Post-translational modifications in proteins

Post-translational modifications of proteins regulate protein functions by causing changes in protein activity, their cellular locations and interactions with other proteins. These kinds of protein modifications are transient and reversible involved in signalling pathways from membrane to nucleus in response to external stimuli.

Besides performing catalytic functions, signaling proteins modified by phosphorylation, myristoylation, farnesylation, cysteine oxidation, ubiquitination, acetylation, methylation, nitrosylation, etc, serve as scaffolds for the assembly of multiprotein signaling complexes, as adaptors, as transcription factors and as signal pathway regulators [67]. Phosphorylation and dephosphorylation on S, T, Y and H residues are the best known modifications involved in reversible, activation and inactivation of enzyme activity and modulation of molecular interactions in signaling pathway [68]. Acetylation regulates many diverse functions, including DNA recognition, protein-protein interaction and protein stability. Acetylation and deacetylation in N-terminal and K-residue are suggested as rival to phosphorylation [69]. As well as phosphorylation, glycosylation is another regulatory post-translational modification which occurs on serine and threonine residues of cytosolic and nuclear proteins [70]. The glycans are distinguished by the type of linkage atom involved as N-, O-, C- or S-glycans. Determination of glycoprotein structure modification is difficult because of the number of monosaccharides and amino acid residues involved in the O-glycan linkages [71]. Previous reports show that there is a sequence consensus for N-glycosylations, Asn-Xaa-Ser/Thr/Cys where the Xaa may be any amino acid except Pro [72].

Furthermore, biological functions of many proteins may be altered by ubiquitination and deubiquitination, sumoylation and desumoylation through covalent attachment to the polypeptide modifiers. Ubiquitin plays a key role in targeting proteins for degradation by the proteasome [73]. In recent reports it is suggested that ubiquitination, sumoylation, acetylation and methylation of lysine residues link specific covalent modification of the transcriptional apparatus to their regulatory function [74]. The most common post-translational modifications are summarized in Table 1.1.

Table 1.1. Summary of the post-translational modification found in proteins PTM types Mass

increament Modified amino

acid residues^a Position^b Remarks

Phosphorylation 79.9 Y, S, T, H, D Anywhere Reversible, regulate protein function Glycosylation Anywhere Reversible, cell-cell interaction O-linked > 800 S, T

(O-Glc-NAc) 203.2

N-linked > 800 N

Acetylation 42.0 S N-term Reversible, protein activity, stability

K Anywhere

Deamidation 0.9 N, Q Anywhere N to D, Q to E

Methylation Anywhere Regulation of gene expression

Monomethylation 14.0 K

Dimethylation 28.0 K

Trimethylation 42.0 K

Acylation Cellular localization to membrane

farnesylations 204.3 C C-term

myristoylation 210.3 G N-term

K Anywhere

Cys oxidation Anywhere Oxidative regulation of proteins Disulfide bond -2.0 C

Glutathionylation 305.3 C Sulfenic acid 16.0 C Sulfinic acid 32.0 C

Ubiquitination K Anywhere Reversible/ireversible

Sumoylation K [ILFV]KD

Hydroxyproline 16.0 P Protein stability

Pyroglutamic

acid -17 Q N-term

a- amino acid residues where the modification has occurred b- the location of the modified amino acid in the protein sequence

A large class of post-tranlational modifications are caused by reactive oxygen species which induce oxidative post-tranlational modifications. ‘‘Oxidative stress’’

occurs when the balance of formation of oxidants exceeds the ability of antioxidant systems to remove reactive oxygen species, when inflammatory phagocytes (e.g., neutrophils and macrophages) are activated to undergo an oxidative burst by exposure to a foreign agent. Under these conditions, biomolecules become subjected to attack by excess reactive oxygen species and significant molecular and physiological damage can occur [75, 76]. Reactive oxygen species encompass a variety of diverse chemical species including superoxide anions, hydroxyl radicals and hydrogen peroxide. Some of these species, such as superoxide or hydroxyl

radicals, are extremely unstable, whereas others, like hydrogen peroxide, are freely diffusible and relatively long-lived. Because there are so many mechanisms for induction of protein oxidation and because all of the amino acyl side chains can become oxidatively modified, there are numerous different types of protein oxidative modification. The thiol (-SH) moiety on the side chain of the amino acid cysteine is particularly sensitive to redox reactions and is an established redox sensor. As for cysteine residue-specific oxidative post-translational modification of the protein (S-thiolation), it is found to act as a switch regulating biological function like phosphorylation-dephosphorylation [77]. With Met residues, the major product under biological conditions is methionine sulfoxide [78]

Direct protein carbonylation can occur through a variety of reactions.

Oxidation of amino acid side chains with metals and hydrogen peroxide is known to cause the formation of semialdehyde amino acids, with the majority of these reactions occurring with lysine, arginine, and proline residues [79]. According to their proposal, lysine, proline and/or arginine from myofibrillar protein are oxidized in the presence of Fe³⁺ and H2O2 to yield aminoadipic semialedehyde. The reaction is initiated by OOH^● radicals derived from the reaction between Fe³⁺ and H2O2. The oxidative deamination from the intermediate radical molecule occurs in the presence of Fe³⁺ and yields the semialdehyde. The resulting Fe²⁺ could propagate the oxidative degradation to new amino acid residues by reacting with H2O2 to form further hydroxyl radicals. A recent study [80] has confirmed the H2O2-activated myoglobin and metal ions such as Cu²⁺ are also able to promote the formation of aminoadipic semialedehyde from myofibrillar proteins. Aminoadipic semialedehyde is thought to account for approximately 70% of the total amount of protein carbonyls formed in oxidized animal proteins [81]. It is worth noticing that the formation of these semialdehydes does not require a previous cleavage of the peptide bond as protein-bound amino acids can be degraded into their corresponding semialdehydes.

Aminoadipic semialedehyde has already been detected and employed as indicators of protein oxidation in raw meat and a large variety of processed muscle foods such as cooked patties, frankfurters and dry-cured meats [82, 83].

Alternatively, protein carbonylation can result from an indirect mechanism involving the hydroxyl radical-mediated oxidation of lipids. Polyunsaturated acyl chains of phospholipids or polyunsaturated fatty acids such as arachidonic acid and

linoleic acid are highly susceptible to peroxidation and breakdown through non-enzymatic Hock cleavage, forming a variety of lipid-derived aldehydes and ketones [84]. Lipid peroxidation products can diffuse across membranes, allowing the reactive aldehyde-containing lipids to covalently modify proteins localized throughout the cell and relatively far away from the initial site of reactive oxygen species formation. The most reactive aldehydes generated from polyunsaturated fatty acid oxidation are 4-hydroxy-2-nonenal (HNE) [84], 4-oxo-2-nonenal [85], and acrolein [86]. Because of the presence of electron-withdrawing functional groups, the double bond of 4-HNE or 4-ONE serves as a site for Michael addition with the sulfur atom of cysteine, the imidizole nitrogen of histidine, and, to a lesser extent, the amine nitrogen of lysine.

The modification can take place by the 1,4-addition (Michael addition) of the nucleophilic groups in cysteine, histidine or lysine residues of the protein, respectively, onto the electrophilic double bond of HNE, giving an increase in the protein’s molecular mass by 156 amu with each molecule of HNE being added. After forming Michael adducts, the aldehyde moiety may in some cases undergo Schiff base formation with amines of adjacent lysines, producing intra- and/or intermolecular cross-linked amino acids [87, 88]. 4-HNE exerts a potentially detrimental effect to proteins by forming covalent adducts, resulting in diminished protein function, altered physicochemical properties [89] and induction of antigenicity [90]. In addition to these reversible oxidative post-translational modifications, it becomes clear that irreversible oxidative modification of histidine, lysine, and cysteine residues involves the generation of abnormal protein associated with the etiology of lifestyle related diseases.

Tyrosine nitration is a covalent protein modification resulting from the addition of a nitro- (NO2) group onto one of the two equivalent carbons CE1 and CE2 in the ortho position relative to the hydroxyl group of tyrosine residue and it is believed to depend on the simultaneous availability of tyrosyl (Tyr^•) and nitrogen dioxide (^•NO2) radicals [91, 92]. Since nitration has the effect of decreasing the pKa of the phenoxyl groups (from ~ 10 to 7.5 in free tyrosine), this modification may not only change the protein conformation, but may also affect the redox and signalling properties of tyrosines, thus contributing to peroxynitrate-mediated cell signalling [93]. There has been increasing interest in the effects of tyrosine nitration on changes in protein structure in diverse pathogenesis [94]. Protein tyrosine nitration usually occurs near

basic residues in loop regions [95] in areas free of steric hindrances. Also, the presence of amino acids that compete for nitrating agents proximal to tyrosine residues, including tryptophan (Trp), cysteine (Cys), and methionine (Met), may prevent tyrosine nitration by removing the nitrating agents [96]. The location of tyrosine residues in favorable environments for nitration within the secondary and tertiary protein structure may also influence site-specific nitration.