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1.
FASEB J ; 38(11): e23716, 2024 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-38847490

RESUMEN

Tumor hypoxia has been associated with cancer progression, angiogenesis, and metastasis via modifications in the release and cargo composition of extracellular vesicles secreted by tumor cells. Indeed, hypoxic extracellular vesicles are known to trigger a variety of angiogenic responses via different mechanisms. We recently showed that hypoxia promotes endosomal signaling in tumor cells via HIF-1α-dependent induction of the guanine exchange factor ALS2, which activates Rab5, leading to downstream events involved in cell migration and invasion. Since Rab5-dependent signaling is required for endothelial cell migration and angiogenesis, we explored the possibility that hypoxia promotes the release of small extracellular vesicles containing ALS2, which in turn activate Rab5 in recipient endothelial cells leading to pro-angiogenic properties. In doing so, we found that hypoxia promoted ALS2 expression and incorporation as cargo within small extracellular vesicles, leading to subsequent transfer to recipient endothelial cells and promoting cell migration, tube formation, and downstream Rab5 activation. Consequently, ALS2-containing small extracellular vesicles increased early endosome size and number in recipient endothelial cells, which was followed by subsequent sequestration of components of the ß-catenin destruction complex within endosomal compartments, leading to stabilization and nuclear localization of ß-catenin. These events converged in the expression of ß-catenin target genes involved in angiogenesis. Knockdown of ALS2 in donor tumor cells precluded its incorporation into small extracellular vesicles, preventing Rab5-downstream events and endothelial cell responses, which depended on Rab5 activity and guanine exchange factor activity of ALS2. These findings indicate that vesicular ALS2, secreted in hypoxia, promotes endothelial cell events leading to angiogenesis. Finally, these events might explain how tumor angiogenesis proceeds in hypoxic conditions.


Asunto(s)
Movimiento Celular , Vesículas Extracelulares , Factores de Intercambio de Guanina Nucleótido , Transducción de Señal , beta Catenina , Proteínas de Unión al GTP rab5 , Humanos , Proteínas de Unión al GTP rab5/metabolismo , Proteínas de Unión al GTP rab5/genética , beta Catenina/metabolismo , Vesículas Extracelulares/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Línea Celular Tumoral
2.
bioRxiv ; 2024 May 26.
Artículo en Inglés | MEDLINE | ID: mdl-38826237

RESUMEN

Drosophila s-LNv circadian pacemaker neurons show dramatic structural plasticity, with their projections expanded at dawn and then retracted by dusk. This predictable plasticity makes s-LNvs ideal to study molecular mechanisms of plasticity. Although s-LNv plasticity is controlled by their molecular clock, changing s-LNv excitability also regulates plasticity. Here, we tested the idea that s-LNvs use activity-regulated genes to control plasticity. We found that inducing expression of either of the activity-regulated transcription factors Hr38 or Sr (orthologs of mammalian Nr4a1 and Egr1) is sufficient to rapidly expand s-LNv projections. Conversely, transiently knocking down expression of either Hr38 or sr blocks expansion of s-LNv projections at dawn. We show that Hr38 rapidly induces transcription of sif, which encodes a Rac1 GEF required for s-LNv plasticity rhythms. We conclude that the s-LNv molecular clock controls s-LNv excitability, which couples to an activity-regulated gene expression program to control s-LNv plasticity.

3.
J Biol Chem ; : 107459, 2024 Jun 08.
Artículo en Inglés | MEDLINE | ID: mdl-38857861

RESUMEN

The dedicator of cytokinesis (DOCK)/engulfment and cell motility (ELMO) complex serves as a guanine nucleotide exchange factor (GEF) for the GTPase Rac. RhoG, another GTPase, activates the ELMO-DOCK-Rac pathway during engulfment and migration. Recent cryo-EM structures of the DOCK2/ELMO1 and DOCK2/ELMO1/Rac1 complexes have identified closed and open conformations that are key to understanding the autoinhibition mechanism. Nevertheless, the structural details of RhoG-mediated activation of the DOCK/ELMO complex remain elusive. Herein, we present cryo-EM structures of DOCK5/ELMO1 alone and in complex with RhoG and Rac1. The DOCK5/ELMO1 structure exhibits a closed conformation similar to that of DOCK2/ELMO1, suggesting a shared regulatory mechanism of the autoinhibitory state across DOCK-A/B subfamilies (DOCK1-5). Conversely, the RhoG/DOCK5/ELMO1/Rac1 complex adopts an open conformation that differs from that of the DOCK2/ELMO1/Rac1 complex, with RhoG binding to both ELMO1 and DOCK5. The alignment of the DOCK5 PIP3 binding site with the RhoG C-terminal lipidation site suggests simultaneous binding of RhoG and DOCK5/ELMO1 to the plasma membrane. Structural comparison of the apo and RhoG-bound states revealed that RhoG facilitates a closed-to-open state conformational change of DOCK5/ELMO1. Biochemical and surface plasmon resonance (SPR) assays confirm that RhoG enhances the Rac GEF activity of DOCK5/ELMO1 and increases its binding affinity for Rac1. Further analysis of structural variability underscored the conformational flexibility of the DOCK5/ELMO1/Rac1 complex core, potentially facilitating the proximity of the DOCK5 GEF domain to the plasma membrane. These findings elucidate the structural mechanism underlying the RhoG-induced allosteric activation and membrane binding of the DOCK/ELMO complex.

5.
J Inflamm Res ; 17: 2513-2530, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38699595

RESUMEN

Purpose: Explore the therapeutic effects and regulatory mechanism of Qingyi Decoction (QYD) on severe acute pancreatitis (SAP) associated acute lung injury (ALI). Methods: We identified the constituents absorbed into the blood of QYD based on a network pharmacological strategy. The differentially expressed genes from the GEO database were screened to identify the critical targets of QYD treatment of SAP-ALI. The SAP-ALI rat model was constructed.Some methods were used to evaluate the efficacy and mechanism of QYD in treating SAP-ALI. LPS-stimulated pulmonary microvascular endothelial cell injury simulated the SAP-induced pulmonary endothelial injury model. We further observed the therapeutic effect of QYD and CDK5 plasmid transfection on endothelial cell injury. Results: 18 constituents were absorbed into the blood, and 764 targets were identified from QYD, 25 of which were considered core targets for treating SAP-ALI. CDK5 was identified as the most critical gene. The results of differential expression analysis showed that the mRNA expression level of CDK5 in the blood of SAP patients was significantly up-regulated compared with that of healthy people. Animal experiments have demonstrated that QYD can alleviate pancreatic and lung injury inflammatory response and reduce the upregulation of CDK5 in lung tissue. QYD or CDK5 inhibitors could decrease the expression of NFAT5 and GEF-H1, and increase the expression of ACE-tub in SAP rat lung tissue. Cell experiments proved that QYD could inhibit the expression of TNF-α and IL-6 induced by LPS. Immunofluorescence results suggested that QYD could alleviate the cytoskeleton damage of endothelial cells, and the mechanism might be related to the inhibition of CDK5-mediated activation of NFAT5, GEF-H1, and ACE-tub. Conclusion: CDK5 has been identified as a critical target for pulmonary endothelial injury of SAP-ALI. QYD may partially alleviate microtubule disassembly by targeting the CDK5/NFAT5/GEF-H1 signaling pathway, thus relieving SAP-induced pulmonary microvascular endothelial cell injury.

6.
J Cell Biol ; 223(7)2024 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-38722278

RESUMEN

Aberrant proteins located in the endoplasmic reticulum (ER) undergo rapid ubiquitination by multiple ubiquitin (Ub) E3 ligases and are retrotranslocated to the cytosol as part of the ER-associated degradation (ERAD). Despite several ERAD branches involving different Ub E3 ligases, the molecular machinery responsible for these ERAD branches in mammalian cells remains not fully understood. Through a series of multiplex knockdown/knockout experiments with real-time kinetic measurements, we demonstrate that HERC3 operates independently of the ER-embedded ubiquitin ligases RNF5 and RNF185 (RNF5/185) to mediate the retrotranslocation and ERAD of misfolded CFTR. While RNF5/185 participates in the ERAD process of both misfolded ABCB1 and CFTR, HERC3 uniquely promotes CFTR ERAD. In vitro assay revealed that HERC3 directly interacts with the exposed membrane-spanning domains (MSDs) of CFTR but not with the MSDs embedded in liposomes. Therefore, HERC3 could play a role in the quality control of MSDs in the cytoplasm and might be crucial for the ERAD pathway of select membrane proteins.


Asunto(s)
Degradación Asociada con el Retículo Endoplásmico , Proteínas de la Membrana , Ubiquitina-Proteína Ligasas , Humanos , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Proteínas de Unión al ADN , Retículo Endoplásmico/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Células HEK293 , Células HeLa , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Unión Proteica , Dominios Proteicos , Pliegue de Proteína , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación
7.
Cardiovasc Diabetol ; 23(1): 166, 2024 May 10.
Artículo en Inglés | MEDLINE | ID: mdl-38730425

RESUMEN

BACKGROUND: Studies have shown that RASGRP1 was potently associated with the onset of type 2 diabetes mellitus (T2DM), and RASGRP1 rs7403531 was significantly correlated with islet function in T2DM patients. However, the effect of RASGRP1 polymorphism on blood glucose and blood pressure in T2DM patients after continuous treatment has yet to be fully elucidated. OBJECTIVE: This study aimed to explore the association between RASGRP1 genetic polymorphism and cardiovascular complications in T2DM patients, so as to provide more evidence for the individualized treatment of T2DM patients. METHODS: We retrospectively analyzed a large-scale multicenter drug clinical study cohort that based on a 2 × 2 factorial (glucose control axis and blood pressure lowering axis) randomized controlled design, with follow-up for 5 years. The major vascular endpoint events included cardiovascular death, non-fatal stroke, coronary heart disease, new-onset or worsening renal disease, and diabetic retinopathy. RASGRP1 rs12593201, rs56254815 and rs7403531 were finally selected as candidate single nucleotide polymorphisms. Mixed linear model and Cox hazard ratio (HR) model were used for data analysis with IBM SPSS (version 20.0 for windows; Chicago, IL). RESULTS: Our study enrolled 1357 patients with high-risk diabetes, with a mean follow-up duration of 4.8 years. RASGRP1 rs7403531 was associated with vascular events in hypoglycemic and antihypertensive therapy. Specifically, compared with CC carriers, patients with CT/TT genotype had fewer major microvascular events (HR = 0.41, 95% confidence interval (CI) 0.21-0.80, P = 0.009), and reduced the risk of major eye disease events (HR = 0.44, 95% CI 0.20-0.94, P = 0.03). For glucose lowering axis, CT/TT carriers had a lower risk of secondary nephropathy (HR = 0.48, 95% CI 0.25-0.92, P = 0.03) in patients with standard glycemic control. For blood pressure lowering axis, all cerebrovascular events (HR = 2.24, 95% CI 1.11-4.51, P = 0.025) and stroke events (HR = 2.07, 95% CI 1.03-4.15, P = 0.04) were increased in patients with CC genotype compared to those with CT/TT genotype in the placebo group, respectively. Furthermore, patients with CC genotype showed a reduced risk of major cerebrovascular events in antihypertensive group (HR = 0.36, 95% CI 0.15-0.86, P = 0.021). For RASGRP1 rs56254815, compared with the AA genotype carriers, the systolic blood pressure of AG/GG carriers in the antihypertensive group decreased by 1.5mmhg on average (P = 0.04). In the placebo group, the blood pressure of AG/GG carriers was 1.7mmHg higher than that of AA carriers (P = 0.02). CONCLUSION: We found that patients with G allele of RASGRP1 (rs56254815) showed a better antihypertensive therapy efficacy in T2DM patients. The rs7403531 T allele could reduce the risk of major microvascular events and major eye diseases in T2DM patients receiving either hypoglycemic or antihypertensive therapy. Our findings suggest that RASGRP1 genetic polymorphism might predict the cardiovascular complications in T2DM patients.


Asunto(s)
Antihipertensivos , Glucemia , Presión Sanguínea , Diabetes Mellitus Tipo 2 , Predisposición Genética a la Enfermedad , Control Glucémico , Factores de Intercambio de Guanina Nucleótido , Polimorfismo de Nucleótido Simple , Humanos , Masculino , Femenino , Persona de Mediana Edad , Antihipertensivos/uso terapéutico , Antihipertensivos/efectos adversos , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/sangre , China/epidemiología , Glucemia/metabolismo , Glucemia/efectos de los fármacos , Anciano , Estudios Retrospectivos , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Riesgo , Resultado del Tratamiento , Control Glucémico/efectos adversos , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/genética , Pueblo Asiatico/genética , Angiopatías Diabéticas/genética , Angiopatías Diabéticas/diagnóstico , Medición de Riesgo , Fenotipo , Hipoglucemiantes/uso terapéutico , Hipoglucemiantes/efectos adversos , Factores de Tiempo , Biomarcadores/sangre , Estudios de Asociación Genética , Hipertensión/genética , Hipertensión/tratamiento farmacológico , Hipertensión/fisiopatología , Hipertensión/diagnóstico , Proteínas de Unión al ADN/genética , Pueblos del Este de Asia
8.
Cell Rep ; 43(6): 114256, 2024 May 24.
Artículo en Inglés | MEDLINE | ID: mdl-38795343

RESUMEN

The decline of motor ability is a hallmark feature of aging and is accompanied by degeneration of motor synaptic terminals. Consistent with this, Drosophila motor synapses undergo characteristic age-dependent structural fragmentation co-incident with diminishing motor ability. Here, we show that motor synapse levels of Trio, an evolutionarily conserved guanine nucleotide exchange factor (GEF), decline with age. We demonstrate that increasing Trio expression in adult Drosophila can abrogate age-dependent synaptic structural fragmentation, postpone the decline of motor ability, and maintain the capacity of motor synapses to sustain high-intensity neurotransmitter release. This preservative activity is conserved in transgenic human Trio, requires Trio Rac GEF function, and can also ameliorate synapse degeneration induced by depletion of miniature neurotransmission. Our results support a paradigm where the structural dissolution of motor synapses precedes and promotes motor behavioral diminishment and where intervening in this process can postpone the decline of motor function during aging.

9.
EMBO J ; 2024 May 20.
Artículo en Inglés | MEDLINE | ID: mdl-38769437

RESUMEN

Microtubules regulate cell polarity and migration via local activation of focal adhesion turnover, but the mechanism of this process is insufficiently understood. Molecular complexes containing KANK family proteins connect microtubules with talin, the major component of focal adhesions. Here, local optogenetic activation of KANK1-mediated microtubule/talin linkage promoted microtubule targeting to an individual focal adhesion and subsequent withdrawal, resulting in focal adhesion centripetal sliding and rapid disassembly. This sliding is preceded by a local increase of traction force due to accumulation of myosin-II and actin in the proximity of the focal adhesion. Knockdown of the Rho activator GEF-H1 prevented development of traction force and abolished sliding and disassembly of focal adhesions upon KANK1 activation. Other players participating in microtubule-driven, KANK-dependent focal adhesion disassembly include kinases ROCK, PAK, and FAK, as well as microtubules/focal adhesion-associated proteins kinesin-1, APC, and αTAT. Based on these data, we develop a mathematical model for a microtubule-driven focal adhesion disruption involving local GEF-H1/RhoA/ROCK-dependent activation of contractility, which is consistent with experimental data.

11.
J Cell Biol ; 223(7)2024 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-38748250

RESUMEN

Dynamic presynaptic actin remodeling drives structural and functional plasticity at synapses, but the underlying mechanisms remain largely unknown. Previous work has shown that actin regulation via Rac1 guanine exchange factor (GEF) Vav signaling restrains synaptic growth via bone morphogenetic protein (BMP)-induced receptor macropinocytosis and mediates synaptic potentiation via mobilization of reserve pool vesicles in presynaptic boutons. Here, we find that Gef26/PDZ-GEF and small GTPase Rap1 signaling couples the BMP-induced activation of Abelson kinase to this Vav-mediated macropinocytosis. Moreover, we find that adenylate cyclase Rutabaga (Rut) signaling via exchange protein activated by cAMP (Epac) drives the mobilization of reserve pool vesicles during post-tetanic potentiation (PTP). We discover that Rap1 couples activation of Rut-cAMP-Epac signaling to Vav-mediated synaptic potentiation. These findings indicate that Rap1 acts as an essential, convergent node for Abelson kinase and cAMP signaling to mediate BMP-induced structural plasticity and activity-induced functional plasticity via Vav-dependent regulation of the presynaptic actin cytoskeleton.


Asunto(s)
Plasticidad Neuronal , Terminales Presinápticos , Transducción de Señal , Animales , Citoesqueleto de Actina/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , AMP Cíclico/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Terminales Presinápticos/metabolismo , Proteínas Proto-Oncogénicas c-vav/metabolismo , Proteínas Proto-Oncogénicas c-vav/genética , Proteínas de Unión al GTP rap1/metabolismo , Proteínas de Unión al GTP rap1/genética , Complejo Shelterina/metabolismo , Pinocitosis , Drosophila
12.
Toxicology ; 505: 153843, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38801936

RESUMEN

Benzene, a widely used industrial chemical, has been clarified to cause hematotoxicity. Our previous study suggested that miR-451a may play a role in benzene-induced impairment of erythroid differentiation. However, the mechanism underlying remains unclear. In this study, we explored the role of miR-451a and its underlying mechanisms in hydroquinone (HQ)-induced suppression of erythroid differentiation in K562 cells. 0, 1.0, 2.5, 5.0, 10.0, and 50 µM HQ treatment of K562 cells resulted in a dose-dependent inhibition of erythroid differentiation, as well as the expression of miR-451a. Bioinformatics analysis was conducted to predict potential target genes of miR-451a and dual-luciferase reporter assays confirmed that miR-451a can directly bind to the 3'-UTR regions of BATF, SETD5, and ARHGEF3 mRNAs. We further demonstrated that over-expression or down-regulation of miR-451a altered the expression of BATF, SETD5, and ARHGEF3, and also modified erythroid differentiation. In addition, BATF, SETD5, and ARHGEF3 were verified to play a role in HQ-induced inhibition of erythroid differentiation in this study. Knockdown of SETD5 and ARHGEF3 reversed HQ-induced suppression of erythroid differentiation while knockdown of BATF had the opposite effect. On the other hand, we also identified c-Jun as a potential transcriptional regulator of miR-451a. Forced expression of c-Jun increased miR-451a expression and reversed the inhibition of erythroid differentiation induced by HQ, whereas knockdown of c-Jun had the opposite effect. And the binding site of c-Jun and miR-451a was verified by dual-luciferase reporter assay. Collectively, our findings indicate that miR-451a and its downstream targets BATF, SETD5, and ARHGEF3 are involved in HQ-induced erythroid differentiation disorder, and c-Jun regulates miR-451a as a transcriptional regulator in this process.


Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Diferenciación Celular , MicroARNs , Factores de Intercambio de Guanina Nucleótido Rho , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Diferenciación Celular/efectos de los fármacos , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Células K562 , Factores de Intercambio de Guanina Nucleótido Rho/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Células Eritroides/efectos de los fármacos , Células Eritroides/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Proteínas Proto-Oncogénicas c-jun/genética , Metiltransferasas/genética , Metiltransferasas/metabolismo
13.
Biosci Rep ; 44(5)2024 May 29.
Artículo en Inglés | MEDLINE | ID: mdl-38808367

RESUMEN

Platelets are small anucleate blood cells supporting vascular function. They circulate in a quiescent state monitoring the vasculature for injuries. Platelets adhere to injury sites and can be rapidly activated to secrete granules and to form platelet/platelet aggregates. These responses are controlled by signalling networks that include G proteins and their regulatory guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Recent proteomics studies have revealed the complete spectrum of G proteins, GEFs, and GAPs present in platelets. Some of these proteins are specific for platelets and very few have been characterised in detail. GEFs and GAPs play a major role in setting local levels of active GTP-bound G proteins in response to activating and inhibitory signals encountered by platelets. Thus, GEFs and GAPs are highly regulated themselves and appear to integrate G protein regulation with other cellular processes. This review focuses on GAPs of small G proteins of the Arf, Rab, Ras, and Rho families, as well as of heterotrimeric G proteins found in platelets.


Asunto(s)
Plaquetas , Proteínas Activadoras de GTPasa , Plaquetas/metabolismo , Humanos , Proteínas Activadoras de GTPasa/metabolismo , Animales , Proteínas de Unión al GTP/metabolismo , Transducción de Señal , Factores de Intercambio de Guanina Nucleótido/metabolismo
14.
Traffic ; 25(5): e12936, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38725127

RESUMEN

Endosomal trafficking of TrkA is a critical process for nerve growth factor (NGF)-dependent neuronal cell survival and differentiation. The small GTPase ADP-ribosylation factor 6 (Arf6) is implicated in NGF-dependent processes in PC12 cells through endosomal trafficking and actin cytoskeleton reorganization. However, the regulatory mechanism for Arf6 in NGF signaling is largely unknown. In this study, we demonstrated that EFA6A, an Arf6-specific guanine nucleotide exchange factor, was abundantly expressed in PC12 cells and that knockdown of EFA6A significantly inhibited NGF-dependent Arf6 activation, TrkA recycling from early endosomes to the cell surface, prolonged ERK1/2 phosphorylation, and neurite outgrowth. We also demonstrated that EFA6A forms a protein complex with TrkA through its N-terminal region, thereby enhancing its catalytic activity for Arf6. Similarly, we demonstrated that EFA6A forms a protein complex with TrkA in cultured dorsal root ganglion (DRG) neurons. Furthermore, cultured DRG neurons from EFA6A knockout mice exhibited disturbed NGF-dependent TrkA trafficking compared with wild-type neurons. These findings provide the first evidence for EFA6A as a key regulator of NGF-dependent TrkA trafficking and signaling.


Asunto(s)
Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP , Endosomas , Factores de Intercambio de Guanina Nucleótido , Factor de Crecimiento Nervioso , Proyección Neuronal , Receptor trkA , Animales , Ratones , Ratas , Factores de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/genética , Endosomas/metabolismo , Ganglios Espinales/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Ratones Noqueados , Factor de Crecimiento Nervioso/metabolismo , Células PC12 , Transporte de Proteínas , Receptor trkA/metabolismo
15.
Viruses ; 16(5)2024 05 08.
Artículo en Inglés | MEDLINE | ID: mdl-38793626

RESUMEN

HBV infection is challenging to cure due to the persistence of viral covalently closed circular viral DNA (cccDNA). The dedicator of cytokinesis 11 (DOCK11) is recognized as a guanine nucleotide exchange factor (GEF) for CDC42 that has been reported to be required for HBV persistence. DOCK11 is expressed in both the cytoplasm and nucleus of human hepatocytes and is functionally associated with retrograde trafficking proteins Arf-GAP with GTPase domain, ankyrin repeat, and pleckstrin homology domain-containing protein 2 (AGAP2), and ADP-ribosylation factor 1 (ARF1), together with the HBV capsid, in the trans-Golgi network (TGN). This opens an alternative retrograde trafficking route for HBV from early endosomes (EEs) to the TGN and then to the endoplasmic reticulum (ER), thereby avoiding lysosomal degradation. DOCK11 also facilitates the association of cccDNA with H3K4me3 and RNA Pol II for activating cccDNA transcription. In addition, DOCK11 plays a crucial role in the host DNA repair system, being essential for cccDNA synthesis. This function can be inhibited by 10M-D42AN, a novel DOCK11-binding peptide, leading to the suppression of HBV replication both in vitro and in vivo. Treatment with a combination of 10M-D42AN and entecavir may represent a promising therapeutic strategy for patients with chronic hepatitis B (CHB). Consequently, DOCK11 may be seen as a potential candidate molecule in the development of molecularly targeted drugs against CHB.


Asunto(s)
Factores de Intercambio de Guanina Nucleótido , Virus de la Hepatitis B , Hepatocitos , Humanos , Virus de la Hepatitis B/fisiología , Virus de la Hepatitis B/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Hepatocitos/virología , Hepatocitos/metabolismo , Internalización del Virus , Replicación Viral , Hepatitis B/virología , Hepatitis B/metabolismo , ADN Viral/metabolismo , ADN Viral/genética , Animales
16.
Commun Biol ; 7(1): 543, 2024 May 07.
Artículo en Inglés | MEDLINE | ID: mdl-38714795

RESUMEN

The Wnt-planar cell polarity (Wnt-PCP) pathway is crucial in establishing cell polarity during development and tissue homoeostasis. This pathway is found to be dysregulated in many pathological conditions, including cancer and autoimmune disorders. The central event in Wnt-PCP pathway is the activation of Weak-similarity guanine nucleotide exchange factor (WGEF) by the adapter protein Dishevelled (Dvl). The PDZ domain of Dishevelled2 (Dvl2PDZ) binds and activates WGEF by releasing it from its autoinhibitory state. However, the actual Dvl2PDZ binding site of WGEF and the consequent activation mechanism of the GEF have remained elusive. Using biochemical and molecular dynamics studies, we show that a unique "internal-PDZ binding motif" (IPM) of WGEF mediates the WGEF-Dvl2PDZ interaction to activate the GEF. The residues at P2, P0, P-2 and P-3 positions of IPM play an important role in stabilizing the WGEFpep-Dvl2PDZ interaction. Furthermore, MD simulations of modelled Dvl2PDZ-WGEFIPM peptide complexes suggest that WGEF-Dvl2PDZ interaction may differ from the reported Dvl2PDZ-IPM interactions. Additionally, the apo structure of human Dvl2PDZ shows conformational dynamics different from its IPM peptide bound state, suggesting an induced fit mechanism for the Dvl2PDZ-peptide interaction. The current study provides a model for Dvl2 induced activation of WGEF.


Asunto(s)
Proteínas Dishevelled , Factores de Intercambio de Guanina Nucleótido , Simulación de Dinámica Molecular , Unión Proteica , Proteínas Dishevelled/metabolismo , Proteínas Dishevelled/química , Proteínas Dishevelled/genética , Humanos , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/genética , Dominios PDZ , Secuencias de Aminoácidos , Vía de Señalización Wnt , Péptidos/metabolismo , Péptidos/química , Sitios de Unión , Proteínas de Microfilamentos , Péptidos y Proteínas de Señalización Intracelular
17.
Commun Biol ; 7(1): 602, 2024 May 18.
Artículo en Inglés | MEDLINE | ID: mdl-38762624

RESUMEN

The role of endothelial cells in promoting cancer cell extravasation to the brain during the interaction of cancer cells with the vasculature is not well characterised. We show that brain endothelial cells activate EGFR signalling in triple-negative breast cancer cells with propensity to metastasise to the brain. This activation is dependent on soluble factors secreted by brain endothelial cells, and occurs via the RAC1 GEF DOCK4, which is required for breast cancer cell extravasation to the brain in vivo. Knockdown of DOCK4 inhibits breast cancer cell entrance to the brain without affecting cancer cell survival or growth. Defective extravasation is associated with loss of elongated morphology preceding intercalation into brain endothelium. We also show that brain endothelial cells promote paracrine stimulation of mesenchymal-like morphology of breast cancer cells via DOCK4, DOCK9, RAC1 and CDC42. This stimulation is accompanied by EGFR activation necessary for brain metastatic breast cancer cell elongation which can be reversed by the EGFR inhibitor Afatinib. Our findings suggest that brain endothelial cells promote metastasis through activation of cell signalling that renders breast cancer cells competent for extravasation. This represents a paradigm of brain endothelial cells influencing the signalling and metastatic competency of breast cancer cells.


Asunto(s)
Neoplasias Encefálicas , Encéfalo , Células Endoteliales , Receptores ErbB , Transducción de Señal , Proteína de Unión al GTP rac1 , Receptores ErbB/metabolismo , Receptores ErbB/genética , Humanos , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rac1/genética , Femenino , Células Endoteliales/metabolismo , Células Endoteliales/patología , Línea Celular Tumoral , Animales , Encéfalo/metabolismo , Encéfalo/patología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/genética , Ratones , Proteínas Activadoras de GTPasa/metabolismo , Proteínas Activadoras de GTPasa/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/genética
18.
Curr Biol ; 34(11): 2387-2402.e5, 2024 Jun 03.
Artículo en Inglés | MEDLINE | ID: mdl-38776905

RESUMEN

The C. elegans hermaphrodite distal tip cell (DTC) leads gonadogenesis. Loss-of-function mutations in a C. elegans ortholog of the Rac1 GTPase (ced-10) and its GEF complex (ced-5/DOCK180, ced-2/CrkII, ced-12/ELMO) cause gonad migration defects related to directional sensing; we discovered an additional defect class of gonad bifurcation in these mutants. Using genetic approaches, tissue-specific and whole-body RNAi, and in vivo imaging of endogenously tagged proteins and marked cells, we find that loss of Rac1 or its regulators causes the DTC to fragment as it migrates. Both products of fragmentation-the now-smaller DTC and the membranous patch of cellular material-localize important stem cell niche signaling (LAG-2 ligand) and migration (INA-1/integrin subunit alpha) factors to their membranes, but only one retains the DTC nucleus and therefore the ability to maintain gene expression over time. The enucleate patch can lead a bifurcating branch off the gonad arm that grows through germ cell proliferation. Germ cells in this branch differentiate as the patch loses LAG-2 expression. While the nucleus is surprisingly dispensable for aspects of leader cell function, it is required for stem cell niche activity long term. Prior work found that Rac1-/-;Rac2-/- mouse erythrocytes fragment; in this context, our new findings support the conclusion that maintaining a cohesive but deformable cell is a conserved function of this important cytoskeletal regulator.


Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Movimiento Celular , Gónadas , Organogénesis , Transducción de Señal , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Gónadas/metabolismo , Gónadas/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Organogénesis/genética , Proteínas de Unión al GTP rac/metabolismo , Proteínas de Unión al GTP rac/genética
19.
BMC Pulm Med ; 24(1): 248, 2024 May 19.
Artículo en Inglés | MEDLINE | ID: mdl-38764064

RESUMEN

BACKGROUND: Neuronal guanine nucleotide exchange factor (NGEF) plays a key role in several cancers; however, its role in lung adenocarcinoma (LUAD) remains unclear. The aim of this study was to evaluate the efficacy of NGEF as a prognostic biomarker and potential therapeutic target for LUAD. METHODS: NGEF expression data for multiple cancers and LUAD were downloaded from multiple databases. The high- and low-NGEF expression groups were constructed based on median NGEF expression in LUAD samples, and then performed Kaplan-Meier survival analysis. Differentially expressed genes (DEGs) from the two NGEF expression groups were screened and applied to construct a protein-protein interaction network. The primary pathways were obtained using gene set enrichment analysis. The associations between NGEF expression and clinical characteristics, immune infiltration, immune checkpoint inhibitors (ICIs), sensitivity to chemotherapy, and tumor mutation burden (TMB) were investigated using R. Levels of NGEF expression in the lung tissue was validated using single-cell RNA sequencing, quantitative polymerase chain reaction (qPCR), immunohistochemical staining, and western blot analysis. RESULTS: The expression of NGEF mRNA was upregulated in multiple cancers. mRNA and protein expression levels of NGEF were higher in patients with LUAD than in controls, as validated using qPCR and western blot. High NGEF expression was an independent prognostic factor for LUAD and was associated with advanced tumor stage, large tumor size, more lymph node metastasis, and worse overall survival (OS). A total of 182 overlapping DEGs were screened between The Cancer Genome Atlas and GSE31210, among which the top 20 hub genes were identified. NGEF expression was mainly enriched in the pathways of apoptosis, cell cycle, and DNA replication. Moreover, elevated NGEF expression were associated with a high fraction of activated memory CD4+ T cells and M0 macrophages; elevated expression levels of the ICIs: programmed cell death 1 and programmed cell death 1 ligand 1 expression; higher TMB; and better sensitivity to bortezomib, docetaxel, paclitaxel, and parthenolide, but less sensitivity to axitinib and metformin. CONCLUSION: NGEF expression is upregulated in LUAD and is significantly associated with tumor stages, OS probability, immune infiltration, immunotherapy response, and chemotherapy response. NGEF may be a potential diagnostic and prognostic biomarker and therapeutic target in LUAD.


Asunto(s)
Adenocarcinoma del Pulmón , Biomarcadores de Tumor , Factores de Intercambio de Guanina Nucleótido , Inmunoterapia , Neoplasias Pulmonares , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/tratamiento farmacológico , Adenocarcinoma del Pulmón/patología , Adenocarcinoma del Pulmón/inmunología , Adenocarcinoma del Pulmón/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Regulación Neoplásica de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inmunoterapia/métodos , Estimación de Kaplan-Meier , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Pronóstico , Mapas de Interacción de Proteínas
20.
Mikrochim Acta ; 191(6): 326, 2024 05 13.
Artículo en Inglés | MEDLINE | ID: mdl-38740583

RESUMEN

Migration is an initial step in tumor expansion and metastasis; suppressing cellular migration is beneficial to cancer therapy. Herein, we designed a novel biogated nanoagents that integrated the migration inhibitory factor into the mesoporous silica nanoparticle (MSN) drug delivery nanosystem to realize cell migratory inhibition and synergistic treatment. Antisense oligonucleotides (Anti) of microRNA-330-3p, which is positively related with cancer cell proliferation, migration, invasion, and angiogenesis, not only acted as the locker for blocking drugs but also acted as the inhibitory factor for suppressing migration via gene therapy. Synergistic with gene therapy, the biogated nanoagents (termed as MSNs-Gef-Anti) could achieve on-demand drug release based on the intracellular stimulus-recognition and effectively kill tumor cells. Experimental results synchronously demonstrated that the migration suppression ability of MSNs-Gef-Anti nanoagents (nearly 30%) significantly contributed to cancer therapy, and the lethality rate of the non-small-cell lung cancer was up to 70%. This strategy opens avenues for realizing efficacious cancer therapy and should provide an innovative way for pursuing the rational design of advanced nano-therapeutic platforms with the combination of cancer cell migratory inhibition.


Asunto(s)
Movimiento Celular , Quimioterapia Combinada , Nanopartículas , Neoplasias , Dióxido de Silicio , Movimiento Celular/efectos de los fármacos , Dióxido de Silicio/química , Quimioterapia Combinada/métodos , Neoplasias/tratamiento farmacológico , Sistema de Administración de Fármacos con Nanopartículas/química , Sistema de Administración de Fármacos con Nanopartículas/uso terapéutico , Nanopartículas/química , Nanopartículas/uso terapéutico , Nanopartículas/ultraestructura , Células A549 , Microscopía Electrónica de Transmisión , Humanos
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