Oxidation of human lens recombinant αA-crystallin and cysteine-deficient mutants

Chen, Shujuan; Sun, Tiffany; Akhtar, Nila J.; Liang, Jinghang

doi:10.1006/jmbi.2000.4348

Cited by 15 publications

(10 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The disulfide bond in the wild-type protein, its reduction by DTT or replacement of the cysteine residue to alanine had negligible effects on either the secondary structure or oligomeric size of the protein. Our data are somewhat similar to the observation that oxidation of the lone cysteine (C141) in Hsp25 did not change its secondary structure [Zavialov et al, 1998] and mutation of two cysteine residues in human αA-crsyatllin had little effect on the secondary structure [Chen et al, 2001]. Thus, it is likely that cysteine residues in small heat shock proteins may not contribute to structural characteristics.…”

Section: Discussionsupporting

confidence: 86%

The role of the cysteine residue in the chaperone and anti‐apoptotic functions of human Hsp27

Pasupuleti

Gangadhariah

Padmanabha

et al. 2010

J of Cellular Biochemistry

View full text Add to dashboard Cite

The small heat shock protein Hsp27 is a molecular chaperone and an anti-apoptotic protein. Human Hsp27 has one cysteine residue at position 137. We investigated the role of this cysteine residue in the chaperone and anti-apoptotic functions of Hsp27 by mutating the cysteine residue to an alanine (Hsp27C137A) and comparing it to wild-type protein (Hsp27WT). Both proteins were multi-subunit oligomers, but subunits of Hsp27WT were disulfide-linked unlike those of Hsp27C137A, which were monomeric. Hsp27C137A was indistinguishable from Hsp27WT with regard to its secondary structure, surface hydrophobicity, oligomeric size and chaperone function. S-thiolation and reductive methylation of the cysteine residue had no apparent effect on the chaperone function of Hsp27WT. The anti-apoptotic function of Hsp27C137A and Hsp27WT was studied by overexpressing them in CHO cells. No difference in the caspase-3 or -9 activity was observed in staurosporine-treated cells. The rate of apoptosis between Hsp27C137A and Hsp27WT overexpressing cells was similar whether the cells were treated with staurosporine or etoposide. However, the mutant protein was less protective relative to the wild-type protein in preventing caspase-3 and caspase-9 activation and apoptosis induced by 1 mM H2O2 in CHO and HeLa cells. These data demonstrate that in human Hsp27, disulfide formation by the lone cysteine does not affect its chaperone function and anti-apoptotic function against chemical toxicants. However, oxidation of the lone cysteine in Hsp27 might at least partially affect the anti-apoptotic function against oxidative stress.

show abstract

Section: Discussionsupporting

confidence: 86%

The role of the cysteine residue in the chaperone and anti‐apoptotic functions of human Hsp27

Pasupuleti

Gangadhariah

Padmanabha

et al. 2010

J of Cellular Biochemistry

View full text Add to dashboard Cite

show abstract

“…Primers used for the substitution of cysteines at positions 131 and 142 with isoleucine and site-directed mutagenesis were as described by Chen et al [15]. Plasmids were sequenced to confirm the presence of mutations.…”

Section: Methodsmentioning

confidence: 99%

“…The proteins were overexpressed in E. coli strain BL21(DE3) as described previously [12]. The recombinant proteins were purified from the supernatant by the following successive steps: fractionation with 30–60% saturated ammonium sulfate, gel filtration on Sephacryl S-300, and ion exchange chromatography on DEAE-Sepharose [15]. The purity of the recombinant α A-crystallins was confirmed by SDS-PAGE and Western blotting using polyclonal rabbit anti- α A-crystallin antibody (Assay Designs, Ann Arbor, MI, USA) (Fig.…”

Section: Methodsmentioning

confidence: 99%

Role of cysteine residues in the enhancement of chaperone function in methylglyoxal-modified human αA-crystallin

2008

View full text Add to dashboard Cite

We have previously demonstrated that the reaction of a physiological dicarbonyl, methylglyoxal (MGO) enhances the chaperone function of human αA-crystallin. MGO can react with cysteine, arginine, and lysine residues in proteins. Although the role of arginine and lysine residues in the enhancement of chaperone function has been investigated, the role of cysteine residues is yet to be determined. In this study, we have investigated the effect of MGO modification on the structure and chaperone function of αA-crystallin mutant proteins in which C131 and C142 were replaced either individually or simultaneously with isoleucine. MGO-modification resulted in improved chaperone function in all three αA-crystallin mutants, including the cysteine-free double mutant. The enhanced chaperone function was due to increased surface hydrophobicity and increased binding of client proteins. These results suggest that the two cysteine residues, even though they could be modified, do not take part in the MGO-induced improvement in the chaperone function of human αA-crystallin.

show abstract

“…As described for a-crystallins, deamidation introduces a negative charge at the modification site that affects the oligomerization [112]. Similarly, modifications by glycation [113], oxidation [114,115], thiolation [116] and the attachment of methylglyoxal have been suggested to influence the oligomerization of HSPBs [117]. Being not really a posttranslational modification, the incorporation of bivalent metal ions, for example, Cu 2+ , represents nevertheless a modification that also changes the local charge pattern, and thus, a potential intra-or inter-molecular interaction site.…”

Section: Understanding Human Shspsmentioning

confidence: 99%

Medical implications of understanding the functions of human small heat shock proteins

Mymrikov

Haslbeck²

2015

Expert Review of Proteomics

View full text Add to dashboard Cite

Small heat shock proteins (sHsps) are ubiquitous molecular chaperones that are implicated in a variety of diseases. Upon stress, they stabilize unfolding proteins and prevent them from aggregating. However, under physiological conditions without severe stress, some sHsps interact with other proteins. In a perspective view, their ability to bind specific client proteins might allow them to fine-tune the availability of the client for other, client-dependent cellular processes. Additionally, some sHsps seem to interact with specific co-chaperones. These co-chaperones are usually part of large protein machineries that are functionally modulated upon sHsps interaction. Finally, secreted human sHsps seem to interact with receptor proteins, potentially as signal molecules transmitting the stress status from one cell to another. This review focuses on the mechanistic description of these different binding modes for human sHsps and how this might help to understand and modulate the function of sHsps in the context of disease.

show abstract

Oxidation of human lens recombinant αA-crystallin and cysteine-deficient mutants

Cited by 15 publications

References 25 publications

The role of the cysteine residue in the chaperone and anti‐apoptotic functions of human Hsp27

The role of the cysteine residue in the chaperone and anti‐apoptotic functions of human Hsp27

Role of cysteine residues in the enhancement of chaperone function in methylglyoxal-modified human αA-crystallin

Medical implications of understanding the functions of human small heat shock proteins

Contact Info

Product

Resources

About