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Mechanism of protein cleavage at asparagine leading to protein-protein cross-links.
Friedrich MG, Wang Z, Schey KL, Truscott RJW
(2019) Biochem J 476: 3817-3834
MeSH Terms: Amino Acid Sequence, Aquaporins, Asparagine, Crystallins, Eye Proteins, Humans, Hydrolysis, Lens, Crystalline, Proteins, Proteolysis
Show Abstract · Added March 24, 2020
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.
© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.
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10 MeSH Terms
MALDI imaging mass spectrometry of β- and γ-crystallins in the ocular lens.
Anderson DM, Nye-Wood MG, Rose KL, Donaldson PJ, Grey AC, Schey KL
(2020) J Mass Spectrom 55: e4473
MeSH Terms: Adult, Age Factors, Animals, Cattle, Humans, Lens, Crystalline, Middle Aged, Molecular Imaging, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, beta-Crystallins, gamma-Crystallins
Show Abstract · Added March 24, 2020
Lens crystallin proteins make up 90% of expressed proteins in the ocular lens and are primarily responsible for maintaining lens transparency and establishing the gradient of refractive index necessary for proper focusing of images onto the retina. Age-related modifications to lens crystallins have been linked to insolubilization and cataractogenesis in human lenses. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) has been shown to provide spatial maps of such age-related modifications. Previous work demonstrated that, under standard protein IMS conditions, α-crystallin signals dominated the mass spectrum and age-related modifications to α-crystallins could be mapped. In the current study, a new sample preparation method was optimized to allow imaging of β- and γ-crystallins in ocular lens tissue. Acquired images showed that γ-crystallins were localized predominately in the lens nucleus whereas β-crystallins were primarily localized to the lens cortex. Age-related modifications such as truncation, acetylation, and carbamylation were identified and spatially mapped. Protein identifications were determined by top-down proteomics analysis of lens proteins extracted from tissue sections and analyzed by LC-MS/MS with electron transfer dissociation. This new sample preparation method combined with the standard method allows the major lens crystallins to be mapped by MALDI IMS.
© 2019 John Wiley & Sons, Ltd.
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11 MeSH Terms
Cleavage C-terminal to Asp leads to covalent crosslinking of long-lived human proteins.
Wang Z, Friedrich MG, Truscott RJW, Schey KL
(2019) Biochim Biophys Acta Proteins Proteom 1867: 831-839
MeSH Terms: Aquaporins, Aspartic Acid, Eye Proteins, Humans, Hydrogen-Ion Concentration, Lens, Crystalline, Models, Molecular, Peptides, alpha-Crystallin A Chain, alpha-Crystallin B Chain
Show Abstract · Added March 24, 2020
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.
Copyright © 2019 Elsevier B.V. All rights reserved.
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Proteomic Analysis of S-Palmitoylated Proteins in Ocular Lens Reveals Palmitoylation of AQP5 and MP20.
Wang Z, Schey KL
(2018) Invest Ophthalmol Vis Sci 59: 5648-5658
MeSH Terms: Animals, Aquaporin 5, Blotting, Western, Cattle, Chromatography, Liquid, Electrophoresis, Polyacrylamide Gel, Eye Proteins, Immunoblotting, Lens, Crystalline, Lipoylation, Membrane Proteins, Palmitates, Proteomics, Tandem Mass Spectrometry
Show Abstract · Added April 4, 2019
Purpose - The purpose of this study was to characterize the palmitoyl-proteome in lens fiber cells. S-palmitoylation is the most common form of protein S-acylation and the reversible nature of this modification functions as a molecular switch to regulate many biological processes. This modification could play important roles in regulating protein functions and protein-protein interactions in the lens.
Methods - The palmitoyl-proteome of bovine lens fiber cells was investigated by combining acyl-biotin exchange (ABE) chemistry and mass-spectrometry analysis. Due to the possibility of false-positive results from ABE experiment, a method was also developed for direct detection of palmitoylated peptides by mass spectrometry for validating palmitoylation of lens proteins MP20 and AQP5. Palmitoylation levels on AQP5 in different regions of the lens were quantified after iodoacetamide (IAA)-palmitate exchange.
Results - The ABE experiment identified 174 potential palmitoylated proteins. These proteins include 39 well-characterized palmitoylated proteins, 92 previously reported palmitoylated proteins in other tissues, and 43 newly identified potential palmitoylated proteins including two important transmembrane proteins in the lens, AQP5 and MP20. Further analysis by direct detection of palmitoylated peptides confirmed palmitoylation of AQP5 on C6 and palmitoylation of MP20 on C159. Palmitoylation of AQP5 was found to only occur in a narrow region of the inner lens cortex and does not occur in the lens epithelium, in the lens outer cortex, or in the lens nucleus.
Conclusions - AQP5 and MP20 are among 174 palmitoylated proteins found in bovine lens fiber cells. This modification to AQP5 and MP20 may play a role in their translocation from the cytoplasm to cell membranes during fiber cell differentiation.
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14 MeSH Terms
Spontaneous cross-linking of proteins at aspartate and asparagine residues is mediated via a succinimide intermediate.
Friedrich MG, Wang Z, Schey KL, Truscott RJW
(2018) Biochem J 475: 3189-3200
MeSH Terms: Aged, Amino Acid Sequence, Asparagine, Aspartic Acid, Humans, Lens, Crystalline, Succinimides
Show Abstract · Added April 4, 2019
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.
© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.
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7 MeSH Terms
Quantification of thioether-linked glutathione modifications in human lens proteins.
Wang Z, Schey KL
(2018) Exp Eye Res 175: 83-89
MeSH Terms: Adolescent, Alanine, Aminobutyrates, Cataract, Cellular Senescence, Chromatography, Liquid, Crystallins, Cysteine, Glutathione, Humans, Lens, Crystalline, Middle Aged, Protein Processing, Post-Translational, Serine, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Sulfides, Threonine, Tissue Donors, Young Adult
Show Abstract · Added April 4, 2019
Dehydroalanine (DHA) and dehydrobutyrine (DHB) intermediates, formed through β-elimination, induce protein irreversible glutathionylation and protein-protein crosslinking in human lens fiber cells. In total, irreversible glutathionylation was detected on 52 sites including cysteine, serine and threonine residues in 18 proteins in human lenses. In this study, the levels of GSH modification on three serine residues and four cysteine residues located in seven different lens proteins isolated from different regions and different aged lenses were quantified. The relative levels of modification (modified/nonmodified) were site-specific and age-related, ranging from less than 0.05% to about 500%. The levels of modification on all of the sites quantified in the lens cortex increased with age and GSH modification also increased from cortex to outer nucleus region suggesting an age-related increase of modification. The levels of modification on sites located in stable regions of the proteins such as Cys117 of βA3, Cys80 of βB1 and Cys27 of γS, continued increasing in inner nucleus, but modification on sites located in regions undergoing degradation with age decreased in the inner nucleus suggesting GSH modified proteins were more susceptible to further modification. Irreversible GSH modification in cataract lenses was typically higher than in age-matched normal lenses, but the difference did not reach statistical significance for a majority of sites, with the exception Cys117 of βA3 crystallin in WSF. Except for S59 of αA and αB crystallins, GSH modification did not induce protein insolubility suggesting a possible role for this modification in protection from protein-protein crosslinking.
Copyright © 2018 Elsevier Ltd. All rights reserved.
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DehydroalanylGly, a new post translational modification resulting from the breakdown of glutathione.
Friedrich MG, Wang Z, Schey KL, Truscott RJW
(2018) Biochim Biophys Acta Gen Subj 1862: 907-913
MeSH Terms: Alanine, Amino Acid Sequence, Crystallins, Dipeptides, Glutathione, Glutathione Disulfide, Humans, Lens, Crystalline, Lysine, Middle Aged, Molecular Structure, Peptides, Protein Conformation, Protein Processing, Post-Translational, Proteins, Tandem Mass Spectrometry, Young Adult
Show Abstract · Added April 3, 2018
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.
Copyright © 2018. Published by Elsevier B.V.
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The Role of Aquaporins in Ocular Lens Homeostasis.
Schey KL, Petrova RS, Gletten RB, Donaldson PJ
(2017) Int J Mol Sci 18:
MeSH Terms: Animals, Aquaporins, Biological Transport, Active, Eye Proteins, Homeostasis, Humans, Lens, Crystalline, Permeability, Protein Isoforms, Water
Show Abstract · Added April 3, 2018
Aquaporins (AQPs), by playing essential roles in the maintenance of ocular lens homeostasis, contribute to the establishment and maintenance of the overall optical properties of the lens over many decades of life. Three aquaporins, AQP0, AQP1 and AQP5, each with distinctly different functional properties, are abundantly and differentially expressed in the different regions of the ocular lens. Furthermore, the diversity of AQP functionality is increased in the absence of protein turnover by age-related modifications to lens AQPs that are proposed to alter AQP function in the different regions of the lens. These regional differences in AQP functionality are proposed to contribute to the generation and directionality of the lens internal microcirculation; a system of circulating ionic and fluid fluxes that delivers nutrients to and removes wastes from the lens faster than could be achieved by passive diffusion alone. In this review, we present how regional differences in lens AQP isoforms potentially contribute to this microcirculation system by highlighting current areas of investigation and emphasizing areas where future work is required.
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10 MeSH Terms
Loss of αB-crystallin function in zebrafish reveals critical roles in the development of the lens and stress resistance of the heart.
Mishra S, Wu SY, Fuller AW, Wang Z, Rose KL, Schey KL, Mchaourab HS
(2018) J Biol Chem 293: 740-753
MeSH Terms: Animals, Cardiomyopathies, Edema, Glucocorticoids, Image Processing, Computer-Assisted, Lens, Crystalline, Molecular Chaperones, Mutation, Myocardium, Pericardium, Phenotype, Receptors, Glucocorticoid, Signal Transduction, Stress, Physiological, Transgenes, Zebrafish, alpha-Crystallin A Chain, alpha-Crystallin B Chain
Show Abstract · Added April 3, 2018
Genetic mutations in the human small heat shock protein αB-crystallin have been implicated in autosomal cataracts and skeletal myopathies, including heart muscle diseases (cardiomyopathy). Although these mutations lead to modulation of their chaperone activity , the functions of αB-crystallin in the maintenance of both lens transparency and muscle integrity remain unclear. This lack of information has hindered a mechanistic understanding of these diseases. To better define the functional roles of αB-crystallin, we generated loss-of-function zebrafish mutant lines by utilizing the CRISPR/Cas9 system to specifically disrupt the two αB-crystallin genes, α and α We observed lens abnormalities in the mutant lines of both genes, and the penetrance of the lens phenotype was higher in α than α mutants. This finding is in contrast with the lack of a phenotype previously reported in αB-crystallin knock-out mice and suggests that the elevated chaperone activity of the two zebrafish orthologs is critical for lens development. Besides its key role in the lens, we uncovered another critical role for αB-crystallin in providing stress tolerance to the heart. The αB-crystallin mutants exhibited hypersusceptibility to develop pericardial edema when challenged by crowding stress or exposed to elevated cortisol stress, both of which activate glucocorticoid receptor signaling. Our work illuminates the involvement of αB-crystallin in stress tolerance of the heart presumably through the proteostasis network and reinforces the critical role of the chaperone activity of αB-crystallin in the maintenance of lens transparency.
© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
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18 MeSH Terms
Dynamic functional contribution of the water channel AQP5 to the water permeability of peripheral lens fiber cells.
Petrova RS, Webb KF, Vaghefi E, Walker K, Schey KL, Donaldson PJ
(2018) Am J Physiol Cell Physiol 314: C191-C201
MeSH Terms: Animals, Aquaporin 5, Aquaporins, Cell Membrane, Epithelial Cells, Eye Proteins, Lens, Crystalline, Mercuric Chloride, Mice, Inbred C57BL, Models, Biological, Organ Culture Techniques, Permeability, Rats, Wistar, Species Specificity, Time Factors, Water
Show Abstract · Added April 3, 2018
Although the functionality of the lens water channels aquaporin 1 (AQP1; epithelium) and AQP0 (fiber cells) is well established, less is known about the role of AQP5 in the lens. Since in other tissues AQP5 functions as a regulated water channel with a water permeability (P) some 20 times higher than AQP0, AQP5 could function to modulate P in lens fiber cells. To test this possibility, a fluorescence dye dilution assay was used to calculate the relative P of epithelial cells and fiber membrane vesicles isolated from either the mouse or rat lens, in the absence and presence of HgCl, an inhibitor of AQP1 and AQP5. Immunolabeling of lens sections and fiber membrane vesicles from mouse and rat lenses revealed differences in the subcellular distributions of AQP5 in the outer cortex between species, with AQP5 being predominantly membranous in the mouse but predominantly cytoplasmic in the rat. In contrast, AQP0 labeling was always membranous in both species. This species-specific heterogeneity in AQP5 membrane localization was mirrored in measurements of P, with only fiber membrane vesicles isolated from the mouse lens, exhibiting a significant Hg-sensitive contribution to P. When rat lenses were first organ cultured, immunolabeling revealed an insertion of AQP5 into cortical fiber cells, and a significant increase in Hg-sensitive P was detected in membrane vesicles. Our results show that AQP5 forms functional water channels in the rodent lens, and they suggest that dynamic membrane insertion of AQP5 may regulate water fluxes in the lens by modulating P in the outer cortex.
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16 MeSH Terms