RESUMO
EDEM-1, EDEM-2 and EDEM-3 are key players for the quality control of newly synthesized proteins in the endoplasmic reticulum (ER) by accelerating disposal and degradation of misfolded proteins through ER Associated Degradation (ERAD). Although many previous studies reported the role of individual ERAD components especially in cell-based systems, still little is known about the consequences of ERAD dysfunction under physiological and ER stress conditions in the context of a multicellular organism. Here we report the first individual and combined characterization and functional interplay of EDEM proteins in Caenorhabditis elegans using single, double, and triple mutant combinations. We found that EDEM-2 has a major role in the clearance of misfolded proteins from ER under physiological conditions, whereas EDEM-1 and EDEM-3 roles become prominent under acute ER stress. In contrast to SEL-1 loss, the loss of EDEMs in an intact organism induces only a modest ER stress under physiological conditions. In addition, chronic impairment of EDEM functioning attenuated both XBP-1 activation and up-regulation of the stress chaperone GRP78/BiP, in response to acute ER stress. We also show that pre-conditioning to EDEM loss in acute ER stress restores ER homeostasis and promotes survival by activating ER hormesis. We propose a novel role for EDEM in fine-tuning the ER stress responsiveness that affects ER homeostasis and survival.
Assuntos
Caenorhabditis elegans , Dobramento de Proteína , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Glicoproteínas/metabolismo , Proteínas de Membrana/metabolismoRESUMO
Iba1 (ionized calcium binding adapter protein 1) is a cytoskeleton protein specific only for microglia and macrophages, where it acts as an actin-cross linking protein. Although frequently regarded as a marker of activation, its involvement in cell migration, membrane ruffling, phagocytosis or in microglia remodeling during immunological surveillance of the brain suggest that Iba1 is not a simple cytoskeleton protein, but a signaling molecule involved in specific signaling pathways. In this study we investigated if Iba1 could also represent a drug target, and tested the hypothesis that its specific silencing with customized Iba1-siRNA can modulate microglia functioning. The results showed that Iba1-silenced BV2 microglia migrate less due to reduced proliferation and cell adhesion, while their phagocytic activity and P2x7 functioning was significantly increased. Our data are the proof of concept that Iba1 protein is a new microglia target, which opens a new therapeutic avenue for modulating microglia behavior.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas do Citoesqueleto/metabolismo , Inativação Gênica , Proteínas dos Microfilamentos/metabolismo , Microglia/metabolismo , Animais , Adesão Celular , Contagem de Células , Linhagem Celular , Movimento Celular , Proliferação de Células , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Proteínas Opsonizantes/metabolismo , Fagocitose , RNA Interferente Pequeno/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Reprodutibilidade dos Testes , Zimosan/metabolismoRESUMO
Endoplasmic reticulum (ER)-associated degradation (ERAD) is the main mechanism of targeting ER proteins for degradation to maintain homeostasis, and perturbations of ERAD lead to pathological conditions. ER-degradation enhancing α-mannosidase-like (EDEM1) was proposed to extract terminally misfolded proteins from the calnexin folding cycle and target them for degradation by ERAD. Here, using mass-spectrometry and biochemical methods, we show that EDEM1 is found in auto-regulatory complexes with ERAD components. Moreover, the N-terminal disordered region of EDEM1 mediates protein-protein interaction with misfolded proteins, whilst the absence of this domain significantly impairs their degradation. We also determined that overexpression of EDEM1 can induce degradation, even when proteasomal activity is severely impaired, by promoting the formation of aggregates, which can be further degraded by autophagy. Therefore, we propose that EDEM1 maintains ER homeostasis and mediates ERAD client degradation via autophagy when either dislocation or proteasomal degradation are impaired.
Assuntos
Retículo Endoplasmático/genética , Proteínas de Membrana/genética , Mapas de Interação de Proteínas/genética , Proteólise , Autofagia/genética , Retículo Endoplasmático/metabolismo , Degradação Associada com o Retículo Endoplasmático/genética , Células HEK293 , Células HeLa , Humanos , Espectrometria de Massas , Complexo de Endopeptidases do Proteassoma/genética , Agregados Proteicos/genética , Dobramento de ProteínaRESUMO
We present here data on EDEM3 network of ER resident interactors and the changes induced upon this network by perturbing the early ER N-glycan processing with mannosidase and glucosidase inhibitors. By coupling immunoprecipitation with mass spectrometry we identified EDEM3 interactors and assigned statistical significance to those most abundant ER-residents that might form functional complexes with EDEM3. We further show that this ER interaction network changes in both content and abundance upon treatment with kifunensine (kif) and N-butyldeoxynojirimycin (NB-DNJ) which suggests that when interfering with the N-glycan processing pathway, the functional complexes involving EDEM3 adapt to maintain the cellular homeostasis. In order to increase the scope of EDEM3 network contenders, the set of MS identified species was further supplemented with putative interactors derived from in silico simulations performed with STRING. Finally, the most interesting candidates to this network were further validated by immunoprecipitation coupled with Western Blotting, which strengthened the confidence in the inferred interactions. The data corroborated herein suggest that besides ER residents, EDEM3 interacts also with proteins involved in the ERAD cargo recognition and targeting to degradation translocation into the cytosol, including UBA1 and UBA2 ubiquitinating enzymes. In addition, the results indicate that this network of EDEM3 interactors is highly sensitive to interfering with early ER N-glycan processing.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica/fisiologia , Manosidases/metabolismo , Polissacarídeos/metabolismo , Transdução de Sinais/fisiologia , Ubiquitinação/fisiologia , Linhagem Celular , Humanos , alfa-ManosidaseRESUMO
Due to their excellent mechanical and biocompatibility properties, titanium-based implants are successfully used as biomedical devices. However, when new bone formation fails for different reasons, impaired fracture healing becomes a clinical problem and affects the patient's quality of life. We aimed to design a new bioactive surface of titanium implants with a synergetic PEG biopolymer-based composition for gradual delivery of growth factors (FGF2, VEGF, and BMP4) during bone healing. The optimal architecture of non-cytotoxic polymeric coatings deposited by dip coating under controlled parameters was assessed both in cultured cells and in a rat tibial defect model (100% viability). Notably, the titanium adsorbed polymer matrix induced an improved healing process when compared with the individual action of each biomolecules. High-performance mass spectrometry analysis demonstrated that recovery after a traumatic event is governed by specific differentially regulated proteins, acting in a coordinated response to the external stimulus. Predicted protein interactions shown by STRING analysis were well organized in hub-based networks related with response to chemical, wound healing and response to stress pathways. The proposed functional polymer coatings of the titanium implants demonstrated the significant improvement of bone healing process after injury.
Assuntos
Regeneração Óssea/efeitos dos fármacos , Próteses e Implantes , Tíbia/fisiopatologia , Titânio/química , Actinas/química , Animais , Biopolímeros , Adesão Celular , Proliferação de Células , Materiais Revestidos Biocompatíveis/química , Biologia Computacional , Consolidação da Fratura/efeitos dos fármacos , Masculino , Espectrometria de Massas , Células-Tronco Mesenquimais , Microscopia de Fluorescência , Osseointegração/efeitos dos fármacos , Desenho de Prótese , Proteômica , Ratos , Ratos Wistar , Propriedades de SuperfícieRESUMO
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 & inibidoresRESUMO
An appreciation of the functional properties of the cytoplasmic fatty acid binding protein 4 (FABP4) has advanced with the recent demonstration that an extracellular form secreted by adipocytes regulates a wide range of physiological functions. Little, however, is known about the mechanisms that mediate the unconventional secretion of FABP4. Here, we demonstrate that FABP4 secretion is mediated by a membrane-bounded compartment, independent of the conventional endoplasmic reticulum-Golgi secretory pathway. We show that FABP4 secretion is also independent of GRASP proteins, autophagy, and multivesicular bodies but involves enclosure within endosomes and secretory lysosomes. We highlight the physiological significance of this pathway with the demonstration that an increase in plasma levels of FABP4 is inhibited by chloroquine treatment of mice. These findings chart the pathway of FABP4 secretion and provide a potential therapeutic means to control metabolic disorders associated with its dysregulated secretion.