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Multi-resistant Staphylococcus aureus (S. aureus) infection is a significant global health concern owing to its high mortality and morbidity rates. Coagulase (Coa), a key enzyme that activates prothrombin to initiate host coagulation, has emerged as a promising target for anti-infective therapeutic approaches. This study identified sinigrin as a potent Coa inhibitor that significantly inhibited S. aureus-induced coagulation at concentration as low as 32 mg/L. Additionally, at a higher concentration of 128 mg/L, sinigrin disrupted the self-protection mechanism of S. aureus. Thermal shift and fluorescence-quenching assays confirmed the direct binding of sinigrin to the Coa protein. Molecular docking analysis predicted specific binding sites for sinigrin in the Coa molecule, and point mutation experiments highlighted the importance of Arg-187 and Asp-222 as critical binding sites for both Coa and sinigrin. In vivo studies demonstrated that the combination of sinigrin with oxacillin exhibited greater antibacterial efficacy than oxacillin alone in the treatment of S. aureus-induced pneumonia in mice. Furthermore, sinigrin was shown to reduce bacterial counts and inflammatory cytokine levels in the lung tissues of S. aureus-infected mice. In summary, sinigrin was shown to directly target Coa, resulting in the attenuation of S. aureus virulence, which suggests the potential of sinigrin as an adjuvant for future antimicrobial therapies.
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AIMS: The main purpose of this study was to study the therapeutical effect of oroxylin A glucuronide (OAG) on methicillin-resistant Staphylococcus aureus (MRSA). METHODS AND RESULTS: By substrate peptide reaction-based fluorescence resonance energy transfer (FRET) screening, we identified that OAG was an efficient inhibitor of Sortase A (SrtA) with an IC50 of 45.61 µg mL-1, and achieved efficacy in the treatment of Staphylococcus aureus (S. aureus) infections. We further demonstrated that OAG inhibited the adhesion of the S. aureus to fibrinogen, the surface protein A anchoring and diminished biofilm formation. Results obtained from fluorescence quenching assay elucidated a direct interaction between OAG and SrtA. Employing molecular dynamics simulations, we proved that OAG binds to the binding sites of R197, G192, E105, and V168 in the SrtA. Notably, OAG exhibited a robust therapeutic effect in a MRSA-induced pneumonia model. CONCLUSIONS: We identified that OAG as a novel class of reversible inhibitors of SrtA, combats MRSA-induced Infections.
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Staphylococcus aureus Resistente à Meticilina , Staphylococcus aureus Resistente à Meticilina/metabolismo , Staphylococcus aureus , Glucuronídeos/farmacologia , Proteínas de Bactérias/metabolismoRESUMO
Staphylococcus aureus (S. aureus) induces a variety of infectious diseases in humans and animals and is responsible for hospital- and community-acquired infections. The aim of this study was to investigate how bilobetin, a natural compound, attenuates S. aureus virulence by inhibiting two key virulence factors, von Willebrand factor-binding protein (vWbp) and staphylocoagulase (Coa). The results showed that bilobetin inhibited Coa- or vWbp-induced coagulation without affecting S. aureus proliferation. The Western blotting and fluorescence quenching assays indicated that bilobetin did not affect the expression of vWbp and Coa but directly bound to the proteins with KA values of 1.66 × 104 L/mol and 1.04 × 104 L/mol, respectively. To gain further insight into the mechanism of interaction of bilobetin with these virulence factors, we performed molecular docking and point mutation assays, which indicated that the TYR-6 and TYR-18 residues on vWbp and the ALA-190 and ASP-189 residues on Coa were essential for the binding of bilobetin. In addition, the in vivo studies showed that bilobetin ameliorated lung tissue damage and inflammation caused by S. aureus, thereby improving the survival of mice. Furthermore, the use of bilobetin as an adjuvant in combination with vancomycin was more effective in the treatment of a mouse model of pneumonia. Taken together, bilobetin had a dual inhibitory effect on vWbp and Coa by reducing the virulence of S. aureus, suggesting that it is a viable lead compound against S. aureus infections.
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Coagulase , Infecções Estafilocócicas , Humanos , Camundongos , Animais , Coagulase/genética , Coagulase/metabolismo , Coagulase/farmacologia , Proteínas de Transporte/metabolismo , Staphylococcus aureus , Virulência , Fator de von Willebrand/metabolismo , Fator de von Willebrand/farmacologia , Simulação de Acoplamento Molecular , Infecções Estafilocócicas/tratamento farmacológico , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Antimicrobial resistance (AMR) poses a major threat to human health globally. Staphylococcus aureus is recognized as a cause of disease worldwide, especially methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA). The enzyme sortase A (SrtA), present on the cell surface of S. aureus, plays a key role in bacterial virulence without affecting the bacterial viability, and SrtA-deficient S. aureus strains do not affect the growth of bacteria. Here, we found that punicalagin, a natural compound, was able to inhibit SrtA activity with a very low half maximal inhibitory concentration (IC50) value of 4.23 µg/mL, and punicalagin is a reversible inhibitor of SrtA. Moreover, punicalagin has no distinct cytotoxicity toward A549, HEK293T, or HepG2 cells at a much higher concentration than the IC50 detected by MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assays. In addition, punicalagin visibly attenuated the virulence-related phenotype of SrtA in vitro by decreasing adhesion of S. aureus to fibrinogen, reducing the ability of protein A (SpA) displayed on the surface of the bacteria and biofilm formation. Fluorescence quenching elucidated the interaction between punicalagin and SrtA. Molecular docking further implied that the inhibitory activity lay in the bond between punicalagin and SrtA residues LYS190, TYR187, ALA104, and GLU106. In In vivo studies, we surprisingly found that punicalagin had a more effective curative effect combined with cefotaxime when mice were infected with pneumonia caused by MRSA. Essentially, punicalagin, a therapeutic compound targeting SrtA, demonstrates great potential for combating MRSA infections.
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Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Aminoaciltransferases , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Proteínas de Bactérias/genética , Cisteína Endopeptidases , Células HEK293 , Humanos , Taninos Hidrolisáveis , Camundongos , Simulação de Acoplamento Molecular , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureusRESUMO
Staphylococcus aureus (S. aureus), a Gram-positive bacteria, is an incurable cause of hospital and community-acquired infections. Inhibition bacterial virulence is a viable strategy against S. aureus infections based on the multiple virulence factors secreted by S. aureus. Alpha-hemolysin (Hla) plays a crucial role in bacteria virulence without affecting bacterial viability. Here, we identified that 7,8-Dihydroxyflavone (7,8-DHF), a natural compound, was able to decrease the expression of and did not affect the in vitro growth of S. aureus USA300 at a concentration of 32 µg/mL. It was verified by western blot and RT-qPCR that the natural compound could inhibit the transcription and translation of Hla. Further mechanism studies revealed that 7,8-DHF has a negative effect on transcriptional regulator agrA and RNAIII, preventing the upregulation of virulence gene. Cytotoxicity assays showed that 7,8-DHF did not produce significant cytotoxicity to A549 cells. Animal experiments showed that the combination of 7,8-DHF and vancomycin had a more significant therapeutic effect on S. aureus infection, reflecting the synergistic effect of 7,8-DHF with antibiotics. In conclusion, 7,8-DHF was able to target Hla to protect host cells from hemolysis while limiting the development of bacterial resistance.
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Toxinas Bacterianas , Flavonas , Infecções Estafilocócicas , Staphylococcus aureus , Células A549 , Animais , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Toxinas Bacterianas/metabolismo , Flavonas/farmacologia , Proteínas Hemolisinas/genética , Humanos , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Background and aim: Lymphocytes are effector cells that fight cancer by killing tumor cells. Here, we aim to explore the prognostic significance of both peripheral and tumor-infiltrating lymphocytes (TILs) in newly diagnosed stage III/IV non-small-cell lung cancer (NSCLC). Materials and methods: In total, 105 cases of newly diagnosed stage III/IV NSCLC from July 2017 to October 2022 at the Tianjin Beichen Hospital were retrospectively investigated. Peripheral blood samples at the time of diagnosis and tumor tissue slices from these patients were collected. General peripheral blood cell composition and TILs were measured and analyzed via an automatic blood analyzer and immunofluorescence staining analysis. The overall survival (OS) time of all patients was also obtained and analyzed. Results: The median overall survival (mOS) of all patients is 12 months. The 1-, 2-, and 3-year overall survival rates were 60.5, 28.4, and 18.6%, respectively. Peripheral lymphocyte and neutrophil percentages, serum C-reactive protein (CRP) expression, tumor size, and tumor pathology are the prognostic factors of OS for newly diagnosed stage III/IV NSCLC patients. Moreover, patients with high tumor CD4+ and CD8+ T cell infiltration survived significantly longer compared to patients with low tumor CD4+ and CD8+ T cell infiltration (p < 0.0001 and p = 0.011, respectively). Compared to low tumor CD33+ cell infiltration, high tumor CD33+ cell infiltration was associated with worse OS (p = 0.018). High tumor CD8+ T cell infiltration was associated with lower peripheral lymphocyte number, lower serum CRP expression, smaller tumor size, and better tumor pathology (p = 0.012, p = 0.040, p = 0.012, and p = 0.029, respectively). Conclusion: Increased numbers of peripheral lymphocytes, CD33+ cells, CD4+ TILs, and CD8+ TILs were significantly associated with OS in newly diagnosed stage III/IV NSCLC patients, which were positively associated with several basic clinical factors.
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Antimicrobial resistance (AMR) is a global health threat. The dramatic increase of Methicillin-resistant Staphylococcus aureus (MRSA) infections emphasizes the need to find new anti-infective agents with a novel mode of action. The Caseinolytic protease (ClpP) is a central virulence factor in stress survival, virulence, and antibiotic resistance of MRSA. Here, we found ayanin, a flavonoid isolated from Callicarpa nudiflora, was an inhibitor of MRSA ClpP with an IC50 of 19.63 µM. Using quantitative real-time PCR, ayanin reduced the virulence of Staphylococcus aureus (S. aureus) by down-regulating the level of some important virulence factors, including agrA, RNAâ ¢, hla, pvl, psmα and spa. The results of cellular thermal shift assay and thermal shift assay revealed a binding between ayanin and ClpP. Molecular docking showed that ASP-168, ASN-173 and ARG-171 were the potential binding sites for ClpP binding to ayanin. ClpP mutagenesis study further indicated that ARG-171 and ASN-173 were the main active sites of ClpP. The affinity constant (KD) value of ayanin with ClpP was 3.15 × 10-5 M measured by surface plasmon resonance. In addition, ayanin exhibited a significant therapeutic effect on pneumonia infection induced by S. aureus in mice in vivo, especially in combination with vancomycin. This is the first report of ayanin with in vivo and in vitro efficacy against S. aureus infection. In conclusion, ayanin is a promising therapeutic agent to combat MRSA infections by targeting ClpP.
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Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Animais , Camundongos , Staphylococcus aureus , Peptídeo Hidrolases/farmacologia , Simulação de Acoplamento Molecular , Flavonoides/farmacologia , Flavonoides/uso terapêutico , Infecções Estafilocócicas/tratamento farmacológico , Fatores de Virulência , Endopeptidases , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Testes de Sensibilidade MicrobianaRESUMO
The activation of NLRC4 is a major host response against intracellular bacteria infection. However, NLRC4 activation after a host senses diverse stimuli is difficult to understand. Here, we found that the lncRNA LNCGM1082 plays a critical role in the activation of NLRC4. LNCGM1082 in macrophages affects the maturation of interleukin (IL)-1ß and pyroptotic cell death only after exposure to an NLRC4 ligand. Similar to NLRC4-/- mice, LNCGM1082-/- mice were highly sensitive to Salmonella Typhimurium (S. T) infection. LNCGM1082 deficiency in mouse or human macrophages inhibited IL-1ß maturation and pyroptosis. Mechanistically, LNCGM1082 induced the binding of PKCδ with NLRC4 in both mice and humans. In contrast, NLRC4 did not bind PKCδ in LNCGM1082-/- macrophages. The activity of the lncRNA LNCGM1082 induced by S. T may be mediated through TLR5 in the macrophages of both mice and humans. In summary, our data indicate that TLR5-mediated LNCGM1082 activity can promote the binding of PKCδ with NLRC4 to activate NLRC4 and induce resistance to bacterial infection.
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RNA Longo não Codificante , Infecções por Salmonella , Animais , Humanos , Camundongos , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Caspase 1/metabolismo , Inflamassomos/metabolismo , Camundongos Endogâmicos C57BL , Receptor 5 Toll-Like/metabolismoRESUMO
Aim: Our primary objective was to investigate the protective effects and mechanisms of isovanillic acid in mice infected with Staphylococcus aureus Newman. Methods: In vitro coagulation assays were used to validate vWbp and Coa as inhibitory targets of isovanillic acid. The binding mechanism of isovanillic acid to vWbp and Coa was investigated using molecular docking and point mutagenesis. Importantly, a lethal pneumonia mouse model was used to assess the effect of isovanillic acid on survival and pathological injury in mice. Results & Conclusion: Isovanillic acid reduced the virulence of S. aureus by directly binding to inhibit the clotting activity of vWbp and Coa, thereby reducing lung histopathological damage and improving the survival rate in mice with pneumonia.
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Coagulase , Infecções Estafilocócicas , Camundongos , Animais , Coagulase/metabolismo , Staphylococcus aureus/metabolismo , Simulação de Acoplamento Molecular , Infecções Estafilocócicas/prevenção & controleRESUMO
Antimicrobial proteins possess a broad spectrum of bactericidal activity and play an important role in shaping the composition of gut microbiota, which is related to multiple diseases such as metabolic syndrome. However, it is incompletely known for the regulation of defensin expression in the gut Paneth cells. Here, we found that FABP4 in the Paneth cells of gut epithelial cells and organoids can downregulate the expression of defensins. FABP4fl/flpvillinCreT mice were highly resistance to Salmonella Typhimurium (S.T) infection and had increased bactericidal ability to pathogens. The FABP4-mediated downregulation of defensins is through degrading PPARγ after K48 ubiquitination. We also demonstrate that high-fat diet (HFD)-mediated downregulation of defensins is through inducing a robust FABP4 in Paneth cells. Firmicutes/Bacteroidetes (F/B) ratio in FABP4fl/flpvillinCreT mice is lower than control mice, which is opposite to that in mice fed HFD, indicating that FABP4 in the Paneth cells could reprogram gut microbiota. Interestingly, FABP4-mediated downregulation of defensins in Paneth cells not only happens in mice but also in human. A better understanding of the regulation of defensins, especially HFD-mediated downregulation of defensin in Paneth cells will provide insights into factor(s) underlying modern diseases.Abbreviations: FABP4: Fatty acid binding protein 4; S. T: Salmonella Typhimurium; HFD: High-fat diet; Defa: α-defensin; 930 HD5: Human α-defensin 5; HD6: Human α-defensin 6; F/B: Firmicutes/Bacteroidetes; SFB: Segmental filamentous bacteria; AMPs: Antimicrobial peptides; PPARγ: Peroxisome proliferator-activated receptor γ; P-PPAR: Phosphorylated PPAR; Dhx15: DEAD-box helicase 15; 935 EGF: Epidermal growth factor; ENR: Noggin and R-spondin 1; CFU: Colony forming unit; Lyz1: Lysozyme 1; Saa1: Serum amyoid A 1; Pla2g2a: Phospholipase A2, group IIA; MMP-7: Matrix metalloproteinase; AU-PAGE: Acid-urea polyacrylamide gel electrophoresis; PA: Palmitic 940 acid; GPR40: G-protein-coupled receptor; GF: Germ-free; EGF: Epidermal growth factor; LP: Lamina propria; KO: Knock out; WT: Wild-type.
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Anti-Infecciosos , Proteínas de Ligação a Ácido Graxo , Microbioma Gastrointestinal , Animais , Humanos , Camundongos , Anti-Infecciosos/metabolismo , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Celulas de Paneth/metabolismo , RNA Helicases/metabolismoRESUMO
BACKGROUND AND AIMS: Increased E. coli in the colon are related to the occurrence and development of multiple diseases. Chemokines are shown to possess potential antimicrobial activity, including against Gram-positive and -negative bacterial pathogens. We here investigated function[s] of chemokine CXCL9 expressed in the gut epithelial cells, and mechanism[s] of CXCL9 by which to kill E. coli. METHODS: We generated CXCL9fl/flpvillin-creT mice [pvillin-cre positive mice] and their control CXCL9fl/flpvillin-crewmice [pvillin-cre negative mice], and then employed a dextran sulphate sodium [DSS]-mediated colitis model to determine the sensitivity of CXCL9fl/flpvillin-creT mice. We analysed the composition of the gut microbiota by using 16S ribosomal RNA [V3-V4 variable region] sequencing and shotgun metagenomic analyses. We generated E. coli ΔFtsX [FtsX-depleted E. coli] and E. coli ΔaceE [aceE-depleted E. coli] by using a bacterium red recombining system to investigate the mechanism[s] of CXCL9 by which to kill E. coli. RESULTS: CXCL9 fl/flpvillin-creTmice were more sensitive to chemically induced colitis than their control littermates, CXCL9fl/flpvillin-crewmice. After DSS treatment, there were markedly increased gut E. coli [Escherichia-Shigella] in the colonic contents of CXCL9fl/flpvillin-creT mice as compared with control CXCL9fl/flpvillin-crew mice. The increased E. coli could promote colitis through NLRC4 and caspase 1/11-mediated IL-18, which was derived from gut epithelial cells. We finally demonstrated that CXCL9 expressed in gut epithelial cells could kill the overgrown E. coli. E. coli expressed Ftsx and PDHc subunits aceE. E.coliΔaceE but not E. coliΔFtsX were resistant to CXCL9-mediated killing. CONCLUSIONS: Gut epithelial cells-derived CXCL9 can kill the expanded E. coli through aceE, to remain gut homeostasis.
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Colite , Escherichia coli , Animais , Quimiocina CXCL9/efeitos adversos , Colite/genética , Colo/microbiologia , Sulfato de Dextrana , Modelos Animais de Doenças , Homeostase , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Hypopharyngeal squamous cell carcinoma (HSCC) is one of the high mortality cancers with a poor prognosis, which is driving the development of new chemotherapeutic agents. We identified the anticancer effects of a natural compound, solamargine (SM), on FaDU cells and explored its mechanism in terms of non-coding RNA. It was observed that SM inhibited the proliferation of FaDU cells with an IC50 of 5.17 µM. High-throughput sequencing data revealed that lncRNA HOXA11-AS was significantly downregulated in cells co-incubated with SM. Further assays demonstrated that SM-induced downregulation of lncRNA HOXA11-AS showed important implications for apoptosis. Given the properties of HOXA11-AS as a miR-155 sponge, we further confirmed that SM upregulated the expression of miR-155 in FaDU cells. C-Myc is a transcription factor that regulates cell differentiation and apoptosis, whose mRNA is considered to be targeted by miR-155. We showed that c-Myc expression was downregulated by SM and accompanied by increased apoptosis, which was consistent with the findings of transcriptome sequencing. Furthermore, SM administration suppressed xenograft tumor growth in a xenograft mouse model in vivo. In the light of the aforementioned findings, our results suggested that SM downregulated the expression of HOXA11-AS, which in turn induces apoptosis by downregulating c-Myc in FaDU, providing evidence for the anticancer effect of SM on HSCC and uncovering the effect of SM on non-coding RNAs as, at least partly, a mechanism of action.
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OBJECTIVE: Central inflammation is generally accepted to be involved in the pathology of depression. We investigated whether liquiritin exerts antidepressant effects by inhibiting central NLRP3 inflammasomes. RESULTS: The behavioral despair model and chronic unpredictable mild stress (CUMS) model in mice were established to evaluate the antidepressant action of liquiritin. In the despair model study, liquiritin (40 mg/kg) administration reduced immobility time in tail suspension test (TST) and forced swimming test (FST) without affecting locomotion activity. In CUMS model study, liquiritin (40 mg/kg, once daily for 4 weeks) significantly increased sucrose consumption and body weight of CUMS mice. The behavioral experiment results showed that liquiritin reduced the immobile time of CUMS mice in TST and FST, respectively, and increased the time spent and open arm entries in the elevated plus-maze (EPM) test. Further, the hippocampal superoxide dismutase (SOD) activity was increased in liquiritin-treated group, while malonaldehyde (MDA) decreased. Additionally, the hippocampal cytokines interleukin-18 (IL-18) and interleukin-1 beta (IL-1ß) contents were reduced in the liquiritin-treated group. Further, liquiritin downregulated the expression of NLRP3 in the hippocampus of CUMS mice rather than TLR4. Besides, NLRP3 inflammasome-associated proteins caspase-1 and ASC were also downregulated. However, liquiritin did not alter the thermal stability of NLRP3 in the cellular thermal shift assay (CETSA), suggesting that its inhibition of NLPR3 was not by direct targeting of NLRP3 protein. CONCLUSIONS: Liquiritin attenuates depression-like behavior of CUMS mice and inhibited cytokines levels triggered by NLRP3 inflammasome, suggesting the antidepressant action is, at least partially, associated with antioxidant stress and inhibition of NLRP3 inflammasome activation.
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BACKGROUND: A dysfunctional gut epithelial barrier allows the augmented permeation of endotoxins, luminal antigens, and bacteria into the bloodstream, causing disease. The maintenance of gut epithelial barrier integrity may be regulated by multiple factors. Herein we analyze the role of leucine-rich repeat-containing protein 19 (LRRC19) in regulating the permeability of the gut epithelial barrier. METHODS: We utilized Lrrc19 knockout (KO) mice and clinical samples through transmission electron, intestinal permeability assay, Western blot, and immunofluorescence staining to characterize the role of LRRC19 in the permeability of the gut epithelial barrier. RESULTS: We found that LRRC19, which is expressed in gut epithelial cells, impairs gut barrier function. Transmission electron micrographs revealed a tighter junction and narrower gaps in the colon epithelium cells in LRRC19 KO mice. There were lower levels of serum lipopolysaccharide and 4 kDa-fluorescein isothiocyanate-dextran after gavage in LRRC19 KO mice than in wild-type mice. We found that LRRC19 could reduce the expression of zonula occludens (ZO)-1, ZO-3, and occludin in the colonic epithelial cells. The decreased expression of ZO-1, ZO-3, and occludin was dependent on degrading protein kinase C (PKC) ζ and PKCι/λ through K48 ubiquitination by LRRC19. The expression of LRRC19 was also negatively correlated with ZO-1, ZO-3, occludin, PKCζ, and PKCι/λ in human colorectal cancers. CONCLUSIONS: The protein LRRC19 can promote the permeability of the gut epithelial barrier through degrading PKC ζ and PKCι/λ to reduce the expression of ZO-1, ZO-3, and occludin.