The hydrophobic cluster on the surface of protein is the key structural basis for the SDS-resistance of chondroitinase VhChlABC.

The application of chondroitinase requires consideration of the complex microenvironment of the target. Our previous research reported a marine-derived sodium dodecyl sulfate (SDS)-resistant chondroitinase VhChlABC. This study further investigated the mechanism of VhChlABC resistance to SDS. Focusing on the hydrophobic cluster on its strong hydrophilic surface, it was found that the reduction of hydrophobicity of surface residues Ala181, Met182, Met183, Ala184, Val185, and Ile305 significantly reduced the SDS resistance and stability. Molecular dynamics (MD) simulation and molecular docking analysis showed that I305G had more conformational flexibility around residue 305 than wild type (WT), which was more conducive to SDS insertion and binding. The affinity of A181G, M182A, M183A, V185A and I305G to SDS was significantly higher than that of WT. In conclusion, the surface hydrophobic microenvironment composed of six residues was the structural basis for SDS resistance. This feature could prevent the binding of SDS and the destruction of hydrophobic packaging by increasing the rigid conformation of protein and reducing the binding force of SDS-protein. The study provides a new idea for the rational design of SDS-resistant proteins and may further promote chondroitinase research in the targeted therapy of lung diseases under the pressure of pulmonary surfactant. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00201-1.

Falcarindiol Isolated from Notopterygium incisum Inhibits the Quorum Sensing of Pseudomonas aeruginosa.

Quorum sensing (QS) is employed by the opportunistic pathogen Pseudomonas aeruginosa to regulate physiological behaviors and virulence. QS inhibitors (QSIs) are potential anti-virulence agents for the therapy of P. aeruginosa infection. During the screening for QSIs from Chinese herbal medicines, falcarindiol (the major constituent of Notopterygium incisum) exhibited QS inhibitory activity. The subinhibitory concentration of falcarindiol exerted significant inhibitory effects on the formation of biofilm and the production of virulence factors such as elastase, pyocyanin, and rhamnolipid. The mRNA expression of QS-related genes (lasB, phzH, rhlA, lasI, rhlI, pqsA, and rhlR) was downregulated by falcarindiol while that of lasR was not affected by falcarindiol. The transcriptional activation of the lasI promoter was inhibited by falcarindiol in the P. aeruginosa QSIS-lasI selector. Further experiments confirmed that falcarindiol inhibited the las system using the reporter strain Escherichia coli MG4/pKDT17. Electrophoretic mobility shift assay (EMSA) showed that falcarindiol inhibited the binding of the transcription factor LasR and the lasI promoter region. Molecular docking showed that falcarindiol interacted with the Tyr47 residue, leading to LasR instability. The decrease of LasR-mediated transcriptional activation was responsible for the reduction of downstream gene expression, which further inhibited virulence production. The inhibition mechanism of falcarindiol to LasR provides a theoretical basis for its medicinal application.

Identification of Crocin as a New hIAPP Amyloid Inhibitor via a Simple Yet Highly Biospecific Screening System.

Amylin (hIAPP) amyloid formation plays an important role in the pathogenesis of type 2 diabetes (T2D), which makes it a promising therapeutic target for T2D. In this study, we established a screening tool for identifying chemicals affecting hIAPP amyloid formation based on a reported genetic tool, which constantly tracks protein aggregates in Saccharomyces cerevisiae. In order to obtain the hIAPP with better aggregation ability, the gene of hIAPP was tandemly ligated to create 1×, 2×, 4× or 6×-hIAPP expressing strains. By measuring the cell density and fluorescence intensity of green fluorescent protein (GFP) regulated by the aggregation status of hIAPP, it was found that four intramolecular ligated hIAPP (4×hIAPP) could form obvious amyloids with mild toxicity. The validity and reliability of the screening tool were verified by testing six reported hIAPP inhibitors, including curcumin, epigallocatechin gallate and so on. Combined with surface plasmon resonance (SPR) and the screening tool, which could be a screening system for hIAPP inhibitors, we found that crocin specifically binds to hIAPP and acts inhibit amyloid formation of hIAPP. The effect of crocin was further confirmed by Thioflavin T (ThT) fluorescence and transmission electron microscopy (TEM) analysis. Thus, a screening system for hIAPP amyloid inhibitors and a new mechanism of crocin on anti-T2D were obtained as a result of this study.

Equisetin as potential quorum sensing inhibitor of Pseudomonas aeruginosa.

OBJECTIVE: To screen for the quorum-sensing (QS) inhibitors from marine-derived fungi and evaluate their anti-QS properties in Pseudomonas aeruginosa. RESULTS: QS inhibitory activity was found in secondary metabolites of a marine fungus Fusarium sp. Z10 using P. aeruginosa QSIS-lasI biosensor. The major active compound of this fungus was isolated by HPLC and identified as equisetin. Subinhibitory concentration of equisetin could inhibit the formation of biofilm, swarming motility, and the production of virulence factors in P. aeruginosa. The inhibition of las, PQS, and rhl system by equisetin were determined using Escherichia coli MG4/pKDT17, E.coli pEAL08-2, and E.coli pDSY, respectively. Real-time RT-PCR assays showed that equisetin could downregulate the mRNA expression of QS-related genes. CONCLUSIONS: Equisetin proved its potential as an inhibitor against P. aeruginosa QS system and might also serve as precursor compound in development of novel therapeutics for infectious diseases by optimal design of structures.

[Research progress of new antibacterial drugs that target bacterial quorum sensing systems].

In recent years, antibiotic resistance of bacteria has become a global health crisis. Especially, the new class of "superbug" was found in South Asia, which is resistant to almost known antibiotics and causes worldwide alarm. Through the underlying mechanisms of bacterial pathogenecity, the expression of many pathogen virulence factors is regulated by the process of quorum sensing. Screening efficient quorum sensing inhibitors is an especially compelling approach to the future treatment of bacterial infections and antibiotic resistance. This article focuses on bacterial quorum sensing system, quorum sensing screening model for in vitro and evaluation of animal models in vivo, recent research of quorum sensing inhibitors and so on.

Construction of an effective screening system for detection of Pseudomonas aeruginosa quorum sensing inhibitors and its application in bioautographic thin-layer chromatography.

In Pseudomonas aeruginosa, quorum sensing (QS) regulates dozens of genes and proteins, many of which contribute to the virulence of this pathogen. QS inhibitory (QSI) compounds have been proposed as potential agents for treatment of bacterial infections. To search for Ps. aeruginosa QS inhibitors, we constructed an effective screening system, QSIS-lasI selector, based on the PlasI-sacB reporter, in which QS could be induced with 20 nM 3-oxo-N-[(3S)-tetrahydro-2-oxo-3-furanyl]-dodecanamide (3-oxo-C(12)-HSL). During screening of the crude extracts from 65 marine fungi, an isolate of Penicillium atramentosum was found to have QSI activity. Thin-layer chromatography assay of the fungal extracts for bioautographic identification of QSIS-lasI indicated that this fungus produced several QSI compounds, including QS inhibitors other than penicillic acid or patulin.

Polypeptide from Chlamys farreri attenuates murine thymocytes damage induced by ultraviolet B.

AIM: Polypeptide from Chlamys farreri (PCF, molecular mass is 879) is a new marine polypeptide compound isolated from Chlamys farreri. This study investigates the possible protective roles and the mechanism of PCF against ultraviolet B (UVB)-induced apoptosis in murine thymocytes. METHODS: The rate of apoptosis and caspase-3 activation was measured by flow cytometry. The expression of stress-response genes c-fos and c-jun was observed by RT-PCR. Western blot analysis was performed to determine the release of cytochrome c. RESULTS: It was found that UVB induced murine thymocyte death. The cells treated with UVB showed an increase in cytochrome c release, caspase-3 activity, as well as in the expression of c-fos and c-jun. In addition, all were involved in UVB-induced cell apoptosis. CONCLUSION: Our present observations pointed to the ability of PCF to avert UVB-induced apoptosis in thymocytes by modulating c-fos and c-jun expression, cytochrome c release, and the consequent activation of caspase-3, which were essential components of the UV-induced cell apoptotic pathway. The results suggested that PCF is a promising protective substance against UV radiation.

[Induction of CuZn-SOD mRNA expression and activity by PGMS in rat liver].

AIM: To study the effect of propylene glycol mannate sulfate (PGMS) on induction of CuZn-SOD. METHODS: Wistar rats were given PGMS p.o. at different doses (0, 18.9, 37.8 and 75.6 mg.kg-1.d) for ten days. Then the rats were sacrificed and the total RNA was extracted from the livers. The total RNA samples were loaded on a 1% agarose gel to detect the quality of total RNA. RT-PCR was applied to study the expression of CuZn-SOD mRNA in rat livers. The amplified products were detected by the 1.5% agarose gel electrophoresis. Simultaneously, the CuZn-SOD activities in rat liver were determined by nitrite method. RESULTS: The total RNA extracted from rat livers was integrated without being decomposed by RNase. The level of CuZn-SOD mRNA of the high-dosage group (75.6 mg.kg-1.d) was higher than that of the control group (0 mg.kg-1.d) (P < 0.01); the CuZn-SOD activities of the high-dosage group were significantly higher than those of the control group (P < 0.001) and the CuZn-SOD activities of the middle- (37.8 mg.kg-1.d) and low-dosage groups (18.9 mg.kg-1.d) were higher than those of the control group (P < 0.01). CONCLUSION: PGMS can increase the CuZn-SOD activities as well as CuZn-SOD on mRNA level. Therefore, it is possible for PGMS to counteract Atherosclerosis (AS) by inducing the expression of CuZn-SOD.