RESUMO
Autophagy is a critical cellular homeostatic mechanism, the dysfunction of which has been linked to a wide variety of disease states. It is regulated through the activity of specific kinases, in particular Unc-51 like autophagy activating kinase 1 (ULK1) and Phosphatidylinositol 3-kinase vacuolar protein sorting 34 (VPS34), which have both been suggested as potential targets for drug development. To identify new chemical compounds that might provide useful chemical tools or act as starting points for drug development, we screened each protein against the Published Kinase Inhibitor Set (PKIS), a library of known kinase inhibitors. In vitro screening and analysis of the published selectivity profiles of the hits informed the selection of three relatively potent ATP-competitive inhibitors against each target that presented the least number of off-target kinases in common. Cellular assays confirmed potent inhibition of autophagy in response to two of the ULK1 inhibitors and all three of the VPS34 inhibitors. These compounds represent not only a new resource for the study of autophagy but also potential chemical starting points for the validation or invalidation of these two centrally important autophagy kinases in disease models.
Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/antagonistas & inibidores , Autofagia , Classe III de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Descoberta de Drogas , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Osteossarcoma/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Osteossarcoma/tratamento farmacológico , Osteossarcoma/metabolismo , Fosforilação , Células Tumorais CultivadasRESUMO
Mitophagy, a conserved intracellular process by which mitochondria are eliminated via the autophagic machinery, is a quality control mechanism which facilitates maintenance of a functional mitochondrial network and cell homeostasis, making it a key process in development and longevity. Mitophagy has been linked to multiple human disorders, especially neurodegenerative diseases where the long-lived neurons are relying on clearance of old/damaged mitochondria to survive. During the past decade, the availability of novel tools to study mitophagy both in vitro and in vivo has significantly advanced our understanding of the molecular mechanisms governing this fundamental process in normal physiology and in various disease models. We here give an overview of the known mitophagy pathways and how they are induced, with a particular emphasis on the early events governing mitophagosome formation.
RESUMO
Autophagy is a crucial homeostatic mechanism that mediates the degradation of damaged or excess intracellular components. Such components are engulfed and sequestered into double membrane autophagosomes, which deliver their contents to lysosomes for degradation. Autophagy plays a role in numerous human disorders and its pharmacological targeting by small molecules offers therapeutic potential. The serine/threonine kinase ULK1 (and its homologue ULK2) is the most upstream component of the autophagic machinery and is required for autophagy initiation. Here, we use the most selective and potent published ULK1 inhibitors to gain insights into ULK1 kinase function during autophagy. Treatment with all inhibitors blocked autophagy but also resulted in the limited formation of initial autophagosome-like structures, which appeared abnormal in size and did not traffic to lysosomes. We found that upon ULK1 inhibition, phosphatidylinositol-3-phosphate-binding proteins are still recruited to forming autophagosomes, implying that ULK1 activity is not essential for VPS34 activation. We conclude that the kinase activity of ULK1 is important in regulating autophagosome maturation, by the phosphorylation of currently unidentified key substrates.
Assuntos
Autofagossomos/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Animais , Autofagossomos/genética , Autofagia/genética , Autofagia/fisiologia , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/antagonistas & inibidores , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/fisiologia , Proteínas Relacionadas à Autofagia/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisossomos/metabolismo , Macroautofagia , Camundongos , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismoRESUMO
The dynamics and coordination between autophagy machinery and selective receptors during mitophagy are unknown. Also unknown is whether mitophagy depends on pre-existing membranes or is triggered on the surface of damaged mitochondria. Using a ubiquitin-dependent mitophagy inducer, the lactone ivermectin, we have combined genetic and imaging experiments to address these questions. Ubiquitination of mitochondrial fragments is required the earliest, followed by auto-phosphorylation of TBK1. Next, early essential autophagy proteins FIP200 and ATG13 act at different steps, whereas ULK1 and ULK2 are dispensable. Receptors act temporally and mechanistically upstream of ATG13 but downstream of FIP200. The VPS34 complex functions at the omegasome step. ATG13 and optineurin target mitochondria in a discontinuous oscillatory way, suggesting multiple initiation events. Targeted ubiquitinated mitochondria are cradled by endoplasmic reticulum (ER) strands even without functional autophagy machinery and mitophagy adaptors. We propose that damaged mitochondria are ubiquitinated and dynamically encased in ER strands, providing platforms for formation of the mitophagosomes.
Assuntos
Retículo Endoplasmático/metabolismo , Mitofagia , Ubiquitinação , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Proteína 3 com Repetições IAP de Baculovírus/metabolismo , Células Cultivadas , Células HEK293 , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Fator 2 Associado a Receptor de TNF/metabolismoRESUMO
Autophagy is a vital lysosomal degradation pathway that serves as a quality control mechanism. It rids the cell of damaged, toxic or excess cellular components, which if left to persist could be detrimental to the cell. It also serves as a recycling pathway to maintain protein synthesis under starvation conditions. A key initial event in autophagy is formation of the autophagosome, a unique double-membrane organelle that engulfs the cytosolic cargo destined for degradation. This step is mediated by the serine/threonine protein kinase ULK1 (unc-51-like kinase 1), which functions in a complex with at least three protein partners: FIP200 (focal adhesion kinase family interacting protein of 200 kDa), ATG (autophagy-related protein) 13 (ATG13), and ATG101. In this artcile, we focus on the regulation of the ULK1 complex during autophagy initiation. The complex pattern of upstream pathways that converge on ULK1 suggests that this complex acts as a node, converting multiple signals into autophagosome formation. Here, we review our current understanding of this regulation and in turn discuss what happens downstream, once the ULK1 complex becomes activated.
Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Humanos , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
The AlamarBlue assay is based on fluorometric detection of metabolic mitochondrial activity of cells. In this study, we determined the methodology for application of the assay to radiation response experiments in 96-well plates. AlamarBlue was added and its reduction measured 7 hours later. Selection of the initial number of plated cells was important so that the number of proliferating cells remains lower than the critical number that produced full AlamarBlue reduction (plateau phase) at the time points of measurements. Culture medium was replaced twice a week to avoid suppression of viability due to nutrient competition and metabolic waste accumulation. There was no need to replace culture medium before adding AlamarBlue. Cell proliferation continued after irradiation and the suppression effect on cell viability was most evident on day 8. At this time point, by comparing measurements from irradiated vs. non-irradiated cells, for various dose levels, a viability dose response curve was plotted. Immediately after the 8(th) day (nadir), cells started to re-grow at a rate inversely related to the radiation dose. By comparing measurements at the time point of nadir vs. a convenient subsequent time point, re-growth dose response abilities were plotted, simulating clonogenic assays.