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1.
Chembiochem ; 25(2): e202300136, 2024 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-37815526

RESUMEN

We developed a high-content image-based screen that utilizes the pro-inflammatory stimulus lipopolysaccharide (LPS) and murine macrophages (RAW264.7) with the goal of enabling the identification of novel anti-inflammatory lead compounds. We screened 2,259 bioactive compounds with annotated mechanisms of action (MOA) to identify compounds that block the LPS-induced phenotype in macrophages. We utilized a set of seven fluorescence microscopy probes to generate images that were used to train and optimize a deep neural network classifier to distinguish between unstimulated and LPS-stimulated macrophages. The top hits from the deep learning classifier were validated using a linear classifier trained on individual cells and subsequently investigated in a multiplexed cytokine secretion assay. All 12 hits significantly modulated the expression of at least one cytokine upon LPS stimulation. Seven of these were allosteric inhibitors of the mitogen-activated protein kinase kinase (MEK1/2) and showed similar effects on cytokine expression. This deep learning morphological assay identified compounds that modulate the innate immune response to LPS and may aid in identifying new anti-inflammatory drug leads.


Asunto(s)
Aprendizaje Profundo , FN-kappa B , Ratones , Animales , Lipopolisacáridos/farmacología , Antiinflamatorios/farmacología , Citocinas , Óxido Nítrico/metabolismo
2.
Mol Cell Proteomics ; 17(2): 216-232, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-29222160

RESUMEN

Mitochondria exert an immense amount of cytophysiological functions, but the structural basis of most of these processes is still poorly understood. Here we use cross-linking mass spectrometry to probe the organization of proteins in native mouse heart mitochondria. Our approach provides the largest survey of mitochondrial protein interactions reported so far. In total, we identify 3,322 unique residue-to-residue contacts involving half of the mitochondrial proteome detected by bottom-up proteomics. The obtained mitochondrial protein interactome gives insights in the architecture and submitochondrial localization of defined protein assemblies, and reveals the mitochondrial localization of four proteins not yet included in the MitoCarta database. As one of the highlights, we show that the oxidative phosphorylation complexes I-V exist in close spatial proximity, providing direct evidence for supercomplex assembly in intact mitochondria. The specificity of these contacts is demonstrated by comparative analysis of mitochondria after high salt treatment, which disrupts the native supercomplexes and substantially changes the mitochondrial interactome.


Asunto(s)
Mitocondrias Cardíacas/metabolismo , Proteínas Mitocondriales/metabolismo , Complejos Multiproteicos/metabolismo , Animales , Masculino , Espectrometría de Masas , Ratones Endogámicos C57BL , Mitocondrias Cardíacas/efectos de los fármacos , Mapas de Interacción de Proteínas , Proteómica , Cloruro de Sodio/farmacología
3.
bioRxiv ; 2024 Apr 03.
Artículo en Inglés | MEDLINE | ID: mdl-38617352

RESUMEN

Circadian (~24 h) rhythms are a fundamental feature of life, and their disruption increases the risk of infectious diseases, metabolic disorders, and cancer1-6. Circadian rhythms couple to the cell cycle across eukaryotes7,8 but the underlying mechanism is unknown. We previously identified an evolutionarily conserved circadian oscillation in intracellular potassium concentration, [K+]i9,10. As critical events in the cell cycle are regulated by intracellular potassium11,12, an enticing hypothesis is that circadian rhythms in [K+]i form the basis of this coupling. We used a minimal model cell, the alga Ostreococcus tauri, to uncover the role of potassium in linking these two cycles. We found direct reciprocal feedback between [K+]i and circadian gene expression. Inhibition of proliferation by manipulating potassium rhythms was dependent on the phase of the circadian cycle. Furthermore, we observed a total inhibition of cell proliferation when circadian gene expression is inhibited. Strikingly, under these conditions a sudden enforced gradient of extracellular potassium was sufficient to induce a round of cell division. Finally, we provide evidence that interactions between potassium and circadian rhythms also influence proliferation in mammalian cells. These results establish circadian regulation of intracellular potassium levels as a primary factor coupling the cell- and circadian cycles across diverse organisms.

4.
Genetics ; 180(2): 857-71, 2008 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-18780725

RESUMEN

Gut granules are specialized lysosome-related organelles that act as sites of fat storage in Caenorhabditis elegans intestinal cells. We identified mutations in a gene, glo-3, that functions in the formation of embryonic gut granules. Some glo-3(-) alleles displayed a complete loss of embryonic gut granules, while other glo-3(-) alleles had reduced numbers of gut granules. A subset of glo-3 alleles led to mislocalization of gut granule contents into the intestinal lumen, consistent with a defect in intracellular trafficking. glo-3(-) embryos lacking gut granules developed into adults containing gut granules, indicating that glo-3(+) function may be differentially required during development. We find that glo-3(+) acts in parallel with or downstream of the AP-3 complex and the PGP-2 ABC transporter in gut granule biogenesis. glo-3 encodes a predicted membrane-associated protein that lacks obvious sequence homologs outside of nematodes. glo-3 expression initiates in embryonic intestinal precursors and persists almost exclusively in intestinal cells through adulthood. GLO-3GFP localizes to the gut granule membrane, suggesting it could play a direct role in the trafficking events at the gut granule. smg-1(-) suppression of glo-3(-) nonsense alleles indicates that the C-terminal half of GLO-3, predicted to be present in the cytoplasm, is not necessary for gut granule formation. Our studies identify GLO-3 as a novel player in the formation of lysosome-related organelles.


Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Genes de Helminto , Lisosomas/metabolismo , Alelos , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/embriología , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Gránulos Citoplasmáticos/metabolismo , Tracto Gastrointestinal/citología , Tracto Gastrointestinal/metabolismo , Datos de Secuencia Molecular , Fenotipo
5.
Mol Biol Cell ; 16(7): 3273-88, 2005 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-15843430

RESUMEN

The intestinal cells of Caenorhabditis elegans embryos contain prominent, birefringent gut granules that we show are lysosome-related organelles. Gut granules are labeled by lysosomal markers, and their formation is disrupted in embryos depleted of AP-3 subunits, VPS-16, and VPS-41. We define a class of gut granule loss (glo) mutants that are defective in gut granule biogenesis. We show that the glo-1 gene encodes a predicted Rab GTPase that localizes to lysosome-related gut granules in the intestine and that glo-4 encodes a possible GLO-1 guanine nucleotide exchange factor. These and other glo genes are homologous to genes implicated in the biogenesis of specialized, lysosome-related organelles such as melanosomes in mammals and pigment granules in Drosophila. The glo mutants thus provide a simple model system for the analysis of lysosome-related organelle biogenesis in animal cells.


Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Factores de Intercambio de Guanina Nucleótido/genética , Lisosomas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Naranja de Acridina/farmacología , Complejo 3 de Proteína Adaptadora , Alelos , Secuencia de Aminoácidos , Animales , Transporte Biológico , Tamaño Corporal , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/fisiología , Proteínas de Unión al ADN/genética , Drosophila , Epistasis Genética , GTP Fosfohidrolasas/metabolismo , Genes de Helminto , Genes Reporteros , Proteínas Fluorescentes Verdes/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/fisiología , Mucosa Intestinal/metabolismo , Melanosomas/metabolismo , Microscopía Fluorescente , Modelos Genéticos , Datos de Secuencia Molecular , Mutación , Fenotipo , Transporte de Proteínas , Homología de Secuencia de Aminoácido , Temperatura , Factores de Transcripción/genética , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/fisiología
6.
PLoS One ; 7(8): e43043, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22916203

RESUMEN

The human disease Hermansky-Pudlak syndrome results from defective biogenesis of lysosome-related organelles (LROs) and can be caused by mutations in subunits of the BLOC-1 complex. Here we show that C. elegans glo-2 and snpn-1, despite relatively low levels of amino acid identity, encode Pallidin and Snapin BLOC-1 subunit homologues, respectively. BLOC-1 subunit interactions involving Pallidin and Snapin were conserved for GLO-2 and SNPN-1. Mutations in glo-2 and snpn-1,or RNAi targeting 5 other BLOC-1 subunit homologues in a genetic background sensitized for glo-2 function, led to defects in the biogenesis of lysosome-related gut granules. These results indicate that the BLOC-1 complex is conserved in C. elegans. To address the function of C. elegans BLOC-1, we assessed the intracellular sorting of CDF-2::GFP, LMP-1, and PGP-2 to gut granules. We validated their utility by analyzing their mislocalization in intestinal cells lacking the function of AP-3, which participates in an evolutionarily conserved sorting pathway to LROs. BLOC-1(-) intestinal cells missorted gut granule cargo to the plasma membrane and conventional lysosomes and did not have obviously altered function or morphology of organelles composing the conventional lysosome protein sorting pathway. Double mutant analysis and comparison of AP-3(-) and BLOC-1(-) phenotypes revealed that BLOC-1 has some functions independent of the AP-3 adaptor complex in trafficking to gut granules. We discuss similarities and differences of BLOC-1 activity in the biogenesis of gut granules as compared to mammalian melanosomes, where BLOC-1 has been most extensively studied for its role in sorting to LROs. Our work opens up the opportunity to address the function of this poorly understood complex in cell and organismal physiology using the genetic approaches available in C. elegans.


Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas Portadoras/metabolismo , Gránulos Citoplasmáticos/metabolismo , Lisosomas/metabolismo , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas Portadoras/genética , Síndrome de Hermanski-Pudlak/genética , Síndrome de Hermanski-Pudlak/metabolismo , Humanos , Unión Proteica , Técnicas del Sistema de Dos Híbridos
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