Effects of Site-directed Mutations on the Chaperone-like Activity of αB-Crystallin

Plater, M. L.; Goode, Derek; Crabbe, M. James C.

doi:10.1074/jbc.271.45.28558

Cited by 140 publications

(131 citation statements)

References 35 publications

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“…Deletion of the two C-terminal lysines (K174X) did not alter significantly the oligomerization of ␣B-crystallin, as previously reported (33). Removal of 5 C-terminal residues, however, reduced the apparent size of the main peak, as did the removal of both 11 (E165X) and 12 (E164X) residues.…”

Section: Expression and Purification Of The C-terminal Extensionmentioning

confidence: 48%

“…The K174X construct removes the two most C-terminal lysine residues and would be expected to retain the secondary structural characteristics and oligomerization characteristics of ␣B-crystallin (33). The electrophoretic mobility of this K174X mutant was increased relative to the wild type ␣B-crystallin (Fig.…”

Section: Expression and Purification Of The C-terminal Extensionmentioning

confidence: 97%

“…The 450delA (arrow) and 464delCT (arrowhead) mutants could not be produced as individual soluble proteins and therefore were refolded in the presence of wild type ␣B-crystallin. Molecular weight markers (track M) are indicated (F) in order of increasing relative mobility (40,33,24, and 17 kDa). The relative molecular weights of the wild type and mutant ␣B-crystallins (C) were confirmed by mass spectrometry and compared with values calculated from the amino acid sequences using the Emboss Pepstats program.…”

Section: Expression and Purification Of The C-terminal Extensionmentioning

confidence: 99%

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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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Section: Expression and Purification Of The C-terminal Extensionmentioning

confidence: 48%

Section: Expression and Purification Of The C-terminal Extensionmentioning

confidence: 97%

Section: Expression and Purification Of The C-terminal Extensionmentioning

confidence: 99%

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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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“…It has been sugthe crystallin proteins, decreased chaperone-like activity [19] gested that far from having a deleterious effect on the lens, the and possibly osmotic stress. Evidence now seems to rule out accumulation of polyol may have a protective effect against AR2 as the principle enzyme of polyol metabolism in the lens.…”

Section: Udp-glucuronyl Transferasementioning

confidence: 99%

Accumulation of xylitol in the mammalian lens is related to glucuronate metabolism

1996

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Cataract remains the major cause of blindness worldwide and a common complication of diabetes. Polyol Preparation of lens lysates accumulation in the lens is associated with cataract formation.Freshly dissected bovine or rat lenses were homgenised in 2 ml of a Here we present evidence for a novel pathway for xylitol suitable ice-chilled assay buffer, using a manual Braun Homogeniser. production in the lens involving glucuronate metabolism. Xylitol Membranes and cell debris were removed by brief centrifugation in a can be produced in rat and bovine lens from glucose, via the benchtop centrifuge (at maximum speed for 2 rain) and the cleared enzymes myo-inositol-oxygen oxidoreductase, D,glucuronate lysates were then stored on ice and assayed immediately for the enreductase, L-gulonate NAD+-3-oxidoreductase and L-iditolzyme of interest. If crude lysates failed to demonstrate significant NAD+-5-oxidoreductase, which have been found in the mammaenzyme activity, samples of lysate were separated by gel-filtration lian lens for the first time. Glucuronate reductase has been chromatography on a Pharmacia Hi Prep Sephacryl S-300 column purified and was inhibited by thiol quenching reagents. UDPand fractions in the predicted molecular weight range of the enzyme glucuronyl transferase is also present in mammalian lenses; this of interest were then assayed individually.enzyme may be an anti-toxic defense mechanism in the lens. Enzyme assaysAll substrates and buffer components, other than those specified Key words." Gulonate; myo-Inositol; Glucuronyl transferase; separately, were obtained from Sigma Biochemicals. SpectrophotoXylulose; Lens; Cataract; Aging metric analysis was carried out using a Beckman DU-70 spectrophotometer with a water-heated cuvette holder. myo-Inositol-l-phosphatesynthetase, myo-Inositol-l-phosphate synthetase was assayed by a modification of the method of Eisenberg [10]. Lens lysates contain many potentially glucose-6-phos-1. Introduction phate-utilising enzymes and addition of labeled G-6-P to a crude lens lysate would therefore yield a large number of different labeled prodXylose-fed young rats accumulate polyols, including xylitol ucts, making it difficult to identify any inositol-l-phosphate produced, myo-Inositol-phosphate synthetase has been reported in [1] and develop cataractous lesions. Kinoshita and colleagues non-lens tissues as having a molecular mass of approx. 215 kDa have proposed that 'sugar' cataract arises through osmotic [10]. HPLC gel filtration was used to isolate fractions of lens lysate damage to the lens as a direct result of polyol accumulation corresponding to that molecular mass range and those fractions were [2,3]. However, whilst xylose-fed adult rats accumulate similar then pooled and assayed for myo-inositol-l-phosphate synthetase acpolyol levels to young rats, they fail to develop cataract sugtivity. Incubations were carried out in 50 mM sodium phosphate buffer containing 1 mM NAD +, 1 mM unlabeled G-6-P and 0.1 gesting that the relationship between polyol levels...

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“…Because current models suggest sHSP subunits must reassemble in some fashion after substrate binding, disruption of oligomeric structure could also impact this step (1,5). Site-directed mutagenesis identified specific N-terminal Phe residues (Phe-24, Phe-27) in human ␣B-crystallin as necessary for chaperone activity and potentially involved in substrate binding (33). Bis-ANS also binds the N-terminal arm, reducing sHSP chaperone activity potentially by blocking substrate interactions (21,32).…”

mentioning

confidence: 99%

The N-terminal Arm of Small Heat Shock Proteins Is Important for Both Chaperone Activity and Substrate Specificity

Basha¹,

Friedrich²,

Vierling

2006

Journal of Biological Chemistry

165

147

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Small heat shock proteins (sHSPs) are a ubiquitous class of molecular chaperones that interacts with substrates to prevent their irreversible insolubilization during denaturation. How sHSPs interact with substrates remains poorly defined. To investigate the role of the conserved C-terminal ␣-crystallin domain versus the variable N-terminal arm in substrate interactions, we compared two closely related dodecameric plant sHSPs, Hsp18.1 and Hsp16.9, and four chimeras of these two sHSPs, in which all or part of the N-terminal arm was switched. The efficiency of substrate protection and formation of sHSPsubstrate complexes by these sHSPs with three different model substrates, firefly luciferase, citrate synthase, and malate dehydrogenase (MDH) provide new insights into sHSP/substrate interactions. Results indicate that different substrates have varying affinities for different domains of the sHSP. For luciferase and citrate synthase, the efficiency of substrate protection was determined by the identity of the N-terminal arm in the chimeric proteins. In contrast, for MDH, efficient protection clearly required interactions with the ␣-crystallin domain in addition to the N-terminal arm. Furthermore, we show that sHSP-substrate complexes with varying stability and composition can protect substrate equally, and substrate protection is not correlated with sHSP oligomeric stability for all substrates. Protection of MDH by the dimeric chimera composed of the Hsp16.9 N-terminal arm and Hsp18.1 ␣-crystallin domain supports the model that a dimeric form of the sHSP can bind and protect substrate. In total, results demonstrate that sHSP-substrate interactions are complex, likely involve multiple sites on the sHSP, and vary depending on substrate.The small heat shock proteins (sHSPs), 4 and the related ␣-crystallins comprise a superfamily of chaperones defined by a conserved C-terminal domain of ϳ90 amino acids referred to as the ␣-crystallin domain (1). Flanking this domain is a short C-terminal extension and an N-terminal arm of variable length and highly divergent sequence (1-3). Although the monomeric size of the sHSPs ranges from 15 to 42 kDa, in their native state most sHSPs assemble into large oligomers of 8 to Ͼ32 subunits, although there are also dimeric and tetrameric sHSPs (1, 4). 5Studies with numerous sHSPs from different organisms have shown that in vitro these proteins act as chaperones by binding to partially unfolded proteins in an ATP-independent manner, preventing their irreversible aggregation (1, 5). Substrates that are denatured in the presence of sHSPs can then be refolded and reactivated by the ATP-dependent chaperone DnaK/ Hsp70 with the participation in some cases of ClpB/Hsp100 and GroEL (1, 6, 7). In vivo, increased expression of these ubiquitous stress proteins can enhance cellular tolerance to a variety of stresses, such as heat, salt, drugs, and oxidants (1). sHSPs have also been reported to act as negative regulators of apoptosis, to modulate cellular redox state, and to be linked to increased orga...

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Effects of Site-directed Mutations on the Chaperone-like Activity of αB-Crystallin

Cited by 140 publications

References 35 publications

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

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

Accumulation of xylitol in the mammalian lens is related to glucuronate metabolism

The N-terminal Arm of Small Heat Shock Proteins Is Important for Both Chaperone Activity and Substrate Specificity

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