RESUMO
Complement C3 (C3) is usually deposited spontaneously on the surfaces of invading bacteria prior to internalization, but the impact of C3 coating on cellular responses is largely unknown. Staphylococcus aureus (S. aureus) is a facultative intracellular pathogen that subverts autophagy and replicates in both phagocytic and nonphagocytic cells. In the present study, we deposited C3 components on the surface of S. aureus by complement opsonization before cell infection and confirmed that C3-coatings remained on the surface of the bacteria after they have invaded the cells, suggesting S. aureus cannot escape or degrade C3 labeling. We found that the C3 deposition on S. aureus notably enhanced cellular autophagic responses, and distinguished these responses as xenophagy, in contrast to LC3-associated phagocytosis (LAP). Furthermore, this upregulation was due to the recruitment of and direct interaction with autophagy-related 16-like 1 (ATG16L1), thereby resulting in autophagy-dependent resistance to bacterial growth within cells. Interestingly, this autophagic effect occurred only after C3 activation by enzymatic cleavage because full-length C3 without cleavage of the complement cascade reaction, although capable of binding to ATG16L1, failed to promote autophagy. These findings demonstrate the biological function of intracellular C3 upon bacterial infection in enhancing autophagy against internalized S. aureus.
Assuntos
Autofagia , Complemento C3 , Fagocitose , Infecções Estafilocócicas , Staphylococcus aureus , Staphylococcus aureus/imunologia , Staphylococcus aureus/fisiologia , Complemento C3/metabolismo , Humanos , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Animais , Interações Hospedeiro-Patógeno , Camundongos , Opsonização , Ativação do ComplementoRESUMO
Amyotrophic lateral sclerosis (ALS) is an adult-onset motor neuron disease with a mean survival time of three years. The 97% of the cases have TDP-43 nuclear depletion and cytoplasmic aggregation in motor neurons. TDP-43 prevents non-conserved cryptic exon splicing in certain genes, maintaining transcript stability, including ATG4B, which is crucial for autophagosome maturation and Microtubule-associated proteins 1A/1B light chain 3B (LC3B) homeostasis. In ALS mice (G93A), Atg4b depletion worsens survival rates and autophagy function. For the first time, we observed an elevation of LC3ylation in the CNS of both ALS patients and atg4b-/- mouse spinal cords. Furthermore, LC3ylation modulates the distribution of ATG3 across membrane compartments. Antisense oligonucleotides (ASOs) targeting cryptic exon restore ATG4B mRNA in TARDBP knockdown cells. We further developed multi-target ASOs targeting TDP-43 binding sequences for a broader effect. Importantly, our ASO based in peptide-PMO conjugates show brain distribution post-IV administration, offering a non-invasive ASO-based treatment avenue for neurodegenerative diseases.
Assuntos
Esclerose Lateral Amiotrófica , Proteínas Relacionadas à Autofagia , Cisteína Endopeptidases , Proteínas de Ligação a DNA , Proteínas Associadas aos Microtúbulos , Animais , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Humanos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases/genética , Masculino , Medula Espinal/metabolismo , Medula Espinal/patologia , Autofagia/fisiologia , Camundongos Knockout , Splicing de RNA/genética , Feminino , Camundongos Transgênicos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Oligonucleotídeos Antissenso/farmacologiaRESUMO
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
Autophagy is a catabolic process that was described to play a critical role in advanced stages of cancer, wherein it maintains tumor cell homeostasis and growth by supplying nutrients. Autophagy is also described to support alternative cellular trafficking pathways, providing a non-canonical autophagy-dependent inflammatory cytokine secretion mechanism. Therefore, autophagy inhibitors have high potential in the treatment of cancer and acute inflammation. In our study, we identified compound 1 as an inhibitor of the ATG12-ATG3 protein-protein interaction. We focused on the systematic modification of the original hit 1, a casein kinase 2 (CK2) inhibitor, to find potent disruptors of ATG12-ATG3 protein-protein interaction. A systematic modification of the hit structure led us to a wide plethora of compounds that maintain its ATG12-ATG3 inhibitory activity, which could act as a viable starting point to design new compounds with diverse therapeutic applications.
Assuntos
Proteínas Relacionadas à Autofagia , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade , Humanos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/síntese química , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/antagonistas & inibidores , Ligação Proteica , Estrutura Molecular , Autofagia/efeitos dos fármacos , Relação Dose-Resposta a Droga , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/metabolismoRESUMO
Autophagy pathway plays a central role in cellular degradation. The proteins involved in the core autophagy process are mostly localised on membranes or interact indirectly with lipid-associated proteins. Therefore, progress in structure determination of 'core autophagy proteins' remained relatively limited. Recent paradigm shift in structural biology that includes cutting-edge cryo-EM technology and robust AI-based Alphafold2 predicted models has significantly increased data points in biology. Here, we developed Autophagy3D, a web-based resource that provides an efficient way to access data associated with 40 core human autophagic proteins (80322 structures), their protein-protein interactors and ortholog structures from various species. Autophagy3D also offers detailed visualizations of protein structures, and, hence deriving direct biological insights. The database significantly enhances access to information as full datasets are available for download. The Autophagy3D can be publicly accessed via https://autophagy3d.igib.res.in. Database URL: https://autophagy3d.igib.res.in.
Assuntos
Autofagia , Bases de Dados de Proteínas , Humanos , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/química , InternetRESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron degeneration. Dysregulation of long non-coding RNAs (lncRNAs) has been implicated in ALS pathogenesis but their roles remain unclear. Previous studies found lnc-ABCA12-3 was downregulated in ALS patients. We aim to characterize the expression and function of lnc-ABCA12-3 in ALS and explore its mechanisms of action. Lnc-ABCA12-3 expression was analyzed in PBMCs from ALS patients and correlated with clinical outcomes. Effect of modulating lnc-ABCA12-3 expression was assessed in cell models using assays of apoptosis, protein homeostasis and pathway analysis. RNA pull-down and interaction studies were performed to identify lnc-ABCA12-3 binding partners. Lnc-ABCA12-3 was downregulated in ALS patients, correlating with faster progression and shorter survival. Overexpression of lnc-ABAC12-3 conferred protection against oxidative stress-induced apoptosis, while knockdown lnc-ABCA12-3 enhanced cell death. Lnc-ABCA12-3 maintained protein quality control pathways, including ubiquitination, autophagy and stress granule formation, by regulating the ubiquitin shuttle protein UBQLN1. This study identified lnc-ABCA12-3 as a novel regulatory lncRNA implicated in ALS pathogenesis by modulating cellular survival and stress responses through interactions with UBQLN1, influencing disease progression. Lnc-ABCA12-3 may influence ALS through regulating protein homeostasis pathways.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Esclerose Lateral Amiotrófica , Apoptose , Proteínas Relacionadas à Autofagia , Regulação para Baixo , RNA Longo não Codificante , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Humanos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Apoptose/genética , Feminino , Proteostase , Masculino , Pessoa de Meia-Idade , Autofagia/genética , Estresse Oxidativo , Regulação da Expressão GênicaRESUMO
ATG9A is the only integral membrane protein among core autophagy-related (ATG) proteins. We previously found that ATG9A does not co-assemble into synaptophysin-positive vesicles, but rather, localizes to a distinct pool of vesicles within synapsin condensates in both fibroblasts and nerve terminals. The endocytic origin of these vesicles further suggests the existence of different intracellular sorting or segregation mechanisms for ATG9A and synaptophysin in cells. However, the precise underlying mechanism remains largely unknown. In this follow-up study, we investigated the endosomal localization of these two proteins by exploiting the advantages of a Rab5 mutant that induces the formation of enlarged endosomes. Notably, ATG9A and synaptophysin intermix perfectly and do not segregate on giant endosomes, indicating that the separation of these two proteins is not solely caused by the inherent properties of the proteins, but possibly by other unknown factors.
Assuntos
Proteínas Relacionadas à Autofagia , Endossomos , Mutação , Sinaptofisina , Proteínas rab5 de Ligação ao GTP , Endossomos/metabolismo , Mutação/genética , Sinaptofisina/metabolismo , Sinaptofisina/genética , Proteínas rab5 de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Animais , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Humanos , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/genética , CamundongosRESUMO
Cells release intraluminal vesicles in multivesicular bodies as exosomes to communicate with other cells. Although recent studies suggest an intimate link between exosome biogenesis and autophagy, the detailed mechanism is not fully understood. Here we employed comprehensive RNA interference screening for autophagy-related factors and discovered that Rubicon, a negative regulator of autophagy, is essential for exosome release. Rubicon recruits WIPI2d to endosomes to promote exosome biogenesis. Interactome analysis of WIPI2d identified the ESCRT components that are required for intraluminal vesicle formation. Notably, we found that Rubicon is required for an age-dependent increase of exosome release in mice. In addition, small RNA sequencing of serum exosomes revealed that Rubicon determines the fate of exosomal microRNAs associated with cellular senescence and longevity pathways. Taken together, our current results suggest that the Rubicon-WIPI axis functions as a key regulator of exosome biogenesis and is responsible for age-dependent changes in exosome quantity and quality.
Assuntos
Envelhecimento , Proteínas Relacionadas à Autofagia , Complexos Endossomais de Distribuição Requeridos para Transporte , Exossomos , MicroRNAs , Exossomos/metabolismo , Exossomos/genética , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , Humanos , Envelhecimento/metabolismo , Envelhecimento/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Autofagia , Camundongos , Senescência Celular , Camundongos Endogâmicos C57BL , Células HEK293 , Endossomos/metabolismo , Camundongos Knockout , MasculinoRESUMO
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
Colorectal cancer (CRC) remains a highly prevalent gastrointestinal neoplasm, presenting significant prevalence and lethality rate. DEAD/H box RNA helicase 10 (DDX10) has been proposed as a potential oncogene in CRC, the specific action mechanism by which DDX10 modulates the aggressive biological cellular events in CRC remains implicitly elucidated, however. During this study, DDX10 expression was detected via RT-qPCR and Western blotting. Cell proliferation was estimated via EDU staining. TUNEL staining and Western blotting appraised cell apoptosis. Cell stemness was evaluated by sphere formation assay, RT-qPCR, Western blotting as well as immunofluorescence staining. Relevant assay kit examined aldehyde dehydrogenase (ALDH) activity. Western blotting and immunofluorescence staining also detected autophagy. DDX10 was hyper-expressed in CRC cells. Down-regulation of DDX10 hampered cell proliferation, aggravated the apoptosis while eliminated the ability to form spheroid cells in CRC. In addition, DDX10 deletion improved ATG10 expression and therefore activated autophagy in CRC cells. Consequently, ATG10 depletion or treatment with autophagy inhibitor 3-Methyladenine (3-MA) partially compensated the influences of DDX10 silencing on the proliferation, apoptosis and stemness of CRC cells. Accordingly, DDX10 deficiency may aggravate autophagy mediated by ATG10 to impede cell proliferation, stemness and facilitate cell apoptosis, hence blocking the progression of CRC.
Assuntos
Apoptose , Proteínas Relacionadas à Autofagia , Autofagia , Proliferação de Células , Neoplasias Colorretais , RNA Helicases DEAD-box , Células-Tronco Neoplásicas , Humanos , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/genética , Autofagia/fisiologia , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/metabolismo , Linhagem Celular Tumoral , Enzimas de Conjugação de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Camundongos , Animais , Proteínas de Transporte VesicularRESUMO
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 initiation is regulated by the ULK1 kinase complex. To gain insights into functions of the holo-complex, we generated a deep interactome by combining affinity purification- and proximity labeling-mass spectrometry of all four complex members: ULK1, ATG13, ATG101, and RB1CC1/FIP200. Under starvation conditions, the ULK1 complex interacts with several protein and lipid kinases and phosphatases, implying the formation of a signalosome. Interestingly, several selective autophagy receptors also interact with ULK1, indicating the activation of selective autophagy pathways by nutrient starvation. One effector of the ULK1 complex is the HSC/HSP70 co-chaperone BAG2, which regulates the subcellular localization of the VPS34 lipid kinase complex member AMBRA1. Depending on the nutritional status, BAG2 has opposing roles. In growth conditions, the unphosphorylated form of BAG2 sequesters AMBRA1, attenuating autophagy induction. In starvation conditions, ULK1 phosphorylates BAG2 on Ser31, which supports the recruitment of AMBRA1 to the ER membrane, positively affecting autophagy.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Autofagia , Peptídeos e Proteínas de Sinalização Intracelular , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células HEK293 , Fosforilação , Proteínas Relacionadas à Autofagia/metabolismo , Ligação Proteica , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Células HeLa , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/genética , Chaperonas MolecularesRESUMO
This research focused on investigating the effects of sevoflurane (Sev) on myocardial autophagy levels after myocardial ischemia reperfusion (I/R) injury via the microRNA-542-3p (miR-542-3p)/ADAM9 axis. Mice underwent 30 min occlusion of the left anterior descending coronary (LAD) followed by 2 h reperfusion. Cardiac infarction was determined by 2,3,5-triphenyltetrazolium chloride triazole (TTC) staining. Cardiac function was examined by echocardiography. Cardiac markers and oxidative stress factors were evaluated by ELISA. Autophagy-associated factors were detected by western blot. Relationship between miR-542-3p and ADAM9 was tested by dual-luciferase reporter gene assay, RT-qPCR, and western blot. Sev treatment ameliorated cardiac dysfunction, myocardial oxidative stress, and histopathological damages, decreased myocardial infarction size and myocardial apoptotic cells after myocardial I/R injury. Sev treatment elevated miR-542-3p expression and decreased ADAM9 expression in myocardial tissues after myocardial I/R injury. miR-542-3p overexpression could enhance the ameliorative effects of Sev on myocardial injury and myocardial autophagy in I/R mice. miR-542-3p targeted and negatively regulated ADAM9 expression. ADAM9 overexpression reversed the ameliorative effects of miR-542-3p up-regulation on myocardial injury and myocardial autophagy in Sev-treated I/R mice. Sev treatment could ameliorate myocardial injury and myocardial autophagy in I/R mice, mediated by mechanisms that include miR-542-3p up-regulation and ADAM9 down-regulation.
Assuntos
Autofagia , Modelos Animais de Doenças , Proteínas de Membrana , Camundongos Endogâmicos C57BL , MicroRNAs , Infarto do Miocárdio , Traumatismo por Reperfusão Miocárdica , Miócitos Cardíacos , Estresse Oxidativo , Sevoflurano , Transdução de Sinais , Animais , MicroRNAs/metabolismo , MicroRNAs/genética , Sevoflurano/farmacologia , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Autofagia/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Miócitos Cardíacos/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/genética , Infarto do Miocárdio/fisiopatologia , Estresse Oxidativo/efeitos dos fármacos , Proteínas ADAM/metabolismo , Proteínas ADAM/genética , Apoptose/efeitos dos fármacos , Camundongos , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genéticaRESUMO
Ubiquitination is one of the most common posttranslational modifications in eukaryotic cells. Depending on the architecture of polyubiquitin chains, substrate proteins can meet different cellular fates, but our understanding of how chain linkage controls protein fate remains limited. UBL-UBA shuttle proteins, such as UBQLN2, bind to ubiquitinated proteins and to the proteasome or other protein quality control machinery elements and play a role in substrate fate determination. Under physiological conditions, UBQLN2 forms biomolecular condensates through phase separation, a physicochemical phenomenon in which multivalent interactions drive the formation of a macromolecule-rich dense phase. Ubiquitin and polyubiquitin chains modulate UBQLN2's phase separation in a linkage-dependent manner, suggesting a possible link to substrate fate determination, but polyubiquitinated substrates have not been examined directly. Using sedimentation assays and microscopy we show that polyubiquitinated substrates induce UBQLN2 phase separation and incorporate into the resulting condensates. This substrate effect is strongest with K63-linked substrates, intermediate with mixed-linkage substrates, and weakest with K48-linked substrates. Proteasomes can be recruited to these condensates, but proteasome activity toward K63-linked and mixed linkage substrates is inhibited in condensates. Substrates are also protected from deubiquitinases by UBQLN2-induced phase separation. Our results suggest that phase separation could regulate the fate of ubiquitinated substrates in a chain-linkage-dependent manner, thus serving as an interpreter of the ubiquitin code.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Relacionadas à Autofagia , Ubiquitinação , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/química , Proteínas Relacionadas à Autofagia/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Humanos , Poliubiquitina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Condensados Biomoleculares/metabolismo , Condensados Biomoleculares/química , Ubiquitina/metabolismo , Ubiquitina/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas Ubiquitinadas/metabolismo , Proteínas Ubiquitinadas/isolamento & purificação , Proteínas Ubiquitinadas/química , Separação de FasesRESUMO
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
Autophagy induction in cancer is involved in cancer progression and the acquisition of resistance to anticancer agents. Therefore, autophagy is considered a potential therapeutic target in cancer therapy. In this study, we found that long-chain fatty acids (LCFAs) have inhibitory effects on Atg4B, which is essential for autophagosome formation, through screening based on the pharmacophore of 21f, a recently developed Atg4B inhibitor. Among these fatty acids, docosahexaenoic acid (DHA), a polyunsaturated fatty acid, exhibited the most potent Atg4B inhibitory activity. DHA inhibited autophagy induced by androgen receptor signaling inhibitors (ARSI) in LNCaP and 22Rv1 prostate cancer cells and significantly increased apoptotic cell death. Furthermore, we investigated the effect of DHA on resistance to ARSI by establishing darolutamide-resistant prostate cancer 22Rv1 (22Rv1/Dar) cells, which had developed resistance to darolutamide, a novel ARSI. At baseline, 22Rv1/Dar cells showed a higher autophagy level than parental 22Rv1 cells. DHA significantly suppressed Dar-induced autophagy and sensitized 22Rv1/Dar cells by inducing apoptotic cell death through mitochondrial dysfunction. These results suggest that DHA supplementation may improve prostate cancer therapy with ARSI.
Assuntos
Proteínas Relacionadas à Autofagia , Autofagia , Cisteína Endopeptidases , Ácidos Docosa-Hexaenoicos , Neoplasias de Próstata Resistentes à Castração , Masculino , Humanos , Ácidos Docosa-Hexaenoicos/farmacologia , Autofagia/efeitos dos fármacos , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/patologia , Neoplasias de Próstata Resistentes à Castração/metabolismo , Linhagem Celular Tumoral , Proteínas Relacionadas à Autofagia/metabolismo , Cisteína Endopeptidases/metabolismo , Apoptose/efeitos dos fármacos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacosRESUMO
Subversion of cellular membranes and membrane proliferation are used by positive-strand RNA viruses to build viral replication organelles (VROs) that support virus replication. The biogenesis of the membranous VROs requires major changes in lipid metabolism and lipid transfer in infected cells. In this work, we show that tomato bushy stunt virus (TBSV) hijacks Atg2 autophagy related protein with bulk lipid transfer activity into VROs via interaction with TBSV p33 replication protein. Deletion of Atg2 in yeast and knockdown of Atg2 in Nicotiana benthamiana resulted in decreased TBSV replication. We found that subversion of Atg2 by TBSV was important to enrich VRO membranes with phosphatidylethanolamine (PE), phosphatidylserine (PS) and PI(3)P phosphoinositide. Interestingly, inhibition of autophagy did not affect the efficient recruitment of Atg2 into VROs, and overexpression of Atg2 enhanced TBSV replication, indicating autophagy-independent subversion of Atg2 by TBSV. These findings suggest that the proviral function of Atg2 lipid transfer protein is in VRO membrane proliferation. In addition, we find that Atg2 interacting partner Atg9 with membrane lipid-scramblase activity is also coopted for tombusvirus replication. Altogether, the subversion of Atg2 bridge-type lipid transfer protein provides a new mechanism for tombusviruses to greatly expand VRO membranes to support robust viral replication.
Assuntos
Proteínas Relacionadas à Autofagia , Autofagia , Nicotiana , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Tombusvirus , Replicação Viral , Tombusvirus/fisiologia , Tombusvirus/metabolismo , Replicação Viral/fisiologia , Proteínas Relacionadas à Autofagia/metabolismo , Nicotiana/virologia , Nicotiana/metabolismo , Autofagia/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fosfolipídeos/metabolismo , Compartimentos de Replicação Viral/metabolismo , Proteínas de Plantas/metabolismo , Fosfatidiletanolaminas/metabolismo , Proteínas Virais/metabolismo , Proteínas de Transporte/metabolismo , Doenças das Plantas/virologia , Membrana Celular/metabolismoRESUMO
OBJECTIVES: This study aims to explore the mechanisms of dual regulation of osteoarthritis (OA) progression by the involvement of estrogen receptor (ER) in autophagy and inflammation. MATERIALS AND METHODS: Bioinformatics methods were used to explore the relationship among associated genes. Western blot assays were used to detect related protein expression of OA in C28I2 and induced OA cellular model. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis were used to detect OA related gene expression in C28I2 and induced OA cellular model. Co-immunoprecipitation (CO-IP) analysis were used to verify the direct interaction between ER and NOD-like receptor thermal protein domain associated protein 3 (NLRP3). RESULTS: The C28I2 cellular model of OA was induced by interleukin-1ß (IL-1ß). The small interfering ribonucleic acid (SiRNA)-mediated knockdown of autophagy-related 16 like 1 (ATG16L1) in C28I2 decreased the expression of MAP1LC3B (LC3B) and NLRP3. Besides, ER-beta (ERß) agonist changed the gene expression of NLRP3 and ATG16L1. Moreover, CO-IP analysis indicated the direct interaction between ER and NLRP3. CONCLUSION: Our study results revealed that ATG16L1, NLRP3, and IL-1ß interacted closely and ERß was involved in OA process by affecting autophagy and inflammatory activation.
Assuntos
Proteínas Relacionadas à Autofagia , Receptor beta de Estrogênio , Proteína 3 que Contém Domínio de Pirina da Família NLR , Osteoartrite , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Osteoartrite/metabolismo , Osteoartrite/patologia , Osteoartrite/genética , Humanos , Receptor beta de Estrogênio/metabolismo , Receptor beta de Estrogênio/genética , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Autofagia , Interleucina-1beta/metabolismo , Interleucina-1beta/genética , Receptor alfa de Estrogênio/metabolismo , Receptor alfa de Estrogênio/genética , Linhagem CelularRESUMO
The characterization of lipid binding to lipid transfer proteins (LTPs) is fundamental to understand their molecular mechanism. However, several structures of LTPs, and notably those proposed to act as bridges between membranes, do not provide the precise location of their endogenous lipid ligands. To address this limitation, computational approaches are a powerful alternative methodology, but they are often limited by the high flexibility of lipid substrates. Here, we develop a protocol based on unbiased coarse-grain molecular dynamics simulations in which lipids placed away from the protein can spontaneously bind to LTPs. This approach accurately determines binding pockets in LTPs and provides a working hypothesis for the lipid entry pathway. We apply this approach to characterize lipid binding to bridge LTPs of the Vps13-Atg2 family, for which the lipid localization inside the protein is currently unknown. Overall, our work paves the way to determine binding pockets and entry pathways for several LTPs in an inexpensive, fast, and accurate manner.
Assuntos
Proteínas de Transporte , Simulação de Dinâmica Molecular , Ligação Proteica , Sítios de Ligação , Proteínas de Transporte/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Lipídeos/química , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/química , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/químicaRESUMO
Autophagy is a complex physiological pathway mediating homeostasis and survival of cells degrading damaged organelles and regulating their recycling. Physiologic autophagy can maintain normal lung function, decrease lung cellular senescence, and inhibit myofibroblast differentiation. It is well known that autophagy is activated in several chronic inflammatory diseases; however, its role in the pathogenesis of chronic obstructive pulmonary disease (COPD) and the expression of autophagy-related genes (ATGs) in lower airways of COPD patients is still controversial. The expression and localization of all ATG proteins that represented key components of the autophagic machinery modulating elongation, closure, and maturation of autophagosome membranes were retrospectively measured in peripheral lungs of patients with stable COPD (n = 10), control smokers with normal lung function (n = 10), and control nonsmoking subjects (n = 8) using immunohistochemical analysis. These results show an increased expression of ATG4 protein in alveolar septa and bronchiolar epithelium of stable COPD patients compared to smokers with normal lung function and non-smoker subjects. In particular, the genes in the ATG4 protein family (including ATG4A, ATG4B, ATG4C, and ATG4D) that have a key role in the modulation of the physiological autophagic machinery are the most important ATGs increased in the compartment of lower airways of stable COPD patients, suggesting that the alteration shown in COPD patients can be also correlated to impaired modulation of autophagic machinery modulating elongation, closure, and maturation of autophagosomes membranes. Statistical analysis was performed by the Kruskal-Wallis test and the Mann-Whitney U test for comparison between groups. A statistically significant increased expression of ATG4A (p = 0.0047), ATG4D (p = 0.018), and ATG5 (p = 0.019) was documented in the bronchiolar epithelium as well in alveolar lining for ATG4A (p = 0.0036), ATG4B (p = 0.0054), ATG4C (p = 0.0064), ATG4D (p = 0.0084), ATG5 (p = 0.0088), and ATG7 (p = 0.018) in patients with stable COPD compared to control groups. The ATG4 isoforms may be considered as additional potential targets for the development of new drugs in COPD.