Characterization of DnaJB6: a molecular chaperone implicated in LGMD type 1D

Thesis

To maintain protein homeostasis, life has developed highly conserved networks of molecular chaperones and degradation machinery to assist in protein synthesis, quality control, maintenance, localization and degradation. J-proteins (HSP40s) are a family of co-chaperones that specifically bind misfolded target proteins for the HSP70 chaperone machinery. DnaJB6 is a human J-protein shown to suppress aggregation of misfolded disease-related proteins such as huntingtin, aβ42 and α-synuclein, either through the HSP70 pathway or independently. Mutations in DnaJB6 are associated with limb-girdle muscular dystrophy type 1D (LGMD1D). DnaJB6 contains a substrate binding domain (SBD) with low sequence complexity, especially enriched in S/T/G/F-residues, and possibly a separate C-terminal domain (CTD), but these regions of the protein remain largely structurally uncharacterized. DnaJB6 is also known to form polydisperse oligomers, but the mechanism and func- tion of oligomerization remains unknown. In this work, DnaJB6 was recombinantly expressed, purified and characterized with focus on the properties of oligomerization, the effect of LGMD1D-associated mutations and structural features of the SBD and CTD. Native mass spectrometry indicated that the polydisperse oligomer- distribution is populated from 10-mer to 15-mer with both odd and even oligomers populated, excluding a homodimer as the smallest subunit of oligomerization. The mutation of 6 phenylalanines in the SBD resulted in a significant decrease in oligomerization propensity, while mutation of a positive charge-block of arginines only resulted in a small decrease, as determined by dynamic light scattering. At ionic strength shown to dissociate the DnaJB6 oligomer, holdase chaperone activity for aggregating insulin was lost, indicating that oligomerization is necessary for chaperone function. ¹H- ¹⁵N correlation NMR spectroscopy of DnaJB6 at oligomeric conditions indicated that regions of the protein remain flexible in the oligomer. Chemical shift perturbations from LGMD1D-associated mutations F89I, F91L and F93L, determined with a DnaJB6 residue 1-130 truncation construct, revealed that these mutations structurally perturb the J-domain of DnaJB6. Helix II and the HPD-motif of the J-domain, known to be important for binding to HSP70 and stimulation of HSP70 ATPase activity, were least structurally perturbed, and stimulation of HSP70 ATPase activity was not significantly affected by LGMD1D- associated mutations.

Authors: Thomasen, F

• DOI: 10.5287/ora-ngzb9bpne
• Type of award: MSc by Research
• Level of award: Masters
• Awarding institution: University of Oxford
• Language: English
• Keywords: Protein; Molecular chaperone
• Subjects: Biochemistry; Biophysics; Structural biology