RESUMO
The fusion of autophagosomes and lysosomes is essential for the prevention of nonalcoholic fatty liver disease (NAFLD). Here, we generate a hepatocyte-specific CHIP knockout (H-KO) mouse model that develops NAFLD more rapidly in response to a high-fat diet (HFD) or high-fat, high-fructose diet (HFHFD). The accumulation of P62 and LC3 in the livers of H-KO mice and CHIP-depleted cells indicates the inhibition of autophagosome-lysosome fusion. AAV8-mediated overexpression of CHIP in the murine liver slows the progression of NAFLD induced by HFD or HFHFD feeding. Mechanistically, CHIP induced K63- and K27-linked polyubiquitination at the lysine 198 residue of STX17, resulting in increased STX17-SNAP29-VAMP8 complex formation. The STX17 K198R mutant was not ubiquitinated by CHIP; it interfered with its interaction with VAMP8, rendering STX17 incapable of inhibiting steatosis development in mice. These results indicate that a signaling regulatory mechanism involving CHIP-mediated non-degradative ubiquitination of STX17 is necessary for autophagosome-lysosome fusion.
Assuntos
Autofagossomos , Lisossomos , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica , Ubiquitina-Proteína Ligases , Ubiquitinação , Animais , Lisossomos/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Hepatopatia Gordurosa não Alcoólica/genética , Autofagossomos/metabolismo , Camundongos , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Dieta Hiperlipídica/efeitos adversos , Masculino , Proteínas Qa-SNARE/metabolismo , Proteínas Qa-SNARE/genética , Hepatócitos/metabolismo , Modelos Animais de Doenças , Fígado/metabolismo , Fígado/patologia , Camundongos Endogâmicos C57BL , Proteínas R-SNARE/metabolismo , Proteínas R-SNARE/genética , Fusão de Membrana , Autofagia , Fator de Transcrição TFIIHRESUMO
In this issue of Developmental Cell, Jiang et al. report that the Arabidopsis HOPS tethering complex subunit VPS41 acts to catalyze the formation of a degradation pathway composed of a hybrid of autophagosomes and late endosomes.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Autofagossomos , Autofagia , Endossomos , Vacúolos , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Autofagossomos/metabolismo , Autofagia/fisiologia , Endossomos/metabolismo , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMO
Impaired tumor cell antigen presentation contributes significantly to immune evasion. This study identifies Berbamine hydrochloride (Ber), a compound derived from traditional Chinese medicine, as an effective inhibitor of autophagy that enhances antigen presentation in tumor cells. Ber increases MHC-I-mediated antigen presentation in melanoma cells, improving recognition and elimination by CD8+ T cells. Mutation of Atg4b, which blocks autophagy, also raises MHC-I levels on the cell surface, and further treatment with Ber under these conditions does not increase MHC-I, indicating Ber's role in blocking autophagy to enhance MHC-I expression. Additionally, Ber treatment leads to the accumulation of autophagosomes, with elevated levels of LC3-II and p62, suggesting a disrupted autophagic flux. Fluorescence staining and co-localization analyses reveal that Ber likely inhibits lysosomal acidification without hindering autophagosome-lysosome fusion. Importantly, Ber treatment suppresses melanoma growth in mice and enhances CD8+ T cell infiltration, supporting its therapeutic potential. Our findings demonstrate that Ber disturbs late-stage autophagic flux through abnormal lysosomal acidification, enhancing MHC-I-mediated antigen presentation and curtailing tumor immune escape.
Assuntos
Autofagia , Benzilisoquinolinas , Melanoma , Evasão Tumoral , Autofagia/efeitos dos fármacos , Animais , Camundongos , Linhagem Celular Tumoral , Humanos , Evasão Tumoral/efeitos dos fármacos , Benzilisoquinolinas/farmacologia , Benzilisoquinolinas/uso terapêutico , Melanoma/tratamento farmacológico , Melanoma/patologia , Melanoma/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Apresentação de Antígeno/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Camundongos Endogâmicos C57BL , Autofagossomos/metabolismo , Autofagossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lisossomos/efeitos dos fármacos , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Melanoma Experimental/tratamento farmacológico , Cisteína EndopeptidasesRESUMO
BACKGROUND: Empagliflozin (EMPA) has demonstrated efficacy in providing cardiovascular benefits in metabolic diseases. However, the direct effect of EMPA on autophagy in obesity-related cardiac dysfunction remains unclear. Therefore, this study aimed to determine changes in cardiac autophagy during diet-induced obesity and clarify the exact mechanism by which EMPA regulates autophagic pathways. METHODS: Male C57BL/6J mice were fed a 12-week high-fat diet (HFD) followed by 8 weeks of EMPA treatment. Body composition analysis and echocardiography were performed to evaluate metabolic alterations and cardiac function. Histological and immunofluorescence staining was used to evaluate potential enhancements in myocardial structure and biological function. Additionally, H9c2 cells were transfected with small interfering RNA targeting sirtuin 3 (SIRT3) and further treated with palmitic acid (PA) with or without EMPA. Autophagy-related targets were analyzed by western blotting and RTâqPCR. RESULTS: EMPA administration effectively ameliorated metabolic disorders and cardiac diastolic dysfunction in HFD-fed mice. EMPA prevented obesity-induced myocardial hypertrophy, fibrosis, and inflammation through the activation of SIRT3-mediated autophagosome formation. The upregulation of SIRT3 triggered by EMPA promoted the initiation of autophagy by activating AMP-activated protein kinase (AMPK) and Beclin1. Furthermore, activated SIRT3 contributed to the elongation of autophagosomes through autophagy-related 4B cysteine peptidase (ATG4B) and autophagy-related 5 (ATG5). CONCLUSIONS: EMPA promotes SIRT3-mediated autophagosome formation to alleviate damage to the cardiac structure and function of obese mice. Activated SIRT3 initiates autophagy through AMPK/Beclin1 and further stimulates elongation of the autophagosome membrane via ATG4B/ATG5. These results provide a new explanation for the cardioprotective benefits of EMPA in obesity.
Assuntos
Autofagossomos , Autofagia , Compostos Benzidrílicos , Dieta Hiperlipídica , Glucosídeos , Camundongos Endogâmicos C57BL , Obesidade , Sirtuína 3 , Animais , Glucosídeos/farmacologia , Compostos Benzidrílicos/farmacologia , Obesidade/tratamento farmacológico , Obesidade/complicações , Obesidade/metabolismo , Masculino , Camundongos , Autofagossomos/metabolismo , Autofagossomos/efeitos dos fármacos , Dieta Hiperlipídica/efeitos adversos , Autofagia/efeitos dos fármacos , Sirtuína 3/metabolismo , Sirtuína 3/genética , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Linhagem Celular , Miocárdio/metabolismo , Miocárdio/patologia , Inibidores do Transportador 2 de Sódio-Glicose/farmacologiaRESUMO
Cytopathology induced by methamphetamine (METH) is reminiscent of degenerative disorders such as Parkinson's disease, and it is characterized by membrane organelles arranged in tubulo-vesicular structures. These areas, appearing as clusters of vesicles, have never been defined concerning the presence of specific organelles. Therefore, the present study aimed to identify the relative and absolute area of specific membrane-bound organelles following a moderate dose (100 µM) of METH administered to catecholamine-containing PC12 cells. Organelles and antigens were detected by immunofluorescence, and they were further quantified by plain electron microscopy and in situ stoichiometry. This analysis indicated an increase in autophagosomes and damaged mitochondria along with a decrease in lysosomes and healthy mitochondria. Following METH, a severe dissipation of hallmark proteins from their own vesicles was measured. In fact, the amounts of LC3 and p62 were reduced within autophagy vacuoles compared with the whole cytosol. Similarly, LAMP1 and Cathepsin-D within lysosomes were reduced. These findings suggest a loss of compartmentalization and confirm a decrease in the competence of cell clearing organelles during catecholamine degeneration. Such cell entropy is consistent with a loss of energy stores, which routinely govern appropriate subcellular compartmentalization.
Assuntos
Autofagossomos , Lisossomos , Metanfetamina , Metanfetamina/farmacologia , Animais , Células PC12 , Ratos , Lisossomos/metabolismo , Lisossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagossomos/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Catepsina D/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismoRESUMO
Autophagy plays a crucial role in cancer cell survival by facilitating the elimination of detrimental cellular components and the recycling of nutrients. Understanding the molecular regulation of autophagy is critical for developing interventional approaches for cancer therapy. In this study, we report that migfilin, a focal adhesion protein, plays a novel role in promoting autophagy by increasing autophagosome-lysosome fusion. We found that migfilin is associated with SNAP29 and Vamp8, thereby facilitating Stx17-SNAP29-Vamp8 SNARE complex assembly. Depletion of migfilin disrupted the formation of the SNAP29-mediated SNARE complex, which consequently blocked the autophagosome-lysosome fusion, ultimately suppressing cancer cell growth. Restoration of the SNARE complex formation rescued migfilin-deficiency-induced autophagic flux defects. Finally, we found depletion of migfilin inhibited cancer cell proliferation. SNARE complex reassembly successfully reversed migfilin-deficiency-induced inhibition of cancer cell growth. Taken together, our study uncovers a new function of migfilin as an autophagy-regulatory protein and suggests that targeting the migfilin-SNARE assembly could provide a promising therapeutic approach to alleviate cancer progression.
Assuntos
Autofagia , Moléculas de Adesão Celular , Proliferação de Células , Lisossomos , Proteínas Qb-SNARE , Proteínas Qc-SNARE , Proteínas R-SNARE , Humanos , Proteínas R-SNARE/metabolismo , Proteínas R-SNARE/genética , Proteínas Qb-SNARE/metabolismo , Proteínas Qb-SNARE/genética , Proteínas Qc-SNARE/metabolismo , Proteínas Qc-SNARE/genética , Lisossomos/metabolismo , Moléculas de Adesão Celular/metabolismo , Moléculas de Adesão Celular/genética , Autofagossomos/metabolismo , Células HeLa , Linhagem Celular Tumoral , Ligação Proteica , Proteínas SNARE/metabolismo , Proteínas SNARE/genética , Fusão de Membrana , Proteínas Qa-SNARERESUMO
Autophagy is a finely orchestrated process required for the lysosomal degradation of cytosolic components. The final degradation step is essential for clearing autophagic cargo and recycling macromolecules. Using a CRISPR/Cas9-based screen, we identify RNAseK, a highly conserved transmembrane protein, as a regulator of autophagosome degradation. Analyses of RNAseK knockout cells reveal that, while autophagosome maturation is intact, cargo degradation is severely disrupted. Importantly, lysosomal protease activity and acidification remain intact in the absence of RNAseK suggesting a specificity to autolysosome degradation. Analyses of lysosome fractions show reduced levels of a subset of hydrolases in the absence of RNAseK. Of these, the knockdown of PLD3 leads to a defect in autophagosome clearance. Furthermore, the lysosomal fraction of RNAseK-depleted cells exhibits an accumulation of the ESCRT-III complex component, VPS4a, which is required for the lysosomal targeting of PLD3. Altogether, here we identify a lysosomal hydrolase delivery pathway required for efficient autolysosome degradation.
Assuntos
Autofagossomos , Autofagia , Complexos Endossomais de Distribuição Requeridos para Transporte , Lisossomos , Lisossomos/metabolismo , Humanos , Autofagossomos/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Sistemas CRISPR-Cas , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , Hidrolases/metabolismo , Hidrolases/genética , Células HeLa , Células HEK293RESUMO
Personalized cancer vaccines based on tumor cell lysates offer promise for cancer immunotherapy yet fail to elicit a robust therapeutic effect due to the weak immunogenicity of tumor antigens. Autophagosomes, obtained from pleural effusions and ascites of cancer patients, have been identified as abundant reservoirs of tumor neoantigens that exhibit heightened immunogenicity. However, their potential as personalized cancer vaccines have been constrained by suboptimal lymphatic-targeting performances and challenges in antigen-presenting cell endocytosis. Here,a reinforced biomimetic autophagosome-based (BAPs) nanovaccine generated by precisely amalgamating autophagosome-derived neoantigens and two types of adjuvants capable of targeting lymph nodes is developed to potently elicit antitumor immunity. The redox-responsive BAPs facilitate cytosolic vaccine opening within antigen-presenting cells, thereby exposing adjuvants and antigens to stimulate a strong immune response. BAPs evoke broad-spectrum T-cell responses, culminating in the effective eradication of 71.4% of established tumors. Notably, BAPs vaccination triggers enduring T-cell responses that confer robust protection, with 100% of mice shielded against tumor rechallenge and a significant reduction in tumor incidence by 87.5%. Furthermore, BAPs synergize with checkpoint blockade therapy to inhibit tumor growth in the poorly immunogenic breast cancer model. The biomimetic approach presents a powerful nanovaccine formula with high versatility for personalized cancer immunotherapy.
Assuntos
Autofagossomos , Materiais Biomiméticos , Vacinas Anticâncer , Animais , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/química , Vacinas Anticâncer/administração & dosagem , Camundongos , Materiais Biomiméticos/química , Autofagossomos/metabolismo , Linhagem Celular Tumoral , Humanos , Imunoterapia , Feminino , Antígenos de Neoplasias/imunologia , Nanopartículas/química , Adjuvantes Imunológicos/química , Biomimética/métodos , NanovacinasRESUMO
One of the key events in autophagy is the formation of a double-membrane phagophore, and many regulatory mechanisms underpinning this remain under investigation. WIPI2b is among the first proteins to be recruited to the phagophore and is essential for stimulating autophagy flux by recruiting the ATG12-ATG5-ATG16L1 complex, driving LC3 and GABARAP lipidation. Here, we set out to investigate how WIPI2b function is regulated by phosphorylation. We studied two phosphorylation sites on WIPI2b, S68 and S284. Phosphorylation at these sites plays distinct roles, regulating WIPI2b's association with ATG16L1 and the phagophore, respectively. We confirm WIPI2b is a novel ULK1 substrate, validated by the detection of endogenous phosphorylation at S284. Notably, S284 is situated within an 18-amino acid stretch, which, when in contact with liposomes, forms an amphipathic helix. Phosphorylation at S284 disrupts the formation of the amphipathic helix, hindering the association of WIPI2b with membranes and autophagosome formation. Understanding these intricacies in the regulatory mechanisms governing WIPI2b's association with its interacting partners and membranes, holds the potential to shed light on these complex processes, integral to phagophore biogenesis.
Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Autofagia , Proteínas de Membrana , Humanos , Autofagossomos/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas de Transporte/metabolismo , Células HEK293 , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Fosforilação , Ligação ProteicaRESUMO
Regulation of autophagy through the 62 kDa ubiquitin-binding protein/autophagosome cargo protein sequestosome 1 (p62/SQSTM1), whose level is generally inversely proportional to autophagy, is crucial in microglial functions. Since autophagy is involved in inflammatory mechanisms, we investigated the actions of pro-inflammatory lipopolysaccharide (LPS) and anti-inflammatory rosuvastatin (RST) in secondary microglial cultures with or without bafilomycin A1 (BAF) pretreatment, an antibiotic that potently inhibits autophagosome fusion with lysosomes. The levels of the microglia marker protein Iba1 and the autophagosome marker protein p62/SQSTM1 were quantified by Western blots, while the number of p62/SQSTM1 immunoreactive puncta was quantitatively analyzed using fluorescent immunocytochemistry. BAF pretreatment hampered microglial survival and decreased Iba1 protein level under all culturing conditions. Cytoplasmic p62/SQSTM1 level was increased in cultures treated with LPS+RST but reversed markedly when BAF+LPS+RST were applied together. Furthermore, the number of p62/SQSTM1 immunoreactive autophagosome puncta was significantly reduced when RST was used but increased significantly in BAF+RST-treated cultures, indicating a modulation of autophagic flux through reduction in p62/SQSTM1 degradation. These findings collectively indicate that the cytoplasmic level of p62/SQSTM1 protein and autophagocytotic flux are differentially regulated, regardless of pro- or anti-inflammatory state, and provide context for understanding the role of autophagy in microglial function in various inflammatory settings.
Assuntos
Autofagossomos , Autofagia , Lipopolissacarídeos , Macrolídeos , Microglia , Proteína Sequestossoma-1 , Animais , Proteína Sequestossoma-1/metabolismo , Microglia/metabolismo , Microglia/efeitos dos fármacos , Macrolídeos/farmacologia , Autofagia/efeitos dos fármacos , Ratos , Autofagossomos/metabolismo , Autofagossomos/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Células Cultivadas , Inflamação/metabolismo , Biomarcadores/metabolismoRESUMO
BACKGROUND: The preservation of autophagosome formation presents a promising strategy for tackling neurological disorders, such as Parkinson's disease (PD). Mitochondria-associated endoplasmic reticulum (ER) membranes (MAM) serve not only as a focal point linked to various neurological disorders but also play a crucial role in supporting the biogenesis of autophagosomes. PURPOSE: This investigation aimed to elucidate the neuroprotective properties of phillyrin against PD and its underlying mechanisms in promoting autophagosome formation. METHODS: ER and mitochondria co-localization was assessed via fluorescent staining. Annexin V-fluorescein isothiocyanate (FITC) fluorescence was employed to quantify accessible cardiolipin (CL) on mitochondrial surfaces. The levels of CL within the MAM fraction of SH-SY5Y cells were evaluated using a CL probe assay kit. Monodansylcadaverine staining was utilized to detect autophagosome formation in SH-SY5Y cells. In an A53T-alpha-synuclein (αSyn)-induced PD mouse model, the anti-PD properties of phillyrin were assessed using open field, pole climbing, and rotarod tests, as well as immunohistochemistry staining of TH+ neurons in the brain sections. RESULTS: In A53T-αSyn-treated SH-SY5Y cells, phillyrin facilitated autophagosome formation by suppressing CL externalization and restoring MAM integrity. Phillyrin enhanced the localization of receptor expression-enhancing protein 1 (REEP1) within MAM and mitochondria, bolstering MAM formation. Increased REEP1 levels in mitochondria, attributed to phillyrin, enhanced the interaction between REEP1 and NDPK-D, thereby reducing CL externalization. Furthermore, phillyrin exhibited a dose-dependent enhancement of motor function in mice, accompanied by an increase in the abundance of dopaminergic neurons within the substantia nigra. CONCLUSIONS: These findings illuminate phillyrin's ability to enhance MAM formation through upregulation of REEP1 expression within MAM, while concurrently attenuating CL externalization via the REEP1-NDPK-D interaction. These mechanisms bolster autophagosome biogenesis, offering resilience against A53T-αSyn-induced PD. Thus, our study advances the understanding of phillyrin's complex mechanisms and underscores its potential as a therapeutic approach for PD, opening new avenues in natural product pharmacology.
Assuntos
Autofagossomos , Mitocôndrias , Doença de Parkinson , alfa-Sinucleína , Animais , alfa-Sinucleína/metabolismo , Humanos , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Camundongos , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fármacos Neuroprotetores/farmacologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Cardiolipinas/metabolismoRESUMO
Autophagy is a highly conserved process from yeast to mammals in which intracellular materials are engulfed by a double-membrane organelle called autophagosome and degrading materials by fusing with the lysosome. The process of autophagy is regulated by sequential recruitment and function of autophagy-related (Atg) proteins. Genetic hierarchical analyses show that the ULK1 complex comprised of ULK1-FIP200-ATG13-ATG101 translocating from the cytosol to autophagosome formation sites as a most upstream ATG factor; this translocation is critical in autophagy initiation. However, how this translocation occurs remains unclear. Here, we show that ULK1 is palmitoylated by palmitoyltransferase ZDHHC13 and translocated to the autophagosome formation site upon autophagy induction. We find that the ULK1 palmitoylation is required for autophagy initiation. Moreover, the ULK1 palmitoylated enhances the phosphorylation of ATG14L, which is required for activating PI3-Kinase and producing phosphatidylinositol 3-phosphate, one of the autophagosome membrane's lipids. Our results reveal how the most upstream ULK1 complex translocates to the autophagosome formation sites during autophagy.
Assuntos
Aciltransferases , Autofagossomos , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Autofagia , Peptídeos e Proteínas de Sinalização Intracelular , Lipoilação , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Autofagia/fisiologia , Humanos , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fosforilação , Aciltransferases/metabolismo , Aciltransferases/genética , Autofagossomos/metabolismo , Células HEK293 , Fosfatos de Fosfatidilinositol/metabolismo , Animais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Transporte Proteico , Proteínas de Transporte VesicularRESUMO
Autophagy refers to a set of degradative mechanisms whereby cytoplasmic contents are targeted to the lysosome. This is best described for macroautophagy, where a double-membrane compartment (autophagosome) is generated to engulf cytoplasmic contents. Autophagosomes are decorated with ubiquitin-like ATG8 molecules (ATG8s), which are recruited through covalent lipidation, catalysed by the E3-ligase-like ATG16L1 complex. LC3 proteins are ATG8 family members that are often used as a marker for autophagosomes. In contrast to canonical macroautophagy, conjugation of ATG8s to single membranes (CASM) describes a group of non-canonical autophagy processes in which ATG8s are targeted to pre-existing single-membrane compartments. CASM occurs in response to disrupted intracellular pH gradients, when the V-ATPase proton pump recruits ATG16L1 in a process called V-ATPase-ATG16L1-induced LC3 lipidation (VAIL). Recent work has demonstrated a parallel, alternative axis for CASM induction, triggered when the membrane recruitment factor TECPR1 recognises sphingomyelin exposed on the cytosolic face of a membrane and forms an alternative E3-ligase-like complex. This sphingomyelin-TECPR1-induced LC3 lipidation (STIL) is independent of the V-ATPase and ATG16L1. In light of these discoveries, this Cell Science at a Glance article summarises these two mechanisms of CASM to highlight how they differ from canonical macroautophagy, and from each other.
Assuntos
Família da Proteína 8 Relacionada à Autofagia , Autofagia , Humanos , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Família da Proteína 8 Relacionada à Autofagia/genética , Animais , Autofagossomos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Membrana Celular/metabolismoRESUMO
Autophagosome formation initiated on the endoplasmic reticulum (ER)-associated omegasome requires LC3. Translational regulation of LC3 biosynthesis is unexplored. Here we demonstrate that LC3 mRNA is recruited to omegasomes by directly binding to the ER transmembrane Sigma-1 receptor (S1R). Cell-based and in vitro reconstitution experiments show that S1R interacts with the 3' UTR of LC3 mRNA and ribosomes to promote LC3 translation. Strikingly, the 3' UTR of LC3 is also required for LC3 protein lipidation, thereby linking the mRNA-3' UTR to LC3 function. An autophagy-defective S1R mutant responsible for amyotrophic lateral sclerosis cannot bind LC3 mRNA or induce LC3 translation. We propose a model wherein S1R de-represses LC3 mRNA via its 3' UTR at the ER, enabling LC3 biosynthesis and lipidation. Because several other LC3-related proteins use the same mechanism, our data reveal a conserved pathway for localized translation essential for autophagosome biogenesis with insights illuminating the molecular basis of a neurodegenerative disease.
Assuntos
Regiões 3' não Traduzidas , Autofagia , Retículo Endoplasmático , Proteínas Associadas aos Microtúbulos , Biossíntese de Proteínas , RNA Mensageiro , Receptores sigma , Receptor Sigma-1 , Receptores sigma/metabolismo , Receptores sigma/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Retículo Endoplasmático/metabolismo , Humanos , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Regiões 3' não Traduzidas/genética , Ribossomos/metabolismo , Animais , Autofagossomos/metabolismo , Células HeLaRESUMO
BACKGROUND: The encystation of Acanthamoeba castellanii has important ecological and medical significance. Blocking encystation is the key to preventing transmission and curing infections caused by A. castellanii. The formation of autophagosomes is one of the most important changes that occur during the encystation of Acanthamoeba. Our previous studies have shown that the heat shock protein 20 of A. castellanii (Ac-HSP20) is involved in its encystation. This study aimed to determine the role and mechanism of Ac-HSP20 in regulating autophagy involved in the encystation of A. castellanii. METHODS: Immunofluorescence assay, western blotting and transmission electron microscopy were used to analyze the dynamic changes in autophagy during the initiation and continuation of encystation. The knockdown of Ac-HSP20 was performed to clarify its regulation of encystation and autophagy and to elucidate the molecular mechanism by which Ac-HSP20 participates in autophagy to promote cyst maturation. RESULTS: The encystation rates and autophagosomes were significantly decreased by treatment with the autophagy inhibitor 3-MA. The autophagy marker LC3B and autophagic lysosomes increased with the induced duration of encystation and reached the maximum at 48 h. The encystation rate, LC3B expression and autophagosomes decreased when Ac-HSP20 was knocked down by siRNA transfection. In addition, the expression levels of Ac-HSP20 and LC3B increased and the expressions of p-AKT and p-mTOR decreased after 48 h of encystation without knockdown. However, the expressions of p-AKT and p-mTOR increased while the expression of LC3B decreased under the knockdown of Ac-HSP20. Furthermore, the protein expression of LC3B increased when the PI3K/AKT/mTOR signaling pathway was inhibited but decreased when the pathway was activated. CONCLUSIONS: The results demonstrated that autophagy is positively correlated with the encystation of A. castellanii, and Ac-HSP20 regulates autophagy to maintain the homeostasis of A. castellanii by inhibiting the PI3K /AKT /mTOR signaling pathway, thus promoting the maturation and stability of encystation.
Assuntos
Acanthamoeba castellanii , Autofagia , Proteínas de Choque Térmico HSP20 , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Serina-Treonina Quinases TOR , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/genética , Acanthamoeba castellanii/fisiologia , Acanthamoeba castellanii/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas de Choque Térmico HSP20/metabolismo , Proteínas de Choque Térmico HSP20/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/genética , Encistamento de Parasitas/fisiologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Autofagossomos/metabolismoRESUMO
Autophagy is a universal degradation system in eukaryotic cells. In plants, although autophagosome biogenesis has been extensively studied, the mechanism of how autophagosomes are transported to the vacuole for degradation remains largely unexplored. In this study, we demonstrated that upon autophagy induction, Arabidopsis homotypic fusion and protein sorting (HOPS) subunit VPS41 converts first from condensates to puncta, then to ring-like structures, termed VPS41-associated phagic vacuoles (VAPVs), which enclose autophagy-related gene (ATG)8s for vacuolar degradation. This process is initiated by ADP ribosylation factor (ARF)-like GTPases ARLA1s and occurs concurrently with autophagy progression through coupling with the synaptic-soluble N-ethylmaleimide-sensitive factor attachment protein rmleceptor (SNARE) proteins. Unlike in other eukaryotes, autophagy degradation in Arabidopsis is largely independent of the RAB7 pathway. By contrast, dysfunction in the condensates-to-VAPVs conversion process impairs autophagosome structure and disrupts their vacuolar transport, leading to a significant reduction in autophagic flux and plant survival rate. Our findings suggest that the conversion pathway might be an integral part of the autophagy program unique to plants.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Autofagossomos , Autofagia , Vacúolos , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Vacúolos/metabolismo , Autofagossomos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/genética , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Família da Proteína 8 Relacionada à Autofagia/genética , Proteínas SNARE/metabolismo , Proteínas SNARE/genética , proteínas de unión al GTP Rab7 , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Potential exposure to cobalt nanoparticles (CoNPs) occurs in various fields, including hard alloy industrial production, the increasing use of new energy lithium-ion batteries, and millions of patients with metal-on-metal joint prostheses. Evidence from human, animal, and in vitro experiments suggests a close relationship between CoNPs and neurotoxicity. However, a systematic assessment of central nervous system (CNS) impairment due to CoNPs exposure and the underlying molecular mechanisms is lacking. In this study, we found that CoNPs induced neurodegenerative damage both in vivo and in vitro, including cognitive impairment, ß-amyloid deposition and Tau hyperphosphorylation. CoNPs promoted the formation of autophagosomes and impeding autophagosomal-lysosomal fusion in vivo and in vitro, leading to toxic protein accumulation. Moreover, CoNPs exposure reduced the level of transcription factor EB (TFEB) and the abundance of lysosome, causing a blockage in autophagosomal-lysosomal fusion. Interestingly, overexpression of long noncoding RNA NR_030777 mitigated CoNPs-induced neurodegenerative damage in both in vivo and in vitro models. Fluorescence in situ hybridization assay revealed that NR_030777 directly binds and stabilizes TFEB mRNA, alleviating the blockage of autophagosomal-lysosomal fusion and ultimately restoring neurodegeneration induced by CoNPs in vivo and in vitro. In summary, our study demonstrates that autophagic dysfunction is the main toxic mechanism of neurodegeneration upon CoNPs exposure and NR_030777 plays a crucial role in CoNPs-induced autophagic dysfunction. Additionally, the proposed adverse outcome pathway contributes to a better understanding of CNS toxicity assessment of CoNPs.
Assuntos
Autofagossomos , Cobalto , Lisossomos , Nanopartículas Metálicas , RNA Longo não Codificante , Lisossomos/metabolismo , Lisossomos/efeitos dos fármacos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Autofagossomos/metabolismo , Autofagossomos/efeitos dos fármacos , Cobalto/química , Cobalto/farmacologia , Animais , Nanopartículas Metálicas/química , Humanos , Camundongos , Masculino , Autofagia/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/induzido quimicamenteRESUMO
We previously reported autophagy-mediated degradation of nuclei, nucleophagy, in the filamentous fungus Aspergillus oryzae. In this study, we examined whether nuclei are degraded as a whole. We generated A. oryzae mutants deleted for orthologs of Saccharomyces cerevisiae YPT7 and ATG15 which are required, respectively, for autophagosome-vacuole fusion and vacuolar degradation of autophagic bodies. Degradation of histone H2B-EGFP under starvation conditions was greatly decreased in the ΔAoypt7 and ΔAoatg15 mutants. Fluorescence and electron microscopic observations showed that autophagosomes and autophagic bodies surrounding the entire nuclei were accumulated in the cytoplasm of ΔAoypt7 and the vacuole of ΔAoatg15, respectively. These results indicate that nuclei are engulfed in the autophagosomes as a whole and transported/released into the vacuolar lumen where they are degraded.
Assuntos
Aspergillus oryzae , Autofagossomos , Proteínas Fúngicas , Vacúolos , Vacúolos/metabolismo , Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Autofagossomos/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Autofagia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Deleção de Genes , Proteínas rab de Ligação ao GTPRESUMO
The selective degradation of nuclear components via autophagy, termed nucleophagy, is an essential process observed from yeasts to mammals and crucial for maintaining nucleus homeostasis and regulating nucleus functions. In the budding yeast Saccharomyces cerevisiae, nucleophagy occurs in two different manners: one involves autophagosome formation for the sequestration and vacuolar transport of nucleus-derived vesicles (NDVs), and the other proceeds with the invagination of the vacuolar membrane for the uptake of NDVs into the vacuole, termed macronucleophagy and micronucleophagy, respectively. This chapter describes methods to analyze and quantify activities of these nucleophagy pathways in yeast.
Assuntos
Autofagia , Núcleo Celular , Saccharomyces cerevisiae , Vacúolos , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Núcleo Celular/metabolismo , Autofagia/fisiologia , Autofagossomos/metabolismoRESUMO
Selective removal of excess or damaged mitochondria is an evolutionarily conserved process that contributes to mitochondrial quality and quantity control. This catabolic event relies on autophagy, a membrane trafficking system that sequesters cytoplasmic constituents into double membrane-bound autophagosomes and delivers them to lysosomes (vacuoles in yeast) for hydrolytic degradation and is thus termed mitophagy. Dysregulation of mitophagy is associated with various diseases, highlighting its physiological relevance. In budding yeast, the pro-mitophagic single-pass membrane protein Atg32 is upregulated under prolonged respiration or nutrient starvation, anchored on the surface of mitochondria, and activated to recruit the autophagy machinery for the formation of autophagosomes surrounding mitochondria. In this chapter, we provide protocols to assess Atg32-mediated mitophagy using fluorescence microscopy and immunoblotting.