledge from scientific publications and reconstruct on this basis associative gene networks describing the molecular genetic mechanisms of biological processes. The aim of this work was prioritization of neuronal apoptosis genes by their involvement in PD pathogenesis, taking into account the structure of the PD associative gene network using ANDSystem. Analysis of the centrality of neuronal apoptosis genes, associated with PD, revealed that mean values of degree, closeness and betweenness centralities statistically significantly exceed such values of all nodes of the PD network. The APOE, CASP3 and GAPDH genes involved in neuronal apoptosis were among the most central genes. Prioritization of neuronal apoptosis genes for which there was no data in ANDSystem on their associations with PD was performed using standard methods (Endeavor and ToppGene) and the criteria of centrality and specificity of genes interactions with the PD gene network. Analysis revealed that genes involved in such processes as positive and negative regulation of neu ronal apoptosis, MAPK and ephrin receptor signaling pathways, are mainly represented among candidate genes with the highest priority (top 50, 70, 100 genes were considered). In particular, TP53, JUN, BCL2, PIK3CA and APP were among candidate genes with the highest priority

Role of apoptosis genes in aggression revealed using combined analysis of ANDSystem gene networks, expression and genomic data in grey rats with aggressive behavior

Aggressive behavior in animals plays an important role in protecting the territory, offspring, establishing social hierarchical relations, etc. Increased aggression is observed in a number of diseases ( schizophrenia, bipolar disorder, brain degenerative disorders). Neuronal apoptosis is crucial in the maintenance of developmental processes during neurogenesis. Alterations in neuronal apoptosis are observed in aging and neuropathologies accompanied by changes in psycho­emo­ tional state (epilepsy, Alzheimer’s disease, neurotrauma). The expression of key neuronal apoptosis genes (Casp3, Bax and Bcl-xl) in the brain of highly aggressive rats is significantly altered. The aim of this work was to analyze associative networks that describe genetic interactions between genes/proteins involved in neuronal apoptosis, differentially expressed genes and genes with polymorphisms in grey rats with aggressive behavior. Analysis revealed 819 differentially expressed genes in the hypothalamus, ventral tegmental region and periaqueductus Sylvii grey matter in grey rats with aggressive and tame behavior. The Stx1a, Mbp and Th genes have the highest index of betweenness centrality in the associative network of differentially expressed genes. Genome analysis revealed 137 polymorphic genes. Three of them (Lig4, Parp1 and Pigt) were involved in neuronal apoptosis. It was shown that polymorphic and differentially expressed genes were statistically significantly overrepresented among ge nes interacting with neuronal apoptosis genes (p value < 0.01). Three molecular­genetic chains describing connections between polymorphic and neuronal apoptosis genes mediated by differentially expressed genes were reconstructed. Chains included the polymorphic genes Tsc1, Adamts4 and Lgals3, differentially expressed genes Ezr, Acan, Th and 19 neuronal apoptosis genes. It was shown that neuronal apoptosis is closely related to aggressive behavior in animals

Computer analysis of regulation of hepatocarcinoma marker genes hypermethylated by HCV proteins

Hepatitis C virus (HCV) is a risk factor that leads to hepatocellular carcinoma (HCC) development. Epigenetic changes are known to play an important role in the molecular genetic mechanisms of virus-induced oncogenesis. Aber rant DNA methylation is a mediator of epigenetic changes that are closely associated with the HCC pathogenesis and considered a biomarker for its early diagnosis. The ANDSystem software package was used to reconstruct and evaluate the statistical significance of the pathways HCV could potentially use to regulate 32 hypermethylated genes in HCC, including both oncosuppressor and protumorigenic ones identified by genome-wide analysis of DNA methylation. The reconstructed pathways included those affecting protein-protein interactions (PPI), gene expression, protein activity, stability, and transport regulations, the expression regulation pathways being statistically significant. It has been shown that 8 out of 10 HCV proteins were involved in these pathways, the HCV NS3 protein being implicated in the largest number of regulatory pathways. NS3 was associated with the regulation of 5 tumor-suppressor genes, which may be the evidence of its central role in HCC pathogenesis. Analysis of the reconstructed pathways has demonstrated that following the transcription factor inhibition caused by binding to viral proteins, the expression of a number of oncosuppressors (WT1, MGMT, SOCS1, P53) was suppressed, while the expression of others (RASF1, RUNX3, WIF1, DAPK1) was activated. Thus, the performed gene-network reconstruction has shown that HCV proteins can influence not only the methylation status of oncosuppressor genes, but also their transcriptional regulation. The results obtained can be used in the search for pharmacological targets to develop new drugs against HCV-induced HCC