RESUMO
Ischaemic diseases such as critical limb ischaemia and myocardial infarction affect millions of people worldwide1. Transplanting endothelial cells (ECs) is a promising therapy in vascular medicine, but engrafting ECs typically necessitates co-transplanting perivascular supporting cells such as mesenchymal stromal cells (MSCs), which makes clinical implementation complicated2,3. The mechanisms that enable MSCs to facilitate EC engraftment remain elusive. Here we show that, under cellular stress, MSCs transfer mitochondria to ECs through tunnelling nanotubes, and that blocking this transfer impairs EC engraftment. We devised a strategy to artificially transplant mitochondria, transiently enhancing EC bioenergetics and enabling them to form functional vessels in ischaemic tissues without the support of MSCs. Notably, exogenous mitochondria did not integrate into the endogenous EC mitochondrial pool, but triggered mitophagy after internalization. Transplanted mitochondria co-localized with autophagosomes, and ablation of the PINK1-Parkin pathway negated the enhanced engraftment ability of ECs. Our findings reveal a mechanism that underlies the effects of mitochondrial transfer between mesenchymal and endothelial cells, and offer potential for a new approach for vascular cell therapy.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Células Endoteliais , Isquemia , Mitocôndrias , Mitofagia , Animais , Humanos , Masculino , Camundongos , Autofagossomos/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células Endoteliais/transplante , Metabolismo Energético , Células Endoteliais da Veia Umbilical Humana/metabolismo , Isquemia/metabolismo , Isquemia/terapia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos Nus , Mitocôndrias/metabolismo , Mitocôndrias/transplante , Proteínas Quinases/deficiência , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/metabolismo , Terapia Baseada em Transplante de Células e Tecidos/métodosRESUMO
Inorganic phosphate (Pi) is one of the essential molecules for life. However, little is known about intracellular Pi metabolism and signalling in animal tissues1. Following the observation that chronic Pi starvation causes hyperproliferation in the digestive epithelium of Drosophila melanogaster, we determined that Pi starvation triggers the downregulation of the Pi transporter PXo. In line with Pi starvation, PXo deficiency caused midgut hyperproliferation. Interestingly, immunostaining and ultrastructural analyses showed that PXo specifically marks non-canonical multilamellar organelles (PXo bodies). Further, by Pi imaging with a Förster resonance energy transfer (FRET)-based Pi sensor2, we found that PXo restricts cytosolic Pi levels. PXo bodies require PXo for biogenesis and undergo degradation following Pi starvation. Proteomic and lipidomic characterization of PXo bodies unveiled their distinct feature as an intracellular Pi reserve. Therefore, Pi starvation triggers PXo downregulation and PXo body degradation as a compensatory mechanism to increase cytosolic Pi. Finally, we identified connector of kinase to AP-1 (Cka), a component of the STRIPAK complex and JNK signalling3, as the mediator of PXo knockdown- or Pi starvation-induced hyperproliferation. Altogether, our study uncovers PXo bodies as a critical regulator of cytosolic Pi levels and identifies a Pi-dependent PXo-Cka-JNK signalling cascade controlling tissue homeostasis.
Assuntos
Drosophila melanogaster , Homeostase , Organelas , Fosfatos , Animais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/metabolismo , Organelas/metabolismo , Fosfatos/deficiência , Fosfatos/metabolismo , Proteômica , Transferência Ressonante de Energia de Fluorescência , Lipidômica , Citosol/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismoRESUMO
Dysregulated mTORC1 signaling alters a wide range of cellular processes, contributing to metabolic disorders and cancer. Defining the molecular details of downstream effectors is thus critical for uncovering selective therapeutic targets. We report that mTORC1 and its downstream kinase S6K enhance eIF4A/4B-mediated translation of Wilms' tumor 1-associated protein (WTAP), an adaptor for the N6-methyladenosine (m6A) RNA methyltransferase complex. This regulation is mediated by 5' UTR of WTAP mRNA that is targeted by eIF4A/4B. Single-nucleotide-resolution m6A mapping revealed that MAX dimerization protein 2 (MXD2) mRNA contains m6A, and increased m6A modification enhances its degradation. WTAP induces cMyc-MAX association by suppressing MXD2 expression, which promotes cMyc transcriptional activity and proliferation of mTORC1-activated cancer cells. These results elucidate a mechanism whereby mTORC1 stimulates oncogenic signaling via m6A RNA modification and illuminates the WTAP-MXD2-cMyc axis as a potential therapeutic target for mTORC1-driven cancers.
Assuntos
Adenosina/análogos & derivados , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Estabilidade de RNA , Adenosina/metabolismo , Animais , Sequência de Bases , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Fatores de Iniciação em Eucariotos/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Modelos Biológicos , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Processamento de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Transdução de SinaisRESUMO
Mechanistic Target of Rapamycin Complex 1 (mTORC1) is a central regulator of cell growth and metabolism that senses and integrates nutritional and environmental cues with cellular responses. Recent studies have revealed critical roles of mTORC1 in RNA biogenesis and processing. Here, we find that the m6A methyltransferase complex (MTC) is a downstream effector of mTORC1 during autophagy in Drosophila and human cells. Furthermore, we show that the Chaperonin Containing Tailless complex polypeptide 1 (CCT) complex, which facilitates protein folding, acts as a link between mTORC1 and MTC. The mTORC1 activates the chaperonin CCT complex to stabilize MTC, thereby increasing m6A levels on the messenger RNAs encoding autophagy-related genes, leading to their degradation and suppression of autophagy. Altogether, our study reveals an evolutionarily conserved mechanism linking mTORC1 signaling with m6A RNA methylation and demonstrates their roles in suppressing autophagy.
Assuntos
Autofagia , Proteínas de Drosophila/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Metiltransferases/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Metilação , Metiltransferases/genética , Receptores Nucleares Órfãos , Estabilidade de RNA , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Repressoras/genéticaRESUMO
The management of persistent postoperative pain still remains a clinical challenge currently. Although ropivacaine (RVC) is widely used for postoperative analgesia as a local anesthetic, the short half-life makes it difficult to achieve the desired duration of analgesia. Herein, a RVC sustained-release microspheres encapsulated by zeolite imidazole framework-8 (RVC@ZIF-8) was synthesized for the first time, which prolonged the sustained-release of RVC and decreased the resulting drug toxicity. RVC can continuously release inâ vitro for at least 96â h with high drug loading of 30.6 % and RVC@ZIF-8 had excellent biocompatibility and low cytotoxicity. In sciatic nerve block model, the sensory block time of RVC@ZIF-8 was significantly prolonged compared with RVC, achieving more than 72â h post injection and no inflammation or lesion were found. Based on high drug loading, ideal sustained-release and superior biological safety, RVC@ZIF-8 will be a novel delivery material for local anesthetic with potential application.
Assuntos
Anestésicos Locais , Zeolitas , Ropivacaina , Amidas , Preparações de Ação Retardada , Microesferas , ImidazóisRESUMO
Antibacterial photodynamic therapy (APDT) has received considerable attention owing to its superiority. ZIF-8 was used to address the poor stability of the photosensitizer Rose Bengal (RB) encapsulation to synthesize RB@ZIF-8 NPs, which were doped into a composite film with poly (ϵ-caprolactone) (PCL) and polyvinyl alcohol-quaternary ammonium chitosan (PVA-QCS) as substrates to form composite films (PQZ). The composite films exhibited excellent photodynamic sterilization and good resistance to bacterial adhesion. The tensile strength of the film increased to 43.4â MPa, which was approximately 1.8â times that of the PCL film. With the addition of SiO2 and RB@ZIF-8 NPs, the film exhibited water repellency and UV-blocking properties. RAW264.7â cells were selected using the MTT method to confirm that the composite films had excellent biocompatibility and had no significant inhibitory effect on cell growth and reproduction. PQZ multifunctional composite films show potential as novel APDT antimicrobial materials for food packaging.
Assuntos
Anti-Infecciosos , Quitosana , Dióxido de Silício , Antibacterianos/farmacologia , Antibacterianos/química , Poliésteres , Anti-Infecciosos/química , Quitosana/química , Embalagem de AlimentosRESUMO
Metabolites are increasingly appreciated for their roles as signaling molecules. To dissect the roles of metabolites, it is essential to understand their signaling pathways and their enzymatic regulations. From an RNA interference (RNAi) screen for regulators of intestinal stem cell (ISC) activity in the Drosophila midgut, we identified adenosine receptor (AdoR) as a top candidate gene required for ISC proliferation. We demonstrate that Ras/MAPK and Protein Kinase A (PKA) signaling act downstream of AdoR and that Ras/MAPK mediates the major effect of AdoR on ISC proliferation. Extracellular adenosine, the ligand for AdoR, is a small metabolite that can be released by various cell types and degraded in the extracellular space by secreted adenosine deaminase. Interestingly, down-regulation of adenosine deaminase-related growth factor A (Adgf-A) from enterocytes is necessary for extracellular adenosine to activate AdoR and induce ISC overproliferation. As Adgf-A expression and its enzymatic activity decrease following tissue damage, our study provides important insights into how the enzymatic regulation of extracellular adenosine levels under tissue-damage conditions facilitates ISC proliferation.
Assuntos
Adenosina Desaminase/metabolismo , Proteínas de Drosophila/metabolismo , Enterócitos/fisiologia , Células-Tronco Multipotentes/fisiologia , Receptores Purinérgicos P1/metabolismo , Adenosina/metabolismo , Animais , Animais Geneticamente Modificados , Diferenciação Celular , Proliferação de Células , Regulação para Baixo , Drosophila , Proteínas de Drosophila/genética , Técnicas de Introdução de Genes , Técnicas de Silenciamento de Genes , Sistema de Sinalização das MAP Quinases/genética , Interferência de RNA , Receptores Purinérgicos P1/genéticaRESUMO
We propose a method for diagnosis of cirrhosis and hepatocellular carcinoma (HCC) by using a terahertz (THz) metamaterial (MM) biosensor. The biosensor has a resonance frequency at about 0.801 THz and can measure the concentration of alpha-fetoprotein (AFP) in serum. The sensitivity of the sensor is 124 GHz/refractive index unit (RIU), and the quality-factor (Q) is 6.913, respectively. When the surface of the biosensor is covered with healthy serum (AFP≤7ng/mL), the maximum resonance frequency shift is 50 GHz. However, when it is covered with serum from patients with cirrhosis and early HCC (AFP>7ng/mL), the resonance frequency shift is more than 59 GHz. Positive correlation exists between the frequency shift of the biosensor and serum levels of the AFP in the HCC patients. This study provides a method for quick diagnosis and prediction of cirrhosis and HCC.
Assuntos
Técnicas Biossensoriais , Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/patologia , alfa-Fetoproteínas , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/patologia , Cirrose Hepática , Biomarcadores TumoraisRESUMO
The mechanistic target of rapamycin (mTOR) complex 1, mTORC1, integrates nutrient and growth factor signals with cellular responses and plays critical roles in regulating cell growth, proliferation, and lifespan. mTORC1 signaling has been reported as a central regulator of autophagy by modulating almost all aspects of the autophagic process, including initiation, expansion, and termination. An increasing number of studies suggest that mTORC1 and autophagy are critical for the physiological function of skeletal muscle and are involved in diverse muscle diseases. Here, we review recent insights into the essential roles of mTORC1 and autophagy in skeletal muscles and their implications in human muscle diseases. Multiple inhibitors targeting mTORC1 or autophagy have already been clinically approved, while others are under development. These chemical modulators that target the mTORC1/autophagy pathways represent promising potentials to cure muscle diseases.
Assuntos
Nutrientes , Transdução de Sinais , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Autofagia/fisiologia , Músculo Esquelético/metabolismoRESUMO
N6-methyladenosine (m6A), the most abundant chemical modification in eukaryotic mRNA, has been implicated in Drosophila sex determination by modifying Sex-lethal (Sxl) pre-mRNA and facilitating its alternative splicing. Here, we identify a sex determination gene, CG7358, and rename it xio according to its loss-of-function female-to-male transformation phenotype. xio encodes a conserved ubiquitous nuclear protein of unknown function. We show that Xio colocalizes and interacts with all previously known m6A writer complex subunits (METTL3, METTL14, Fl(2)d/WTAP, Vir/KIAA1429, and Nito/Rbm15) and that loss of xio is associated with phenotypes that resemble other m6A factors, such as sexual transformations, Sxl splicing defect, held-out wings, flightless flies, and reduction of m6A levels. Thus, Xio encodes a member of the m6A methyltransferase complex involved in mRNA modification. Since its ortholog ZC3H13 (or KIAA0853) also associates with several m6A writer factors, the function of Xio in the m6A pathway is likely evolutionarily conserved.
Assuntos
Adenosina/análogos & derivados , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Metiltransferases/metabolismo , Precursores de RNA/metabolismo , RNA/metabolismo , Processos de Determinação Sexual/genética , Adenosina/metabolismo , Processamento Alternativo , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Feminino , Masculino , Metiltransferases/genética , RNA/genética , Precursores de RNA/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Autophagy is a membrane-mediated degradation process of macromolecule recycling. Although the formation of double-membrane degradation vesicles (autophagosomes) is known to have a central role in autophagy, the mechanism underlying this process remains elusive. The serine/threonine kinase Atg1 has a key role in the induction of autophagy. In this study, we show that overexpression of Drosophila Atg1 promotes the phosphorylation-dependent activation of the actin-associated motor protein myosin II. A novel myosin light chain kinase (MLCK)-like protein, Spaghetti-squash activator (Sqa), was identified as a link between Atg1 and actomyosin activation. Sqa interacts with Atg1 through its kinase domain and is a substrate of Atg1. Significantly, myosin II inhibition or depletion of Sqa compromised the formation of autophagosomes under starvation conditions. In mammalian cells, we found that the Sqa mammalian homologue zipper-interacting protein kinase (ZIPK) and myosin II had a critical role in the regulation of starvation-induced autophagy and mammalian Atg9 (mAtg9) trafficking when cells were deprived of nutrients. Our findings provide evidence of a link between Atg1 and the control of Atg9-mediated autophagosome formation through the myosin II motor protein.
Assuntos
Autofagia/fisiologia , Proteínas de Drosophila/fisiologia , Miosina Tipo II/metabolismo , Fagossomos/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Inanição/metabolismo , Animais , Animais Geneticamente Modificados , Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Células Cultivadas , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Expressão Gênica/fisiologia , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Quinase de Cadeia Leve de Miosina/genética , Quinase de Cadeia Leve de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/fisiologia , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Inanição/genética , Distribuição TecidualRESUMO
The commonality between various muscle diseases is the loss of muscle mass, function, and regeneration, which severely restricts mobility and impairs the quality of life. With muscle stem cells (MuSCs) playing a key role in facilitating muscle repair, targeting regulators of muscle regeneration has been shown to be a promising therapeutic approach to repair muscles. However, the underlying molecular mechanisms driving muscle regeneration are complex and poorly understood. Here, we identified a new regulator of muscle regeneration, Deaf1 (Deformed epidermal autoregulatory factor-1) - a transcriptional factor downstream of foxo signaling. We showed that Deaf1 is transcriptionally repressed by FOXOs and that DEAF1 targets to Pik3c3 and Atg16l1 promoter regions and suppresses their expression. Deaf1 depletion therefore induces macroautophagy/autophagy, which in turn blocks MuSC survival and differentiation. In contrast, Deaf1 overexpression inactivates autophagy in MuSCs, leading to increased protein aggregation and cell death. The fact that Deaf1 depletion and its overexpression both lead to defects in muscle regeneration highlights the importance of fine tuning DEAF1-regulated autophagy during muscle regeneration. We further showed that Deaf1 expression is altered in aging and cachectic MuSCs. Manipulation of Deaf1 expression can attenuate muscle atrophy and restore muscle regeneration in aged mice or mice with cachectic cancers. Together, our findings unveil an evolutionarily conserved role for DEAF1 in muscle regeneration, providing insights into the development of new therapeutic strategies against muscle atrophy.Abbreviations: DEAF1: Deformed epidermal autoregulatory factor-1; FOXO: Forkhead box O; MuSC: Muscle Stem Cell; PAX7: Paired box 7; PIK3C3: Phosphatidylinositol 3-kinase catalytic subunit type 3.
RESUMO
Although stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics was first developed as a cell culture-based technique, stable isotope-labeled amino acids have since been successfully introduced in vivo into select multicellular model organisms by manipulating the feeding diets. An earlier study by others has demonstrated that heavy lysine labeled Drosophila melanogaster can be derived by feeding with an exclusive heavy lysine labeled yeast diet. In this work, we have further evaluated the use of heavy lysine and/or arginine for metabolic labeling of fruit flies, with an aim to determine its respective quantification accuracy and versatility. In vivo conversion of heavy lysine and/or heavy arginine to several nonessential amino acids was observed in labeled flies, leading to distorted isotope pattern and underestimated heavy to light ratio. These quantification defects can nonetheless be rectified at protein level using the normalization function. The only caveat is that such a normalization strategy may not be suitable for every biological application, particularly when modified peptides need to be individually quantified at peptide level. In such cases, we showed that peptide ratios calculated from the summed intensities of all isotope peaks are less affected by the heavy amino acid conversion and therefore less sequence-dependent and more reliable. Applying either the single Lys8 or double Lys6/Arg10 metabolic labeling strategy to flies, we quantitatively mapped the proteomic changes during the onset of metamorphosis and upon amino acid deprivation. The expression of a number of steroid hormone 20-hydroxyecdysone regulated proteins was found to be changed significantly during larval-pupa transition, while several subunits of the V-ATPase complex and components regulating actomyosin were up-regulated under starvation-induced autophagy conditions.
Assuntos
Aminoácidos/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteoma/metabolismo , Aminoácidos/química , Animais , Proteínas de Drosophila/química , Drosophila melanogaster/crescimento & desenvolvimento , Corpo Adiposo/crescimento & desenvolvimento , Corpo Adiposo/metabolismo , Privação de Alimentos , Marcação por Isótopo/métodos , Masculino , Metamorfose Biológica , Proteoma/química , Proteômica , Pupa/crescimento & desenvolvimento , Pupa/metabolismo , Estresse Fisiológico , Espectrometria de Massas em TandemRESUMO
Endonuclease G (ENDOG), a nuclear-encoded mitochondrial intermembrane space protein, is well known to be translocated into the nucleus during apoptosis. Recent studies have shown that ENDOG might enter the mitochondrial matrix to regulate mitochondrial genome cleavage and replication. However, little is known about the role of ENDOG in the cytosol. Our previous work showed that cytoplasmic ENDOG competitively binds with 14-3-3γ, which released TSC2 to repress mTORC1 signaling and induce autophagy. Here, we demonstrate that cytoplasmic ENDOG could also release Rictor from 14-3-3γ to activate the mTORC2-AKT-ACLY axis, resulting in acetyl-CoA production. Importantly, we observe that ENDOG could translocate to the ER, bind with Bip, and release IRE1a/PERK to activate the endoplasmic reticulum stress response, promoting lipid synthesis. Taken together, we demonstrate that loss of ENDOG suppresses acetyl-CoA production and lipid synthesis, along with reducing endoplasmic reticulum stress, which eventually alleviates high-fat diet-induced nonalcoholic fatty liver disease in female mice.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Feminino , Camundongos , Animais , Proteínas Proto-Oncogênicas c-akt/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina , Citosol/metabolismo , Acetilcoenzima A , Estresse do Retículo Endoplasmático , Lipídeos , Apoptose/genéticaRESUMO
Generating reference maps of interactome networks illuminates genetic studies by providing a protein-centric approach to finding new components of existing pathways, complexes, and processes. We apply state-of-the-art methods to identify binary protein-protein interactions (PPIs) for Drosophila melanogaster. Four all-by-all yeast two-hybrid (Y2H) screens of > 10,000 Drosophila proteins result in the 'FlyBi' dataset of 8723 PPIs among 2939 proteins. Testing subsets of data from FlyBi and previous PPI studies using an orthogonal assay allows for normalization of data quality; subsequent integration of FlyBi and previous data results in an expanded binary Drosophila reference interaction network, DroRI, comprising 17,232 interactions among 6511 proteins. We use FlyBi data to generate an autophagy network, then validate in vivo using autophagy-related assays. The deformed wings (dwg) gene encodes a protein that is both a regulator and a target of autophagy. Altogether, these resources provide a foundation for building new hypotheses regarding protein networks and function.
Assuntos
Proteínas de Drosophila , Mapas de Interação de Proteínas , Animais , Mapas de Interação de Proteínas/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Drosophila/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Mapeamento de Interação de Proteínas/métodos , Técnicas do Sistema de Duplo-HíbridoRESUMO
BACKGROUND: Autophagy and molecular chaperones both regulate protein homeostasis and maintain important physiological functions. Atg7 (autophagy-related gene 7) and Hsp27 (heat shock protein 27) are involved in the regulation of neurodegeneration and aging. However, the genetic connection between Atg7 and Hsp27 is not known. METHODS: The appearances of the fly eyes from the different genetic interactions with or without polyglutamine toxicity were examined by light microscopy and scanning electronic microscopy. Immunofluorescence was used to check the effect of Atg7 and Hsp27 knockdown on the formation of autophagosomes. The lifespan of altered expression of Hsp27 or Atg7 and that of the combination of the two different gene expression were measured. RESULTS: We used the Drosophila eye as a model system to examine the epistatic relationship between Hsp27 and Atg7. We found that both genes are involved in normal eye development, and that overexpression of Atg7 could eliminate the need for Hsp27 but Hsp27 could not rescue Atg7 deficient phenotypes. Using a polyglutamine toxicity assay (41Q) to model neurodegeneration, we showed that both Atg7 and Hsp27 can suppress weak, toxic effect by 41Q, and that overexpression of Atg7 improves the worsened mosaic eyes by the knockdown of Hsp27 under 41Q. We also showed that overexpression of Atg7 extends lifespan and the knockdown of Atg7 or Hsp27 by RNAi reduces lifespan. RNAi-knockdown of Atg7 expression can block the extended lifespan phenotype by Hsp27 overexpression, and overexpression of Atg7 can extend lifespan even under Hsp27 knockdown by RNAi. CONCLUSIONS: We propose that Atg7 acts downstream of Hsp27 in the regulation of eye morphology, polyglutamine toxicity, and lifespan in Drosophila.
Assuntos
Proteínas de Drosophila/genética , Drosophila , Olho , Proteínas de Choque Térmico HSP27/genética , Animais , Autofagia/genética , Proteína 7 Relacionada à Autofagia , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Epistasia Genética/genética , Olho/anatomia & histologia , Olho/crescimento & desenvolvimento , Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Choque Térmico HSP27/fisiologia , Peptídeos/toxicidade , Interferência de RNARESUMO
Lung cancer is the leading cause of cancer death in the world. In particular, non-small-cell lung cancer (NSCLC) represents the majority of the lung cancer population. Advances in DNA sequencing technologies have significantly contributed to revealing the roles, functions and mechanisms of gene mutations. However, the driver mutations that cause cancers and their pathologies remain to be explored. Here, we performed next-generation sequencing (NGS) on tumor tissues isolated from 314 Chinese NSCLC patients and established the mutational landscape in NSCLC. Among 656 mutations, we identified TP53-p.Glu358Val as a driver mutation in lung cancer and found that it activates mitophagy to sustain cancer cell growth. In support of this finding, mice subcutaneously implanted with NSCLC cells expressing TP53-p.Glu358Val developed larger tumors compared to wild-type cells. The pharmaceutical inhibition of autophagy/mitophagy selectively suppresses the cell proliferation of TP53-null or TP53-p.Glu358Val-expressing lung cancer cells. Together, our study characterizes a new TP53 mutation identified from Chinese lung cancer patients and uncovers its roles in regulating mitophagy, providing a new insight into NSCLC treatment.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Animais , Camundongos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Genes p53 , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mitofagia/genética , Mutação/genética , Proteína Supressora de Tumor p53/genética , HumanosRESUMO
Endonuclease G (ENDOG), a mitochondrial nuclease, is known to participate in many cellular processes, including apoptosis and paternal mitochondrial elimination, while its role in autophagy remains unclear. Here, we report that ENDOG released from mitochondria promotes autophagy during starvation, which we find to be evolutionally conserved across species by performing experiments in human cell lines, mice, Drosophila and C. elegans. Under starvation, Glycogen synthase kinase 3 beta-mediated phosphorylation of ENDOG at Thr-128 and Ser-288 enhances its interaction with 14-3-3γ, which leads to the release of Tuberin (TSC2) and Phosphatidylinositol 3-kinase catalytic subunit type 3 (Vps34) from 14-3-3γ, followed by mTOR pathway suppression and autophagy initiation. Alternatively, ENDOG activates DNA damage response and triggers autophagy through its endonuclease activity. Our results demonstrate that ENDOG is a crucial regulator of autophagy, manifested by phosphorylation-mediated interaction with 14-3-3γ, and its endonuclease activity-mediated DNA damage response.
Assuntos
Autofagia/fisiologia , Dano ao DNA/fisiologia , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Proteínas 14-3-3/metabolismo , Animais , Apoptose , Caenorhabditis elegans , Linhagem Celular , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Drosophila , Técnicas de Inativação de Genes , Células Hep G2 , Humanos , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Fosforilação , Transcriptoma , Proteína 2 do Complexo Esclerose Tuberosa/metabolismoRESUMO
The ubiquitin-proteasome system (UPS) and autophagy are two major quality control processes whose impairment is linked to a wide variety of diseases. The coordination between UPS and autophagy remains incompletely understood. Here, we show that ubiquitin ligase UBE3C and deubiquitinating enzyme TRABID reciprocally regulate K29/K48-branched ubiquitination of VPS34. We find that this ubiquitination enhances the binding of VPS34 to proteasomes for degradation, thereby suppressing autophagosome formation and maturation. Under ER and proteotoxic stresses, UBE3C recruitment to phagophores is compromised with a concomitant increase of its association with proteasomes. This switch attenuates the action of UBE3C on VPS34, thereby elevating autophagy activity to facilitate proteostasis, ER quality control and cell survival. Specifically in the liver, we show that TRABID-mediated VPS34 stabilization is critical for lipid metabolism and is downregulated during the pathogenesis of steatosis. This study identifies a ubiquitination type on VPS34 and elucidates its cellular fate and physiological functions in proteostasis and liver metabolism.
Assuntos
Autofagia/fisiologia , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Fígado/metabolismo , Proteostase/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Ubiquitinação/fisiologia , Animais , Autofagossomos/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/genética , Dieta Hiperlipídica/efeitos adversos , Células HEK293 , Células HeLa , Humanos , Masculino , Camundongos Endogâmicos C57BL , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genéticaRESUMO
Peptide therapeutics, unlike small-molecule drugs, display crucial advantages of target specificity and the ability to block large interacting interfaces, such as those of transcription factors. The transcription co-factor of the Hippo pathway, YAP/Yorkie (Yki), has been implicated in many cancers, and is dependent on its interaction with the DNA-binding TEAD/Sd proteins via a large Ω-loop. In addition, the mammalian vestigial-like (VGLL) proteins, specifically their TONDU domain, competitively inhibit YAP-TEAD interaction, resulting in arrest of tumor growth. Here, we show that overexpression of the TONDU peptide or its oral uptake leads to suppression of Yki-driven intestinal stem cell tumors in the adult Drosophila midgut. In addition, comparative proteomic analyses of peptide-treated and untreated tumors, together with chromatin immunoprecipitation analysis, reveal that integrin pathway members are part of the Yki-oncogenic network. Collectively, our findings establish Drosophila as a reliable in vivo platform to screen for cancer oral therapeutic peptides and reveal a tumor suppressive role for integrins in Yki-driven tumors.This article has an associated First Person interview with the first author of the paper.