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1.
Cell ; 176(6): 1461-1476.e23, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30849374

RESUMO

Maintaining the optimal performance of cell processes and organelles is the task of auto-regulatory systems. Here we describe an auto-regulatory device that helps to maintain homeostasis of the endoplasmic reticulum (ER) by adjusting the secretory flux to the cargo load. The cargo-recruiting subunit of the coatomer protein II (COPII) coat, Sec24, doubles as a sensor of folded cargo and, upon cargo binding, acts as a guanine nucleotide exchange factor to activate the signaling protein Gα12 at the ER exit sites (ERESs). This step, in turn, activates a complex signaling network that activates and coordinates the ER export machinery and attenuates proteins synthesis, thus preventing large fluctuations of folded and potentially active cargo that could be harmful to the cell or the organism. We call this mechanism AREX (autoregulation of ER export) and expect that its identification will aid our understanding of human physiology and diseases that develop from secretory dysfunction.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Transporte Biológico , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/fisiologia , Linhagem Celular , Proteína Coatomer/metabolismo , Retículo Endoplasmático/fisiologia , Estresse do Retículo Endoplasmático/fisiologia , Feminino , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Complexo de Golgi/metabolismo , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Células HeLa , Humanos , Masculino , Dobramento de Proteína , Transporte Proteico , Proteostase/fisiologia , Transdução de Sinais
2.
EMBO J ; 40(20): e107766, 2021 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-34516001

RESUMO

The Golgi apparatus, the main glycosylation station of the cell, consists of a stack of discontinuous cisternae. Glycosylation enzymes are usually concentrated in one or two specific cisternae along the cis-trans axis of the organelle. How such compartmentalized localization of enzymes is achieved and how it contributes to glycosylation are not clear. Here, we show that the Golgi matrix protein GRASP55 directs the compartmentalized localization of key enzymes involved in glycosphingolipid (GSL) biosynthesis. GRASP55 binds to these enzymes and prevents their entry into COPI-based retrograde transport vesicles, thus concentrating them in the trans-Golgi. In genome-edited cells lacking GRASP55, or in cells expressing mutant enzymes without GRASP55 binding sites, these enzymes relocate to the cis-Golgi, which affects glycosphingolipid biosynthesis by changing flux across metabolic branch points. These findings reveal a mechanism by which a matrix protein regulates polarized localization of glycosylation enzymes in the Golgi and controls competition in glycan biosynthesis.


Assuntos
Glicoesfingolipídeos/metabolismo , Complexo de Golgi/metabolismo , Proteínas da Matriz do Complexo de Golgi/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Brefeldina A/farmacologia , Ceramidas/metabolismo , Toxina da Cólera/farmacologia , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Expressão Gênica , Glicosilação/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/genética , Proteínas da Matriz do Complexo de Golgi/genética , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Toxina Shiga/farmacologia
3.
Int J Mol Sci ; 24(5)2023 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-36901886

RESUMO

The World Health Organization has indicated Helicobacter pylori as a high-priority pathogen whose infections urgently require an update of the antibacterial treatments pipeline. Recently, bacterial ureases and carbonic anhydrases (CAs) were found to represent valuable pharmacological targets to inhibit bacterial growth. Hence, we explored the underexploited possibility of developing a multiple-targeted anti-H. pylori therapy by assessing the antimicrobial and antibiofilm activities of a CA inhibitor, carvacrol (CAR), amoxicillin (AMX) and a urease inhibitor (SHA), alone and in combination. Minimal Inhibitory (MIC) and Minimal Bactericidal (MBC) Concentrations of their different combinations were evaluated by checkerboard assay and three different methods were employed to assess their capability to eradicate H. pylori biofilm. Through Transmission Electron Microscopy (TEM) analysis, the mechanism of action of the three compounds alone and together was determined. Interestingly, most combinations were found to strongly inhibit H. pylori growth, resulting in an additive FIC index for both CAR-AMX and CAR-SHA associations, while an indifferent value was recorded for the AMX-SHA association. Greater antimicrobial and antibiofilm efficacy of the combinations CAR-AMX, SHA-AMX and CAR-SHA against H. pylori were found with respect to the same compounds used alone, thereby representing an innovative and promising strategy to counteract H. pylori infections.


Assuntos
Infecções por Helicobacter , Helicobacter pylori , Humanos , Amoxicilina/farmacologia , Antibacterianos/farmacologia , Infecções por Helicobacter/microbiologia , Biofilmes , Testes de Sensibilidade Microbiana
4.
EMBO J ; 36(12): 1736-1754, 2017 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-28495678

RESUMO

Sphingolipids are membrane lipids globally required for eukaryotic life. The sphingolipid content varies among endomembranes with pre- and post-Golgi compartments being poor and rich in sphingolipids, respectively. Due to this different sphingolipid content, pre- and post-Golgi membranes serve different cellular functions. The basis for maintaining distinct subcellular sphingolipid levels in the presence of membrane trafficking and metabolic fluxes is only partially understood. Here, we describe a homeostatic regulatory circuit that controls sphingolipid levels at the trans-Golgi network (TGN). Specifically, we show that sphingomyelin production at the TGN triggers a signalling pathway leading to PtdIns(4)P dephosphorylation. Since PtdIns(4)P is required for cholesterol and sphingolipid transport to the trans-Golgi network, PtdIns(4)P consumption interrupts this transport in response to excessive sphingomyelin production. Based on this evidence, we envisage a model where this homeostatic circuit maintains a constant lipid composition in the trans-Golgi network and post-Golgi compartments, thus counteracting fluctuations in the sphingolipid biosynthetic flow.


Assuntos
Fosfatidilinositóis/metabolismo , Esfingolipídeos/metabolismo , Rede trans-Golgi/metabolismo , Células HeLa , Homeostase , Humanos , Modelos Biológicos
5.
Proc Natl Acad Sci U S A ; 112(17): E2174-81, 2015 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-25870285

RESUMO

The organization and mutual interactions between endoplasmic reticulum (ER) and mitochondria modulate key aspects of cell pathophysiology. Several proteins have been suggested to be involved in keeping ER and mitochondria at a correct distance. Among them, in mammalian cells, mitofusin 2 (Mfn2), located on both the outer mitochondrial membrane and the ER surface, has been proposed to be a physical tether between the two organelles, forming homotypic interactions and heterocomplexes with its homolog Mfn1. Recently, this widely accepted model has been challenged using quantitative EM analysis. Using a multiplicity of morphological, biochemical, functional, and genetic approaches, we demonstrate that Mfn2 ablation increases the structural and functional ER-mitochondria coupling. In particular, we show that in different cell types Mfn2 ablation or silencing increases the close contacts between the two organelles and strengthens the efficacy of inositol trisphosphate (IP3)-induced Ca(2+) transfer from the ER to mitochondria, sensitizing cells to a mitochondrial Ca(2+) overload-dependent death. We also show that the previously reported discrepancy between electron and fluorescence microscopy data on ER-mitochondria proximity in Mfn2-ablated cells is only apparent. By using a different type of morphological analysis of fluorescent images that takes into account (and corrects for) the gross modifications in mitochondrial shape resulting from Mfn2 ablation, we demonstrate that an increased proximity between the organelles is also observed by confocal microscopy when Mfn2 levels are reduced. Based on these results, we propose a new model for ER-mitochondria juxtaposition in which Mfn2 works as a tethering antagonist preventing an excessive, potentially toxic, proximity between the two organelles.


Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/genética , GTP Fosfo-Hidrolases/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Animais , Retículo Endoplasmático/diagnóstico por imagem , GTP Fosfo-Hidrolases/genética , Células HeLa , Humanos , Transporte de Íons/fisiologia , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/ultraestrutura , Membranas Mitocondriais , Proteínas Mitocondriais/genética , Ultrassonografia
7.
Bioact Mater ; 35: 99-121, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38283385

RESUMO

Recently, the term theragenerative has been proposed for biomaterials capable of inducing therapeutic approaches followed by repairing/regenerating the tissue/organ. This study is focused on the design of a new theragenerative nanocomposite composed of an amphiphilic non-ionic surfactant (Pluronic F127), bioactive glass (BG), and black phosphorus (BP). The nanocomposite was prepared through a two-step synthetic strategy, including a microwave treatment that turned BP nanosheets (BPNS) into quantum dots (BPQDs) with 5 ± 2 nm dimensions in situ. The effects of surfactant and microwave treatment were assessed in vitro: the surfactant distributes the ions homogenously throughout the composite and the microwave treatment chemically stabilizes the composite. The presence of BP enhanced bioactivity and promoted calcium phosphate formation in simulated body fluid. The inherent anticancer activity of BP-containing nanocomposites was tested against osteosarcoma cells in vitro, finding that 150 µg mL-1 was the lowest concentration which prevented the proliferation of SAOS-2 cells, while the counterpart without BP did not affect the cell growth rate. Moreover, the apoptosis pathways were evaluated and a mechanism of action was proposed. NIR irradiation was applied to induce further proliferation suppression on SAOS-2 cells through hyperthermia. The inhibitory effects of bare BP nanomaterials and nanocomposites on the migration and invasion of bone cancer, breast cancer, and prostate cancer cells were assessed in vitro to determine the anticancer potential of nanomaterials against primary and secondary bone cancers. The regenerative behavior of the nanocomposites was tested with healthy osteoblasts and human mesenchymal stem cells; the BPQDs-incorporated nanocomposite significantly promoted the proliferation of osteoblast cells and induced the osteogenic differentiation of stem cells. This study introduces a new multifunctional theragenerative platform with promising potential for simultaneous bone cancer therapy and regeneration.

8.
Methods Mol Biol ; 2557: 225-234, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36512218

RESUMO

The Golgi apparatus is a highly dynamic organelle that controls lipid and protein sorting in the endocytic and exocytic cellular pathways. Perturbation of the lipid homeostasis or of the molecular machineries that regulate membrane remodeling/trafficking events on the Golgi membranes can dramatically change the morphology and functions of the Golgi apparatus. So far, several approaches have been described to characterize and define the Golgi morphology in intact cells and in vitro. Here, we describe the application of negative staining (NS) electron microscopy (EM) on purified Golgi membranes from HeLa cells. This approach allows to quantify and functionally characterize membrane remodeling events upon specific treatments that alter the Golgi morphology.


Assuntos
Complexo de Golgi , Lipídeos , Animais , Humanos , Células HeLa , Coloração Negativa , Complexo de Golgi/metabolismo , Microscopia Eletrônica , Mamíferos
9.
Membranes (Basel) ; 13(11)2023 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-37999346

RESUMO

Outer membrane vesicles (OMVs) are spherical, lipid-based nano-structures, which are released by Gram-negative bacteria in both in vitro and in vivo conditions. The size and composition of OMVs depend on not only the producer bacterial species but also cells belonging to the same strain. The mechanism of vesicles' biogenesis has a key role in determining their cargo and the pattern of macromolecules exposed on their surface. Thus, the content of proteins, lipids, nucleic acids, and other biomolecules defines the properties of OMVs and their beneficial or harmful effects on human health. Many studies have provided evidence that OMVs can be involved in a plethora of biological processes, including cell-to-cell communication and bacteria-host interactions. Moreover, there is a growing body of literature supporting their role in horizontal gene transfer (HGT). During this process, OMVs can facilitate the spreading of genes involved in metabolic pathways, virulence, and antibiotic resistance, guaranteeing bacterial proliferation and survival. For this reason, a deeper understanding of this new mechanism of genetic transfer could improve the development of more efficient strategies to counteract infections sustained by Gram-negative bacteria. In line with this, the main aim of this mini-review is to summarize the latest evidence concerning the involvement of OMVs in HGT.

10.
EMBO J ; 27(7): 970-81, 2008 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-18354494

RESUMO

Membrane fission is an essential process in membrane trafficking and other cellular functions. While many fissioning and trafficking steps are mediated by the large GTPase dynamin, some fission events are dynamin independent and involve C-terminal-binding protein-1/brefeldinA-ADP ribosylated substrate (CtBP1/BARS). To gain an insight into the molecular mechanisms of CtBP1/BARS in fission, we have studied the role of this protein in macropinocytosis, a dynamin-independent endocytic pathway that can be synchronously activated by growth factors. Here, we show that upon activation of the epidermal growth factor receptor, CtBP1/BARS is (a) translocated to the macropinocytic cup and its surrounding membrane, (b) required for the fission of the macropinocytic cup and (c) phosphorylated on a specific serine that is a substrate for p21-activated kinase, with this phosphorylation being essential for the fission of the macropinocytic cup. Importantly, we also show that CtBP1/BARS is required for macropinocytic internalization and infection of echovirus 1. These results provide an insight into the molecular mechanisms of CtBP1/BARS activation in membrane fissioning, and extend the relevance of CtBP1/BARS-induced fission to human viral infection.


Assuntos
Oxirredutases do Álcool/metabolismo , Proteínas de Ligação a DNA/metabolismo , Pinocitose , Quinases Ativadas por p21/metabolismo , Actinas/metabolismo , Oxirredutases do Álcool/ultraestrutura , Linhagem Celular Tumoral , Extensões da Superfície Celular/efeitos dos fármacos , Extensões da Superfície Celular/metabolismo , Proteínas de Ligação a DNA/ultraestrutura , Enterovirus Humano B/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Humanos , Integrina alfa2beta1/metabolismo , Fosforilação/efeitos dos fármacos , Pinocitose/efeitos dos fármacos , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Quinases Ativadas por p21/química
11.
Nat Cell Biol ; 7(6): 570-80, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15880102

RESUMO

Membrane fission is a fundamental step in membrane transport. So far, the only fission protein machinery that has been implicated in in vivo transport involves dynamin, and functions in several, but not all, transport pathways. Thus, other fission machineries may exist. Here, we report that carboxy-terminal binding protein 3/brefeldin A-ribosylated substrate (CtBP3/BARS) controls fission in basolateral transport from the Golgi to the plasma membrane and in fluid-phase endocytosis, whereas dynamin is not involved in these steps. Conversely, CtBP3/BARS protein is inactive in apical transport to the plasma membrane and in receptor-mediated endocytosis, both steps being controlled by dynamin. This indicates that CtBP3/BARS controls membrane fission in endocytic and exocytic transport pathways, distinct from those that require dynamin.


Assuntos
Proteínas de Transporte/metabolismo , Dinaminas/metabolismo , Membranas Intracelulares/metabolismo , Organelas/metabolismo , Fatores de Transcrição/metabolismo , Vesículas Transportadoras/metabolismo , Animais , Células COS , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Chlorocebus aethiops , Cães , Endocitose/fisiologia , Exocitose/fisiologia , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Membranas Intracelulares/ultraestrutura , Microscopia Eletrônica de Transmissão , Organelas/ultraestrutura , Transporte Proteico/fisiologia , Receptores de Superfície Celular/metabolismo , Vesículas Transportadoras/ultraestrutura
12.
Traffic ; 9(12): 2221-35, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18764928

RESUMO

The activation state of the EGF receptor (EGF-R) is tightly controlled in the cell so as to prevent excessive signalling, with the dangerous consequences that this would have on cell growth and proliferation. This control occurs at different levels, with a key level being the trafficking and degradation of the EGF-R itself. Multiple guanosine triphosphatases belonging to the Arf, Rab and Rho families and their accessory proteins have key roles in these processes. In this study, we have identified ARAP1, a multidomain protein with both Arf GTPase-activating protein (GAP) and Rho GAP activities, as a novel component of the machinery that controls the trafficking and signalling of the EGF-R. We show that ARAP1 localizes to multiple cell compartments, including the Golgi complex, as previously reported, and endosomal compartments, where it is enriched in the internal membranes of multivesicular bodies. ARAP1 distribution is controlled by its phosphorylation and by its interactions with the 3- and 4-phosphorylated phosphoinositides through its five PH domains. We provide evidence that ARAP1 controls the late steps of the endocytic trafficking of the EGF-R, with ARAP1 knockdown leading to EGF-R accumulation in a sorting/late endosomal compartment and to the inhibition of EGF-R degradation that is accompanied by prolonged signalling.


Assuntos
Proteínas de Transporte/metabolismo , Receptores ErbB/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Transdução de Sinais , Animais , Biomarcadores , Proteínas de Transporte/genética , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Chlorocebus aethiops , Endossomos/metabolismo , Endossomos/ultraestrutura , Fator de Crescimento Epidérmico/metabolismo , Proteínas Ativadoras de GTPase/genética , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Humanos , Transporte Proteico
13.
Sci Signal ; 13(631)2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32398348

RESUMO

Understanding the costimulatory signaling that enhances the activity of cytotoxic T cells (CTLs) could identify potential targets for immunotherapy. Here, we report that CD2 costimulation plays a critical role in target cell killing by freshly isolated human CD8+ T cells, which represent a challenging but valuable model to gain insight into CTL biology. We found that CD2 stimulation critically enhanced signaling by the T cell receptor in the formation of functional immune synapses by promoting the polarization of lytic granules toward the microtubule-organizing center (MTOC). To gain insight into the underlying mechanism, we explored the CD2 signaling network by phosphoproteomics, which revealed 616 CD2-regulated phosphorylation events in 373 proteins implicated in the regulation of vesicular trafficking, cytoskeletal organization, autophagy, and metabolism. Signaling by the master metabolic regulator AMP-activated protein kinase (AMPK) was a critical node in the CD2 network, which promoted granule polarization toward the MTOC in CD8+ T cells. Granule trafficking was driven by active AMPK enriched on adjacent lysosomes, revealing previously uncharacterized signaling cross-talk between vesicular compartments in CD8+ T cells. Our results thus establish CD2 signaling as key for mediating cytotoxic killing and granule polarization in freshly isolated CD8+ T cells and strengthen the rationale to choose CD2 and AMPK as therapeutic targets to enhance CTL activity.


Assuntos
Proteínas Quinases Ativadas por AMP/imunologia , Antígenos CD2/imunologia , Fosfoproteínas/imunologia , Vesículas Secretórias/imunologia , Transdução de Sinais/imunologia , Linfócitos T Citotóxicos/imunologia , Humanos , Fosforilação/imunologia , Proteômica
14.
Dev Cell ; 49(1): 145-155.e4, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30880003

RESUMO

Signal-sequence-lacking interleukin (IL)-1ß, is cleaved by caspase-1 to mature mIL-1ß, which is secreted, without entering the endoplasmic reticulum. We report that macrophages of GRASP55-/- mice are defective in mIL-1ß secretion and retain it as intracellular aggregates. Intriguingly, GRASP55-/- macrophages are defective in the IRE1α branch of the unfolded protein response. This finding fits well with our data that inhibition of IRE1α also impairs mIL-1ß secretion and causes its accumulation in intracellular aggregates. PERK inhibition, on the other hand, controls caspase-1-mediated conversion of proIL-1ß to mIL-1ß. These findings reveal translation-independent functions of PERK and IRE1α: PERK controls the production of mIL-1ß, which is then followed by GRASP55 and IRE1α activity to keep mIL-1ß in a secretion-competent form.


Assuntos
Endorribonucleases/genética , Proteínas da Matriz do Complexo de Golgi/genética , Interleucina-1beta/genética , Agregados Proteicos/genética , Proteínas Serina-Treonina Quinases/genética , eIF-2 Quinase/genética , Animais , Caspase 1/genética , Proteínas de Ligação a DNA/genética , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/genética , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Resposta a Proteínas não Dobradas/genética , eIF-2 Quinase/antagonistas & inibidores
16.
Elife ; 62017 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-28500756

RESUMO

The flat Golgi cisterna is a highly conserved feature of eukaryotic cells, but how is this morphology achieved and is it related to its function in cargo sorting and export? A physical model of cisterna morphology led us to propose that sphingomyelin (SM) metabolism at the trans-Golgi membranes in mammalian cells essentially controls the structural features of a Golgi cisterna by regulating its association to curvature-generating proteins. An experimental test of this hypothesis revealed that affecting SM homeostasis converted flat cisternae into highly curled membranes with a concomitant dissociation of membrane curvature-generating proteins. These data lend support to our hypothesis that SM metabolism controls the structural organization of a Golgi cisterna. Together with our previously presented role of SM in controlling the location of proteins involved in glycosylation and vesicle formation, our data reveal the significance of SM metabolism in the structural organization and function of Golgi cisternae.


Assuntos
Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Esfingomielinas/metabolismo , Células HeLa , Humanos
17.
Sci Rep ; 7(1): 14035, 2017 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-29070863

RESUMO

Poly-ADP-ribose-polymerases (PARPs) 1 and 2 are nuclear enzymes that catalyze the poly-ADP-ribosylation of nuclear proteins transferring poly-ADP-ribose (PAR) polymers to specific residues. PARPs and PAR intervene in diverse functions, including DNA repair in the nucleus and stress granule assembly in the cytoplasm. Stress granules contribute to the regulation of translation by clustering and stabilizing mRNAs as well as several cytosolic PARPs and signaling proteins to modulate cell metabolism and survival. Our study is focused on one of these PARPs, PARP12, a Golgi-localized mono-ADP-ribosyltransferase that under stress challenge reversibly translocates from the Golgi complex to stress granules. PARP1 activation and release of nuclear PAR drive this translocation by direct PAR binding to the PARP12-WWE domain. Thus, PAR formation functionally links the activity of the nuclear and cytosolic PARPs during stress response, determining the release of PARP12 from the Golgi complex and the disassembly of the Golgi membranes, followed by a block in anterograde-membrane traffic. Notably, these functions can be rescued by reverting the stress condition (by drug wash-out). Altogether these data point at a novel, reversible nuclear signaling that senses stress to then act on cytosolic PARP12, which in turn converts the stress response into a reversible block in intracellular-membrane traffic.


Assuntos
Complexo de Golgi/fisiologia , Poli(ADP-Ribose) Polimerases/fisiologia , Linhagem Celular , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Modelos Moleculares , Estresse Oxidativo , Poli(ADP-Ribose) Polimerases/metabolismo , Domínios Proteicos , Transporte Proteico , Transdução de Sinais , Estresse Fisiológico
18.
Cell Rep ; 15(10): 2226-2238, 2016 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-27239030

RESUMO

Communication between organelles plays key roles in cell biology. In particular, physical and functional coupling of the endoplasmic reticulum (ER) and mitochondria is crucial for regulation of various physiological and pathophysiological processes. Here, we demonstrate that Presenilin 2 (PS2), mutations in which underlie familial Alzheimer's disease (FAD), promotes ER-mitochondria coupling only in the presence of mitofusin 2 (Mfn2). PS2 is not necessary for the antagonistic effect of Mfn2 on organelle coupling, although its abundance can tune it. The two proteins physically interact, whereas their homologues Mfn1 and PS1 are dispensable for this interplay. Moreover, PS2 mutants associated with FAD are more effective than the wild-type form in modulating ER-mitochondria tethering because their binding to Mfn2 in mitochondria-associated membranes is favored. We propose a revised model for ER-mitochondria interaction to account for these findings and discuss possible implications for FAD pathogenesis.


Assuntos
Retículo Endoplasmático/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Presenilina-2/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Cálcio/metabolismo , Linhagem Celular , Retículo Endoplasmático/ultraestrutura , Fibroblastos/metabolismo , Camundongos Endogâmicos C57BL , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Presenilina-1/metabolismo , Ligação Proteica
19.
Elife ; 52016 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-27115345

RESUMO

The unconventional secretory pathway exports proteins that bypass the endoplasmic reticulum. In Saccharomyces cerevisiae, conditions that trigger Acb1 secretion via this pathway generate a Grh1 containing compartment composed of vesicles and tubules surrounded by a cup-shaped membrane and collectively called CUPS. Here we report a quantitative assay for Acb1 secretion that reveals requirements for ESCRT-I, -II, and -III but, surprisingly, without the involvement of the Vps4 AAA-ATPase. The major ESCRT-III subunit Snf7 localizes transiently to CUPS and this was accelerated in vps4Δ cells, correlating with increased Acb1 secretion. Microscopic analysis suggests that, instead of forming intraluminal vesicles with the help of Vps4, ESCRT-III/Snf7 promotes direct engulfment of preexisting Grh1 containing vesicles and tubules into a saccule to generate a mature Acb1 containing compartment. This novel multivesicular / multilamellar compartment, we suggest represents the stable secretory form of CUPS that is competent for the release of Acb1 to cells exterior.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Deleção de Genes , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
20.
Nat Commun ; 7: 12148, 2016 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-27401954

RESUMO

Membrane fission is an essential cellular process by which continuous membranes split into separate parts. We have previously identified CtBP1-S/BARS (BARS) as a key component of a protein complex that is required for fission of several endomembranes, including basolateral post-Golgi transport carriers. Assembly of this complex occurs at the Golgi apparatus, where BARS binds to the phosphoinositide kinase PI4KIIIß through a 14-3-3γ dimer, as well as to ARF and the PKD and PAK kinases. We now report that, when incorporated into this complex, BARS binds to and activates a trans-Golgi lysophosphatidic acid (LPA) acyltransferase type δ (LPAATδ) that converts LPA into phosphatidic acid (PA); and that this reaction is essential for fission of the carriers. LPA and PA have unique biophysical properties, and their interconversion might facilitate the fission process either directly or indirectly (via recruitment of proteins that bind to PA, including BARS itself).


Assuntos
Aciltransferases/metabolismo , Oxirredutases do Álcool/metabolismo , Citocinese/fisiologia , Proteínas de Ligação a DNA/metabolismo , Complexo de Golgi/metabolismo , Membranas Intracelulares/metabolismo , Proteínas 14-3-3/metabolismo , Células HeLa , Humanos , Lisofosfolipídeos/metabolismo , Ácidos Fosfatídicos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteína Quinase C/metabolismo , Quinases Ativadas por p21/metabolismo
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