Jun Xu

The seeds of Cassia obtusifolia L. or Cassia tora L., known as Cassiae semen (juemingzi in Chinese), has been traditionally utilized for its hepatoprotective, lipid-lowering, anti-inflammatory, and immunomodulatory properties. However, their effects on hepatitis B virus (HBV), which causes chronic infection leading to liver cirrhosis and hepatocellular carcinoma, remain largely unexplored. In this study, we prepared Cassiae semen water extract (CWE) and evaluated its impact on HBV replication using HepAD38 cells. Our results showed that CWE suppressed HBV replication in a concentration-dependent manner. To identify the bioactive components, several compounds derived from Cassiae semen, including cassiaside C, rubrofusarin-6-O-β-gentiobioside, aurantioobtusin, and aloe emodin, were tested for their anti-HBV activity. Among them, aloe emodin exhibited the most potent anti-HBV activity. Further mechanistic investigations indicated that the antiviral effects of CWE and aloe emodin were associated with the interferon (IFN) signaling pathway. Treatment with both CWE and aloe emodin markedly upregulated the expression of type I IFN and the IFN-stimulated gene (ISG) IFITM1. Knockout of IFITM1 expression significantly weakened the anti-HBV activity of CWE and aloe emodin, demonstrating that the IFN-IFITM1 axis serves as a key pathway mediating the inhibitory effects of Cassiae semen on HBV replication. Collectively, our findings demonstrate for the first time that Cassiae semen extract and its bioactive constituent aloe emodin exert potent anti-HBV efficacy by modulating the interferon signaling pathway, thereby highlighting their potential as novel therapeutic agents for the treatment of chronic hepatitis B. IMPORTANCE: CHB caused by HBV is a major global health threat, often progressing to liver cirrhosis and hepatocellular carcinoma, with limited effective therapies. Cassiae semen, a widely used food-medicine homology herb in traditional Chinese medicine, is known for hepatoprotective properties, but its anti-HBV potential remains unexploited. This study identifies aloe emodin as the key bioactive component of Cassiae semen water extract that suppresses HBV replication via activating the host interferon-IFITM1 pathway with low cytotoxicity. These findings not only uncover a novel natural anti-HBV agent but also bridge traditional herbal medicine with modern antiviral drug development, offering a promising, safe candidate for HBV-associated disease treatment.

The broad-spectrum antiviral functions of interferon-inducible transmembrane 1 (IFITM1) rely on S-palmitoylation post-translational modification. α/β-hydrolase domain-containing 17A (ABHD17A) has been reported to be responsible for protein depalmitoylation over the past decade, but whether and how ABHD17A regulates the dynamic S-palmitoylation modification of IFITM1 remains unknown. Here, we demonstrated that ABHD17A physically interacts with IFITM1 and increases the S-palmitoylation level of IFITM1. Sequence alignment revealed that ABHD17A lacked the DHHC motif, which is capable of catalyzing the S-palmitoylation modification. Thus, we screened multiple candidate palmitoylating and depalmitoylating enzymes that may contribute to ABHD17A-induced upregulation of IFITM1 S-palmitoylation. The recently discovered depalmitoylase ABHD16A was significantly downregulated by ABHD17A, which counteracted the palmitate-removing reactions of ABHD16A on IFITM1 and subsequently upregulated the S-palmitoylation level and antiviral activity of IFITM1. Our work therefore elucidated the unconventional role of depalmitoylase ABHD17A in elevating the S-palmitoylation modification, expanded the biological functions of ABHD17A in innate immunity, and provided potential targets for viral disease therapy.

Interferon-inducible transmembrane (IFITM) proteins have been widely reported as antiviral factors against various viral pathogens. However, the mechanisms of IFITM regulation on hepatitis B virus (HBV), which induces chronic infection resulting in cirrhosis as well as liver cancer, remain poorly characterized. In the present study, we identified that HBV infection significantly increased the expression of IFITM1. To dissect the role of IFITM1 in HBV infection, we overexpressed IFITM1 in HepG2.215 cells and revealed that the replication of HBV was restricted by IFITM1. Recently, we demonstrated that the anti-RNA virus activity of IFITM proteins depends on palmitoylation modification, and the depalmitoylase α/β-hydrolase domain-containing 16A (ABHD16A) negatively regulates IFITM1 against RNA virus infection in human and porcine cells. However, whether ABHD16A-IFITM1 regulates DNA virus infection has not been researched. Here, by using co-immunoprecipitation (Co-IP) and acyl-PEGyl exchange gel-shift assay, ABHD16A-catalyzed depalmitoylation of IFITM1 was verified in HepG2.215 cells. In addition, we respectively knocked out IFITM1 and ABHD16A via CRISPR/Cas9 and showed that the anti-HBV activity of IFITM1 was negatively regulated by ABHD16A through depalmitoylation. Collectively, our findings demonstrated for the first time that ABHD16A catalyzes the depalmitoylation of IFITM1 to regulate its antiviral activity against HBV, which expands the biological functions of ABHD16A in immune regulation and provides potential targets for HBV infection-related disease therapy. IMPORTANCE: Nowadays, hepatitis B virus (HBV) infection remains a major global public health problem, with over 375 million people worldwide having been infected. Chronic HBV infection leads to serious liver diseases, such as liver cirrhosis and hepatocellular carcinoma. Therefore, it is urgent to reveal the mechanism of HBV infection and uncover novel drug targets. Interferon and interferon-stimulated genes are responsible for the inhibition of HBV infection. Interferon-inducible transmembrane (IFITM) is distributed on plasma membrane, restricting various virus invasions. Nevertheless, whether and how IFITM regulates HBV infection remains unclear. Here, we show that IFITM1 inhibited the replication of HBV, which depended on palmitoylation modification. In addition, the depalmitoylase α/β-hydrolase domain-containing 16A (ABHD16A) negatively regulates the anti-HBV activity of IFITM1. Overall, our findings provided ABHD16A as a potential target for interfering with HBV replication.

Interferon-inducible transmembrane (IFITM) proteins are broad-spectrum antiviral factors that confer cellular resistance to virus invasion. α/β-Hydrolase domain-containing 16A (ABHD16A) has recently been identified as a novel depalmitoylase that can inhibit the antiviral activity of IFITM proteins by catalyzing the depalmitoyl reaction; this pattern may be crucial for the host to avoid damage caused by excessive immune response. However, it remains largely elusive about how host cells regulate the activity of ABHD16A. In the present study, we performed the AlphaFold2-based protein-protein interaction prediction and identified swine E3 ubiquitin ligase ring finger protein 5 (sRNF5) as a sABHD16A-interacting protein and negatively regulated the stability of sABHD16A. Using immunofluorescence and co-immunoprecipitation techniques, we uncovered that sRNF5 targeted sABHD16A for ubiquitination and degradation via the proteasomal pathway at residues K3 and K452. Furthermore, sABHD16A catalyzed the depalmitoylation of sIFITM1, which obstructed the antiviral function of sIFITM1, while sRNF5 caused ubiquitination of sABHD16A, which attenuated the depalmitoylation effect on sIFITM1, and consequently restored the antiviral activity of sIFITM1. Collectively, our findings demonstrate for the first time that sRNF5 positively regulates the antiviral function of sIFITM1 by mediating the degradation of sABHD16A, which expands the biological functions of RNF5 and ABHD16A in immune regulation. Moreover, our work highlights the well-designed interplay between RNF5, ABHD16A, and IFITM, which balances antiviral immune responses to avoid the disorders induced by excessive immune response. IMPORTANCE: Interferon and interferon-stimulated genes play significant and protective roles in the host’s defense against viral infection. IFITM family proteins, which can be strongly induced by interferon, have been identified as the first line of defense to prevent invasion of various viruses. Further analysis reveals the antiviral activity of IFITMs depends on palmitoylation/depalmitoylation. Recently, we reported that ABHD16A, as the first depalmitoylase of IFITMs, negatively regulated the antiviral activity of IFITMs. However, these raise crucial questions: how ABHD16A is regulated and remained in a balanced manner? Here, we show that swine RNF5 attenuates the negative regulation of sIFITM1 against virus invasion by modifying sABHD16A through ubiquitination and guiding sABHD16A for degradation. Thus, sRNF5-sABHD16A interplay plays an indispensable role in regulating immune response and avoiding the disorders induced by elevated interferon levels. Overall, our findings extend the upstream subtle regulatory molecular mechanism of IFITMs and provide potential targets for viral disease therapy.

Phosphatidylserine (PS) has significant biological and nutritional effects and finds wide applications in the food, pharmaceutical, and chemical industries. To produce high-value PS efficiently, phospholipase D (PLD)-induced transphosphatidylation of low-value phosphatidylcholine (PC) with L-serine has been explored. In this research, we purified recombinant PLD from Streptomyces antibioticus SK-3 using ion exchange chromatography and gel filtration chromatography. Subsequently, we thoroughly characterized the purified enzyme and optimized the transphosphatidylation conditions to identify the most favorable settings for synthesizing PS in a biphasic system. The purified recombinant PLD displayed a robust transphosphatidylation function, facilitating efficient catalysis in the synthesis of PS. Under the optimal conditions (butyl acetate/enzyme solution 1:1, L-serine 160 mg/mL, soybean lecithin 2 mg/mL, and MgCl₂ 15 mM, at 50 °C for 2.5 h with shaking), we achieved a conversion rate of 91.35% and a productivity of 0.73 g/L/h. These results demonstrate the applicability of the process optimization strategy for using the candidate enzyme in the efficient synthesis of PS. Overall, this study presents a novel and scalable approach for the efficient large-scale synthesis of PS.

IFITM protein is the cells first line of antiviral defense that blocks early stages of viral replication; the underlying mechanism might be associated with the proper distribution in cells. The palmitoylation/depalmitoylation cycle can dynamically regulate protein localization, stability, and function.

Phospholipase D (PLD) is highly valuable in the food and medicine industries, where it is used to convert low-cost phosphatidylcholine into high-value phospholipids (PLs). Despite being overexpressed in Streptomyces, PLD production requires expensive thiostrepton feeding during fermentation, limiting its industrialization. To address this issue, we propose a new thiostrepton-free system. We developed a system using a combinatorial strategy containing the constitutive promoter kasOp* and PLD G215S mutation fused to a signal peptide sigcin of Streptoverticillium cinnamoneum pld. To find a candidate vector, we first expressed PLD using the integrative vector pSET152 and then built three autonomously replicating vectors by substituting Streptomyces replicons to increase PLD expression. According to our findings, replicon 3 with stability gene (sta) inserted had an ideal result. The retention rate of the plasmid pOJ260-rep3-pld* was 99% after five passages under non-resistance conditions. In addition, the strain SK-3 harboring plasmid pOJ260-rep3-pld* produced 62 U/mL (3.48 mg/g) of PLD, which further improved to 86.8 U/mL (7.51 mg/g) at 32 °C in the optimized medium, which is the highest activity achieved in the PLD secretory expression to date. This is the first time that a thiostrepton-free PLD production system has been reported in Streptomyces. The new system produced stable PLD secretion and lays the groundwork for the production of PLs from fermentation stock. Meanwhile, in the Streptomyces expression system, we present a highly promising solution for producing other complex proteins.

Protein palmitoylation—a lipid modification in which one or more cysteine thiols on a substrate protein are modified to form a thioester with a palmitoyl group—is a significant post-translational biological process. This process regulates the trafficking, subcellular localization, and stability of different proteins in cells. Since palmitoylation participates in various biological processes, it is related to the occurrence and development of multiple diseases. It has been well evidenced that the proteins whose functions are palmitoylation-dependent or directly involved in key proteins’ palmitoylation/depalmitoylation cycle may be a potential source of novel therapeutic drugs for the related diseases. Many researchers have reported palmitoylation of proteins, which are crucial for host-virus interactions during viral infection. Quite a few explorations have focused on figuring out whether targeting the acylation of viral or host proteins might be a strategy to combat viral diseases. All these remarkable achievements in protein palmitoylation have been made to technological advances. This paper gives an overview of protein palmitoylation modification during viral infection and the methods for palmitoylated protein detection. Future challenges and potential developments are proposed.