Peptide Self-Regulation of Living Systems (Facts and Hypotheses) - Shataeva L. K. 2003

Interaction of peptides with cell nucleus chromatin
Intermolecular interactions of regulatory peptides and DNA

Protein-Nucleic Acid Interactions take place at all stages of METABOLISM/36.html">DNA Replication and expression, as well as during numerous processes of cellular autoregulation.

An Analysis of the polypeptide backbone Structure and its side groups has revealed four potential types of interactions between Peptides and Nucleic Acids (Claud, 1971; Hippel, McGhee, 1972; Nucleic acid-protein recognition, 1977):

1) Salt bridges between phosphate groups and positively charged side chains of amino acid residues (Lysine, the guanidine group of Arginine, and the protonated Histidine residue);

2) Hydrogen Bonds between phosphate groups, NUCLEOTIDES, carbohydrate moieties of nucleic acids, and the proton-donor and proton-acceptor groups of the peptide;

3) stacking interactions between the side groups of aromatic amino acid residues (Tryptophan, Tyrosine, phenylalanine, histidine) and nucleotides via a mechanism similar to intercalation;

4) hydrophobic interactions of 5-methylcytosine and thymine with the nonpolar side groups of peptides.

The energy of these four interaction types decreases in the order they are listed. However, when examining such systems, one must keep in mind that all these interactions occur simultaneously as a complex.

DNA exhibits high reactivity in binding to Polypeptides and Polyamines. Considerable attention has been focused on studying DNA complexation with protamines. These peptides lie along The surface of The Double Helix in such a way that the positive charges of adjacent side chains point in opposite directions, simultaneously binding to the phosphate groups of both strands of the DNA double helix (Zenger, 1987). Such interactions have been shown to drive the association of complexes into supramolecular structures, where the distribution of hydrophobic polypeptide groups along the chain plays a crucial role. Detailed crystallographic studies of The structure of these complexes have been carried out to model DNA-histone interactions.

Histones are evolutionarily conserved, single-stranded alkaline peptides rich in lysine and Proline residues that perform structural Functions in Chromatin. A DNA fragment of about 200 Base Pairs and an octamer composed of four types of histones (H2A, H2B, H3, H4) form a nucleosome, the fundamental structural unit of chromatin (McGhee, Felsenfeld, 1980). Nucleosomes resemble flat disks approximately 11 nm in diameter and 5.7 nm in thickness, positioned along DNA chains like “birds on a wire.” On average, 146 base pairs are wrapped in a superhelix around a core of eight histone molecules (the core particle), thereby protecting them from Transcription. Between adjacent nucleosomes lie 50–150 base pairs of linker DNA that remain accessible for transcription (Van Holde, 1988).

All other polypeptide and protein components of chromatin are generally referred to as non-histone Proteins. Their number reaches 500 and includes dozens of Enzymes, polymerases, repressors, and activators, alongside 15–20 polypeptides whose structures remain insufficiently characterized. Furthermore, components involved in maintaining the Spatial Structure of chromatin have been isolated from the nucleoplasm, including Proteins of the intranuclear fibrillar-granular network such as Actin, troponin, tubulin, DNA-relaxing proteins, and nucleoporins.

It is important to note the substantial methodological challenges involved in studying intermolecular interactions between DNA and peptides. These difficulties stem from a notable disparity in The Development of Methods for Investigating highly purified DNA versus its complexes with peptides. On the one hand, large DNA macromolecules can be easily visualized, oriented in an electric field, positioned within a groove on a Glass surface, and cleaved at specific sites by Nucleases. All these manipulations are performed under the control of atomic force and tunneling Cell/15.html">Microscopy, resembling molecular surgery (Yuqiu et al., 1992). On the other hand, developing methods to separate polypeptides from DNA while preserving their Native State is far from complete. Typically, Separation relies on extraction, complexation with polyethyleneimine, and fractional precipitation or reprecipitation of polypeptides (Burgess, 1991). Peptides specifically bound to DNA are exceptionally difficult to dissociate and frequently lose their

specific activity in the process. For instance, tissue Homeostasis is known to be regulated by a negative feedback mechanism involving chalones, which are produced in mature and differentiated Cells. These tissue-specific inhibitors of cell proliferation are isolated from DNA preparations through fractional alcohol precipitation (Baláž, Blažek, 1982). Like cytostatics, chalones could potentially be used as antitumor agents. However, the resulting preparations of highly purified chalones are unstable, which hinders not only their medical application but also their investigation in model systems (Romanov et al., 1984).

In principle, Transcription can be initiated by lifting the histone blockade (Dynlacht, 1997). Ubiquitin plays a significant role in this mechanism. It was observed long ago that the nuclear protein A24 consists of two covalently linked polypeptides: histone H2A and ubiquitin (Goldknopf et al., 1980). The removal of histone H2A from the nucleosome via ubiquitination (analogous to the internalization and removal of Membrane Receptors) and its transport to proteasomes serve as the initial step in preparing that specific DNA region for transcription (Boarends, 1999).

In addition to ubiquitin itself, an entire family of ubiquitin-like regulatory peptides has been discovered in chromatin. These peptides control DNA conformation at various Stages of Transcription and, by inhibiting topoisomerase, prevent DNA damage (Suzuki et al., 1999; Mao et al., 2000).

Chromatin also operates via a “receptor-mediated” transcriptional activation mechanism. The intrinsic chromatin receptor system consists of Nuclear Proteins firmly bound to DNA, whose conformation depends on the presence of a specific Ligand. They are referred to as ligand-dependent transcription factors. In the absence of a ligand, the nuclear receptor acts as a repressor. Upon binding its specific ligand, the receptor undergoes a conformational change and activates transcription (provided no other repressor is present). Such nuclear receptors have been identified and characterized for vitamin A, thyroxine, and Steroid Hormones (Horlein et al., 1995).

Histones and non-histone chromatin proteins regulate the Cell Cycle at every stage, from Cell Division to the end of a differentiated cell's lifespan. The normal

ratio between dividing and quiescent cells—that is, the mitotic equilibrium in continuously functioning Organs and Tissues—is maintained by tissue-specific transcription factors. Within The Scope of the present Discussion on autoregulation, we will focus exclusively on the mechanisms of peptide-mediated Transcriptional Regulation.

The transcription of each Gene is controlled by its regulatory region, located near the transcription start site in the 5'- to 3'-direction. This region consists of a segment of the DNA double helix and its complementary regulatory peptides. In other words, gene regulatory regions are nucleoprotein complexes (NPCs). NPCs comprise Two Types of proteins: repressors (which prevent gene transcription) and transcription factors, which switch transcription on (Jacob, Monod, 1961). These proteins control Gene Expression and the synthesis of proteins required by The Cell at a given stage of its existence without unwinding the DNA double helix. As it turns out, peptide transcription factors (TFs) do not need to penetrate the interior of the double helix, because the proton-donor, proton-acceptor, and hydrophobic groups of each base pair are exposed on its outer surface.

During The formation of the double helix, G–C and C–G base pairs form three internal hydrogen bonds, whereas A–T and T–A pairs form only two. At the same time, G–C and C–G pairs expose two proton-donor groups in the major groove, while A–T and T–A pairs additionally expose the hydrophobic CH₃ group of thymine. Methylated cytosine groups are of particular importance (Fig. 11). They occur at elevated concentrations in DNA regions commonly referred to as CpG islands (Antequera, Bird, 1993). These regions can be up to 1000 base pairs long (i.e., longer than exons and shorter than introns) and are located in regulatory regions near the 5' end of genes. Experiments have shown that methylated DNA binds peptides much more strongly (Bird, 1992). Apparently, the methyl group of 5-methylcytosine (mC) contributes both to the configuration of the characteristic recognition site and to the binding energy between the peptide transcription factor and DNA.

As we will see later, it is precisely the spatial arrangement (metrics) of Hydrogen bonds and hydrophobic groups On the surface of the major groove of the double helix that dictates the selective binding of regulatory peptides to DNA (Alberts et al., 1994).

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Fig. 12. Relative arrangement of Functional groups of complementary nucleotides in cross-section of the DNA double helix (A) and the combination of functional groups exposed on the surface along the major groove of the double helix for the CGGmCAT sequence (B).

Figure 12 shows diagrams of A–T, G–C, and G–mC nucleotide pair associations and the characteristic layout of functional groups along the major groove surface of the double helix for the coding strand sequence CGGmCAT. Figure 12B illustrates the fact that each nucleotide sequence generates a unique pattern (arrangement) of functional groups on the surface of the major groove. Notably, the functional groups exposed on the surface belong to nucleotides from both the coding and non-coding strands of DNA. The layout of these groups forms the basis for the specific intermolecular “recognition” of the DNA site by peptide TFs—that is, the regulatory peptides of chromatin.

Regulatory functions are primarily carried out by polypeptides responsible for local bending and twisting of DNA chains. They recognize specific sites by the characteristic distribution of hydrogen bonds and hydrophobic groups in the major groove of the double helix, establishing strong ion-ion interactions with DNA phosphate groups. All of this induces high torsional stress within the macromolecule, allowing rigid segments of the DNA chain to bend. It is precisely these local structural changes that switch DNA from a repressed state to an active state, i.e., readiness for transcription (Tang et al., 2000). The subsequent unwinding of the double helix required for transcription occurs only upon the interaction of DNA with high mobility group (HMG) peptides. These polypeptides possess a heterogeneous structure consisting of globular domains and extended regions rich in polyglutamic and polyaspartic acids (up to 22 amino acid residues). Unlike more alkaline peptides, they facilitate helix unwinding and the separation of double-stranded DNA (Van Holde, 1988). Evidently, the decisive factor in this intermolecular interaction is not merely the mutual electrostatic repulsion between the negatively charged DNA phosphates and the carboxyl groups of the polypeptides, but rather the formation of strong hydrogen bonds between the peptide carboxyl groups and the nitrogen atoms of the nucleic bases (Luisi et al., 1998).

Investigations into the structures of numerous TFs functioning in chromatin are far from complete. It was previously shown that specific binding sites for peptide TFs consist of short sequences on the DNA helix up to 20 base pairs long. This is the fundamental building block of the genetic “switch.” Hundreds of such sequences are known for various regulatory proteins (Mitchell, Tjian, 1989). For instance, the DNA region governing the Transcription of the secretory protein Sp-1 gene contains the sequence GGGCGG. The diagram presented in Fig. 12B makes it possible to construct a map of the functional group arrangement on the double helix surface for this sequence, as well as to deduce the likely Amino Acid Sequence of the peptide transcription factor complementary to this layout.

The high Specificity of the interaction between transcription factors and the DNA double helix observed in nature cannot be explained solely by simple complementary binding of a peptide within the major groove of DNA. The conformation and spatial orientation of the binding site, as well as the matching arrangement of hydrophilic, hydrophobic, and donor-acceptor regions in both macromolecules, are of critical importance. Experimental studies have demonstrated the existence of protein factors in chromatin that induce a conformational change in DNA, thereby facilitating the subsequent binding of regulatory peptides to promoter regions and the activation of gene transcription. These are the so-called chromatin architectural factors (Wolffe, 1994). Because the interaction between peptides and nucleic acids regulates the state of DNA within chromatin and the physiological condition of the cell, numerous studies have investigated The impact of such complexation on complex mobility (Katan-Khaykovich, Shaul, 1998), enzyme activity (Bell et al., 1997), and the physical characteristics of macromolecules, such as complex stability and shifts in the melting Temperature of the double-stranded chain (Cattau et al., 1969; Tanatani et al., 1998).

The most extensively studied Structural motifs of peptide transcription factors consist of “helix–turn–helix” and “helix–loop–helix” geometric sequences, in which the helical segments of the peptide chain are rotated relative to each other by 120°. The peptide chain typically adopts a right-handed helical shape, even when it lacks a true α-helical conformation. It is generally assumed that a portion of the peptide chain, likely a loop, fits snugly into the major groove of DNA (Harrison, 1991). The DNA double helix is also right-handed, allowing their respective surfaces to make tight, complementary contact when these macromolecules interact.

Another structural variant of the transcription factor binding site is the basic leucine zipper motif, which consists of two principal flanking sequences forming an α-Helix that selectively binds to the 5'ATTTGCAT3' nucleotide octamer. Obviously, the hydrophobic leucine groups interact directly with the hydrophobic CH₃ groups of thymidine in this process (Alberts et al., 1994; Leonard et al., 1997).

Another well-documented type of Peptide Structure responsible for recognizing specific DNA sites to initiate transcription comprises an α-helix and a β-sheet held together by a zinc ion and coordinated with a sequence of C–C and H–H amino acid residues. This structure binds to the GGG nucleotide sequence, with arginine and histidine residues actively participating in the binding process (Coleman, 1992).

However, current “recognition” models are largely variations of geometric surface matching at the DNA–peptide contact interface. To date, there is no clearly established correlation between The nucleotide sequence of the binding site and The amino acid sequence of the regulatory transcription factor peptide (Pabo, Sauer, 1992). One can only hypothesize that the modulation of cellular genetic activity by oligopeptides of the cytomedin group is driven by their site-specific binding to promoter regions of chromatin DNA (Morozov, Khavinson, 1985).

In addition to peptides associated with DNA promoter regions, there are known peptides that control post-transcriptional processes involving newly synthesized mRNA. Notably, their Amino acid sequences contain multiple repeats of the KH dipeptide block (up to 15 per molecule), although further research is still required to identify the exact specific sites on the mRNA chain responsible for binding these blocks (Adinolfi et al., 1999).

Over the past decade, significant progress has been made in understanding the molecular mechanisms that distinguish normal cell division from pathological proliferation. Across various cell cultures, a family of division factors has been discovered that act similarly to ubiquitin while also regulating telomere length (Tanaka et al., 1999). Telomeres are the terminal DNA structures of Chromosomes whose functioning relies on the ability of guanosine to form self-associates. As specialized DNA-polypeptide complexes, telomeres protect chromosomes from end-to-end fusions and the action of endonucleases. Some authors also suggest that telomeres may serve to recognize homologous chromosomes during Meiosis. Telomere length varies across different Stages of the CELL CYCLE AND among tissues, yet it shortens with every replication cycle, limiting cells to a finite number of divisions. At their termini, telomeres feature a protruding (unpaired) region of a G-rich DNA strand. Using this “tail” as a template, telomerase initiates the Synthesis of the second strand, thereby preserving chromosome length (Ilyicheva, Florentiev, 1992). It was originally hypothesized that telomerase, the DNA polymerase involved in telomere formation, could significantly extend cellular lifespan by increasing the number of normal divisions. Experiments in tissue culture demonstrated that introduced telomerase can maintain a constant telomere length, enabling cells to undergo unlimited yet non-malignant proliferation. Nevertheless, secondary effects — such as incomplete telomere replication and the inhibition of leading-strand DNA Repair — cast doubt on whether telomerase alone can fully solve The problem of cellular longevity (Egorov, 1999).

The normal Life Cycle of a cell concludes after its final division with apoptosis, which is an organized and programmed cell destruction. Alternatively, necrosis may occur, representing the pathological degradation and breakdown of cellular elements driven by external factors such as infection, poisoning, metabolic arrest, or mechanical injury.

Normal apoptosis proceeds as an orderly process governed by a genetic program. The primary regulator of mammalian apoptosis is the bcl-2 gene, whose active expression inhibits apoptotic progression (Khavinson, Kvetnoy, 2000). The apoptotic process begins within the Cell Nucleus with characteristic chromatin Condensation and fragmentation, followed by The breakdown of the nuclear envelope such that each chromatin fragment becomes enclosed in its own membrane shell. Throughout this phase, The Plasma Membrane remains intact, and no changes are observed in the Cytoplasm. Next, the segregation of the cytoplasm and intracellular Organelles begins, encapsulating portions of them into membrane vesicles until the cell ultimately disappears. This self-destruction occurs without the release of toxins and conserves cytoplasmic material for future reuse. Evidently, the remaining cellular vesicles can be engulfed via endocytosis by surrounding populations of cells, including daughter cells (Re et al., 1994). This sequence of events highlights the fundamental difference between apoptosis and necrosis. In necrosis, the Components of the membrane, nucleus, and cytoplasm undergo profound Hydrolysis, and enzymes released from Lysosomes exert a toxic effect on the microenvironment of the dying cell, necessitating the complete removal of necrotic debris from the Organism (Kutsiy et al., 1999). Necrosis can lead to the death not only of a single cell, but of an entire organ or organism. Age-related involution of key organs and tissues disrupts The regulation of physiological cell death, consequently increasing the proportion of necrotic cells (Khavinson, Kvetnoy, 2000). Currently, assessing the number of apoptotic cells is of significant importance in medical practice during Clinical and immunological evaluations (Yarilin et al., 2000).