Regulation of gene expression with Khavinson peptides

It was revealed that fluorescein isothiocyanate -labeled (FITC) small peptides penetrate into cytoplasm, nucleus and nucleolus of the HeLa cell. It is known that the nucleus of eukaryotic cells has a system of nucleopores formed by protein complexes - nucleoporins. The inner diameter of the nucleopores is about 50 nm. Thus, they are permeable for the diffusing molecules with a molecular weight up to 3,5 kDa. Thus, small peptides by their physical-chemical characteristics, (charge, size, hydrophobicity) can penetrate the cytoplasmic and nuclear membrane of the cell and possibly interact with DNA.

According to the data obtained by physical methods (UV spectroscopy, circular dichroism, viscosimetry, atomic force microscopy) and molecular modelling; signal peptides are capable of binding to DNA in solution. This process takes several hours without almost any electrostatic force. The formation between the major groove of DNA with nitrogenous bases and peptide leads to a destabilization of the secondary structure of macromolecule. According to results from the spectrophotometry, in the ultraviolet region of the spectrum in the mixture of the peptide (AEDG and double-stranded DNA) there was registered a concentration-dependent hyperchromic effect (an increase in the optical density of the DNA solution at a wavelength of 260 nm). This hyperchromic effect proves that there is a partial destruction of hydrogen bonds between the nucleotide pairs of the double helix and local separation of the DNA chains (allosteric conformational change).

It was established through experiments that the separation of chains (melting) of free synthetic DNA occurs at a temperature of 69.50 C. Nevertheless in the DNA-peptide (AEDG) system this melting took place at a temperature of 280 C and was characterized by an almost two-fold decrease in the enthalpy and entropy values. This shows that there is a thermodynamically easier way to melt DNA strands at the temperature, which is adequate to the thermal regimes of biochemical processes in the majority of living organisms. It also shows that separation of DNA strands at physiological temperatures is not a denaturation, instead it is the initiation of protein synthesis process.

These theoretical and experimental data allowed to propose a model of peptide-DNA interactions in case of Khavinson peptides. Moreover these data enable to create a stable peptide-DNA complex. Analysis of the main physical and chemical parameters of this complex, (the number of hydrogen bonds, hydrophobic and electrostatic interactions, energy of minimization of the DNA-peptide complex) was performed using molecular modelling. It allowed to determine quantitative characteristics of the DNA-peptide complex [Molecular Operating Environment; Chemical Computing Group Inc (2012) 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2012]. Based on these calculations a three-dimensional model of the interaction of peptide AEDG with the DNA ATTTC site was created.

The experiments revealed a specific binding of peptides with oligonucleotides which may be particularly important for the epigenetic mechanism for regulation of gene expression. Interaction of small peptides, particularly with single stranded DNA regions, may specifically control expression of genes.

Short peptides were found to modulate the activity of wheat seedling endonucleases. This modulation of endonucleases activity probably happens due to the site-specific DNA-peptide binding which protects DNA against enzymatic hydrolysis. Then in turn, the modulation of endonucleases activity by peptides is modulated by histones. Chromatin histones in the nucleus may affect binding of DNA to small peptides. In addition, some peptides appear to control hydrolysis of DNA by endonucleases, at the level of interaction for a peptide with an enzyme.

It was revealed that small peptides activate heterochromatin in cell nuclei in elderly people and contribute to the 'release' of genes, repressed as a result of heterochromatinization of euchromatic regions of chromosomes that occurs with aging.

Structural condensation of chromatin is in close correlation with the functional heterogeneity. It was revealed that with aging the heterochromatinization becomes more intensive and this correlates with inactivation of previously active genes. Tightly condensed heterochromatic regions of chromosomes are genetically inactivated and have a slower replication. The decondensed (euchromatic) regions of chromosomes are always actively functioning. Regulatory peptides increase concentration of euchromatin in the nucleus. It means that more genes become available for transcription, which also occurs faster. As a result the protein synthesis also increases. The more euchromatin there is in the nucleus, the more intensive the protein synthesis in the cell is. The results of this experiment lead to the conclusion that heterochromatinization is a reversible process, with important biological outcomes.

It was revealed that administration of peptides KE and AEDG to transgenic mice caused a 2-3.6-fold suppression of HER-2/neu gene expression (human breast cancer) when compared to the control group. This suppression is accompanied by a significan treduction of the tumor diameter (Fig. 2).

It was shown that the addition of peptide AEDG to the cultural medium of human lung fibroblasts induces telomerase gene expression and contributes to a 2.4-fold lengthening of telomeres. Activation of gene expression is accompanied by a growing number of cellular divisions by 42.5% (Fig. 3). It was revealed that addition of tetrapeptide Ala-Glu-Asp-Gly to the cultural medium of human lung fibroblasts induces telomerase gene expression and contributes to a 2.4-fold lengthening of telomeres. Activation of gene expression is accompanied by a growing number of cellular divisions (by 42.5%), which is the evidence of Hayflick's limit overcoming. This fact fully correlates with earlier stated maximum increase of animal life span (43,3%) after administration of this peptide.

The effect of peptides KE, EW, AEDG, AEDP on the expression of 15,247 murine heart and brain genes was studied with the employment of DNA-microarray technology. It was revealed that each peptide specifically regulates a particular group of genes. The result of this experiment strengthens the theory that peptides have a regulatory function for gene activity. It was also shown that the dipeptide KE, had an immunomodulating activity, regulating gene interleukin-2 expression in blood lymphocyte.

In the cell cultures of human bronchial epithelium, tetrapeptide AEDL activates the expression of genes of bronchial epithelium differentiation Nkx2.1, SCGB1A1, SCGB3A2, FoxA1, FoxA2. This peptide also increases expression of genes MUC4, MUC5АС, SftpA1, which reduces the frequency of chronic bronchitis. In the cell cultures of human pancreas tetrapeptide KEDW increased expression of differentiation genes PDX1, NGN3, PAX6, FOXA2, NKX2.2, NKX6.1, PAX4 and decreased expression of genes MNX1 HOXA3 Methylation profile of PDX1, PAX6, NGN3, NKX2-1, SCGB1A1 genes promoter regions in pancreas and bronchial epithelium cells changes with aging and under the influence of peptides which correlates with alterations in the levels of the genes expression. Methylation of gene promoter regions may be modulated under the influence of peptides. Promoter zone of gene FOXA2 in pancreas cells contains small number of methylated CpG-sites which methylation process changes with aging and is under the direct influence of peptide KEDW. The changes in the character of methylation of promoter zones may serve the reason for age-related and peptide induced alterations in the levels of PDX1, PAX6, NGN3 genes expression in pancreas cells and NKX2-1, SCGB1A genes expression in bronchial cells.

It was revealed that the tripeptide EDG regulates mRNA expression of various genes in the model of induced gastric ulcers in rats. Peptide EDG decreased synthesis of mRNA genes which encode cellular metabolism proteins SOD, TNFα, Cox-2.

Thus, specific (complementary) DNA-peptide interactions may have played an important role for the earliest stages of the origin of life and its evolution by epigenetic control of cell functioning.