RESUMO
The Endoplasmic Reticulum (ER) is a membranous organelle with diverse structural and functional domains. Peripheral ER includes interconnected tubules, and dense tubular arrays called "ER matrices" together with bona fide flat cisternae. Transitions between these states are regulated by membrane-associated proteins and cytosolic factors. Recently, the small GTPases Rab10 and Rab18 were reported to control ER shape by regulating ER dynamics and fusion. Here, we present evidence that another Rab protein, Rab7a, modulates the ER morphology by controlling the ER homeostasis and ER stress. Indeed, inhibition of Rab7a expression by siRNA or expression of the dominant negative mutant Rab7aT22â¯N, leads to enlargement of sheet-like ER structures and spreading towards the cell periphery. Notably, such alterations are ascribable neither to a direct modulation of the ER shaping proteins Reticulon-4b and CLIMP63, nor to interactions with Protrudin, a Rab7a-binding protein known to affect the ER organization. Conversely, depletion of Rab7a leads to basal ER stress, in turn causing ER membrane expansion. Both ER enlargement and basal ER stress are reverted in rescue experiments by Rab7a re-expression, as well as by the ER chemical chaperone tauroursodeoxycholic acid (TUDCA). Collectively, these findings reveal a new role of Rab7a in ER homeostasis, and indicate that genetic and pharmacological ER stress manipulation may restore ER morphology in Rab7a silenced cells.
Assuntos
Estresse do Retículo Endoplasmático/genética , Retículo Endoplasmático/genética , Homeostase/genética , Proteínas rab de Ligação ao GTP/genética , Regulação da Expressão Gênica , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteínas Nogo/genética , Ligação Proteica , RNA Interferente Pequeno/genética , Ácido Tauroquenodesoxicólico/farmacologia , Proteínas de Transporte Vesicular/genética , proteínas de unión al GTP Rab7RESUMO
Cystatin B (CSTB), an inhibitor of the cysteine proteases, belongs to the cathepsin family and it is known to interact with a number of proteins involved in cytoskeletal organization. CSTB has an intrinsic tendency to form aggregates depending on the redox environment. The gene encoding for CSTB is frequently mutated in association with the rare neurodegenerative condition progressive myoclonus epilepsy. Increased levels of CSTB have been observed in the spinal cord of transgenic mice modeling SOD1-linked familial amyotrophic lateral sclerosis, a fatal neurodegenerative disease affecting motoneurons. In the present study, we have investigated the relationship occurring between the expression of SOD1 and CSTB either wild-type or double-cysteine substitution mutant (Cys 3 and Cys 64). Whether or not there is a physical interaction between the two proteins was also investigated in overexpression experiments using a human neuroblastoma cell line and mouse-immortalized motoneurons. Here we report evidences for a reciprocal influence of CSTB and SOD1 at the gene expression level and for a direct interaction of the two proteins.
Assuntos
Cistatina B/metabolismo , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Superóxido Dismutase/metabolismo , Animais , Western Blotting , Linhagem Celular , Células Clonais , Cistatina B/genética , Regulação da Expressão Gênica , Humanos , Imunoprecipitação , Camundongos , Proteínas Mutantes/metabolismo , Ligação Proteica , Ratos , Solubilidade , Superóxido Dismutase/genética , Superóxido Dismutase-1RESUMO
The role of autophagy is known to be highly complex and context-dependent, leading to both cancer suppression and progression in several tumors including melanoma, breast and prostate cancer. In the present review, recent advances in an understanding of the involvement of autophagy in prostate cancer treatment are described. The regulatory effects of androgens on prostate cancer cell autophagy are particularly discussed in order to highlight the effects of autophagy modulation during androgen deprivation. A critical evaluation of the studies examined in the present review suggests the attractive possibility of autophagy inhibition combined with hormonal therapy as a promising approach for prostate cancer treatment.
Assuntos
Androgênios/farmacologia , Androgênios/uso terapêutico , Autofagia/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Animais , Progressão da Doença , Humanos , Imunidade/efeitos dos fármacos , Masculino , Modelos Biológicos , Neoplasias da Próstata/imunologiaRESUMO
TNF-alpha levels in prostate cancer correlate with the extent of disease and are significantly elevated in the metastatic stage. TNF receptor superfamily controls two distinct signalling cascades, leading to opposite effects, i.e. apoptosis and survival; in prostate cancer TNF-alpha-mediated signalling induces cell survival and resistance to therapy. The apoptosis of prostate epithelial cancer cells LNCaP and PC3 was investigated upon treatment with the autophagy inhibitor 3-methyladenine and the autophagy inducer rapamycin, in combination with TNF-alpha. Cells were exposed to these molecules for 18, 24 and 48 h. Autophagy was assessed via LC3 Western blot analysis; propidium iodide and TUNEL stainings followed by flow cytometry or caspase-8 and caspase-3 activation assays were performed to evaluate apoptosis. TNF-alpha-induced apoptosis was potentiated by 3-methyladenine in the androgen-responsive LNCaP cells, whereas no effect was observed in the androgen-insensitive PC3 cells. Interestingly such pro-apoptosis effect in LNCaP cells was associated with reduced c-Flip levels through proteasomal degradation via increased reactive oxygen species production and p38 activation; such c-Flip reduction was reversed in the presence of either the proteasome inhibitor MG132 or the reactive oxygen species scavenger N-acetyl-cysteine. Conversely in PC3 but not in LNCaP cells, rapamycin stimulated TNF-alpha-dependent apoptosis; such effect was associated with reduced c-Flip promoter activity and FoxO3a activation. We conclude that TNF-alpha-induced apoptosis may be potentiated, in prostate cancer epithelial cells, through autophagy modulators. Increased sensitivity to TNF-alpha-dependent apoptosis correlates with reduced c-Flip levels which are consequent to a post-transcriptional and a transcriptional mechanism in LNCaP and PC3 cells respectively.
Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Células Epiteliais/patologia , Neoplasias da Próstata/patologia , Fator de Necrose Tumoral alfa/farmacologia , Adenina/análogos & derivados , Western Blotting , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Ativação Enzimática/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/metabolismo , Humanos , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Regiões Promotoras Genéticas/genética , Neoplasias da Próstata/enzimologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sirolimo/farmacologia , Transcrição Gênica/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Mitophagy is a highly specialized process to remove dysfunctional or superfluous mitochondria through the macroautophagy/autophagy pathway, aimed at protecting cells from the damage of disordered mitochondrial metabolism and apoptosis induction. PINK1, a neuroprotective protein mutated in autosomal recessive Parkinson disease, has been implicated in the activation of mitophagy by selectively accumulating on depolarized mitochondria, and promoting PARK2/Parkin translocation to them. While these steps have been characterized in depth, less is known about the process and site of autophagosome formation upon mitophagic stimuli. A previous study reported that, in starvation-induced autophagy, the proautophagic protein BECN1/Beclin1 (which we previously showed to interact with PINK1) relocalizes at specific regions of contact between the endoplasmic reticulum (ER) and mitochondria called mitochondria-associated membranes (MAM), from which the autophagosome originates. Here we show that, following mitophagic stimuli, autophagosomes also form at MAM; moreover, endogenous PINK1 and BECN1 were both found to relocalize at MAM, where they promoted the enhancement of ER-mitochondria contact sites and the formation of omegasomes, that represent autophagosome precursors. PARK2 was also enhanced at MAM following mitophagy induction. However, PINK1 silencing impaired BECN1 enrichment at MAM independently of PARK2, suggesting a novel role for PINK1 in regulating mitophagy. MAM have been recently implicated in many key cellular events. In this light, the observed prevalent localization of PINK1 at MAM may well explain other neuroprotective activities of this protein, such as modulation of mitochondrial calcium levels, mitochondrial dynamics, and apoptosis.
Assuntos
Autofagossomos/metabolismo , Proteína Beclina-1/metabolismo , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Mitofagia , Proteínas Quinases/metabolismo , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Compartimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Regulação para Baixo , HumanosRESUMO
Cellular-Flice-like inhibitory protein (c-FLIP) is an apoptosis modulator known to inhibit the extrinsic apoptotic pathway thus blocking Caspase-8 processing in the Death Inducing Signalling Complex (DISC). We previously demonstrated that c-FLIP localizes at the endoplasmic reticulum (ER) and that c-FLIP-deficient mouse embryonic fibroblasts (MEFs) display an enlarged ER morphology. In the present study, we have addressed the consequences of c-FLIP ablation in the ER stress response by investigating the effects of pharmacologically-induced ER stress in Wild Type (WT) and c-FLIP-/- MEFs. Surprisingly, c-FLIP-/- MEFs were found to be strikingly more resistant than WT MEFs to ER stress-mediated apoptosis. Analysis of Unfolded Protein Response (UPR) pathways revealed that Pancreatic ER Kinase (PERK) and Inositol-Requiring Enzyme 1 (IRE1) branch signalling is compromised in c-FLIP-/- cells when compared with WT cells. We found that c-FLIP modulates the PERK pathway by interfering with the activity of the serine threonine kinase AKT. Indeed, c-FLIP-/- MEFs display higher levels of active AKT than WT MEFs upon ER stress, while treatment with a specific AKT inhibitor of c-FLIP-/- MEFs subjected to ER stress restores the PERK but not the IRE1 pathway. Importantly, the AKT inhibitor or dominant negative AKT transfection sensitizes c-FLIP-/- cells to ER stress-induced cell death while the expression of a constitutively active AKT reduces WT cells sensitivity to ER stress-induced death. Thus, our results demonstrate that c-FLIP modulation of AKT activity is crucial in controlling PERK signalling and sensitivity to ER stress, and highlight c-FLIP as a novel molecular player in PERK and IRE1-mediated ER stress response.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Estresse do Retículo Endoplasmático , Animais , Apoptose , Autofagia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/deficiência , Embrião de Mamíferos/citologia , Ativação Enzimática , Fibroblastos/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , eIF-2 Quinase/metabolismoRESUMO
Parkinson disease (PD) is a multifactorial neurodegenerative disease characterized by the progressive loss of specific neuronal populations and accumulation of Lewy bodies in the brain, leading to motor and non-motor symptoms. In a small subset of patients, PD is dominantly or recessively inherited, while a number of susceptibility genetic loci have been identified through genome wide association studies. The discovery of genes mutated in PD and functional studies on their protein products have provided new insights into the molecular events leading to neurodegeneration, suggesting that few interconnected molecular pathways may be deranged in all forms of PD, triggering neuronal loss. Here, we summarize the most relevant findings implicating the main PD-related proteins in biological processes such as mitochondrial dysfunction, misfolded protein damage, alteration of cellular clearance systems, abnormal calcium handling and altered inflammatory response, which represent key targets for neuroprotection.
Assuntos
Autofagia/genética , Estudos de Associação Genética/métodos , Doença de Parkinson/diagnóstico , Doença de Parkinson/genética , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla/métodos , HumanosRESUMO
Polyinosinic-polycytidylic acid (poly(I:C)) is a synthetic double-stranded RNA (dsRNA) analog able to induce apoptosis in different cancer cells by the activation of toll-like receptor 3 (TLR3) and cytosolic helicases, retinoic acid inducible gene I (RIG-I) like receptors. In this work, we have synthesized and thoroughly characterized a core-shell liposome-silica hybrid (LSH) nanoparticle (NP) made of a silica core surrounded by a multicomponent cationic lipid bilayer. In view of in vivo applications, a variant with polyethyleneglycol (PEG) grafted onto the lipid surface was also synthesized. Poly(I:C)-loaded LSH NPs were characterized and optimized in terms of their chemical-physical properties by using dynamic light scattering (DLS), micro-electrophoresis and transmission electron microscopy (TEM). The ability of this new technology to kill cancer cells was validated in PC3 prostate cancer and MCF7 breast cancer cells by MTT proliferation assay, flow cytometry and fluorescence confocal microscopy. We found that negatively charged poly(I:C)-loaded LSH NPs are more efficient than their liposome counterpart in eliminating cancer cells, thus representing excellent candidates for both in vitro and in vivo drug delivery applications.