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1.
J Med Chem ; 66(4): 2457-2476, 2023 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-36749313

RESUMO

One possible strategy for modulating autophagy is to disrupt the critical protein-protein interactions (PPIs) formed during this process. Our attention is on the autophagy-related 12 (ATG12)-autophagy-related 5 (ATG5)-autophagy-related 16-like 1 (ATG16L1) heterotrimer complex, which is responsible for ATG8 translocation from ATG3 to phosphatidylethanolamine. In this work, we discovered a compound with an (E)-3-(2-furanylmethylene)-2-pyrrolidinone core moiety (T1742) that blocked the ATG5-ATG16L1 and ATG5-TECAIR interactions in the in vitro binding assay (IC50 = 1-2 µM) and also exhibited autophagy inhibition in cellular assays. The possible binding mode of T1742 to ATG5 was predicted through molecular modeling, and a batch of derivatives sharing essentially the same core moiety were synthesized and tested. The outcomes of the in vitro binding assay and the flow cytometry assay of those newly synthesized compounds were generally consistent. This work has validated our central hypothesis that small-molecule inhibitors of the PPIs involving ATG5 can tune down autophagy effectively, and their pharmaceutical potential may be further explored.


Assuntos
Antineoplásicos , Proteína 12 Relacionada à Autofagia , Proteína 5 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Autofagia , Complexos Multiproteicos , Autofagia/efeitos dos fármacos , Proteína 12 Relacionada à Autofagia/antagonistas & inibidores , Proteína 12 Relacionada à Autofagia/química , Proteína 5 Relacionada à Autofagia/antagonistas & inibidores , Proteína 5 Relacionada à Autofagia/química , Proteínas Relacionadas à Autofagia/antagonistas & inibidores , Proteínas Relacionadas à Autofagia/química , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Moleculares , Conformação Proteica , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Humanos , Animais
2.
Aging Cell ; 16(5): 1180-1190, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28782874

RESUMO

Mitophagy is thought to be a critical mitochondrial quality control mechanism in neurons and has been extensively studied in neurological disorders such as Parkinson's disease. However, little is known about how mitochondria are maintained in the lengthy neuronal axons in the context of physiological aging. Here, we utilized the unique Drosophila wing nerve model and in vivo imaging to rigorously profile changes in axonal mitochondria during aging. We revealed that mitochondria became fragmented and accumulated in aged axons. However, lack of Pink1 or Parkin did not lead to the accumulation of axonal mitochondria or axonal degeneration. Further, unlike in in vitro cultured neurons, we found that mitophagy rarely occurred in intact axons in vivo, even in aged animals. Furthermore, blocking overall mitophagy by knockdown of the core autophagy genes Atg12 or Atg17 had little effect on the turnover of axonal mitochondria or axonal integrity, suggesting that mitophagy is not required for axonal maintenance; this is regardless of whether the mitophagy is PINK1-Parkin dependent or independent. In contrast, downregulation of mitochondrial fission-fusion genes caused age-dependent axonal degeneration. Moreover, Opa1 expression in the fly head was significantly decreased with age, which may underlie the accumulation of fragmented mitochondria in aged axons. Finally, we showed that adult-onset, neuronal downregulation of the fission-fusion, but not mitophagy genes, dramatically accelerated features of aging. We propose that axonal mitochondria are maintained independently of mitophagy and that mitophagy-independent mechanisms such as fission-fusion may be central to the maintenance of axonal mitochondria and neural integrity during normal aging.


Assuntos
Envelhecimento/genética , Axônios/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas de Membrana/genética , Mitocôndrias/genética , Mitofagia/genética , Envelhecimento/metabolismo , Animais , Proteína 12 Relacionada à Autofagia/antagonistas & inibidores , Proteína 12 Relacionada à Autofagia/genética , Proteína 12 Relacionada à Autofagia/metabolismo , Axônios/ultraestrutura , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Dinâmica Mitocondrial/genética , Imagem Óptica , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética , Asas de Animais/citologia , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/inervação , Asas de Animais/metabolismo , Proteína Vermelha Fluorescente
3.
Toxicol Sci ; 156(1): 72-83, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28013216

RESUMO

Epidemiological studies suggest that an increase of PM2.5 diesel exhaust particles (DEP) in ambient air corresponds to increased myocardial infarctions and atherosclerosis. When exposed to DEP, endothelial cells exhibit increases in oxidative stress and apoptosis, but the role of autophagy in this DEP-induced cell death remains unclear. Here, we suggest that acute DEP exposure produces intracellular reactive oxygen species (ROS) leading to induction of DEP internalization, endothelial dysfunction, and pro-inflammation in an in vitro human umbilical vein endothelial cells (HUVEC) model. This study found that increases in intracellular oxidative stress and cellular internalization of DEP occurred within 2 h of exposure to DEP. After 2 h of DEP exposure, Mdm2 expression was increased, which triggered cellular autophagy after 4 h of DEP exposure and suppressed cellular senescence. Unfortunately, phagocytized DEP could not be eliminated by cellular autophagy, which led to a continuous buildup of ROS, an increased release of cytokines, and an increased expression of anchoring molecules. After 12 h of DEP exposure, HUVEC reduced Mdm2 expression leading to increased p53 expression, which triggered apoptosis and ultimately resulted in endothelial dysfunction. On the other hand, when cells lacked the ability to induce autophagy, DEP was unable to induce cell senescence and most of the cells survived with only a small percentage of the cells undergoing necrosis. The results presented in this study clearly demonstrate the role cellular autophagy plays in DEP-induced atherosclerosis.


Assuntos
Poluentes Atmosféricos/toxicidade , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Material Particulado/toxicidade , Emissões de Veículos/toxicidade , Poluentes Atmosféricos/química , Poluentes Atmosféricos/isolamento & purificação , Poluentes Atmosféricos/metabolismo , Proteína 12 Relacionada à Autofagia/antagonistas & inibidores , Proteína 12 Relacionada à Autofagia/genética , Proteína 12 Relacionada à Autofagia/metabolismo , Biomarcadores/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Ensaio Cometa , Endotélio Vascular/imunologia , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Tamanho da Partícula , Material Particulado/química , Material Particulado/isolamento & purificação , Material Particulado/metabolismo , Fagocitose/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/química , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Interferência de RNA , Espécies Reativas de Oxigênio/agonistas , Espécies Reativas de Oxigênio/metabolismo , Fuligem/química , Fuligem/toxicidade , Tóquio , Vasculite/induzido quimicamente , Vasculite/imunologia , Vasculite/metabolismo , Vasculite/patologia , Emissões de Veículos/análise
4.
PLoS One ; 11(11): e0165701, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27828984

RESUMO

Fusobacterium nucleatum (F. nucleatum) plays a critical role in gastrointestinal inflammation. However, the exact mechanism by which F. nucleatum contributes to inflammation is unclear. In the present study, it was revealed that F. nucleatum could induce the production of proinflammatory cytokines (IL-8, IL-1ß and TNF-α) and reactive oxygen species (ROS) in Caco-2 colorectal) adenocarcinoma cells. Furthermore, ROS scavengers (NAC or Tiron) could decrease the production of proinflammatory cytokines during F. nucleatum infection. In addition, we observed that autophagy is impaired in Caco-2 cells after F. nucleatum infection. The production of proinflammatory cytokines and ROS induced by F. nucleatum was enhanced with either autophagy pharmacologic inhibitors (3-methyladenine, bafilomycin A1) or RNA interference in essential autophagy genes (ATG5 or ATG12) in Caco-2 cells. Taken together, these results indicate that F. nucleatum-induced impairment of autophagic flux enhances the expression of proinflammatory cytokines via ROS in Caco-2 Cells.


Assuntos
Células Epiteliais/imunologia , Infecções por Fusobacterium/imunologia , Fusobacterium nucleatum/imunologia , Interleucina-1beta/imunologia , Interleucina-8/imunologia , Espécies Reativas de Oxigênio/imunologia , Fator de Necrose Tumoral alfa/imunologia , Sal Dissódico do Ácido 1,2-Di-Hidroxibenzeno-3,5 Dissulfônico/farmacologia , Acetilcisteína/farmacologia , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Autofagia/efeitos dos fármacos , Proteína 12 Relacionada à Autofagia/antagonistas & inibidores , Proteína 12 Relacionada à Autofagia/genética , Proteína 12 Relacionada à Autofagia/imunologia , Proteína 5 Relacionada à Autofagia/antagonistas & inibidores , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/imunologia , Células CACO-2 , Linhagem Celular Tumoral , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Sequestradores de Radicais Livres/farmacologia , Infecções por Fusobacterium/genética , Infecções por Fusobacterium/microbiologia , Infecções por Fusobacterium/patologia , Regulação da Expressão Gênica , Humanos , Interleucina-1beta/genética , Interleucina-8/genética , Macrolídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/genética
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