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1.
Biol Pharm Bull ; 47(6): 1136-1143, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38866522

RESUMEN

Ceramide (Cer) is synthesized de novo in the bilayer of the endoplasmic reticulum and transported to the cytosolic leaflet of the trans-Golgi apparatus for sphingomyelin (SM) synthesis. As the active site of SM synthase (SMS) is located on the luminal side of the Golgi membrane, Cer translocates to the lumen via transbilayer movement for SM synthesis. However, the mechanism of transbilayer movement is not fully understood. As the Cer-related translocases seem to localize near the SMS, the protein was identified using proximity-dependent biotin identification proteomics. Phospholipid scramblase 1 (PLSCR1), which is thought to act as a scramblase for phosphatidylserine and phosphatidylethanolamine, was identified as a protein proximal to the SMS isoforms SMS1 and SMS2. Although five isoforms of PLSCR have been reported in humans, only PLSCR1, PLSCR3, and PLSCR4 are expressed in HEK293T cells. Confocal microscopic analysis showed that PLSCR1 and PLSCR4 partially co-localized with p230, a trans-Golgi network marker, where SMS isoforms are localized. We established CRISPR/Cas9-mediated PLSCR1, PLSCR3, and PLSCR4 single-knockout cells and PLSCR1, 3, 4 triple knockout HEK293T cells. Liquid chromatography-tandem mass spectrometry revealed that the levels of species with distinct acyl chains in Cer and SM were not significantly different in single knockout cells or in the triple knockout cells compared to the wild-type cells. Our findings suggest that PLSCR1 is localized in the vicinity of SMS isoforms, however is not involved in the transbilayer movement of Cer for SM synthesis.


Asunto(s)
Proteínas de Transferencia de Fosfolípidos , Esfingomielinas , Transferasas (Grupos de Otros Fosfatos Sustitutos) , Humanos , Proteínas de Transferencia de Fosfolípidos/metabolismo , Proteínas de Transferencia de Fosfolípidos/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Células HEK293 , Esfingomielinas/metabolismo , Esfingomielinas/biosíntesis , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Isoenzimas/metabolismo , Isoenzimas/genética , Aparato de Golgi/metabolismo , Aparato de Golgi/enzimología
2.
Anal Chem ; 96(23): 9737-9743, 2024 Jun 11.
Artículo en Inglés | MEDLINE | ID: mdl-38825763

RESUMEN

Various signal molecules mediate complex physiological processes collectively in the Golgi. However, most currently accessible probes are questionable in illuminating the functions of these reactive species in Golgi because of the inability to irradiate these probes only at the desired Golgi location, which compromises specificity and accuracy. In this study, we rationally designed the first photocontrollable and Golgi-targeted fluorescent probe to in situ visualize the Golgi alkaline phosphatase (ALP). The designed probe with natural yellow fluorescence can provide access into Golgi and monitor the exact timing of accumulation in Golgi. On-demand photoactivation at only the desired Golgi location affords a significant emission response to ALP with illuminating red fluorescence at 710 nm. Through the photocontrollable fluorescence responsiveness to ALP, precise spatiotemporal recognition of Golgi ALP fluctuations is successfully performed. With this probe, for the first time, we revealed the Golgi ALP levels during cisplatin-induced acute kidney injury (AKI), which will further facilitate and complement the comprehensive exploration of ALP kinetics during physiological and pathological processes.


Asunto(s)
Fosfatasa Alcalina , Colorantes Fluorescentes , Aparato de Golgi , Aparato de Golgi/metabolismo , Fosfatasa Alcalina/metabolismo , Humanos , Animales , Colorantes Fluorescentes/química , Células HeLa , Ratones , Cisplatino/farmacología
3.
FASEB J ; 38(13): e23739, 2024 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-38884157

RESUMEN

Arf6 is a member of ADP-ribosylation factor (Arf) family, which is widely implicated in the regulation of multiple physiological processes including endocytic recycling, cytoskeletal organization, and membrane trafficking during mitosis. In this study, we investigated the potential relationship between Arf6 and aging-related oocyte quality, and its roles on organelle rearrangement and cytoskeleton dynamics in porcine oocytes. Arf6 expressed in porcine oocytes throughout meiotic maturation, and it decreased in aged oocytes. Disruption of Arf6 led to the failure of cumulus expansion and polar body extrusion. Further analysis indicated that Arf6 modulated ac-tubulin for meiotic spindle organization and microtubule stability. Besides, Arf6 regulated cofilin phosphorylation and fascin for actin assembly, which further affected spindle migration, indicating the roles of Arf6 on cytoskeleton dynamics. Moreover, the lack of Arf6 activity caused the dysfunction of Golgi and ER for protein synthesis and signal transduction. Mitochondrial dysfunction was also observed in Arf6-deficient porcine oocytes, which was supported by the increased ROS level and abnormal membrane potential. In conclusion, our results reported that insufficient Arf6 was related to aging-induced oocyte quality decline through spindle organization, actin assembly, and organelle rearrangement in porcine oocytes.


Asunto(s)
Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP , Oocitos , Animales , Oocitos/metabolismo , Factores de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/genética , Porcinos , Femenino , Meiosis/fisiología , Huso Acromático/metabolismo , Envejecimiento/metabolismo , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo
4.
PLoS One ; 19(6): e0304001, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38885274

RESUMEN

The plant BEACH-domain protein SPIRRIG (SPI) is involved in regulating cell morphogenesis and salt stress responses in Arabidopsis thaliana, Arabis alpina, and Marchantia polymorpha and was reported to function in the context of two unrelated cellular processes: vesicular trafficking and P-body mediated RNA metabolism. To further explore the molecular function of SPI, we isolated a second-site mutant, specifically rescuing the spi mutant trichome phenotype. The molecular analysis of the corresponding gene revealed a dominant negative mutation in RABE1C, a ras-related small GTP-binding protein that localizes to Golgi. Taken together, our data identified the genetic interaction between RABE1C and SPI, which is beneficial for further dissecting the function of SPI in vesicle trafficking-associated cell morphogenesis.


Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Mutación , Fenotipo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Aparato de Golgi/metabolismo , Aparato de Golgi/genética , Tricomas/genética
5.
Cell Death Dis ; 15(6): 417, 2024 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-38879509

RESUMEN

Chemotherapy is a crucial treatment for colorectal tumors. However, its efficacy is restricted by chemoresistance. Recently, Golgi dispersal has been suggested to be a potential response to chemotherapy, particularly to drugs that induce DNA damage. However, the underlying mechanisms by which Golgi dispersal enhances the capacity to resist DNA-damaging agents remain unclear. Here, we demonstrated that DNA-damaging agents triggered Golgi dispersal in colorectal cancer (CRC), and cancer stem cells (CSCs) possessed a greater degree of Golgi dispersal compared with differentiated cancer cells (non-CSCs). We further revealed that Golgi dispersal conferred resistance against the lethal effects of DNA-damaging agents. Momentously, Golgi dispersal activated the Golgi stress response via the PKCα/GSK3α/TFE3 axis, resulting in enhanced protein and vesicle trafficking, which facilitated drug efflux through ABCG2. Identification of Golgi dispersal indicated an unexpected pathway regulating chemoresistance in CRC.


Asunto(s)
Neoplasias Colorrectales , Resistencia a Antineoplásicos , Aparato de Golgi , Células Madre Neoplásicas , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Humanos , Aparato de Golgi/metabolismo , Aparato de Golgi/efectos de los fármacos , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Animales , Línea Celular Tumoral , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Daño del ADN , Ratones , Ratones Desnudos , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/genética , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico
6.
Elife ; 122024 Jun 05.
Artículo en Inglés | MEDLINE | ID: mdl-38837189

RESUMEN

The organelles of eukaryotic cells maintain distinct protein and lipid compositions required for their specific functions. The mechanisms by which many of these components are sorted to their specific locations remain unknown. While some motifs mediating subcellular protein localization have been identified, many membrane proteins and most membrane lipids lack known sorting determinants. A putative mechanism for sorting of membrane components is based on membrane domains known as lipid rafts, which are laterally segregated nanoscopic assemblies of specific lipids and proteins. To assess the role of such domains in the secretory pathway, we applied a robust tool for synchronized secretory protein traffic (RUSH, Retention Using Selective Hooks) to protein constructs with defined affinity for raft phases. These constructs consist solely of single-pass transmembrane domains (TMDs) and, lacking other sorting determinants, constitute probes for membrane domain-mediated trafficking. We find that while raft affinity can be sufficient for steady-state PM localization, it is not sufficient for rapid exit from the endoplasmic reticulum (ER), which is instead mediated by a short cytosolic peptide motif. In contrast, we find that Golgi exit kinetics are highly dependent on raft affinity, with raft preferring probes exiting the Golgi ~2.5-fold faster than probes with minimal raft affinity. We rationalize these observations with a kinetic model of secretory trafficking, wherein Golgi export can be facilitated by protein association with raft domains. These observations support a role for raft-like membrane domains in the secretory pathway and establish an experimental paradigm for dissecting its underlying machinery.


Asunto(s)
Retículo Endoplásmico , Aparato de Golgi , Microdominios de Membrana , Transporte de Proteínas , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Microdominios de Membrana/metabolismo , Vías Secretoras , Humanos , Cinética , Membrana Celular/metabolismo , Proteínas de la Membrana/metabolismo , Células HeLa
7.
Bioorg Chem ; 148: 107476, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38788368

RESUMEN

Depression is a debilitating mental illness that poses a serious threat to human health. Nitric Oxide (NO), as an important gasotransmitter, is closely associated with the pathogenesis of depressive disorders. Effective monitoring of NO fluctuation is beneficial for the diagnosis of depression and therapy assessment of antidepressants. Currently, there is a lack of effective methods for rapidly and sensitively identifying NO and elucidating its relationship with depression diseases. Herein, we developed a NIR dye TJ730-based fluorescent probe TJ730-Golgi-NO incorporating benzenesulfonamide as a Golgi-targeted moiety and the thiosemicarbazide group for NO detection. The probe exhibited turn-on fluorescence ability and a large Stokes shift of 158 nm, which shows high sensitivity, selectivity, and rapid response (<1 min) for NO detection. TJ730-Golgi-NO could detect exogenous and endogenous NO in cells stimulated by Glu and LPS, and target Golgi apparatus. Moreover, we disclose a significant increase of NO in the depression model and a weak fluorescence evidenced in the fluoxetine-treated depression mice. This study provides a competent tool for studying the function of NO and helping improve the effective treatment of depression diseases.


Asunto(s)
Depresión , Colorantes Fluorescentes , Aparato de Golgi , Óxido Nítrico , Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/farmacología , Animales , Óxido Nítrico/metabolismo , Óxido Nítrico/análisis , Ratones , Aparato de Golgi/metabolismo , Depresión/tratamiento farmacológico , Estructura Molecular , Humanos , Modelos Animales de Enfermedad , Masculino , Relación Estructura-Actividad , Rayos Infrarrojos , Relación Dosis-Respuesta a Droga , Imagen Óptica , Células RAW 264.7
8.
Biotechnol J ; 19(5): e2400098, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38797728

RESUMEN

Human carboxypeptidase B1 (hCPB1) is vital for recombinant insulin production, holding substantial value in the pharmaceutical industry. Current challenges include limited hCPB1 enzyme activity. In this study, recombinant hCPB1 efficient expression in Pichia pastoris was achieved. To enhance hCPB1 secretion, we conducted signal peptides screening and deleted the Vps10 sortilin domain, reducing vacuolar mis-sorting. Overexpression of Sec4p increased the fusion of secretory vesicles with the plasma membrane and improved hCPB1 secretion by 20%. Rational protein engineering generated twenty-two single-mutation mutants and identified the A178L mutation resulted in a 30% increase in hCPB1 specific activity. However, all combinational mutations that increased specific activities decreased protein expression levels. Therefore, computer-aided global protein design with PROSS was employed for the aim of improving specific activities and preserving good protein expression. Among the six designed mutants, hCPB1-P6 showed a remarkable 114% increase in the catalytic rate constant (kcat), a 137% decrease in the Michaelis constant (Km), and a 490% increase in catalytic efficiency. Most mutations occurred on the surface of hCPB1-P6, with eight sites mutated to proline. In a 5 L fermenter, hCPB1-P6 was produced by the secretion-enhanced P. pastoris chassis to 199.6 ± 20 mg L-1 with a specific activity of 96 ± 0.32 U mg-1, resulting in a total enzyme activity of 19137 ± 1131 U L-1, demonstrating significant potential for industrial applications.


Asunto(s)
Carboxipeptidasa B , Membrana Celular , Aparato de Golgi , Ingeniería de Proteínas , Proteínas Recombinantes , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ingeniería de Proteínas/métodos , Carboxipeptidasa B/genética , Carboxipeptidasa B/metabolismo , Membrana Celular/metabolismo , Membrana Celular/genética , Aparato de Golgi/metabolismo , Aparato de Golgi/genética , Aparato de Golgi/enzimología , Saccharomycetales/genética , Saccharomycetales/enzimología , Mutación , Pichia/genética , Pichia/metabolismo , Señales de Clasificación de Proteína/genética , Transporte de Proteínas
9.
Nat Commun ; 15(1): 4514, 2024 May 27.
Artículo en Inglés | MEDLINE | ID: mdl-38802491

RESUMEN

Knowledge on the distribution and dynamics of glycosylation enzymes in the Golgi is essential for better understanding this modification. Here, using a combination of CRISPR/Cas9 knockin technology and super-resolution microscopy, we show that the Golgi complex is assembled by a number of small 'Golgi units' that have 1-3 µm in diameter. Each Golgi unit contains small domains of glycosylation enzymes which we call 'zones'. The zones of N- and O-glycosylation enzymes are colocalised. However, they are less colocalised with the zones of a glycosaminoglycan synthesizing enzyme. Golgi units change shapes dynamically and the zones of glycosylation enzymes rapidly move near the rim of the unit. Photobleaching analysis indicates that a glycosaminoglycan synthesizing enzyme moves between units. Depletion of giantin dissociates units and prevents the movement of glycosaminoglycan synthesizing enzymes, which leads to insufficient glycosaminoglycan synthesis. Thus, we show the structure-function relationship of the Golgi and its implications in human pathogenesis.


Asunto(s)
Glicosaminoglicanos , Aparato de Golgi , Aparato de Golgi/metabolismo , Glicosilación , Humanos , Glicosaminoglicanos/metabolismo , Células HeLa , Sistemas CRISPR-Cas , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Matriz de Golgi
10.
Life Sci Alliance ; 7(8)2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-38782601

RESUMEN

Complexes of ERLIN1 and ERLIN2 (ER lipid raft-associated 1 and 2) form large ring-like cup-shaped structures on the endoplasmic reticulum (ER) membrane and serve as platforms to bind cholesterol and E3 ubiquitin ligases, potentially defining functional nanodomains. Here, we show that ERLIN scaffolds mediate the interaction between the full-length isoform of TMUB1 (transmembrane and ubiquitin-like domain-containing 1) and RNF170 (RING finger protein 170). We identify a luminal N-terminal conserved region in TMUB1 and RNF170, which is required for this interaction. Three-dimensional modelling shows that this conserved motif binds the stomatin/prohibitin/flotillin/HflKC domain of two adjacent ERLIN subunits at different interfaces. Protein variants that preclude these interactions have been previously linked to hereditary spastic paraplegia. Using omics-based approaches in combination with phenotypic characterization of HeLa cells lacking both ERLINs, we demonstrate a role of ERLIN scaffolds in limiting cholesterol esterification, thereby favouring cholesterol transport from the ER to the Golgi apparatus and regulating Golgi morphology and the secretory pathway.


Asunto(s)
Colesterol , Retículo Endoplásmico , Aparato de Golgi , Proteínas de la Membrana , Vías Secretoras , Ubiquitina-Proteína Ligasas , Humanos , Proteínas de la Membrana/metabolismo , Colesterol/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Retículo Endoplásmico/metabolismo , Células HeLa , Aparato de Golgi/metabolismo , Unión Proteica , Proteínas del Tejido Nervioso
11.
Mol Cell Biol ; 44(4): 123-137, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38747374

RESUMEN

SREBP transcription factors are central regulators of lipid metabolism. Their proteolytic activation requires ER to the Golgi translocation and subsequent cleavage by site-1-protease (S1P). Produced as a proprotein, S1P undergoes autocatalytic cleavage from its precursor S1PA to mature S1PC form. Here, we report that SPRING (previously C12ORF29) and S1P interact through their ectodomains, and that this facilitates the autocatalytic cleavage of S1PA into its mature S1PC form. Reciprocally, we identified a S1P recognition-motif in SPRING and demonstrate that S1P-mediated cleavage leads to secretion of the SPRING ectodomain in cells, and in liver-specific Spring knockout (LKO) mice transduced with AAV-mSpring. By reconstituting SPRING variants into SPRINGKO cells we show that the SPRING ectodomain supports proteolytic maturation of S1P and SREBP signaling, but that S1P-mediated SPRING cleavage is not essential for these processes. Absence of SPRING modestly diminishes proteolytic maturation of S1PA→C and trafficking of S1PC to the Golgi. However, despite reaching the Golgi in SPRINGKO cells, S1PC fails to rescue SREBP signaling. Remarkably, whereas SREBP signaling was severely attenuated in SPRINGKO cells and LKO mice, that of ATF6, another S1P substrate, was unaffected in these models. Collectively, our study positions SPRING as a dedicated licensing factor for SREBP-specific activation by S1P.


Asunto(s)
Aparato de Golgi , Ratones Noqueados , Proproteína Convertasas , Animales , Ratones , Aparato de Golgi/metabolismo , Humanos , Proproteína Convertasas/metabolismo , Proproteína Convertasas/genética , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , Transducción de Señal , Células HEK293 , Hígado/metabolismo , Proteolisis , Retículo Endoplásmico/metabolismo , Proteínas de Unión a los Elementos Reguladores de Esteroles/metabolismo , Proteínas de Unión a los Elementos Reguladores de Esteroles/genética
12.
Commun Biol ; 7(1): 596, 2024 May 18.
Artículo en Inglés | MEDLINE | ID: mdl-38762629

RESUMEN

Apicomplexan parasites harbor a complex endomembrane system as well as unique secretory organelles. These complex cellular structures require an elaborate vesicle trafficking system, which includes Rab GTPases and their regulators, to assure the biogenesis and secretory of the organelles. Here we exploit the model apicomplexan organism Toxoplasma gondii that encodes a family of Rab GTPase Activating Proteins, TBC (Tre-2/Bub2/Cdc16) domain-containing proteins. Functional profiling of these proteins in tachyzoites reveals that TBC9 is the only essential regulator, which is localized to the endoplasmic reticulum (ER) in T. gondii strains. Detailed analyses demonstrate that TBC9 is required for normal distribution of proteins targeting to the ER, and the Golgi apparatus in the parasite, as well as for the normal formation of daughter inner membrane complexes (IMCs). Pull-down assays show a strong protein interaction between TBC9 and specific Rab GTPases (Rab11A, Rab11B, and Rab2), supporting the role of TBC9 in daughter IMC formation and early vesicular transport. Thus, this study identifies the only essential TBC domain-containing protein TBC9 that regulates early vesicular transport and IMC formation in T. gondii and potentially in closely related protists.


Asunto(s)
Retículo Endoplásmico , Proteínas Activadoras de GTPasa , Proteínas Protozoarias , Toxoplasma , Proteínas de Unión al GTP rab , Toxoplasma/metabolismo , Toxoplasma/genética , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Retículo Endoplásmico/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas Activadoras de GTPasa/metabolismo , Proteínas Activadoras de GTPasa/genética , Aparato de Golgi/metabolismo , Transporte de Proteínas , Animales , Vesículas Transportadoras/metabolismo
13.
Sci Rep ; 14(1): 10160, 2024 05 03.
Artículo en Inglés | MEDLINE | ID: mdl-38698045

RESUMEN

How information flow is coordinated for managing transit of 1/3 of the genome through endomembrane pathways by the coat complex II (COPII) system in response to human variation remains an enigma. By examining the interactome of the COPII cage-assembly component Sec13, we show that it is simultaneously associated with multiple protein complexes that facilitate different features of a continuous program of chromatin organization, transcription, translation, trafficking, and degradation steps that are differentially sensitive to Sec13 levels. For the trafficking step, and unlike other COPII components, reduction of Sec13 expression decreased the ubiquitination and degradation of wild-type (WT) and F508del variant cargo protein cystic fibrosis transmembrane conductance regulator (CFTR) leading to a striking increase in fold stability suggesting that the events differentiating export from degradation are critically dependent on COPII cage assembly at the ER Golgi intermediate compartment (ERGIC) associated recycling and degradation step linked to COPI exchange. Given Sec13's multiple roles in protein complex assemblies that change in response to its expression, we suggest that Sec13 serves as an unanticipated master regulator coordinating information flow from the genome to the proteome to facilitate spatial covariant features initiating and maintaining design and function of membrane architecture in response to human variation.


Asunto(s)
Vesículas Cubiertas por Proteínas de Revestimiento , Proteínas Portadoras , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Transporte de Proteínas , Proteínas de Transporte Vesicular , Humanos , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Aparato de Golgi/metabolismo , Retículo Endoplásmico/metabolismo , Ubiquitinación , Proteolisis
14.
Nat Commun ; 15(1): 4469, 2024 May 25.
Artículo en Inglés | MEDLINE | ID: mdl-38796472

RESUMEN

To facilitate inter-tissue communication and the exchange of proteins, lipoproteins, and metabolites with the circulation, hepatocytes have an intricate and efficient intracellular trafficking system regulated by small Rab GTPases. Here, we show that Rab30 is induced in the mouse liver by fasting, which is amplified in liver-specific carnitine palmitoyltransferase 2 knockout mice (Cpt2L-/-) lacking the ability to oxidize fatty acids, in a Pparα-dependent manner. Live-cell super-resolution imaging and in vivo proximity labeling demonstrates that Rab30-marked vesicles are highly dynamic and interact with proteins throughout the secretory pathway. Rab30 whole-body, liver-specific, and Rab30; Cpt2 liver-specific double knockout (DKO) mice are viable with intact Golgi ultrastructure, although Rab30 deficiency in DKO mice suppresses the serum dyslipidemia observed in Cpt2L-/- mice. Corresponding with decreased serum triglyceride and cholesterol levels, DKO mice exhibit decreased circulating but not hepatic ApoA4 protein, indicative of a trafficking defect. Together, these data suggest a role for Rab30 in the selective sorting of lipoproteins to influence hepatocyte and circulating triglyceride levels, particularly during times of excessive lipid burden.


Asunto(s)
Carnitina O-Palmitoiltransferasa , Ayuno , Hepatocitos , Homeostasis , Metabolismo de los Lípidos , Hígado , Ratones Noqueados , Proteínas de Unión al GTP rab , Animales , Masculino , Ratones , Carnitina O-Palmitoiltransferasa/metabolismo , Carnitina O-Palmitoiltransferasa/genética , Colesterol/metabolismo , Ayuno/metabolismo , Aparato de Golgi/metabolismo , Hepatocitos/metabolismo , Hígado/metabolismo , Ratones Endogámicos C57BL , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genética , Triglicéridos/metabolismo , Triglicéridos/sangre
15.
Biomed Pharmacother ; 175: 116646, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38692058

RESUMEN

The Golgi apparatus plays a crucial role in mediating the modification, transport, and sorting of intracellular proteins and lipids. The morphological changes occurring in the Golgi apparatus are exceptionally important for maintaining its function. When exposed to external pressure or environmental stimulation, the Golgi apparatus undergoes adaptive changes in both structure and function, which are known as Golgi stress. Although certain signal pathway responses or post-translational modifications have been observed following Golgi stress, further research is needed to comprehensively summarize and understand the related mechanisms. Currently, there is evidence linking Golgi stress to neurodegenerative diseases; however, the role of Golgi stress in the progression of neurodegenerative diseases such as Alzheimer's disease remains largely unexplored. This review focuses on the structural and functional alterations of the Golgi apparatus during stress, elucidating potential mechanisms underlying the involvement of Golgi stress in regulating immunity, autophagy, and metabolic processes. Additionally, it highlights the pivotal role of Golgi stress as an early signaling event implicated in the pathogenesis and progression of neurodegenerative diseases. Furthermore, this study summarizes prospective targets that can be therapeutically exploited to mitigate neurodegenerative diseases by targeting Golgi stress. These findings provide a theoretical foundation for identifying novel breakthroughs in preventing and treating neurodegenerative diseases.


Asunto(s)
Aparato de Golgi , Enfermedades Neurodegenerativas , Humanos , Aparato de Golgi/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Animales , Transducción de Señal , Autofagia/fisiología , Estrés Fisiológico/fisiología
16.
J Cell Sci ; 137(10)2024 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-38770683

RESUMEN

Membrane trafficking, a fundamental cellular process encompassing the transport of molecules to specific organelles, endocytosis at the plasma membrane and protein secretion, is crucial for cellular homeostasis and signalling. Cancer cells adapt membrane trafficking to enhance their survival and metabolism, and understanding these adaptations is vital for improving patient responses to therapy and identifying therapeutic targets. In this Review, we provide a concise overview of major membrane trafficking pathways and detail adaptations in these pathways, including COPII-dependent endoplasmic reticulum (ER)-to-Golgi vesicle trafficking, COPI-dependent retrograde Golgi-to-ER trafficking and endocytosis, that have been found in cancer. We explore how these adaptations confer growth advantages or resistance to cell death and conclude by discussing the potential for utilising this knowledge in developing new treatment strategies and overcoming drug resistance for cancer patients.


Asunto(s)
Carcinogénesis , Membrana Celular , Neoplasias , Humanos , Neoplasias/metabolismo , Neoplasias/patología , Carcinogénesis/metabolismo , Carcinogénesis/patología , Animales , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Endocitosis , Transporte de Proteínas , Aparato de Golgi/metabolismo
17.
Biophys J ; 123(12): 1531-1541, 2024 Jun 18.
Artículo en Inglés | MEDLINE | ID: mdl-38698644

RESUMEN

The emergence of phase separation phenomena among macromolecules has identified biomolecular condensates as fundamental cellular organizers. These condensates concentrate specific components and accelerate biochemical reactions without relying on membrane boundaries. Although extensive studies have revealed a large variety of nuclear and cytosolic membraneless organelles, we are witnessing a surge in the exploration of protein condensates associated with the membranes of the secretory pathway, such as the endoplasmic reticulum and the Golgi apparatus. This review focuses on protein condensates in the secretory pathway and discusses their impact on the organization and functions of this cellular process. Moreover, we explore the modes of condensate-membrane association and the biophysical and cellular consequences of protein condensate interactions with secretory pathway membranes.


Asunto(s)
Vías Secretoras , Humanos , Animales , Condensados Biomoleculares/metabolismo , Condensados Biomoleculares/química , Aparato de Golgi/metabolismo , Fenómenos Biofísicos , Retículo Endoplásmico/metabolismo
18.
Curr Opin Cell Biol ; 88: 102365, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38705050

RESUMEN

Vesicle transport at the Golgi apparatus is a well-described process, and the major protein components involved have been identified. This includes the coat proteins that function in cargo sorting and vesicle formation, and the proteins that mediate the downstream events of vesicle tethering and membrane fusion. However, despite this knowledge, there remain significant gaps in our mechanistic understanding of these processes which includes how they are coordinated in space and time. In this review we discuss recent advances that have provided new insights into the mechanisms of Golgi trafficking, focussing on vesicle formation and cargo sorting, and vesicle tethering and fusion. These studies point to a high degree of spatial organisation of trafficking components at the Golgi and indicate an inherent plasticity of trafficking. Going forward, further advancements in technology and more sophisticated functional assays are expected to yield greater understanding of the mechanisms that govern Golgi trafficking events.


Asunto(s)
Aparato de Golgi , Aparato de Golgi/metabolismo , Humanos , Animales , Transporte de Proteínas , Transporte Biológico , Vesículas Transportadoras/metabolismo , Fusión de Membrana
19.
EMBO Rep ; 25(6): 2610-2634, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38698221

RESUMEN

GABAB receptors (GBRs), the G protein-coupled receptors for GABA, regulate synaptic transmission throughout the brain. A main synaptic function of GBRs is the gating of Cav2.2-type Ca2+ channels. However, the cellular compartment where stable GBR/Cav2.2 signaling complexes form remains unknown. In this study, we demonstrate that the vesicular protein synaptotagmin-11 (Syt11) binds to both the auxiliary GBR subunit KCTD16 and Cav2.2 channels. Through these dual interactions, Syt11 recruits GBRs and Cav2.2 channels to post-Golgi vesicles, thus facilitating assembly of GBR/Cav2.2 signaling complexes. In addition, Syt11 stabilizes GBRs and Cav2.2 channels at the neuronal plasma membrane by inhibiting constitutive internalization. Neurons of Syt11 knockout mice exhibit deficits in presynaptic GBRs and Cav2.2 channels, reduced neurotransmitter release, and decreased GBR-mediated presynaptic inhibition, highlighting the critical role of Syt11 in the assembly and stable expression of GBR/Cav2.2 complexes. These findings support that Syt11 acts as a vesicular scaffold protein, aiding in the assembly of signaling complexes from low-abundance components within transport vesicles. This mechanism enables insertion of pre-assembled functional signaling units into the synaptic membrane.


Asunto(s)
Ratones Noqueados , Transducción de Señal , Sinaptotagminas , Animales , Sinaptotagminas/metabolismo , Sinaptotagminas/genética , Ratones , Humanos , Neuronas/metabolismo , Transmisión Sináptica , Receptores de GABA-B/metabolismo , Receptores de GABA-B/genética , Terminales Presinápticos/metabolismo , Canales de Calcio Tipo N/metabolismo , Canales de Calcio Tipo N/genética , Aparato de Golgi/metabolismo , Unión Proteica , Células HEK293
20.
Nat Commun ; 15(1): 3978, 2024 May 10.
Artículo en Inglés | MEDLINE | ID: mdl-38729926

RESUMEN

A key mechanism employed by plants to adapt to salinity stress involves maintaining ion homeostasis via the actions of ion transporters. While the function of cation transporters in maintaining ion homeostasis in plants has been extensively studied, little is known about the roles of their anion counterparts in this process. Here, we describe a mechanism of salt adaptation in plants. We characterized the chloride channel (CLC) gene AtCLCf, whose expression is regulated by WRKY transcription factor under salt stress in Arabidopsis thaliana. Loss-of-function atclcf seedlings show increased sensitivity to salt, whereas AtCLCf overexpression confers enhanced resistance to salt stress. Salt stress induces the translocation of GFP-AtCLCf fusion protein to the plasma membrane (PM). Blocking AtCLCf translocation using the exocytosis inhibitor brefeldin-A or mutating the small GTPase gene AtRABA1b/BEX5 (RAS GENES FROM RAT BRAINA1b homolog) increases salt sensitivity in plants. Electrophysiology and liposome-based assays confirm the Cl-/H+ antiport function of AtCLCf. Therefore, we have uncovered a mechanism of plant adaptation to salt stress involving the NaCl-induced translocation of AtCLCf to the PM, thus facilitating Cl- removal at the roots, and increasing the plant's salinity tolerance.


Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Membrana Celular , Canales de Cloruro , Aparato de Golgi , Estrés Salino , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/fisiología , Arabidopsis/efectos de los fármacos , Membrana Celular/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Aparato de Golgi/metabolismo , Canales de Cloruro/metabolismo , Canales de Cloruro/genética , Regulación de la Expresión Génica de las Plantas , Transporte de Proteínas/efectos de los fármacos , Tolerancia a la Sal/genética , Cloruro de Sodio/farmacología , Plantas Modificadas Genéticamente
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