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The cGAS-STING pathway plays an important role in host defense by sensing pathogen DNA, inducing type I IFNs, and initiating autophagy. However, the molecular mechanism of autophagosome formation in cGAS-STING pathway-induced autophagy is still unclear. Here, we report that STING directly interacts with WIPI2, which is the key protein for LC3 lipidation in autophagy. Binding to WIPI2 is necessary for STING-induced autophagosome formation but does not affect STING activation and intracellular trafficking. In addition, the specific interaction between STING and the PI3P-binding motif of WIPI2 leads to the competition of WIPI2 binding between STING and PI3P, and mutual inhibition between STING-induced autophagy and canonical PI3P-dependent autophagy. Furthermore, we show that the STING-WIPI2 interaction is required for the clearance of cytoplasmic DNA and the attenuation of cGAS-STING signaling. Thus, the direct interaction between STING and WIPI2 enables STING to bypass the canonical upstream machinery to induce LC3 lipidation and autophagosome formation.
Assuntos
Autofagossomos , Autofagia , Proteínas de Membrana , Autofagossomos/metabolismo , Autofagia/fisiologia , DNA/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , HumanosRESUMO
mTORC1, the major homeostatic sensor and responder, regulates cell catabolism mainly by targeting autophagy. Here, we show that mTORC1 directly controls autophagosome formation via phosphorylation of WIPI2, a critical protein in isolation membrane growth and elongation. mTORC1 phosphorylates Ser395 of WIPI2, directing WIPI2 to interact specifically with the E3 ubiquitin ligase HUWE1 for ubiquitination and proteasomal degradation. Physiological or pharmacological inhibition of mTORC1 in cells promotes WIPI2 stabilization, autophagosome formation, and autophagic degradation. In mouse liver, fasting significantly increases the WIPI2 protein level, while silencing HUWE1 enhances autophagy, and introducing WIPI2 improves lipid clearance. Thus, regulation of the intracellular WIPI2 protein level by mTORC1 and HUWE1 is a key determinant of autophagy flux and may coordinate the initiation, progression, and completion of autophagy.
Assuntos
Autofagia/fisiologia , Proteínas de Transporte/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Ligação a Fosfato , Ubiquitinação/fisiologiaRESUMO
PROPPINs constitute a conserved protein family with multiple members being expressed in many eukaryotes. PROPPINs have mainly been investigated for their role in autophagy, where they co-operate with several core factors for autophagosome formation. Recently, novel functions of these proteins on endo-lysosomal compartments have emerged. PROPPINs support the division of these organelles and the formation of tubulo-vesicular cargo carriers that mediate protein exit from them, such as those generated by the Retromer coat. In both cases, PROPPINs provide membrane fission activity. Integrating information from yeast and human cells this review summarizes the most important molecular features that allow these proteins to facilitate membrane fission and thus provide a critical element to endo-lysosomal protein traffic.
Assuntos
Autofagia , Endossomos , Lisossomos , Humanos , Lisossomos/metabolismo , Autofagia/fisiologia , Endossomos/metabolismo , Animais , Transporte Proteico , Membranas Intracelulares/metabolismoRESUMO
Hydra possesses three distinct stem cell populations that continuously self-renew and prevent aging in Hydra vulgaris However, sexual animals from the H. oligactis cold-sensitive strain Ho_CS develop an aging phenotype upon gametogenesis induction, initiated by the loss of interstitial stem cells. Animals stop regenerating, lose their active behaviors and die within 3â months. This phenotype is not observed in the cold-resistant strain Ho_CR To dissect the mechanisms of Hydra aging, we compared the self-renewal of epithelial stem cells in these two strains and found it to be irreversibly reduced in aging Ho_CS but sustained in non-aging Ho_CR We also identified a deficient autophagy in Ho_CS epithelial cells, with a constitutive deficiency in autophagosome formation as detected with the mCherry-eGFP-LC3A/B autophagy sensor, an inefficient response to starvation as evidenced by the accumulation of the autophagosome cargo protein p62/SQSTM1, and a poorly inducible autophagy flux upon proteasome inhibition. In the non-aging H. vulgaris animals, the blockade of autophagy by knocking down WIPI2 suffices to induce aging. This study highlights the essential role of a dynamic autophagy flux to maintain epithelial stem cell renewal and prevent aging.
Assuntos
Envelhecimento/fisiologia , Autofagia , Células Epiteliais/citologia , Água Doce , Hydra/fisiologia , Células-Tronco/citologia , Animais , Autofagia/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Temperatura Baixa , Epiderme/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Gametogênese/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Hydra/efeitos dos fármacos , Hydra/genética , Imageamento Tridimensional , Fenótipo , Inibidores de Proteassoma/farmacologia , Sirolimo/farmacologia , Células-Tronco/efeitos dos fármacos , Análise de SobrevidaRESUMO
The potential impact of mask-wearing specifically on early-childhood speech and language development in classrooms has not been widely reported yet, although face masks are compulsory even in educational settings during the COVID-19 pandemic. This study investigated the combined effects of face-mask usage (no mask, surgical and KF94 masks) and room acoustics (RT 0.6 s and 1.2 s, SNR 12 dB and 22 dB) on speech recognition (KS-MWL-P) in preschool children (N = 67) in realistic classroom-acoustic settings using the auralisation technique. The face mask and reverberation time affected pre-schoolers' speech recognition scores. Reducing RT in the classroom improved the pre-schoolers' speech recognition that was reduced by face masks. Children aged 4 and 5 years were affected by face masks and RT more significantly than children aged 6 years. Appropriate room acoustics for classrooms and clear speech of teachers are recommended for better speech recognition in preschool, where pre-schoolers' language and speech development usually occur.
RESUMO
TRAPPC11 has been implicated in membrane traffic and lipid-linked oligosaccharide synthesis, and mutations in TRAPPC11 result in neuromuscular and developmental phenotypes. Here, we show that TRAPPC11 has a role upstream of autophagosome formation during macroautophagy. Upon TRAPPC11 depletion, LC3-positive membranes accumulate prior to, and fail to be cleared during, starvation. A proximity biotinylation assay identified ATG2B and its binding partner WIPI4/WDR45 as TRAPPC11 interactors. TRAPPC11 depletion phenocopies that of ATG2 and WIPI4 and recruitment of both proteins to membranes is defective upon reduction of TRAPPC11. We find that a portion of TRAPPC11 and other TRAPP III proteins localize to isolation membranes. Fibroblasts from a patient with TRAPPC11 mutations failed to recruit ATG2B-WIPI4, suggesting that this interaction is physiologically relevant. Since ATG2B-WIPI4 is required for isolation membrane expansion, our study suggests that TRAPPC11 plays a role in this process. We propose a model whereby the TRAPP III complex participates in the formation and expansion of the isolation membrane at several steps.
Assuntos
Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas de Transporte/metabolismo , Distrofia Muscular do Cíngulo dos Membros/genética , Proteínas de Transporte Vesicular/metabolismo , Autofagia , Células Cultivadas , Fibroblastos/metabolismo , Células HeLa , Humanos , Membranas Intracelulares/metabolismo , Mutação , Ligação Proteica , Transporte Proteico , Proteínas de Transporte Vesicular/genéticaRESUMO
Atg2 is one of the essential factors for autophagy. Recent advance of structural and biochemical study on yeast Atg2 proposed that Atg2 tethers the edge of the isolation membrane (IM) to the endoplasmic reticulum and mediates direct lipid transfer (LT) from ER to IM for IM expansion. In mammals, two Atg2 orthologs, ATG2A and ATG2B, participate in autophagic process. Here we showed that human ATG2B possesses the membrane tethering (MT) and LT activity that was promoted by negatively charged membranes and an Atg18 ortholog WIPI4. By contrast, negatively charged membranes reduced the yeast Atg2 activities in the absence of Atg18. These results suggest that the MT/LT activity of Atg2 is evolutionally conserved although their regulation differs among species.
Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Autofagia , Proteínas Relacionadas à Autofagia/fisiologia , Transporte Biológico , Proteínas de Transporte/fisiologia , Retículo Endoplasmático/metabolismo , Humanos , Metabolismo dos Lipídeos/fisiologia , Lipídeos/fisiologia , Proteínas de Ligação a Fosfato/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/fisiologiaRESUMO
Autophagy is a critical cytoprotective mechanism against stress, which is initiated by the protein kinase Unc-51-like kinase 1 (ULK1) complex. Autophagy plays a role in both inhibiting the progression of diseases and facilitating pathogenesis, so it is critical to elucidate the mechanisms regulating individual components of the autophagy machinery under various conditions. Here, we examined whether ULK1 complex component autophagy-related protein 101 (ATG101) is downregulated via ubiquitination, and whether this in turn suppresses autophagy activity in cancer cells. Knockout of ATG101 in cancer cells using CRISPR resulted in severe growth retardation and lower survival under nutrient starvation. Transfection of mutant ATG101 revealed that the C-terminal region is a key domain of ubiquitination, while co-immunoprecipitation and knockdown experiments revealed that HECT, UBA and WWE domain containing E3 ubiquitin protein ligase 1(HUWE1) is a major E3 ubiquitin ligase targeting ATG101. Protein levels of ATG101 was more stable and the related-autophagy activity was higher in HUWE1-depleted cancer cells compared to wild type (WT) controls, indicating that HUWE1-mediated ubiquitination promotes ATG101 degradation. Moreover, enhanced autophagy in HUWE1-depleted cancer cells was reversed by siRNA-mediated ATG101 knockdown. Stable ATG101 level in HUWE1-depleted cells was a strong driver of autophagosome formation similar to upregulation of the known HUWE1 substrate WD repeat domain, phosphoinositide interacting 2 (WIPI2). Cellular survival rates were higher in HUWE1-knockdown cancer cells compared to controls, while concomitant siRNA-mediated ATG101 knockdown tends to increase apoptosis rate. Collectively, these results suggest that HUWE1 normally serves to suppress autophagy by ubiquitinating and triggering degradation of ATG101 and WIPI2, which in turn represses the survival of cancer cells. Accordingly, ATG101-mediated autophagy may play a critical role in overcoming metabolic stress, thereby contributing to the growth, survival, and treatment resistance of certain cancers.
Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Neoplasias/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas de Transporte Vesicular/metabolismo , Proteínas Relacionadas à Autofagia/genética , Sobrevivência Celular , Células HEK293 , Células HeLa , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMO
Mammalian cells deploy autophagy to defend their cytosol against bacterial invaders. Anti-bacterial autophagy relies on the core autophagy machinery, cargo receptors, and "eat-me" signals such as galectin-8 and ubiquitin that label bacteria as autophagy cargo. Anti-bacterial autophagy also requires the kinase TBK1, whose role in autophagy has remained enigmatic. Here we show that recruitment of WIPI2, itself essential for anti-bacterial autophagy, is dependent on the localization of catalytically active TBK1 to the vicinity of cytosolic bacteria. Experimental manipulation of TBK1 recruitment revealed that engagement of TBK1 with any of a variety of Salmonella-associated "eat-me" signals, including host-derived glycans and K48- and K63-linked ubiquitin chains, suffices to restrict bacterial proliferation. Promiscuity in recruiting TBK1 via independent signals may buffer TBK1 functionality from potential bacterial antagonism and thus be of evolutionary advantage to the host.
Assuntos
Autofagia , Proteínas de Transporte/metabolismo , Citosol/microbiologia , Imunidade Inata , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Salmonella typhimurium/imunologia , Animais , Humanos , Camundongos , Proteínas de Ligação a FosfatoRESUMO
We describe a large consanguineous pedigree from a remote area of Northern Pakistan, with a complex developmental disorder associated with wide-ranging symptoms, including mental retardation, speech and language impairment and other neurological, psychiatric, skeletal and cardiac abnormalities. We initially carried out a genetic study using the HumanCytoSNP-12 v2.1 Illumina gene chip on nine family members and identified a single region of homozygosity shared amongst four affected individuals on chromosome 7p22 (positions 3059377-5478971). We performed whole-exome sequencing on two affected individuals from two separate branches of the extended pedigree and identified a novel nonsynonymous homozygous mutation in exon 9 of the WIPI2 (WD-repeat protein interacting with phosphoinositide 2) gene at position 5265458 (c.G745A;pV249M). WIPI2 plays a critical role in autophagy, an evolutionary conserved cellular pathway implicated in a growing number of medical conditions. The mutation is situated in a highly conserved and critically important region of WIPI2, responsible for binding PI(3)P and PI(3,5)P2, an essential requirement for autophagy to proceed. The mutation is absent in all public databases, is predicted to be damaging and segregates with the disease phenotype. We performed functional studies in vitro to determine the potential effects of the mutation on downstream pathways leading to autophagosome assembly. Binding of the V231M mutant of WIPI2b to ATG16L1 (as well as ATG5-12) is significantly reduced in GFP pull-down experiments, and fibroblasts derived from the patients show reduced WIPI2 puncta, reduced LC3 lipidation and reduced autophagic flux.
Assuntos
Autofagia/genética , Deficiências do Desenvolvimento/diagnóstico , Deficiências do Desenvolvimento/genética , Proteínas de Membrana/genética , Mutação/genética , Proteínas de Ligação a Fosfato/genética , Adulto , Sequência de Aminoácidos , Células Cultivadas , Feminino , Células HEK293 , Humanos , Masculino , Proteínas de Membrana/química , Pessoa de Meia-Idade , Linhagem , Proteínas de Ligação a Fosfato/química , Estrutura Secundária de ProteínaRESUMO
Autophagy is a quality-control mechanism that helps to maintain cellular homeostasis by removing damaged proteins and organelles through lysosomal degradation. During autophagy, signaling events lead to the formation of a cup-shaped structure called the phagophore that matures into the autophagosome. Recruitment of the autophagy-associated Atg12-5-16L1 complex to Wipi2-positive phagophores is crucial for producing microtubule-associated protein 1 light chain 3-II (LC3-II), which is required for autophagosome formation. Here, we explored the role of the autophagy receptor optineurin (Optn) in autophagosome formation. Fibroblasts from Optn knock-out mouse showed reduced LC3-II formation and a lower number of autophagosomes and autolysosomes during both basal and starvation-induced autophagy. However, the number of Wipi2-positive phagophores was not decreased in Optn-deficient cells. We also found that the number of Atg12/16L1-positive puncta and recruitment of the Atg12-5-16L1 complex to Wipi2-positive puncta are reduced in Optn-deficient cells. Of note, Optn was recruited to Atg12-5-16L1-positive puncta, and interacted with Atg5 and also with Atg12-5 conjugate. A disease-associated Optn mutant, E478G, defective in ubiquitin binding, was also defective in autophagosome formation and recruitment to the Atg12-5-16L1-positive puncta. Moreover, we noted that Optn phosphorylation at Ser-177 was required for autophagosome formation but not for Optn recruitment to the phagophore. These results suggest that Optn potentiates LC3-II production and maturation of the phagophore into the autophagosome, by facilitating the recruitment of the Atg12-5-16L1 complex to Wipi2-positive phagophores.
Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Proteínas de Transporte/metabolismo , Proteínas do Olho/metabolismo , Proteínas de Membrana/metabolismo , Fator de Transcrição TFIIIA/metabolismo , Animais , Autofagossomos/metabolismo , Autofagia/fisiologia , Proteínas de Ciclo Celular , Feminino , Células HEK293 , Humanos , Masculino , Proteínas de Membrana Transportadoras , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Ligação a Fosfato , Ligação ProteicaRESUMO
Autophagy is a highly conserved process and is essential for the maintenance of cellular homeostasis. Autophagy occurs at a basal level in all cells, but it can be up-regulated during stress, starvation, or infection. Misregulation of autophagy has been linked to various disorders, including cancer, neurodegeneration, and immune diseases. Here, we discuss the essential proteins acting in the formation of an autophagosome, with a focus on the ULK and VPS34 kinase complexes, phosphatidylinositol 3-phosphate effector proteins, and the transmembrane autophagy-related protein ATG9. The function and regulation of these and other autophagy-related proteins acting during formation will be addressed, in particular during amino acid starvation.
Assuntos
Autofagia , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/genética , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Humanos , Doenças do Sistema Imunitário/genética , Doenças do Sistema Imunitário/metabolismo , Doenças do Sistema Imunitário/patologia , Infecções/genética , Infecções/metabolismo , Infecções/patologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Doenças Neurodegenerativas , Inanição/genética , Inanição/metabolismo , Inanição/patologiaRESUMO
Autophagosome biogenesis requires two ubiquitin-like conjugation systems. One couples ubiquitin-like Atg8 to phosphatidylethanolamine, and the other couples ubiquitin-like Atg12 to Atg5. Atg12~Atg5 then forms a heterodimer with Atg16. Membrane recruitment of the Atg12~Atg5/Atg16 complex defines the Atg8 lipidation site. Lipidation requires a PI3P-containing precursor. How PI3P is sensed and used to coordinate the conjugation systems remained unclear. Here, we show that Atg21, a WD40 ß-propeller, binds via PI3P to the preautophagosomal structure (PAS). Atg21 directly interacts with the coiled-coil domain of Atg16 and with Atg8. This latter interaction requires the conserved F5K6-motif in the N-terminal helical domain of Atg8, but not its AIM-binding site. Accordingly, the Atg8 AIM-binding site remains free to mediate interaction with its E2 enzyme Atg3. Atg21 thus defines PI3P-dependently the lipidation site by linking and organising the E3 ligase complex and Atg8 at the PAS.
Assuntos
Endopeptidases/metabolismo , Fosfatos de Inositol/metabolismo , Lipoilação/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Motivos de Aminoácidos , Família da Proteína 8 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Endopeptidases/genética , Fosfatos de Inositol/genética , Proteínas Associadas aos Microtúbulos/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Enzimas de Conjugação de Ubiquitina/genéticaRESUMO
Autophagy is a pivotal cytoprotective process that secures cellular homeostasis, fulfills essential roles in development, immunity and defence against pathogens, and determines the lifespan of eukaryotic organisms. However, autophagy also crucially contributes to the development of age-related human pathologies, including cancer and neurodegeneration. Macroautophagy (hereafter referred to as autophagy) clears the cytoplasm by stochastic or specific cargo recognition and destruction, and is initiated and executed by autophagy related (ATG) proteins functioning in dynamical hierarchies to form autophagosomes. Autophagosomes sequester cytoplasmic cargo material, including proteins, lipids and organelles, and acquire acidic hydrolases from the lysosomal compartment for cargo degradation. Prerequisite and essential for autophagosome formation is the production of phosphatidylinositol 3-phosphate (PtdIns3P) by phosphatidylinositol 3-kinase class III (PI3KC3, also known as PIK3C3) in complex with beclin 1, p150 (also known as PIK3R4; Vps15 in yeast) and ATG14L. Members of the human WD-repeat protein interacting with phosphoinositides (WIPI) family play an important role in recognizing and decoding the PtdIns3P signal at the nascent autophagosome, and hence function as autophagy-specific PtdIns3P-binding effectors, similar to their ancestral yeast Atg18 homolog. The PtdIns3P effector function of human WIPI proteins appears to be compromised in cancer and neurodegeneration, and WIPI genes and proteins might present novel targets for rational therapies. Here, we summarize the current knowledge on the roles of the four human WIPI proteins, WIPI1-4, in autophagy. This article is part of a Focus on Autophagosome biogenesis. For further reading, please see related articles: 'ERES: sites for autophagosome biogenesis and maturation?' by Jana Sanchez-Wandelmer et al. (J. Cell Sci. 128, 185-192) and 'Membrane dynamics in autophagosome biogenesis' by Sven R. Carlsson and Anne Simonsen (J. Cell Sci. 128, 193-205).
Assuntos
Autofagia/genética , Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Fagossomos/genética , Fosfatos de Fosfatidilinositol/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Relacionadas à Autofagia , Proteína Beclina-1 , Classe III de Fosfatidilinositol 3-Quinases/genética , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas Nucleares/metabolismo , Fagossomos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fatores de Transcrição/metabolismo , Proteína VPS15 de Distribuição Vacuolar/metabolismoRESUMO
Central to the process of macroautophagy (hereafter autophagy) is the formation of autophagosomes, double-membrane vesicles that sequester cytoplasmic cargo, including proteins, lipids and organelles, for lysosomal degradation and macromolecule recycling. Tight regulation of both autophagic activity and capacity is crucial to secure cellular homeostasis and aberrant autophagy is tightly linked to the development of many human diseases. Hence it is of great importance to accurately measure autophagy progression in health and disease. Members of the human WIPI ß-propeller proteins associate with autophagosomal membranes due to specific phosphatidylinositol 3-phosphate (PtdIns3P) binding at the onset of autophagy. The specific autophagosomal localization of both WIPI1 and WIPI2 (refered to as WIPI puncta) has been employed to assess autophagy using fluorescence microscopy methods, such as confocal and live-cell video microscopy and was extended for automated high-throughput image acquisition and analyses procedures. We here provide an overview on the employment of human WIPI members for the assessment of autophagy in higher eukaryotic cells, suitable for systems biology approaches such as mathematical modelling.
Assuntos
Autofagia/genética , Proteínas de Transporte/isolamento & purificação , Proteínas de Membrana/isolamento & purificação , Proteínas Relacionadas à Autofagia , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Fluorescência , Humanos , Proteínas de Membrana/genética , Biologia Molecular/métodos , Fagossomos/genética , Fagossomos/metabolismo , Fagossomos/ultraestrutura , Proteínas de Ligação a Fosfato , Fosfatos de Fosfatidilinositol/metabolismo , Biologia de SistemasRESUMO
Autophagy is a lysosomal bulk degradation pathway for cytoplasmic cargo, such as long-lived proteins, lipids, and organelles. Induced upon nutrient starvation, autophagic degradation is accomplished by the concerted actions of autophagy-related (ATG) proteins. Here we demonstrate that two ATGs, human Atg2A and Atg14L, colocalize at cytoplasmic lipid droplets (LDs) and are functionally involved in controlling the number and size of LDs in human tumor cell lines. We show that Atg2A is targeted to cytoplasmic ADRP-positive LDs that migrate bidirectionally along microtubules. The LD localization of Atg2A was found to be independent of the autophagic status. Further, Atg2A colocalized with Atg14L under nutrient-rich conditions when autophagy was not induced. Upon nutrient starvation and dependent on phosphatidylinositol 3-phosphate [PtdIns(3)P] generation, both Atg2A and Atg14L were also specifically targeted to endoplasmic reticulum-associated early autophagosomal membranes, marked by the PtdIns(3)P effectors double-FYVE containing protein 1 (DFCP1) and WD-repeat protein interacting with phosphoinositides 1 (WIPI-1), both of which function at the onset of autophagy. These data provide evidence for additional roles of Atg2A and Atg14L in the formation of early autophagosomal membranes and also in lipid metabolism.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Membrana Celular/metabolismo , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Relacionadas à Autofagia/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Membrana Celular/genética , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fosfatos de Fosfatidilinositol/genética , Fosfatos de Fosfatidilinositol/metabolismoRESUMO
Leucine Rich Repeat Kinase 2 (LRRK2) is one of the most important genetic contributors to Parkinson's disease. LRRK2 has been implicated in a number of cellular processes, including macroautophagy. To test whether LRRK2 has a role in regulating autophagy, a specific inhibitor of the kinase activity of LRRK2 was applied to human neuroglioma cells and downstream readouts of autophagy examined. The resulting data demonstrate that inhibition of LRRK2 kinase activity stimulates macroautophagy in the absence of any alteration in the translational targets of mTORC1, suggesting that LRRK2 regulates autophagic vesicle formation independent of canonical mTORC1 signaling. This study represents the first pharmacological dissection of the role LRRK2 plays in the autophagy/lysosomal pathway, emphasizing the importance of this pathway as a marker for LRRK2 physiological function. Moreover it highlights the need to dissect autophagy and lysosomal activities in the context of LRRK2 related pathologies with the final aim of understanding their aetiology and identifying specific targets for disease modifying therapies in patients.
Assuntos
Autofagia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Astrócitos/enzimologia , Autofagia/efeitos dos fármacos , Benzodiazepinonas/farmacologia , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Imuno-Histoquímica , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Proteínas Associadas aos Microtúbulos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico/efeitos dos fármacos , Pirimidinas/farmacologia , Ratos , Proteína Sequestossoma-1RESUMO
The four human WIPI ß-propellers, WIPI1 through WIPI4, belong to the ancient PROPPIN family and fulfill scaffold functions in the control of autophagy. In this context, WIPI ß-propellers function as PI3P effectors during autophagosome formation and loss of WIPI function negatively impacts autophagy and contributes to neurodegeneration. Of particular interest are mutations in WDR45, the human gene that encodes WIPI4. Sporadic WDR45 mutations are the cause of a rare human neurodegenerative disease called BPAN, hallmarked by high brain iron accumulation. Here, we discuss the current understanding of the functions of human WIPI ß-propellers and address unanswered questions with a particular focus on the role of WIPI4 in autophagy and BPAN.
Assuntos
Proteínas de Transporte , Doenças Neurodegenerativas , Humanos , Proteínas de Transporte/genética , Doenças Neurodegenerativas/genética , Mutação , Proteínas Relacionadas à Autofagia/genética , Autofagia/genéticaRESUMO
The human WDR33 gene encodes three major isoforms. The canonical isoform WDR33v1 (V1) is a well-characterized nuclear mRNA polyadenylation factor, while the other two, WDR33v2 (V2) and WDR33v3 (V3), have not been studied. Here, we report that V2 and V3 are generated by alternative polyadenylation, and neither protein contains all seven WD (tryptophan-aspartic acid) repeats that characterize V1. Surprisingly, V2 and V3 are not polyadenylation factors but localize to the endoplasmic reticulum and interact with stimulator of interferon genes (STING), the immune factor that induces the cellular response to cytosolic double-stranded DNA. V2 suppresses interferon-ß induction by preventing STING disulfide oligomerization but promotes autophagy, likely by recruiting WIPI2 isoforms. V3, on the other hand, functions to increase STING protein levels. Our study has not only provided mechanistic insights into STING regulation but also revealed that protein isoforms can be functionally completely unrelated, indicating that alternative mRNA processing is a more powerful mechanism than previously appreciated.
Assuntos
Poliadenilação , Fatores de Poliadenilação e Clivagem de mRNA , Humanos , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Membrana/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Imunidade InataRESUMO
Induction of autophagy is a primordial function of the cGAS-STING pathway. However, the molecular mechanisms regulating autophagosome formation during STING-induced autophagy remain largely unknown. Recently, we reported that STING directly interacts with WIPI2 to recruit WIPI2 onto STING-positive vesicles for LC3 lipidation and autophagosome formation. We found that STING and PtdIns3P competitively bind to the FRRG motif of WIPI2, resulting in a mutual inhibition between STING-induced and PtdIns3P-dependent autophagy. We also showed that STING-WIPI2 interaction is necessary for cells to clear cytoplasmic DNA and attenuate activated cGAS-STING signaling. In summary, by identifying the interaction between STING and WIPI2, our study revealed a mechanism that allows STING to bypass the canonical upstream machinery to induce autophagosome formation.Abbreviations: ATG: autophagy-related; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; cGAMP: cyclic GMP-AMP; cGAS: cyclic GMP-AMP synthase; ER: endoplasmic reticulum; ERGIC: ER-Golgi intermediate compartment; IRF3: interferon regulatory factor 3; PtdIns3P: phosphatidylinositol-3-phosphate; SQSTM1: sequestosome 1; STING: stimulator of interferon genes; TBK1: TANK-binding kinase 1; ULK1: unc-51 like autophagy activating kinase 1; WIPI2: WD repeat domain, phosphoinositide interacting 2.