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Biogenesis of hepatitis B virus e antigen is driven by translocon-associated protein complex and regulated by conserved cysteine residues within its signal peptide sequence

ABSTRACT: Hepatitis B virus (HBV) uses e antigen (HBe) which is dispensable for virus infectivity to modulate host immune responses and to achieve viral persistence in human hepatocytes. The HBe precursor (p25) is directed to the endoplasmic reticulum (ER), where cleavage of the signal peptide (sp) gives rise to the first processing product, p22. The p22 can be retro-translocated back to the cytosol or it can enter the secretory pathway and undergo a second cleavage event resulting in secreted p17 (HBe). Here, we report that translocation of p25 precursor is promoted by translocon-associated protein complex (TRAP). We found that p25 is not completely translocated into the ER since a fraction of p25 is phosphorylated and remains present in the cytoplasm and the nucleus. Within the sp sequence of p25 we identified three cysteine residues which control the efficiency of sp cleavage and correct subcellular distribution of the precore pool. Highlights • A fraction of the HBV e antigen precursor p25 is not translocated into the ER and is phosphorylated in cytosol. • Cysteine residues in the signal peptide sequence regulate the p25 N-terminal processing and resulting p22 intracellular level. • Mutations of all three Cys residues within the signal peptide lead to a mislocalization of intracellular precore protein. • P25 translocation requires assistance of TRAP complex for an efficient mature HBe secretion. • The depletion of individual TRAP subunits results in a change of precore subcellular distribution.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human) Hepatitis B Virus

TISSUE(S): Hepatocyte, Hepg2 Cell

DISEASE(S): Hepatitis B

SUBMITTER: Martin Hubalek

LAB HEAD: Martin Hubálek

PROVIDER: PXD025430 | Pride | 2022-07-01

REPOSITORIES: Pride

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Project description:Background: Atherosclerosis leads high mortality, highlighting an urgent need for new therapeutic strategies. Protein kinases orchestrate multiple cellular events in atherosclerosis and may provide new therapeutic targets for atherosclerosis. Here, we identified a protein kinase, WEE1 G2 checkpoint kinase (WEE1), promoting inflammatory response in atherosclerosis. Methods: The ApoE-/- mice without macrophage-specific WEE1 deletion (ApoE-/-WEE1f/f) and ApoE-/- mice with macrophage-specific WEE1 deletion (ApoE-/-WEE1MCKO) were generated using bone marrow transplantation. These mice and WEE1 inhibitor MK1775 were used in a high-fat diet (HFD)-induced atherosclerotic model. Human atherosclerotic tissues, mouse primary peritoneal macrophages (MPMs), 293T cells, and recombinant proteins were utilized to investigate the pathogenic role and underlying mechanisms of WEE1. Results: We identified up-regulated p-WEE1 in macrophages in atherosclerotic lesions of HFD-fed ApoE-/- mice. Transcriptome sequencing analysis indicated that WEE1 promotes oxLDL-induced inflammation in macrophages. We then demonstrated that macrophage-specific deletion or pharmacological inhibition of WEE1 attenuates atherosclerosis by reducing inflammation both in vivo and in vitro. The overexpression of wild-type WEE1 or activating-mutant WEE1, but not inactivating-mutant WEE1, exacerbates inflammation in macrophages. Mechanistically, transcriptome sequencing analysis and co-immunoprecipitation followed by quantitative proteomics analysis identified p65 as a binding protein of WEE1. We confirmed that WEE1 directly interacts with the RHD domain of p65 via kinase domain and phosphorylates p65 at S536, thereby facilitating subsequent NF-κB activation and inflammatory response in macrophages. Conclusions: Our findings demonstrated that macrophage WEE1 promotes inflammatory atherosclerosis by directly binding to p65 and phosphorylate it at S536. This study provides WEE1 as a new p65 regulator in inflammation and a potential therapeutic target for atherosclerosis.

			Action	DRS
	210105_C3A_E1-B.mzML	Mzml
	210105_C3A_E2_B.mzML	Mzml
	210105_C3A_E3_B.mzML	Mzml
	210105_CTRL_0-2_B.mzML	Mzml
	210105_CTRL_1_B.mzML	Mzml

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Biogenesis of hepatitis B virus e antigen is driven by translocon-associated protein complex and regulated by conserved cysteine residues within its signal peptide sequence

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