Changes in Phospholipid Composition of the Human Cerebellum and Motor Cortex during Normal Ageing.

(1) Background: Changes in phospholipid (phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine, i.e., PC, PE and PS) composition with age in the mitochondrial and microsomal membranes of the human cerebellum and motor cortex were examined and compared to previous analyses of the prefrontal cortex, hippocampus and entorhinal cortex. (2) Methods: Nano-electrospray ionization on a hybrid triple quadrupole−linear ion trap mass spectrometer was used to analyse the brain regions of subjects aged 18−104 years. (3) Results: With age, the cerebellum showed many changes in the major phospholipids (>10% of the phospholipid class). In both membrane types, these included increases in PE 18:0_22:6 and PS 18:0_22:6, decreases in PE 18:0_20:4 and PS 18:0_18:1 and an increase in PC 16:0_16:0 (microsomal membrane only). In addition, twenty-one minor phospholipids also changed. In the motor cortex, only ten minor phospholipids changed with age. With age, the acyl composition of the membranes in the cerebellum increased in docosahexaenoic acid (22:6) and decreased in the arachidonic (20:4) and adrenic (22:4) acids. A comparison of phospholipid changes in the cerebellum, motor cortex and other brain areas is provided. (4) Conclusions: The cerebellum is exceptional in the large number of major phospholipids that undergo changes (with consequential changes in acyl composition) with age, whereas the motor cortex is highly resistant to change.

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.

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.

Tau Is Truncated in Five Regions of the Normal Adult Human Brain.

The truncation of Tau is thought to be important in promoting aggregation, with this feature characterising the pathology of dementias such as Alzheimer disease. Antibodies to the C-terminal and N-terminal regions of Tau were employed to examine Tau cleavage in five human brain regions: the entorhinal cortex, prefrontal cortex, motor cortex, hippocampus, and cerebellum. These were obtained from normal subjects ranging in age from 18 to 104 years. Tau fragments of approximately 40 kDa and 45 kDa with an intact N-terminus retained were found in soluble and insoluble brain fractions. In addition, smaller C-terminal Tau fragments ranging in mass from 17 kDa to 25 kDa were also detected. These findings are consistent with significant Tau cleavage taking place in brain regions from 18 years onwards. It appears that site-specific cleavage of Tau is widespread in the normal human brain, and that large Tau fragments that contain the N-terminus, as well as shorter C-terminal Tau fragments, are present in brain cells across the age range.

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 Aß in the human brain is crosslinked via Glu 22 and the N-terminal amino group.

Profiling and Bioinformatic Analysis of Differentially Expressed Cytokines in Aqueous Humor of High Myopic Eyes - Clues for Anti-VEGF Injections.

Purpose: To investigate the aqueous humor growth factor profile in high myopic eyes and analyze the interaction of differentially expressed cytokines.Methods: A case-control study including aqueous humor samples from 36 high myopic patients and 32 controls was conducted. Quantibody® Human Growth Factor Array was used to screen the presence of 40 growth factors in aqueous humor. Expressions of differential growth factors were validated by Bio-Plex ProTM multiplex bead-based immunoassay. Protein-protein interaction (PPI) and gene ontology (GO) analyses were performed.Results: Growth differentiation factor 15 (GDF-15), hepatocyte growth factor (HGF), and platelet-derived growth factor (PDGF)-AA were found to be significantly higher and vascular endothelial growth factor (VEGF) was detected to be lower in high myopic eyes (all P = .03). Multi-plex bead-based assay further validated the differential expressions of four growth factors and all of them were significantly correlated with axial length (P < .001). Twenty-six proteins were mapped into PPI network and positive regulation of cell migration, cellular component movement, and cell motility were the most enriched biological processes based on GO analysis.Conclusions: Differential expressed cytokines that indicates a distinctive intraocular microenvironment in high myopic eyes might provide clues for pathological changes within high myopic eyes after anti-VEGF injections.

Spatiotemporal changes in the human lens proteome: Critical insights into long-lived proteins.

The ocular lens is a unique tissue that contains an age gradient of cells and proteins ranging from newly differentiated cells containing newly synthesized proteins to cells and proteins that are as old as the organism. Thus, the ocular lens is an excellent model for studying long-lived proteins (LLPs) and the effects of aging and post-translational modifications on protein structure and function. Given the architecture of the lens, with young fiber cells in the outer cortex and the oldest cells in the lens nucleus, spatially-resolved studies provide information on age-specific protein changes. In this review, experimental strategies and proteomic methods that have been used to examine age-related and cataract-specific changes to the human lens proteome are described. Measured spatio-temporal changes in the human lens proteome are summarized and reveal a highly consistent, time-dependent set of modifications observed in transparent human lenses. Such measurements have led to the discovery of cataract-specific modifications and the realization that many animal systems are unsuitable to study many of these modifications. Mechanisms of protein modifications such as deamidation, racemization, truncation, and protein-protein crosslinking are presented and the implications of such mechanisms for other long-lived proteins in other tissues are discussed in the context of age-related neurological diseases. A comprehensive understanding of LLP modifications will enhance our ability to develop new therapies for the delay, prevention or reversal of age-related diseases.

Molecular Processes Implicated in Human Age-Related Nuclear Cataract.

Human age-related nuclear cataract is commonly characterized by four biochemical features that involve modifications to the structural proteins that constitute the bulk of the lens: coloration, oxidation, insolubility, and covalent cross-linking. Each of these is progressive and increases as the cataract worsens. Significant progress has been made in understanding the origin of the factors that underpin the loss of lens transparency. Of these four hallmarks of cataract, it is protein-protein cross-linking that has been the most intransigent, and it is only recently, with the advent of proteomic methodology, that mechanisms are being elucidated. A diverse range of cross-linking processes involving several amino acids have been uncovered. Although other hypotheses for the etiology of cataract have been advanced, it is likely that spontaneous decomposition of the structural proteins of the lens, which do not turn over, is responsible for the age-related changes to the properties of the lens and, ultimately, for cataract. Cataract may represent the first and best characterized of a number of human age-related diseases where spontaneous protein modification leads to ongoing deterioration and, ultimately, a loss of tissue function.

Mechanism of protein cleavage at asparagine leading to protein-protein cross-links.

Long-lived proteins (LLPs) are present in numerous tissues within the human body. With age, they deteriorate, often leading to the formation of irreversible modifications such as peptide bond cleavage and covalent cross-linking. Currently understanding of the mechanism of formation of these cross-links is limited. As part of an ongoing study, proteomics was used to characterise sites of novel covalent cross-linking in the human lens. In this process, Lys residues were found cross-linked to C-terminal aspartates that had been present in the original protein as Asn residues. Cross-links were identified in major lens proteins such as αA-crystallin, αB-crystallin and aquaporin 0. Quantification of the level of an AQP0/AQP0 cross-linked peptide showed increased cross-linking with age and in cataract lenses. Using model peptides, a mechanism of cross-link formation was elucidated that involves spontaneous peptide bond cleavage on the C-terminal side of Asn residues resulting in the formation of a C-terminal succinimide. This succinimide does not form cross-links, but can hydrolyse to a mixture of C-terminal Asn and C-terminal Asp amide peptides. The C-terminal Asp amide is unstable at neutral pH and decomposes to a succinic anhydride. If the side chain of Lys attacks the anhydride, a covalent cross-link will be formed. This multi-step mechanism represents a link between two spontaneous events: peptide bond cleavage at Asn and covalent cross-linking. Since Asn deamidation and cleavage are abundant age-related modifications in LLPs, this finding suggests that such susceptible Asn residues should also be considered as potential sites for spontaneous covalent cross-linking.

Cleavage C-terminal to Asp leads to covalent crosslinking of long-lived human proteins.

With age, long-lived proteins in the human body deteriorate, which can have consequences both for aging and disease. The aging process is often associated with the formation of covalently crosslinked proteins. Currently our knowledge of the mechanism of formation of these crosslinks is limited. In this study, proteomics was used to characterize sites of covalent protein-protein crosslinking and identify a novel mechanism of protein-protein crosslinking in the adult human lens. In this mechanism, Lys residues are crosslinked to C-terminal Asp residues that are formed by non-enzymatic protein truncation. Ten different crosslinks were identified in major lens proteins such as αA-crystallin, αB-crystallin and AQP0. Crosslinking in AQP0 increased significantly with age and also increased significantly in cataract lenses compared with normal lenses. Using model peptides, a mechanism of formation of the Lys-Asp crosslink was elucidated. The mechanism involves spontaneous peptide cleavage on the C-terminal side of Asp residues which can take place in the pH range 5-7.4. Cleavage appears to involve attack by the side chain carboxyl group on the adjacent peptide bond, resulting in the formation of a C-terminal Asp anhydride. This anhydride intermediate can then either react with water to form Asp, or with a nucleophile, such as a free amine group to form a crosslink. If an ε-amino group of Lys or an N-terminal amine group attacks the anhydride, a covalent protein-protein crosslink will be formed. This bi-phasic mechanism represents the first report to link two spontaneous events: protein cleavage and crosslinking that are characteristic of long-lived proteins.

Can the Fact That Myelin Proteins Are Old and Break down Explain the Origin of Multiple Sclerosis in Some People?

Recent discoveries may change the way that multiple sclerosis (MS) is viewed, particularly with regard to the reasons for the untoward immune response. The fact that myelin proteins are long-lived, and that by the time we are adults, they are extensively degraded, alters our perspective on the reasons for the onset of autoimmunity and the origin of MS. For example, myelin basic protein (MBP) from every human brain past the age of 20 years, is so greatly modified, that it is effectively a different protein from the one that was laid down in childhood. Since only a subset of people with such degraded MBP develop MS, a focus on understanding the mechanism of immune responses to central nervous system (CNS) antigens and cerebral immune tolerance appear to be worthwhile avenues to explore. In accord with this, it will be productive to examine why all people, whose brains contain large quantities of a "foreign antigen", do not develop MS. Importantly for the potential causation of MS, MBP from MS patients breaks down differently from the MBP in aged controls. If the novel structures formed in these MS-specific regions are particularly antigenic, it could help explain the origin of MS. If verified, these findings could provide an avenue for the rational synthesis of drugs to prevent and treat MS.

Spontaneous cross-linking of proteins at aspartate and asparagine residues is mediated via a succinimide intermediate.

The breakdown of long-lived proteins (LLPs) is associated with aging, as well as disease; however, our understanding of the molecular processes involved is still limited. Of particular relevance, cross-linked proteins are often reported in aged tissues but the mechanisms for their formation are poorly understood. In the present study, sites of protein cross-linking in human ocular lenses were characterized using proteomic techniques. In long-lived lens proteins, several sites of cross-linking were found to involve the addition of Lys to Asp or Asn residues. Using model peptides containing Asp or Asn, a mechanism was elucidated that involves a succinimide intermediate. Succinimides formed readily from Asn at neutral pH, whereas a higher rate of formation from Asp peptides was observed at more acidic pHs. Succinimides were found to be relatively stable in the absence of nucleophiles. Since racemization of Asp residues, as well as deamidation of Asn, involves a succinimide intermediate, sites of d-Asp and isoAsp in LLPs should also be considered as potential sites of protein covalent cross-linking.

Proteostasis and the Regulation of Intra- and Extracellular Protein Aggregation by ATP-Independent Molecular Chaperones: Lens α-Crystallins and Milk Caseins.

Molecular chaperone proteins perform a diversity of roles inside and outside the cell. One of the most important is the stabilization of misfolding proteins to prevent their aggregation, a process that is potentially detrimental to cell viability. Diseases such as Alzheimer's, Parkinson's, and cataract are characterized by the accumulation of protein aggregates. In vivo, many proteins are metastable and therefore under mild destabilizing conditions have an inherent tendency to misfold, aggregate, and hence lose functionality. As a result, protein levels are tightly regulated inside and outside the cell. Protein homeostasis, or proteostasis, describes the network of biological pathways that ensures the proteome remains folded and functional. Proteostasis is a major factor in maintaining cell, tissue, and organismal viability. We have extensively investigated the structure and function of intra- and extracellular molecular chaperones that operate in an ATP-independent manner to stabilize proteins and prevent their misfolding and subsequent aggregation into amorphous particles or highly ordered amyloid fibrils. These types of chaperones are therefore crucial in maintaining proteostasis under normal and stress (e.g., elevated temperature) conditions. Despite their lack of sequence similarity, they exhibit many common features, i.e., extensive structural disorder, dynamism, malleability, heterogeneity, oligomerization, and similar mechanisms of chaperone action. In this Account, we concentrate on the chaperone roles of α-crystallins and caseins, the predominant proteins in the eye lens and milk, respectively. Intracellularly, the principal ATP-independent chaperones are the small heat-shock proteins (sHsps). In vivo, sHsps are the first line of defense in preventing intracellular protein aggregation. The lens proteins αA- and αB-crystallin are sHsps. They play a crucial role in maintaining solubility of the crystallins (including themselves) with age and hence in lens proteostasis and, ultimately, lens transparency. As there is little metabolic activity and no protein turnover in the lens, crystallins are very long lived proteins. Lens proteostasis is therefore very different to that in normal, metabolically active cells. Crystallins undergo extensive post-translational modification (PTM), including deamidation, racemization, phosphorylation, and truncation, which can alter their stability. Despite this, the lens remains transparent for tens of years, implying that lens proteostasis is intimately integrated with crystallin PTMs. Many PTMs do not significantly alter crystallin stability, solubility, and functionality, which thereby facilitates lens transparency. In the long term, however, extensive accumulation of crystallin PTMs leads to large-scale crystallin aggregation, lens opacification, and cataract formation. Extracellularly, various ATP-independent molecular chaperones exist that exhibit sHsp-like structural and functional features. For example, caseins, the major milk proteins, exhibit chaperone ability by inhibiting the amorphous and amyloid fibrillar aggregation of a diversity of destabilized proteins. Caseins maintain proteostasis within milk by preventing deleterious casein amyloid fibril formation via incorporation of thousands of individual caseins into an amorphous structure known as the casein micelle. Hundreds of nanoclusters of calcium phosphate are sequestered within each casein micelle through interactions with short, highly phosphorylated casein sequences. This results in a stable biofluid that contains a high concentration of potentially amyloidogenic caseins and concentrations of calcium and phosphate that can be far in excess of the solubility of calcium phosphate. Casein micelle formation therefore performs vital roles in neonatal nutrition and calcium homeostasis in the mammary gland.

DehydroalanylGly, a new post translational modification resulting from the breakdown of glutathione.

BACKGROUND: The human body contains numerous long-lived proteins which deteriorate with age, typically by racemisation, deamidation, crosslinking and truncation. Previously we elucidated one reaction responsible for age-related crosslinking, the spontaneous formation of dehydroalanine (DHA) intermediates from phosphoserine and cysteine. This resulted in non-disulphide covalent crosslinks. The current paper outlines a novel posttranslational modification (PTM) in human proteins, which involves the addition of dehydroalanylglycine (DHAGly) to Lys residues. METHODS: Human lens digests were examined by mass spectrometry for the presence of (DHA)Gly (+144.0535 Da) adducts to Lys residues. Peptide model studies were undertaken to elucidate the mechanism of formation. RESULTS: In the lens, this PTM was detected at 18 lysine sites in 7 proteins. Using model peptides, a pathway for its formation was found to involve initial formation of the glutathione degradation product, γ-Glu(DHA)Gly from oxidised glutathione (GSSG). Once the Lys adduct formed, the Glu residue was lost in a hydrolytic mechanism apparently catalysed by the ε-amino group of the Lys. CONCLUSIONS: This discovery suggests that within cells, the functional groups of amino acids in proteins may be susceptible to modification by reactive metabolites derived from GSSG. GENERAL SIGNIFICANCE: Our finding demonstrates a novel +144.0535 Da PTM arising from the breakdown of oxidised glutathione.

Hotspots of age-related protein degradation: the importance of neighboring residues for the formation of non-disulfide crosslinks derived from cysteine.

Over time, the long-lived proteins that are present throughout the human body deteriorate. Typically, they become racemized, truncated, and covalently cross-linked. One reaction responsible for age-related protein cross-linking in the lens was elucidated recently and shown to involve spontaneous formation of dehydroalanine (DHA) intermediates from phosphoserine. Cys residues are another potential source of DHA, and evidence for this was found in many lens crystallins. In the human lens, some sites were more prone to forming non-disulfide covalent cross-links than others. Foremost among them was Cys5 in ßA4 crystallin. The reason for this enhanced reactivity was investigated using peptides. Oxidation of Cys to cystine was a prerequisite for DHA formation, and DHA production was accelerated markedly by the presence of a Lys, one residue separated from Cys5. Modeling and direct investigation of the N-terminal sequence of ßA4 crystallin, as well as a variety of homologous peptides, showed that the epsilon amino group of Lys can promote DHA production by nucleophilic attack on the alpha proton of cystine. Once a DHA residue was generated, it could form intermolecular cross-links with Lys and Cys. In the lens, the most abundant cross-link involved Cys5 of ßA4 crystallin attached via a thioether bond to glutathione. These findings illustrate the potential of Cys and disulfide bonds to act as precursors for irreversible covalent cross-links and the role of nearby amino acids in creating 'hotpsots' for the spontaneous processes responsible for protein degradation in aged tissues.

The phospholipid composition of the human entorhinal cortex remains relatively stable over 80 years of adult aging.

Membrane lipid composition is altered in the brain during the pathogenesis of several age-related neurodegenerative diseases, including Alzheimer's disease. The entorhinal cortex is one of the first regions of the brain to display the neuropathology typical of Alzheimer's disease, yet little is known about the changes that occur in membrane lipids within this brain region during normal aging (i.e., in the absence of dementia). In the present study, the phospholipid composition of mitochondrial and microsomal membranes from human entorhinal cortex was examined for any changes over the adult lifespan (18-98 years). Overall, changes in several molecular phospholipids were seen with age in the entorhinal cortex across both membranes. The proportion of total phosphatidylcholine within the mitochondrial fraction increased within the entorhinal cortex with age, while total mitochondrial phosphatidylethanolamine decreased. Many mitochondrial phosphatidylethanolamines containing docosahexaenoic acid increased with age; however, this did not translate into an overall age-related increase in total mitochondrial docosahexaenoic acid. The most abundant phospholipid present within the human brain, PC 16:0_18:1, also increased with age within the mitochondrial membranes of the entorhinal cortex. When compared to other regions of the brain, the phospholipid composition of the entorhinal cortex remains relatively stable in adults over the lifespan in the absence of dementia.

Isoaspartic acid is present at specific sites in myelin basic protein from multiple sclerosis patients: could this represent a trigger for disease onset?

Multiple sclerosis (MS) is associated with breakdown of the myelin sheath that coats neurons in the central nervous system. The cause of MS is not known, although the pathogenesis involves destruction of myelin by the immune system. It was the aim of this study to examine the abundant myelin protein, myelin basic protein (MBP), to determine if there are sites of modification that may be characteristic for MS. MBP from the cerebellum was examined from controls and MS patients across the age range using mass spectrometry and amino acid analysis. Amino acid racemization data indicated that myelin basic protein is long-lived and proteomic analysis of MBP showed it to be highly modified. A common modification of MBP was racemization of Asp and this was significantly greater in MS patients. In long-lived proteins, L-Asp and L-Asn can racemize to three other isomers, D-isoAsp, L-isoAsp and D-Asp and this is significant because isoAsp formation in peptides renders them immunogenic.Proteomic analysis revealed widespread modifications of MBP with two surface regions that are altered in MS. In particular, isoAsp was significantly elevated at these sites in MS patients. The generation of isoAsp could be responsible for eliciting an immune response to modified MBP and therefore be implicated in the etiology of MS.

Old Proteins in Man: A Field in its Infancy.

It has only recently been appreciated that the human body contains many long-lived proteins (LLPs). Their gradual degradation over time contributes to human aging and probably also to a range of age-related disorders. Indeed, the role of progressive damage of proteins in aging may be indicated by the fact that many neurological diseases do not appear until after middle age. A major factor responsible for the deterioration of old proteins is the spontaneous breakdown of susceptible amino acid residues resulting in racemization, truncation, deamidation, and crosslinking. When proteins decompose in this way, their structures and functions may be altered and novel epitopes can be formed that can induce an autoimmune response.

Cross-linking of lens crystallin proteins induced by tryptophan metabolites and metal ions: implications for cataract development.

Long-wavelength solar UV radiation is implicated in photodamage to the human eye. The human lens contains multiple tryptophan-derived compounds that have significant absorbance bands in the UVA region (λ 315-400 nm) that act as efficient physical filters for these wavelengths. The concentrations of many of these UV filter compounds decrease with increase in age, resulting in diminished protection, increased oxidative damage and the accumulation of modified proteins implicated in nuclear cataract formation. This damage may arise via the formation of α,ß-unsaturated carbonyls from the UV filter compounds, adduction to lens proteins and subsequent action as photosensitizers, and/or via the reactions of redox-active transition metal ions that accumulate in aged human lenses. The latter may promote the oxidation of free, or protein-bound, o-aminophenols, such as the UV filter compounds 3-hydroxykynurenine (3OHKyn) and 3-hydroxyanthranilic acid (3OHAA). It is shown here that Cu(II), and to a lesser extent Fe(III), enhance oxidation of free 3OHKyn, 3OHAA and 3OHKyn bound to specific amino acids and lens proteins, with this resulting in increased cross-linking of lens proteins. These data indicate that elevated levels of transition metal ions in aging lenses can enhance the loss of protective UV filter compounds, and contribute to the formation of high-molecular-mass dysfunctional crystallin proteins in a light-independent manner. These reactions may contribute to the formation of lens cataracts in humans.

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.