RESUMEN
As a significant member of the immune checkpoint, programmed cell death 1 ligand 1 (PD-L1) plays a critical role in cancer immune escape and has become an important target for cancer immunotherapy. Clinically approved drugs mainly target the extracellular domain of PD-L1. Recently, the small cytoplasmic domain of PD-L1 has been reported to regulate PD-L1 stability and function through multiple pathways. Therefore, the intracellular domain of PD-L1 and its regulatory pathways could be promising targets for cancer therapy, expanding available strategies for combined immunotherapy. Here, we summarize the emerging roles of the PD-L1 cytoplasmic domain and its regulatory pathways. The conserved motifs, homodimerization, and posttranslational modifications of the PD-L1 cytoplasmic domain have been reported to regulate the membrane anchoring, degradation, nuclear translocation, and glycosylation of PD-L1, etc. This summary provides a comprehensive understanding of the functions of the PD-L1 cytoplasmic domain and evaluates the broad prospects for targeted therapy.
RESUMEN
Profiling membrane proteins' interacting networks is crucial for understanding their regulatory mechanisms and functional characteristics, but it remains a challenging task. Here, by combining genetic incorporation of crosslinkers, tandem denatured purification, and proteomics, we added interaction partners for PD-L1, a cancer cell surface protein that inhibits T cell activity. The site-specifically incorporated crosslinker mediates the covalent capture of interactions under physiological conditions and enabled the PD-L1 complexes to withstand the harsh extraction conditions of membrane proteins. Subsequent experiments led to the identification of potential PD-L1 interaction candidates and verified membrane-associated progesterone receptor component 1 as a novel PD-L1 interaction partner in mammalian cells. Importantly, we demonstrated that PGRMC1 positively regulates PD-L1 expression by regulating GSK3ß-mediated PD-L1 degradation in cancer cells. Furthermore, PGRMC1 knockdown results in dramatically enhanced T cell-mediated cytotoxicity in cancer cells. In conclusion, our study elucidated the interactome of PD-L1 and uncovered a new player in the PD-L1 regulation mechanism.
Asunto(s)
Antígeno B7-H1 , Linfocitos T , Animales , Línea Celular Tumoral , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mamíferos/metabolismoRESUMEN
How pathogens manipulate host UPRER to mediate immune evasion is largely unknown. Here, we identify the host zinc finger protein ZPR1 as an interacting partner of the enteropathogenic E. coli (EPEC) effector NleE using proximity-enabled protein crosslinking. We show that ZPR1 assembles via liquid-liquid phase separation (LLPS) in vitro and regulates CHOP-mediated UPRER at the transcriptional level. Interestingly, in vitro studies show that the ZPR1 binding ability with K63-ubiquitin chains, which promotes LLPS of ZPR1, is disrupted by NleE. Further analyses indicate that EPEC restricts host UPRER pathways at the transcription level in a NleE-ZPR1 cascade-dependent manner. Together, our study reveals the mechanism by which EPEC interferes with CHOP-UPRER via regulating ZPR1 to help pathogens escape host defense.
Asunto(s)
Escherichia coli Enteropatógena , Infecciones por Escherichia coli , Proteínas de Escherichia coli , Humanos , Células HeLa , Factores de Virulencia/metabolismo , Escherichia coli Enteropatógena/metabolismo , Proteínas de Escherichia coli/metabolismoRESUMEN
Whether membrane-anchored PD-L1 homodimerizes in living cells is controversial. The biological significance of the homodimer waits to be expeditiously explored. However, characterization of the membrane-anchored full-length PD-L1 homodimer is challenging, and unconventional approaches are needed. By using genetically incorporated crosslinkers, we showed that full length PD-L1 forms homodimers and tetramers in living cells. Importantly, the homodimerized intracellular domains of PD-L1 play critical roles in its complex glycosylation. Further analysis identified three key arginine residues in the intracellular domain of PD-L1 as the regulating unit. In the PD-L1/PD-L1-3RE homodimer, mutations result in a decrease in the membrane abundance and an increase in the Golgi of wild-type PD-L1. Notably, PD-1 binding to abnormally glycosylated PD-L1 on cancer cells was attenuated, and subsequent T-cell induced toxicity increased. Collectively, our study demonstrated that PD-L1 indeed forms homodimers in cells, and the homodimers play important roles in PD-L1 complex glycosylation and T-cell mediated toxicity.
Asunto(s)
Antígeno B7-H1 , Linfocitos T , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Glicosilación , Linfocitos T/metabolismoRESUMEN
Autophagy acts as a pivotal innate immune response against infection. Some virulence effectors subvert the host autophagic machinery to escape the surveillance of autophagy. The mechanism by which pathogens interact with host autophagy remains mostly unclear. However, traditional strategies often have difficulty identifying host proteins that interact with effectors due to the weak, dynamic, and transient nature of these interactions. Here, we found that Enteropathogenic Escherichia coli (EPEC) regulates autophagosome formation in host cells dependent on effector NleE. The 26S Proteasome Regulatory Subunit 10 (PSMD10) was identified as a direct interaction partner of NleE in living cells by employing genetically incorporated crosslinkers. Pairwise chemical crosslinking revealed that NleE interacts with the N-terminus of PSMD10. We demonstrated that PSMD10 homodimerization is necessary for its interaction with ATG7 and promotion of autophagy, but not necessary for PSMD10 interaction with ATG12. Therefore, NleE-mediated PSMD10 in monomeric state attenuates host autophagosome formation. Our study reveals the mechanism through which EPEC attenuates host autophagy activity.
Asunto(s)
Autofagia/inmunología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Proteína 12 Relacionada con la Autofagia , Proteína 7 Relacionada con la Autofagia , Escherichia coli Enteropatógena , Infecciones por Escherichia coli/inmunología , Proteínas de Escherichia coli/química , Células HeLa , Humanos , Interleucina-6 , Lipopolisacáridos , Modelos Moleculares , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/genética , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Virulencia/genética , Factores de Virulencia/químicaRESUMEN
Highly oxygenated schitriterpenoids are interesting for study of their structures, bioactivities and synthesis. From Kadsura angustifolia fermented by an associated symbiotic endophytic fungus, Penicillium sp. SWUKD4.1850, nine undescribed triterpenoids, kadhenrischinins A-H, and 7ß-schinalactone C together with four known triterpenoids, henrischinins A and B, schinalactone C and nigranoic acid were isolated and established by the extensive 1D-, 2D-NMR, HR-ESI-MS and ECD data analysis. Except nigranoic acid, all these metabolites have been first detected in non-fermented K. angustifolia. Structurally, kadhenrischinins A-D belong to the relatively rare class of highly oxygenated schitriterpenoids that contain a unique 3-one-2-oxabicyclo [3,2,1]-octane motif, while kadhenrischinins E-H feature a cyclopentane ring in a side chain rarely found in the family Schisandraceae. These results indicated that fermentation of K. angustifolia with SWUKD4.1850 induced the production of highly oxygenated schitriterpenoids from nigranoic acid, which provided a guidance to obtain desired compounds from those plants initially thought not to produce. This is the first report on the fermentation of K. angustifolia medical plant and the first discovery of highly oxygenated schitriterpenoids by microbial technology.
Asunto(s)
Kadsura/metabolismo , Kadsura/microbiología , Penicillium/fisiología , Triterpenos/química , Triterpenos/metabolismo , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Endófitos/fisiología , Fermentación , Células Hep G2 , Humanos , Kadsura/química , Espectroscopía de Resonancia Magnética , Estructura Molecular , Espectrometría de Masa por Ionización de Electrospray , Simbiosis , Triterpenos/farmacologíaRESUMEN
With increasing incidence and mortality, hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. In this study, microRNA-122 (miR-122) mimics and relevant control oligonucleotides were transfected into HepG2 cells in vitro, followed by coptisine (COP) and sorafenib treatments. Cell proliferation, migration, and apoptosis were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and colony formation assay, wound-healing assay, Hoechst 33258 staining and flow cytometry, respectively. Histopathology and miR-122 were analyzed by haemotoxylin and eosin (H&E) staining and real-time RT-PCR, respectively; whereas, the relevant protein expressions were detected by western blot. In vivo, COP enhanced the expression of miR-122 by 160% compared to control in male BALB/c nude mice; COP not only protected the liver morphology but also showed a significant anti-cancer effect. Further, there was no remarkable difference between the tumor weights in the COP and sorafenib groups, but there was a striking difference to the tumor control group (pâ¯<â¯0.05). Hence, COP inhibited the proliferation, migration and promoted apoptosis of HCC cells; moreover, it inhibited the tumor growth in nude mice by up-regulating the expression of miR-122.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Berberina/análogos & derivados , Medicamentos Herbarios Chinos/química , Neoplasias Hepáticas/tratamiento farmacológico , MicroARNs/genética , Animales , Antineoplásicos Fitogénicos/análisis , Apoptosis/efectos de los fármacos , Berberina/análisis , Berberina/farmacología , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Coptisine (COP), one of the main active ingredients of Rhizoma Coptidis, reportedly has anti-inflammatory, anti-colon cancer properties, but it remains elusive whether COP owns hepatoprotective activity. Mice were pretreated with COP for 7d prior to lipopolysaccharide/d-galactosamine (LPS/D-GalN) administration to detect the hepatic protective effects of COP. The mechanism was explored in using HepG2 cells with low level of miR-122 and LO2 cells with high level of miR-122, combining with miR-122 agomir transfection by means of detecting the expression of miR-122 and proteins, clinical index and apoptosis. COP ameliorated the LPS/D-GalN-induced liver failure by lowering serum levels of ALT and AST, raising hepatic GSH and SOD levels, and maintaining the morphology of hepatocytes, along with an increase in miR-122 expression in mice. The results in vitro indicated that, after miR-122 mimic administration, the alone treatment of COP and the co-treatment of COP and LPS transfection obviously promoted the apoptosis of HepG2, which was increased by 152.67% and 113.97% compared with NC (P < 0.05 vs NC). LPS significantly induced the apoptosis of L02 cells, but COP treatment attenuated that of L02 cells. Further analysis showed that COP increased the miR-122 level and the expression of Bax, cleaved-casp3 and decreased Bcl-2, Bcl-xL in LPS-treated HepG2 cells. COP increased the miR-122 level but decreased the expression of TLR4, Bcl-2, Bcl-xL in LPS-treated L02 cells. COP attenuated LPS/D-GalN-induced ALF by up-regulating the level of miR-122, synergistically promoting apoptosis, and suggesting COP which showed a potential protective effect on ALF.
Asunto(s)
Berberina/análogos & derivados , Medicamentos Herbarios Chinos/uso terapéutico , Galactosamina/toxicidad , Lipopolisacáridos/toxicidad , Fallo Hepático Agudo/prevención & control , MicroARNs/biosíntesis , Animales , Berberina/farmacología , Berberina/uso terapéutico , Coptis chinensis , Relación Dosis-Respuesta a Droga , Medicamentos Herbarios Chinos/farmacología , Expresión Génica , Células Hep G2 , Humanos , Fallo Hepático Agudo/inducido químicamente , Fallo Hepático Agudo/metabolismo , Ratones , MicroARNs/genética , Sustancias Protectoras/farmacología , Sustancias Protectoras/uso terapéutico , Distribución Aleatoria , Resultado del Tratamiento , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
The aim of this study was to investigate the antihypercholesterolemic activity and potential molecular mechanism of columbamine (COL) that was prepared by extraction from Rhizoma Coptidis in hamsters and HepG2 cells. The results displayed that the COL from Rhizoma Coptidis was a safe natural compound with a LD50 0f 1524.6mg/kg and no detectable toxic symptoms during the observation of chronic toxicity. COL dose-dependently reversed the abnormal lipid levels induced by HFHC diet. Specifically, COL(M) and COL(H) significantly reduced the blood lipid levels(TC, TG and LDL-c) and enhanced the fecal contents of TBA by 21.8% and 25.1% respectively in hamsters. COL up-regulated the genes of CYP8B1, CYP7A1 and LDLR in mRNA and protein level, and down-regulated those of HMGCR to a different degree. Especially, CYP7A1 were significantly up-regulated by COL in hamsters (p<0.01). Further analysis indicated that COL obviously activated the mRNA and protein expression of the transcription factors FTF, HNF-4α, and inhibited those of SHP. Promoter luciferase assay showed that COL induced the expression of FTF and HNF-4α, further transactivating CYP7A1, which accelerated the conversion of liver cholesterol to bile acids. It concluded that the COL showed high lipid-lowering activities through indirectly transactivating CYP7A1 by upregulating FTF and HNF-4α, and directly activating CYP7A1 catalytic activity by strongly interacting with receptor and ligand, therefore promoting cholesterol catabolism and accelerating the excretion of bile acids.