RESUMEN
The NACHT-, leucine-rich-repeat- (LRR), and pyrin domain-containing protein 3 (NLRP3) is emerging to be a critical intracellular inflammasome sensor of membrane integrity and a highly important clinical target against chronic inflammation. Here, we report that an endogenous, stimulus-responsive form of full-length mouse NLRP3 is a 12- to 16-mer double-ring cage held together by LRR-LRR interactions with the pyrin domains shielded within the assembly to avoid premature activation. Surprisingly, this NLRP3 form is predominantly membrane localized, which is consistent with previously noted localization of NLRP3 at various membrane organelles. Structure-guided mutagenesis reveals that trans-Golgi network dispersion into vesicles, an early event observed for many NLRP3-activating stimuli, requires the double-ring cages of NLRP3. Double-ring-defective NLRP3 mutants abolish inflammasome punctum formation, caspase-1 processing, and cell death. Thus, our data uncover a physiological NLRP3 oligomer on the membrane that is poised to sense diverse signals to induce inflammasome activation.
Asunto(s)
Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/química , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Secuencia de Aminoácidos , Animales , Membrana Celular/metabolismo , Microscopía por Crioelectrón , Células HEK293 , Humanos , Ratones , Modelos Biológicos , Modelos Moleculares , Mutación/genética , Quinasas Relacionadas con NIMA/genética , Proteína con Dominio Pirina 3 de la Familia NLR/aislamiento & purificación , Proteína con Dominio Pirina 3 de la Familia NLR/ultraestructura , Nigericina/farmacología , Unión Proteica , Dominios Proteicos , Multimerización de Proteína , Red trans-Golgi/metabolismoRESUMEN
Through a network of progressively maturing vesicles, the endosomal system connects the cell's interior with extracellular space. Intriguingly, this network exhibits a bilateral architecture, comprised of a relatively immobile perinuclear vesicle "cloud" and a highly dynamic peripheral contingent. How this spatiotemporal organization is achieved and what function(s) it curates is unclear. Here, we reveal the endoplasmic reticulum (ER)-located ubiquitin ligase Ring finger protein 26 (RNF26) as the global architect of the entire endosomal system, including the trans-Golgi network (TGN). To specify perinuclear vesicle coordinates, catalytically competent RNF26 recruits and ubiquitinates the scaffold p62/sequestosome 1 (p62/SQSTM1), in turn attracting ubiquitin-binding domains (UBDs) of various vesicle adaptors. Consequently, RNF26 restrains fast transport of diverse vesicles through a common molecular mechanism operating at the ER membrane, until the deubiquitinating enzyme USP15 opposes RNF26 activity to allow vesicle release into the cell's periphery. By drawing the endosomal system's architecture, RNF26 orchestrates endosomal maturation and trafficking of cargoes, including signaling receptors, in space and time.
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Retículo Endoplásmico/metabolismo , Endosomas/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Neoplasias/metabolismo , Línea Celular Tumoral , Células Dendríticas/citología , Células Dendríticas/metabolismo , Humanos , Macrófagos/citología , Macrófagos/metabolismo , Proteína Sequestosoma-1/metabolismo , Vesículas Transportadoras/metabolismo , Proteasas Ubiquitina-Específicas/metabolismoRESUMEN
The trans-Golgi Network (TGN) sorts molecular "addresses" and sends newly synthesized proteins to their destination via vesicular transport carriers. Despite the functional significance of packaging processes at the TGN, the sorting of soluble proteins remains poorly understood. Recent research has shown that the Golgi resident protein Cab45 is a significant regulator of secretory cargo sorting at the TGN. Cab45 oligomerizes upon transient Ca2+ influx, recruits soluble cargo molecules (clients), and packs them in sphingomyelin-rich transport carriers. However, the identity of client molecules packed into Cab45 vesicles is scarce. Therefore, we used a precise and highly efficient secretome analysis technology called hiSPECs. Intriguingly, we observed that Cab45 deficient cells manifest hypersecretion of lysosomal hydrolases. Specifically, Cab45 deficient cells secrete the unprocessed precursors of prosaposin (PSAP) and progranulin (PGRN). In addition, lysosomes in these cells show an aberrant perinuclear accumulation suggesting a new role of Cab45 in lysosomal positioning. This work uncovers a yet unknown function of Cab45 in regulating lysosomal function.
Asunto(s)
Proteínas , Saposinas , Humanos , Transporte Biológico , Lisosomas/metabolismo , Progranulinas/metabolismo , Transporte de Proteínas/fisiología , Proteínas/metabolismo , Saposinas/genética , Saposinas/metabolismo , Red trans-Golgi/metabolismoRESUMEN
In hepatocytes, the Wilson disease protein ATP7B resides on the trans-Golgi network (TGN) and traffics to peripheral lysosomes to export excess intracellular copper through lysosomal exocytosis. We found that in basal copper or even upon copper chelation, a significant amount of ATP7B persists in the endolysosomal compartment of hepatocytes but not in non-hepatic cells. These ATP7B-harbouring lysosomes lie in close proximity of ~10 nm to the TGN. ATP7B constitutively distributes itself between the sub-domain of the TGN with a lower pH and the TGN-proximal lysosomal compartments. The presence of ATP7B on TGN-lysosome colocalising sites upon Golgi disruption suggested a possible exchange of ATP7B directly between the TGN and its proximal lysosomes. Manipulating lysosomal positioning significantly alters the localisation of ATP7B in the cell. Contrary to previous understanding, we found that upon copper chelation in a copper-replete hepatocyte, ATP7B is not retrieved back to TGN from peripheral lysosomes; rather, ATP7B recycles to these TGN-proximal lysosomes to initiate the next cycle of copper transport. We report a hitherto unknown copper-independent lysosomal localisation of ATP7B and the importance of TGN-proximal lysosomes but not TGN as the terminal acceptor organelle of ATP7B in its retrograde pathway.
Asunto(s)
Cobre , Lisosomas , Cobre/metabolismo , ATPasas Transportadoras de Cobre/metabolismo , Transporte de Proteínas , Lisosomas/metabolismo , ExocitosisRESUMEN
The epsin-related adaptor proteins Ent3p and Ent5p participate in budding of clathrin coated vesicles in transport between trans-Golgi network and endosomes in yeast. Transport of the arginine permease Can1p was analyzed, which recycles between plasma membrane and endosomes and can be targeted to the vacuole for degradation. ent3∆ cells accumulate Can1p-GFP in endosomes. Can1p-GFP is transported faster to the vacuole upon induction of degradation in ent5∆ cells than in wild type cells. The C-terminal domain of Ent5p was sufficient to restore recycling of the secretory SNARE GFP-Snc1p between plasma membrane and TGN in ent3∆ ent5∆ cells. The SNARE Tlg2p was identified as interaction partner of the Ent5p ENTH domain by in vitro binding assays and the interaction site on Ent5p was mapped. Tlg2p functions in transport from early endosomes to the trans-Golgi network and in homotypic fusion of these organelles. Tlg2p is partially shifted to denser fractions in sucrose density gradients of organelles from ent5∆ cells while distribution of Kex2p is unaffected demonstrating that Ent5p acts as cargo adaptor for Tlg2p in vivo. Taken together we show that Ent3p and Ent5p have different roles in transport and function as cargo adaptors for distinct SNAREs.
Asunto(s)
Proteínas SNARE , Proteínas de Saccharomyces cerevisiae , Proteínas SNARE/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Saccharomyces cerevisiae/metabolismo , Red trans-Golgi/metabolismo , Endosomas/metabolismoRESUMEN
Normal receptor tyrosine kinases (RTKs) need to reach the plasma membrane (PM) for ligand-induced activation, whereas its cancer-causing mutants can be activated before reaching the PM in organelles, such as the Golgi/trans-Golgi network (TGN). Inhibitors of protein export from the endoplasmic reticulum (ER), such as brefeldin A (BFA) and 2-methylcoprophilinamide (M-COPA), can suppress the activation of mutant RTKs in cancer cells, suggesting that RTK mutants cannot initiate signaling in the ER. BFA and M-COPA block the function of ADP-ribosylation factors (ARFs) that play a crucial role in ER-Golgi protein trafficking. However, among ARF family proteins, the specific ARFs inhibited by BFA or M-COPA, that is, the ARFs involved in RTKs transport from the ER, remain unclear. In this study, we showed that M-COPA blocked the export of not only KIT but also PDGFRA/EGFR/MET RTKs from the ER. ER-retained RTKs could not fully transduce anti-apoptotic signals, thereby leading to cancer cell apoptosis. Moreover, a single knockdown of ARF1, ARF3, ARF4, ARF5, or ARF6 could not block ER export of RTKs, indicating that BFA/M-COPA treatment cannot be mimicked by the knockdown of only one ARF member. Interestingly, simultaneous transfection of ARF1, ARF4, and ARF5 siRNAs mirrored the effect of BFA/M-COPA treatment. Consistent with these results, in vitro pulldown assays showed that BFA/M-COPA blocked the function of ARF1, ARF4, and ARF5. Taken together, these results suggest that BFA/M-COPA targets at least ARF1, ARF4, and ARF5; in other words, RTKs require the simultaneous activation of ARF1, ARF4, and ARF5 for their ER export.
Asunto(s)
Factor 1 de Ribosilacion-ADP , Factores de Ribosilacion-ADP , Brefeldino A , Retículo Endoplásmico , Transporte de Proteínas , Humanos , Factores de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/genética , Retículo Endoplásmico/metabolismo , Factor 1 de Ribosilacion-ADP/metabolismo , Factor 1 de Ribosilacion-ADP/genética , Brefeldino A/farmacología , Transporte de Proteínas/efectos de los fármacos , Receptores ErbB/metabolismo , Receptores ErbB/genética , Células HeLaRESUMEN
Both constitutive and regulated secretion require cell organelles that are able to store and release the secretory cargo. During development, the larval salivary gland of Drosophila initially produces high amount of glue-containing small immature secretory granules, which then fuse with each other and reach their normal 3-3.5 µm in size. Following the burst of secretion, obsolete glue granules directly fuse with late endosomes or lysosomes by a process called crinophagy, which leads to fast degradation and recycling of the secretory cargo. However, hindering of endosome-to-TGN retrograde transport in these cells causes abnormally small glue granules which are not able to fuse with each other. Here, we show that loss of function of the SNARE genes Syntaxin 16 (Syx16) and Synaptobrevin (Syb), the small GTPase Rab6 and the GARP tethering complex members Vps53 and Scattered (Vps54) all involved in retrograde transport cause intense early degradation of immature glue granules via crinophagy independently of the developmental program. Moreover, silencing of these genes also provokes secretory failure and accelerated crinophagy during larval development. Our results provide a better understanding of the relations among secretion, secretory granule maturation and degradation and paves the way for further investigation of these connections in other metazoans.
Asunto(s)
Drosophila , Vesículas Secretoras , Animales , Larva , Vesículas Secretoras/metabolismo , Aparato de Golgi/metabolismo , Glándulas Salivales/metabolismoRESUMEN
The intracellular trafficking of ß-site amyloid precursor protein (APP) cleaving enzyme (BACE1) and APP regulates amyloid-ß production. Our previous work demonstrated that newly synthesized BACE1 and APP are segregated into distinct trafficking pathways from the trans-Golgi network (TGN), and that alterations in their trafficking lead to an increase in Aß production in non-neuronal and neuronal cells. However, it is not known whether BACE1 and APP are transported through the Golgi stacks together and sorted at the TGN or segregated prior to arrival at the TGN. To address this question, we have used high-resolution Airyscan technology followed by Huygens deconvolution to quantify the overlap of BACE1 and APP in Golgi subcompartments in HeLa cells and primary neurons. Here, we show that APP and BACE1 are segregated, on exit from the endoplasmic reticulum and in the cis-Golgi and throughout the Golgi stack. In contrast, the transferrin receptor, which exits the TGN in AP-1 mediated transport carriers as for BACE1, colocalizes with BACE1, but not APP, throughout the Golgi stack. The segregation of APP and BACE1 is independent of the Golgi ribbon structure and the cytoplasmic domain of the cargo. Overall, our findings reveal the segregation of different membrane cargoes early in the secretory pathway, a finding relevant to the regulation of APP processing events.
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Enfermedad de Alzheimer , Precursor de Proteína beta-Amiloide , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Aparato de Golgi/metabolismo , Células HeLa , Humanos , Transporte de Proteínas/fisiologíaRESUMEN
Here, we report that human lactoferrin (hLF), known for its anticancer properties, induced intracellular activation of the Na+/H+ exchanger (NHE) 7 in human lung cancer PC-9 cells. Compared to non-fused hLF, the fusion of human serum albumin (HSA) with hLF (hLF-HSA) facilitated its internalization into PC-9 cells in a caveolae-mediated manner, thereby exhibiting enhanced anti-proliferative effects. Although hLF alone did not exhibit any discernible effects, hLF-HSA resulted in organelle alkalization as detected using an acidotropic pH indicator. hLF-HSA-induced elevation of organelle pH and inhibition of cancer growth were abolished by NHE7 siRNA. hLF-HSA upregulated NHE7. Thus, upon cellular uptake, hLF-HSA triggers proton leakage through the upregulation of NHE7. This process led to organelle alkalization, probably in the trans-Golgi network (TGN) as suggested by the localization of NHE7 in PC-9 cells, thereby suppressing lung cancer cell growth. Forcing the cellular uptake of hLF alone using a caveolae-mediated endocytosis activator led to an increase in organelle pH. Furthermore, cell entry of hLF also activated proton-loading NHE7, leading to organelle acidification in the pancreatic cancer cell line MIA PaCa-2. Therefore, the intracellularly delivered hLF functions as an activator of NHE7.
Asunto(s)
Lactoferrina , Neoplasias Pulmonares , Intercambiadores de Sodio-Hidrógeno , Humanos , Lactoferrina/metabolismo , Lactoferrina/farmacología , Neoplasias Pulmonares/metabolismo , Protones , Intercambiadores de Sodio-Hidrógeno/metabolismo , Red trans-Golgi/metabolismoRESUMEN
Formation of transport vesicles requires the coordinate activity of the coating machinery that selects cargo into the nascent vesicle and the membrane bending machinery that imparts curvature to the forming bud. Vesicle coating at the trans-Golgi Network (TGN) involves AP1, GGA2 and clathrin, which are recruited to membranes by activated ARF GTPases. The ARF activation at the TGN is mediated by the BIG1 and BIG2 guanine nucleotide exchange factors (GEFs). Membrane deformation at the TGN has been shown to be mediated by lipid flippases, including ATP8A1, that moves phospholipids from the inner to the outer leaflet of the TGN membrane. We probed a possible coupling between the coating and deformation machineries by testing for an interaction between BIG1, BIG2 and ATP8A1, and by assessing whether such an interaction may influence coating efficiency. Herein, we document that BIG1 and BIG2 co-localize with ATP8A1 in both, static and highly mobile TGN elements, and that BIG1 and BIG2 bind ATP8A1. We show that the interaction involves the catalytic Sec7 domain of the GEFs and the cytosolic C-terminal tail of ATP8A1. Moreover, we report that the expression of ATP8A1, but not ATP8A1 lacking the GEF-binding cytosolic tail, increases the generation of activated ARFs at the TGN and increases the selective recruitment of AP1, GGA2 and clathrin to TGN membranes. This occurs without increasing BIG1 or BIG2 levels at the TGN, suggesting that the binding of the ATP8A1 flippase tail to the Sec7 domain of BIG1/BIG2 increases their catalytic activity. Our results support a model in which a flippase component of the deformation machinery impacts the activity of the GEF component of the coating machinery.
Asunto(s)
Factores de Ribosilacion-ADP , Factores de Intercambio de Guanina Nucleótido , Red trans-Golgi , Red trans-Golgi/metabolismo , Humanos , Factores de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Adenosina Trifosfatasas/metabolismo , Células HeLa , Unión Proteica , Proteínas de la Membrana , Proteínas de Transferencia de FosfolípidosRESUMEN
BACKGROUND: Therapeutic drug monitoring is effective for optimizing anti-tumor necrosis factor therapies in inflammatory bowel disease, but for vedolizumab, a gut-selective leucocyte migration inhibitor, data are scarce. METHODS: Observational cohort study including 116 bio-experienced inflammatory bowel disease patients treated with vedolizumab for active luminal disease. Biobanked trough blood samples (n = 676) covering 96% of patients were analyzed using a drug-binding immunofluorometric assay. Steroid-free treatment outcomes were classified by clinical disease activity indices and objective findings, primarily endoscopy. RESULTS: Patients with clinical remission to vedolizumab induction therapy (37%) had significantly higher trough levels than those without at weeks 6 (mean 34.1 vs 28.0 µg/mL, P = 0.03) and 10 (34.8 vs 27.5 µg/mL, P = 0.01). Optimal thresholds for discrimination were 32.4 µg/mL (AUCROC 0.66, P = 0.04) and 23.5 (AUCROC 0.67, P = 0.01), respectively. This positive association persisted during maintenance phase with 11.9 µg/mL (AUCROC 0.69, P < 0.01) associated with clinical remission (37%) and 15.3 (AUCROC 0.74, P < 0.001) for objective remission (46%). Stratification by temporal evolution of treatment effects revealed higher induction and maintenance vedolizumab levels in persistent and slow responders as compared to secondary or persistent failures. Pharmacokinetics was influenced by rare formation of anti-vedolizumab antibodies (2%), and to a lesser extent gender and albumin during induction, but not disease severity, concomitant steroids, or thiopurine metabolites. Switching to subcutaneous administrations resulted in 2.3-fold increase in steady-state trough levels. CONCLUSION: Our study supports maintaining adequate drug exposure being essential for sustained positive outcomes of vedolizumab and emphasizes individualized, therapeutic drug monitoring-based treatment regimens. Controlled trials and pharmacokinetic modeling are, however, needed.
Asunto(s)
Anticuerpos Monoclonales Humanizados , Monitoreo de Drogas , Fármacos Gastrointestinales , Enfermedades Inflamatorias del Intestino , Humanos , Anticuerpos Monoclonales Humanizados/farmacocinética , Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales Humanizados/uso terapéutico , Monitoreo de Drogas/métodos , Masculino , Femenino , Fármacos Gastrointestinales/administración & dosificación , Fármacos Gastrointestinales/farmacocinética , Adulto , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Persona de Mediana Edad , Resultado del Tratamiento , Estudios de Cohortes , Inducción de Remisión , Quimioterapia de InducciónRESUMEN
Cellulose is synthesized at the plasma membrane by cellulose synthase (CESA) complexes (CSCs), which are assembled in the Golgi and secreted to the plasma membrane through the trans-Golgi network (TGN) compartment. However, the molecular mechanisms that guide CSCs through the secretory system and deliver them to the plasma membrane are poorly understood. Here, we identified an uncharacterized gene, TRANVIA (TVA), that is transcriptionally coregulated with the CESA genes required for primary cell wall synthesis. The tva mutant exhibits enhanced sensitivity to cellulose synthesis inhibitors; reduced cellulose content; and defective dynamics, density, and secretion of CSCs to the plasma membrane as compared to wild type. TVA is a plant-specific protein of unknown function that is detected in at least two different intracellular compartments: organelles labeled by markers for the TGN and smaller compartments that deliver CSCs to the plasma membrane. Together, our data suggest that TVA promotes trafficking of CSCs to the plasma membrane by facilitating exit from the TGN and/or interaction of CSC secretory vesicles with the plasma membrane.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Celulosa/metabolismo , Glucosiltransferasas/metabolismo , Aparato de Golgi/metabolismo , Red trans-Golgi/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Citocinesis , Glucosiltransferasas/genética , Microtúbulos , Transporte de ProteínasRESUMEN
The trans-Golgi network/early endosome (TGN/EE) serves as the central hub in which exocytic and endocytic trafficking pathways converge and specificity of cargo routing needs to be achieved. Acidification is a hallmark of the TGN/EE and is maintained by the vacuolar H+-ATPase (V-ATPase) with support of proton-coupled antiporters. We show here that ClCd and ClCf, two distantly related members of the Arabidopsis Cl- channel (ClC) family, colocalize in the TGN/EE, where they act redundantly, and are essential for male gametophyte development. Combining an inducible knockdown approach and in vivo pH measurements, we show here that reduced ClC activity does not affect pH in the TGN/EE but causes hyperacidification of trans-Golgi cisternae. Taken together, our results show that ClC-mediated anion transport into the TGN/EE is essential and affects spatiotemporal aspects of TGN/EE maturation as well as its functional separation from the Golgi stack.
Asunto(s)
Proteínas de Arabidopsis , Red trans-Golgi , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Endosomas/metabolismo , Fluoresceínas , Concentración de Iones de Hidrógeno , Transporte de Proteínas , Red trans-Golgi/metabolismoRESUMEN
Previous analysis of postentry events revealed that human cytomegalovirus (HCMV) displays a unique, extended nuclear translocation pattern in monocytes. We determined that c-Src signaling through pentamer engagement of integrins is required upon HCMV entry to avoid sorting of the virus into late endosomes and subsequent degradation. To follow up on this previous study, we designed experiments to investigate how HCMV-induced signaling through the other major axis-the epidermal growth factor receptor (EGFR) kinase-regulates viral postentry events. Here we show that HCMV induces chronic and functional EGFR signaling that is distinct to the virus as compared to the natural EGFR ligand: EGF. This chronic EGFR kinase activity in infected monocytes is required for the proper subcellular localization of the viral particle during trafficking events, as well as for promoting translocation of viral DNA into the host nucleus. Our data indicate that HCMV glycoprotein B (gB) binds to EGFR at the monocyte surface, the virus and EGFR are internalized together, and gB remains bound to EGFR throughout viral postentry events until de-envelopment to promote the chronic EGFR kinase activity required for viral trafficking and nuclear translocation. These data highlight how initial EGFR signaling via viral binding is necessary for entry, but not sufficient to promote each viral trafficking event. HCMV appears to manipulate the EGFR kinase postentry, via gB-EGFR interaction, to be active at the critical points throughout the trafficking process that leads to nuclear translocation and productive infection of peripheral blood monocytes.
Asunto(s)
Núcleo Celular/metabolismo , Citomegalovirus/fisiología , Monocitos/virología , Proteínas del Envoltorio Viral/metabolismo , Núcleo Celular/virología , Células Cultivadas , ADN Viral/metabolismo , Endosomas/metabolismo , Endosomas/virología , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Interacciones Huésped-Patógeno , Humanos , Monocitos/metabolismo , Unión Proteica , Transducción de Señal , Red trans-Golgi/metabolismo , Red trans-Golgi/virologíaRESUMEN
In eukaryotic cells, protein sorting is a highly regulated mechanism important for many physiological events. After synthesis in the endoplasmic reticulum and trafficking to the Golgi apparatus, proteins sort to many different cellular destinations including the endolysosomal system and the extracellular space. Secreted proteins need to be delivered directly to the cell surface. Sorting of secreted proteins from the Golgi apparatus has been a topic of interest for over thirty years, yet there is still no clear understanding of the machinery that forms the post-Golgi carriers. Most evidence points to these post-Golgi carriers being tubular pleomorphic structures that bud from the trans-face of the Golgi. In this review, we present the background studies and highlight the key components of this pathway, we then discuss the machinery implicated in the formation of these carriers, their translocation across the cytosol, and their fusion at the plasma membrane.
Asunto(s)
Membrana Celular/metabolismo , Aparato de Golgi/metabolismo , Animales , Humanos , Metabolismo de los Lípidos , Fusión de Membrana , Transporte de Proteínas , Vías SecretorasRESUMEN
Historically, the trans-Golgi network (TGN) has been recognized as a sorting center of newly synthesized proteins, whereas the recycling endosome (RE) is a compartment where endocytosed materials transit before being recycled to the plasma membrane. However, recent findings revealed that both the TGN and RE connect endocytosis and exocytosis and, thus, are functionally overlapping. Here we report, in both Drosophila and microtubule-disrupted HeLa cells, that REs are interconvertible between two distinct states, namely Golgi-associated REs and free REs. Detachment and reattachment of REs and Golgi stacks are often observed, and newly synthesized glycosylphosphatidylinositol-anchored cargo protein but not vesicular stomatitis virus G protein is transported through these two types of RE. In plants, there are two types of TGN - Golgi-associated TGN and Golgi-independent TGN. We show that dynamics of REs in both Drosophila and mammalian cells are very similar compared with those of plant TGNs. And, together with the similarity on the molecular level, our results indicate that fly and mammalian REs are organelles that are equivalent to TGNs in plants. This suggests that the identities and functional relationships between REs and TGNs should be reconsidered.
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Drosophila , Aparato de Golgi , Animales , Endosomas/metabolismo , Aparato de Golgi/metabolismo , Células HeLa , Humanos , Transporte de Proteínas , Red trans-Golgi/metabolismoRESUMEN
Activator of G-protein signaling 3 (AGS3, also known as GPSM1) regulates the trans-Golgi network. The AGS3 GoLoco motif binds to Gαi and thereby regulates the transport of proteins to the plasma membrane. Compaction of early embryos is based on the accumulation of E-cadherin (Cdh1) at cell-contacted membranes. However, how AGS3 regulates the transport of Cdh1 to the plasma membrane remains undetermined. To investigate this, AGS3 was knocked out using the Cas9-sgRNA system. Both trans-Golgi network protein 46 (TGN46, also known as TGOLN2) and transmembrane p24-trafficking protein 7 (TMED7) were tracked in early mouse embryos by tagging these proteins with a fluorescent protein label. We observed that the majority of the AGS3-edited embryos were developmentally arrested and were fragmented after the four-cell stage, exhibiting decreased accumulation of Cdh1 at the membrane. The trans-Golgi network and TMED7-positive vesicles were also dispersed and were not polarized near the membrane. Additionally, increased Gαi1 (encoded by GNAI1) expression could rescue AGS3-overexpressed embryos. In conclusion, AGS3 reinforces the dynamics of the trans-Golgi network and the transport of TMED7-positive cargo containing Cdh1 to the cell-contact surface during early mouse embryo development.
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Inhibidores de Disociación de Guanina Nucleótido/genética , Transporte de Proteínas , Red trans-Golgi , Animales , Membrana Celular/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Proteínas de Unión al GTP/metabolismo , Regulación del Desarrollo de la Expresión Génica , Ratones , Transducción de Señal , Red trans-Golgi/metabolismoRESUMEN
Protein sorting at the trans-Golgi network (TGN) usually requires the assistance of cargo adaptors. However, it remains to be examined how the same complex can mediate both the export and retention of different proteins or how sorting complexes interact among themselves. In Saccharomyces cerevisiae, the exomer complex is involved in the polarized transport of some proteins from the TGN to the plasma membrane (PM). Intriguingly, exomer and its cargos also show a sort of functional relationship with TGN clathrin adaptors that is still unsolved. Here, using a wide range of techniques, including time-lapse and BIFC microscopy, we describe new molecular implications of the exomer complex in protein sorting and address its different layers of functional interaction with clathrin adaptor complexes. Exomer mutants show impaired amino acid uptake because it facilitates not only the polarized delivery of amino acid permeases to the PM but also participates in their endosomal traffic. We propose a model for exomer where it modulates the recruitment of TGN clathrin adaptors directly or indirectly through the Arf1 function. Moreover, we describe an in vivo competitive relationship between the exomer and AP-1 complexes for the model cargo Chs3. These results highlight a broad role for exomer in regulating protein sorting at the TGN that is complementary to its role as cargo adaptor and present a model to understand the complexity of TGN protein sorting.
Asunto(s)
Factor 1 de Ribosilacion-ADP/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Quitina Sintasa/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Red trans-Golgi/metabolismo , Membrana Celular/metabolismo , Endosomas/metabolismo , Transporte de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
Aquaporin is a membrane channel protein widely expressed in body tissues, which can control the input and output of water in cells. AQPs are differentially expressed in different cardiovascular tissues and participate in water transmembrane transport, cell migration, metabolism, inflammatory response, etc. The aberrant expression of AQPs highly correlates with the onset of ischemic heart disease, myocardial ischemia-reperfusion injury, heart failure, etc. Despite much attention to the regulatory role of AQPs in the cardiovascular system, the translation of AQPs into clinical application still faces many challenges, including clarification of the localization of AQPs in the cardiovascular system and mechanisms mediating cardiovascular pathophysiology, as well as the development of cardiovascular-specific AQPs modulators.Therefore, in this study, we comprehensively reviewed the critical roles of AQP family proteins in maintaining cardiovascular homeostasis and described the underlying mechanisms by which AQPs mediated the outcomes of cardiovascular diseases. Meanwhile, AQPs serve as important therapeutic targets, which provide a wide range of opportunities to investigate the mechanisms of cardiovascular diseases and the treatment of those diseases.
Asunto(s)
Acuaporinas , Enfermedades Cardiovasculares , Acuaporinas/metabolismo , Transporte Biológico , Corazón , Humanos , AguaRESUMEN
AIMS: The effect of the Dutch nationwide adjustment of reducing 6-thioguanine nucleotide (6-TGN) target values (from 600-1200 to 320-630 pmol/8 × 108 red blood cells [RBC]) on toxicity and clinical outcome of thiopurine treatment in patients with inflammatory bowel disease has not yet been established. Therefore, the authors determined the incidence of toxicity-induced discontinuations and efficacy at both target concentrations. METHODS: This retrospective study was performed in inflammatory bowel disease patients treated with azathioprine or mercaptopurine. Two groups were defined: the former target (FT) group with target concentrations of 600-1200 pmol/8 × 108 RBC and the adjusted target (AT) group with target concentrations of 320-630 pmol/8 × 108 RBC. Patients were followed for maximum 52 weeks or until discontinuation of thiopurine therapy. Data were collected from the local hospital electronic health software of Rijnstate Hospital. RESULTS: In total, 151 patients were included, 76 in the FT group and 75 in the AT group. At week 52, 100 out of 151 patients (66%) of the total population discontinued thiopurine therapy. Forty-eight of the discontinuations were due toxicity (48%). The incidence of toxicity induced discontinuations was 35% in the AT group vs. 47% in the FT group (P = .25). No loss of efficacy was seen in the AT group. CONCLUSION: After reduction of the target range, there was a trend towards fewer toxicity-induced discontinuations, albeit not statistically significant. In addition, this study did not find any indication that the reduction of the target range diminished efficacy.