Structural and functional consequences of phosphorylation on the small heat-shock protein Hsp27

Structural and functional consequences of phosphorylation on the small heat-shock protein Hsp27

Structural and functional consequences of phosphorylation on the small heat-shock protein Hsp27 (#372)

Blagojce Jovcevski ¹ , Megan Kelly ¹ , Andrew Aquilina ¹ , Heath Ecroyd ¹

IHMRI, University Of Wollongong, NSW, Australia

Background: Small heat shock proteins (sHSPs) are molecular chaperones that are one of the cells first lines of defence against protein aggregation. They do so by binding to destabilised proteins and prevent downstream misfolding and aggregation. These dynamic proteins exist as large polydisperse oligomeric species, which undergo rapid subunit exchange. Hsp27 has previously been shown to exist as large polydisperse oligomers ranging from 18mers to 30mers, with the 18mer being the most abundant¹ .Phosphorylation of Hsp27 is thought to decrease oligomer size and polydispersity and, in doing so, enhance the chaperone activity of this sHSP.

Aim: Our goal was to perform a detailed investigation of the effect phosphorylation has on the structure and chaperone function of Hsp27.

Methodology: We utilised phosphomimics of Hsp27 in which phosphorylatable serine residues (S15, S78 and S82) were replaced with aspartic acid. Mass spectrometry (MS), native-PAGE and SEC-MALS were used to determine the oligomeric size and polydispersity of these phosphomimics, compared to the wild-type protein. The chaperone function of the phosphomimics was assessed in amorphous and amyloid fibril aggregation assays.

Results: The single phosphomimics of Hsp27 (S15D, S78D and S82D) were found to form oligomers ranging from 12mers to 24mers, with a 14mer being the most abundant. Increasing the degree of phosphorylation lead to a decrease in size and polydispersity such that the double phosphomimics (S15/78D, S15/82D and S78/82D) had an oligomeric range from 12mers to 22mers, and the triple phosphomimics (S15/78/82D, 3D) was exclusively a dimer. These findings were confirmed by SEC-MALS. As the number or phosphomimicking residues increased, from none in wild-type Hsp27 to three in Hsp27-3D, there was an increase in the ability of Hsp27 to prevent both amorphous and fibrillar protein aggregation.

Conclusion: Phosphorylation of Hsp27 acts as a ‘switch’, decreasing oligomeric size, polydispersity and increasing its chaperone activity. Our data supports a model in which dimers are the chaperone-active component of Hsp27.

Aquilina J. A. A., Shrestha S., Morris A. M., Ecroyd H. (2013) Structural and Functional Aspects of Hetero-oligomers Formed by the Small Heat Shock Proteins αB-Crystallin and HSP27. J. Biol. Chem. 288, 13602-13609

Authors contributing to this presentation.

Jovcevski, B

Blagojce (BJ) is a postdoctoral researcher with expertise in utilising molecular biology and biophysical approaches to rationalise protein function and dysfunction in the context of protein homeostasis. In 2017, he completed his PhD at the University of Wollongong under the supervision of Prof. Heath Ecroyd and Dr Andrew Aquilina, as well as working with in the laboratories of Prof. Justin Benesch and Prof. Dame Carol Robinson FRS at the University of Oxford, focusing on the structure and dynamics of molecular chaperone proteins by native mass spectrometry. His work has gained both national and international recognition at both Lorne Proteins and American Society of Mass Spectrometry conferences. At the end of 2017, BJ subsequently moved to the University of Adelaide to join the research group led by Assoc. Prof. Tara Pukala. His work centres on utilising a range of MS-based approaches to the structural dynamics of amyloidogenic proteins. In addition to developing novel peptide-based protein aggregation inhibitors and enzymatic-targetting antimicrobials, structure-activity relationships to guide antifungal drug design and vaccine design against infectious disease. BJ is also an Associate Investigator for the Research Consortia of Agricultural Product Development, funded by the SA Government Department of Innovation and Skills, bringing together academic and industry partners from the agriculture and food sector to develop high-value products from agricultural and food waste. In 2023, Blagojce joined the School of Biological Sciences and established the Proteostasis and Disease Research Group - a multidisciplinary team focusing on the mechanisms and molecular networks regulating protein homeostasis (proteostasis) in several cell types (gut and the brain) and their contribution to neurodegenerative diseases including Parkinson’s disease and ALS, through funding from The Hospital Research Foundation.

Structural and functional consequences of phosphorylation on the small heat-shock protein Hsp27 (#372)

Jovcevski, B

Kelly, M

Aquilina, A

Ecroyd, H

Structural and functional consequences of phosphorylation on the small heat-shock protein Hsp27 (#372)

Add notes

Jovcevski, B

Kelly, M

Aquilina, A

Ecroyd, H

Login