Mifepristone increases mRNA translation rate, triggers the unfolded protein response, increases autophagic flux, and kills ovarian cancer cells in combination with proteasome or lysosome inhibitors

The synthetic steroid mifepristone blocks the growth of ovarian cancer cells, yet the mechanism driving such effect is not entirely understood. Unbiased genomic and proteomic screenings using ovarian cancer cell lines of different genetic backgrounds and sensitivities to platinum led to the identification of two key genes upregulated by mifepristone and involved in the unfolded protein response (UPR): the master chaperone of the endoplasmic reticulum (ER), glucose regulated protein (GRP) of 78 kDa, and the CCAAT/enhancer binding protein homologous transcription factor (CHOP). GRP78 and CHOP were upregulated by mifepristone in ovarian cancer cells regardless of p53 status and platinum sensitivity. Further studies revealed that the three UPR-associated pathways, PERK, IRE1α, and ATF6, were activated by mifepristone. Also, the synthetic steroid acutely increased mRNA translation rate, which, if prevented, abrogated the splicing of XBP1 mRNA, a non-translatable readout of IRE1α activation. Moreover, mifepristone increased LC3-II levels due to increased autophagic flux. When the autophagic–lysosomal pathway was inhibited with chloroquine, mifepristone was lethal to the cells. Lastly, doses of proteasome inhibitors that are inadequate to block the activity of the proteasomes, caused cell death when combined with mifepristone; this phenotype was accompanied by accumulation of poly-ubiquitinated proteins denoting proteasome inhibition. The stimulation by mifepristone of ER stress and autophagic flux offers a therapeutic opportunity for utilizing this compound to sensitize ovarian cancer cells to proteasome or lysosome inhibitors.Fil: Zhang, Lei. University Of South Dakota; Estados UnidosFil: Hapon, María Belén. University Of South Dakota; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Goyeneche, Alicia A.. University Of South Dakota; Estados Unidos. McGill University; CanadáFil: Srinivasan, Rekha. University Of South Dakota; Estados UnidosFil: Gamarra Luques, Carlos Diego. University Of South Dakota; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Callegari, Eduardo A.. University Of South Dakota; Estados UnidosFil: Drappeau, Donis D.. University Of South Dakota; Estados UnidosFil: Terpstra, Erin J.. University Of South Dakota; Estados UnidosFil: Pan, Bo. University Of South Dakota; Estados UnidosFil: Knapp, Jennifer R.. University of Kansas; Estados UnidosFil: Chien, Jeremy. University of Kansas; Estados UnidosFil: Wang, Xuejun. University Of South Dakota; Estados UnidosFil: Eyster, Kathleen M.. University Of South Dakota; Estados UnidosFil: Telleria, Carlos Marcelo. University Of South Dakota; Estados Unidos. McGill University; Canadá. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

SIRT1 - a new mammalian substrate of nuclear autophagy

Macroautophagic/autophagic degradation of nuclear components (or nuclear autophagy) is a poorly understood area in autophagy research. We previously reported the nuclear lamina protein LMNB1 (lamin B1) as a nuclear autophagy substrate in primary human cells, stimulating the investigation of nuclear autophagy in the mammalian system. We recently reported the sirtuin protein SIRT1 as a new selective substrate of nuclear autophagy in senescence and aging. Upon senescence of primary human cells, SIRT1 degradation is mediated by a direct nuclear SIRT1-LC3 interaction, followed by nucleus-to-cytoplasm shuttling of SIRT1 and autophagosome-lysosome degradation. In vivo, SIRT1 is downregulated by lysosomes in hematopoietic and immune organs upon natural aging in mice and in aged human T cells. Our study identified another substrate of nuclear autophagy and suggests a new strategy to promote SIRT1-mediated health benefits by suppressing its autophagic degradation

Mouse skeletal muscle fiber-type-specific macroautophagy and muscle wasting are regulated by a Fyn/STAT3/Vps34 signaling pathway

Skeletal muscle atrophy induced by aging (sarcopenia), inactivity, and prolonged fasting states (starvation) is predominantly restricted to glycolytic type II muscle fibers and typical spares oxidative type I fibers. However, the mechanisms accounting for muscle fiber-type specificity of atrophy have remained enigmatic. In the current study, although the Fyn tyrosine kinase activated the mTORC1 signaling complex, it also induced marked atrophy of glycolytic fibers with relatively less effect on oxidative muscle fibers. This was due to inhibition of macroautophagy via an mTORC1-independent but STAT3-dependent reduction in Vps34 protein levels and decreased Vps34/p150/Beclin1/Atg14 complex 1. Physiologically, in the fed state endogenous Fyn kinase activity was increased in glycolytic but not oxidative skeletal muscle. In parallel, Y705-STAT3 phosphorylation increased with decreased Vps34 protein levels. Moreover, fed/starved regulation of Y705-STAT3 phosphorylation and Vps34 protein levels was prevented in skeletal muscle of Fyn null mice. These data demonstrate a Fyn/STAT3/Vps34 pathway that is responsible for fiber-type-specific regulation of macroautophagy and skeletal muscle atrophy

Insight into Parkinson’s Disease and α-Synuclein Degradation via the Lysosome: α-Synuclein Localization Changes in Vps28Δ

The neurodegeneration pathology in Parkinson’s disease patients predominantly targets dopaminergic neurons in the midbrain. These neurons accumulate aggregated alpha-synuclein, which may be linked to cell death. The misfolding and buildup of α-synuclein is thought to trigger its accumulation and aggregation. An attractive hypothesis states that excess amounts of α-synuclein are due to dysfunctional degradation of the protein. Until recently, the proteasome was considered the major site for degrading alpha-synuclein, but recent studies suggest that the lysosome may also be involved. To test this latter hypothesis, we employed a budding yeast model for α-synuclein aggregation and toxicity to genetically evaluate the role of the multivesicular body (MVB) pathway, which is a major route used by proteins to target the yeast vacuole for degradation. ESCRT-1 is a major protein complex in the MVB pathway. We asked whether alpha-synuclein would accumulate and increase toxicity in yeast that lacked important ESRCT-1 components, in this case vps28 or mvb12. We demonstrate that the absence of vps28 altered wildtype, A53T, and E46K α-synuclein localization. Specifically,ƒn α-synuclein shifted from being localized primarily on the plasma membrane to being diffuse and aggregated within the cytoplasm. In contrast, the mvb12∆ strain retained plasma membrane α-synuclein localization. Our preliminary data indicates that the MVB pathway is involved in α-synuclein degradation, but not all proteins within ESRCT-1 participate. Complete analysis of the remaining ESCRT-I proteins and other ESCRT complexes is needed to fully understand the role of sorting proteins and MVBs in the α-synuclein lysosome degradation pathway

Proteomic Characterization of the E3 Ubiquitin-Ligase Hakai: Biological Insights and New Therapeutic Strategies

Programa Oficial de Doutoramento en Bioloxía Celular e Molecular . 5004V01[Resumen] El carcinoma es el tipo más común de cáncer y surge de las células epiteliales. La transición del adenoma al carcinoma se asocia con la pérdida de E-cadherina y, en consecuencia, de los contactos intercelulares. La E-cadherina es un supresor tumoral que está regulado negativamente durante la transición epitelio-mesénquima (EMT) y su pérdida es un marcador de mal pronóstico durante la progresión tumoral. Hakai es una E3 ubiquitina-ligasa que media en la ubiquitinación de la E-cadherina, su endocitosis y consecuente degradación. Aunque la E-cadherina es el sustrato más conocido de Hakai, otras dianas moleculares reguladas por Hakai pueden estar involucradas en la plasticidad celular durante la progresión tumoral. En este trabajo, empleamos la técnica iTRAQ para explorar nuevas rutas moleculares involucradas en la EMT inducida por Hakai. Nuestros resultados muestran que Hakai puede tener una influencia importante sobre proteínas relacionadas con el citoesqueleto, proteínas extracelulares asociadas con el exosoma, proteínas relacionadas con el ARN y proteínas involucradas en metabolismo. Entre las proteínas reguladas por Hakai, describimos la Anexina A2 como un nuevo posible sustrato de Hakai. Además, nuestros resultados revelan que la inhibición farmacológica de Hsp90 con geldanamicina resulta en la degradación de Hakai vía lisosoma. Así, proponemos a Hakai como una nueva proteína cliente de la chaperona Hsp90, destacando un mecanismo novedoso por el cual los inhibidores de Hsp90 pueden influir en el proceso EMT mediado por Hakai y el tratamiento del cáncer.[Resumo] O carcinoma é o tipo de cancro máis común e xorde das células epiteliais. A transición do adenoma ao carcinoma está asociada á perda de E-cadherina e, en consecuencia, aos contactos intercelulares. A E-cadherina é un supresor tumoral que se encontra regulado negativamente durante a transición epitelio-mesénquima (EMT), e a súa perda é un marcador de mala prognose durante a progresión do tumor. Hakai é unha E3 ubiquitina-ligasa que media a ubiquitinización da E-cadherina, a súa endocitose e a súa conseguinte degradación. Aínda que a E-cadherina é o substrato máis coñecido de Hakai, outras dianas moleculares reguladas por Hakai poden estar implicadas na plasticidade celular durante a progresión tumoral. Neste traballo empregamos a técnica iTRAQ para explorar novas vías moleculares implicadas na EMT inducida por Hakai. Os nosos resultados mostran que Hakai pode ter unha influencia importante sobre proteínas relacionadas co citoesqueleto, proteínas extracelulares asociadas co exosoma, proteínas relacionadas co ARN e proteínas implicadas no metabolismo. Entre as proteínas reguladas por Hakai, describimos a Anexina A2 coma un novo posible substrato de Hakai. Ademáis, describimos una relación entre Hakai e o complexo chaperona da proteína Heat shock protein 90 (Hsp90). Tamén, os nosos resultados revelan que a inhibición farmacolóxica de Hsp90 con geldanamicina resulta na degradación de Hakai vía lisosoma. Así, propoñemos a Hakai como unha nova proteína cliente da chaperona Hsp90, destacando un novo mecanismo polo cal os inhibidores de Hsp90 poden influir no proceso de EMT mediado por Hakai e no tratamento do cancro.[Abstract] Carcinoma is the most common type of cancer and arises from epithelial cells. Transition from adenoma to carcinoma is associated with the loss of E-cadherin and, in consequence, the disruption of cell−cell contacts. E-cadherin is a tumor suppressor which is down-regulated during epithelial-to-mesenchymal transition (EMT), and its loss is a predictor of poor prognosis during tumor progression. Hakai is an E3 ubiquitin-ligase that mediates E-cadherin ubiquitination, endocytosis and consequent degradation. Although E-cadherin is the most established substrate for Hakai activity, other regulated molecular targets for Hakai may be involved in cancer cell plasticity during tumor progression. In this work we employed an iTRAQ approach to explore novel molecular pathways involved in Hakai-driven EMT. Our results show that Hakai may have an important influence on cytoskeleton-related proteins, extracellular exosome-associated proteins, RNA-related proteins and proteins involved in metabolism. Among Hakai-down-regulated proteins, we describe Annexin A2 as a new possible susbtrate for Hakai. Moreover, we also report an interaction between Hakai and the heat shock protein 90 (Hsp90) chaperone complex. Besides, our results reveal that the pharmacological inhibition of Hsp90 with geldanamycin results in the degradation of Hakai in a lysosome-dependent manner. Based on that, we propose Hakai as a new client protein of Hsp90 chaperone highlighting a new mechanism by which Hsp90 inhibitors may influence Hakai-mediated EMT process and cancer treatment

Atg9b Deficiency Suppresses Autophagy and Potentiates Endoplasmic Reticulum Stress-Associated Hepatocyte Apoptosis in Hepatocarcinogenesis

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Annealing novel nucleobase-lipids with oligonucleotides or plasmid DNA based on H-bonding or π-π interaction:Assemblies and transfections

Lipid derivatives of nucleoside analogs have been highlighted for their potential for effective gene delivery. A novel class of nucleobase-lipids are rationally designed and readily synthesized, comprising thymine/cytosine, an ester/amide linker and an oleyl lipid. The diversity of four nucleobase-lipids termed DXBAs (DOTA, DNTA, DOCA and DNCA) is investigated. Besides, DNCA is demonstrated to be an effective neutral transfection material for nucleic acid delivery, which enbles to bind to oligonucleotides via H-bonding and π-π stacking with reduced toxicity in vitro and in vivo. Several kinds of nucleic acid drugs including aptamer, ssRNA, antisense oligonucleotide, and plasmid DNAs can be delivered by DXBAs, especially DNCA. In particular, G4-aptamer AS1411 encapsulated by DNCA exhibits cellular uptake enhancement, lysosome degradation reduction, cell apoptosis promotion, cell cycle phase alteration in vitro and duration prolongation in vivo, resulting in significant anti-proliferative activity. Our results demonstrate that DNCA is a promising transfection agent for G4-aptamers and exhibites bright application prospects in the permeation improvement of single-stranded oligonucleotides or plasmid DNAs

A murine model of variant late infantile ceroid lipofuscinosis recapitulates behavioral and pathological phenotypes of human disease.

Neuronal ceroid lipofuscinoses (NCLs; also known collectively as Batten Disease) are a family of autosomal recessive lysosomal storage disorders. Mutations in as many as 13 genes give rise to ∼10 variants of NCL, all with overlapping clinical symptomatology including visual impairment, motor and cognitive dysfunction, seizures, and premature death. Mutations in CLN6 result in both a variant late infantile onset neuronal ceroid lipofuscinosis (vLINCL) as well as an adult-onset form of the disease called Type A Kufs. CLN6 is a non-glycosylated membrane protein of unknown function localized to the endoplasmic reticulum (ER). In this study, we perform a detailed characterization of a naturally occurring Cln6 mutant (Cln6(nclf)) mouse line to validate its utility for translational research. We demonstrate that this Cln6(nclf) mutation leads to deficits in motor coordination, vision, memory, and learning. Pathologically, we demonstrate loss of neurons within specific subregions and lamina of the cortex that correlate to behavioral phenotypes. As in other NCL models, this model displays selective loss of GABAergic interneuron sub-populations in the cortex and the hippocampus with profound, early-onset glial activation. Finally, we demonstrate a novel deficit in memory and learning, including a dramatic reduction in dendritic spine density in the cerebral cortex, which suggests a reduction in synaptic strength following disruption in CLN6. Together, these findings highlight the behavioral and pathological similarities between the Cln6(nclf) mouse model and human NCL patients, validating this model as a reliable format for screening potential therapeutics