RESUMEN
BACKGROUND & AIMS: In the classic form of α1-antitrypsin deficiency (ATD), the misfolded α1-antitrypsin Z (ATZ) variant accumulates in the endoplasmic reticulum (ER) of liver cells. A gain-of-function proteotoxic mechanism is responsible for chronic liver disease in a subgroup of homozygotes. Proteostatic response pathways, including conventional endoplasmic reticulum-associated degradation and autophagy, have been proposed as the mechanisms that allow cellular adaptation and presumably protection from the liver disease phenotype. Recent studies have concluded that a distinct lysosomal pathway called endoplasmic reticulum-to-lysosome completely supplants the role of the conventional macroautophagy pathway in degradation of ATZ. Here, we used several state-of-the-art approaches to characterize the proteostatic responses more fully in cellular systems that model ATD. METHODS: We used clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing coupled to a cell selection step by fluorescence-activated cell sorter to perform screening for proteostasis genes that regulate ATZ accumulation and combined that with selective genome editing in 2 other model systems. RESULTS: Endoplasmic reticulum-associated degradation genes are key early regulators and multiple autophagy genes, from classic as well as from ER-to-lysosome and other newly described ER-phagy pathways, participate in degradation of ATZ in a manner that is temporally regulated and evolves as ATZ accumulation persists. Time-dependent changes in gene expression are accompanied by specific ultrastructural changes including dilation of the ER, formation of globular inclusions, budding of autophagic vesicles, and alterations in the overall shape and component parts of mitochondria. CONCLUSIONS: Macroautophagy is a critical component of the proteostasis response to cellular ATZ accumulation and it becomes more important over time as ATZ synthesis continues unabated. Multiple subtypes of macroautophagy and nonautophagic lysosomal degradative pathways are needed to respond to the high concentrations of misfolded protein that characterizes ATD and these pathways are attractive candidates for genetic variants that predispose to the hepatic phenotype.
Asunto(s)
Degradación Asociada con el Retículo Endoplásmico , Retículo Endoplásmico , Lisosomas , Macroautofagia , Proteostasis , Deficiencia de alfa 1-Antitripsina , alfa 1-Antitripsina , Deficiencia de alfa 1-Antitripsina/patología , Deficiencia de alfa 1-Antitripsina/genética , Deficiencia de alfa 1-Antitripsina/metabolismo , Humanos , Lisosomas/metabolismo , alfa 1-Antitripsina/metabolismo , alfa 1-Antitripsina/genética , Retículo Endoplásmico/metabolismo , Sistemas CRISPR-Cas , Autofagia/genética , Edición GénicaRESUMEN
MYC oncoprotein is a multifunctional transcription factor that regulates the expression of a large number of genes involved in cellular growth, proliferation and metabolism. Altered MYC protein level lead to cellular transformation and tumorigenesis. MYC is deregulated in > 50% of human cancers, rendering it an attractive drug target. However, direct inhibition of this class of proteins using conventional small molecules is challenging due to their intrinsically disordered state. To discover novel posttranslational regulators of MYC protein stability and turnover, we established a genetic screen in mammalian cells by combining a fluorescent protein-based MYC abundance sensor, CRISPR/Cas9-based gene knockouts and next-generation sequencing. Our screen identifies UBR5, an E3 ligase of the HECT-type family, as a novel regulator of MYC degradation. Even in the presence of the well-described and functional MYC ligase, FBXW7, UBR5 depletion leads to accumulation of MYC in cells. We demonstrate interaction of UBR5 with MYC and reduced K48-linked ubiquitination of MYC upon loss of UBR5 in cells. Interestingly, in cancer cell lines with amplified MYC expression, depletion of UBR5 resulted in reduced cell survival, as a consequence of MYC stabilization. Finally, we show that MYC and UBR5 are co-amplified in more than 40% of cancer cells and that MYC copy number amplification correlates with enhanced transcriptional output of UBR5. This suggests that UBR5 acts as a buffer in MYC amplified settings and protects these cells from apoptosis.
Asunto(s)
Sistemas CRISPR-Cas , Neoplasias/patología , Proteolisis , Proteínas Proto-Oncogénicas c-myc/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Apoptosis , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/genética , Células Tumorales Cultivadas , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Kendomycin is a small-molecule natural product that has gained significant attention due to reported cytotoxicity against pathogenic bacteria and fungi as well as a number of cancer cell lines. Despite significant biomedical interest and attempts to reveal its mechanism of action, the cellular target of kendomycin remains disputed. Herein it is shown that kendomycin induces cellular responses indicative of cation stress comparable to the effects of established iron chelators. Furthermore, addition of excess iron and copper attenuated kendomycin cytotoxicity in bacteria, yeast, and mammalian cells. Finally, NMR analysis demonstrated a direct interaction with cations, corroborating a close link between the observed kendomycin polypharmacology across different species and modulation of iron and/or copper levels.
Asunto(s)
Antibacterianos/farmacología , Antibióticos Antineoplásicos/farmacología , Antifúngicos/farmacología , Bacterias/efectos de los fármacos , Quelantes/farmacología , Hongos/efectos de los fármacos , Rifabutina/análogos & derivados , Cationes , Línea Celular , Cobre/metabolismo , Hierro/metabolismo , Leupeptinas/farmacología , Pruebas de Sensibilidad Microbiana , Mutagénesis , Rifabutina/farmacología , Levaduras/efectos de los fármacosRESUMEN
Invasive infections by fungal pathogens cause more deaths than malaria worldwide. We found the ergoline compound NGx04 in an antifungal screen, with selectivity over mammalian cells. High-resolution chemogenomics identified the lipid transfer protein Sec14p as the target of NGx04 and compound-resistant mutations in Sec14p define compound-target interactions in the substrate binding pocket of the protein. Beyond its essential lipid transfer function in a variety of pathogenic fungi, Sec14p is also involved in secretion of virulence determinants essential for the pathogenicity of fungi such as Cryptococcus neoformans, making Sec14p an attractive antifungal target. Consistent with this dual function, we demonstrate that NGx04 inhibits the growth of two clinical isolates of C. neoformans and that NGx04-related compounds have equal and even higher potency against C. neoformans. Furthermore NGx04 analogues showed fungicidal activity against a fluconazole resistant C. neoformans strain. In summary, we present genetic evidence that NGx04 inhibits fungal Sec14p and initial data supporting NGx04 as a novel antifungal starting point.
Asunto(s)
Proteínas Portadoras/química , Criptococosis/tratamiento farmacológico , Cryptococcus neoformans/efectos de los fármacos , Ergolinas/farmacología , Metabolismo de los Lípidos/efectos de los fármacos , Antifúngicos/farmacología , Proteínas Portadoras/genética , Criptococosis/microbiología , Cryptococcus neoformans/genética , Cryptococcus neoformans/patogenicidad , Ergolinas/química , Humanos , Pruebas de Sensibilidad Microbiana , Conformación Proteica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genéticaRESUMEN
A new cyclic decadepsipeptide was isolated from Chaetosphaeria tulasneorum with potent bioactivity on mammalian and yeast cells. Chemogenomic profiling in S. cerevisiae indicated that the Sec61 translocon complex, the machinery for protein translocation and membrane insertion at the endoplasmic reticulum, is the target. The profiles were similar to those of cyclic heptadepsipeptides of a distinct chemotype (including HUN-7293 and cotransin) that had previously been shown to inhibit cotranslational translocation at the mammalian Sec61 translocon. Unbiased, genome-wide mutagenesis followed by full-genome sequencing in both fungal and mammalian cells identified dominant mutations in Sec61p (yeast) or Sec61α1 (mammals) that conferred resistance. Most, but not all, of these mutations affected inhibition by both chemotypes, despite an absence of structural similarity. Biochemical analysis confirmed inhibition of protein translocation into the endoplasmic reticulum of both co- and post-translationally translocated substrates by both chemotypes, demonstrating a mechanism independent of a translating ribosome. Most interestingly, both chemotypes were found to also inhibit SecYEG, the bacterial Sec61 translocon homolog. We suggest 'decatransin' as the name for this new decadepsipeptide translocation inhibitor.
Asunto(s)
Productos Biológicos/farmacología , Retículo Endoplásmico/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Transporte de Proteínas/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Animales , Ascomicetos/metabolismo , Células COS , Células Cultivadas , Chlorocebus aethiops , Células HCT116 , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Péptidos Cíclicos/farmacología , Polimorfismo de Nucleótido Simple/genética , Canales de Translocación SEC , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
Due to evolutionary conservation of biology, experimental knowledge captured from genetic studies in eukaryotic model organisms provides insight into human cellular pathways and ultimately physiology. Yeast chemogenomic profiling is a powerful approach for annotating cellular responses to small molecules. Using an optimized platform, we provide the relative sensitivities of the heterozygous and homozygous deletion collections for nearly 1800 biologically active compounds. The data quality enables unique insights into pathways that are sensitive and resistant to a given perturbation, as demonstrated with both known and novel compounds. We present examples of novel compounds that inhibit the therapeutically relevant fatty acid synthase and desaturase (Fas1p and Ole1p), and demonstrate how the individual profiles facilitate hypothesis-driven experiments to delineate compound mechanism of action. Importantly, the scale and diversity of tested compounds yields a dataset where the number of modulated pathways approaches saturation. This resource can be used to map novel biological connections, and also identify functions for unannotated genes. We validated hypotheses generated by global two-way hierarchical clustering of profiles for (i) novel compounds with a similar mechanism of action acting upon microtubules or vacuolar ATPases, and (ii) an un-annotated ORF, YIL060w, that plays a role in respiration in the mitochondria. Finally, we identify and characterize background mutations in the widely used yeast deletion collection which should improve the interpretation of past and future screens throughout the community. This comprehensive resource of cellular responses enables the expansion of our understanding of eukaryotic pathway biology.
Asunto(s)
Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Antifúngicos/farmacología , Vías Biosintéticas , Farmacorresistencia Fúngica , Regulación Fúngica de la Expresión Génica , Ensayos Analíticos de Alto Rendimiento , Datos de Secuencia Molecular , Filogenia , Saccharomyces cerevisiae/clasificación , Saccharomyces cerevisiae/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Translation initiation is an emerging target in oncology and neurobiology indications. Naturally derived and synthetic rocaglamide scaffolds have been used to interrogate this pathway; however, there is uncertainty regarding their precise mechanism(s) of action. We exploited the genetic tractability of yeast to define the primary effect of both a natural and a synthetic rocaglamide in a cellular context and characterized the molecular target using biochemical studies and in silico modeling. Chemogenomic profiling and mutagenesis in yeast identified the eIF (eukaryotic Initiation Factor) 4A helicase homologue as the primary molecular target of rocaglamides and defined a discrete set of residues near the RNA binding motif that confer resistance to both compounds. Three of the eIF4A mutations were characterized regarding their functional consequences on activity and response to rocaglamide inhibition. These data support a model whereby rocaglamides stabilize an eIF4A-RNA interaction to either alter the level and/or impair the activity of the eIF4F complex. Furthermore, in silico modeling supports the annotation of a binding pocket delineated by the RNA substrate and the residues identified from our mutagenesis screen. As expected from the high degree of conservation of the eukaryotic translation pathway, these observations are consistent with previous observations in mammalian model systems. Importantly, we demonstrate that the chemically distinct silvestrol and synthetic rocaglamides share a common mechanism of action, which will be critical for optimization of physiologically stable derivatives. Finally, these data confirm the value of the rocaglamide scaffold for exploring the impact of translational modulation on disease.