Understanding the α-crystallin cell membrane conjunction

PURPOSE. It is well established that levels of soluble α-crystallin in the lens cytoplasm fall steadily with age, accompanied by a corresponding increase in the amount of membrane-bound α-crystallin. Less well understood, is the mechanism driving this age-dependent membrane association. The aim of this study was to investigate the role of the membrane and its associated proteins and peptides in the binding of α-crystallin. METHODS. Fibre cell membranes from human and bovine lenses were separated from soluble proteins by centrifugation. Membranes were stripped of associated proteins with successive aqueous, urea and alkaline solutions. Protein constituents of the respective membrane isolates were examined by SDS-PAGE and Western immunoblotting. Recombinant αA- and αB-crystallins were fluorescently-labeled with Alexa350® dye and incubated with the membrane isolates and the binding capacity of membrane for α-crystallin was determined. RESULTS. The binding capacity of human membranes was consistently higher than that of bovine membranes. Urea- and alkali-treated membranes from the nucleus had similar binding capacities for αA-crystallin, which were significantly higher than both cortical membrane extracts. αB-Crystallin also had a higher affinity for nuclear membrane. However, urea-treated nuclear membrane had three times the binding capacity for αB-crystallin as compared to the alkali-treated nuclear membrane. Modulation of the membrane-crystallin interaction was achieved by the inclusion of an N-terminal peptide of αB-crystallin in the assays, which significantly increased the binding. Remarkably, following extraction with alkali, full length αA- and αB-crystallins were found to remain associated with both bovine and human lens membranes. CONCLUSIONS. Fiber cell membrane isolated from the lens has an inherent capacity to bind α-crystallin. For αB-crystallin, this binding was found to be proportional to the level of extrinsic membrane proteins in cells isolated from the lens nucleus, indicating these proteins may play a role in the recruitment of αB-crystallin. No such relationship was evident for αA-crystallin in the nucleus, or for cortical membrane binding. Intrinsic lens peptides, which increase in abundance with age, may also function to modulate the interaction between soluble α-crystallin and the membrane. In addition, the tight association between α-crystallin and the lens membrane suggests that the protein may be an intrinsic component of the membrane structure

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

Protein-bound kynurenine decreases with the progression of age-related nuclear cataract

PURPOSE. Posttranslational modification by UV filters is a key event in human lenses that appears to be largely responsible for normal age-dependent yellowing. It has been proposed that subsequent reactions of these covalently bound UV filters may also be involved in the genesis of age-related nuclear cataract. To examine this hypothesis, the levels of kynurenine-lysine and kynurenine-histidine were measured in both normal and cataractous human lenses. METHODS. Proteins isolated from the nuclei of normal lenses and lenses with and types I to IV nuclear cataract were hydrolyzed in 6 M HCl, and the levels of kynurenine-lysine and kynurenine-histidine were determined by HPLC. RESULTS. The content of kynurenine-lysine and kynurenine-histidine decreased substantially with the progression of age-related nuclear cataract. On average, levels of both kynurenine adducts were four times lower in advanced cataract (type IV) than in normal lenses. Simple autoxidation of the derivatives did not appear to be responsible for this decrease, because incubation in the presence of oxygen or H2O2 did not affect adduct stability. CONCLUSIONS. Although protein-bound kynurenine accumulates over time in normal lenses, the levels attached to the proteins decrease significantly with the progression of age-related nuclear cataract. This finding suggests that in cataract there is a breakdown of the protein-bound adducts. Such further reactions of bound UV filters may contribute to the etiology of age-related nuclear cataract.</p

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 degrees celsius, 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

New insights into the mechanisms of age-related protein-protein crosslinking in the human lens

Although protein crosslinking is often linked with aging as well as some age-related diseases, very few molecular details are available on the nature of the amino acids involved, or mechanisms that are responsible for crosslinking. Recent research has shown that several amino acids are able to generate reactive intermediates that ultimately lead to covalent crosslinking through multiple non-enzymatic mechanisms. This information has been derived from proteomic investigations on aged human lenses and the mechanisms of crosslinking, in each case, have been elucidated using model peptides. Residues involved in spontaneous protein-protein crosslinking include aspartic acid, asparagine, cysteine, lysine, phosphoserine, phosphothreonine, glutamic acid and glutamine. It has become clear, therefore, that several amino acids can act as potential sites for crosslinking in the long-lived proteins that are present in aged individuals. Moreover, the lens has been an invaluable model tissue and source of crosslinked proteins from which to determine crosslinking mechanisms that may lead to crosslinking in other human tissues

Spontaneous Cleavage at Glu and Gln Residues in Long-Lived Proteins

Long-lived proteins (LLPs) are prone to deterioration with time, and one prominent breakdown process is the scission of peptide bonds. These cleavages can either be enzymatic or spontaneous. In this study, human lens proteins were examined and many were found to have been cleaved on the C-terminal side of Glu and Gln residues. Such cleavages could be reproduced experimentally by in vitro incubation of Glu- or Gln-containing peptides at physiological pHs. Spontaneous cleavage was dependent on pH and amino acid sequence. These model peptide studies suggested that the mechanism involves a cyclic intermediate and is therefore analogous to that characterized for cleavage of peptide bonds adjacent to Asp and Asn residues. An increased amount of some Glu/Gln cleaved peptides in the insoluble fraction of human lenses suggests that cleavage may act to destabilize proteins. Spontaneous cleavage at Glu and Gln, as well as recently described cross-linking at these residues, can therefore be added to the similar processes affecting long-lived proteins that have already been documented for Asn and Asp residues

Spontaneous protein–protein crosslinking at glutamine and glutamic acid residues in long-lived proteins

Long-lived proteins (LLPs) are susceptible to the accumulation of both enzymatic and spontaneous post-translational modifications (PTMs). A prominent PTM observed in LLPs is covalent protein–protein crosslinking. In this study, we examined aged human lenses and found several proteins to be crosslinked at Glu and Gln residues. This new covalent bond involves the amino group of Lys or an α-amino group. A number of these crosslinks were found in intermediate filament proteins. Such crosslinks could be reproduced experimentally by incubation of Glu- or Gln-containing peptides and their formation was consistent with an amino group attacking a glutarimide intermediate. These findings show that both Gln and Glu residues can act as sites for spontaneous covalent crosslinking in LLPs and they provide a mechanistic explanation for an otherwise puzzling observation, that a major fraction of Aβ in the human brain is crosslinked via Glu 22 and the N-terminal amino group

Structural characterization of piperidine alkaloids from Pandanus amaryllifolius by inverse-detected 2D NMR techniques

Three novel piperidine alkaloids were isolated and identified from the leaves of Pandanus amaryllifolius. The structures of pandamarilactone-1, pandamarilactone-32 and pandamarilactone-31 were elucidated using 2D NMR techniques including inverse-detected Heteronuclear Multiple Bond Correlation (HMBC) and Heteronuclear Multiple Quantum Coherence (HMQC) spectroscopy. All of these piperidine alkaloids have a C-N-C skeleton and could be derived biogenetically from 4-hydroxy-4-methylglutamic acid

A study of kynurenine fragmentation using electrospray tandem mass spectrometry

A combination of accurate mass measurement and tandem mass spectrometry (both product ion and precursor ion scans) have been used to characterize the major fragment ions observed in the ESI mass spectrum of kynurenine. Kynurenine is a metabolite of tryptophan found in the human lens and is thought to play a role in protecting the retina from UV-induced damage. Three major fragmentation pathways were evident, following initial elimination either of ammonia, H2O and CO or the imine form of glycine. The latter is proposed to occur via the formation of an ion-molecule complex. In the case of loss of H2O and CO from deaminated kynurenine, there is evidence for an acylium ion intermediate, which is not observed for the loss of H2O and CO directly from protonated kynurenine. Product ion scans of deuterated kynurenine enabled the elucidation of structural rearrangements that were not evident in the spectra of the native compound. Since UV filter compounds can often only be isolated in small quantities from the lens, this study forms the basis for the characterization of novel UV filter compounds using mass spectrometry. The approach presented here may also be useful for the characterization of related classes of small molecules.</p

Production of truncated enzymically active human indoleamine 2,3-dioxygenase using site-directed mutagenesis

Indoleamine 2,3-dioxygenase (IDO), the first enzyme of the kynurenine pathway of tryptophan metabolism, has been implicated in numerous disease states. Site-directed mutagenesis was undertaken using the expression plasmid pQE9-IDO, to incorporate a stop codon at lysine 389. This produced a C-terminally truncated protein, similar to that previously reported as a minor product during purification of the recombinant protein. Initial studies here, show that the Lys389 mutant retains enzymatic activity. Purification of this truncated protein, which lacks a presumably mobile terminal region, may increase the likelihood of crystallisation of human IDO