USP14 negatively regulates IFN signaling by dampening K63-linked ubiquitination of TBK1 in black carp.

USP14 regulates the immune related pathways by deubiquitinating the signaling molecules in mammals. In teleost, USP14 is also reported to inhibit the antiviral immune response through TBK1, but its regulatory mechanism remains obscure. To elucidate the role of USP14 in the RLR/IFN antiviral pathway in teleost, the homolog USP14 (bcUSP14) of black carp (Mylopharyngodon piceus) has been cloned and characterize in this paper. bcUSP14 contains 490 amino acids (aa), and the sequence is well conserved among in vertebrates. Over-expression of bcUSP14 in EPC cells attenuated SVCV-induced transcription activity of IFN promoters and enhanced SVCV replication. Knockdown of bcUSP14 in MPK cells led to the increased transcription of IFNs and decreased SVCV replication, suggesting the improved antiviral activity of the host cells. The interaction between bcUSP14 and bcTBK1 was identified by both co-immunoprecipitation and immunofluorescent staining. Co-expressed bcUSP14 obviously inhibited bcTBK1-induced IFN production and antiviral activity in EPC cells. K63-linked polyubiquitination of bcTBK1 was dampened by co-expressed bcUSP14, and bcTBK1-mediated phosphorylation and nuclear translocation of IRF3 were also inhibited by this deubiquitinase. Thus, all the data demonstrated that USP14 interacts with and inhibits TBK1 through deubiquitinating TBK1 in black carp.

USP46 promotes the interferon antiviral signaling in black carp by deubiquitinating TBK1.

Ubiquitin-specific peptidase 46 (USP46) functions as a deubiquitinating enzyme, facilitating the removal of ubiquitin molecules attached to substrate proteins and playing a critical role in cancer and neurodegenerative diseases. However, its function in innate antiviral immunity is unknown. In this study we cloned and identified bcUSP46, a homolog of USP46 from black carp. We discovered that overexpression of bcUSP46 enhanced the transcription of interferon (IFN) promoters and increased the expression of IFN, PKR, and Mx1. In addition, bcUSP46 knockdown significantly inhibited the expression of ISG genes, as well as the antiviral activity of the host cells. Interestingly, when bcUSP46 was co-expressed with the RLR factors, it significantly enhanced the activity of the IFN promoter mediated by these factors, especially TANK-binding kinase 1 (TBK1). The subsequent co-immunoprecipitation (co-IP) and immunofluorescence (IF) assay confirmed the association between bcUSP46 and bcTBK1. Noteworthily, co-expression of bcUSP46 with bcTBK1 led to an elevation of bcTBK1 protein level. Further analysis revealed that bcUSP46 stabilized bcTBK1 by eliminating the K48-linked ubiquitination of bcTBK1. Overall, our findings highlight the unique role of USP46 in modulating TBK1/IFN signaling and enrich our knowledge of the function of deubiquitination in regulating innate immunity in vertebrates.

Black carp LGP2 suppresses RIG-I mediated IFN signaling during the antiviral innate immunity.

Laboratory of genetics and physiology 2 (LGP2), a member of retinoic acid-inducible gene (RIG)-I-like receptors (RLRs), has been reported to play different roles in IFN signaling in both mammals and teleost fish. In our previous study, black carp (Mylopharyngodon piceus) LGP2 (bcLGP2) has been characterized to positively regulate melanoma differentiation-associated gene 5 (MDA5). In this study, knockdown of bcLGP2 decreased the expression of host genes, including bcIFNb, bcPKR, bcMx1, and bcViperin, and also attenuated the antiviral capability of host cells. The relationship between bcLGP2 and black carp RIG-Ib (bcRIG-Ib) has been explored. Dual-luciferase reporter assay and qRT-PCR assay indicated that bcLGP2 dampened bcRIG-Ib induced transcription of type I interferons (IFNs) and interferon-stimulated genes (ISGs), including PKR, ISG15, and Viperin. Consistently, the plaque assay identified that bcLGP2 attenuated bcRIG-Ib mediated antiviral ability against spring viremia of carp virus (SVCV). Co-immunoprecipitation assay identified the interaction between bcLGP2 and bcRIG-Ib, as well as bcLGP2 and bcRIG-Ib-CARD. And bcRIG-Ib-CARD mediated antiviral ability was also attenuated by bcLGP2. Truncation mutation analysis showed DExD/H-box Helicase domain of bcLGP2 possessed a similar inhibitory effect on bcRIG-Ib to that of bcLGP2, while the C-terminus repressor domain (CTD) presented little impact on bcRIG-Ib. Furthermore, bcLGP2 enhanced the K48-linked ubiquitination of bcRIG-Ib, promoting proteasome-dependent degradation of bcRIG-Ib. Thus, our data supported the conclusion that bcLGP2 interacted with and induced degradation of bcRIG-Ib through proteasome, leading to the dampened antiviral signaling mediated by bcRIG-Ib.

TRIM25 negatively regulates IKKε-mediated interferon signaling in black carp.

IKKε plays an important role in the activation of IRF3/IRF7 and the production of interferon (IFN), however, its regulation remains obscure in human. E3 ligase TRIM25 has been reported to manipulate the K63-linked ubiquitination of RIG-I, leading to the activation of RIG-I/IFN signaling. To elucidate the role of TRIM25 in teleost, a TRIM25 homolog (bcTRIM25) was cloned and characterized from black carp (Mylopharyngodon piceus). bcTRIM25 contains 653 amino acids, possessing conservative RING, B-box and SPRY domain, which is highly expressed in muscle, spleen and skin. bcTRIM25 knock-down enhanced the antiviral ability of host cells. bcTRIM25 over-expression alone in EPC cells attenuated bcIFNa promoter transcription in the reporter assays and impeded PKR and MX1 expression in qRT-PCR. Interestingly, co-IP assays indicated that bcTRIM25 interacted with bcIKKε and the induced bcIFNa promoter transcription by bcIKKε was notably hindered by bcTRIM25. Furthermore, bcIKKε-induced expression of interferon stimulated genes (ISGs) and antiviral activity were dampened by bcTRIM25. Further exploration showed that bcTRIM25 visibly enhanced the ubiquitination of bcIKKε but significantly attenuated the phosphorylation of bcIKKε. Thus, our data demonstrate for the first time in vertebrate that TRIM25 negatively regulates IKKε through enhancing its ubiquitination, which sheds a light on the regulation of IKKε/IFN signaling.

The phenylalanine-28 is crucial for black carp RIG-I mediated antiviral signaling.

Retinoic acid-inducible gene I (RIG-I) functions as a cytosolic sensor to recognize RNA products of the invading microorganisms and induce the production of type I interferons（IFNs）. In this study, two RIG-I variants, named as bcRIG-Ia and bcRIG-Ib, were characterized in black carp (Mylopharyngodon piceus) respectively. RNA pull-down assay revealed that both bcRIG-Ia and bcRIG-Ib could bind to synthetic poly(I:C) and the RD domain was crucial for RNA binding of these two molecules. However, over-expression of bcRIG-Ib, but not bcRIG-Ia, induced the transcription of IFN promoter, and led to the improved antiviral activity against both spring viremia of carp virus (SVCV) and grass carp reovirus (GCRV). And knockdown of bcRIG-I dampened the transcription of bcViperin and bcIFNb in host cells. Truncation mutation and site mutation analysis identified that phenylalanine (F)- 28 was crucial for bcRIG-Ib oligomerization and its mediated IFN signaling. Interestingly, F28 was conserved among teleost RIG-Is and site mutation analysis revealed that F28 was essential for RIG-I mediated IFN signaling in the cyprinid fish. Thus, our study concludes that F28 is crucial for black carp RIG-I mediated antiviral signaling and suggests F28 is also essential for the activation of IFN signaling by RIG-Is from other teleost fish.

Rosmarinic Acid Inhibits Platelet Aggregation and Neointimal Hyperplasia In Vivo and Vascular Smooth Muscle Cell Dedifferentiation, Proliferation, and Migration In Vitro via Activation of the Keap1-Nrf2-ARE Antioxidant System.

The activation of platelets and proliferation of vascular smooth muscle cells (VSMCs) in the vascular intima play an essential role in the pathological mechanism of vascular restenosis (RS). Rosmarinic acid (RA) is a natural phenolic acid compound. However, its mechanism of action on platelets and VSMCs is still unclear. This study investigated the effects of RA on platelet function, VSMCs phenotypic conversion, proliferation, and migration in vascular remodeling with a specific focus on the Keap1-Nrf2-ARE signaling pathway. RA inhibited platelet aggregation and Ca2+ release and significantly reduced the release of platelet microvesicles. In addition, RA inhibited the phenotypic transition of VSMCs in vitro and in vivo. In vitro experiments showed that RA could effectively inhibit the proliferation and migration of VSMCs induced by the platelet-derived growth factor (PDGF)-BB. PDGF-BB triggered ROS generation and a decrease in mitochondrial membrane potential, which were inhibited by RA. Mechanistically, after artery injury or treatment with PDGF-BB, VSMCs presented with inhibition of the Nrf2/antioxidant response element (ARE) signaling pathway. RA treatment reversed this profile by activating the Nrf2/ARE signaling pathway; stabilizing Keap1 protein; upregulating HO-1, NQO1, GCLM, and GST protein levels; promoting typical Nrf2 nuclear translocation; and preventing VSMCs from oxidative stress damage. On the other hand, RA also inhibited the NF-κB pathway to reduce inflammation. In summary, these results indicate that RA inhibits platelet function and attenuates the proliferation, migration, and phenotypic transition of VSMCs induced by PDGF-BB in vitro and vascular remodeling in vivo. Therefore, RA treatment may be a potential therapy for preventing or treating RS.

A study of the antibacterial mechanism of pinocembrin against multidrug-resistant Aeromonas hydrophila.

Aeromonas hydrophila is a common pathogen in fish that has caused severe economic losses in aquaculture worldwide. With the emergence of bacterial resistance, it is necessary to develop new drugs to combat bacterial infection, particularly for multidrug-resistant bacteria. In this study, the antibacterial activity of pinocembrin was investigated by observing bacterial growth and microscopic structure, and its mechanism of action was identified by investigating its effect on protein and DNA. The antibacterial susceptibility test indicated that pinocembrin inhibits A. hydrophila growth. The minimal inhibitory concentration and minimum bactericidal concentration were 256 µg/mL and 512 µg/mL, respectively. Ultrastructurally, the bacteria treated with pinocembrin showed surface roughness and plasmolysis. When bacteria were treated with 512 µg/mL pinocembrin, lactate dehydrogenase activity and soluble protein content decreased significantly, and electrical conductivity and DNA exosmosis levels increased by 4.21 ± 0.64% and 15.98 ± 1.93 mg/L, respectively. Staining with 4', 6-Diamidino-2-phenylindole showed that the nucleic acid fluorescence intensity and density decreased after the treatment with pinocembrin. Pinocembrin may inhibit the growth of A. hydrophila by increasing cell membrane permeability and affecting protein and DNA metabolism. Thus, pinocembrin is a candidate drug for the treatment of A. hydrophila infection in aquaculture.

Evaluation of efficacy and safety after replacement of methyl hydrogen with deuterium at methyl formate of Clopidogrel.

BACKGROUND AND PURPOSE: Despite being a first-line clinical drug, thienopyridines have many unsatisfactory aspects, including the low bioavailability of clopidogrel(CLP) and the high bleeding risk of prasugrel. We synthesized deuterium clopidogrel(D-CL, patented in China) to alleviate the deficiency of CLP in clinical, such as a slow onset, a greater influence of gene polymorphism, and a high frequency of drug-drug interaction. EXPERIMENTAL APPROACH: Molecular docking was used to analyze the affinity between D-CL and the P2Y12 receptor. The levels of active metabolites of D-CL were detected using HPLC/MS-MS and the activities of main metabolic enzymes were analyzed; Subsequently, platelet aggregation function, thrombus model were used to evaluate the pharmacodynamics of D-CL. Finally, the safety of D-CL were evaluated through examination of blood routine, PT, APTT, bleeding time, serological tests, liver pathological biopsy, liver cell apoptosis and detection of apoptosis-related proteins. KEY RESULTS: The introduction of deuterium made the binding of CLP to P2Y12 receptor more stable, improved the concentration of active metabolites, and substantially reduced the inhibition of major metabolic enzymes, including CYP2B6, CYP2C9, and CYP2C19, thereby, exerting better antiplatelet effects without increasing the risk of bleeding, along with a concomitant decrease in the apoptosis of hepatocytes.

Study on the Interaction between Deuterium Clopidogrel and Omeprazole.

INTRODUCTION: Dual antiplatelet therapy with aspirin in combination with a P2Y12 receptor antagonist is a cornerstone for treating patients with acute coronary syndrome and in percutaneous coronary intervention. However, as this combination of antiplatelet therapy increases the risk of bleeding, proton pump inhibitors (PPIs) are currently recommended to prevent gastrointestinal ulcers and bleeding. The cytochrome P450 (CYP450) isoenzyme system metabolizes both clopidogrel (CLP) and PPIs. Unfortunately, omeprazole (OM) reduce the antiplatelet activity of CLP and increases the probability of recurrence of cardiovascular events by competitively inhibiting the CYP450 isoenzyme CYP2C19. METHODS: To address these abovementioned problems, we designed and synthesized deuterium CLP (D-CL) using selective deuterium technology. Our previous research results showed that D-CL had better pharmacokinetic and pharmacodynamic properties. Thus, the HPLC-MS/MS method, cocktail method, Born method, electro-stimulated thrombus generation, and thrombus elastography were used to detect the production of thiol active metabolites (AM), CYP450 enzyme activities, platelet aggregation, time and length of thrombus formation, and the maximum clot strength after combination therapy. We investigated the pharmacokinetics and pharmacodynamics properties of D-CL combined with OM. RESULTS: As compared to CLP, D-CL was less affected when combined with OM, which was reflected in lower inhibitory effects of CYP450 enzyme activities, a greater area under the curve of AM, and better antiplatelet and antithrombotic effects. CONCLUSION: D-CL may reduce drug-drug interactions and address the clinical disadvantages of CLP.

Interplay between transglutaminase treatment and changes in digestibility of dietary proteins.

The changes in digestibility of TG-treated myofibrillar protein (MP), soybean protein isolate (SPI) and mixed proteins were evaluated by measuring liberation of primary amino groups, monitoring structural changes and investigating peptide fingerprints. TG treatment generally increased gastric digestion of treated proteins, possibly due to the structural changes occurred during TG treatment. In contrast, the initial intestinal digestion was suppressed by TG treatment. Compared with MP, the digestibility and peptide composition of SPI were affected by TG treatment to a larger degree, possibly due to the higher level of glutamine in SPI. Peptidomics analysis indicated that the changes in peptide composition of digests of TG-treated samples were related with the loss of Lys residues during TG treatment. Larger quantities of bioactive peptides KIEFEQFLPM, EVHEPEEKPRPK and TVKEDQVFPMNPPK were released after digestion of TG-treated MP. These results highlighted the complex and substantial influence of TG treatment on the digestibility of dietary proteins.

A Pharmacodynamic Study of CN-218, a Novel Antiplatelet and Antithrombotic Agent Primarily Targeting the P2Y12 Receptor.

PURPOSE: Drugs inhibiting the platelet P2Y12 receptor, such as clopidogrel and prasugrel, are potent antithrombotic agents and are widely used in cardiovascular disease. However, the adverse effects of these drugs have limited their clinical use. For example, clopidogrel resistance occurs in approximately one third of patients, while prasugrel increases the risk of major bleeding. Therefore, new generations of such drugs are of clinical interest. METHODS: In this study, the pharmacodynamics of a new P2Y12 antagonist, CN-218, was compared with that of clopidogrel and prasugrel in rats and mice. The differences between CN-218 and clopidogrel include deuteration of the 7-position methyl carboxylate and the introduction of cinnamate in the 2-position of thiophene. RESULTS: CN-218 had an antiaggregatory efficacy that was at least five times more potent than that of clopidogrel but not as potent as that of prasugrel. It had a significant impact on activated partial thromboplastin time (APTT), whereby the APTT of CN-218-treated rats was approximately 9 s longer than that of the vehicle- or clopidogrel-treated group, while it had no impact on prothrombin time (PT) in rats. CN-218 had a similar potent antithrombotic effect to that of prasugrel and clopidogrel and also reduced the risk of bleeding compared to prasugrel. CONCLUSION: CN-218 may be a promising antithrombotic agent, with potent antiplatelet and significant anticoagulant activity, as well as a lower risk of bleeding compared to clopidogrel and prasugrel.