Brian Lyons

Human protein aging: modification and crosslinking through dehydroalanine and dehydrobutyrine intermediates

Nonenzymatic post‐translational modification (PTM) of proteins is a fundamental molecular process of aging. The combination of various modifications and their accumulation with age not only affects function, but leads to crosslinking and protein aggregation. In this study, aged human lens proteins were examined using HPLC–tandem mass spectrometry and a blind PTM search strategy. Multiple thioether modifications of Ser and Thr residues by glutathione (GSH) and its metabolites were unambiguously identified. Thirty‐four of 36 sites identified on 15 proteins were found on known phosphorylation sites, supporting a mechanism involving dehydroalanine (DHA) and dehydrobutyrine (DHB) formation through β‐elimination of phosphoric acid from phosphoserine and phosphothreonine with subsequent nucleophilic attack by GSH. In vitro incubations of phosphopeptides demonstrated that this process can occur spontaneously under physiological conditions. Evidence that this mechanism can also lead to protein–protein crosslinks within cells is provided where five crosslinked peptides were detected in a human cataractous lens. Nondisulfide crosslinks were identified for the first time in lens tissue between βB2‐ & βB2‐, βA4‐ & βA3‐, γS‐ & βB1‐, and βA4‐ & βA4‐crystallins and provide detailed structural information on in vivo crystallin complexes. These data suggest that phosphoserine and phosphothreonine residues represent susceptible sites for spontaneous breakdown in long‐lived proteins and that DHA‐ and DHB‐mediated protein crosslinking may be the source of the long‐sought after nondisulfide protein aggregates believed to scatter light in cataractous lenses. Furthermore, this mechanism may be a common aging process that occurs in long‐lived proteins of other tissues leading to protein aggregation diseases.

Molecular signatures of long‐lived proteins: autolytic cleavage adjacent to serine residues

The centre of the human lens, which is composed of proteins that were synthesized prior to birth, is an ideal model for the evaluation of long‐term protein stability and processes responsible for the degradation of macromolecules. By analysing the sequences of peptides present in human lens nuclei, characteristic features of intrinsic protein instability were determined. Prominent was the cleavage on the N‐terminal side of serine residues. Despite accounting for just 9% of the amino acid composition of crystallins, peptides with N‐terminal Ser represented one‐quarter of all peptides. Nonenzymatic cleavage at Ser could be reproduced by incubating peptides at elevated temperatures. Serine residues may thus represent susceptible sites for autolysis in polypeptides exposed to physiological conditions over a period of years. Once these sites are cleaved, other chemical processes result in progressive removal or ‘laddering’ of amino acid residues from newly exposed N‐ and C‐termini. As N‐terminal Ser peptides originated from several crystallins with unrelated sequences, this may represent a general feature of long‐lived proteins.

Is protein methylation in the human lens a result of non-enzymatic methylation by S-adenosylmethionine?

Since crystallins in the human lens do not turnover, they are susceptible to modification by reactive molecules over time. Methylation is a major post-translational lens modification, however the source of the methyl group is not known and the extent of modification across all crystallins has yet to be determined. Sites of methylation in human lens proteins were determined using HPLC/mass spectrometry following digestion with trypsin. The overall extent of protein methylation increased with age, and there was little difference in the extent of modification between soluble and insoluble crystallins. Several different cysteine and histidine residues in crystallins from adult lenses were found to be methylated with one cysteine (Cys 110 in γD crystallin) at a level approaching 70%, however, methylation of crystallins was not detected in fetal or newborn lenses. S-adenosylmethionine (SAM) was quantified at significant (10-50 μM) levels in lenses, and in model experiments SAM reacted readily with N-α-tBoc-cysteine and N-α-tBoc-histidine, as well as βA3-crystallin. The pattern of lens protein methylation seen in the human lens was consistent with non-enzymatic alkylation. The in vitro data shows that SAM can act directly to methylate lens proteins and SAM was present in significant concentrations in human lens. Thus, non-enzymatic methylation of crystallins by SAM offers a possible explanation for this major human lens modification.

Spontaneous Cleavage of Proteins at Serine Residues

Long-lived proteins are found at several sites in the body and they undergo numerous changes as a result of prolonged exposure to physiological conditions. Truncation is a common modification and many cleavages appear to be non-enzymatic, however little is known about the processes involved. In this study we demonstrate, using synthetic peptides that incorporate the sequence of a protein that is known to cleave in older lenses, that truncation on the N-terminal side of serine residues can occur at neutral pH. A mechanism that incorporates an N,O-acyl shift, which is analogous to intein cleavage, is proposed. Such cleavages may explain the origin of abundant peptides derived from crystallins in aged human lenses.

Amyloid Plaque in the Human Brain Can Decompose from Aβ(1-40/1-42) by Spontaneous Nonenzymatic Processes

The degradation of long-lived proteins in the body is an important aspect of aging, and much of the breakdown is due to the intrinsic instability of particular amino acids. In this study, peptides were examined to discover if spontaneous nonenzymatic reactions could be responsible for the composition of Alzheimers (AD) plaque in the human brain. The great majority of AD plaque consists of N-terminally truncated versions of Aβ(1-40/1-42), with the most abundant peptide commencing with Glu (residue 3 in Aβ1-40/1-42) that is present as pyroGlu. Several Asp residues are racemized in Aβ plaque, with residue 1 being predominantly l-isoAsp and peptide bond cleavage next to Ser 8 is also evident. In peptides, loss of the two N-terminal amino acids as a diketopiperazine was demonstrated at pH 7. For the Aβ N-terminal hexapeptide, AspAlaGluPheArgHis, this resulted in the removal of AspAla diketopiperazine and the generation of Glu as the new N-terminal residue. The Glu cyclized readily to pyroGlu. This pathway was altered significantly by zinc, which promoted pyroGlu formation but decreased AspAla diketopiperazine release. Zinc also facilitated cleavage on the N-terminal side of Ser 8. Racemization of the original N-terminal Asp to l-isoAsp was also detected and loss of one amino acid from the N-terminus. These data are therefore entirely consistent with plaque in the human brain forming from deposition of Aβ(1-40/1-42) and, over time, decomposing spontaneously. Since amyloid plaque is present in the human brain for years prior to the onset of AD, gradual spontaneous changes to the polypeptides within it will alter its properties and those of the oligomers that can diffuse from it. Such incremental changes in composition may therefore contribute to the origin of AD-associated cytotoxicity.

Proteome changes induced by laser in diabetic retinopathy.

Diabetic macular oedema (DMO) is the commonest cause of vision loss in people with diabetes. Laser photocoagulation can be effective in the treatment of DMO; however, its mechanism of action is still poorly understood. A better understanding of these mechanisms may allow the development of therapeutic approaches that could avoid the deleterious adverse events associated with photocoagulation.

Spontaneous cyclization of polypeptides with a penultimate Asp, Asn or isoAsp at the N‐terminus and implications for cleavage by aminopeptidase

A cyclic product that forms spontaneously from peptides that contain a penultimate Asp, Asn or isoAsp residue at the N‐terminus has been characterized. This 2,5‐diketopiperazine derivative forms under physiological conditions and is stable, showing little degradation even following heating at 60 °C. A mechanism for its formation from Asn and Asp peptides is proposed that involves a succinimide or isoaspartate intermediate. A diketopiperazine‐modified peptide was also detected in human lens extracts. Since peptides that contain the diketopiperazine moiety are not readily hydrolysed by leucine aminopeptidase, it is hypothesized that proteins and peptides modified in this way in the body may not readily be digested by the normal proteolytic machinery of cells.

Separate mechanisms for age-related truncation and racemisation of peptide-bound serine

Some amino acids are particularly susceptible to degradation in long-lived proteins. Foremost among these are asparagine, aspartic acid and serine. In the case of serine residues, cleavage of the peptide bond on the N-terminal side, as well as racemisation, has been observed. To investigate the role of the hydroxyl group, and whether cleavage and racemisation are linked by a common mechanism, serine peptides with a free hydroxyl group were compared to analogous peptides where the serine hydroxyl group was methylated. Peptide bond cleavage adjacent to serine was increased when the hydroxyl group was present, and this was particularly noticeable when it was present as the hydroxide ion. Adjacent amino acid residues also had a pronounced affect on cleavage at basic pH, with the SerPro motif being especially susceptible to scission. Methylation of the serine hydroxyl group abolished truncation, as did insertion of a bulky amino acid on the N-terminal side of serine. By contrast, racemisation of serine occurred to a similar extent in both O-methylated and unmodified peptides. On the basis of these data, it appears that racemisation of Ser, and cleavage adjacent to serine, occur via separate mechanisms. Addition of water across the double bond of dehydroalanine was not detected, suggesting that this mechanism was unlikely to be responsible for conversion of l-serine to d-serine. Abstraction of the alpha proton may account for the majority of racemisation of serine in proteins.

Age‐dependent modification of proteins: N‐terminal racemization

Age‐dependent deterioration of long‐lived proteins in humans may have wide‐ranging effects on health, fitness and diseases of the elderly. To a large extent, denaturation of old proteins appears to result from the intrinsic instability of certain amino acids; however, these reactions are incompletely understood. One method to investigate these reactions involves exposing peptides to elevated temperatures at physiological pH. Incubation of PFHSPSY, which corresponds to a region of human αB‐crystallin that is susceptible to age‐related modification, resulted in the appearance of a major product. NMR spectroscopy confirmed that this novel peptide formed via racemization of the N‐terminal Pro. This phenomenon was not confined to Pro, because peptides with N‐terminal Ser and Ala residues also underwent racemization. As N‐terminal racemization occurred at 37 °C, a long‐lived protein was examined. LC‐MS/MS analysis revealed that approximately one third of aquaporin 0 polypeptides in the centre of aged human lenses were racemized at the N‐terminal methionine.

Spontaneous cleavage of proteins at serine and threonine is facilitated by zinc

Old proteins are widely distributed in the body. Over time, they deteriorate and many spontaneous reactions, for example isomerisation of Asp and Asn, can be replicated by incubation of peptides under physiological conditions. One of the signatures of long‐lived proteins that has proven to be difficult to replicate in vitro is cleavage on the N‐terminal side of Ser residues, and this is important since cleavage at Ser, and also Thr, has been observed in a number of human proteins. In this study, the autolysis of Ser‐ and Thr‐containing peptides was investigated with particular reference to discovering factors that promote cleavage adjacent to Ser/Thr at neutral pH. It was found that zinc catalyses cleavage of the peptide bond on the N‐terminal side of Ser residues and further that this process is markedly accelerated if a His residue is adjacent to the Ser. NMR analysis indicated that the imidazole group co‐ordinates zinc and that once zinc is co‐ordinated, it can polarize the carbonyl group of the peptide bond in a manner analogous to that observed in the active site of the metalloexopeptidase, carboxypeptidase A. The hydroxyl side chain of Ser/Thr is then able to cleave the adjacent peptide bond. These observations enable an understanding of the origin of common truncations observed in long‐lived proteins, for example truncation on the N‐terminal side of Ser 8 in Abeta, Ser 19 in alpha B crystallin and Ser 66 in alpha A crystallin. The presence of zinc may therefore significantly affect the long‐term stability of cellular proteins.