Biosynthetic incorporation of oxidized amino acids into proteins and their cellular proteolysis

We demonstrate that oxidized amino acids can be incorporated into proteins by protein synthesis. The level of incorporation into protein was dependent on the concentration of oxidized amino acid supplied to the cells. At low levels of incorporation, the oxidized amino acids examined increased the degradation rate of the cell proteins. Degradation of certain proteins containing high levels of DOPA (but not ortho or meta tyrosine) was decreased to below the basal degradation rates suggesting that DOPA may contribute to proteins becoming resistant to proteolysis. Changes in the degradation rates of the oxidized amino acid-containing proteins was shown to have no impact on the degradation rates of native proteins, indicating that the activity of the degradative machinery was not affected. We demonstrate that oxidized proteins are selectively degraded by the proteasomes and provide evidence to suggest that the proteasomes and the endosomal-lysosomal systems may act in sequence as well as in parallel. The incorporation approach, unlike cell studies in which an exogenous oxidant is used, allows the degradation rates of the oxidatively modified proteins to be selectively measured, offering a greater sensitivity as well as greatly reducing toxicity to the cell and avoiding oxidative modification of other cell components.     

Misincorporation of amino acid analogues into proteins by biosynthesis

Despite astounding diversity in their structure and function, proteins are constructed from 22 protein or ‘canonical’ amino acids. Hundreds of amino acid analogues exist; many occur naturally in plants, some are synthetically produced or can be produced in vivo by oxidation of amino acid side-chains. Certain structural analogues of the protein amino acids can escape detection by the cellular machinery for protein synthesis and become misincorporated into the growing polypeptide chain of proteins to generate non-native proteins. In this review we seek to provide a comprehensive overview of the current knowledge on the biosynthetic incorporation of amino acid analogues into proteins by mammalian cells. We highlight factors influencing their incorporation and how the non-native proteins generated can alter cell function. We examine the ability of amino acid analogues, representing those commonly found in damaged proteins in pathological tissues, to be misincorporated into proteins by cells in vitro, providing us with a useful tool in the laboratory to generate modified proteins representing those present in a wide-range of pathologies. We also discuss the evidence for amino acid analogue incorporation in vivo and its association with autoimmune symptoms. We confine the review to studies in which the synthetic machinery of cell has not been modified to accept non-protein amino acids.     

Incorporation of non-natural amino acids into proteins

Chemical and biological diversity of protein structures and functions can be widely expanded by position-specific incorporation of non-natural amino acids carrying a variety of specialty side groups. After the pioneering works of Schultz's group and Chamberlin's group in 1989, noticeable progress has been made in expanding types of amino acids, in finding novel methods of tRNA aminoacylation and in extending genetic codes for directing the positions. Aminoacylation of tRNA with non-natural amino acids has been achieved by directed evolution of aminoacyl-tRNA synthetases or some ribozymes. Codons have been extended to include four-base codons or non-natural base pairs. Multiple incorporation of different non-natural amino acids has been achieved by the use of a different four-base codon for each tRNA. The combination of these novel techniques has opened the possibility of synthesising non-natural mutant proteins in living cells.     

Splicing of unnatural amino acids into proteins: a peptide model study

S-Ethyl 2-azidohexanethioate (N3-Hex-SEt), an unnatural amino acid analog of leucine, is coupled with L-cysteine ethyl ester (NH2-Cys-OEt) to obtain N3-Hex-Cys-OEt by native chemical ligation. Coupling of this dipeptide with N-t-butoxycarbonyl-2-diphenylphosphinoethanethioglycinate produces the tripeptide, t-Boc-Gly-Hex-Cys-OEt, in high yield. These reactions suggest an approach for the incorporation of unnatural amino acids into proteins by successive native chemical ligation and Staudinger ligation.     

Five-base codons for incorporation of nonnatural amino acids into proteins

Extension of the genetic code for the introduction of nonnatural amino acids into proteins was examined by using five-base codon–anticodon pairs. A streptavidin mRNA containing a CGGUA codon at the Tyr54 position and a tRNA_UACCG chemically aminoacylated with a nonnatural amino acid were added to an Escherichia coli in vitro translation system. Western blot analysis indicated that the CGGUA codon is decoded by the aminoacyl-tRNA containing the UACCG anticodon. HPLC analysis of the tryptic fragment of the translation product revealed that the nonnatural amino acid was incorporated corresponding to the CGGUA codon without affecting the reading frame adjacent to the CGGUA codon. Another 15 five-base codons CGGN₁N₂, where N₁ and N₂ indicate one of four nucleotides, were also successfully decoded by aminoacyl-tRNAs containing the complementary five-base anticodons. These results provide a novel strategy for nonnatural mutagenesis as well as a novel insight into the mechanism of frameshift suppression.     

Free radical-mediated oxidation of free amino acids and amino acid residues in proteins

Summary. We summarize here results of studies designed to elucidate basic mechanisms of reactive oxygen (ROS)-mediated oxidation of proteins and free amino acids. These studies have shown that oxidation of proteins can lead to hydroxylation of aromatic groups and aliphatic amino acid side chains, nitration of aromatic amino acid residues, nitrosylation of sulfhydryl groups, sulfoxidation of methionine residues, chlorination of aromatic groups and primary amino groups, and to conversion of some amino acid residues to carbonyl derivatives. Oxidation can lead also to cleavage of the polypeptide chain and to formation of cross-linked protein aggregates. Furthermore, functional groups of proteins can react with oxidation products of polyunsaturated fatty acids and with carbohydrate derivatives (glycation/glycoxidation) to produce inactive derivatives. Highly specific methods have been developed for the detection and assay of the various kinds of protein modifications. Because the generation of carbonyl derivatives occurs by many different mechanisms, the level of carbonyl groups in proteins is widely used as a marker of oxidative protein damage. The level of oxidized proteins increases with aging and in a number of age-related diseases. However, the accumulation of oxidized protein is a complex function of the rates of ROS formation, antioxidant levels, and the ability to proteolytically eliminate oxidized forms of proteins. Thus, the accumulation of oxidized proteins is also dependent upon genetic factors and individual life styles. It is noteworthy that surface-exposed methionine and cysteine residues of proteins are particularly sensitive to oxidation by almost all forms of ROS; however, unlike other kinds of oxidation the oxidation of these sulfur-containing amino acid residues is reversible. It is thus evident that the cyclic oxidation and reduction of the sulfur-containing amino acids may serve as an important antioxidant mechanism, and also that these reversible oxidations may provide an important mechanism for the regulation of some enzyme functions.     

Incorporation of tellurium into amino acids and proteins in a tellurium-tolerant fungi

Aspergillus fumigatus, Aspergillus terreus, and Penicillium chrysogenum, a tellurium tolerant fungi, are able to grow on sulfur free medium amended with 0.2% (w/v) tellurite. Tellurium was incorporated into several types of low and high molecular weight proteins. The newly detected telluro-proteins contained an extraordinary high level of tellurium, as well as telluro-cysteine, telluro-cystine, telluro-methionine, and serine.     

G2A as a receptor for oxidized free fatty acids

G2A was identified as a G protein-coupled receptor that can be induced by different classes of DNA-damaging agents and block cell cycle progression in lymphocytes. We recently reported that G2A functions as a receptor for oxidized free fatty acids derived from linoleic and arachidonic acids. When ectopically expressed in CHO cells, G2A mediates intracellular signaling events such as intracellular calcium mobilization and JNK activation in response to oxidized free fatty acids. In human epidermal keratinocytes, G2A mediates the secretion of cytokines including interleukin-6 and -8, and blocks cell cycle progression at the G1 phase in response to ligands. G2A might function as a sensor that monitors the oxidative states and mediates appropriate cellular responses such as secretion of paracrine signals and attenuation of proliferation.     

Atypical incorporation of single amino acids into myelin proteins.

—Purified myelin incorporated l -[¹⁴C]leucine and l -[¹⁴C]lysine into myelin proteins in an enzymatic process similar to that of renal brush border membranes. The system was not inhibited by cycloheximide or puromycin or by pretreatment with ribonuclease; the reaction was inhibited by cetophenicol. ATP was an effector, shifting the optimal pH from 7.2 to 8.3. In the presence of ATP, myelin was less dense in a sucrose gradient. Ammonia was released from the membrane during the incorporation of amino acids. Myelin preloaded with cold leucine did not incorporate [¹⁴C]leucine but did incorporate [¹⁴C]lysine; there was no cross inhibition between the two amino acids. The incorporation was into or onto proteins of the Wolfgram proteolipid fraction of myelin. The incorporation was of the high affinity type with a K_m of 10^?7m and was restricted to the natural amino acids.     

Site-specific incorporation of PEGylated amino acids into proteins using nonnatural amino acid mutagenesis

Site-directed incorporation of PEGylated nonnatural amino acids with 4, 8, and 12 repeated ethylene glycol units was examined in a cell-free translation system. PEGylated aminophenylalanine derivatives were successfully incorporated into proteins, whereas PEGylated lysines were not. The incorporation efficiency of the PEGylated amino acids decreased with an increase in PEG chain length. The present method will be useful for preparation of proteins which are PEGylated in a site-specific and quantitative manner.     

Residue-specific incorporation of non-canonical amino acids into proteins: recent developments and applications

Residue-specific incorporation of non-canonical amino acids into proteins allows facile alteration and enhancement of protein properties. In this review, we describe recent technical developments and applications of residue-specific incorporation to problems ranging from elucidation of biochemical mechanisms to engineering of protein-based biomaterials. We hope to inform the reader of the ease and broad utility of residue-specific non-canonical amino acid incorporation with the goal of inspiring investigators outside the field to consider applying this tool to their own research.