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1.
Nature ; 558(7708): 136-140, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29849149

RESUMEN

Autophagy increases the lifespan of model organisms; however, its role in promoting mammalian longevity is less well-established1,2. Here we report lifespan and healthspan extension in a mouse model with increased basal autophagy. To determine the effects of constitutively increased autophagy on mammalian health, we generated targeted mutant mice with a Phe121Ala mutation in beclin 1 (Becn1F121A/F121A) that decreases its interaction with the negative regulator BCL2. We demonstrate that the interaction between beclin 1 and BCL2 is disrupted in several tissues in Becn1 F121A/F121A knock-in mice in association with higher levels of basal autophagic flux. Compared to wild-type littermates, the lifespan of both male and female knock-in mice is significantly increased. The healthspan of the knock-in mice also improves, as phenotypes such as age-related renal and cardiac pathological changes and spontaneous tumorigenesis are diminished. Moreover, mice deficient in the anti-ageing protein klotho 3 have increased beclin 1 and BCL2 interaction and decreased autophagy. These phenotypes, along with premature lethality and infertility, are rescued by the beclin 1(F121A) mutation. Together, our data demonstrate that disruption of the beclin 1-BCL2 complex is an effective mechanism to increase autophagy, prevent premature ageing, improve healthspan and promote longevity in mammals.


Asunto(s)
Envejecimiento/fisiología , Autofagia/fisiología , Beclina-1/metabolismo , Longevidad/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Envejecimiento/genética , Animales , Autofagosomas/metabolismo , Beclina-1/genética , Células Cultivadas , Femenino , Fibroblastos/citología , Técnicas de Sustitución del Gen , Glucuronidasa/deficiencia , Glucuronidasa/genética , Células HeLa , Salud , Humanos , Proteínas Klotho , Longevidad/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación
2.
Nature ; 561(7723): E30, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-29921925

RESUMEN

In this Letter, the graphs in Fig. 2a and c were inadvertently the same owing to a copy and paste error from the original graphs in Prism. The Source Data files containing the raw data were correct. Fig. 2c has been corrected online.

3.
FASEB J ; 35(2): e21361, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33522017

RESUMEN

Bcl-2-associated athanogen-6 (BAG6) is a nucleocytoplasmic shuttling protein involved in protein quality control. We previously demonstrated that BAG6 is essential for autophagy by regulating the intracellular localization of the acetyltransferase EP300, and thus, modifying accessibility to its substrates (TP53 in the nucleus and autophagy-related proteins in the cytoplasm). Here, we investigated BAG6 localization and function in the cytoplasm. First, we demonstrated that BAG6 is localized in the mitochondria. Specifically, BAG6 is expressed in the mitochondrial matrix under basal conditions, and translocates to the outer mitochondrial membrane after mitochondrial depolarization with carbonyl cyanide m-chlorophenyl hydrazine, a mitochondrial uncoupler that induces mitophagy. In SW480 cells, the deletion of BAG6 expression abrogates its ability to induce mitophagy and PINK1 accumulation. On the reverse, its ectopic expression in LoVo colon cancer cells, which do not express endogenous BAG6, reduces the size of the mitochondria, induces mitophagy, leads to the activation of the PINK1/PARKIN pathway and to the phospho-ubiquitination of mitochondrial proteins. Finally, BAG6 contains two LIR (LC3-interacting Region) domains specifically found in receptors for selective autophagy and responsible for the interaction with LC3 and for autophagosome selectivity. Site-directed mutagenesis showed that BAG6 requires wild-type LIRs domains for its ability to stimulate mitophagy. In conclusion, we propose that BAG6 is a novel mitophagy receptor or adaptor that induces PINK1/PARKIN signaling and mitophagy in a LIR-dependent manner.


Asunto(s)
Mitofagia , Chaperonas Moleculares/metabolismo , Proteínas Quinasas/metabolismo , Transducción de Señal , Sitios de Unión , Línea Celular Tumoral , Humanos , Mitocondrias/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Unión Proteica , Ubiquitina-Proteína Ligasas/metabolismo
4.
FASEB J ; 34(2): 3129-3150, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31908069

RESUMEN

Aging-related organ degeneration is driven by multiple factors including the cell maintenance mechanisms of autophagy, the cytoprotective protein αKlotho, and the lesser known effects of excess phosphate (Pi), or phosphotoxicity. To examine the interplay between Pi, autophagy, and αKlotho, we used the BK/BK mouse (homozygous for mutant Becn1F121A ) with increased autophagic flux, and αKlotho-hypomorphic mouse (kl/kl) with impaired urinary Pi excretion, low autophagy, and premature organ dysfunction. BK/BK mice live longer than WT littermates, and have heightened phosphaturia from downregulation of two key NaPi cotransporters in the kidney. The multi-organ failure in kl/kl mice was rescued in the double-mutant BK/BK;kl/kl mice exhibiting lower plasma Pi, improved weight gain, restored plasma and renal αKlotho levels, decreased pathology of multiple organs, and improved fertility compared to kl/kl mice. The beneficial effects of heightened autophagy from Becn1F121A was abolished by chronic high-Pi diet which also shortened life span in the BK/BK;kl/kl mice. Pi promoted beclin 1 binding to its negative regulator BCL2, which impairs autophagy flux. Pi downregulated αKlotho, which also independently impaired autophagy. In conclusion, Pi, αKlotho, and autophagy interact intricately to affect each other. Both autophagy and αKlotho antagonizes phosphotoxicity. In concert, this tripartite system jointly determines longevity and life span.


Asunto(s)
Envejecimiento/metabolismo , Autofagia , Glucuronidasa/metabolismo , Fosfatos/metabolismo , Animales , Beclina-1/deficiencia , Beclina-1/genética , Femenino , Glucuronidasa/genética , Células HEK293 , Humanos , Riñón/metabolismo , Proteínas Klotho , Masculino , Ratones , Unión Proteica , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo
5.
Proc Natl Acad Sci U S A ; 111(11): 4115-20, 2014 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-24591579

RESUMEN

Autophagy is regulated by posttranslational modifications, including acetylation. Here we show that HLA-B-associated transcript 3 (BAT3) is essential for basal and starvation-induced autophagy in embryonic day 18.5 BAT3(-/-) mouse embryos and in mouse embryonic fibroblasts (MEFs) through the modulation of p300-dependent acetylation of p53 and ATG7. Specifically, BAT3 increases p53 acetylation and proautophagic p53 target gene expression, while limiting p300-dependent acetylation of ATG7, a mechanism known to inhibit autophagy. In the absence of BAT3 or when BAT3 is located exclusively in the cytosol, autophagy is abrogated, ATG7 is hyperacetylated, p53 acetylation is abolished, and p300 accumulates in the cytosol, indicating that BAT3 regulates the nuclear localization of p300. In addition, the interaction between BAT3 and p300 is stronger in the cytosol than in the nucleus and, during starvation, the level of p300 decreases in the cytosol but increases in the nucleus only in the presence of BAT3. We conclude that BAT3 tightly controls autophagy by modulating p300 intracellular localization, affecting the accessibility of p300 to its substrates, p53 and ATG7.


Asunto(s)
Autofagia/fisiología , Proteína p300 Asociada a E1A/metabolismo , Embrión de Mamíferos/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Acetilación , Animales , Autofagia/genética , Proteína 7 Relacionada con la Autofagia , Fraccionamiento Celular , Núcleo Celular/metabolismo , Citosol/metabolismo , Cartilla de ADN/genética , Embrión de Mamíferos/metabolismo , Inmunoprecipitación , Ratones , Ratones Noqueados , Chaperonas Moleculares/genética , Proteínas Nucleares/genética , Reacción en Cadena en Tiempo Real de la Polimerasa
6.
J Clin Invest ; 134(1)2024 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-37917202

RESUMEN

Virophagy, the selective autophagosomal engulfment and lysosomal degradation of viral components, is crucial for neuronal cell survival and antiviral immunity. However, the mechanisms leading to viral antigen recognition and capture by autophagic machinery remain poorly understood. Here, we identified cyclin-dependent kinase-like 5 (CDKL5), known to function in neurodevelopment, as an essential regulator of virophagy. Loss-of-function mutations in CDKL5 are associated with a severe neurodevelopmental encephalopathy. We found that deletion of CDKL5 or expression of a clinically relevant pathogenic mutant of CDKL5 reduced virophagy of Sindbis virus (SINV), a neurotropic RNA virus, and increased intracellular accumulation of SINV capsid protein aggregates and cellular cytotoxicity. Cdkl5-knockout mice displayed increased viral antigen accumulation and neuronal cell death after SINV infection and enhanced lethality after infection with several neurotropic viruses. Mechanistic studies demonstrated that CDKL5 directly binds the canonical selective autophagy receptor p62 and phosphorylates p62 at T269/S272 to promote its interaction with viral capsid aggregates. We found that CDKL5-mediated phosphorylation of p62 facilitated the formation of large p62 inclusion bodies that captured viral capsids to initiate capsid targeting to autophagic machinery. Overall, these findings identify a cell-autonomous innate immune mechanism for autophagy activation to clear intracellular toxic viral protein aggregates during infection.


Asunto(s)
Agregado de Proteínas , Virus , Ratones , Animales , Autofagia/genética , Fosforilación , Ratones Noqueados , Proteínas de la Cápside , Antígenos Virales , Proteínas Serina-Treonina Quinasas/genética
7.
Autophagy ; 19(3): 957-965, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-35993269

RESUMEN

Macroautophagy/autophagy is necessary for lifespan extension in multiple model organisms and autophagy dysfunction impacts age-related phenotypes and diseases. Introduction of an F121A mutation into the essential autophagy protein BECN1 constitutively increases basal autophagy in young mice and reduces cardiac and renal age-related changes in longer lived Becn1F121A mutant mice. However, both autophagic and lysosomal activities decline with age. Thus, whether autophagic flux is maintained during aging and whether it is enhanced in Becn1F121A mice is unknown. Here, we demonstrate that old wild-type mice maintained functional autophagic flux in heart, kidney and skeletal muscle but not liver, and old Becn1F121A mice had increased autophagic flux in those same organs compared to wild type. In parallel, Becn1F121A mice were not protected against age-associated hepatic phenotypes but demonstrated reduced skeletal muscle fiber atrophy. These findings identify an organ-specific role for the ability of autophagy to impact organ aging phenotypes.


Asunto(s)
Envejecimiento , Autofagia , Ratones , Animales , Autofagia/fisiología , Envejecimiento/metabolismo , Mutación , Fenotipo , Atrofia Muscular , Beclina-1/metabolismo
8.
Autophagy ; 18(2): 409-422, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-34101533

RESUMEN

Macroautophagy/autophagy is emerging as a major pathway that regulates both aging and stem cell function. Previous studies have demonstrated a positive correlation of autophagy with longevity; however, these studies did not directly address the consequence of altered autophagy in stem cells during aging. In this study, we used Becn1F121A/F121A knockin mice (designated as Becn1 KI mice) with the F121A allele in the autophagy gene Becn1 to investigate the consequences of enhanced autophagy in postnatal neural stem cells (NSCs) during aging. We found that increased autophagy protected NSCs from exhaustion and promoted neurogenesis in old (≥18-months-old) mice compared with age-matched wild-type (WT) mice, although it did not affect NSCs in young (3-months-old) mice. After pharmacologically-induced elimination of proliferative cells in the subventricular zone (SVZ), there was enhanced re-activation of quiescent NSCs in old Becn1 KI mice as compared to those in WT mice, with more efficient exit from quiescent status to generate proliferative cells and neuroblasts. Moreover, there was also improved maintenance and increased neuronal differentiation of NSCs isolated from the SVZ of old Becn1 KI mice in in vitro assays. Lastly, the increased neurogenesis in Becn1 KI mice was associated with better olfactory function in aged animals. Together, our results suggest a protective role of increased autophagy in aging NSCs, which may help the development of novel strategies to treat age-related neurodegeneration.Abbreviations: ATG: autophagy related; Baf A1: bafilomycin A1; Becn1: beclin 1; BrdU: bromodeoxyuridine/5-bromo-2'-deoxyuridine; DCX: doublecortin; GFAP: glial fibrillary acidic protein; GFP: green fluorescent protein; H&E: hematoxylin and eosin; HSCs: hematopoietic stem cells; KI: knockin; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; mo: month; NSCs: neural stem cells; OB: olfactory bulb; RB1CC1: RB1-inducible coiled-coil 1; ROS: reactive oxygen species; SOX2: SRY (sex determining region Y)-box 2; SGZ: subgranular zone; SVZ: subventricular zone; TMZ: temozolomide; WT: wild type.


Asunto(s)
Autofagia , Células-Madre Neurales , Envejecimiento , Animales , Autofagia/genética , Beclina-1/genética , Beclina-1/metabolismo , Ratones , Células-Madre Neurales/metabolismo , Neurogénesis
9.
Front Cell Dev Biol ; 10: 891332, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35832792

RESUMEN

With great sadness, the scientific community received the news of the loss of Beth Levine on 15 June 2020. Dr. Levine was a pioneer in the autophagy field and work in her lab led not only to a better understanding of the molecular mechanisms regulating the pathway, but also its implications in multiple physiological and pathological conditions, including its role in development, host defense, tumorigenesis, aging or metabolism. This review does not aim to provide a comprehensive view of autophagy, but rather an outline of some of the discoveries made by the group of Beth Levine, from the perspective of some of her own mentees, hoping to honor her legacy in science.

10.
Cardiovasc Res ; 116(3): 483-504, 2020 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-31504266

RESUMEN

Autophagy is a highly conserved recycling mechanism essential for maintaining cellular homeostasis. The pathophysiological role of autophagy has been explored since its discovery 50 years ago, but interest in autophagy has grown exponentially over the last years. Many researchers around the globe have found that autophagy is a critical pathway involved in the pathogenesis of cardiac diseases. Several groups have created novel and powerful tools for gaining deeper insights into the role of autophagy in the aetiology and development of pathologies affecting the heart. Here, we discuss how established and emerging methods to study autophagy can be used to unravel the precise function of this central recycling mechanism in the cardiac system.


Asunto(s)
Autofagia , Cardiopatías/patología , Mitocondrias Cardíacas/ultraestructura , Miocardio/ultraestructura , Animales , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Autofagia Mediada por Chaperones , Modelos Animales de Enfermedad , Cardiopatías/genética , Cardiopatías/metabolismo , Humanos , Mitocondrias Cardíacas/metabolismo , Mitofagia , Miocardio/metabolismo , Transducción de Señal
11.
EBioMedicine ; 2(3): 255-263, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25825707

RESUMEN

Both BRCA1 and Beclin 1 (BECN1) are tumor suppressor genes, which are in close proximity on the human chromosome 17q21 breast cancer tumor susceptibility locus and are often concurrently deleted. However, their importance in sporadic human breast cancer is not known. To interrogate the effects of BECN1 and BRCA1 in breast cancer, we studied their mRNA expression patterns in breast cancer patients from two large datasets: The Cancer Genome Atlas (TCGA) (n=1067) and the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) (n=1992). In both datasets, low expression of BECN1 was more common in HER2-enriched and basal-like (mostly triple-negative) breast cancers compared to luminal A/B intrinsic tumor subtypes, and was also strongly associated with TP53 mutations and advanced tumor grade. In contrast, there was no significant association between low BRCA1 expression and HER2-enriched or basal-like subtypes, TP53 mutations or tumor grade. In addition, low expression of BECN1 (but not low BRCA1) was associated with poor prognosis, and BECN1 (but not BRCA1) expression was an independent predictor of survival. These findings suggest that decreased mRNA expression of the autophagy gene BECN1 may contribute to the pathogenesis and progression of HER2-enriched, basal-like, and TP53 mutant breast cancers.

12.
Oncotarget ; 6(29): 28084-103, 2015 Sep 29.
Artículo en Inglés | MEDLINE | ID: mdl-26183398

RESUMEN

The lysosomal protease cathepsin D (Cath-D) is overproduced in breast cancer cells (BCC) and supports tumor growth and metastasis formation. Here, we describe the mechanism whereby Cath-D is accumulated in the nucleus of ERα-positive (ER+) BCC. We identified TRPS1 (tricho-rhino-phalangeal-syndrome 1), a repressor of GATA-mediated transcription, and BAT3 (Scythe/BAG6), a nucleo-cytoplasmic shuttling chaperone protein, as new Cath-D-interacting nuclear proteins. Cath-D binds to BAT3 in ER+ BCC and they partially co-localize at the surface of lysosomes and in the nucleus. BAT3 silencing inhibits Cath-D accumulation in the nucleus, indicating that Cath-D nuclear targeting is controlled by BAT3. Fully mature Cath-D also binds to full-length TRPS1 and they co-localize in the nucleus of ER+ BCC where they are associated with chromatin. Using the LexA-VP16 fusion co-activator reporter assay, we then show that Cath-D acts as a transcriptional repressor, independently of its catalytic activity. Moreover, microarray analysis of BCC in which Cath-D and/or TRPS1 expression were silenced indicated that Cath-D enhances TRPS1-mediated repression of several TRPS1-regulated genes implicated in carcinogenesis, including PTHrP, a canonical TRPS1 gene target. In addition, co-silencing of TRPS1 and Cath-D in BCC affects the transcription of cell cycle, proliferation and transformation genes, and impairs cell cycle progression and soft agar colony formation. These findings indicate that Cath-D acts as a nuclear transcriptional cofactor of TRPS1 to regulate ER+ BCC proliferation and transformation in a non-proteolytic manner.


Asunto(s)
Neoplasias de la Mama/genética , Catepsina D/genética , Ciclo Celular/genética , Proteínas de Unión al ADN/genética , Proteínas Represoras/genética , Factores de Transcripción/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Catepsina D/metabolismo , Núcleo Celular/metabolismo , Proliferación Celular/genética , Proteínas de Unión al ADN/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Immunoblotting , Células MCF-7 , Microscopía Fluorescente , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteína Relacionada con la Hormona Paratiroidea , Unión Proteica , Interferencia de ARN , Receptores de Estrógenos/metabolismo , Proteínas Represoras/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/metabolismo , Transcripción Genética , Técnicas del Sistema de Dos Híbridos
13.
Autophagy ; 10(7): 1341-2, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24852146

RESUMEN

We recently reported that BAG6/BAT3 (BCL2-associated athanogene 6) is essential for basal and starvation-induced autophagy in E18.5 bag6(-/-) mouse embryos and in mouse embryonic fibroblasts (MEFs) through the modulation of the EP300/p300-dependent acetylation of TRP53 and autophagy-related (ATG) proteins. We observed that BAG6 increases TRP53 acetylation during starvation and pro-autophagic TRP53-target gene expression. BAG6 also decreases the EP300 dependent-acetylation of ATG5, ATG7, and LC3-I, posttranslational modifications that inhibit autophagy. In addition, in the absence of BAG6 or when using a mutant of BAG6 exclusively located in the cytoplasm, autophagy is inhibited, ATG7 is hyperacetylated, TRP53 acetylation is abrogated, and EP300 accumulates in the cytoplasm indicating that BAG6 is involved in the regulation of the nuclear localization of EP300. We also reported that the interaction between BAG6 and EP300 occurs in the cytoplasm rather than the nucleus. Moreover, during starvation, EP300 is transported to the nucleus in a BAG6-dependent manner. We concluded that BAG6 regulates autophagy by controlling the localization of EP300 and its accessibility to nuclear (TRP53) and cytoplasmic (ATGs) substrates.


Asunto(s)
Autofagia , Proteína p300 Asociada a E1A/metabolismo , Espacio Intracelular/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/metabolismo , Acetilación , Animales , Ratones , Modelos Biológicos , Transporte de Proteínas , Proteína p53 Supresora de Tumor/metabolismo
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