Intracellular Degradation and Deamidation of α‐Crystallin Subunits

Kleef, Frans S.M. van; Willems‐Thijssen, Willy; Hoenders, Herman J.

doi:10.1111/j.1432-1033.1976.tb10572.x

Cited by 79 publications

(27 citation statements)

References 12 publications

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“…The major components of peak 5 were aB1 and aBo; aB2 was a minor component. Both peaks 4 and 5 contained minor components with molecular weights that are consistent with the degradation products reported by Van Kleef et al (1976).…”

Section: Identification Of Crystallins By Esimssupporting

confidence: 70%

“…The nomenclature of the m-and P-crystalIins describes whether they are acidic (e.g., aA2, and PA3) or basic (aB2 and f3B2). Early investigators of the lens crystallins attributed the altered electrophoretic mobility of several a-crystallins to deamidation of asparagine (Bloemendal et al, 1972;Stauffer et al, 1974) and C-terminal degradation (Van Kleef et al, 1976). In 1985, Spector et al (1985) showed that radiolabeled phosphate could be incorporated into both &A-and aB-crystallins, with evidence that serine was the only phosphorylated residue.…”

Section: Introductionmentioning

confidence: 99%

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Identification of the posttranslational modifications of bovine lens αB‐crystallins by mass spectrometry

et al. 1992

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A combination of mass spectrometric techniques has been used to investigate the amino acid sequence and posttranslational modifications of aB-crystallin isolated from bovine lenses by gel filtration chromatography and reversed-phase high performance liquid chromatography. Chromatographic fractions were analyzed by electrospray ionization mass spectrometry to determine the homogeneity and molecular weights of proteins in the fractions. The aB-crystallin primary gene product, its mono-and diphosphorylated forms, its N-and C-terminal truncated forms, as well as other lens proteins unrelated to the aB-crystallins were identified by their molecular weights.Detailed information about the sites of phosphorylation, as well as evidence supporting reassignment of Asn to Asp at position 80, was obtained by analyzing proteolytic digests of these proteins by fast atom bombardment mass spectrometry. Results of this investigation indicate that aB-crystallin is phosphorylated in vivo at Ser 45, Ser 59, and either Ser 19 or 21. From the specificity of phosphorylation of a-crystallins, it appears that there may be two different kinases responsible for their phosphorylation.Keywords: crystallins; lens proteins; mass spectrometry; protein phosphorylationThe lens crystallins are long-lived structural proteins of particular interest in understanding cataractogenesis (Harding, 1985) and chemical processes associated with aging (Harding & Dilley, 1976). There are three primary groups of lens crystallins, a-, P-, and y-crystallins, which can be separated by the size of aggregates they form. a-crystallins form aggregates with molecular weights over 500,000; &crystallin aggregates have molecular weights of 40,000-200,000; y-crystallins remain as monomers of 20,000. The molecular weights of the monomers of aand P-crystallins are also approximately 20,000. The nomenclature of the m-and P-crystalIins describes whether they are acidic (e.g., aA2, and PA3) or basic (aB2 and f3B2). Early investigators of the lens crystallins attributed the altered electrophoretic mobility of several a-crystallins to deamidation of asparagine (Bloemendal et al., 1972;Stauffer et al., 1974) and C-terminal degradation (Van Kleef et al., 1976). In 1985, Spector et al. (1985) showed that radiolabeled phosphate could be incorporated into both &A-and aB-crystallins, with evidence that serine was the only phosphorylated residue. Their as- sumption that electrophoretic behavior originally attributed to deamidation is due to phosphorylation (Chiesa et al., 1987(Chiesa et al., , 1988) was substantiated by quantification of phosphorus in a-crystallins (Voorter et al., 1989). The C-terminaI degradation and phosphorylation of aA-crystallins were confirmed recently by mass spectrometry . The fact that the a-crystallins could be phosphorylated suggested that they might have biological functions other than their contribution to lens structure. Using radioactively labeled phosphate Chiesa et al. (1987Chiesa et al. ( , 1988 determined that the phosphorylation of aBcrystal...

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Section: Identification Of Crystallins By Esimssupporting

confidence: 70%

Section: Introductionmentioning

confidence: 99%

Identification of the posttranslational modifications of bovine lens αB‐crystallins by mass spectrometry

et al. 1992

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“…One manner in which proteins can be marked for degradation is via deamidation [155][156][157]. Acting as a "molecular clock" during physiological conditions, there is suggestion that deamidation may also signal proteolysis with pathogenesis [158,159]. Several studies on individual proteins have shown a significant role for protein deamidation in pathogenesis, including the deamidation of Asp 146 in a B-crystalline, which has been significantly correlated with cataractogenesis [158,159].…”

Section: Mitochondrial Signalingmentioning

confidence: 99%

“…Acting as a "molecular clock" during physiological conditions, there is suggestion that deamidation may also signal proteolysis with pathogenesis [158,159]. Several studies on individual proteins have shown a significant role for protein deamidation in pathogenesis, including the deamidation of Asp 146 in a B-crystalline, which has been significantly correlated with cataractogenesis [158,159]. Proteomics has been used to show that deamidated proteins undergo differential modifications with I/R injury, where fragmentation of deamidated myosin light chain 2 (MLC-2) resulted from ROS generation during reperfusion [88], whilst deami-dated a B-crystalline decreased during the preceding ischemia [160], suggesting further differential proteolysis.…”

Section: Mitochondrial Signalingmentioning

confidence: 99%

Mitochondria: A mirror into cellular dysfunction in heart disease

White

Edwards

Cordwell

et al. 2008

Proteomics Clinical Apps

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Cardiovascular (CV) disease is the single most significant cause of morbidity and mortality worldwide. The emerging global impact of CV disease means that the goals of early diagnosis and a wider range of treatment options are now increasingly pertinent. As such, there is a greater need to understand the molecular mechanisms involved and potential targets for intervention. Mitochondrial function is important for physiological maintenance of the cell, and when this function is altered, the cell can begin to suffer. Given the broad range and significant impacts of the cellular processes regulated by the mitochondria, it becomes important to understand the roles of the proteins associated with this organelle. Proteomic investigations of the mitochondria are hampered by the intrinsic properties of the organelle, including hydrophobic mitochondrial membranes; high proportion of basic proteins (pI greater than 8.0); and the relative dynamic range issues of the mitochondria. For these reasons, many proteomic studies investigate the mitochondria as a discrete subproteome. Once this has been achieved, the alterations that result in functional changes with CV disease can be observed. Those alterations that lead to changes in mitochondrial function, signaling and morphology, which have significant implications for the cardiomyocyte in the development of CV disease, are discussed.

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“…1B) and is in agreement with previous observations for C-terminal-deleted forms of ␣B-crystallin (34 -37). Removal of the 5 most C-terminal residues generated the A171X mutant and is equivalent to the truncated form of ␣B-crystallin found in normal rat lenses (38), bovine lenses (39), and in rat models of diabetic cataracts (40). Another C-terminal-truncated form of ␣B-crystallin (E164X) has 12 residues removed from the C terminus and this has been detected in normal rat lenses (41).…”

Section: Expression and Purification Of The C-terminal Extensionmentioning

confidence: 99%

Truncation of αB-Crystallin by the Myopathy-causing Q151X Mutation Significantly Destabilizes the Protein Leading to Aggregate Formation in Transfected Cells

Hayes

Devlin

Quinlan

2008

Journal of Biological Chemistry

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Here we investigate the effects of a myopathy-causing mutation in ␣B-crystallin, Q151X, upon its structure and function. This mutation removes the C-terminal domain of ␣B-crystallin, which is expected to compromise both its oligomerization and chaperone activity. We compared this to two other ␣B-crystallin mutants (450delA, 464delCT) and also to a series of C-terminal truncations (E164X, E165X, K174X, and A171X). We find that the effects of the Q151X mutation were not always as predicted. Specifically, we have found that although the Q151X mutation decreased oligomerization of ␣B-crystallin and even increased some chaperone activities, it also significantly destabilized ␣B-crystallin causing it to self-aggregate. This conclusion was supported by our analyses of both the other disease-causing mutants and the series of C-terminal truncation constructs of ␣B-crystallin. The 450delA and 464delCT mutants could only be refolded and assayed as a complex with wild type ␣B-crystallin, which was not the case for Q151X ␣B-crystallin. From these studies, we conclude that all three disease-causing mutations (450delA, 464delCT, and Q151X) in the C-terminal extension destabilize ␣B-crystallin and increase its tendency to self-aggregate. We propose that it is this, rather than a catastrophic loss of chaperone activity, which is a major factor in the development of the reported diseases for the three disease-causing mutations studied here. In support of this hypothesis, we show that Q151X ␣B-crystallin is found mainly in the insoluble fraction of cell extracts from transient transfected cells, due to the formation of cytoplasmic aggregates.The crystallins were first identified as the major proteins of the eye lens and their subsequent classification into ␣-, ␤-, and ␥-crystallins (1) allowed different functional properties to be assigned to the ␣-and ␤␥-crystallin groups (2). The ␣-crystallins are two distinct proteins derived from two separate genes, ␣A-and ␣B-crystallin (2). Whereas ␣A-crystallin is almost entirely lens specific (3), ␣B-crystallin is expressed widely in other tissues, most notably astrocytes (4) and muscle (5). In the early 1980s, pioneering work from Klemenz and co-workers (6) and Horwitz (7) laboratories established that ␣B-crystallin was a protein chaperone and a member of the small heat shock protein (sHSP) 2 family, that is now known to comprise 10 different human proteins (8).When the first mutation (R120G) in ␣B-crystallin was reported, it was found to cause both cataract and desmin-related myopathy in the affected patients (9). Characteristic histopathological aggregates of the muscle intermediate filament protein desmin were observed (9), strongly suggesting that there is an important functional interaction between intermediate filaments and ␣B-crystallin. Subsequent analyses showed that the R120G ␣B-crystallin mutation induced increased binding for desmin intermediate filaments (10), providing an explanation for the formation of the desmin aggregates in the muscles of the affected individuals. Coinci...

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Intracellular Degradation and Deamidation of α‐Crystallin Subunits

Cited by 79 publications

References 12 publications

Identification of the posttranslational modifications of bovine lens αB‐crystallins by mass spectrometry

Identification of the posttranslational modifications of bovine lens αB‐crystallins by mass spectrometry

Mitochondria: A mirror into cellular dysfunction in heart disease

Truncation of αB-Crystallin by the Myopathy-causing Q151X Mutation Significantly Destabilizes the Protein Leading to Aggregate Formation in Transfected Cells

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