Identification of the Hsp40 Chaperone DNAJB12 in the Functional Mechanism of the Nucleic Acid Polymer REP 2139

Angelo, Léna; Boulon, Richard; Blanchet, Matthieu; Vaillant, Andrew et Labonté, Patrick ORCID: https://orcid.org/0000-0001-7262-3125 (2022). Identification of the Hsp40 Chaperone DNAJB12 in the Functional Mechanism of the Nucleic Acid Polymer REP 2139 In: AASLD: The Liver Meeting, November, 4-8 2022, Washington D.C., USA and Virtual.

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Résumé

Background: During hepatitis B viral (HBV) infection, subviral particles (SVP) are produced in large excess over virions and constitute > 99.99% of synthesized and secreted HBs antigen (HBsAg). HBsAg is postulated to be important in allowing HBV to chronically persist by interfering with immune function. Nucleic acid polymers (NAPs) interfere with the assembly / secretion of HBV SVP without affecting the release of HBeAg or Dane particles. Combination therapy with the NAP REP 2139, pegylated interferon and tenofovir disopropxil fumarate leads to high rates of HBsAg loss and functional cure of HBV and HDV. Ongoing experimental verification of the molecular mechanism of NAPs driving inhibition of SVP assembly and secretion are presented. Methods: MS/MS interactome analysis with biotinylated NAPs in HepG2.2.15 lysates was used to identify potential NAP interactors. ShRNA-mediated knockdown of identified interactors was confirmed by western blotting and used to confirm effects on secreted viral proteins. Effects on secretion of HBsAg (GS EIA 3.0, Biorad) and HBeAg (ETI-EBK PLUS N0140, Diasorin) were monitored by ELISA and normalized to total cellular protein (as determined by BCA assay). NAP target interaction was further confirmed by co-immunoprecipitation experiments and western blotting using biotinylated REP 2139. Results: MS/MS interactome analysis identified the HSP40 J-protein DNAJB12 as NAP interactor conforming to the known structure activity relationship for NAP antiviral activity. Silencing of DNAJB12 by shRNA knock-down experiments showed a selective effect on viral protein secretion, inhibiting up to 90% of HBsAg secretion inhibition with no effect on HBeAg secretion. Using co-immunoprecipitation of biotinylated REP 2139, DNAJB12 was readily detected in the pull-down extract confirming interaction between REP 2139 and DNAJB12. Conclusion: The inhibition of SVP assembly and secretion involves the interaction of NAPs with DNAJB12. The normal function of DNAJB12 is to assist Hsc70/Hsp70 in the folding of transmembrane proteins. Indeed, DNAJB12 is located at the endoplasmic reticulum membrane where its function determines the fate of nascent transmembrane proteins which in turn may lead to the degradation of misfolded proteins. We hypothesize that the interaction of REP 2139 with DNAJB12, prevents DNAJB12 from folding nascent HBsAg, leading to its proteasomal/lysosomal degradation.

Type de document:	Document issu d'une conférence ou d'un atelier
Informations complémentaires:	Affiche scientifique 1224 Hepatology 76(S1):329-329
Mots-clés libres:	-
Centre:	Centre INRS-Institut Armand Frappier
Date de dépôt:	25 juill. 2023 04:17
Dernière modification:	25 juill. 2023 04:17
URI:	https://espace.inrs.ca/id/eprint/13330

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