Defining The Mechanism-of-Action of the Hsp40 Chaperones DNAJB12 and DNAJB14 in the Assembly and Secretion of Hepatitis B Subviral Particles

Angelo, Léna; Blanchet, Matthieu; Vaillant, Andrew et Labonté, Patrick ORCID: https://orcid.org/0000-0001-7262-3125 (2022). Defining The Mechanism-of-Action of the Hsp40 Chaperones DNAJB12 and DNAJB14 in the Assembly and Secretion of Hepatitis B Subviral Particles In: AASLD: The Liver Meeting, Novembre 4-8, 2022, Washington D.C., USA and Virtual.

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Résumé

Background: The hepatitis B surface antigen (HBsAg) is implicated in maintaining the chronicity of HBV infection by interfering with immune function. In the blood, > 99.99% of HBsAg is in the form of non-infectious subviral particles (SVP). The molecular mechanisms underlying the morphogenesis of SVP are largely unknown. Recently, we identified HSP40 chaperone DNAJB12 as a novel protein involved in SVP morphogenesis by interaction analysis with the nucleic acid polymer (NAP) REP 2139 and hypothesized that REP 2139-DNAJB12 interaction prevented the proper folding of nascent HBsAg, directing it toward proteasomal/lysosomal degradation. DNAJB14 is a chaperone with a highly similar structure to DNAJB12, which shares some of DNAJB12’s client proteins and may form heterodimers with DNAJB12. DNAJB14 may be a required co-chaperone to assist DNAJB12 in folding transmembrane protein. Using DNAJB12 / DNAJB14 as starting points, the molecular machinery of HBV SVP morphogenesis is being dissected. Methods: As DNAJB12 is a co-chaperone with Hsc70/Hsp70, immunofluorescence experiments were performed in HepG2.2.15 cells and analyzed by confocal microscopy in order to determine the presence of colocalization between DNAJB12, Hsp70 and HBsAg. Sh-RNA mediated knock-down of DNAJB12 and DNAJB14 were conducted and confirmed by western blot to assess the role of each co-chaperone in HBsAg secretion. Effects on HBsAg secretion were monitored by ELISA (GS EIA 3.0, Biorad) and normalized to total cellular proteins (BCA assay). Results: Our results from immunofluorescence experiments demonstrated a strong colocalization of DNAJB12, HSP70 and HBsAg. Using DNAJB14 and/or DNAJB12 silencing experiments, we observed that DNAJB14 is not involved in the secretion of HBsAg, in comparison to the strong implication of DNAJB12 which reaches up to 90% secretion inhibition. Conclusion: DNAJB12 appears to exist in a complex with HBsAg and HSP70, consistent with its role in the assembly of HBV SVP. DNAJB12 does not appear to require DNAJB14 as a co-chaperone in its role on SVP morphogenesis. Additional confirmatory experiments using a proximity ligation assay and interference assay with REP 2139 are ongoing to further confirm the existence of the DNAJB12-HBsAg-HSP70 complex and evaluate the role of DNAJB12 and DNAJB14 in the assembly of HBV SVP.

Type de document:	Document issu d'une conférence ou d'un atelier
Informations complémentaires:	Affiche scientifique Hepatology 76(S1):329-330
Mots-clés libres:	-
Centre:	Centre INRS-Institut Armand Frappier
Date de dépôt:	25 juill. 2023 04:16
Dernière modification:	25 juill. 2023 04:17
URI:	https://espace.inrs.ca/id/eprint/13329

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