Research Article: Chemical and Functional Aspects of Posttranslational Modification of Proteins

Date Published: October , 2009

Publisher: A.I. Gordeyev

Author(s): D.G. Knorre, N.V. Kudryashova, T.S. Godovikova.

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Abstract

This paper reviews the chemical and functional aspects of the posttranslational modifications of proteins, which are achieved by the addition of various groups to the side chain of the amino acid residue backbone of proteins. It describes the main prosthetic groups and the interaction of these groups and the apoenzyme in the process of catalysis, using pyridoxal catalysis as an example. Much attention is paid to the role of posttranslational modification of proteins in the regulation of biochemical processes in live organisms, and especially to the role of protein kinases and their respective phosphotases. Methylation and acetylation reactions and their role in the “histone code”, which regulates genome expression on the transcription level, are also reviewed. This paper also describes the modification of proteins by large hydrophobic residues and their role in the function of membrane-associated proteins. Much attention is paid to the glycosylation of proteins, which leads to the formation of glycoproteins. We also describe the main non-enzymatic protein modifications such as glycation, homocysteination, and desamida-tion of amide residues in dibasic acids.

Partial Text

Template biosynthesis of polypeptide chains on ribosomes most often does not immediately produce a fully functional protein. The newly formed polypeptide chain must undergo certain chemical modifications outside the ribosome. These modifications are most often driven by enzymes and take place after all the information supplied by the template RNA (mRNA) has been read, that is after mRNA translation: thus, these additional processes are called posttranslational modifications.

In some cases, the last step in the biosynthesis of a functional protein is the covalent binding of a prosthetic group, which forms part of the active site [1, 2]. Table 1 shows the structural formulas of side chain modification products after the covalent binding of certain cofactors to proteins, as well as the types of reactions in which the corresponding prosthetic groups take part.

The central role in reactions responsible for rearrangement of all intracellular processes eventually signaling either cell division or cell death is played by a large group of enzymes called protein kinases (phosphotransferases, EC 2.7.). These enzymes can add phosphate groups to the side chains of amino acids in various proteins [5-12]. γ-phosphate ATP is the donor of a phosphate group in all such reactions. Kinases are grouped according to the amino acid to which they add the phosphate into tyrosine kinases (E.C. 2.7.10.2) and serine/threonine kinases (E.C. 2.7.11.1) [5]. Also, histidine kinases are often found in bacteria, plants, and fungi. The latter enzymes function in a two-step signal transduction system [13]. The inorganic phosphate residue, which is attached to a histidine in the enzyme itself, is then transferred onto an aspartate residue in the target protein. Phosphorylation of the aspartate results in further signal transduction [13]. Figure 2 shows the structures of amino acid phosphorylation products in proteins [1].

One of the widely spread types of posttranslational modification that plays an important role in living organisms is acetylation [30-38]. The reaction takes place at the ε-aminogroups of lysine residues, and acetyl coenzyme A acts as a donor of acetyl groups. The positive charge of the amino group disappears after this reaction, causing a redistribution of charge in the whole protein molecule, and also increasing the hydrophobicity and size of the modified amino acid’s side chain. Among other things, histones use this as a binding signal for transcription factors and associated proteins, i.e. transcription initiation. A very important feature of the proteins that can be acetylated is a so-called bromodomain, a conservative 110 amino acid module [30, 31].

The most widespread modifications by addition of fatty acid residues are myristoylation, which is the addition of a CH3-(CH2)12-CO- residue to the amino group of an N-terminal glycine [1, 41, 42], and palmitoylation, which is the addition of a CH3-(CH2)14-CO- residue at the SH-group of a cysteine residue [1, 43, 44]. In both cases, the acylation is accomplished by the appropriate acyl coenzyme A, which is produced during oxidative decay of longer fatty acids.

Acylation of proteins by the activated C-terminal carboxyl group of glycine in ubiquitin, an 8kDa peptide consisting of 76 amino acid residues, is of great biological importance [52-59]. The main, although not the only, purpose of this reaction is the marking of proteins for degradation. These include various damaged proteins, as well as ordinary proteins which fulfill their functions in certain phases of the cell cycle and whose activity is unfavorable during other phases.

Anoter often-seen posttranslational modification is alkylation. This type of modification includes the methylation of lysine and arginine residues [26, 30, 33-38, 39, 65-72] and prenylation (addition of pharnesyl and geranyl-geranyl moieties to cysteine side chains) [47, 73-80] (Fig. 7).

Some cases of posttranslational modification are the addition of isoprenoid moieties onto a cysteine residue. These moieties are formed from isoprene residues – farnesyl and geranyl-geranyl (Fig. 11). Modification of proteins with these radicals is catalyzed by proteinfarnesyl and proteingeranyl-geranyl transferases, respectively (E.C. 2.5.1.58 and E.C. 2.5.1.59 or E.C. 2.5.1.60; Type I and II geranyl-geranyl transferases). Type I enzymes catalyze the transfer of a gernayl-geranyl residue onto a cysteine residue in a Cys-A-A-X sequence, while type II use the Cys-Cys-X-X, X-X-Cys-Cys or X-Cys-X-Cys sequences [47, 73-80], where A is a small aliphatic amino acid, and X are various amino acids.

Glycosylation of proteins plays a very important role in the functioning of eukaryotic cells. Glycosylation modifies the OH-groups of serine and threonine residues (O-glycosylation) and the functional groups of asparagine residue side chains (N-glycosylation) (Fig. 13).

Another posttranslational modification of protein molecules is the addition of a sulfate residue at the OH-group of tyrosine. Phosphoadenosylphosphosulfate acts as a sulfate donor (Fig. 16). The reaction is catalyzed by the sulfotransferase enzyme (E.C. 2.8.2.20) [103, 104].

Many cellular processes, such as DNA reparation, apoptosis, and the functioning of the spindle during cell division, use mono- and poly(ADP-ribosyl)ation as an important regulating mechanism [107]. Various pathogenic bacteria secrete toxins that ADP-rybosylate human proteins, thus causing severe diseases, such as cholera, diphtheria, pertussis, and botulism [108-111].

A large number of proteins are characterized by the formation of disulfide bonds in a reaction between cysteine residues either inside a single polypeptide chain or between different polypeptide molecules. Such bonds fulfill a structural function and determine the tertiary and quaternary structure of the protein, which are vital for the protein’s metabolic functions in the organism. This modification is also involved in the regulation of the cell’s reduction-oxidation status, which affects numerous aspects of cellular processes, such as proliferation, differentiation, and apoptosis by changing the functioning of proteins via a reversible modification of cysteine residues [143-147].

Another type of posttranslational modification is the oxidative hydroxylation reaction. This reaction takes place at non-nucleophilic amino acid residue side chains: the CH2-groups of proline, lysine and asparagine form 3-hydroxyproline, 4-hydroxyproline, 5-hydroxyproline, and 3-hydroxyasparagine, and this process is catalyzed by iron-containing monooxygenases of the E.C. 1.14.16 subclass [155, 156, 157] (Fig. 22).

Most protein factors, which are involved in blood clotting in mammals, contain several residues of γ-carboxyglutamic acid (Gla). This residue appears in blood clotting factors as a result of posttranslational modification; namely the fixation of CO2 by the γ-methylene carbon atom of glutamic acid (Glu) during the factor’s progress down the secretion pathways [164-166]. The Gla residue side chain, which bears two negatively charged carboxyl groups, has a capacity to form chelate complexes with bivalent cations, which is especially important for interaction with the Ca2+ ion [164].

Protein glycation is an endogenous non-enzymatic addition of reducing sugar residues present in the bloodstream to the side chains of either lysine or arginine residues in proteins. A schematic representation of the glycation process, which can be divided into the early and late stages, is shown on Fig. 25. The first stage of glycation involves the nucleophilic attack of the glucose carbonyl group by an ε-amino group of lysine or a guanidine moiety of arginine, which results in the formation of a labile Schiff base – N-glycosylimine (1). The formation of the Schiff base is a relatively rapid and reversible process [168]. Next, the glycosylimine regroups and forms an Amadori product, 1-amino-1-deoxyfructose (2). This process happens more slowly than the formation of glycosylimine, but much quicker if compared to the rate of Schiff base hydrolysis. This is why proteins bearing 1-amino-1-deoxyfructose residues tend to accumulate in blood. Modification of lysine residues at the early glycation steps is thought to be facilitated by the close proximity of histidine or lysine residues, which catalyze this process [169].

A very impressive type of posttranslational modification is the autocatalytic restructuring of the peptide backbone in the folded protein during GFP (green fluorescent protein) maturation. This protein is encoded by a single gene, and the chromofore is made up of three amino acid residues, Ser65-Tyr66-Gly67, capable of posttranslational autocatalytic cyclization, which does not require any cofactors or substrates [181-183].

The majority of methylation processes in live organisms use S-adenosylmethionine, thus forming S-adenosylhomocysteine. The latter is hydrolyzed by the adensylhomocysteinase (E.C. 3.3.1.1) enzyme into adenosine and homocysteine. This reaction catalyzed by methionyl-tRNA synthetase (E.C. 6.1.1.1) turns homocysteine into thiolactone (this is a side reaction for this enzyme) [199]. Homocysteine thiolactone is an acylating agent and can react with the functional groups of lysine residues [200-203]. The ε-amino group of lysine performs a nucleophilic attack of the carbonyl carbon atom of the thiolactone, which results in decyclization of the lactone and the formation of an additional sulfhydryl moeity (Fig. 28).

One of the types of posttranslational modification, which plays an important role in cellular functions, is the deamidation of the amides of dicarbonic acids. Many authors believe these reactions to be non-enzymatic cleavage of ammonia from the amide group of asparagine or glutamine, resulting in an intermediate product, a cyclic imide (Fig. 30) [211-215]. The rate of this product’s formation is determined by the local amino acid surroundings and the characteristics of the solution (pH and ingredients) [213, 214]. Asparagine residues in proteins are deamidated 40 times more often than glutamine residues. Furthermore, the rate of asparagine deamidation is 100-fold greater than the rate of glutamine deamidation [214].

This work on the effects of chemical modification of human serum albumin on the RNA-hydrolytic activity of the protein was performed with support from the Interdisciplinary Integration Project for Basic Research Siberian Branch RAS №88 and the Russian Foundation for Basic Research (grant №09-04-01483a).

 

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