PIN1 and PIN4 inhibition via parvulin impeders Juglone, PiB, ATRA, 6,7,4'-THIF, KPT6566, and EGCG thwarted hepatitis B virus replication.

Introduction: Human parvulin peptidyl prolyl cis/trans isomerases PIN1 and PIN4 play important roles in cell cycle progression, DNA binding, protein folding and chromatin remodeling, ribosome biogenesis, and tubulin polymerization. In this article, we found that endogenous PIN1 and PIN4 were upregulated in selected hepatocellular carcinoma (HCC) cell lines. Methods: In this study, we inhibited PIN1 and PIN4 via parvulin inhibitors (Juglone, PiB, ATRA, 6,7,4'-THIF, KPT6566, and EGCG). The native agarose gel electrophoresis (NAGE) immunoblotting analysis revealed that upon PIN1 and/ or PIN4 inhibition, the HBc protein expression and core particle or capsid synthesis reduced remarkably. The effects of PIN4 inhibition on hepatitis B virus (HBV) replication were more pronounced as compared to that of PIN1. The Northern and Southern blotting revealed reduced HBV RNA and DNA levels. Results: During the HBV course of infection, Juglone, PiB, ATRA, 6,7,4'-THIF, KPT6566, and EGCG-mediated inhibition of PIN1 and PIN4 significantly lowered HBV transcriptional activities without affecting total levels of covalently closed circular DNA (cccDNA). Similar to the inhibitory effects of PIN1 and PIN4 on HBV replication, the knockdown of PIN1 and PIN4 in HBV infection cells revealed significantly reduced amounts of intracellular HBc, HBs, HBV pgRNA, SmRNAs, core particles, and HBV DNA synthesis. Similarly, PIN1 and PIN4 KD abrogated extracellular virion release, naked capsid levels, and HBV DNA levels. In comparison with PIN1 KD, the PIN4 KD showed reduced HBc and/or core particle stabilities, indicating that PIN4 is more critically involved in HBV replication. Chromatin immunoprecipitation (ChIP) assays revealed that in contrast to DNA binding PIN4 proteins, the PIN1 did not show binding to cccDNA. Similarly, upon PIN1 KD, the HBc recruitment to cccDNA remained unaffected. However, PIN4 KD significantly abrogated PIN4 binding to cccDNA, followed by HBc recruitment to cccDNA and restricted HBV transcriptional activities. These effects were more pronounced in PIN4 KD cells upon drug treatment in HBV-infected cells. Conclusion: The comparative analysis revealed that in contrast to PIN1, PIN4 is more critically involved in enhancing HBV replication. Thus, PIN1 and PIN4 inhibition or knockdown might be novel therapeutic targets to suppress HBV infection. targets to suppress HBV infection.

Leucine-rich repeats and immunoglobulin-like domains protein 1 (LRIG1) is downregulated in Invasive ductal carcinoma and potential prognostic marker of breast cancer.

BACKGROUND: LRIG1 belongs to the family of transmembrane proteins containing leucine-rich repeats. LRIGs are considered as tumor suppressors as they negatively regulate receptor tyrosine kinases. The role of LRIG1 as an EGFR regulator makes it an important marker to be studied in various epithelial-derived cancers. METHODS: LRIG1 expression was determined in Erbb2 + cell lines by western blotting, and cell motility was examined by cell migration assay. The AKT/GSK3-ß/ß-catenin pathway was determined in the presence of LRIG1 and Erbb2 by using western blotting. RESULTS: So far, no study has reported the expression of LRIG1 in benign forms of tumor such as fibroadenoma. The current study aims to analyze LRIG1 expression in fibroadenoma and invasive ductal carcinoma (IDC) tissues. In this study, we compared the LRIG1 expression with different clinicopathological parameters of patients having IDC or fibroadenoma. LRIG1 expression was low in Erbb2+ cell lines, and more cell motility was observed. The AKT/GSK3-ß/ß-catenin pathway was activated when LRIG1 was downregulated; consequently, Erbb2 was upregulated. Our results indicated that LRIG1 expression can be significantly correlated with age, Nottingham index, and Her2/neu status of cancer. The expression of LRIG1 in IDC and fibroadenoma were found to be significantly different. CONCLUSION: The fibroadenoma tissue sections were found to express LRIG1 more intensely as compared to the IDC sections, which are in line with the studies reporting reduced copy number of the gene either due to gene deletion or transcriptional inhibition. This further supports that the downregulation of LRIG1 may lead to malignant tumor acting as a tumor suppressor.

Decoding the multifaceted interventions between human sirtuin 2 and dynamic hepatitis B viral proteins to confirm their roles in HBV replication.

The human sirtuin 2 gene (SIRT2) encodes a full-length Sirt2 protein (i.e., the Sirt2 isoform 1), which primarily functions as a cytoplasmic α-tubulin deacetylase, and which promotes the growth of hepatocellular carcinoma (HCC). Hepatitis B virus (HBV) replication itself, or HBV X (HBx) protein-mediated transcriptional transactivation, enhances Sirt2.1 expression; therefore, Sirt2.1 itself is capable of positively increasing HBV transcription and replication. Sirt2.1 is linked to liver fibrosis and epithelial-to-mesenchymal transition and, consequently, augments the risk of HCC. The Sirt2.1 protein enhances the HBV replication cycle by activating the AKT/glycogen synthase kinase 3 beta (GSK3ß)/ß-catenin pathway. It also activates the transcription of the viral enhancer I/HBx promoter (EnI/Xp) and enhancer II/HBc promoter (EnII/Cp) by targeting the transcription factor p53. The Sirt2 isoform 2 (Sirt2.2) is mainly localized in the cytoplasm, and the N-terminus is shorter by 37 amino acids than that of Sirt2.1. Despite the truncation of the N-terminal region, Sirt2.2 is still capable of enhancing HBV replication and activating the AKT/GSK3ß/ß-catenin signaling pathway. The Sirt2 isoform 5 (Sirt2.5) is primarily localized to the nucleus, it lacks a nuclear export signal (NES), and the catalytic domain (CD) is truncated. Upon HBV replication, expression of the Sirt2 isoforms is also enhanced, which further upregulates the HBV replication, and, therefore, supports the vicious cycle of viral replication and progression of the disease. Sirt2 diversely affects HBV replication such that its isoform 1 intensely augments HBV replication and isoform 2 (despite of the truncated N-terminal region) moderately enhances HBV replication. Isoform 5, on the other hand, tends to protect the cell (for smooth long-term continued viral replication) from HBV-induced extreme damage or death via a discrete set of regulatory mechanisms impeding viral mRNAs, the hepatitis B core/capsid protein (HBc), core particles, replicative intermediate (RI) DNAs, and covalently closed circular DNA (cccDNA) levels, and, consequently, limiting HBV replication. In contrast to Sirt2.1 and Sirt 2.2, the Sirt2.5-mediated HBV replication is independent of the AKT/GSK3ß/ß-catenin signaling cascade. Sirt2.5 is recruited more at cccDNA than the recruitment of Sirt2.1 onto the cccDNA. This recruitment causes the deposition of more histone lysine methyltransferases (HKMTs), including SETDB1, SUV39H1, EZH2, and PR-Set7, along with the respective corresponding transcriptional repressive markers such as H3K9me3, H3K27me3, and H4K20me1 onto the HBV cccDNA. In HBV-replicating cells, Sirt2.5 can also make complexes with PR-Set7 and SETDB1. In addition, Sirt2.5 has the ability to turn off transcription from cccDNA through epigenetic modification via either direct or indirect interaction with HKMTs.

SARS-CoV-2 induced hepatic injuries and liver complications.

Background: Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which is resilient, highly pathogenic, and rapidly transmissible. COVID-19 patients have been reported to have underlying chronic liver abnormalities linked to hepatic dysfunction. Discussion: Viral RNAs are detectable in fecal samples by RT-PCR even after negative respiratory samples, which suggests that SARS-CoV-2 can affect the gastrointestinal tract and the liver. The case fatality rates are higher among the elderly and those with underlying comorbidities such as hypertension, diabetes, liver abnormality, and heart disease. There is insufficient research on signaling pathways. Identification of molecular mechanisms involved in SARS-CoV-2-induced damages to hepatocytes is challenging. Herein, we demonstrated the multifactorial effects of SARS-CoV-2 on liver injury such as psychological stress, immunopathogenesis, systemic inflammation, ischemia and hypoxia, drug toxicity, antibody-dependent enhancement (ADE) of infection, and several others which can significantly damage the liver. Conclusion: During the COVID-19 pandemic, it is necessary for clinicians across the globe to pay attention to SARS-CoV-2-mediated liver injury to manage the rising burden of hepatocellular carcinoma. To face the challenges during the resumption of clinical services for patients with pre-existing liver abnormalities and HCC, the impact of SARS-CoV-2 on hepatocytes should be investigated both in vitro and in vivo.

An Alternatively Spliced Sirtuin 2 Isoform 5 Inhibits Hepatitis B Virus Replication from cccDNA by Repressing Epigenetic Modifications Made by Histone Lysine Methyltransferases.

Sirtuin 2 (Sirt2), an NAD+-dependent protein deacetylase, deacetylates tubulin, AKT, and other proteins. Previously, we showed that Sirt2 isoform 1 (Sirt2.1) increased replication of hepatitis B virus (HBV). Here, we show that HBV replication upregulates the expression of Sirt2 primary and alternatively spliced transcripts and their respective isoforms, 1, 2, and 5. Since Sirt2 isoform 5 (Sirt2.5) is a catalytically inactive nuclear protein with a spliced-out nuclear export signal (NES), we speculated that its different localization affects its activity. The overexpression of Sirt2.5 reduced expression of HBV mRNAs, replicative intermediate DNAs, and covalently closed circular DNA (cccDNA), an activity opposite that of Sirt2.1 and Sirt2.2. Unlike the Sirt2.1-AKT interaction, the Sirt2.5-AKT interaction was weakened by HBV replication. Unlike Sirt2.1, Sirt2.5 activated the AKT/GSK-3ß/ß-catenin signaling pathway very weakly and independently of HBV replication. When the NES and an N-terminal truncated catalytic domain were added to the Sirt2.5 construct, it localized in the cytoplasm and increased HBV replication (like Sirt2.1 and Sirt2.2). Chromatin immunoprecipitation assays revealed that more Sirt2.5 was recruited to cccDNA than Sirt2.1. The recruitment of histone lysine methyltransferases (HKMTs), such as SETDB1, SUV39H1, EZH2, and PR-Set7, and their respective transcriptional repressive markers, H3K9me3, H3K27me3, and H4K20me1, to cccDNA also increased in Sirt2.5-overexpressing cells. Among these, the Sirt2.5-PR-Set7 and -SETDB1 interactions increased upon HBV replication. These results demonstrate that Sirt2.5 reduces cccDNA levels and viral transcription through epigenetic modification of cccDNA via direct and/or indirect association with HKMTs, thereby exhibiting anti-HBV activity.IMPORTANCE Sirt2, a predominant cytoplasmic α-tubulin deacetylase, promotes the growth of hepatocellular carcinoma; indeed, HBV replication increases Sirt2 expression, and overexpression of Sirt2 is associated with hepatic fibrosis and epithelial-to-mesenchymal transition. Increased amounts of Sirt2 isoforms 1, 2, and 5 upon HBV replication might further upregulate HBV replication, leading to a vicious cycle of virus replication/disease progression. However, we show here that catalytically inactive nuclear Sirt2.5 antagonizes the effects of Sirt2.1 and Sirt2.2 on HBV replication, thereby inhibiting cccDNA level, transcription of cccDNA, and subsequent synthesis of replicative intermediate DNA. More Sirt2.5 was recruited to cccDNA than Sirt2.1, thereby increasing epigenetic modification by depositing transcriptional repressive markers, possibly through direct and/or indirect association with histone lysine methyltransferases, such as SETDB1, SUV39H1, EZH2, and/or PR-Set7, which represses HBV transcription. Thus, Sirt2.5 might provide a functional cure for HBV by silencing the transcription of HBV.

Parvulin 14 and Parvulin 17 Bind to HBx and cccDNA and Upregulate Hepatitis B Virus Replication from cccDNA to Virion in an HBx-Dependent Manner.

The parvulin 14 (Par14) and parvulin 17 (Par17) proteins, which are both encoded by the PIN4 gene, play roles in protein folding, chromatin remodeling, DNA binding, ribosome biogenesis, and cell cycle progression. However, the effects of Par14 and Par17 on viral replication have never been explored. In this study, we found that, in the presence of HBx, either Par14 or Par17 could upregulate hepatitis B virus (HBV) replication, whereas in the absence of HBx, neither Par14 nor Par17 had any effect on replication. Overexpression of Par14/Par17 markedly increased the formation of covalently closed circular DNA (cccDNA), synthesis of HBV RNA and DNA, and virion secretion. Conversely, PIN4 knockdown significantly decreased HBV replication in HBV-transfected and -infected cells. Coimmunoprecipitation revealed that Par14/Par17 engaged in direct physical interactions with HBx in the cytoplasm, nucleus, and mitochondria, possibly mediated through substrate-binding residues on Par14/Par17 (E46/D74 and E71/D99, respectively) and conserved 19R20P-28R29P motifs on HBx. Furthermore, these interactions enhanced HBx stability, promoted HBx translocation to the nuclear and mitochondrial fractions, and increased HBV replication. Chromatin immunoprecipitation assays revealed that, in the presence of HBx, Par14/Par17 were efficiently recruited to cccDNA and promoted transcriptional activation via specific DNA-binding residues (S19/44). In contrast, in the absence of HBx, Par14/Par17 bound cccDNA only at the basal level and did not promote transcriptional activation. Taken together, our results demonstrate that Par14 and Par17 upregulate HBV RNA transcription and DNA synthesis, thereby increasing the HBV cccDNA level, through formation of the cccDNA-Par14/17-HBx complex.IMPORTANCE The HBx protein plays an essential regulatory role in HBV replication. We found that substrate-binding residues on the human parvulin peptidylprolyl cis/trans isomerase proteins Par14 and Par17 bound to conserved arginine-proline (RP) motifs on HBx in the cytoplasm, nucleus, and mitochondria. The HBx-Par14/Par17 interaction stabilized HBx; promoted its translocation to the nucleus and mitochondria; and stimulated multiple steps of HBV replication, including cccDNA formation, HBV RNA and DNA synthesis, and virion secretion. In addition, in the presence of HBx, the Par14 and Par17 proteins bound to cccDNA and promoted its transcriptional activation. Our results suggest that inhibition or knockdown of Par14 and Par17 may represent a novel therapeutic option against HBV infection.

Sirtuin 2 Isoform 1 Enhances Hepatitis B Virus RNA Transcription and DNA Synthesis through the AKT/GSK-3ß/ß-Catenin Signaling Pathway.

Sirtuin 2 (Sirt2), a NAD+-dependent protein deacetylase, is overexpressed in many hepatocellular carcinomas (HCCs) and can deacetylate many proteins, including tubulins and AKT, prior to AKT activation. Here, we found that endogenous Sirt2 was upregulated in wild-type hepatitis B virus (HBV WT)-replicating cells, leading to tubulin deacetylation; however, this was not the case in HBV replication-deficient-mutant-transfected cells and 1.3-mer HBV WT-transfected and reverse transcriptase inhibitor (entecavir or lamivudine)-treated cells, but all HBV proteins were expressed. In HBV WT-replicating cells, upregulation of Sirt2 induced AKT activation, which consequently downregulated glycogen synthase kinase 3ß (GSK-3ß) and increased ß-catenin levels; however, downregulation of Sirt2 in HBV-nonreplicating cells impaired AKT/GSK-3ß/ß-catenin signaling. Overexpression of Sirt2 isoform 1 stimulated HBV transcription and consequently HBV DNA synthesis, which in turn activated AKT and consequently increased ß-catenin levels, possibly through physical interactions with Sirt2 and AKT. Knockdown of Sirt2 by short hairpin RNAs (shRNAs), inhibition by 2-cyano-3-[5-(2,5-dichlorophenyl)-2-furanyl]-N-5-quinolinyl-2-propenamide (AGK2), or dominant negative mutant expression inhibited HBV replication, reduced AKT activation, and decreased ß-catenin levels. Through HBV infection, we demonstrated that Sirt2 knockdown inhibited HBV replication from transcription. Although HBx itself activates AKT and upregulates ß-catenin, Sirt2-mediated signaling and upregulated HBV replication were HBx independent. Since constitutively active AKT inhibits HBV replication, the results suggest that upregulated Sirt2 and activated AKT may balance HBV replication to prolong viral replication, eventually leading to the development of HCC. Also, the results indicate that Sirt2 inhibition may be a new therapeutic option for controlling HBV infection and preventing HCC.IMPORTANCE Even though Sirt2, a NAD+-dependent protein deacetylase, is overexpressed in many HCCs, and overexpressed Sirt2 promotes hepatic fibrosis and associates positively with vascular invasion by primary HCCs through AKT/GSK-3ß/ß-catenin signaling, the relationship between Sirt2, HBV, HBx, and/or HBV-associated hepatocarcinogenesis is unclear. Here, we show that HBV DNA replication, not HBV expression, correlates positively with Sirt2 upregulation and AKT activation. We demonstrate that overexpression of Sirt2 further increases HBV replication, increases AKT activation, downregulates GSK-3ß, and increases ß-catenin levels. Conversely, inhibiting Sirt2 decreases HBV replication, reduces AKT activation, and decreases ß-catenin levels. Although HBx activates AKT to upregulate ß-catenin, Sirt2-mediated effects were not dependent on HBx. The results also indicate that a Sirt2 inhibitor may control HBV infection and prevent the development of hepatic fibrosis and HCC.