RESUMEN
To select the core target (RAB13) in sepsis patients' peripheral blood and investigate its molecular functions and possible mechanisms. The peripheral blood of sepsis patients (n = 21) and healthy individuals (n = 9) within 24 h after admission were collected for RNA-seq, and differential gene screening was performed by iDEP online analysis software (P < 0.01; log2FC ≥ 2) and enrichment analysis, the potential core target RAB13 was screened out. The association between RAB13 expression and sepsis severity was explored using multiple datasets in the GEO database, and survival analysis was conducted. Subsequently, peripheral blood mononuclear cells (PBMCs) from sepsis and control groups were isolated, and 10 × single-cell sequencing was used to identify the main RAB13-expressing cell types. Finally, LPS was used to stimulate THP1 cells to construct a sepsis model to explore the function and possible mechanism of RAB13. We found that RAB13 was a potential core target, and RAB13 expression level was positively associated with sepsis severity and negatively correlated with survival based on multiple public datasets. A single-cell sequencing indicated that RAB13 is predominantly localized in monocytes. Cell experiments validated that RAB13 is highly expressed in sepsis, and the knockdown of RAB13 promotes the polarization of macrophages towards the M2 phenotype. This mechanism may be associated with the ECM-receptor interaction signaling pathway. The upregulation of RAB13 in sepsis patients promotes the polarization of M2-like macrophages and correlates positively with the severity of sepsis.
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Macrófagos , Sepsis , Proteínas de Unión al GTP rab , Humanos , Sepsis/metabolismo , Sepsis/genética , Sepsis/patología , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genética , Macrófagos/metabolismo , Masculino , Femenino , Persona de Mediana Edad , Células THP-1 , Anciano , Estudios de Casos y Controles , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos/farmacologíaRESUMEN
Studies have indicated that RAB17 expression levels are associated with tumor malignancy, and RAB17 is more highly expressed in endometrial cancer (EC) tissues than in peritumoral tissues. However, the roles and potential mechanisms of RAB17 in EC remain undefined. The present study confirmed that the expression of RAB17 facilitates EC progression by suppressing cellular ferroptosis-like alterations. Mechanistically, RAB17 attenuated ferroptosis in EC cells by inhibiting transferrin receptor (TFRC) protein expression in a ubiquitin proteasome-dependent manner. Because EC is a blood-deprived tumor with a poor energy supply, the relationship between RAB17 and hypoglycemia was investigated. RAB17 expression was increased in EC cells incubated in low-glucose medium. Moreover, low-glucose medium limited EC cell ferroptosis and promoted EC progression through the RAB17-TFRC axis. The in vitro results were corroborated by in vivo studies and clinical data. Overall, the present study revealed that increased RAB17 promotes the survival of EC cells during glucose deprivation by inhibiting the onset of TFRC-dependent ferroptosis.
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Progresión de la Enfermedad , Neoplasias Endometriales , Ferroptosis , Receptores de Transferrina , Proteínas de Unión al GTP rab , Animales , Femenino , Humanos , Ratones , Antígenos CD , Línea Celular Tumoral , Neoplasias Endometriales/patología , Neoplasias Endometriales/metabolismo , Neoplasias Endometriales/genética , Ferroptosis/genética , Glucosa/metabolismo , Ratones Desnudos , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genética , Receptores de Transferrina/metabolismo , Receptores de Transferrina/genéticaRESUMEN
How are Rab GTPases regulated during lysosome-related organelle (LRO) biogenesis? Li et al. (https://doi.org/10.1083/jcb.202402016) identify LYSMD proteins as crucial activators of Rab32-family GTPases in LRO development, shedding light on the previously ambiguous mechanisms governing Rab functionality in this process.
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Lisosomas , Biogénesis de Organelos , Proteínas de Unión al GTP rab , Lisosomas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genética , Humanos , Animales , Orgánulos/metabolismoRESUMEN
Endosomal Toll-like receptors (eTLRs) are essential for the sensing of non-self through RNA and DNA detection. Here, using spatiotemporal analysis of vesicular dynamics, super-resolution microscopy studies, and functional assays, we show that endomembrane defects associated with the deficiency of the small GTPase Rab27a cause delayed eTLR ligand recognition, defective early signaling, and impaired cytokine secretion. Rab27a-deficient neutrophils show retention of eTLRs in amphisomes and impaired ligand internalization. Extracellular signal-regulated kinase (ERK) signaling and ß2-integrin upregulation, early responses to TLR7 and TLR9 ligands, are defective in Rab27a deficiency. CpG-stimulated Rab27a-deficient neutrophils present increased tumor necrosis factor alpha (TNF-α) secretion and decreased secretion of a selected group of mediators, including interleukin (IL)-10. In vivo, CpG-challenged Rab27a-null mice show decreased production of type I interferons (IFNs) and IFN-γ, and the IFN-α secretion defect is confirmed in Rab27a-null plasmacytoid dendritic cells. Our findings have significant implications for immunodeficiency, inflammation, and CpG adjuvant vaccination.
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Citocinas , Receptor Toll-Like 9 , Proteínas rab27 de Unión a GTP , Animales , Proteínas rab27 de Unión a GTP/metabolismo , Proteínas rab27 de Unión a GTP/genética , Ratones , Citocinas/metabolismo , Receptor Toll-Like 9/metabolismo , Receptor Toll-Like 9/deficiencia , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/deficiencia , Proteínas de Unión al GTP rab/genética , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 7/deficiencia , Receptor Toll-Like 7/genética , Neutrófilos/metabolismo , Neutrófilos/inmunología , Endosomas/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Factor de Necrosis Tumoral alfa/metabolismo , Ácidos Nucleicos/metabolismo , Transducción de Señal , Interferón gamma/metabolismo , Glicoproteínas de MembranaRESUMEN
Background: As one of the most common cancer, colorectal cancer (CRC) is with high morbidity and mortality. Peritoneal metastasis (PM) is a fatal state of CRC, and few patients may benefit from traditional therapies. There is a complex interaction between PM and immune cell infiltration. Therefore, we aimed to determine biomarkers associated with colorectal cancer peritoneal metastasis (CRCPM) and their relationship with immune cell infiltration. Methods: By informatic analysis, differently expressed genes (DEGs) were selected and hub genes were screened out. RAB13, one of the hub genes, was identificated from public databases and validated in CRC tissues. The ESTIMATE, CEBERSORT and TIMER algorithms were applied to analyze the correlation between RAB13 and immune infiltration in CRC. RAB13's expression in different cells were analyzed at the single-cell level in scRNA-Seq. The Gene Set Enrichment Analysis (GSEA) was performed for RAB13 enrichment and further confirmed. Using oncoPredict algorithm, RAB13's impact on drug sensitivity was evaluated. Results: High RAB13 expression was identified in public databases and led to a poor prognosis. RAB13 was found to be positively correlated with the macrophages and other immune cells infiltration and from scRNA-Seq, RAB13 was found to be located in CRC cells and macrophages. GSEA revealed that high RAB13 expression enriched in a various of biological signaling, and oncoPredict algorithm showed that RAB13 expression was correlated with paclitaxel sensitivity. Conclusion: Our study indicated clinical role of RAB13 in CRC-PM, suggesting its potential as a therapeutic target in the future.
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Biomarcadores de Tumor , Neoplasias Colorrectales , Neoplasias Peritoneales , Proteínas de Unión al GTP rab , Humanos , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/genética , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Neoplasias Peritoneales/secundario , Neoplasias Peritoneales/genética , Neoplasias Peritoneales/inmunología , Regulación Neoplásica de la Expresión Génica , Pronóstico , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Femenino , Masculino , Perfilación de la Expresión GénicaRESUMEN
The transmembrane protein ß-amyloid precursor protein (APP) is central to the pathophysiology of Alzheimer's disease (AD). The ß-amyloid hypothesis posits that aberrant processing of APP forms neurotoxic ß-amyloid aggregates, which lead to the cognitive impairments observed in AD. Although numerous additional factors contribute to AD, there is a need to better understand the synaptic function of APP. We have found that Drosophila APP-like (APPL) has both shared and non-shared roles at the synapse with Kismet (Kis), a chromatin helicase binding domain (CHD) protein. Kis is the homolog of CHD7 and CHD8, both of which are implicated in neurodevelopmental disorders including CHARGE Syndrome and autism spectrum disorders, respectively. Loss of function mutations in kis and animals expressing human APP and BACE in their central nervous system show reductions in the glutamate receptor subunit, GluRIIC, the GTPase Rab11, and the bone morphogenetic protein (BMP), pMad, at the Drosophila larval neuromuscular junction (NMJ). Similarly, processes like endocytosis, larval locomotion, and neurotransmission are deficient in these animals. Our pharmacological and epistasis experiments indicate that there is a functional relationship between Kis and APPL, but Kis does not regulate appl expression at the larval NMJ. Instead, Kis likely influences the synaptic localization of APPL, possibly by promoting rab11 transcription. These data identify a potential mechanistic connection between chromatin remodeling proteins and aberrant synaptic function in AD.
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Precursor de Proteína beta-Amiloide , Proteínas de Drosophila , Unión Neuromuscular , Proteínas de Unión al GTP rab , Animales , Unión Neuromuscular/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genética , Precursor de Proteína beta-Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Transmisión Sináptica , Sinapsis/metabolismo , Receptores de Glutamato/metabolismo , Receptores de Glutamato/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Humanos , ADN Helicasas/metabolismo , ADN Helicasas/genética , Proteínas de la Membrana , Proteínas del Tejido Nervioso , Proteínas de Homeodominio , Receptores Ionotrópicos de GlutamatoRESUMEN
Breast cancer is the most frequently diagnosed cancer worldwide, and the incidence rate has increased enormously over the last three decades. Rab proteins are members of the Rab GTPase superfamily. The aberrant function of these proteins leads to the development of tumors. Mentha longifolia var. asiatica and Zygophyllum arabicum have been known for their therapeutic potential for ages. The present study aimed to synthesize ZnO nanoparticles encapsulated with the extracts of M. longifolia var. asiatica and Z. arabicum and evaluating their therapeutic potential against breast cancer, targeting the Rab22A gene and its protein. UV-Vis spectrophotometer showed characteristic absorbance peaks at 295 nm and 345 nm for Z. arabicum and M. longifolia var. asiatica ZnONPs, respectively. The FTIR bands of Z. arabicum nanoparticles suggested the presence of aldehydes, alcohols, and polyols whereas bands of M. longifolia var. asiatica ZnONPs suggested the presence of carboxyl groups, hydroxyl groups, alkynes, and amines. SEM revealed the size of Z. arabicum ZnO NPs to be 25 ± 4 nm with a spherical shape as compared to nanoparticles of M. longifolia var. asiatica having a size of 35 ± 6 nm with a hexagonal shape. EDX determined the elemental composition of both particles. The cytotoxicity of both plant extracts and respective NPs was determined against the MCF-7 breast cancer cell line, which was found to be significant with an IC50 value of 51.68 µM for Z. arabicum and 88.02 µM for M. longifolia var. asiatica ZnO compared to plant extracts (64.01 µM and 107.9 µM for Z. arabicum and M. longifolia var. asiatica). The gene expression and protein levels of Rab22A were decreased in nanoparticle-treated cells as compared to the control group. The apoptotic role of synthesized nanoparticles against the MCF-7 cell line was also determined by the expression of apoptotic pathway genes and proteins (bax, caspase 3, caspase 8 and caspase 9). All samples showed significant apoptotic activity by activating intrinsic and extrinsic pathway genes. The activity of Z. arabicum was more eminent as compared to M. longifolia var. asiatica which was evident by the greater expression of studied genes and proteins as determined by Real-time qPCR and ELISA. This is the first-ever report describing the comparative analysis of the efficacy of Z. arabicum and M. longifolia var. asiatica ZnONPs against breast cancer.
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Neoplasias de la Mama , Mentha , Extractos Vegetales , Óxido de Zinc , Proteínas de Unión al GTP rab , Humanos , Óxido de Zinc/farmacología , Óxido de Zinc/química , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Femenino , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genética , Mentha/química , Extractos Vegetales/farmacología , Extractos Vegetales/química , Células MCF-7 , Nanopartículas/química , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Nanopartículas del Metal/químicaRESUMEN
Most bacteria will use their toxins to interact with the host cell, causing damage to the cell and then escaping from it. When bacteria enter the cell, they will be transported via the endosomal pathway. Rab GTPases are involved in bacterial transport as major components of endosomes that bind to their downstream effector proteins. The bacteria manipulate some Rab GTPases, escape the cell, and get to survive. In this review, we will focus on summarizing the many processes of how bacteria manipulate Rab GTPases to control their escape.
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Bacterias , Endosomas , Interacciones Huésped-Patógeno , Proteínas de Unión al GTP rab , Proteínas de Unión al GTP rab/metabolismo , Bacterias/metabolismo , Bacterias/enzimología , Bacterias/genética , Endosomas/metabolismo , Humanos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Transporte de Proteínas , Animales , Transporte BiológicoRESUMEN
Lysosomes play a pivotal role in coordinating macromolecule degradation and regulating cell growth and metabolism. Despite substantial progress in identifying lysosomal signaling proteins, understanding the pathways that synchronize lysosome functions with changing cellular demands remains incomplete. This study uncovers a role for TANK-binding kinase 1 (TBK1), well known for its role in innate immunity and organelle quality control, in modulating lysosomal responsiveness to nutrients. Specifically, we identify a pool of TBK1 that is recruited to lysosomes in response to elevated amino acid levels. This lysosomal TBK1 phosphorylates Rab7 on serine 72. This is critical for alleviating Rab7-mediated inhibition of amino acid-dependent mTORC1 activation. Furthermore, a TBK1 mutant (E696K) associated with amyotrophic lateral sclerosis and frontotemporal dementia constitutively accumulates at lysosomes, resulting in elevated Rab7 phosphorylation and increased mTORC1 activation. This data establishes the lysosome as a site of amino acid regulated TBK1 signaling that is crucial for efficient mTORC1 activation. This lysosomal pool of TBK1 has broader implications for lysosome homeostasis, and its dysregulation could contribute to the pathogenesis of ALS-FTD.
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Aminoácidos , Lisosomas , Diana Mecanicista del Complejo 1 de la Rapamicina , Proteínas Serina-Treonina Quinasas , Proteínas de Unión al GTP rab , Proteínas de Unión a GTP rab7 , Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Humanos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Aminoácidos/metabolismo , Fosforilación , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genética , Células HEK293 , Transducción de Señal , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/genética , Demencia Frontotemporal/patología , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patologíaRESUMEN
Peritoneal metastasis is one of the most common risk factors contributing to the poor prognosis of gastric cancer. We previously reported that extracellular vesicles from gastric cancer cells could facilitate peritoneal metastasis. However, their impact on gastric cancer-induced peritoneal metastasis under hypoxic conditions remains unclear. This study aims to elucidate how hypoxia-resistant gastric cancer cell-derived extracellular vesicles affect the peritoneal metastasis of normoxic gastric cancer cells. Proteomic analysis revealed elevated levels of Caveolin1 and Laminin ß2 in hypoxia-resistant gastric cancer cells and their corresponding extracellular vesicles. Importantly, Caveolin1 was found to play a central role in mediating Laminin ß2 sorting into extracellular vesicles derived from hypoxia-resistant gastric cancer cells, and subsequently, extracellular vesicle-associated Laminin ß2 promoted peritoneal metastasis in normoxic gastric cancer cells by activating the AKT pathway. Further investigation confirmed that Caveolin1 activation by Rho-related Coiled-coil kinase 1-mediated phosphorylation of Y14 residue is a key factor facilitating Laminin ß2 sorting into extracellular vesicles. Moreover, Y14 phosphorylated- Caveolin1 enhanced Laminin ß2 sorting by activating Rab11. Finally, our study demonstrated that a combined assessment of plasma extracellular vesicle-associated Caveolin1 and extracellular vesicle-associated Laminin ß2 could provide an accurate predictive tool for peritoneal metastasis occurrence in gastric cancer.
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Caveolina 1 , Vesículas Extracelulares , Neoplasias Peritoneales , Neoplasias Gástricas , Proteínas de Unión al GTP rab , Quinasas Asociadas a rho , Neoplasias Gástricas/patología , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/genética , Humanos , Caveolina 1/metabolismo , Caveolina 1/genética , Neoplasias Peritoneales/secundario , Neoplasias Peritoneales/metabolismo , Animales , Quinasas Asociadas a rho/metabolismo , Vesículas Extracelulares/metabolismo , Ratones , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genética , Línea Celular Tumoral , Transducción de Señal , Masculino , FemeninoRESUMEN
Orai1 is a plasma membrane Ca2+ channel involved in store operated calcium entry (SOCE). SOCE can regulate cell growth, exocytosis, gene expression and inflammation. We previously found that short palate lung and nasal epithelial clone 1's (SPLUNC1) sixth α-helix (α6) bound Orai1 to inhibit SOCE. SPLUNC1 was not proteolytically stable, so we developed ELD607, an 11 amino acid peptide based on SPLUNC1's α6 region which was more stable and more potent than SPLUNC1/α6. Here, we studied ELD607's mechanism of action. We overexpressed either Orai1-HA or Orai1-YFP in HEK293T cells to probe ELD607-Orai1 interactions by confocal microscopy. We also measured changes in Fluo-4 fluorescence in a multiplate reader as a marker of cytoplasmic Ca2+ levels. ELD607 internalized Orai1 independently of STIM1. Both 15 min and 3 h exposure to ELD607 similarly depleted Orai1 in the plasma membrane. However, 3 h exposure to ELD607 yielded greater inhibition of SOCE. ELD607 continued to colocalize with Orai1 after internalization and this process was dependent on the presence of the ubiquitin ligase NEDD4.2. Similarly, ELD607 increased the colocalization between Orai1 and ubiquitin. ELD607 also increased the colocalization between Orai1 and Rab5 and 7, but not Rab11, suggesting that Orai1 trafficked through early and late but not recycling endosomes. Finally, ELD607 caused Orai1, but not Orai2, Orai3, or STIM1 to traffic to lysosomes. We conclude that ELD607 rapidly binds to Orai1 and works in an identical fashion as full length SPLUNC1 by internalizing Orai1 and sending it to lysosomes, leading to a decrease in SOCE.
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Calcio , Lisosomas , Proteína ORAI1 , Humanos , Proteína ORAI1/metabolismo , Células HEK293 , Calcio/metabolismo , Lisosomas/metabolismo , Canales de Calcio/metabolismo , Transporte de Proteínas , Membrana Celular/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Péptidos/metabolismo , Péptidos/farmacología , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Autophagy is a universal degradation system in eukaryotic cells. In plants, although autophagosome biogenesis has been extensively studied, the mechanism of how autophagosomes are transported to the vacuole for degradation remains largely unexplored. In this study, we demonstrated that upon autophagy induction, Arabidopsis homotypic fusion and protein sorting (HOPS) subunit VPS41 converts first from condensates to puncta, then to ring-like structures, termed VPS41-associated phagic vacuoles (VAPVs), which enclose autophagy-related gene (ATG)8s for vacuolar degradation. This process is initiated by ADP ribosylation factor (ARF)-like GTPases ARLA1s and occurs concurrently with autophagy progression through coupling with the synaptic-soluble N-ethylmaleimide-sensitive factor attachment protein rmleceptor (SNARE) proteins. Unlike in other eukaryotes, autophagy degradation in Arabidopsis is largely independent of the RAB7 pathway. By contrast, dysfunction in the condensates-to-VAPVs conversion process impairs autophagosome structure and disrupts their vacuolar transport, leading to a significant reduction in autophagic flux and plant survival rate. Our findings suggest that the conversion pathway might be an integral part of the autophagy program unique to plants.
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Proteínas de Arabidopsis , Arabidopsis , Autofagosomas , Autofagia , Vacuolas , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Vacuolas/metabolismo , Autofagosomas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/genética , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Familia de las Proteínas 8 Relacionadas con la Autofagia/genética , Proteínas SNARE/metabolismo , Proteínas SNARE/genética , Proteínas de Unión a GTP rab7 , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genéticaRESUMEN
Herpes simplex virus 1 (HSV-1) is an alpha herpesvirus that infects a majority of the world population. The mechanisms and cellular host factors involved in the intracellular transport and exocytosis of HSV-1 particles are not fully understood. To elucidate these late steps in the replication cycle, we developed a live-cell fluorescence microscopy assay of HSV-1 virion intracellular trafficking and exocytosis. This method allows us to track individual virus particles and identify the precise moment and location of particle exocytosis using a pH-sensitive reporter. We show that HSV-1 uses the host cell's post-Golgi secretory pathway during egress. The small GTPase, Rab6, binds to nascent secretory vesicles at the trans-Golgi network and plays important, but non-essential, roles in vesicle traffic and exocytosis at the plasma membrane, therefore making it a useful marker of the Golgi and post-Golgi secretory pathway. We show that HSV-1 particles colocalize with Rab6a in the region of the Golgi, cotraffic with Rab6a to the cell periphery, and undergo exocytosis from Rab6a vesicles. Consistent with previous reports, we find that HSV-1 particles accumulate at preferential egress sites in infected cells. The secretory pathway mediates this preferential/polarized egress, since Rab6a vesicles accumulate near the plasma membrane similarly in uninfected cells. These data suggest that, following particle envelopment, HSV-1 egress follows a pre-existing cellular secretory pathway to exit infected cells rather than novel, virus-induced mechanisms. IMPORTANCE: Herpes simplex virus 1 (HSV-1) infects a majority of people. It establishes a life-long latent infection and occasionally reactivates, typically causing characteristic oral or genital lesions. Rarely in healthy natural hosts, but more commonly in zoonotic infections and in elderly, newborn, or immunocompromised patients, HSV-1 can cause severe herpes encephalitis. The precise cellular mechanisms used by HSV-1 remain an important area of research. In particular, the egress pathways that newly assembled virus particles use to exit from infected cells are unclear. In this study, we used fluorescence microscopy to visualize individual virus particles exiting from cells and found that HSV-1 particles use the pre-existing cellular secretory pathway.
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Exocitosis , Aparato de Golgi , Herpesvirus Humano 1 , Vías Secretoras , Liberación del Virus , Proteínas de Unión al GTP rab , Herpesvirus Humano 1/fisiología , Herpesvirus Humano 1/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Humanos , Animales , Aparato de Golgi/metabolismo , Aparato de Golgi/virología , Células Vero , Red trans-Golgi/metabolismo , Red trans-Golgi/virología , Chlorocebus aethiops , Herpes Simple/virología , Herpes Simple/metabolismo , Virión/metabolismo , Células HeLa , Membrana Celular/metabolismo , Membrana Celular/virologíaRESUMEN
BACKGROUND: GTPases of the Rab family are important orchestrators of membrane trafficking, and their dysregulation has been linked to a variety of neuropathologies. In 2017, we established a causal link between RAB11A variants and developmental and epileptic encephalopathy. In this study, we expand the phenotype of RAB11A-associated neurodevelopmental disorder and explore genotype-phenotype correlations. METHODS: We assessed 16 patients with pathogenic or likely pathogenic RAB11A variants, generally de novo, heterozygous missense variants. One individual had a homozygous nonsense variant, although concomitant with a pathogenic LAMA2 variant, which made their respective contributions to the phenotype difficult to discriminate. RESULTS: We reinforce the finding that certain RAB11A missense variants lead to intellectual disability and developmental delays. Other clinical features might include gait disturbances, hypotonia, magnetic resonance imaging abnormalities, visual anomalies, dysmorphisms, early adrenarche, and obesity. Epilepsy seems to be less common and linked to variants outside the binding sites. Individuals with variants in the binding sites seem to have a more multisystemic, nonepileptic phenotype. CONCLUSIONS: Similar to other Rab-related disorders, RAB11A-associated neurodevelopmental disorder can also impact gait, tonus, brain anatomy and physiology, vision, adrenarche, and body weight and structure. Epilepsy seems to affect the minority of patients with variants outside the binding sites.
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Estudios de Asociación Genética , Trastornos del Neurodesarrollo , Proteínas de Unión al GTP rab , Humanos , Proteínas de Unión al GTP rab/genética , Masculino , Niño , Femenino , Trastornos del Neurodesarrollo/genética , Preescolar , Adolescente , Estudios de Cohortes , Mutación Missense , Fenotipo , Discapacidad Intelectual/genética , Discapacidad Intelectual/diagnóstico por imagen , Epilepsia/genética , Epilepsia/fisiopatología , Epilepsia/diagnóstico por imagen , Lactante , Discapacidades del Desarrollo/genética , Discapacidades del Desarrollo/diagnóstico por imagen , Discapacidades del Desarrollo/etiologíaRESUMEN
BACKGROUND: Recycling of integrin via endosomal vesicles is critical for the migration of cancer cells, which leads to the metastasis of pancreatic cancer and devastating cancer-related death. So, new diagnostic and therapeutic molecules which target the recycling of endosomal vesicles need to be developed. METHODS: Public databases including TCGA, ICGC, GSE21501, GSE28735, and GENT are analyzed to derive diagnostic and therapeutic targets. To reveal biological roles and underlying mechanisms of molecular targets, various molecular biological experiments were conducted. RESULTS: First, we identified UNC13D's overexpression in patients with pancreatic cancer (n = 824) and its prognostic significance and high hazard ratio (HR) in four independent pancreatic cancer cohorts (TCGA, n = 178, p = 0.014, HR = 3.629; ICGC, n = 91, p = 0.000, HR = 4.362; GSE21501, n = 102, p = 0.002, HR = 2.339; GSE28735, n = 45, p = 0.022, HR = 2.681). Additionally, its expression is associated with the clinicopathological progression of pancreatic cancer. Further biological studies have shown that UNC13D regulates the migration of pancreatic cancer cells by coupling the exocytosis of recycling endosomes with focal adhesion turnover via the regulation of FAK phosphorylation. Immunoprecipitation and immunocytochemistry showed the formation of the RAB11-UNC13D-FAK axis in endosomes during integrin recycling. We observed that UNC13D directly interacted with the FERM domain of FAK and regulated FAK phosphorylation in a calcium-dependent manner. Finally, we found co-expression of UNC13D and FAK showed the poorest survival (TCGA, p = 0.000; ICGC, p = 0.036; GSE28735, p = 0.006). CONCLUSIONS: We highlight that UNC13D, a novel prognostic factor, promotes pancreatic cancer progression by coupling integrin recycling with focal adhesion turnover via the RAB11-UNC13D-FAK axis for the migration of pancreatic cancer cells.
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Movimiento Celular , Adhesiones Focales , Integrinas , Neoplasias Pancreáticas , Proteínas de Unión al GTP rab , Humanos , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/genética , Proteínas de Unión al GTP rab/metabolismo , Línea Celular Tumoral , Adhesiones Focales/metabolismo , Integrinas/metabolismo , Quinasa 1 de Adhesión Focal/metabolismo , Femenino , Masculino , Transducción de Señal , Persona de Mediana Edad , Pronóstico , Regulación Neoplásica de la Expresión Génica , Endosomas/metabolismo , Progresión de la EnfermedadRESUMEN
We previously reported autophagy-mediated degradation of nuclei, nucleophagy, in the filamentous fungus Aspergillus oryzae. In this study, we examined whether nuclei are degraded as a whole. We generated A. oryzae mutants deleted for orthologs of Saccharomyces cerevisiae YPT7 and ATG15 which are required, respectively, for autophagosome-vacuole fusion and vacuolar degradation of autophagic bodies. Degradation of histone H2B-EGFP under starvation conditions was greatly decreased in the ΔAoypt7 and ΔAoatg15 mutants. Fluorescence and electron microscopic observations showed that autophagosomes and autophagic bodies surrounding the entire nuclei were accumulated in the cytoplasm of ΔAoypt7 and the vacuole of ΔAoatg15, respectively. These results indicate that nuclei are engulfed in the autophagosomes as a whole and transported/released into the vacuolar lumen where they are degraded.
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Aspergillus oryzae , Autofagosomas , Proteínas Fúngicas , Vacuolas , Vacuolas/metabolismo , Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Autofagosomas/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Autofagia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Eliminación de Gen , Proteínas de Unión al GTP rabRESUMEN
MICAL proteins represent a unique family of actin regulators crucial for synapse development, membrane trafficking, and cytokinesis. Unlike classical actin regulators, MICALs catalyze the oxidation of specific residues within actin filaments to induce robust filament disassembly. The potent activity of MICALs requires tight control to prevent extensive damage to actin cytoskeleton. However, the molecular mechanism governing MICALs' activity regulation remains elusive. Here, we report the cryo-EM structure of MICAL1 in the autoinhibited state, unveiling a head-to-tail interaction that allosterically blocks enzymatic activity. The structure also reveals the assembly of C-terminal domains via a tripartite interdomain interaction, stabilizing the inhibitory conformation of the RBD. Our structural, biochemical, and cellular analyses elucidate a multi-step mechanism to relieve MICAL1 autoinhibition in response to the dual-binding of two Rab effectors, revealing its intricate activity regulation mechanisms. Furthermore, our mutagenesis study of MICAL3 suggests the conserved autoinhibition and relief mechanisms among MICALs.
Asunto(s)
Actinas , Microscopía por Crioelectrón , Oxigenasas de Función Mixta , Humanos , Actinas/metabolismo , Oxigenasas de Función Mixta/metabolismo , Oxigenasas de Función Mixta/química , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/química , Unión Proteica , Citoesqueleto de Actina/metabolismo , Modelos Moleculares , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Dominios Proteicos , CalponinasRESUMEN
Purpose: Glaucoma is the primary cause of permanent vision loss worldwide. However, the pathogenesis of primary open-angle glaucoma (POAG), the main type of glaucoma, has not yet been completely understood. Methods: In our study, the POAG cohorts were obtained from the Gene Expression Omnibus (GEO) database (GSE45570). Biomarkers with diagnostic utility for POAG were identified through combining differentially expressed analysis, enrichment analysis, machine learning algorithms, and receiver operating characteristic (ROC) analysis. The regulatory networks (including a competing endogenous RNA (ceRNA) regulatory network and a small molecule compounds-mRNA network) were created. In addition, the Mendelian randomization (MR) analysis was used to identify exposures causally associated with POAG. Finally, the expression of the biomarkers was validated via real-time quantitative polymerase chain reaction (RT-qPCR). Results: The Gene Ontology (GO) items that the differentially expressed genes (DEGs) between POAG and control groups enriched were relevant to light stimulation and DNA methylation. A total of three light stimulation-related biomarkers (RAB8A, PRG3, and SMAD3) were identified, which had diagnostic value for POAG patients. Besides, the ceRNA regulatory network contained 88 nodes and 93 edges, and a small molecule compounds-mRNA network included 66 nodes and 76 edges. The MR results indicated a causal association between DNA methylation GrimAge acceleration and POAG. Additionally, the results of RT-qPCR revealed that the expression trend of RAB8A was consistent with that of GSE45570. Conclusions: Taken together, this study provides three light stimulation-related biomarkers (RAB8A, PRG3, and SMAD3) for the diagnosis of POAG, providing scientifically valuable insights for further studies of POAG. Translational Relevance: Discovering biomarkers that possess diagnostic significance for POAG has the potential to offer new insights into the pathogenesis of POAG and present novel objectives for clinical intervention.
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Biomarcadores , Biología Computacional , Redes Reguladoras de Genes , Glaucoma de Ángulo Abierto , Análisis de la Aleatorización Mendeliana , Humanos , Glaucoma de Ángulo Abierto/genética , Glaucoma de Ángulo Abierto/diagnóstico , Biomarcadores/metabolismo , Proteína smad3/genética , Proteína smad3/metabolismo , Nervio Óptico/metabolismo , Proteínas de Unión al GTP rab/genética , Curva ROC , Proteoglicanos/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Metilación de ADNRESUMEN
BACKGROUND: Lovastatin, a type of statin usually considered as a lipid-lowering drug that lowers blood cholesterol and low-density lipoprotein cholesterol levels, has been rediscovered to have anticancer activity. Fewer studies exist regarding the effect of lovastatin on esophageal squamous cell carcinoma (ESCC). METHODS: Here, we report that lovastatin shows anticancer effect on ESCC By affecting the mitochondrial autophagy pathway. Moreover, based on proteomics and computer molecular simulations found that RAB38 and RAB27A may be a target of lovastatin. RESULTS: We observed that autophagy of mitochondria is inhibited by lovastatin, affecting esophageal squamous cell proliferation. There is a possible link between the expression of RAB38, RAB27A and immune cell invasion in esophageal cancer. CONCLUSIONS: These results demonstrate the huge potential of lovastatin as an RAB38, RAB27A inhibitor in esophageal cancer chemotherapy and chemoprevention.
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Autofagia , Proliferación Celular , Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Lovastatina , Proteómica , Lovastatina/farmacología , Humanos , Carcinoma de Células Escamosas de Esófago/tratamiento farmacológico , Carcinoma de Células Escamosas de Esófago/metabolismo , Carcinoma de Células Escamosas de Esófago/patología , Proliferación Celular/efectos de los fármacos , Proteómica/métodos , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , Línea Celular Tumoral , Autofagia/efectos de los fármacos , Proteínas de Unión al GTP rab/metabolismo , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Simulación del Acoplamiento MolecularRESUMEN
Phagocytosis is an important immune response that protects the host from pathogen invasion. Rit1 GTPase is known to be involved in diverse cellular processes. However, its role in FcγR-mediated phagocytosis remains unclear. Our live-cell imaging analysis revealed that Rit1 was localized to the membranes of F-actin-rich phagocytic cups in RAW264 macrophages. Rit1 knockout and expression of the GDP-locked Rit1 mutant suppressed phagosome formation. We also found that TBC1D10B, a GAP for the Rab family GTPases, colocalizes with Rit1 in the membranes of phagocytic cups. Expression and knockout studies have shown that TBC1D10B decreases phagosome formation in both Rab-GAP activity-dependent and -independent manners. Notably, the expression of the GDP-locked Rit1 mutant or Rit1 knockout inhibited the dissociation of TBC1D10B from phagocytic cups. In addition, the expression of the GTP-locked Rit1 mutant promoted the dissociation of TBC1D10B in phagocytic cups and restored the rate of phagosome formation in TBC1D10B-expressing cells. These data suggest that Rit1-TBC1D10B signaling regulates FcγR-mediated phagosome formation in macrophages.