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The influence of the N-terminal region proximal to the core domain on the assembly and chaperone activity of αB-crystallin

Journal Article


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Abstract


  • αB-Crystallin (HSPB5) is a small heat-shock protein that is composed of dimers that then assemble into a polydisperse ensemble of oligomers. Oligomerisation is mediated by heterologous interactions between the C-terminal tail of one dimer and the core “α-crystallin” domain of another and stabilised by interactions made by the N-terminal region. Comparatively little is known about the latter contribution, but previous studies have suggested that residues in the region 54–60 form contacts that stabilise the assembly. We have generated mutations in this region (P58A, S59A, S59K, R56S/S59R and an inversion of residues 54–60) to examine their impact on oligomerisation and chaperone activity in vitro. By using native mass spectrometry, we found that all the αB-crystallin mutants were assembly competent, populating similar oligomeric distributions to wild-type, ranging from 16-mers to 30-mers. However, circular dichroism spectroscopy, intrinsic tryptophan and bis-ANS fluorescence studies demonstrated that the secondary structure differs to wild type, the 54–60 inversion mutation having the greatest impact. All the mutants exhibited a dramatic decrease in exposed hydrophobicity. We also found that the mutants in general were equally active as the wild-type protein in inhibiting the amorphous aggregation of insulin and seeded amyloid fibrillation of α-synuclein in vitro, except for the 54–60 inversion mutant, which was significantly less effective at inhibiting insulin aggregation. Our data indicate that alterations in the part of the N-terminal region proximal to the core domain do not drastically affect the oligomerisation of αB-crystallin, reinforcing the robustness of αB-crystallin in functioning as a molecular chaperone.

Authors


  •   Jovcevski, Blagojce (external author)
  •   Aquilina, John Andrew. (external author)
  •   Benesch, Justin (external author)
  •   Ecroyd, Heath

Publication Date


  • 2018

Citation


  • Jovcevski, B., Aquilina, J., Benesch, J. L. P. & Ecroyd, H. (2018). The influence of the N-terminal region proximal to the core domain on the assembly and chaperone activity of αB-crystallin. Cell Stress and Chaperones, 23 (5), 827-836.

Scopus Eid


  • 2-s2.0-85043396889

Ro Full-text Url


  • http://ro.uow.edu.au/cgi/viewcontent.cgi?article=2261&context=ihmri

Ro Metadata Url


  • http://ro.uow.edu.au/ihmri/1234

Number Of Pages


  • 9

Start Page


  • 827

End Page


  • 836

Volume


  • 23

Issue


  • 5

Place Of Publication


  • Netherlands

Abstract


  • αB-Crystallin (HSPB5) is a small heat-shock protein that is composed of dimers that then assemble into a polydisperse ensemble of oligomers. Oligomerisation is mediated by heterologous interactions between the C-terminal tail of one dimer and the core “α-crystallin” domain of another and stabilised by interactions made by the N-terminal region. Comparatively little is known about the latter contribution, but previous studies have suggested that residues in the region 54–60 form contacts that stabilise the assembly. We have generated mutations in this region (P58A, S59A, S59K, R56S/S59R and an inversion of residues 54–60) to examine their impact on oligomerisation and chaperone activity in vitro. By using native mass spectrometry, we found that all the αB-crystallin mutants were assembly competent, populating similar oligomeric distributions to wild-type, ranging from 16-mers to 30-mers. However, circular dichroism spectroscopy, intrinsic tryptophan and bis-ANS fluorescence studies demonstrated that the secondary structure differs to wild type, the 54–60 inversion mutation having the greatest impact. All the mutants exhibited a dramatic decrease in exposed hydrophobicity. We also found that the mutants in general were equally active as the wild-type protein in inhibiting the amorphous aggregation of insulin and seeded amyloid fibrillation of α-synuclein in vitro, except for the 54–60 inversion mutant, which was significantly less effective at inhibiting insulin aggregation. Our data indicate that alterations in the part of the N-terminal region proximal to the core domain do not drastically affect the oligomerisation of αB-crystallin, reinforcing the robustness of αB-crystallin in functioning as a molecular chaperone.

Authors


  •   Jovcevski, Blagojce (external author)
  •   Aquilina, John Andrew. (external author)
  •   Benesch, Justin (external author)
  •   Ecroyd, Heath

Publication Date


  • 2018

Citation


  • Jovcevski, B., Aquilina, J., Benesch, J. L. P. & Ecroyd, H. (2018). The influence of the N-terminal region proximal to the core domain on the assembly and chaperone activity of αB-crystallin. Cell Stress and Chaperones, 23 (5), 827-836.

Scopus Eid


  • 2-s2.0-85043396889

Ro Full-text Url


  • http://ro.uow.edu.au/cgi/viewcontent.cgi?article=2261&context=ihmri

Ro Metadata Url


  • http://ro.uow.edu.au/ihmri/1234

Number Of Pages


  • 9

Start Page


  • 827

End Page


  • 836

Volume


  • 23

Issue


  • 5

Place Of Publication


  • Netherlands