Types and meanings of post-translational modification of proteins. Post-translational modification of proteins

Chemical modification of proteins is carried out with the help of acid or alkaline hydrolysis, stabilization of proteins by salt formation, acylation, and plastein reaction.

Alkaline and acid hydrolysis. These methods of protein modification are widely used to solubilize fish proteins in the process of obtaining fish protein concentrates, resulting in increased solubility, emulsification and foaming properties of proteins.

The depth of hydrolysis depends on the type and concentration of alkali or acid, the ratio of substrate and reagent, temperature, duration of treatment. To achieve a certain result, the process must be optimized for a specific protein of a specific raw material.

With the complete hydrolysis of proteins, a mixture of amino acids is formed. This is used in the latest technologies. The degree of hydrolysis can be controlled and adjusted. But it must be taken into account that along with the positive consequences of hydrolysis, there are also negative ones. For example, the formation of racemates of acids - peptides with a bitter taste.

One example of such a modification is the destruction of 11-S-globulin, which is characteristic of legumes, in particular soybeans, and has a globular molecule. Moreover, its quaternary structure is characterized by the fact that several subunits are combined into a globule using intermolecular bonds. Such structures are not capable of gelation, as well as imitation of meat-like systems. Controlled hydrolysis makes it possible to obtain a protein with the properties of a gelling agent, which is more typical for a number of fibrillar proteins, for example, gelatin.

This principle of modification of protein properties has found wide application in the technological process of obtaining structured products by the spinning method.

Similarly, the structure of a protein can be changed by heating. Protein breakdown into subunits, their partial destruction and aggregation lead to the formation of protein jelly. The stability of the resulting gels depends on the formation of disulfide bridges between the polypeptide chains.

Solubilization of proteins by salt formation. The possibility of such modification follows from the basic property of proteins as polymeric ampholytes capable of interacting with both cations and anions. Two types of interaction are possible: the formation of salt bridges and the specific sorption of ions on the protein surface. In this case, proteinates are formed, which are more soluble than native proteins.

The formation of proteinates is widely used in the isolation of proteins from soy (soy proteinates) and from milk (caseinate and sodium coprecipitate).

The most widely used modifier salts are sodium chloride and inorganic phosphates. Thus, by regulating the ability of meat formulations to retain water, sodium chloride, pyro- or sodium tripolyphosphate are used, which increase the solubility of myofibrillar proteins. At the same time, it is known that polyphosphates in relation to proteins are characterized by anti-denaturation, antiseptic, and antioxidant properties.

Every year the use of proteinates in the food industry and catering is expanding.

Acylation. Acylation with acetic or succinic anhydrides is one of the widely used methods of chemical modification of proteins. The result of this modification is a shift in the isoelectric point of the protein to a more acidic zone. Under the action of succinic anhydride, this process takes place to a greater extent. This makes it possible, even with a low degree of modification, to significantly improve such technological characteristics as solubility, emulsifying and foaming abilities.

The introduction of acyl residues (such as R-COO-) contributes to the unfolding and, ultimately, destruction of the protein globule, which leads to a change in the electrostatic equilibrium characteristic of the native protein due to the blocking of positively charged amino groups in globulins and an increase in the role of electrostatic repulsion of like-charged groups . The consequence of this is a change in protein conformation and its dissociation. At the same time, such technological effects as the ability to gel formation are achieved.

In practice, it has been proven that by acylation it is possible to obtain modified vegetable proteins with improved gel-forming ability, and this ability and the structural and mechanical characteristics of the resulting gels depend on the degree of acylation. So, at a very high degree, its excess of negative charges causes such a strong repulsion of polypeptide chains that the aggregation necessary for gel formation will be impossible. That is, the degree of acylation acts as an indicator of the functional properties of the protein, and acylation itself is a method of regulating these properties.

Acylated milk casein is used as an emulsifier and emulsion stabilizer, thickener for drinks, sauces, fruit and vegetable purees. Fish proteins are used as emulsifiers, binders, as substances that form jelly during heat treatment.

Enzymatic modification of proteins. Using enzymes, it is possible to purposefully change the structure of a protein in a variety of ways. Thanks to the partial hydrolysis of the protein, it is possible to provide an increase in solubility, emulsifying activity, foaming ability, stabilization of foams and emulsions. The specificity of enzymes allows you to influence only certain areas or groups of the protein molecule. It is also important that most enzymatic processes take place in an aquatic environment and, as a rule, under conditions close to physiological. However, not all enzymatic changes in proteins are important for food technology.

So, recently, partial hydrolysis of connective tissue proteins by proteases, meat tenderization has been used to improve its quality indicators. In fish proteins, under the action of enzymes of microbial origin amylosubtilin, protosubtilin, bromelain at pH 6.5-7.0 and a temperature of about 30 ° C, the emulsifying activity increases by 1.5 times, the solubility increases by 20%.

A special effect is achieved by combining the enzymatic process and chemical modification, for example, with succination. Thus, the products of enzymatic hydrolysis of fish proteins, which are characterized by a high foaming ability, lose their characteristic fishy taste as a result of succination, which allows them to be used in the production of confectionery products, ice cream, and drinks.

Very good prospects are given by the recently opened plasticine reaction- a process reverse to enzymatic cleavage, when peptide bonds are re-formed under the action of enzymes. Using this reaction, it is possible to create polypeptide chains with a molecular weight of about 3,000 Daltons from the products of protein hydrolysis. Due to the fact that individual amino acids, including essential ones, are able to react in the form of esters, they can be purposefully incorporated into polypeptides and proteins. By incorporating tryptophan, lysine, and methionine into maize zein, it was possible to obtain plastein with good biological value.

The biological value of soy proteins is low due to their low content of sulfur-containing amino acids. By partial hydrolysis of soy protein with pepsin, mixing it with the same wool keratin hydrolyzate containing many sulfur-containing amino acids, and subsequent plastein reaction under the action of nagarase (Bacilus subtilis protease), plastein with a nutritional value close to casein is obtained.

Plasteins obtained by incorporating glutamic acids obtained from soy proteins into proteins have very good properties. First, these glutamic acids are soluble at all pH values and resistant to thermal coagulation. Secondly, they have a pronounced taste of heat-treated meat.

The plastein reaction has great prospects for the extraction of undesirable amino acids from proteins. The latter include phenylalanine, the presence of which causes serious consequences in patients with phenyloketonuria. Partial enzymatic hydrolysis with pepsin, extraction of phenylalanine peptides by gel filtration, and subsequent plastein synthesis in the presence of ethyl esters of tyrosine and tryptophan under the action of papain plant protease leads to the production of phenylalanine-free plasteins, but balanced in other amino acids.

Physical and chemical methods of modification. Physicochemical methods of influencing protein systems combine the following methods: complex formation with natural polymers (proteins, polysaccharides, etc.), as well as with monomers (carbohydrates, fats), mechanical effects of various kinds, heat treatment, etc.

Complexation by type protein-protein interaction found practical application even earlier, but now there is a scientific interpretation of this phenomenon. So it was found that the joint drying of proteins of different nature - fish and cereals - not only leads to the production of valuable protein mixtures, but also preserves the functional properties of the original proteins. As grain additives to minced fish, wheat, rice or other flour can be used in an amount of 10% to 30%.

The addition of vegetable proteins to semi-finished meat products, due to complex formation, ensures a minimal decrease in water-holding capacity during heat treatment.

Conjugates of proteins and carbohydrates are characterized by high functional properties, which is traditionally used in technological processes. Thus, the ability of sucrose to increase the coagulation temperature of egg proteins is widely used in the technology of sweet dishes and confectionery. The ability of carbohydrates to stabilize animal proteins to the action of low and high temperature denaturation is known.

When fish proteins are dried together with monosaccharides, highly soluble complexes are formed, the solubility of which depends on the nature of sugars and their concentration in minced fish. In terms of the effect on the solubility of the resulting product, glucose is most effective, and sucrose and fructose are less effective. Similarly, glycerol and modified starch stabilize fish proteins. But it should be borne in mind that in this case, conditions are created for the Maillard reaction to occur, which will lead to a decrease in the nutritional value of proteins.

The addition of glucono-delta-lactone stabilizes minced meats.

There are also known methods of "strengthening" flour gluten in the formation of its complexes with acidic polysaccharides, such as pectin derivatives, as well as in the presence of microbial xanthan polysaccharide in the amount of 0.1-0.5%.

Increase the resistance of proteins to denaturation and lipids, which are also able to form complexes with the former. The nature of this phenomenon has not been sufficiently clarified, but nevertheless it is used in the production of minced sausages, the semi-finished products of which are protein-fat emulsions.

Physical methods of exposure also play a role in modifying the properties of protein substances. Thus, the intensity, method and degree of grinding are key ceteris paribus in shaping the quality of wheat flour. By setting certain temperature conditions, they regulate the water-holding capacity, tenderness, juiciness of meat systems. The temperature and duration of processing regulate the quality indicators of milk curd. Simultaneous mechanical mixing of the mass leads to the formation of "casein grain", which differs significantly in organoleptic characteristics from curd obtained by thermal acid coagulation, but without mixing.

A high degree of minced meat and fish grinding, especially in colloid mills, leads to mechanical degradation of myofibril sarcomeres, resulting in an increase in water-holding capacity and protein solubility.

Partial thermal coagulation of fish proteins or brewing flour, resulting in denaturation of gluten proteins, changes cohesion, allows you to adjust the ability to form and organoleptic properties of systems.

PROTEIN MODIFICATION

(from late Latin modificatio-change) biogenic, occurs after completion broadcasts matrix ribonucleic acid, or mRNA, (protein synthesis on an mRNA template) or until it is completed. In the first case, M. b. called post t and n s l a t i n o n y, in the second - to o t r a n c l a t ion n o y. It is carried out thanks to districts decomp. funkt. groups of amino acid residues, as well as peptide bonds and determines the final form of the protein molecule, its fiziol. , stability, movement within the cell.

Extracellular (secreted), as well as many others. cytoplasmic proteins. membranes and intracellular compartments (isolated parts of the cell) undergo glycosylation, as a result of which glycoproteins. Naib. mannose-containing chains attached to polypeptides by an N-glycosidic bond are complexly organized. The initial stage of the formation of such chains proceeds co-translationally according to the scheme:

Dol-dolichol (polyprenol), DolHRChR-dolichol pyrophosphate, Glc-glucose, GlcNAc-N-acetyl-D-glucosamine, Man-mannose

Afterbirth. stages are carried out post-translationally with the participation of several. enzymes located in different subcellular compartments. So, for the G-protein of the vesicular stomatitis virus, glycosidic chains to-rogo are built from 15 carbohydrate residues, such a sequence of events has been established. First, in the endoplasmic reticulum occurs in two stages, the separation of terminal glucose residues with the participation of two different glucosidases. Then, mannosidases (I and II) remove 6 mannose residues, and N-acetyl-D-glucosamine transferase adds three GlcNAc residues to the glycoprotein mannose residues. Finally, in the Golgi complex, the residues of fucose, galactose, and sialic acid bind to these residues with the participation of the corresponding transferases. Monosaccharide residues can undergo phosphorylation, sulfonation, and other modifications.

Glycosylation of secreted proteins is preceded by proteolytic. processing - separation from the N-terminus of the polypeptide chain "signal" amino acid sequence. In eukaryotic cells (cells of all organisms, with the exception of bacteria and blue-green algae), this process is carried out by translation, in prokaryotes. cells (cells of bacteria and blue-green algae) it can proceed post-translationally. Naib. common signal sequences include 23 amino acid residues. The characteristic features of these sequences are the presence at the end of a short positively charged section, followed by a hydrophobic section containing from 7 to 14 amino acid residues. The signal sequences end with a hydrophilic region, conservative in length (5-7 residues), at the C-terminus of which most often there are residues of alanine, glycine, serine, threonine, cysteine or glutamine.

Almost all functions. classes of extracellular proteins (, hormones, etc.) contain disulfide bonds. They are formed from the cystene SH groups in a multi-step process involving the enzyme disulfide isomerase. Mean appears in its early stages. number of "wrong" disulfide bridges, to-rye are eliminated as a result of thiol-disulfide exchange, in Krom, apparently, cystamine (H 2 NCH 2 CH 2 S) 2 is involved. It is assumed that such a "enumeration" of connections occurs until the most. stable tertiary structure, in which the disulfide bridges are "buried" and therefore inaccessible to reagents.

To the max. common modifications of intracellular proteins include phosphorylation and dephosphorylation in the OH group of serine, tyrosine and threonine residues, which are carried out with the participation of protein kinase and phosphatase enzymes according to the scheme:

ATP - adenosine triphosphate, ADP - adenosine diphosphate, P - phosphoric acid or its residue

Phosphorylation is accompanied by activation or inactivation of enzymes, for example. glycosyltransferases, and also change fiz.-chem. St. in non-enzymatic proteins. Reversible proteins control, for example, such important processes as translation, lipids, gluconeogenesis, and muscle contraction.

Proteins of mitochondria and chloroplasts encoded by nuclear DNA have excess amino acid sequences at the N-terminus, to-rye selectively direct polypeptide chains to certain compartments of organelles, after which they are cleaved off as a result of proteolysis with specific participation. endopeptidases. The excess sequences of precursors of mitochondrial proteins differ significantly in the number of amino acid residues; they can be from 22 to 80. Short sequences are characterized by a high (20-25%) content of positively charged amino acid residues evenly spaced along the polypeptide chain. Long sequences additionally include a section consisting of hydrophobic amino acids, to-ry "anchoring" the precursor in the lipid bilayer of mitochondrial membranes.

Precursors for a number of hormones are known (eg, for gastrin, glucagon and insulin), to-rye pass into an active form by means of splitting of a polypeptide chain in the sites containing two consecutively located remains of the main amino acids (and lysine). Cleavage is carried out with the participation of specific. endopeptidase acting in ensemble with a second enzyme having carboxypeptidase. The latter removes the residues of the terminal basic amino acids, completing the transformation. peptide into an active hormone. To proteins undergoing proteolytic. activation, also include proteinases (, trypsin,), procollagen, proteins of the blood coagulation system, etc. In some cases, inactive forms of enzymes (zymogens) are necessary for temporary "preservation" of enzymes. So, the zymogens trypsin and chymotrypsin (respectively, trypsinogen and chymotrypsinogen) are synthesized in the pancreas, secreted into the small intestine, and only there under the action of specific. enzymes convert. into active form.

A wide range of proteins (myosin, actin, ribosomal proteins, etc.) are methylated post-translationally at lysine, arginine and histidine residues (N-methylation), as well as at glutamic and aspartic acid residues (O-methylation). Usually acts as a methylating agent S-adenosylmethionine.

In some eukaryotic In cells, more than half of p-rimy proteins are acetylated at the N-terminus. This process can be carried out co- and post-translationally (indicated in the diagram respectively. K. T. and P. T.), for example:

HSCoA-coenzyme A, AcCoA - acetyl coenzyme A, Met-methionine, Asp - aspartic acid

For peptides containing from 3 to 64 amino acid residues and secreted in decomp. organs (, secretin, etc.), found post-translation. amidation of the C-terminal residue (with the exception of the terminal residues of arginine and asparagine).

Nek-ry types of modifications are characteristic of separate proteins or small groups of proteins. In particular, in collagen and several. other proteins with similar amino acid sequences have been found to contain 4- and 3-hydroxyproline, as well as 5-hydroxylysine. Hydroxylation of proline and lysine residues proceeds co-translationally and is important for the formation of a unique collagen structure. Hydroxylysine is involved in the formation of covalent crosslinks between the polypeptide chains of collagen according to the scheme:

Nuclear proteins (histones, non-histone proteins) undergo adenosine diphosphate ribosylation and polyadenosine diphosphate ribosylation, during which adenosine diphosphate-tribosyl residues are transferred from the coenzyme nicotinamide adenine dinucleotide (NAD) to acceptor proteins:

These two districts are different in many ways. aspects. In particular, polyadenosine diphosphate ribosylation proceeds in the presence of a day. DNA. Most adenosine diphosphate ribosyl groups are attached to proteins via an ether bond formed by the OH group in position 5 "of the ribose residue and the COOH group of the C-terminal amino acid or glutamic acid, located inside the polypeptide chain.

Of great importance is the residue of glutamine to - you with the formation of g-carboxyglutamine to - you in the precursor of prothrombin. This district is catalyzed by a K-dependent carboxylase localized in endoplasmic membranes. reticulum. A similar district proceeds during the maturation of certain other coagulation factors.

Lit.: Fundamentals of biochemistry, trans. from English, vol. 1, M., 1981, p. 277-80; General, trans. from English, vol. 10, M., 1986, p. 543-70; The enzymology of post-translational modification of proteins, v. 1, L.-N. Y., 1980; The biochemistry of glycoproteins and proteoglycans, N. Y.-L., 1980; cell biology. A comprehensive treatise, v. 4-Translation and the behavior of proteins, N. Y., 1980; Methods in enzymology, v. 106, N.Y., 1984; Hurt E.G., Loon A.P.G.M. van, "Trends in Biochem. Sci.", 1986, v. 11, no. 5. n. 204-07. V. N. Luzikov.

Chemical encyclopedia. - M.: Soviet Encyclopedia. Ed. I. L. Knunyants. 1988 .

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At this stage, the formation of the tertiary structure and processing of the polypeptide molecule takes place.

A polypeptide protein molecule synthesized on a ribosome carries information and is called conformational , i.e. she undergoes transformation processing ) into a strictly defined three-dimensional body that already carries functional information.

This is true for proteins with a structural function, but not for biologically inactive protein precursor molecules. Their functional activity is manifested as a result of transformations called postsynthetic or post-translational modification . In the process of synthesis, 20 amino acids can be included in its composition. After translation, post-translational modification expands the functional composition of the protein:

Cleavage of the N-terminus of formylmethionine or methionine;

Cleavage of signal peptides;

Attachment of a prosthetic group;

Phosphorylation of histones and non-histone chromatin proteins;

Methylation of lysine and arginine radicals;

Attachment of oligosaccharide fragments to the radicals of asparagine, serine;

The choice of the correct protein structure occurs with the participation of proteins - chaperones . Hydrophobic regions on the surface of the chaperone-70 globule interact with hydrophobic regions of the synthesized chain, protecting it from incorrect interactions with other cytosolic proteins. Chaperones-60 are involved in correcting the spatial structure of an incorrectly folded or damaged chain.

Transport of synthesized proteins across membranes

In eukaryotes, mRNA is produced in the nucleus and enters the ribosome located in the cytosol of the cell. The synthesized protein moves from the ribosome to the cytosol. If it is not used for the needs of the cell itself, i.e. refers to exported (secreted) proteins , then it is transported through the cell membrane with the help of low molecular weight peptides (15-30 amino acid residues) containing hydrophobic radicals. it leading or signal peptides . Signal peptide sequences are formed in ribosomes from the N-terminus during protein synthesis by signal codons located immediately after the initiator one and are recognized by receptor sites of the endoplasmic reticulum. A channel is formed in the membrane through which the signal peptide penetrates into the cistern of the endoplasmic reticulum and drags the synthesized protein molecule along with it. Under the influence signal peptidase The N-terminal signal sequence is cleaved off, and the protein exits the cell through the Golgi apparatus in the form of a secretory vesicle.

Regulation of protein synthesis

The concentration of many proteins in a cell is not constant and changes depending on the state of the cell and external conditions. This occurs as a result of the regulation of the rates of protein synthesis and degradation.

Genes- DNA segments encoding the synthesis of tRNA, rRNA, mRNA.

The genes that code for mRNA synthesis are called protein genes .

Transcriptional regulation(formation of the primary transcript) is the most common mechanism for the regulation of protein synthesis.

Distinguish two forms of regulation – fusion induction (positive regulation) and synthesis repression (negative regulation).

Concepts of induction and repression suggest a change in the rate of protein synthesis with respect to initial (basal) level .

Synthesis in the basal state - constitutive synthesis .

If the rate of constitutive protein synthesis is high, then the protein is regulated by the mechanism of synthesis repression, and, conversely, at a low basal rate, synthesis is induced.

In the genetic apparatus of the cell there are operons - DNA segments containing structural genes of certain proteins and regulatory regions.

The reading of the genetic code begins with a promoter located next to the operator gene.

Operator gene located on the extreme segment of the structural gene. It either prohibits or permits the replication of mRNA to DNA.

At eukaryote positive regulatory mechanisms prevail. The main regulatory point is the stage of transcription initiation. Regulatory elements that stimulate transcription are called enhancers , and suppressing it - sealers (silencers) . They can selectively associate with regulatory proteins : enhancers - with inducer proteins , silencers - with repressor proteins .

Repressor protein communicates between the operon and the regulator gene. The repressor is formed in the ribosomes of the nucleus on mRNA synthesized on the regulator gene. It forms a complex with the operator gene and blocks the synthesis of mRNA, and, consequently, protein. The repressor can bind to low molecular weight substances - inductors or effectors. After that, it loses its ability to bind to the operator gene, the operator gene gets out of control of the regulator gene, and mRNA synthesis begins.

in mammalian cells exist two types of regulation of protein biosynthesis :

- short-term , providing adaptation of the body to environmental changes;

- long lasting, stable , which determines the differentiation of cells and the different protein composition of organs and tissues.

(from late Latin modificatio-change) biogenic, occurs after completion broadcasts matrix ribonucleic acid, or mRNA, (protein synthesis on an mRNA template) or until it is completed. In the first case, M.b. called post t and n s l a t i n o n y, in the second - to o t r a n c l a t ion n o y. It is carried out due to reactions decomp. functional groups of acid residues, as well as peptide bonds, and determines the final form of the protein molecule, its physiological activity, stability, and movement within the cell.

Extracellular (secreted) proteins, as well as many others. cytoplasmic proteins. membranes and intracellular compartments (isolated parts of the cell) undergo glycosylation, as a result of which glycoproteins. Naib. mannose-containing chains attached to polypeptides by an N-glycosidic bond are complexly organized. The initial stage of the formation of such chains proceeds co-translationally according to the scheme:

Dol-dolichol (polyprenol), Dol-P-P-dolichol pyrophosphate, Glc-glucose, GlcNAc-N-acetyl-D-glucosamine, Man-mannose

Almost all functions. classes of extracellular proteins (enzymes, hormones, immunoglobulins, etc.) contain disulfide bonds. They are formed from the cystene SH groups in a multi-step process involving the enzyme disulfide isomerase. Mean appears in its early stages. number of "wrong" disulfide bridges, to-rye are eliminated as a result of thiol-disulfide exchange, in Krom, apparently, cystamine (H 2 NCH 2 CH 2 S) 2 is involved. It is assumed that such a "enumeration" of connections occurs until the most. stable tertiary structure, in which the disulfide bridges are "buried" and therefore inaccessible to reagents.

ATP - adenosine triphosphate, ADP - adenosine diphosphate, P - phosphoric acid or its residue

Phosphorylation is accompanied by activation or inactivation of enzymes, for example. glycosyltransferases, as well as a change in the physicochemical properties of non-enzymatic proteins. Reversible protein phosphorylation controls, for example, important processes such as transcription and translation, lipid metabolism, gluconeogenesis, and muscle contraction.

Precursors for a number of hormones are known (eg, for gastrin, glucagon and insulin), to-rye pass into an active form by means of splitting of a polypeptide chain in the sites containing two consecutively located remains of the main amino acids (arginine and lysine). Cleavage is carried out with the participation of specific. endopeptidase acting in ensemble with a second enzyme having carboxypeptidase activity. The latter removes the residues of the terminal basic amino acids, completing the transformation. peptide into an active hormone. To proteins undergoing proteolytic. activation, also include proteinases (pepsin, trypsin, chymotrypsin), albumins, procollagen, proteins of the blood coagulation system, etc. In some cases, inactive forms of enzymes (zymogens) are necessary for temporary "preservation" of enzymes. So, the zymogens trypsin and chymotrypsin (respectively, trypsinogen and chymotrypsinogen) are synthesized in the pancreas, secreted into the small intestine, and only there under the action of specific. enzymes convert. into active form.

A wide range of proteins (histones, myosin, actin, ribosomal proteins, etc.) are methylated post-translationally at lysine, arginine and histidine residues (N-methylation), as well as at glutamic and aspartic acid residues (O-methylation) . Usually acts as a methylating agent S-adenosylmethionine.

HSCoA-coenzyme A, AcCoA - acetyl coenzyme A, Met-methionine, Asp - aspartic acid

For peptides containing from 3 to 64 amino acid residues and secreted in decomp. organs (gastrin, secretin, cholecystokinin, etc.), posttranslations were found. amidation of the C-terminal amino acid residue (with the exception of the terminal residues of arginine and asparagine).

Of great importance is the carboxylation of glutamine residues to - you with the formation of g-carboxyglutamine to - you in the precursor of prothrombin. This reaction is catalyzed by a vitamin K-dependent carboxylase localized in endoplasmic membranes. reticulum. A similar reaction occurs during the maturation of certain other coagulation factors.

Lit.: Fundamentals of biochemistry, trans. from English, vol. 1, M., 1981, p. 277-80; General organic chemistry, trans. from English, vol. 10, M., 1986, p. 543-70; The enzymology of post-translational modification of proteins, v. 1, L.-N. Y., 1980; The biochemistry of glycoproteins and proteoglycans, N. Y.-L., 1980; cell biology. A comprehensive treatise, v. 4-Translation and the behavior of proteins, N. Y., 1980; Methods in enzymology, v. 106, N.Y., 1984; Hurt E.G., Loon A.P.G.M. van, "Trends in Biochem. Sci.", 1986, v. 11, no. 5. n. 204-07. V. N. Luzikov.

A monograph by a well-known Indian specialist in the field of gerontology, dedicated to the changes that occur with aging in the structure and functions of chromatin, enzyme activity, collagen structure and synthesis, and the activity of the immune and endocrine systems. Cell aging and modern theories of aging are also considered.

It is intended for biologists, biochemists, gerontologists, geriatricians.

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Post-translational covalent modification occurs at the side groups of amino acid residues of several proteins. Chromosomal proteins, both histones and NGPs, are synthesized in the cytoplasm and then transferred to the nucleus, where they bind to DNA. These proteins, especially histones, undergo a variety of post-translational covalent modifications: phosphorylation, acetylation, methylation, and ADPribosylation. Acetylation of the NH 2 -terminal serine residue of histones H1, H2A and H4 occurs during translation and is a stable modification. Acetylation of internal lysine residues of H3 and H4 histones and phosphorylation of internal serine residues occur in the cytoplasm. These histones then pass into the nuclei and bind to DNA. Acetylation of internal lysines is reversible. In addition, reversible modification of lysine residues occurs after histone binding to DNA. Through covalent modifications of four types, the ionic composition of histones and their steric properties, and hence the interaction with DNA, change (Fig. 2.4).

Rice. 2.4. The structure of chromatin, indicating the binding sites for histones and NGPs with DNA. Covalent modifications of histones are presented, as a result of which their binding to DNA changes

With modifications such as phosphorylation and ADPribosylation, the number of negative charges on histones increases, and this can lead to their separation from DNA, allowing its transcription or replication. When acetylated, the total positive charge on the histones decreases. This can also lead to their separation from DNA. At the same time, during methylation, the positive charge on histone molecules can increase, which leads to their stronger binding to DNA and, as a result, to the suppression of gene activity. Specific amino acids are subject to specific modifications. Certain modifications predominantly occur in certain histones and also at specific phases of the cell cycle and cell growth. Thus, it is possible that modifications of the side groups of chromosomal proteins are the mechanism of fine regulation of gene expression. In table. 2.3 shows some characteristics of these modifications.

Table 2.3.Parameters of covalent modifications of histone chains

Phosphorylation

Phosphorylation of chromosomal proteins is an energy-dependent postsynthetic modification. It occurs both in the cytoplasm and in the nucleus. Ord and Stocken demonstrated 32P incorporation into histones in vivo. More recently, histone H1 was shown to be more phosphorylated than other histones. The main centers of phosphorylation by specific AMP-dependent protein kinases are the side groups of serine and threonine residues of histones and NGBs. Lysine, histidine and arginine residues are phosphorylated to a small extent. Kinases are present in the NGB fraction of chromatin. Specific histone kinases seem to be involved in the phosphorylation of specific centers. From the bovine thymus chromatin, it was possible to isolate an AMP-dependent kinase that phosphorylates a single center in the H3 histone. Dephosphorylation of these residues is carried out by phosphatases, which are also present in the NGB fraction. It has been reliably established that phosphorylation and dephosphorylation of enzymes are one of the main mechanisms for regulating their activity, since these modifications, causing conformational changes, transfer enzymes from an active state to an inactive one, and vice versa. With such modifications, structural changes occur in the molecules of chromosomal proteins, which can lead to functional changes in chromatin. The histone phosphorylation-dephosphorylation reaction is shown below:

Two types of addition of phosphate groups were found in the molecules of chromosomal proteins. One of them, including the P-O bond, is characteristic of serine and threonine residues, and this bond is acid-resistant. The second type includes the P-N bond, which is formed in lysine, histidine and arginine residues. This bond is unstable in acidic media.

Histone phosphorylation

The process of phosphorylation - dephosphorylation of internal residues of chromosomal proteins occurs at a high speed. Rapid phosphorylation is observed not only in dividing, but also in non-dividing cells after their stimulation by various effectors. Histone H1 is mainly subject to phosphorylation, i.e., histone phosphorylation, apparently, does not affect the transcriptional activity of chromatin.

Histone H1 phosphorylation centers are different at different stages of the cell cycle. Ser-37 is phosphorylated in the G 1 phase, Ser-114 in the S and G 2 phases, Ser-180 in the M phase. Apparently, this is due to the multiplicity of H1 kinases, each of which is specific. Fast growing cells have been shown to contain a specific histone kinase that catalyzes the phosphorylation of threonine residues, but not Ser-37 and Ser-105. When phosphorylation of one of the Ser-37 and Ser-105 residues or both, the degree of binding of histone H1 to DNA is significantly reduced. Such differences in the properties of different phosphorylation centers can explain the functional role of histone H1 in chromatin condensation. When different sites are phosphorylated, chromatin decondenses in different ways, which opens up different sections of DNA.

It has been shown that histone phosphorylation is associated with changes in chromatin structure, especially during mitosis. A high rate of phosphorylation is observed during mitosis of Chinese hamster ovary cells. The same modifications are observed in HeLa cells. The centers of histone H1 phosphorylation during mitosis (Him) differ from the centers during interphase (NI). It has been proposed that histone H1 phosphorylation is necessary for the condensation of interphase chromatin into chromosomes. This is consistent with data showing that triple phosphorylated histone H1 binds to DNA more strongly than dephosphorylated one. In the slimy fungus Prysarum polycephalum phosphorylation of histone H1 increases in the middle of the G 2 phase and increases sharply up to prophase. Dephosphorylation occurs in the last stage of mitosis.

Phosphorylation of various centers is also observed in histone H3, but it is not found in histones H2A, H2B, and H4. In detailed studies of the phosphorylation of H1 and H3 histones from the Chinese hamster ovary during mitosis, it was shown that in most eukaryotic cells, 2–4 centers in H1 histone are phosphorylated in the S phase and additional centers during mitosis (M) . Phosphorylation centers in the S and M phases appear to be independent. Moreover, these centers are different from those involved in the response to the action of the hormone. In the early stages of preprophase, when chromatin aggregation begins, histone H1 has 1–3 phosphate groups per molecule, and histone H3 is not phosphorylated. During prometa- and anaphase, when chromatin aggregates, all H1 histone molecules as well as H3 are superphosphorylated and have 3–6 phosphate groups per molecule. This may be due to a 6-10-fold increase in the content of specific ATP-histone phosphotransferase in mitotic cells. Due to superphosphorylation, chromatin fibrils are able to twist into supercoils. In telophase, when chromatin is disaggregated, both histones, H1 and H3, are dephosphorylated. When cells enter the G 1 phase, histone H1 is completely dephosphorylated. Thus, H1m histone superphosphorylation and H3 histone phosphorylation are mitotic events that occur only when the chromosomes are fully condensed. Therefore, chromatin condensation during mitosis requires a high degree of phosphorylation of H1 and H3 histones, while dephosphorylation limits this process in interphase. Surprising, however, is the fact that at a high degree of phosphorylation, when it would seem that the complex of histones H1 and H3 should dissociate from DNA due to an increase in negative charges on them, an increase in condensation is observed. Further research is needed to elucidate the fundamental mechanism by which chromosome condensation and decondensation occur. In the developmental rat liver, histone H1 phosphorylation is significant, but it is negligible in the liver of adult animals. However, phosphorylation is increased when liver cells divide after partial hepatectomy. It was shown that under these conditions the ratio of the number of P-O to P-N bonds in the liver changes. P-N bonds are found mainly in the histones H1 and H4. The content of Hl-kinase does not change during the cell cycle, but the amount of H4-kinase increases during DNA synthesis. Histone H4 is also maximally phosphorylated in the S phase; let's assume that histidine residues are the centers of attack. Thus, as a result of phosphorylation, histone H4 can be separated from DNA, which promotes its replication. From this, one can apparently conclude that histone phosphorylation is necessary for DNA replication, followed by cell division. It has been shown that transcription of isolated rat liver nucleosomes is enhanced after phosphorylation. Phosphorylated and non-phosphorylated H1 histones differ from each other in their ability to suppress chromatin matrix activity. Phosphorylated histones were found to have an altered conformation using the circular dichroism method. This may explain the fact that modified histones cause chromatin derepression.

Histone H5 is also subject to phosphorylation. In avian erythrocytes, histone H5 is phosphorylated shortly after its synthesis and then dephosphorylated as the cell matures. Unlike other histones, histone H5 is dephosphorylated during the period of genome inactivation and chromosome condensation. Phosphorylated histone H5 is not as effective in inducing a change in DNA conformation as its dephosphorylated form. Phosphorylated residues (series) are found in areas of the H5 histone that are strongly basic and associated with DNA. 50% of the phosphates are in the 1-28 region, and the rest are in the 100-200 region. During the process of spermatogenesis in some mammalian species, protamines undergo phosphorylation–dephosphorylation, which appears to be necessary for DNA packaging.

Phosphorylation of NGB

NGBs are highly phosphorylated and contain both P-O and P-N bonds. Their phosphorylation is believed to be catalyzed by kinases other than those that catalyze histone phosphorylation. Essential for phosphorylation is the content of protein kinases and cAMP. Calcitonin stimulates phosphorylation of bone cell NHPs in culture, especially low molecular weight proteins (10,000-45,000), but inhibits phosphorylation of high molecular weight NHPs. At the same time, the parathyroid hormone stimulates the phosphorylation of NGP with a large molecular weight. Thus, two peptide hormones that affect calcium metabolism in opposite ways can realize their action with the help of phosphorylated NHPs. Steroid hormones also induce NHP phosphorylation.

Phosphorylation of NGBs depends on the type of cells and their physiological state. The rate of this process varies in synchronized HeLa cells in vitro, with the highest rate observed in the G1 and S phases. When resting LC cells begin to proliferate rapidly, one of the earliest events is phosphorylation of NHPs. When polyamines, spermine and spermidine are added to the nuclei of rat liver cells, the activity of nuclear protein kinase increases by 2–3 times, and the rate of phosphorylation of NGB is many times higher. At Physarlum polyamines stimulate the phosphorylation of several unique NGPs.

Unlike phosphorylated histones, which influence chromatin structure, phosphorylated NHPs are involved in gene expression. Phosphorylated NHPs of HeLa cells in the G1 phase stimulate the transcription of histone genes in the G1 phase, although these genes are inactive in this phase. Phosphorylated NGPs increase transcription, while dephosphorylated ones decrease it. The mechanism of this effect probably lies in the direct interaction of proteins with DNA. This conclusion follows from the following observations: NGBs have different phosphorylation abilities, the way they are phosphorylated depends on the type of tissue; with changes in their phosphorylation, the structure of chromatin and the activity of genes change, and phosphorylated NGPs specifically bind to DNA. NGB of breast carcinoma cells in the stage of hormone-dependent growth and during regression are phosphorylated differently. When human W1-38 fibroblast chromatin is remodeled from DNA and unphosphorylated or phosphorylated NGPs, the transcription rate is much higher in the latter case.

Acetylation

There is a report of the presence of acetyl groups in histones. Histone acetylation in isolated nuclei was first described by Allfrey et al. Two types of acetylated amino acid residues were found in histones: a) the NH2-terminal series of histones H1, H2A and H4 is acetylated to N-acetylserine; it is an irreversible postsynthetic modification catalyzed by an enzyme contained in the cytosol; b) acetylated internal lysine residues are formed as a result of a postsynthetic reaction that occurs in the cytosol and in the nucleus after the histones move from the cytosol to the nucleus and bind to DNA. Acetylation of internal residues in histone H1 is either insignificant or absent. One center is acetylated in the histone H2A and four centers in each of the histones H2B, H3 and H4. Acetylation of lysine residues is catalyzed by acetyltransferase, which is a component of NGB. ?-NH 2 -groups of internal lysine residues located at the NH 2 ends of half of the histones are acetylated to form ?-N-acetyllisine, and one molecule can contain up to four acetyl groups. This is an energy dependent reaction in which the source of the acetyl group is acetyl-CoA. Deacetylation is catalyzed by deacetylase, which is also present in chromatin. The reaction scheme is shown below:

Internal lysine residues 9, 14, 18, and 23 of histone H3 and 5, 8, 12, and 16 of histone H4 are acetylated. These residues are located in the NH 2 -terminal region of the polypeptide chain, which is strongly basic and interacts with the acidic groups of DNA. Acetylated histones bind to DNA less efficiently than deacetylated ones. Acetylation of internal lysine residues is a reversible process and occurs very quickly under a variety of conditions. The half-life of the acetylation reaction is very short, only about 3 min. Two histone acetyltransferases have been isolated having different pH optima. Acetylation is inhibited by Mn 2+ ions. This fact seems to be important, since divalent cations are necessary for the functioning of DNA-dependent RNA polymerase. cAMP, which affects phosphorylation, does not affect acetylation. The maximum rate of acetylation is reached in the interphase; as cells enter mitosis, it decreases. The minimum reaction rate is observed in prophase and metaphase, when the chromosomes are most condensed. As cells enter telophase and chromosomes increase in size, the rate of histone H4 acetylation increases. Minimal RNA synthesis is observed in prophase and metaphase, when the chromosomes are highly condensed. It is important that H4 histone acetylation is also minimal precisely in these two phases of the cell cycle. Acetylation of histones H3 and H4 in avian erythroblasts decreases as they develop into mature erythrocytes, in which chromatin is highly condensed and inactive in either transcription or replication. Thus, histone deacetylation correlates with transcription inhibition, and vice versa, acetylation stimulates transcription. Since nucleosomal histones are less positively charged when acetylated, they can be separated from DNA, making the DNA available for transcription.

If chromatin is deproteinized, then its transcription is enhanced. It has been shown that when RNA synthesis is stimulated in lymphocytes by mitogens, in target tissues by hormones, and in the liver, histone acetylation occurs first after partial hepatectomy. As a result of acetylation of nucleosomal histones, transcription of calf thymus chromatin is enhanced. If histones H2A and H2B are added to DNA lacking chromatin, then transcription is suppressed. However, if these two histones are then acetylated, then the repression stops. Acetylation of H3 and H4 histones also stimulates transcription. Acetylation has been shown to precede an increase in RNA synthesis. Histone acetylation occurs not only in dividing, but also in non-dividing cells, in which a significant part of the chromatin is inactive with respect to transcription. Histone acetylation stimulates chain elongation during transcription. It is possible that the transcription of genes that are specifically “turned on” by effectors is controlled by the degree of histone and/or NGP acetylation.

The above conclusions are confirmed by the following facts. Transcriptionally inactive ciliate heterochromatin has a low degree of acetylation, while euchromatin is transcriptionally active and highly acetylated. Transcriptionally active macronuclei Tetrahymena pyriformis contain acetylated histones, while repressed micronuclei do not. The active maternal chromosomes of the mealybug contain significantly more acetyl groups than the inactive paternal chromosomes. In studies performed on cells Drosophila in culture, it was shown that 14 C-acetate is included mainly in the histones H3, H4 and H2B, and 32 P-phosphate is included in the histones H1, H3 and H4. The highest content of both 14 C-acetate and 32 P is observed in histone H3. When matrix active and matrix inactive regions of chromatin were separated after digestion with DNase II, the former were found to contain more of both marks (14 C and 32 P). These data support the suggestion that differences between transcribed and non-transcribed chromatin regions are partly due to specific histone modifications at certain loci.

The degree of histone acetylation from trout testis cells was studied by incubating them with 14 C-acetate. Histones H2A, H2B and H3 are acetylated in one position, while histone H4 is acetylated in one, two, three and four positions. When the nucleosomes obtained after acetylation were treated with trypsin, the NH 2 -terminal regions of four histones containing lysine residues and associated with DNA were removed. These lysine residues were just acetylated. If the nucleosomes are then cleaved with nuclease, then DNA fragments are released, usually in the presence of nuclease-resistant NH 2 -terminal regions. Acetylation leads to an increase in the rate of chromatin cleavage by DNase I. Chromatin containing highly acetylated histones is more readily cleaved by DNase I, but not by micrococcal nuclease. When trout testis chromatin was digested with DNase II, a transcriptionally active fraction was obtained containing highly acetylated H4 histone. Histones H3 and H4 of HeLa cells are highly acetylated when grown in butyrate. The nucleosomal DNA of such cells is hydrolyzed by DNase I 5-10 times faster. In this case, DNA is specifically cleaved at a site where, under normal conditions, a break does not occur. It has also been shown that DNase I preferentially cleaves DNA in those regions of chromatin that are highly acetylated. Apparently, nucleosomes in these regions undergo conformational changes after acetylation. Since such changes are necessary for RNA and DNA polymerases to use DNA as a template, it is possible that acetylation is one of them. ways in which histones are partially separated from DNA, making the latter available to enzymes. Thus, acetylation is important both for the functioning of chromatin and for its structure and conformation. It has been suggested that histone H4 binds to DNA by a mechanism, in the first stages of which acetylation occurs. Then, deacetylation can occur, leading to electrostatic interactions that fix the conformation. In the sea urchin spermatid, which does not synthesize RNA, histone H4 is completely deacetylated, while in the embryo, where high gene activity is observed, it is acetylated. Thus, there is a direct correlation between H4 histone acetylation and chromatin activity.

The study of the role of histone acetylation in the functioning of chromatin was facilitated by the discovery of the fact that in the presence of butyrate this modification is enhanced and histones H3 and H4 are specifically hyperacetylated, since butyrate inhibits histone deacetylase. Butyrate inhibits preferentially endogenous H3 and H4 histone deacetylase, while it does not affect the rate of acetylation. Apparently, histone deacetylase may play an important role in the metabolic control of acetylation. When histones are hyperacetylated, the DNA associated with them in HeLa cells becomes more accessible to DNase I, but not to staphylococcal nuclease. DNase I also promotes the removal of H3 and H4 histones from the complex. Simultaneously, DNA synthesis is inhibited. It can be assumed that histone acetylation is specifically required for transcription and not required for replication. Unlike phosphorylation, which is characteristic of histone H1 and only dividing cells, acetalization occurs mainly in histones H3 and H4 and in dividing as well as non-dividing cells that are metabolically active. This confirms the assumption that acetylation of nucleosomal histones plays an important role in transcription. Very little is known about the acetylation of NGB; one paper reports that HMG proteins from calf thymus nuclei and duck erythrocytes are acetylated.

Methylation

Histone methylation is a post-synthetic irreversible modification that is catalyzed by histone methyltransferase III present in the NGB fraction of chromatin. This modification was first discovered by Alfrey et al. Histone methyltransferase III catalyzes the transition of the CH 3 group from S-adenosylmethionine to the α-NH 2 group of the lysine residue, as shown below:

This modification of histones occurs after their binding to DNA. Histone methyl groups, unlike phosphoryl and acetyl groups, do not enter into further reactions. As a result, methylation is a stable process. The cytoplasmic enzyme methylase I methylates arginine before the histone enters the nucleus. NGBs are methylated by a special enzyme. One, two, or three methyl groups can be linked to the atom of the α-N-lysine residue, which are sequentially introduced by the same enzyme. Therefore, methyllysine residues can be mono-, di- or trimethyllysine. Histones H3 and H4 are mainly methylated. In histone H3, the ratio of mono-, di- and trimethyllysines is 1.8:1.0:0.45. In histone H4, the ratio of mono- to dimethyllysine is 0.7:1.0. Thus, histone H3 is more methylated than histone H4. Purified histones, as opposed to chromatin-bound histones, are unsuitable substrates for methylation. Methylated histones H3 and H4 after isolation can be further methylated at centers different from those at which the reaction occurs for chromatin-bound histones. Obviously, these additional centers are inaccessible for methylation due to a specific conformation in chromatin. Methyllysins are located near acetylated lysines.

Methylation of H3 and H4 histones occurs only in the NH 2 -terminal region. Histone H3 from calf thymus is methylated at Lys-9 and Lys-27, and histone H4 is methylated at Lys-20. Both lysines in histone H3 can be mono-, di- and trimethylated, but trimethyllysine is not formed in histone H4. Histone H3 has an additional methylation center - Lys-4. Histone H3 and H4 methylation centers, as well as their amino acid sequences, are extremely conserved. K m and V max for methylation of H3 and H4 histones are different. S-adenosylhomocysteine, a reaction product catalyzed by methyltransferase III, is a competitive substrate inhibitor.

Histone methylation in HeLa cells occurs primarily in S-phase. In tissue culture, methylation proceeds throughout the entire cell cycle, but the maximum rate is observed between the S and G 2 phases before the onset of mitosis. Possibly, methylation is necessary to prepare chromatin for mitosis. Following partial hepatectomy, histone methylation occurs during the important post-S-phase period for the cell. The degree of methylation of histones H3 and H4 seems to be the same in all organs, but changes with age. In ten-day-old rats, the molar ratio of mono-, di- and trimethyllysines in histone H3 is 0.55:1.0:0.35. The molar ratio of mono- and dimethyllysines in H4 histone at the same age is 0.1:0.9. With increasing age, there is a gradual shift towards more methylated forms. Histones H3 and H4 in the brain of adult rats are not updated, just as their methyl groups are not updated, regardless of the polypeptide chains. Honda et al. have shown that histone methylation is irreversible in other tissues as well. When young rats were injected with labeled lysine and methionine, significant amounts of each label were found in brain histones. However, only traces of these marks were found in adults. If the nuclei from the brain cells of adult rats are incubated with S-adenosylmethionine, then not a single methyl group is included in the H3 and H4 histones. These results indicate that histone methylation ends before maturity. Methylated histones may have several functions. 1) During methylation of lysine residues, in particular, during the formation of triphenyllysines, the pK of the α-NH 2 group of lysine increases and the basicity of histones increases. This may enhance the binding of histones to DNA. 2) Methylation of histones H3 and H4 may play an important role in the structure of nucleosomes. 3) Methylated histones can irreversibly "lock" DNA and prevent replication. Perhaps this explains the transition of cells to the postmitotic state. 4) Methylated histones can inhibit transcription. Further studies are needed to evaluate the role of histone methylation in the structure and functions of chromatin.

ADPribosylation

There are reports that poly-ADPribose binds covalently to nuclear proteins. This reaction has been shown to be catalyzed by chromatin-associated poly-ADPribose polymerase. The enzyme molecule from bovine thymus consists of one polypeptide chain with a mol. weighing 130,000; this enzyme is only fully active in the presence of DNA. It is associated with the internucleosomal region of chromatin and appears to promote the formation of higher order chromatin structures. The substrate in this reaction is NAD+. The enzyme is inhibited by nicotinamide, thymidine, cytokinins, and methylxanthine. Histone H1 and, to some extent, histone H2B undergo ADPribosylation both in vivo and in vitro. Other nucleosomal histones in rat liver are modified extremely weakly, if at all. The site of ADPribosylation in histone H1 has not yet been conclusively identified. It has been suggested that it is ether-attached to glutamate. With the help of an enzyme, from two to eleven ADPribose molecules are sequentially introduced into the histone. The presence of 65-residue branched chains of ADPribose has been reported in the nuclei of rat liver cells. The modification seems to go like this:

Phosphorylation of serine interferes with ADPribosylation. The series can also be ADPribosylated. Histone H 6 (trout sperm HMG protein), protamines and some NGP components are also ADPribosylated. The bond with ADPribose is unstable in an alkaline environment. Poly(ADPribose)glycohydrolase cleaves the polymer from the histone. ADPribosylated histones are more easily separated from chromatin than other modified histones. Apparently, the binding of histones to DNA weakens after ADPribosylation. This is due to an increase in the negative charges of histones and, in addition, due to the large size of the molecule, which is able to deform the chromatin structure. The function of ADPribose polymers, which covalently attach not only to histones, but also to HBs, has not yet been established. It is assumed that they are involved in the synthesis and repair of DNA, in the formation of the structure of chromosomes, and in cell differentiation. The content of ADPribose polymerase increases by 3–4 times in the G1 phase and in the process of differentiation of mouse erythroleukemia cells. In HeLa cells, the most intense poly-ADPribosylation is observed in the G1 phase, and the weakest - in the S phase. As a result of ADPribosylation, nuclear proteins can be separated from DNA, which promotes its replication in the S-phase. Thus, the modification in question may be necessary for DNA replication. This is supported by the fact that DNA synthesis in nuclei isolated from chick liver increases after ADPribosylation.

The polyamines spermine, spermidine and putrescine stimulate the ADPribosylation of nuclear proteins in that order. Mn 2+ also has a stimulating effect. ADPribosylation of H1 histones in HeLa cells increases almost 3-fold in the presence of polyamines. Spermine stimulates ADPribosylation of NGB cells in culture, and Mn2+ stimulates ADPribosylation of histones. If both spermine and Mn2+ are present in the incubation medium, then NGBs are modified to a greater extent than histones. Thus, polyamines and metal ions appear to have a regulatory effect on ADPribosylation of nuclear proteins.

Summing up, we can say that covalent modifications of chromosomal proteins, in particular histones, can have a significant effect on the structure and functions of chromatin. The main characteristics of modifying reactions are shown in fig. 2.4 and are as follows. 1) Phosphorylation and ADPribosylation occur mainly in histone H1, while acetylation and methylation occur in nucleosomal histones. 2) Phosphorylation, ADPribosylation and acetylation lead to a decrease in the overall positive charge of histones and to their separation from DNA, while methylation leads to an increase in positive charge, which makes the bond with DNA stronger. 3) Histones can undergo all these changes at the same time, which leads to significant changes in their ionic structure. 4) Phosphorylation is obviously a general phenomenon: it is less specific than other modifications and occurs in dividing cells throughout the entire cell cycle. Apparently, phosphorylation is necessary for DNA replication and cell division. The other three modifications are probably more specific. Acetylation occurs mainly in metabolically active cells and appears to be involved in the transcription process. Methylation, being an irreversible process, may be important for the repression of gene activity and for differentiation. 5) All these modifications are specifically modulated by special endogenous effectors, including hormones. Specific and differential modifications of chromosomal proteins can occur at different periods of life and lead to selective gene expression.

While a significant amount of data has been accumulated on covalent modifications of histones, relatively little is known about such modifications of NGPs. Information on the rates and sequence of dephosphorylation, deacetylation, and de-ADPribosylation is also scarce.

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