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Regulation of microtubule dynamics is required to properly control various steps of neurodevelopment. In this study, we identified granule cell antiserum-positive 14 (Gcap14) as a microtubule plus-end-tracking protein and as a regulator of microtubule dynamics during neurodevelopment. Gcap14 knockout mice exhibited impaired cortical lamination. Gcap14 deficiency resulted in defective neuronal migration. Moreover, nuclear distribution element nudE-like 1 (Ndel1), an interacting partner of Gcap14, effectively corrected the downregulation of microtubule dynamics and the defects in neuronal migration caused by Gcap14 deficiency. Finally, we found that the Gcap14-Ndel1 complex participates in the functional link between microtubule and actin filament, thereby regulating their crosstalks in the growth cones of cortical neurons. Taken together, we propose that the Gcap14-Ndel1 complex is fundamental for cytoskeletal remodeling during neurodevelopmental processes such as neuronal processes elongation and neuronal migration.
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Actinas , Proteínas Associadas aos Microtúbulos , Neurônios , Animais , Camundongos , Actinas/metabolismo , Movimento Celular/fisiologia , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neuritos/metabolismo , Neurônios/metabolismoRESUMO
The ER regulates the spatiotemporal organization of endolysosomal systems by membrane contact. In addition to tethering via heterotypic interactions on both organelles, we present a novel ER-endosome tethering mechanism mediated by homotypic interactions. The single-pass transmembrane protein SCOTIN is detected in the membrane of the ER and endosomes. In SCOTIN-knockout (KO) cells, the ER-late endosome contacts are reduced, and the perinuclear positioning of endosomes is disturbed. The cytosolic proline-rich domain (PRD) of SCOTIN forms homotypic assemblies in vitro and is necessary for ER-endosome membrane tethering in cells. A region of 28 amino acids spanning 150-177 within the SCOTIN PRD is essential to elicit membrane tethering and endosomal dynamics, as verified by reconstitution in SCOTIN-KO cells. The assembly of SCOTIN (PRD) is sufficient to mediate membrane tethering, as purified SCOTIN (PRD), but not SCOTIN (PRDΔ150-177), brings two different liposomes closer in vitro. Using organelle-specific targeting of a chimeric PRD domain shows that only the presence on both organellar membranes enables the ER-endosome membrane contact, indicating that the assembly of SCOTIN on heterologous membranes mediates organelle tethering.
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Retículo Endoplasmático , Membranas Intracelulares , Membranas Intracelulares/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Endossomos/metabolismoRESUMO
G protein-coupled receptors (GPCRs) play crucial roles in numerous physiological and pathological processes. Mutations in GPCRs that result in loss of function or alterations in signaling can lead to inherited or acquired diseases. Herein, studying prokineticin receptor 2 (PROKR2), we initially identify distinct interactomes for wild-type (WT) versus a mutant (P290S) PROKR2 that causes hypogonadotropic hypogonadism. We then find that both the WT and mutant PROKR2 are targeted for endoplasmic reticulum (ER)-associated degradation, but the mutant is degraded to a greater extent. Further analysis revealed that both forms can also leave the ER to reach the Golgi. However, whereas most of the WT is further transported to the cell surface, most of the mutant is retrieved to the ER. Thus, the post-ER itinerary plays an important role in distinguishing the ultimate fate of the WT versus the mutant. We have further discovered that this post-ER itinerary reduces ER stress induced by the mutant PROKR2. Moreover, we extend the core findings to another model GPCR. Our findings advance the understanding of disease pathogenesis induced by a mutation at a key residue that is conserved across many GPCRs and thus contributes to a fundamental understanding of the diverse mechanisms used by cellular quality control to accommodate misfolded proteins.
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Estresse do Retículo Endoplasmático/fisiologia , Proteostase/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/metabolismo , Animais , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Retículo Endoplasmático/metabolismo , Degradação Associada com o Retículo Endoplasmático , Complexo de Golgi/metabolismo , Células HEK293 , Células HeLa , Humanos , Hipogonadismo/metabolismo , Mutação de Sentido Incorreto/genética , Transporte Proteico/genética , Transporte Proteico/fisiologia , Receptores Acoplados a Proteínas G/genética , Receptores de Peptídeos/genética , Transdução de SinaisRESUMO
Although large-scale genome-wide association studies (GWAS) have identified an association between MAD1L1 (Mitotic Arrest Deficient-1 Like 1) and the pathology of schizophrenia, the molecular mechanisms underlying this association remain unclear. In the present study, we aimed to address these mechanisms by examining the role of MAD1 (the gene product of MAD1L1) in key neurodevelopmental processes in mice and human organoids. Our findings indicated that MAD1 is highly expressed during active cortical development and that MAD1 deficiency leads to impairments in neuronal migration and neurite outgrowth. We also observed that MAD1 is localized to the Golgi apparatus and regulates vesicular trafficking from the Golgi apparatus to the plasma membrane, which is required for the growth and polarity of migrating neurons. In this process, MAD1 physically interacts and collaborates with the kinesin-like protein KIFC3 (kinesin family member C3) to regulate the morphology of the Golgi apparatus and neuronal polarity, thereby ensuring proper neuronal migration and differentiation. Consequently, our findings indicate that MAD1 is an essential regulator of neuronal development and that alterations in MAD1 may underlie schizophrenia pathobiology.
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Neocórtex , Esquizofrenia , Animais , Humanos , Camundongos , Proteínas de Ciclo Celular/genética , Estudo de Associação Genômica Ampla , Cinesinas/genética , Cinesinas/metabolismo , Neocórtex/metabolismo , Neurônios/metabolismo , Esquizofrenia/genética , Esquizofrenia/metabolismoRESUMO
Starvation poses a fundamental challenge to cell survival. Whereas the role of autophagy in promoting energy homeostasis in this setting has been extensively characterized1, other mechanisms are less well understood. Here we reveal that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) inhibits coat protein I (COPI) transport by targeting a GTPase-activating protein (GAP) towards ADP-ribosylation factor 1 (ARF1) to suppress COPI vesicle fission. GAPDH inhibits multiple other transport pathways, also by targeting ARF GAPs. Further characterization suggests that this broad inhibition is activated by the cell during starvation to reduce energy consumption. These findings reveal a remarkable level of coordination among the intracellular transport pathways that underlies a critical mechanism of cellular energy homeostasis.
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Metabolismo Energético , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Homeostase , Adenilato Quinase/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/metabolismo , Animais , Autofagia , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Linhagem Celular , Chlorocebus aethiops , Cricetulus , Fibroblastos , Proteínas Ativadoras de GTPase/antagonistas & inibidores , Proteínas Ativadoras de GTPase/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/química , Humanos , Camundongos , Fosforilação , Ribonucleotídeos/metabolismo , InaniçãoRESUMO
The Golgi complex has a central role in the intracellular sorting of secretory proteins. Anterograde transport through the Golgi has been explained by the movement of Golgi cisternae, known as cisternal maturation. Because this explanation is now appreciated to be incomplete, interest has developed in understanding tubules that connect the Golgi cisternae. Here we show that the coat protein I (COPI) complex sorts anterograde cargoes into these tubules in human cells. Moreover, the small GTPase CDC42 regulates bidirectional Golgi transport by targeting the dual functions of COPI in cargo sorting and carrier formation. CDC42 also directly imparts membrane curvature to promote COPI tubule formation. Our findings further reveal that COPI tubular transport complements cisternal maturation in explaining how anterograde Golgi transport is achieved, and that bidirectional COPI transport is modulated by environmental cues through CDC42.
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Complexo I de Proteína do Envoltório/metabolismo , Complexo de Golgi/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Proteína Coatomer/metabolismo , Células HeLa , Humanos , Glicoproteínas de Membrana/metabolismo , Transporte Proteico , Receptores de LDL/metabolismo , Proteínas do Envelope Viral/metabolismoRESUMO
BACKGROUND: Children may be exposed to harmful chemicals from their products. Accurate exposure factors are critical for exposure assessment of children's products. Product usage pattern parameters are relatively limited compared with the chemical concentration, children's physiological and behavioral parameters. OBJECTIVE: The aim of this study was to determine nationally representative Korean exposure factors for the usage patterns of children's products by sex, age, and season. METHODS: Using proportional quota sampling, a survey of 10,000 households with children aged 0-12 years was conducted twice, once in summer and winter. The children's ages were divided into four groups: infant (0-2 years old), toddler (3-6), lower-grade elementary student (7-9), and higher-grade elementary student (10-12). Data on exposure factors such as use rate, use frequency, and use duration of 57 children's products were collected. RESULTS: The 57 products were classified into five categories: baby products (13), toys (12), daily products (10), sporting goods (8), and stationery (14). The use rates of products in the daily products and stationery category were >90% in both seasons. Two of the 57 products showed significant sex differences in all three exposure factors (p < 0.001). Twenty-five of the 44 non-baby products showed significant age differences for all three exposure factors. Twenty-three of the 57 products varied significantly with season for all three exposure factors. IMPACT: This study generated a nationally representative exposure factor database for the usage patterns of children's products in Korea. The exposure factors for 57 children's products were investigated through twice survey with quota sampling with each 10,000 children nationwide. Sex, age, and seasonal differences for children's products were identified. These accurate exposure factors by sex, age, and season can be used as input parameters for refined exposure assessment.
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Dynamic changes in mechanical microenvironments, such as cell crowding, regulate lineage fates as well as cell proliferation. Although regulatory mechanisms for contact inhibition of proliferation have been extensively studied, it remains unclear how cell crowding induces lineage specification. Here we found that a well-known oncogene, ETS variant transcription factor 4 (ETV4), serves as a molecular transducer that links mechanical microenvironments and gene expression. In a growing epithelium of human embryonic stem cells, cell crowding dynamics is translated into ETV4 expression, serving as a pre-pattern for future lineage fates. A switch-like ETV4 inactivation by cell crowding derepresses the potential for neuroectoderm differentiation in human embryonic stem cell epithelia. Mechanistically, cell crowding inactivates the integrin-actomyosin pathway and blocks the endocytosis of fibroblast growth factor receptors (FGFRs). The disrupted FGFR endocytosis induces a marked decrease in ETV4 protein stability through ERK inactivation. Mathematical modelling demonstrates that the dynamics of cell density in a growing human embryonic stem cell epithelium precisely determines the spatiotemporal ETV4 expression pattern and, consequently, the timing and geometry of lineage development. Our findings suggest that cell crowding dynamics in a stem cell epithelium drives spatiotemporal lineage specification using ETV4 as a key mechanical transducer.
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Diferenciação Celular , Linhagem da Célula , Células-Tronco Embrionárias Humanas , Proteínas Proto-Oncogênicas c-ets , Fatores de Transcrição , Humanos , Proteínas Proto-Oncogênicas c-ets/metabolismo , Proteínas Proto-Oncogênicas c-ets/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Endocitose , Proliferação de Células , Integrinas/metabolismo , Integrinas/genética , Transdução de Sinais , Mecanotransdução CelularRESUMO
Intracellular transport among organellar compartments occurs in two general ways, by membrane-bound carriers or membrane contacts. Specific circumstances that involve the coordination of these two modes of transport remain to be defined. Studying Coat Protein I (COPI) transport, we find that phosphatidylcholine with short acyl chains (sPC) is delivered through membrane contact from the endoplasmic reticulum (ER) to sites of COPI vesicle formation at the Golgi to support the fission stage. Phosphatidylinositol transfer protein beta (PITPß) plays a key role in this process, with the elucidation of this role advancing a new understanding of how PITPß acts, providing a mechanistic understanding of a specific circumstance when vesicular transport requires membrane contact, and contributing to a basic understanding of how transport carriers in a model intracellular pathway are formed.
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BACKGROUND: Globoside (Gb4), a globo-series glycosphingolipid (GSL), has been characterized as a stage-specific embryonic antigen (SSEA), and is highly expressed during embryogenesis as well as in cancer tissues. However, the functional role and molecular mechanism of Gb4 are so far unknown. METHODS: GSLs were preferentially inhibited by treatment with D-threo-1-ethylenedioxyphenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (EtDO-P4), a nanomolar inhibitor of GSL synthesis, in two carcinoma cell lines, HCT116 and MCF7. The effect of EtDO-P4 was examined by MTT assay, FACS, wound assay, western blotting, and RTK array analysis. The functional role of Gb4 was determined by the exogenous addition of various GSLs, and an assay utilizing GSL-coated latex beads. RESULTS: Both cell lines contained higher levels of neutral GSLs than of sialic acid-containing GSLs. Gb4 was one of the major neutral GSLs. The depletion of total GSLs caused significant reduction of cell proliferation, but had less effect on cell apoptosis or motility. EtDO-P4 treatment also suppressed activation of the epidermal growth factor receptor (EGFR)-induced ERK pathway and various receptor tyrosine kinases (RTKs). The reduced activation of ERK was restored by the exogenous addition of Gb4, but not by the addition of gangliosides (GM1, GM2, GM3, and GD1a). The GSL-coated bead assay indicated that Gb4 forms a complex with EGFR, but not with other RTKs. Taken together, Gb4 promotes activation of EGFR-induced ERK signaling through direct interaction with EGFR. GENERAL SIGNIFICANCE: A globo-series GSL, Gb4, promotes EGFR-induced MAPK signaling, resulting in cancer cell proliferation. These findings suggest a possible application of Gb4 in cancer diagnostics and drug targeting.
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Neoplasias da Mama/metabolismo , Neoplasias do Colo/metabolismo , Receptores ErbB/metabolismo , Globosídeos/farmacologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Western Blotting , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Comunicação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cromatografia em Camada Fina , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Glicoesfingolipídeos/farmacologia , Humanos , Fosforilação , Células Tumorais CultivadasRESUMO
The Coat Protein I (COPI) complex forms vesicles from Golgi membrane for retrograde transport among the Golgi stacks, and also from the Golgi to the endoplasmic reticulum (ER). We have been elucidating the mechanistic details of COPI vesicle formation through a reconstitution system that involves the incubation of Golgi membrane with purified components. This approach has enabled us recently to gain new insight into how certain lipids are critical for the fission stage of COPI vesicle formation. Lipid geometry has been proposed to act in the formation of transport carriers by promoting membrane curvature. However, evidence for this role has come from studies using simplified membranes, while confirmation in the more physiologic setting of native membranes has been challenging, as such membranes contain a complex composition of lipids and proteins. We have recently refined the COPI reconstitution system to overcome this experimental obstacle. This has led us to identify an unanticipated type of lipid geometry needed for COPI vesicle fission. This chapter describes the approach that we have developed to enable this discovery. The methodologies include: (i) preparation Golgi membrane from cells that are deficient in a particular lipid enzyme activity and (ii) functional rescue of this deficiency by introducing the product of the lipid enzyme, with experiments being performed at the in vitro level to gain mechanistic clarity and at the in vivo level to confirm physiologic relevance.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório , Complexo de Golgi , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Complexo de Golgi/metabolismo , Retículo Endoplasmático/metabolismo , Complexo I de Proteína do Envoltório/metabolismo , LipídeosRESUMO
Subungual melanoma (SUM) is a rare type of malignant melanoma that arises beneath the nails. SUM is categorized as a type of acral melanoma (AM), which occurs on the hands and feet. SUM is an aggressive type of cutaneous melanoma that is most common among Asian patients. Recent studies reveal that SUM and AM might have different molecular characteristics. Treatment of melanoma relies on analysis of both clinical and molecular data. Therefore, the clinical and molecular characteristics of SUM need to be established, especially during metastasis. To define the mutation profiles of SUM and compare them with those of AM, we performed next-generation sequencing of primary and metastatic tumors of SUM and AM patients. Subungual location was a better independent prognostic factor than acral location for better overall survival (p = 0.001). Patients with SUM most commonly had the triple wild-type (75%) driven by GNAQ (58%) and KIT (25%) mutations, whereas patients with AM had BRAF (28.6%) and RAF (14.3%) molecular types of mutations. Single-nucleotide variations (SNVs) were more common in SUM than in AM, whereas copy number alterations (CNAs) were more common metastatic lesions of AM. Metastatic tumors in patients with SUM and AM showed increases in CNAs (43% and 80%, respectively), but not in SNVs. The number of CNAs increased during metastasis. When compared with AM, SUM has distinct clinical and molecular characteristics.
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Background: Cholangiocarcinoma (CCA) is a silent tumor with a high mortality rate due to the difficulty of early diagnosis and prediction of recurrence even after timely surgery. Serologic cancer biomarkers have been used in clinical practice, but their low specificity and sensitivity have been problematic. In this study, we aimed to identify CCA-specific glycan epitopes that can be used for diagnosis and to elucidate the mechanisms by which glycosylation is altered with tumor progression. Methods: The serum of patients with various cancers was fractioned into membrane-bound and soluble components using serial ultracentrifugation. Lectin blotting was conducted to evaluate glycosylation. Proteins having altered glycosylation were identified using proteomic analysis and further confirmed using immunoblotting analysis. We performed HPLC, gene analysis, real-time cargo tracking, and immunohistochemistry to determine the origin of CCA glycosylation and its underlying mechanisms. Extracellular vesicles (EV) were isolated from the sera of 62 patients with CCA at different clinical stages and inflammatory conditions and used for glycan analysis to assess their clinical significance. Results: The results reveal that glycosylation patterns between soluble and membrane-bound fractions differ significantly even when obtained from the same donor. Notably, glycans with α1-3/4 fucose and ß1-6GlcNAc branched structures increase specifically in membrane-bound fractions of CCA. Mechanically, it is primarily due to ß-haptoglobin (ß-Hp) originating from CCA expressing fucosyltransferase-3/4 (FUT 3/4) and N-acetylglucosaminyltransferase-V (MGAT5). Newly synthesized ß-Hp is loaded into EVs in early endosomes via a KFERQ-like motif and then secreted from CCA cells to induce tumor progression. In contrast, ß-Hp expressed by hepatocytes is secreted in a soluble form that does not affect CCA progression. Moreover, evaluation of EV glycosylation in CCA patients shows that fucosylation level of EV-Hp gradually increases with tumor progression and decreases markedly when the tumors are eliminated by surgery. Conclusion: This study suggests that terminal fucosylation of Hp in cancer-derived exosomes can be a novel glycan marker for diagnosis and prognosis of CCA. These findings highlight the potential of glycan analysis in different fractions of serum for biomarker discover for other diseases. Further research is needed to understand the role of fucosylated EVs on CCA progression.
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Newly synthesized proteins in the endoplasmic reticulum (ER) are sorted by coat protein complex II (COPII) at the ER exit site en route to the Golgi. Under cellular stresses, COPII proteins become targets of regulation to control the transport. Here, we show that the COPII outer coat proteins Sec31 and Sec13 are selectively sequestered into the biomolecular condensate of SCOTIN/SHISA-5, which interferes with COPII vesicle formation and inhibits ER-to-Golgi transport. SCOTIN is an ER transmembrane protein with a cytosolic intrinsically disordered region (IDR), which is required and essential for the formation of condensates. Upon IFN-γ stimulation, which is a cellular condition that induces SCOTIN expression and condensation, ER-to-Golgi transport was inhibited in a SCOTIN-dependent manner. Furthermore, cancer-associated mutations of SCOTIN perturb its ability to form condensates and control transport. Together, we propose that SCOTIN impedes the ER-to-Golgi transport through its ability to form biomolecular condensates at the ER membrane.
Assuntos
Retículo Endoplasmático , Proteínas de Transporte Vesicular , Proteínas de Transporte Vesicular/metabolismo , Transporte Biológico , Transporte Proteico/fisiologia , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismoRESUMO
The ADP-Ribosylation Factor (ARF) small GTPases have been found to act in vesicle fission through a direct ability to tubulate membrane. Here, we have used cryo-electron microscopy (EM) to solve the structure of an ARF6 protein lattice assembled on tubulated membrane to 3.9 Å resolution. ARF6 forms tetramers that polymerize into helical arrays to form this lattice. We identify, and confirm functionally, protein contacts critical for this lattice formation. The solved structure also suggests how the ARF amphipathic helix is positioned in the lattice for membrane insertion, and how a GTPase-activating protein (GAP) docks onto the lattice to catalyze ARF-GTP hydrolysis in completing membrane fission. As ARF1 and ARF6 are structurally conserved, we have also modeled ARF1 onto the ARF6 lattice, which has allowed us to pursue the reconstitution of Coat Protein I (COPI) vesicles to confirm more definitively that the ARF lattice acts in vesicle fission. Our findings are notable for having achieved the first detailed glimpse of how a small GTPase bends membrane and having provided a molecular understanding of how an ARF protein acts in vesicle fission.
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Aberrant glycosylation has been observed in many types of cancer, but the mechanism of glycosylation change is still poorly understood. To elucidate relationships between glycosylation and colon cancer progression, we analyzed glycosylation status of ß-haptoglobin (ß-Hp) obtained from 46 cancer patients, 14 inflammatory bowel disease patients and 38 normal subjects. Aleuria aurantia lectin reactivity with cancer ß-Hp was much higher than in the other two study groups. These results were confirmed by lectin blotting and microarray assay using other lectins directed to fucosyl residues. Levels of such glycans were correlated with stage of colon cancer progression. Reactivity with fucosylated glycans was eliminated by treatment with α1-3/4 fucosidase but not α1-6 fucosidase, indicating that enhanced lectin reactivity with the fucose moiety of colon cancer ß-Hp is due to Fucα1-3/4GlcNAc. Moreover, site-specific glycan occupancy was determined by sequential LC/MS analysis. Mass spectrometric analysis showed that fucosylation of ß-Hp was higher in colon cancer patients than in other subjects. In particular, fucosylation at Asn 241 of ß-Hp in sera of colon cancer patients was clearly higher than in the other groups, and the ratio of fucosylated glycopeptides containing Asn 241 decreased greatly after treatment with α1-3/4 fucosidase. In conclusion, the level of α1-3/4 fucosyl epitope at Asn 241 of ß-Hp is potentially useful as a novel marker for colon cancer.
Assuntos
Neoplasias do Colo/metabolismo , Haptoglobinas/metabolismo , Idoso , Asparagina , Biomarcadores Tumorais/análise , Colo/metabolismo , Neoplasias do Colo/diagnóstico , Feminino , Fucose/metabolismo , Glicosilação , Haptoglobinas/química , Humanos , Doenças Inflamatórias Intestinais/metabolismo , Masculino , Polissacarídeos/metabolismoRESUMO
The Golgi apparatus regulates the process of modification and subcellular localization of macromolecules, including proteins and lipids. Aberrant protein sorting caused by defects in the Golgi leads to various diseases in mammals. However, the role of the Golgi apparatus in organismal longevity remained largely unknown. By employing a quantitative proteomic approach, we demonstrated that MON-2, an evolutionarily conserved Arf-GEF protein implicated in Golgi-to-endosome trafficking, promotes longevity via upregulating macroautophagy/autophagy in C. elegans. Our data using cultured mammalian cells indicate that MON2 translocates from the Golgi to the endosome under starvation conditions, subsequently increasing autophagic flux by binding LGG-1/GABARAPL2. Thus, Golgi-to-endosome trafficking appears to be an evolutionarily conserved process for the upregulation of autophagy, which contributes to organismal longevity.
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Autofagia , Longevidade , Animais , Autofagia/fisiologia , Caenorhabditis elegans , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Mamíferos , ProteômicaRESUMO
Activation of receptor tyrosine kinase signaling inactivates capicua (CIC), a transcriptional repressor that functions as a tumor suppressor, via degradation and/or cytoplasmic translocation. Although CIC is known to be inactivated by phosphorylation, the mechanisms underlying the cytoplasmic translocation of CIC remain poorly understood. Therefore, we aimed to evaluate the roles of extracellular signal-regulated kinase (ERK), p90RSK, and c-SRC in the epidermal growth factor receptor (EGFR) activation-induced cytoplasmic translocation of CIC and further investigated the molecular basis for this process. We found that nuclear ERK induced the cytoplasmic translocation of CIC-S. We identified 12 serine and threonine (S/T) residues within CIC, including S173 and S301 residues that are phosphorylated by p90RSK, which contribute to the cytoplasmic translocation of CIC-S when phosphorylated. The amino-terminal (CIC-S-N) and carboxyl-terminal (CIC-S-C) regions of CIC-S were found to interact with each other to promote their nuclear localization. EGF treatment disrupted the interaction between CIC-S-N and CIC-S-C and induced their cytoplasmic translocation. Alanine substitution for the 12 S/T residues blocked the cytoplasmic translocation of CIC-S and consequently enhanced the tumor suppressor activity of CIC-S. Our study demonstrates that ERK-mediated disruption of intramolecular interaction of CIC is critical for the cytoplasmic translocation of CIC, and suggests that the nuclear retention of CIC may represent a strategy for cancer therapy.
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Dimerization of beta 2-adrenergic receptor (ß2-AR) has been observed across various physiologies. However, the function of dimeric ß2-AR is still elusive. Here, we revealed that dimerization of ß2-AR is responsible for the constitutive activity of ß2-AR generating inverse agonism. Using a co-immunoimmobilization assay, we found that transient ß2-AR dimers exist in a resting state, and the dimer was disrupted by the inverse agonists. A Gαs preferentially interacts with dimeric ß2-AR, but not monomeric ß2-AR, in a resting state, resulting in the production of a resting cAMP level. The formation of ß2-AR dimers requires cholesterol on the plasma membrane. The cholesterol did not interfere with the agonist-induced activation of monomeric ß2-AR, unlike the inverse agonists, implying that the cholesterol is a specific factor regulating the dimerization of ß2-AR. Our model not only shows the function of dimeric ß2-AR but also provides a molecular insight into the mechanism of the inverse agonism of ß2-AR.
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Transdução de Sinais , Dimerização , Membrana Celular/metabolismoRESUMO
Despite the great potential of disease modeling using human pluripotent stem cells (hPSCs) derived from patients with mutations, lack of an appropriate isogenic control hinders a precise phenotypic comparison due to the bias arising from the dissimilar genetic backgrounds between the control and diseased hPSCs. Herein, we took advantage of currently available base editors (BEs) to epitomize the isogenic disease model from hPSCs. Using this method, we established multiple isogenic GNE myopathy disease models that harbor point mutations on the GNE gene, including four different mutations found in GNE myopathy patients. Four different mutations in the epimerase or kinase domains of GNE revealed mutation-specific hyposialylation and hyposialylation dependent gene signature, which was closely correlated to pathological clinical phenotypes. GNE protein structure modeling based on the mutations, addressed these mutation-specific hyposialylation patterns. Furthermore, treatment with a drug candidate currently under clinical trials showed a mutation-specific drug response in GNE myopathy disease models. These data suggest that derivation of multiple isogenic disease models from hPSCs by using genome editing can enable translationally relevant studies on the pathophysiology of GNE myopathy and drug responses.