Unraveling the efficacy of verbascoside in thwarting MRSA pathogenicity by targeting sortase A.

In the fight against hospital-acquired infections, the challenge posed by methicillin-resistant Staphylococcus aureus (MRSA) necessitates the development of novel treatment methods. This study focused on undermining the virulence of S. aureus, especially by targeting surface proteins crucial for bacterial adherence and evasion of the immune system. A primary aspect of our approach involves inhibiting sortase A (SrtA), a vital enzyme for attaching microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) to the bacterial cell wall, thereby reducing the pathogenicity of S. aureus. Verbascoside, a phenylethanoid glycoside, was found to be an effective SrtA inhibitor in our research. Advanced fluorescence quenching and molecular docking studies revealed a specific interaction between verbascoside and SrtA, pinpointing the critical active sites involved in this interaction. This molecular interaction significantly impedes the SrtA-mediated attachment of MSCRAMMs, resulting in a substantial reduction in bacterial adhesion, invasion, and biofilm formation. The effectiveness of verbascoside has also been demonstrated in vivo, as shown by its considerable protective effects on pneumonia and Galleria mellonella (wax moth) infection models. These findings underscore the potential of verbascoside as a promising component in new antivirulence therapies for S. aureus infections. By targeting crucial virulence factors such as SrtA, agents such as verbascoside constitute a strategic and potent approach for tackling antibiotic resistance worldwide. KEY POINTS: • Verbascoside inhibits SrtA, reducing S. aureus adhesion and biofilm formation. • In vivo studies demonstrated the efficacy of verbascoside against S. aureus infections. • Targeting virulence factors such as SrtA offers new avenues against antibiotic resistance.

Therapeutic potential activity of quercetin complexes against Streptococcus pneumoniae.

This study investigates quercetin complexes as potential synergistic agents against the important respiratory pathogen Streptococcus pneumoniae. Six quercetin complexes (QCX1-6) were synthesized by reacting quercetin with various metal salts and boronic acids and characterized using FTIR spectroscopy. Their antibacterial activity alone and in synergism with antibiotics was evaluated against S. pneumoniae ATCC 49619 using disc diffusion screening, broth microdilution MIC determination, and checkerboard assays. Complexes QCX-3 and QCX-4 demonstrated synergy when combined with levofloxacin via fractional inhibitory concentration indices ≤ 0.5 as confirmed by time-kill kinetics. Molecular docking elucidated interactions of these combinations with virulence enzymes sortase A and sialidase. A biofilm inhibition assay found the synergistic combinations more potently reduced biofilm formation versus monotherapy. Additionally, gene-gene interaction networks, biological activity predictions and in-silico toxicity profiling provided insights into potential mechanisms of action and safety.

N6-methyladenosine-modified SENP1, identified by IGF2BP3, is a novel molecular marker in acute myeloid leukemia and aggravates progression by activating AKT signal via de-SUMOylating HDAC2.

BACKGROUND: Elevated evidence suggests that the SENPs family plays an important role in tumor progression. However, the role of SENPs in AML remains unclear. METHODS: We evaluated the expression pattern of SENP1 based on RNA sequencing data obtained from OHSU, TCGA, TARGET, and MILE datasets. Clinical samples were used to verify the expression of SENP1 in the AML cells. Lentiviral vectors shRNA and sgRNA were used to intervene in SENP1 expression in AML cells, and the effects of SENP1 on AML proliferation and anti-apoptosis were detected using in vitro and in vivo models. Chip-qPCR, MERIP-qPCR, CO-IP, RNA pulldown, and dual-luciferase reporter gene assays were used to explore the regulatory mechanisms of SNEP1 in AML. RESULTS: SENP1 was significantly upregulated in high-risk AML patients and closely related to poor prognosis. The AKT/mTOR signaling pathway is a key downstream pathway that mediates SENP1's regulation of AML proliferation and anti-apoptosis. Mechanistically, the CO-IP assay revealed binding between SENP1 and HDAC2. SUMO and Chip-qPCR assays suggested that SENP1 can desumoylate HDAC2, which enhances EGFR transcription and activates the AKT pathway. In addition, we found that IGF2BP3 expression was upregulated in high-risk AML patients and was positively correlated with SENP1 expression. MERIP-qPCR and RIP-qPCR showed that IGF2BP3 binds SENP1 3-UTR in an m6A manner, enhances SENP1 expression, and promotes AKT pathway conduction. CONCLUSIONS: Our findings reveal a distinct mechanism of SENP1-mediated HDAC2-AKT activation and establish the critical role of the IGF2BP3/SENP1signaling axis in AML development.

OTUB1 contributes to the stability and function of Influenza A virus NS2.

The influenza A virus (IAV) consists of 8 single-stranded, negative-sense viral RNA (vRNA) segments. After infection, vRNA is transcribed, replicated, and wrapped by viral nucleoprotein (NP) to form viral ribonucleoprotein (vRNP). The transcription, replication, and nuclear export of the viral genome are regulated by the IAV protein, NS2, which is translated from spliced mRNA transcribed from viral NS vRNA. This splicing is inefficient, explaining why NS2 is present in low abundance after IAV infection. The levels of NS2 and its subsequent accumulation are thought to influence viral RNA replication and vRNP nuclear export. Here we show that NS2 is ubiquitinated at the K64 and K88 residues by K48-linked and K63-linked polyubiquitin (polyUb) chains, leading to the degradation of NS2 by the proteasome. Additionally, we show that a host deubiquitinase, OTUB1, can remove polyUb chains conjugated to NS2, thereby stabilizing NS2. Accordingly, knock down of OTUB1 by siRNA reduces the nuclear export of vRNP, and reduces the overall production of IAV. These results collectively demonstrate that the levels of NS2 in IAV-infected cells are regulated by a ubiquitination-deubiquitination system involving OTUB1 that is necessary for optimal IAV replication.

SENP1-Mediated deSUMOylation Regulates the Tumor Remodeling of Glioma Stem Cells Under Hypoxic Stress.

Objectives: This study aimed to investigate the effect of specific small ubiquitin-like modifier (SUMO) proteases 1 (SENP1)-mediated deSUMOylation on the malignant behavior of glioma stem cells (GSCs) under hypoxia conditions and evaluate the clinical value of prevention in glioma patients. Introductions: Under hypoxic conditions, upregulated hypoxia-inducible factor 1α (HIF1α) expression in GSCs activates Wnt/ß-catenin signaling pathways, which provide rich nutritional support for glioblastoma (GBM). SENP1-mediated deSUMOylation stabilizes the expression of HIF1α and ß-catenin, leading to the occurrence of GSCs-initiated tumorigenesis. Targeting SENP1-mediated deSUMOylation may suppress the malignancy of GSCs and disrupt GBM progression. Methods: The expression of SENP1 in different World Health Organization grades was observed by immunohistochemistry and western blot. Lentivirus-packaged SENP1shRNA downregulated the expression of SENP1 in GSCs, and the downregulated results were verified by western blotting and polymerase chain reaction. The effects of LV-SENP1shRNA on the migration and proliferation of GSCs were detected by scratch and cloning experiments. The effect of LV-SENP1shRNA on the tumor formation ability of GSCs was observed in nude mice. Immunoprecipitation clarified the mechanism of SENP1 regulating the malignant behavior of GSCs under hypoxia. The correlation between the expression level of SENP1 and the survival of glioma patients was determined by statistical analysis. Results: SENP1 expression in GSCs derived from clinical samples was upregulated in GBM. SUMOylation was observed in GSCs in vitro, and deSUMOylation, accompanied by an increase in SENP1 expression, was induced by hypoxia. SENP1 expression was downregulated in GSCs with lentivirus-mediated stable transfection, which attenuated the proliferation and differentiation of GSCs, thus diminishing tumorigenesis. Mechanistically, HIF1α induced activation of Wnt/ß-catenin, which depended on SENP1-mediated deSUMOylation, promoting GSC-driven GBM growth under the hypoxia microenvironment. Conclusion: Our findings indicate that SENP1-mediated deSUMOylation as a feature of GSCs is essential for GBM maintenance, suggesting that targeting SENP1 against GSCs may effectively improve GBM therapeutic efficacy.

SUMO-specific protease 1 regulates germinal center B cell response through deSUMOylation of PAX5.

Humoral immunity depends on the germinal center (GC) reaction where B cells are tightly controlled for class-switch recombination and somatic hypermutation and finally generated into plasma and memory B cells. However, how protein SUMOylation regulates the process of the GC reaction remains largely unknown. Here, we show that the expression of SUMO-specific protease 1 (SENP1) is up-regulated in GC B cells. Selective ablation of SENP1 in GC B cells results in impaired GC dark and light zone organization and reduced IgG1-switched GC B cells, leading to diminished production of class-switched antibodies with high-affinity in response to a TD antigen challenge. Mechanistically, SENP1 directly binds to Paired box protein 5 (PAX5) to mediate PAX5 deSUMOylation, sustaining PAX5 protein stability to promote the transcription of activation-induced cytidine deaminase. In summary, our study uncovers SUMOylation as an important posttranslational mechanism regulating GC B cell response.

Seneca valley virus 3C protease blocks EphA2-Mediated mTOR activation to facilitate viral replication.

The Seneca Valley virus (SVV) is a recently discovered porcine pathogen that causes vesicular diseases and poses a significant threat to the pig industry worldwide. Erythropoietin-producing hepatoma receptor A2 (EphA2) is involved in the activation of the AKT/mTOR signaling pathway, which is involved in autophagy. However, the regulatory relationship between SVV and EphA2 remains unclear. In this study, we demonstrated that EphA2 is proteolysed in SVV-infected BHK-21 and PK-15 cells. Overexpression of EphA2 significantly inhibited SVV replication, as evidenced by decreased viral protein expression, viral titers, and viral load, suggesting an antiviral function of EphA2. Subsequently, viral proteins involved in the proteolysis of EphA2 were screened, and the SVV 3C protease (3Cpro) was found to be responsible for this cleavage, depending on its protease activity. However, the protease activity sites of 3Cpro did not affect the interactions between 3Cpro and EphA2. We further determined that EphA2 overexpression inhibited autophagy by activating the mTOR pathway and suppressing SVV replication. Taken together, these results indicate that SVV 3Cpro targets EphA2 for cleavage to impair its EphA2-mediated antiviral activity and emphasize the potential of the molecular interactions involved in developing antiviral strategies against SVV infection.

SENP1 regulates intermittent hypoxia-induced microglia mediated inflammation and cognitive dysfunction via wnt/ß-catenin pathway.

Obstructive sleep apnea syndrome (OSAS), characterized by repeated narrow or collapse of the upper airway during sleep, resulting in periodic reductions or cessations in ventilation, consequent hypoxia, hypercapnia, increased sympathetic activity and sleep fragmentation, places a serious burden on society and health care. Intermittent hypoxia (IH), which cause central nervous system (CNS) inflammation, and ultimately lead to neuropathy, is thought to be a crucial contributor to cognitive impairment in OSAS. Wnt signaling pathway exerts an important role in the regulation of CNS disorders. Particularly, it may be involved in the regulation of neuroinflammation and cognitive dysfunction. However, its underlying mechanism remains poorly understood. Accumulating evidence demonstrated that Wnt signaling pathway may inhibited in a variety of neurological disorders. Recently studies revealed that SUMOylation was participated in the regulation of neuroinflammation. Members of Wnt/ß-catenin pathway may be targets of SUMOylation. In vitro and in vivo molecular biology experiments explored the regulatory mechanism of SUMOylation on Wnt/ß-catenin in IH-induced neuroinflammation and neuronal injury, which demonstrated that IH induced the SUMOylation of ß-catenin, microglia mediated inflammation and neuronal damage. Moreover, SENP1 regulated the de-SUMOylation of ß-catenin, triggered Wnt/ß-catenin pathway, and alleviated neuroinflammation and neuronal injury, thus improving IH-related mice cognitive dysfunction.

Sortase A-Based Post-translational Modifications on Encapsulin Nanocompartments.

Protein-based encapsulin nanocompartments, known for their well-defined structures and versatile functionalities, present promising opportunities in the fields of biotechnology and nanomedicine. In this investigation, we effectively developed a sortase A-mediated protein ligation system in Escherichia coli to site-specifically attach target proteins to encapsulin, both internally and on its surfaces without any further in vitro steps. We explored the potential applications of fusing sortase enzyme and a protease for post-translational ligation of encapsulin to a green fluorescent protein and anti-CD3 scFv. Our results demonstrated that this system could attach other proteins to the nanoparticles' exterior surfaces without adversely affecting their folding and assembly processes. Additionally, this system enabled the attachment of proteins inside encapsulins which varied shapes and sizes of the nanoparticles due to cargo overload. This research developed an alternative enzymatic ligation method for engineering encapsulin nanoparticles to facilitate the conjugation process.

A new point mutation in the HC-Pro of potato virus Y is involved in tobacco vein necrosis.

Tobacco vein necrosis (TVN) is a complex phenomenon regulated by different genetic determinants mapped in the HC-Pro protein (amino acids N330, K391 and E410) and in two regions of potato virus Y (PVY) genome, corresponding to the cytoplasmic inclusion (CI) protein and the nuclear inclusion protein a-protease (NIa-Pro), respectively. A new determinant of TVN was discovered in the MK isolate of PVY which, although carried the HC-Pro determinants associated to TVN, did not induce TVN. The HC-Pro open reading frame (ORF) of the necrotic infectious clone PVY N605 was replaced with that of the non-necrotic MK isolate, which differed only by one amino acid at position 392 (T392 instead of I392). The cDNA clone N605_MKHCPro inoculated in tobacco induced only weak mosaics at the systemic level, demostrating that the amino acid at position 392 is a new determinant for TVN. No significant difference in accumulation in tobacco was observed between N605 and N605_MKHCPro. Since phylogenetic analyses showed that the loss of necrosis in tobacco has occurred several times independently during PVY evolution, these repeated evolutions strongly suggest that tobacco necrosis is a costly trait in PVY.

Non-canonical deubiquitination of OTUB1 induces IFNγ-mediated cell cycle arrest via regulation of p27 stability.

The deubiquitinase OTUB1, implicated as a potential oncogene in various tumors, lacks clarity in its regulatory mechanism in tumor progression. Our study investigated the effects and underlying mechanisms of OTUB1 on the breast cancer cell cycle and proliferation in IFNγ stimulation. Loss of OTUB1 abrogated IFNγ-induced cell cycle arrest by regulating p27 protein expression, whereas OTUB1 overexpression significantly enhanced p27 expression even without IFNγ treatment. Tyr26 phosphorylation residue of OTUB1 directly bound to p27, modulating its post-translational expression. Furthermore, we identified crucial lysine residues (K134, K153, and K163) for p27 ubiquitination. Src downregulation reduced OTUB1 and p27 expression, suggesting that IFNγ-induced cell cycle arrest is mediated by the Src-OTUB1-p27 signaling pathway. Our findings highlight the pivotal role of OTUB1 in IFNγ-induced p27 expression and cell cycle arrest, offering therapeutic implications.

SARS-CoV-2 NSP5 antagonizes MHC II expression by subverting histone deacetylase 2.

SARS-CoV-2 interferes with antigen presentation by downregulating major histocompatibility complex (MHC) II on antigen-presenting cells, but the mechanism mediating this process is unelucidated. Herein, analysis of protein and gene expression in human antigen-presenting cells reveals that MHC II is downregulated by the SARS-CoV-2 main protease, NSP5. This suppression of MHC II expression occurs via decreased expression of the MHC II regulatory protein CIITA. CIITA downregulation is independent of the proteolytic activity of NSP5, and rather, NSP5 delivers HDAC2 to the transcription factor IRF3 at an IRF-binding site within the CIITA promoter. Here, HDAC2 deacetylates and inactivates the CIITA promoter. This loss of CIITA expression prevents further expression of MHC II, with this suppression alleviated by ectopic expression of CIITA or knockdown of HDAC2. These results identify a mechanism by which SARS-CoV-2 limits MHC II expression, thereby delaying or weakening the subsequent adaptive immune response.

The Transpeptidase Sortase A Binds Nucleic Acids and Mediates Mammalian Cell Labeling.

Wild-type sortase A is an important virulence factor displaying a diverse array of proteins on the surface of bacteria. This protein display relies on the transpeptidase activity of sortase A, which is widely engineered to allow protein ligation and protein engineering based on the interaction between sortase A and peptides. Here an unknown interaction is found between sortase A from Staphylococcus aureus and nucleic acids, in which exogenously expressed engineered sortase A binds oligonucleotides in vitro and is independent of its canonical transpeptidase activity. When incubated with mammalian cells, engineered sortase A further mediates oligonucleotide labeling to the cell surface, where sortase A attaches itself and is part of the labeled moiety. The labeling reaction can also be mediated by many classes of wild-type sortases as well. Cell surface GAG appears involved in sortase-mediated oligonucleotide cell labeling, as demonstrated by CRISPR screening. This interaction property is utilized to develop a technique called CellID to facilitate sample multiplexing for scRNA-seq and shows the potential of using sortases to label cells with diverse oligonucleotides. Together, the binding between sortase A and nucleic acids opens a new avenue to understanding the virulence of wild-type sortases and exploring the application of sortases in biotechnology.

USP26 promotes colorectal cancer tumorigenesis by restraining PRKN-mediated mitophagy.

Deubiquitinating enzymes (DUBs) are promising targets for cancer therapy because of their pivotal roles in various physiological and pathological processes. Among these, ubiquitin-specific peptidase 26 (USP26) is a protease with crucial regulatory functions. Our study sheds light on the upregulation of USP26 in colorectal cancer (CRC), in which its increased expression correlates with an unfavorable prognosis. Herein, we evidenced the role of USP26 in promoting CRC tumorigenesis in a parkin RBR E3 ubiquitin-protein ligase (PRKN) protein-dependent manner. Our investigation revealed that USP26 directly interacted with PRKN protein, facilitating its deubiquitination, and subsequently reducing its activity. Additionally, we identified the K129 site on PRKN as a specific target for USP26-mediated deubiquitination. Our research highlights that a K-to-R mutation at the site on PRKN diminishes its potential for activation and ability to mediate mitophagy. In summary, our findings underscore the significance of USP26-mediated deubiquitination in restraining the activation of the PRKN-mediated mitophagy pathway, ultimately driving CRC tumorigenesis. This study not only elucidated the multifaceted role of USP26 in CRC but also introduced a promising avenue for therapeutic exploration through the development of small molecule inhibitors targeting USP26. This strategy holds promise as a novel therapeutic approach for CRC.

A flexible 'plug and play' bicistronic construct and its application in the screening of protein expression system in Escherichia coli.

The bicistronic expression system that utilizes fluorescent reporters has been demonstrated to be a straightforward method for detecting recombinant protein expression levels, particularly when compared to polyacrylamide gel electrophoresis and immunoblot analysis, which are tedious and labor-intensive. However, existing bicistronic reporter systems are less capable of quantitative measurement due to the lag in reporter expression and its negative impact on target protein. In this work, a plug and play bicistronic construct using mCherry as reporter was applied in the screening of optimal replicon and promoter for Sortase expression in Escherichia coli (E. coli). The bicistronic construct allowed the reporter gene and target open reading frame (ORF) to be co-transcribed under the same promoter, resulting in a highly positive quantitative correlation between the expression titer of Sortase and the fluorescent intensity (R2 > 0.97). With the correlation model, the titer of target protein can be quantified by noninvasively measuring the fluorescent intensity. On top of this, the expression of reporter has no significant effect on the yield of target protein, thus favoring a plug and play design for removing reporter gene to generate a plain plasmid for industrial use.

Development of an attenuated potato virus Y mutant carrying multiple mutations in helper-component protease for cross-protection.

Tobacco (Nicotiana tabacum) is one of the major cash crops in China. Potato virus Y (PVY), a representative member of the genus Potyvirus, greatly reduces the quality and yield of tobacco leaves by inducing veinal necrosis. Mild strain-mediated cross-protection is an attractive method of controlling diseases caused by PVY. Currently, there is a lack of effective and stable attenuated PVY mutants. Potyviral helper component-protease (HC-Pro) is a likely target for the development of mild strains. Our previous studies showed that the residues lysine at positions 124 and 182 (K124 and K182) in HC-Pro were involved in PVY virulence, and the conserved KITC motif in HC-Pro was involved in aphid transmission. In this study, to improve the stability of PVY mild strains, K at position 50 (K50) in KITC motif, K124, and K182 were separately substituted with glutamic acid (E), leucine (L), and arginine (R), resulting in a triple-mutant PVY-HCELR. The mutant PVY-HCELR had attenuated virulence and did not induce leaf veinal necrosis symptoms in tobacco plants and could not be transmitted by Myzus persicae. Furthermore, PVY-HCELR mutant was genetically stable after six serial passages, and only caused mild mosaic symptoms in tobacco plants even at 90 days post inoculation. The tobacco plants cross-protected by PVY-HCELR mutant showed high resistance to the wild-type PVY. This study showed that PVY-HCELR mutant was a promising mild mutant for cross-protection to control PVY.

Ssc-miR-7139-3p suppresses foot-and-mouth disease virus replication by promoting degradation of 3Cpro through targeting apoptosis-negative regulatory gene Bcl-2.

Foot-and-mouth disease is a highly contagious and infectious disease affecting cloven-hoofed animals. Disease control is complicated by its highly contagious nature and antigenic diversity. Host microRNAs (miRNAs) are post-transcriptional regulators that either promote or repress viral replications in virus infection. In the present study, we found that ssc-miR-7139-3p (Sus scrofa miR-7139-3p) was significantly up-regulated in host cells during foot-and-mouth disease virus (FMDV) infection. Overexpression of miR-7139-3p attenuated FMDV replication, whereas inhibition promoted FMDV replication. In addition, the survival rate of FMDV infected suckling mice was increased through injection of miR-7139-3p agomiR. Further studies revealed that miR-7139-3p targets Bcl-2 to initiate the apoptotic pathway and caspase-3 cleaved 3Cpro behind the 174th aspartic acid (D174), which eventually promotes the degradation of 3Cpro. Overall, our findings demonstrate that miR-7139-3p suppresses FMDV replication by promoting degradation of 3Cpro through targeting the apoptosis-negative regulatory gene Bcl-2.

Legumain-Triggered Macrocyclization of Radiofluorinated Tracer for Enhanced PET Imaging.

Enhancing the accumulation and retention of small-molecule probes in tumors is an important way to achieve accurate cancer diagnosis and therapy. Enzyme-stimulated macrocyclization of small molecules possesses great potential for enhanced positron emission tomography (PET) imaging of tumors. Herein, we reported an 18F-labeled radiotracer [18F]AlF-RSM for legumain detection in vivo. The tracer was prepared by a one-step aluminum-fluoride-restrained complexing agent ([18F]AlF-RESCA) method with high radiochemical yield (RCY) (88.35 ± 3.93%) and radiochemical purity (RCP) (>95%). More notably, the tracer can be transformed into a hydrophobic macrocyclic molecule under the joint action of legumain and reductant. Simultaneously, the tracer could target legumain-positive tumors and enhance accumulation and retention in tumors, resulting in the amplification of PET imaging signals. The enhancement of radioactivity enables PET imaging of legumain activity with high specificity. We envision that, by combining this highly efficient 18F-labeled strategy with our intramolecular macrocyclization reaction, a range of radiofluorinated tracers can be designed for tumor PET imaging and early cancer diagnosis in the future.

Development of Novel Peptidyl Nitriles Targeting Rhodesain and Falcipain-2 for the Treatment of Sleeping Sickness and Malaria.

In recent decades, neglected tropical diseases and poverty-related diseases have become a serious health problem worldwide. Among these pathologies, human African trypanosomiasis, and malaria present therapeutic problems due to the onset of resistance, toxicity problems and the limited spectrum of action. In this drug discovery process, rhodesain and falcipain-2, of Trypanosoma brucei rhodesiense and Plasmodium falciparum, are currently considered the most promising targets for the development of novel antitrypanosomal and antiplasmodial agents, respectively. Therefore, in our study we identified a novel lead-like compound, i.e., inhibitor 2b, which we proved to be active against both targets, with a Ki = 5.06 µM towards rhodesain and an IC50 = 40.43 µM against falcipain-2.

Sortase-Modified Cholera Toxoids Show Specific Golgi Localization.

Cholera toxoid is an established tool for use in cellular tracing in neuroscience and cell biology. We use a sortase labeling approach to generate site-specific N-terminally modified variants of both the A2-B5 heterohexamer and B5 pentamer forms of the toxoid. Both forms of the toxoid are endocytosed by GM1-positive mammalian cells, and while the heterohexameric toxoid was principally localized in the ER, the B5 pentamer showed an unexpectedly specific localization in the medial/trans-Golgi. This study suggests a future role for specifically labeled cholera toxoids in live-cell imaging beyond their current applications in neuronal tracing and labeling of lipid rafts in fixed cells.