Role of the Conserved SRLFDQFFG Region of α-Crystallin, a Small Heat Shock Protein

Pasta, Saloni; Raman, Bakthisaran; Ramakrishna, T.; Rao, Ch. Mohan

doi:10.1074/jbc.m307523200

Cited by 73 publications

(34 citation statements)

References 66 publications

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“…3), which is consistent with previously reported data (42)(43)(44)(45)(46). The Q151X mutation had the most significant effect upon the far UV CD characteristics of ␣B-crystallin (Fig.…”

Section: Expression and Purification Of The C-terminal Extensionsupporting

confidence: 82%

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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“…3), which is consistent with previously reported data (42)(43)(44)(45)(46). The Q151X mutation had the most significant effect upon the far UV CD characteristics of ␣B-crystallin (Fig.…”

Section: Expression and Purification Of The C-terminal Extensionsupporting

confidence: 82%

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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“…These two probes have been previously characterized and used to monitor exchange of subunits. 50,51,54,55,75 FðU The subunit exchange reaction was initiated 50 by mixing an equimolar concentration of the donor (AIAS) with the acceptor (LYI) labeled mutant at 378C. AIAS emission intensity at 415 nm is decreased in a time dependent manner (Figures 8A and 8B) with a concomitant increase in intensity at 525 nm indicating FRET because of the close proximity of LYI and AIAS.…”

Section: Effect Of N-terminal Deletion On Subunit Exchangementioning

confidence: 97%

“…The fluorescence spectrum of different samples was taken from 360 to 600 nm using excitation wavelength of 335 nm at room temperature using a Hitachi 4500 spectrofluorimeter. The subunit exchange rate was calculated 50,54,55 from the Eq. (1).…”

Section: Fluorescence Measurementsmentioning

confidence: 99%

Structure, stability, and chaperone function of αA‐crystallin: Role of N‐terminal region

2007

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Small heat shock protein alphaA-crystallin, the major protein of the eye lens, is a molecular chaperone. It consists of a highly conserved central domain flanked by the N-terminal and C-terminal regions. In this article we studied the role of the N-terminal domain in the structure and chaperone function of alphaA-crystallin. Using site directed truncation we raised several deletion mutants of alphaA-crystallin and their protein products were expressed in Escherichia coli. Size exclusion chromatography of these purified proteins showed that deletion from the N-terminal beyond the first 20 residues drastically reduced the oligomeric association of alphaA-crystallin and its complete removal resulted in a tetramer. Chaperone activity of alphaA-crystallin, determined by thermal and nonthermal aggregation and refolding assay, decreased with increasing length of deletion and little activity was observed for the tetramer. However it was revealed that N-terminal regions were not responsible for specific recognition of natural substrates and that low affinity substrate binding sites existed in other part of the molecule. The number of exposed hydrophobic sites and the affinity of binding hydrophobic probe bis-ANS as well as protein substrates decreased with N-terminal deletion. The stability of the mutant proteins decreased with increase in the length of deletion. The role of thermodynamic stability, oligomeric size, and surface hydrophobicity in chaperone function is discussed. Detailed analysis showed that the most important role of N-terminal region is to control the oligomerization, which is crucial for the stability and in vivo survival of this protein molecule.

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“…This domain forms the C-terminal part of the protein, and is folded into a β-sandwich structure (4)(5)(6). The N-terminal part is more variable in both length and sequence, and largely determines the association properties of the homo-or heteromeric oligomers of sHSPs (7)(8)(9). The α-crystallin domain generally has a short and flexible C-terminal extension which modulates the chaperone-like activity, possessed by most sHSPs (10)(11)(12).…”

Section: Introductionmentioning

confidence: 99%

The Small Heat-Shock Proteins HSPB2 and HSPB3 Form Well-defined Heterooligomers in a Unique 3 to 1 Subunit Ratio

Engelsman

Boros

Dankers

et al. 2009

Journal of Molecular Biology

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Various mammalian small heat-shock proteins (sHSPs) can interact with one another to form large polydisperse assemblies. In muscle cells, HSPB2/MKBP (myotonic dystrophy protein kinase-binding protein) and HSPB3 have been shown to form an independent complex. To date, the biochemical properties of this complex have not been thoroughly characterized. In this study, we show that recombinant HSPB2 and HSPB3 can be successfully purified from E.coli cells co-expressing both proteins. Nanoelectrospray ionization mass spectrometry and sedimentation velocity analytical ultracentrifugation analysis showed that HSPB2/B3 forms a series of well defined hetero-oligomers, consisting of 4,8,12,16,20 and 24 subunits, each maintaining a strict 3:1 HSPB2:HSPB3 subunit ratio. These complexes are thermally stable up to 40 °C, as determined by far-UV circular dichroism spectroscopy. Surprisingly, HSPB2/B3 exerted a poor chaperone-like and thermoprotective activity, which is likely related to the low surface hydrophobicity, as revealed by its interaction with the hydrophobic probe 1-anilino-8-naphthalenesulfonic acid. Co-immunoprecipitation experiments demonstrated that the HSPB2/B3 oligomer cannot interact with HSP20, HSP27 or αB-crystallin, whereas the homomeric form of HSPB2, thus not in complex with HSPB3, could efficiently associate with HSP20. Taken altogether, this study brings evidence that despite the high sequence homology within the sHSP family, the biochemical properties of the HSPB2/B3 complex are distinctly different from other sHSPs, indicating that the HSPB2/B3 assembly likely possesses other cellular functions than its family members. Various mammalian small heat shock proteins (sHSPs) can interact with one another to form large polydisperse assemblies. In muscle cells, HSPB2/MKBP (myotonic dystrophy protein kinase-binding protein) and HSPB3 have been shown to form an independent complex. To date, the biochemical properties of this complex have not been thoroughly characterized. In this study, we show that recombinant HSPB2 and HSPB3 can be successfully purified from E.coli cells co-expressing both proteins. Nanoelectrospray ionization mass spectrometry and sedimentation velocity analytical ultracentrifugation analysis showed that HSPB2/B3 forms a series of well defined hetero-oligomers, consisting of 4, 8, 12, 16, 20 and 24 subunits, each maintains a strict 3:1 HSPB2:HSPB3 subunit ratio. Analyzing the thermal stability of the HSPB2/B3 assembly by far-UV circular dichroism spectroscopy revealed subtle structural changes, occurring slightly above 40 °C, and an unfolding curve with an inflection point at approximately 56 °C. Furthermore, HSPB2/B3 exerted poor chaperone-like and thermoprotective activity, which is likely related to the low surface hydrophobicity, as revealed by its interaction with the hydrophobic probe 1-anilino-8-naphthalenesulfonic acid. Finally, coimmunoprecipitation experiments demonstrated that the HSPB2/B3 oligomer does not interact with HSP20, HSP27 or αB-crystallin. However, HSPB2 that ...

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Role of the Conserved SRLFDQFFG Region of α-Crystallin, a Small Heat Shock Protein

Cited by 73 publications

References 66 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

Structure, stability, and chaperone function of αA‐crystallin: Role of N‐terminal region

The Small Heat-Shock Proteins HSPB2 and HSPB3 Form Well-defined Heterooligomers in a Unique 3 to 1 Subunit Ratio

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