RESUMEN
Gastric cancer (GC) remains a global challenge due to the lack of early detection and precision therapies. Genkwadaphnin (DD1), a natural diterpene isolated from the bud of Flos GenkWa (Thymelaeaceae), serves as a Karyopherin ß1 (KPNB1) inhibitor. In this study, we investigated the anti-tumor effect of DD1 in both cell culture and animal models. Our findings reveal that KPNB1, a protein involved in nuclear import, was highly expressed in GC tissues and associated with a poor prognosis in patients. We demonstrated that DD1, alongside the established KPNB1 inhibitor importazole (IPZ), inhibited GC cell proliferation and tumor growth by enhancing both genomic and non-genomic activity of Nur77. DD1 and IPZ reduced the interaction between KPNB1 and Nur77, resulting in Nur77 cytoplasmic accumulation and triggering mitochondrial apoptosis. The inhibitors also increased the expression of the Nur77 target apoptotic genes ATF3, RB1CC1 and PMAIP1, inducing apoptosis in GC cell. More importantly, loss of Nur77 effectively rescued the inhibitory effect of DD1 and IPZ on GC cells in both in vitro and in vivo experiments. In this study, we for the first time explored the relationship between KPNB1 and Nur77, and found KPNB1 inhibition could significantly increase the expression of Nur77. Moreover, we investigated the function of KPNB1 in GC for the first time, and the results suggested that KPNB1 could be a potential target for cancer therapy, and DD1 might be a prospective therapeutic candidate.
Asunto(s)
Apoptosis , Proliferación Celular , Diterpenos , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Transducción de Señal , Neoplasias Gástricas , beta Carioferinas , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/patología , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Humanos , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Animales , Diterpenos/farmacología , Diterpenos/uso terapéutico , Transducción de Señal/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Línea Celular Tumoral , Apoptosis/efectos de los fármacos , Ratones , beta Carioferinas/metabolismo , beta Carioferinas/genética , Progresión de la Enfermedad , Masculino , Ratones Desnudos , Ensayos Antitumor por Modelo de Xenoinjerto , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Femenino , Ratones Endogámicos BALB CRESUMEN
Golgin tethers are known to mediate vesicular transport in the secretory pathway, whereas it is relatively unknown whether they may mediate cellular stress response within the cell. Here, we describe a cellular stress response during heat shock stress via SUMOylation of a Golgin tether, Golgin45. We found that Golgin45 is a SUMOylated Golgin via SUMO1 under steady state condition. Upon heat shock stress, the Golgin enters the nucleus by interacting with Importin-ß2 and gets further modified by SUMO3. Importantly, SUMOylated Golgin45 appears to interact with PML and SUMO-deficient Golgin45 mutant functions as a dominant negative for PML-NB formation during heat shock stress, suppressing transcription of lipid metabolism genes. These results indicate that Golgin45 may play a role in heat stress response by transcriptional regulation of lipid metabolism genes in SUMOylation-dependent fashion.
Asunto(s)
Respuesta al Choque Térmico , Metabolismo de los Lípidos , Sumoilación , Ubiquitinas , Humanos , Metabolismo de los Lípidos/genética , Respuesta al Choque Térmico/genética , Regulación de la Expresión Génica , Proteína de la Leucemia Promielocítica/metabolismo , Proteína de la Leucemia Promielocítica/genética , Células HeLa , Proteína SUMO-1/metabolismo , Proteína SUMO-1/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Células HEK293 , Transcripción Genética , beta Carioferinas/metabolismo , beta Carioferinas/genéticaRESUMEN
Nucleocytoplasmic transport of macromolecules is essential in eukaryotic cells. In this process, the karyopherins play a central role when they transport cargoes across the nuclear pore complex. Importin 4 belongs to the karyopherin ß family. Many studies have focused on finding substrates for importin 4, but no direct mechanism studies of its precise transport function have been reported. Therefore, this paper mainly aimed to study the mechanism of nucleoporins in mediating nuclear import and export of importin 4. To address this question, we constructed shRNAs targeting Nup358, Nup153, Nup98, and Nup50. We found that depletion of Nup98 resulted in a shift in the subcellular localization of importin 4 from the cytoplasm to the nucleus. Mutational analysis demonstrated that Nup98 physically and functionally interacts with importin 4 through its N-terminal phenylalanine-glycine (FG) repeat region. Mutation of nine of these FG motifs to SG motifs significantly attenuated the binding of Nup98 to importin 4, and we further confirmed the essential role of the six FG motifs in amino acids 121-360 of Nup98 in binding with importin 4. In vitro transport assay also confirmed that VDR, the substrate of importin 4, could not be transported into the nucleus after Nup98 knockdown. Overall, our results showed that Nup98 is required for efficient importin 4-mediated transport. This is the first study to reveal the mechanism of importin 4 in transporting substrates into the nucleus.
Asunto(s)
Transporte Activo de Núcleo Celular , Proteínas de Complejo Poro Nuclear , beta Carioferinas , Humanos , beta Carioferinas/metabolismo , beta Carioferinas/genética , Núcleo Celular/metabolismo , Células HeLa , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/genética , Unión ProteicaRESUMEN
BACKGROUND: Myocarditis is an important clinical issue which lacks specific treatment by now. Ivermectin (IVM) is an inhibitor of importin α/ß-mediated nuclear translocation. This study aimed to explore the therapeutic effects of IVM on acute myocarditis. METHODS: Mouse models of coxsackie B3 virus (CVB3) infection-induced myocarditis and experimental autoimmune myocarditis (EAM) were established to evaluate the effects of IVM. Cardiac functions were evaluated by echocardiography and Millar catheter. Cardiac inflammatory infiltration was assessed by histological staining. Cytometric bead array and quantitative real-time PCR were used to detect the levels of pro-inflammatory cytokines. The macrophages and their M1/M2 polarization were analyzed via flow cytometry. Protein expression and binding were detected by co-immunoprecipitation, Western blotting and histological staining. The underlying mechanism was verified in vitro using CVB3-infected RAW264.7 macrophages. Cyclic polypeptide (cTN50) was synthesized to selectively inhibit the nuclear translocation of NF-κB/p65, and CVB3-infected RAW264.7 cells were treated with cTN50. RESULTS: Increased expression of importin ß was observed in both models. IVM treatment improved cardiac functions and reduced the cardiac inflammation associated with CVB3-myocarditis and EAM. Furthermore, the pro-inflammatory cytokine (IL-1ß/IL-6/TNF-α) levels were downregulated via the inhibition of the nuclear translocation of NF-κB/p65 in macrophages. IVM and cTN50 treatment also inhibited the nuclear translocation of NF-κB/p65 and downregulated the expression of pro-inflammatory cytokines in RAW264.7 macrophages. CONCLUSIONS: Ivermectin inhibits the nuclear translocation of NF-κB/p65 and the expression of major pro-inflammatory cytokines in myocarditis. The therapeutic effects of IVM on viral and non-viral myocarditis models suggest its potential application in the treatment of acute myocarditis.
Asunto(s)
Ivermectina , Miocarditis , Factor de Transcripción ReIA , Animales , Humanos , Masculino , Ratones , Enfermedades Autoinmunes/tratamiento farmacológico , beta Carioferinas/metabolismo , Infecciones por Coxsackievirus/tratamiento farmacológico , Citocinas/metabolismo , Modelos Animales de Enfermedad , Enterovirus Humano B , Ivermectina/uso terapéutico , Ivermectina/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones Endogámicos BALB C , Miocarditis/tratamiento farmacológico , Miocarditis/virología , Miocardio/patología , Miocardio/metabolismo , Células RAW 264.7 , Factor de Transcripción ReIA/metabolismoRESUMEN
The nuclear translocation of YAP1 is significantly implicated in the proliferation, stemness, and metastasis of cancer cells. Although the molecular basis underlying YAP1 subcellular distribution has been extensively explored, it remains to be elucidated how the nuclear localization signal guides YAP1 to pass through the nuclear pore complex. Here, we define a globular type of nuclear localization signal composed of folded WW domains, named as WW-NLS. It directs YAP1 nuclear import through the heterodimeric nuclear transport receptors KPNA-KPNB1, bypassing the canonical nuclear localization signal that has been well documented in KPNA/KPNB1-mediated nuclear import. Strikingly, competitive interference with the function of the WW-NLS significantly attenuates YAP1 nuclear translocation and damages stemness gene activation and sphere formation in malignant breast cancer cells. Our findings elucidate a novel globular type of nuclear localization signal to facilitate nuclear entry of WW-containing proteins including YAP1.
Asunto(s)
Núcleo Celular , Señales de Localización Nuclear , Proteínas Señalizadoras YAP , Humanos , Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , Señales de Localización Nuclear/genética , Señales de Localización Nuclear/metabolismo , Proteínas/metabolismo , Dominios WW , Proteínas Señalizadoras YAP/química , Proteínas Señalizadoras YAP/metabolismo , alfa Carioferinas/metabolismo , beta Carioferinas/metabolismoRESUMEN
Regulator of TElomere Length Helicase 1 (RTEL1) is a helicase required for telomere maintenance and genome replication and repair. RTEL1 has been previously shown to participate in the nuclear export of small nuclear RNAs. Here we show that RTEL1 deficiency leads to a nuclear envelope destabilization exclusively in cells entering S-phase and in direct connection to origin firing. We discovered that inhibiting protein import also leads to similar, albeit non-cell cycle-related, nuclear envelope disruptions. Remarkably, overexpression of wild-type RTEL1, or of its C-terminal part lacking the helicase domain, protects cells against nuclear envelope anomalies mediated by protein import inhibition. We identified distinct domains in the C-terminus of RTEL1 essential for the interaction with KPNB1 (importin ß) and NUP153, respectively, and we demonstrated that, on its own, the latter domain can promote the dynamic nuclear internalization of peptides that freely diffuse through the nuclear pore. Consistent with putative functions exerted in protein import, RTEL1 can be visualized on both sides of the nuclear pore using high-resolution microscopy. In all, our work points to an unanticipated, helicase-independent, role of RTEL1 in connecting both nucleocytoplasmic trafficking and nuclear envelope integrity to genome replication initiation in S-phase.