Structural and Biochemical Characterization of Porcine Epidemic Diarrhea Virus Papain-Like Protease 2.

Coronaviral papain-like proteases (PLpros) are essential enzymes that mediate not only the proteolytic processes of viral polyproteins during virus replication but also the deubiquitination and deISGylation of cellular proteins that attenuate host innate immune responses. Therefore, PLpros are attractive targets for antiviral drug development. Here, we report the crystal structure of papain-like protease 2 (PLP2) of porcine epidemic diarrhea virus (PEDV) in complex with ubiquitin (Ub). The X-ray structural analyses reveal that PEDV PLP2 interacts with the Ub substrate mainly through the Ub core region and C-terminal tail. Mutations of Ub-interacting residues resulted in a moderately or completely abolished deubiquitinylating function of PEDV PLP2. In addition, our analyses also indicate that 2-residue-extended blocking loop 2 at the S4 subsite contributes to the substrate selectivity and binding affinity of PEDV PLP2. Furthermore, the PEDV PLP2 Glu99 residue, conserved in alphacoronavirus PLpros, was found to govern the preference of a positively charged P4 residue of peptidyl substrates. Collectively, our data provided structure-based information for the substrate binding and selectivity of PEDV PLP2. These findings may help us gain insights into the deubiquitinating (DUB) and proteolytic functions of PEDV PLP2 from a structural perspective. IMPORTANCE Current challenges in coronaviruses (CoVs) include a comprehensive understanding of the mechanistic effects of associated enzymes, including the 3C-like and papain-like proteases. We have previously reported that the PEDV PLP2 exhibits a broader substrate preference, superior DUB function, and inferior peptidase activity. However, the structural basis for these functions remains largely unclear. Here, we show the high-resolution X-ray crystal structure of PEDV PLP2 in complex with Ub. Integrated structural and biochemical analyses revealed that (i) three Ub core-interacting residues are essential for DUB function, (ii) 2-residue-elongated blocking loop 2 regulates substrate selectivity, and (iii) a conserved glutamate residue governs the substrate specificity of PEDV PLP2. Collectively, our findings provide not only structural insights into the catalytic mechanism of PEDV PLP2 but also a model for developing antiviral strategies.

Investigating the impact of probiotic on neurological outcomes in Rett syndrome: A randomized, double-blind, and placebo-controlled pilot study.

LAY ABSTRACT: Rett syndrome often involves gastrointestinal symptoms and gut microbiota imbalances. We conducted a study to explore the feasibility of probiotic Lactobacillus plantarum PS128 and the impact on neurological functions in Rett syndrome. The results of our investigation demonstrated that the supplementation of probiotic L. plantarum PS128 was feasible and well tolerated, with 100% retention rate and 0% withdrawal rate. In addition, there was only one participant who had loose stool after taking L. plantarum PS128. Further, there was a tendency to enhance overall cognitive developmental level, as assessed using Mullen Scales of Early Learning. In addition, it significantly improved dystonia, as assessed using the Burke-Fahn-Marsden Movement Scale, in comparison with the placebo group. This study provides a strong foundation for future research and clinical trials exploring the potential of L. plantarum PS128 probiotics as a complementary therapy for individuals with Rett syndrome.

Exopolysaccharide is the potential effector of Lactobacillus fermentum PS150, a hypnotic psychobiotic strain.

Psychobiotics are a class of probiotics that confer beneficial effects on the mental health of the host. We have previously reported hypnotic effects of a psychobiotic strain, Lactobacillus fermentum PS150 (PS150), which significantly shortens sleep latency in experimental mice, and effectively ameliorate sleep disturbances caused by either caffeine consumption or a novel environment. In the present study, we discovered a L. fermentum strain, GR1009, isolated from the same source of PS150, and found that GR1009 is phenotypically distinct but genetically similar to PS150. Compared with PS150, GR1009 have no significant hypnotic effects in the pentobarbital-induced sleep test in mice. In addition, we found that heat-killed PS150 exhibited hypnotic effects and altered the gut microbiota in a manner similar to live bacteria, suggesting that a heat-stable effector, such as exopolysaccharide (EPS), could be responsible for these effects. Our comparative genomics analysis also revealed distinct genetic characteristics in EPS biosynthesis between GR1009 and PS150. Furthermore, scanning electron microscopy imaging showed a sheet-like EPS structure in PS150, while GR1009 displayed no apparent EPS structure. Using the phenol-sulfate assay, we found that the sugar content value of the crude extract containing EPS (C-EPS) from PS150 was approximately five times higher than that of GR1009, indicating that GR1009 has a lower EPS production activity than PS150. Through the pentobarbital-induced sleep test, we confirmed the hypnotic effects of the C-EPS isolated from PS150, as evidenced by a significant reduction in sleep latency and recovery time following oral administration in mice. In summary, we utilized a comparative approach to delineate differences between PS150 and GR1009 and proposed that EPS may serve as a key factor that mediates the observed hypnotic effect.

Lactobacillus fermentum PS150 promotes non-rapid eye movement sleep in the first night effect of mice.

The first night effect (FNE) is a type of sleep disturbance caused by an unfamiliar environment, which leads to difficulty falling asleep and reduced sleep duration. Previously, we reported that Lactobacillus fermentum PS150 (PS150) improves sleep conditions in a pentobarbital-induced sleep mouse model. In this study, we aimed to evaluate the effect of PS150 on the FNE in mice. Briefly, mice were implanted with electrodes and orally administered PS150 for four weeks, and then the FNE was induced by cage changing. Analysis of polysomnographic signals revealed that intervention with PS150 restored non-rapid eye movement (NREM) sleep length under the FNE. Compared to diphenhydramine, a commonly used sleep aid, PS150 had no unwanted side effects, such as rapid eye movement (REM) sleep deprivation and fragmented sleep. Moreover, temporal analysis revealed that PS150 efficiently reduced both sleep latency and time spent restoring normal levels of REM sleep. Taken together, these results suggest that PS150 efficiently ameliorates sleep disturbance caused by the FNE. Additionally, V3-V4 16S rRNA sequencing revealed significant increases in Erysipelotrichia, Actinobacteria, and Coriobacteriia in fecal specimens of the PS150-treated group, indicating that PS150 induces gut microbiota remodeling.

Disulfiram and 6-Thioguanine synergistically inhibit the enzymatic activities of USP2 and USP21.

Disulfiram is a promising repurposed drug that, combining with radiation and chemotherapy, exhibits effective anticancer activities in several preclinical models. The cellular metabolites of disulfiram have been established, however, the intracellular targets of disulfiram remain largely unexplored. We have previously reported that disulfiram suppresses the coronaviral papain-like proteases through attacking their zinc-finger domains, suggesting an inhibitory function potentially on other proteases with similar catalytic structures. Ubiquitin-specific proteases (USPs) share a highly-conserved zinc-finger subdomain that structurally similar to the papain-like proteases and are attractive anticancer targets as upregulated USPs levels are found in a variety of tumors. Here, we report that disulfiram functions as a competitive inhibitor for both USP2 and USP21, two tumor-related deubiquitinases. In addition, we also observed a synergistic inhibition of USP2 and USP21 by disulfiram and 6-Thioguanine (6TG), a clinical drug for acute myeloid leukemia. Kinetic analyses revealed that both drugs exhibited a slow-binding mechanism, moderate inhibitory parameters, and a synergistically inhibitory effect on USP2 and USP21, suggesting the potential combinatory use of these two drugs for USPs-related tumors. Taken together, our study provides biochemical evidence for repurposing disulfiram and 6TG as a combinatory treatment in clinical applications.

FNR-Dependent RmpA and RmpA2 Regulation of Capsule Polysaccharide Biosynthesis in Klebsiella pneumoniae.

Fumarate nitrate reduction regulator (FNR) is a direct oxygen-responsive transcriptional regulator containing an iron-sulfur (Fe-S) cluster. During anaerobic growth, the [4Fe-4S] cluster in FNR (holo-FNR) binds specifically to DNA, whereas exposure to oxygen results in the loss of its DNA-binding activity via oxidation of the [4Fe-4S] cluster. In this study, we aimed to investigate the role of FNR in regulation of capsular polysaccharide (CPS) biosynthesis, serum resistance, and anti-phagocytosis of K. pneumoniae. We found that the CPS amount in K. pneumoniae increased in anaerobic conditions, compared to that in aerobic conditions. An fnr deletion mutant and a site-directed mutant (fnr 3 CA), with the three cysteines (C20, C23, and C29) replaced with alanines to mimic an FNR lacking the [4Fe-4S] cluster, showed marked increase in CPS amount under anaerobic conditions. A promoter-reporter assay and qRT-PCR confirmed that the transcription of the cps genes was repressed by holo-FNR. In addition, we found that holo-FNR could repress the transcription of rmpA and rmpA2, encoding cps transcriptional activators. Deletion of rmpA or rmpA2 in the Δfnr strain reduced CPS biosynthesis, suggesting that RmpA and RmpA2 participated in the holo-FNR-mediated repression of cps transcription, thereby regulating the CPS amount, serum resistance, and anti-phagocytosis. Taken together, our results provided evidence that RmpA and RmpA2 participated in the holo-FNR-mediated repression of CPS biosynthesis, and resistance to the host defense in response to oxygen availability.

Porcine epidemic diarrhea virus papain-like protease 2 can be noncompetitively inhibited by 6-thioguanine.

Porcine epidemic diarrhea virus (PEDV) is a coronavirus (CoV) discovered in the 1970s that infects the intestinal tract of pigs, resulting in diarrhea and vomiting. It can cause extreme dehydration and death in neonatal piglets. In Asia, modified live attenuated vaccines have been used to control PEDV infection in recent years. However, a new strain of PEDV that belongs to genogroup 2a appeared in the USA in 2013 and then quickly spread to Canada and Mexico as well as Asian and European countries. Due to the less effective protective immunity provided by the vaccines against this new strain, it has caused considerable agricultural and economic loss worldwide. The emergence of this new strain increases the importance of understanding PEDV as well as strategies for inhibiting it. Coronaviral proteases, including main proteases and papain-like proteases, are ideal antiviral targets because of their essential roles in viral maturation. Here we provide a first description of the expression, purification and structural characteristics of recombinant PEDV papain-like protease 2, moreover present our finding that 6-thioguanine, a chemotherapeutic drug, in contrast to its competitive inhibition on SARS- and MERS-CoV papain-like proteases, is a noncompetitive inhibitor of PEDV papain-like protease 2.