RESUMO
Coordinated regulation of the ubiquitin-proteasome system (UPS) is crucial for the cell to adjust its protein degradation capacity to changing proteolytic requirements. We have shown previously that mammalian cells upregulate proteasome gene expression in response to proteasome inhibition. Here, we report the identification of the transcription factor TCF11 (long isoform of Nrf1) as a key regulator for 26S proteasome formation in human cells to compensate for reduced proteolytic activity. Under noninducing conditions, TCF11 resides in the endoplasmic reticulum (ER) membrane. There, TCF11 is targeted to ER-associated protein degradation requiring the E3 ubiquitin ligase HRD1 and the AAA ATPase p97. Proteasome inhibitors trigger the accumulation of oxidant-damaged proteins and promote the nuclear translocation of TCF11 from the ER, permitting activation of proteasome gene expression by binding to antioxidant response elements in their promoter regions. Thus, we uncovered the transcriptional control loop regulating human proteasome-dependent protein degradation to counteract proteotoxic stress caused by proteasome inhibition.
Assuntos
Retículo Endoplasmático/metabolismo , Células Endoteliais/enzimologia , Fator 1 Relacionado a NF-E2/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Transcrição Gênica , ATPases Associadas a Diversas Atividades Celulares , Transporte Ativo do Núcleo Celular , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Antioxidantes/metabolismo , Linhagem Celular , Células Endoteliais/efeitos dos fármacos , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Homeostase , Humanos , Dados de Sequência Molecular , Fator 1 Relacionado a NF-E2/genética , Proteínas Nucleares/metabolismo , Estresse Oxidativo , Inibidores de Proteases/farmacologia , Complexo de Endopeptidases do Proteassoma/genética , Inibidores de Proteassoma , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/genética , Interferência de RNA , RNA Mensageiro/metabolismo , Elementos de Resposta , Transcrição Gênica/efeitos dos fármacos , Transfecção , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
BACKGROUND: ADAM23 is widely expressed in the embryonic central nervous system and plays an important role in tissue formation. RESULTS: In this study, we showed that ADAM23 contributes to cell survival and is involved in neuronal differentiation during the differentiation of human neural progenitor cells (hNPCs). Upregulation of ADAM23 in hNPCs was found to increase the number of neurons and the length of neurite, while its downregulation decreases them and triggers cell apoptosis. RNA microarray analysis revealed mechanistic insights into genes and pathways that may become involved in multiple cellular processes upon up- or downregulation of ADAM23. CONCLUSIONS: Our results suggest that ADAM23 regulates neuronal differentiation by triggering specific signaling pathways during hNPC differentiation.
Assuntos
Proteínas ADAM/fisiologia , Células-Tronco Neurais/metabolismo , Neurogênese , Transdução de Sinais , Proteínas ADAM/metabolismo , Humanos , Células-Tronco Neurais/fisiologiaRESUMO
Proteasomes recognize and degrade poly-ubiquitinylated proteins. In infectious disease, cells activated by interferons (IFNs) express three unique catalytic subunits ß1i/LMP2, ß2i/MECL-1 and ß5i/LMP7 forming an alternative proteasome isoform, the immunoproteasome (IP). The in vivo function of IPs in pathogen-induced inflammation is still a matter of controversy. IPs were mainly associated with MHC class I antigen processing. However, recent findings pointed to a more general function of IPs in response to cytokine stress. Here, we report on the role of IPs in acute coxsackievirus B3 (CVB3) myocarditis reflecting one of the most common viral disease entities among young people. Despite identical viral load in both control and IP-deficient mice, IP-deficiency was associated with severe acute heart muscle injury reflected by large foci of inflammatory lesions and severe myocardial tissue damage. Exacerbation of acute heart muscle injury in this host was ascribed to disequilibrium in protein homeostasis in viral heart disease as indicated by the detection of increased proteotoxic stress in cytokine-challenged cardiomyocytes and inflammatory cells from IP-deficient mice. In fact, due to IP-dependent removal of poly-ubiquitinylated protein aggregates in the injured myocardium IPs protected CVB3-challenged mice from oxidant-protein damage. Impaired NFκB activation in IP-deficient cardiomyocytes and inflammatory cells and proteotoxic stress in combination with severe inflammation in CVB3-challenged hearts from IP-deficient mice potentiated apoptotic cell death in this host, thus exacerbating acute tissue damage. Adoptive T cell transfer studies in IP-deficient mice are in agreement with data pointing towards an effective CD8 T cell immune. This study therefore demonstrates that IP formation primarily protects the target organ of CVB3 infection from excessive inflammatory tissue damage in a virus-induced proinflammatory cytokine milieu.
Assuntos
Infecções por Coxsackievirus/imunologia , Enterovirus Humano B , Miocardite/imunologia , Complexo de Endopeptidases do Proteassoma/deficiência , Complexo de Endopeptidases do Proteassoma/imunologia , Subunidades Proteicas/deficiência , Animais , Apoptose , Infecções por Coxsackievirus/patologia , Camundongos , Miocardite/patologia , Miocardite/virologia , Poliubiquitina/imunologiaRESUMO
The galectins, a family of lectins, modulate distinct cellular processes, such as cancer progression, immune response and cellular development, through their specific binding to extracellular or intracellular ligands. In the past few years, research has unravelled interactions of different galectins with lipids and glycoproteins in the outer milieu or in the secretory pathway of cells. Interestingly, these lectins do not possess a signalling sequence to enter the endoplasmic reticulum as a starting point for the classical secretory pathway. Instead they use a so-called non-classical mechanism for translocation across the plasma membrane and/or into the lumen of transport vesicles. Here, they stabilize transport platforms for apical trafficking or sort apical glycoproteins into specific vesicle populations. Modes of ligand interaction as well as the modulation of binding activities and trafficking pathways are discussed in this review.
Assuntos
Membrana Celular/metabolismo , Galectinas/fisiologia , Glicoproteínas/metabolismo , Animais , Galectinas/metabolismo , Humanos , Ligantes , Ligação Proteica , Transporte ProteicoRESUMO
The mammalian dynamin-like protein DLP1/Drp1 has been shown to mediate both mitochondrial and peroxisomal fission. In this study, we have examined whether hFis1, a mammalian homologue of yeast Fis1, which has been shown to participate in mitochondrial fission by an interaction with DLP1/Drp1, is also involved in peroxisomal growth and division. We show that hFis1 localizes to peroxisomes in addition to mitochondria. Through differential tagging and deletion experiments, we demonstrate that the transmembrane domain and the short C-terminal tail of hFis1 is both necessary and sufficient for its targeting to peroxisomes and mitochondria, whereas the N-terminal region is required for organelle fission. hFis1 promotes peroxisome division upon ectopic expression, whereas silencing of Fis1 by small interfering RNA inhibited fission and caused tubulation of peroxisomes. These findings provide the first evidence for a role of Fis1 in peroxisomal fission and suggest that the fission machinery of mitochondria and peroxisomes shares common components.
Assuntos
Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/fisiologia , Peroxissomos/metabolismo , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Dinaminas , GTP Fosfo-Hidrolases/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Mitocondriais/metabolismo , RNA Interferente Pequeno/farmacologia , Ratos , TransfecçãoRESUMO
AIMS: Precise regulation of cellular protein degradation is essential for maintaining protein and redox homeostasis. The ubiquitin proteasome system (UPS) represents one of the major degradation machineries, and UPS disturbances are strongly associated with neurodegeneration. We have previously shown that the transcription factor TCF11/Nrf1 induces antioxidant response element-mediated upregulation of UPS components in response to proteotoxic stress. Knockout of TCF11/Nrf1 is embryonically lethal, and therefore, the present investigation describes the role of oxidative stress in regulating TCF11/Nrf1-dependent proteasome expression in a model system relevant to Parkinson's disease. RESULTS: Using the human dopaminergic neuroblastoma cell line SH-SY5Y and mouse nigrostriatal organotypic slice cultures, gene and protein expression analysis and functional assays revealed oxidative stress is induced by the proteasome inhibitor epoxomicin or the mitochondrial complex I inhibitor rotenone and promotes the upregulation of proteasome expression and function mediated by TCF11/Nrf1 activation. In addition, we show that these stress conditions induce the unfolded protein response. TCF11/Nrf1, thus, has a cytoprotective function in response to oxidative and proteotoxic stress. Innovation and Conclusion: We here demonstrate that adaption of the proteasome system in response to oxidative stress is dependent on TCF11/Nrf1 in this model system. We conclude that TCF11/Nrf1, therefore, plays a vital role in maintaining redox and protein homeostasis. This work provides a vital insight into the molecular mechanisms of neurodegeneration due to oxidative stress by rotenone, and further studies investigating the role of TCF11/Nrf1 in the human condition would be of considerable interest. Antioxid. Redox Signal. 25, 870-885.
Assuntos
Fator 1 Relacionado a NF-E2/metabolismo , Fator 1 Nuclear Respiratório/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Rotenona/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Pré-Escolar , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Modelos Biológicos , Fator 1 Relacionado a NF-E2/genética , Fator 1 Nuclear Respiratório/genética , Oligopeptídeos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Transporte Proteico/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
It is well established that mutual relations exist between the oxidative status of a cell and the ubiquitin proteasome system (UPS). Oxidation of proteins leads to misfolding and thus claims a higher proteolytic activity of the UPS to dispose these proteins. Any disturbance of the proteotoxic-protection machinery may result in protein aggregation and thus in disorders like neurodegenerative diseases or cancer. In this article we describe the molecular mechanisms regulating the ubiquitin proteasome system, thereby focussing on Nrf2 as well as on the recently identified transcription factor (TF) TCF11. In response to proteasome inhibition TCF11 plays a central role in upregulating the proteasome system via an ERAD-dependent feedback loop.
Assuntos
Fator 1 Relacionado a NF-E2 , Fator 2 Relacionado a NF-E2/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Dobramento de Proteína , Ubiquitina/metabolismo , Animais , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica , Inativação Gênica , Humanos , Camundongos , Camundongos Knockout , Fator 1 Relacionado a NF-E2/genética , Fator 1 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genética , Neoplasias/metabolismo , Neoplasias/patologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Oxirredução , Estresse Oxidativo , Inibidores de Proteassoma , Processamento de Proteína Pós-Traducional/genética , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio , Ativação Transcricional , Células Tumorais CultivadasRESUMO
In their natural environments, cells are regularly exposed to oxidizing conditions that may lead to protein misfolding. If such misfolded proteins are allowed to linger, they may form insoluble aggregates and pose a serious threat to the cell. Accumulation of misfolded, oxidatively damaged proteins is characteristic of many diseases and during aging. To counter the adverse effects of oxidative stress, cells can initiate an antioxidative response in an attempt to repair the damage, or rapidly channel the damaged proteins for degradation by the ubiquitin-proteasome system (UPS). Recent studies have shown that elements of the oxidative stress response and the UPS are linked on many levels. To manage the extra burden of misfolded proteins, the UPS is induced by oxidative stress, and special proteasome subtypes protect cells against oxidative damage. In addition, the proteasome is directly associated with a thioredoxin and other cofactors that may adjust the particle's response during an oxidative challenge. Here, we give an overview of the UPS and a detailed description of the degradation of oxidized proteins and of the crosstalk between oxidative stress and protein degradation in health and disease.
Assuntos
Estresse Oxidativo/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Animais , Humanos , Oxirredução , Estresse Oxidativo/genética , Proteínas/metabolismoRESUMO
Galectin-3 is a beta-galactoside-binding protein widely expressed in all epithelia where it is involved in tissue homeostasis and cancer progression. We recently reported unique abnormalities in the identity of membrane domains in galectin-3 null mutant mice, suggesting that galectin-3 may participate in epithelial polarity program. We investigated the potential role of galectin-3 on early events in polarization of epithelial renal cells, using three-dimensional cultures of MDCK cells and also galectin-3 null mutant mouse kidneys. We show that depletion in galectin-3 systematically leads to severe perturbations of microtubular network associated with defects in membrane compartimentation, both in vitro and in vivo. Moreover, the absence of galectin-3 impinges on the morphology of the primary cilium, which is three times longer and unusually shaped. By immunological and biochemical approaches, we could demonstrate that endogenous galectin-3 is normally associated with basal bodies and centrosomes, where it closely interacts with core proteins, such as centrin-2. However, this association transiently occurs during the process of epithelial polarization. Interestingly, galectin-3-depleted cells contain numerous centrosome-like structures, demonstrating an unexpected function of this protein in the formation and/or stability of the centrosomes. Collectively, these data establish galectin-3 as a key determinant in epithelial morphogenesis via its effect on centrosome biology.
Assuntos
Centrossomo/metabolismo , Epitélio/crescimento & desenvolvimento , Galectina 3/metabolismo , Morfogênese , Animais , Centríolos/ultraestrutura , Centrossomo/ultraestrutura , Cílios/metabolismo , Cães , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Células Epiteliais/ultraestrutura , Epitélio/metabolismo , Epitélio/ultraestrutura , Inativação Gênica , Camundongos , RNA Interferente Pequeno/metabolismoRESUMO
In epithelial cells, the ß-galactoside-binding lectin galectin-3 mediates the non-raft-dependent glycoprotein targeting to the apical membrane domain. In this study, we aimed to identify intracellular compartments involved in the trafficking of galectin-3. By studying fluorescent fusion proteins in living cells, we could show that galectin-3 accumulates intracellularly in acidified endosomes. Total internal reflection fluorescence microscopy studies of the apical surface of polarized MDCK cells revealed that galectin-3 is enriched in tubular and vesicular Rab11-positive recycling endosomes in the vicinity of the apical cell surface. These endosomal organelles are candidate compartments for the association between galectin-3 and exocytic apical cargo.
Assuntos
Endossomos/metabolismo , Galectina 3/metabolismo , Organelas/metabolismo , Animais , Células COS , Linhagem Celular , Membrana Celular/metabolismo , Polaridade Celular/genética , Chlorocebus aethiops , Cães , Endossomos/genética , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Galectina 3/genética , Microscopia de Fluorescência , Organelas/genética , Transporte ProteicoRESUMO
Epithelial cells are characterised by distinct apical and basolateral membrane domains that are separated by tight junctions. Establishment and maintenance of this polarity depend on specific gene expression and protein targeting to their correct location. Our former studies, performed with renal epithelial MDCK cells, revealed a new function for galectin-3, a member of a conserved family of lectins. There, galectin-3 is required for intracellular sorting and correct targeting of non-raft-associated glycoproteins to the apical plasma membrane. In the present study, we found transport defects of the intestinal brush border hydrolases lactase-phlorizin hydrolase (LPH) and dipeptidylpeptidase IV (DPPIV) in galectin-3-null mutant mice. We could show that, in enterocytes of wild-type mice, both glycoproteins directly interact with galectin-3 and transit through non-raft-dependent apical transport platforms. Therefore, this genetic analysis provides definitive evidence for the involvement of galectin-3 in protein intracellular trafficking in vivo. Further investigations revealed that gal3-null enterocytes also exhibit striking cytoarchitecture defects, with the presence of numerous and regular protrusions located along basolateral membranes. Moreover, beta-actin and villin, two characteristic markers of brush borders, become abnormally distributed along these atypical basolateral membranes in gal3(-/-) mice. Taken together, our results demonstrate that, in addition to a pivotal role in apical trafficking, galectin-3 also participates in epithelial morphogenesis in mouse enterocytes.
Assuntos
Membrana Celular/metabolismo , Enterócitos/metabolismo , Galectina 3/fisiologia , Actinas/metabolismo , Animais , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Enterócitos/ultraestrutura , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Galectina 3/genética , Galectina 3/metabolismo , Imunoprecipitação , Lactase-Florizina Hidrolase/metabolismo , Camundongos , Camundongos Endogâmicos , Camundongos Mutantes , Proteínas dos Microfilamentos/metabolismo , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , MutaçãoRESUMO
Epithelial cells are characterized by their polarized organization based on an apical membrane that is separated from the basolateral membrane domain by tight junctions. Maintenance of this morphology is guaranteed by highly specific sorting machinery that separates lipids and proteins into different carrier populations for the apical or basolateral cell surface. Lipid-raft-independent apical carrier vesicles harbour the beta-galactoside-binding lectin galectin-3, which interacts directly with apical cargo in a glycan-dependent manner. These glycoproteins are mistargeted to the basolateral membrane in galectin-3-depleted cells, dedicating a central role to this lectin in raft-independent sorting as apical receptor. Here, we demonstrate that high-molecular-weight clusters are exclusively formed in the presence of galectin-3. Their stability is sensitive to increased carbohydrate concentrations, and cluster formation as well as apical sorting are perturbed in glycosylation-deficient Madin-Darby canine kidney (MDCK) II cells. Together, our data suggest that glycoprotein cross-linking by galectin-3 is required for apical sorting of non-raft-associated cargo.
Assuntos
Galectina 3/fisiologia , Glicoproteínas/metabolismo , Animais , Linhagem Celular , Cães , Glicosilação , Transporte Proteico , Receptor de Fator de Crescimento Neural/genética , Receptor de Fator de Crescimento Neural/metabolismoRESUMO
The interaction of the adaptor protein p11, also denoted S100A10, with the C-terminus of the two-pore-domain K+ channel TASK-1 was studied using yeast two-hybrid analysis, glutathione S-transferase pull-down, and co-immunoprecipitation. We found that p11 interacts with a 40 amino-acid region in the proximal C-terminus of the channel. In heterologous expression systems, deletion of the p11-interacting domain enhanced surface expression of TASK-1. Attachment of the p11-interacting domain to the cytosolic tail of the reporter protein CD8 caused retention/retrieval of the construct in the endoplasmic reticulum (ER). Attachment of the last 36 amino acids of p11 to CD8 also caused ER localization, which was abolished by removal or mutation of a putative retention motif (H/K)xKxxx, at the C-terminal end of p11. Imaging of EGFP-tagged TASK-1 channels in COS cells suggested that wild-type TASK-1 was largely retained in the ER. Knockdown of p11 with siRNA enhanced trafficking of TASK-1 to the surface membrane. Our results suggest that binding of p11 to TASK-1 retards the surface expression of the channel, most likely by virtue of a di-lysine retention signal at the C-terminus of p11. Thus, the cytosolic protein p11 may represent a 'retention factor' that causes localization of the channel to the ER.
Assuntos
Anexina A2/metabolismo , Retículo Endoplasmático/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Proteínas S100/metabolismo , Proteínas 14-3-3/química , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Sequência de Aminoácidos , Animais , Anexina A2/química , Anexina A2/genética , Sítios de Ligação/genética , Antígenos CD8/química , Antígenos CD8/genética , Antígenos CD8/metabolismo , Células CHO , Células COS , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Feminino , Humanos , Técnicas In Vitro , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas do Tecido Nervoso , Oócitos/metabolismo , Canais de Potássio de Domínios Poros em Tandem/química , Canais de Potássio de Domínios Poros em Tandem/genética , Estrutura Terciária de Proteína , Interferência de RNA , RNA Interferente Pequeno/genética , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas S100/química , Proteínas S100/genética , Homologia de Sequência de Aminoácidos , Técnicas do Sistema de Duplo-Híbrido , XenopusRESUMO
The mammalian dynamin-like protein DLP1 belongs to the dynamin family of large GTPases, which have been implicated in tubulation and fission events of cellular membranes. We have previously shown that the expression of a dominant-negative DLP1 mutant deficient in GTP hydrolysis (K38A) inhibited peroxisomal division in mammalian cells. In this study, we conducted RNA interference experiments to 'knock down' the expression of DLP1 in COS-7 cells stably expressing a GFP construct bearing the C-terminal peroxisomal targeting signal 1. The peroxisomes in DLP1-silenced cells were highly elongated with a segmented morphology. Ultrastructural and quantitative studies confirmed that the tubular peroxisomes induced by DLP1-silencing retained the ability to constrict their membranes but were not able to divide into spherical organelles. Co-transfection of DLP1 siRNA with Pex11pbeta, a peroxisomal membrane protein involved in peroxisome proliferation, induced further elongation and network formation of the peroxisomal compartment. Time-lapse microscopy of living cells silenced for DLP1 revealed that the elongated peroxisomes moved in a microtubule-dependent manner and emanated tubular projections. DLP1-silencing in COS-7 cells also resulted in a pronounced elongation of mitochondria, and in more dispersed, elongated Golgi structures, whereas morphological changes of the rER, lysosomes and the cytoskeleton were not detected. These observations clearly demonstrate that DLP1 acts on multiple membranous organelles. They further indicate that peroxisomal elongation, constriction and fission require distinct sets of proteins, and that the dynamin-like protein DLP1 functions primarily in the latter process.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Peroxissomos/metabolismo , Animais , Células COS , Membrana Celular/metabolismo , Proliferação de Células , Dinaminas/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Inativação Gênica , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Proteínas de Fluorescência Verde/metabolismo , Immunoblotting , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Microtúbulos/metabolismo , Modelos Biológicos , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
Syncollin, a novel pancreatic zymogen granule protein, is present on the luminal side of the granule membrane. To address the function of syncollin, we searched for putative binding partners. Cross-linking experiments with purified syncollin, and granule content and membrane proteins revealed a direct interaction between syncollin and GP-2, a major glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein. An interaction was also observed when cross-linking was performed with recombinant GP-2. In addition, syncollin could be cross-linked to itself, supporting the suggestion that it exists as a homo-oligomer. Cleavage of the GPI anchor of GP-2 by treatment of granule membranes with phosphatidylinositol-specific phospholipase C had no effect on the membrane attachment of syncollin, indicating that it is not mediated exclusively via an interaction with GP-2. Syncollin was found to be associated with detergent-insoluble cholesterol/glycolipid-enriched complexes. These complexes floated to the lighter fractions of sucrose-density gradients and also contained GP-2, the lectin ZG16p, sulphated matrix proteoglycans and the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) syntaxin 3 and synaptobrevin 2. Our results indicate that membrane-associated syncollin is a component of lipid rafts, where it interacts both with GP-2 and membrane lipids. We suggest that the syncollin-GP-2 complex might play a role in signal transduction across the granule membrane.
Assuntos
Proteínas de Transporte/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Pâncreas/metabolismo , Vesículas Secretórias/metabolismo , Animais , Sítios de Ligação , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/ultraestrutura , Reagentes de Ligações Cruzadas , Glicosilfosfatidilinositóis/metabolismo , Glicoproteínas de Membrana/isolamento & purificação , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/ultraestrutura , Camundongos , Microscopia Eletrônica , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Vesículas Secretórias/ultraestruturaRESUMO
The mammalian dynamin-like protein 1 (DLP1), a member of the dynamin family of large GTPases, possesses mechanochemical properties known to constrict and tubulate membranes. In this study, we have combined two experimental approaches, induction of peroxisome proliferation by Pex11pbeta and expression of dominant-negative mutants, to test whether DLP1 plays a role in peroxisomal growth and division. We were able to localize DLP1 in spots on tubular peroxisomes in HepG2 cells. In addition, immunoblot analysis revealed the presence of DLP1 in highly purified peroxisomal fractions from rat liver and an increase of DLP1 after treatment of rats with the peroxisome proliferator bezafibrate. Expression of a dominant negative DLP1 mutant deficient in GTP hydrolysis (K38A) either alone or in combination with Pex11pbeta caused the appearance of tubular peroxisomes but had no influence on their intracellular distribution. In co-expressing cells, the formation of tubulo-reticular networks of peroxisomes was promoted, and peroxisomal division was completely inhibited. These findings were confirmed by silencing of DLP1 using siRNA. We propose a direct role for the dynamin-like protein DLP1 in peroxisomal fission and in the maintenance of peroxisomal morphology in mammalian cells.