RESUMEN
Intracellular accumulation of misfolded proteins causes toxic proteinopathies, diseases without targeted therapies. Mucin 1 kidney disease (MKD) results from a frameshift mutation in the MUC1 gene (MUC1-fs). Here, we show that MKD is a toxic proteinopathy. Intracellular MUC1-fs accumulation activated the ATF6 unfolded protein response (UPR) branch. We identified BRD4780, a small molecule that clears MUC1-fs from patient cells, from kidneys of knockin mice and from patient kidney organoids. MUC1-fs is trapped in TMED9 cargo receptor-containing vesicles of the early secretory pathway. BRD4780 binds TMED9, releases MUC1-fs, and re-routes it for lysosomal degradation, an effect phenocopied by TMED9 deletion. Our findings reveal BRD4780 as a promising lead for the treatment of MKD and other toxic proteinopathies. Generally, we elucidate a novel mechanism for the entrapment of misfolded proteins by cargo receptors and a strategy for their release and anterograde trafficking to the lysosome.
Asunto(s)
Benzamidas/metabolismo , Compuestos Bicíclicos con Puentes/farmacología , Heptanos/farmacología , Lisosomas/efectos de los fármacos , Proteínas de Transporte Vesicular/metabolismo , Factor de Transcripción Activador 6/metabolismo , Animales , Benzamidas/química , Benzamidas/farmacología , Compuestos Bicíclicos con Puentes/uso terapéutico , Células Epiteliales/citología , Células Epiteliales/metabolismo , Femenino , Mutación del Sistema de Lectura , Heptanos/uso terapéutico , Humanos , Receptores de Imidazolina/antagonistas & inhibidores , Receptores de Imidazolina/genética , Receptores de Imidazolina/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Riñón/citología , Riñón/metabolismo , Riñón/patología , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Lisosomas/metabolismo , Masculino , Ratones , Ratones Transgénicos , Mucina-1/química , Mucina-1/genética , Mucina-1/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Respuesta de Proteína Desplegada/efectos de los fármacos , Proteínas de Transporte Vesicular/químicaRESUMEN
Cellular exposure to free fatty acids (FFAs) is implicated in the pathogenesis of obesity-associated diseases. However, there are no scalable approaches to comprehensively assess the diverse FFAs circulating in human plasma. Furthermore, assessing how FFA-mediated processes interact with genetic risk for disease remains elusive. Here, we report the design and implementation of fatty acid library for comprehensive ontologies (FALCON), an unbiased, scalable, and multimodal interrogation of 61 structurally diverse FFAs. We identified a subset of lipotoxic monounsaturated fatty acids associated with decreased membrane fluidity. Furthermore, we prioritized genes that reflect the combined effects of harmful FFA exposure and genetic risk for type 2 diabetes (T2D). We found that c-MAF-inducing protein (CMIP) protects cells from FFA exposure by modulating Akt signaling. In sum, FALCON empowers the study of fundamental FFA biology and offers an integrative approach to identify much needed targets for diverse diseases associated with disordered FFA metabolism.
Asunto(s)
Diabetes Mellitus Tipo 2 , Ácidos Grasos no Esterificados , Humanos , Ácidos Grasos no Esterificados/metabolismo , Ácidos Grasos , Transducción de Señal , BiologíaRESUMEN
Cellular exposure to free fatty acids (FFA) is implicated in the pathogenesis of obesity-associated diseases. However, studies to date have assumed that a few select FFAs are representative of broad structural categories, and there are no scalable approaches to comprehensively assess the biological processes induced by exposure to diverse FFAs circulating in human plasma. Furthermore, assessing how these FFA- mediated processes interact with genetic risk for disease remains elusive. Here we report the design and implementation of FALCON (Fatty Acid Library for Comprehensive ONtologies) as an unbiased, scalable and multimodal interrogation of 61 structurally diverse FFAs. We identified a subset of lipotoxic monounsaturated fatty acids (MUFAs) with a distinct lipidomic profile associated with decreased membrane fluidity. Furthermore, we developed a new approach to prioritize genes that reflect the combined effects of exposure to harmful FFAs and genetic risk for type 2 diabetes (T2D). Importantly, we found that c-MAF inducing protein (CMIP) protects cells from exposure to FFAs by modulating Akt signaling and we validated the role of CMIP in human pancreatic beta cells. In sum, FALCON empowers the study of fundamental FFA biology and offers an integrative approach to identify much needed targets for diverse diseases associated with disordered FFA metabolism. Highlights: FALCON (Fatty Acid Library for Comprehensive ONtologies) enables multimodal profiling of 61 free fatty acids (FFAs) to reveal 5 FFA clusters with distinct biological effectsFALCON is applicable to many and diverse cell typesA subset of monounsaturated FAs (MUFAs) equally or more toxic than canonical lipotoxic saturated FAs (SFAs) leads to decreased membrane fluidityNew approach prioritizes genes that represent the combined effects of environmental (FFA) exposure and genetic risk for diseaseC-Maf inducing protein (CMIP) is identified as a suppressor of FFA-induced lipotoxicity via Akt-mediated signaling.
RESUMEN
Mutations affecting mitochondrial coenzyme Q (CoQ) biosynthesis lead to kidney failure due to selective loss of podocytes, essential cells of the kidney filter. Curiously, neighboring tubular epithelial cells are spared early in disease despite higher mitochondrial content. We sought to illuminate noncanonical, cell-specific roles for CoQ, independently of the electron transport chain (ETC). Here, we demonstrate that CoQ depletion caused by Pdss2 enzyme deficiency in podocytes results in perturbations in polyunsaturated fatty acid (PUFA) metabolism and the Braf/Mapk pathway rather than ETC dysfunction. Single-nucleus RNA-Seq from kidneys of Pdss2kd/kd mice with nephrotic syndrome and global CoQ deficiency identified a podocyte-specific perturbation of the Braf/Mapk pathway. Treatment with GDC-0879, a Braf/Mapk-targeting compound, ameliorated kidney disease in Pdss2kd/kd mice. Mechanistic studies in Pdss2-depleted podocytes revealed a previously unknown perturbation in PUFA metabolism that was confirmed in vivo. Gpx4, an enzyme that protects against PUFA-mediated lipid peroxidation, was elevated in disease and restored after GDC-0879 treatment. We demonstrate broader human disease relevance by uncovering patterns of GPX4 and Braf/Mapk pathway gene expression in tissue from patients with kidney diseases. Our studies reveal ETC-independent roles for CoQ in podocytes and point to Braf/Mapk as a candidate pathway for the treatment of kidney diseases.
Asunto(s)
Ataxia/metabolismo , Indenos/farmacología , Enfermedades Renales/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Enfermedades Mitocondriales/metabolismo , Debilidad Muscular/metabolismo , Podocitos/metabolismo , Proteínas Proto-Oncogénicas B-raf/metabolismo , Pirazoles/farmacología , Ubiquinona/deficiencia , Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Animales , Ataxia/tratamiento farmacológico , Ataxia/genética , Ataxia/patología , Sistemas de Liberación de Medicamentos , Células HEK293 , Humanos , Enfermedades Renales/tratamiento farmacológico , Enfermedades Renales/genética , Enfermedades Renales/patología , Peroxidación de Lípido/genética , Sistema de Señalización de MAP Quinasas/genética , Ratones , Enfermedades Mitocondriales/tratamiento farmacológico , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/patología , Debilidad Muscular/tratamiento farmacológico , Debilidad Muscular/genética , Debilidad Muscular/patología , Podocitos/patología , Proteínas Proto-Oncogénicas B-raf/genética , RNA-Seq , Ubiquinona/genética , Ubiquinona/metabolismoRESUMEN
Drug repurposing is the only method capable of delivering treatments on the shortened time-scale required for patients afflicted with lung disease arising from SARS-CoV-2 infection. Mucin-1 (MUC1), a membrane-bound molecule expressed on the apical surfaces of most mucosal epithelial cells, is a biochemical marker whose elevated levels predict the development of acute lung injury (ALI) and respiratory distress syndrome (ARDS), and correlate with poor clinical outcomes. In response to the pandemic spread of SARS-CoV-2, we took advantage of a high content screen of 3,713 compounds at different stages of clinical development to identify FDA-approved compounds that reduce MUC1 protein abundance. Our screen identified Fostamatinib (R788), an inhibitor of spleen tyrosine kinase (SYK) approved for the treatment of chronic immune thrombocytopenia, as a repurposing candidate for the treatment of ALI. In vivo , Fostamatinib reduced MUC1 abundance in lung epithelial cells in a mouse model of ALI. In vitro , SYK inhibition by Fostamatinib promoted MUC1 removal from the cell surface. Our work reveals Fostamatinib as a repurposing drug candidate for ALI and provides the rationale for rapidly standing up clinical trials to test Fostamatinib efficacy in patients with COVID-19 lung injury.
RESUMEN
Drug repurposing has the advantage of identifying potential treatments on a shortened timescale. In response to the pandemic spread of SARS-CoV-2, we took advantage of a high-content screen of 3,713 compounds at different stages of clinical development to identify FDA-approved compounds that reduce mucin-1 (MUC1) protein abundance. Elevated MUC1 levels predict the development of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) and correlate with poor clinical outcomes. Our screen identifies fostamatinib (R788), an inhibitor of spleen tyrosine kinase (SYK) approved for the treatment of chronic immune thrombocytopenia, as a repurposing candidate for the treatment of ALI. In vivo, fostamatinib reduces MUC1 abundance in lung epithelial cells in a mouse model of ALI. In vitro, SYK inhibition by the active metabolite R406 promotes MUC1 removal from the cell surface. Our work suggests fostamatinib as a repurposing drug candidate for ALI.
RESUMEN
Human iPSC-derived kidney organoids have the potential to revolutionize discovery, but assessing their consistency and reproducibility across iPSC lines, and reducing the generation of off-target cells remain an open challenge. Here, we profile four human iPSC lines for a total of 450,118 single cells to show how organoid composition and development are comparable to human fetal and adult kidneys. Although cell classes are largely reproducible across time points, protocols, and replicates, we detect variability in cell proportions between different iPSC lines, largely due to off-target cells. To address this, we analyze organoids transplanted under the mouse kidney capsule and find diminished off-target cells. Our work shows how single cell RNA-seq (scRNA-seq) can score organoids for reproducibility, faithfulness and quality, that kidney organoids derived from different iPSC lines are comparable surrogates for human kidney, and that transplantation enhances their formation by diminishing off-target cells.
Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Riñón/citología , Organoides/citología , Animales , Diferenciación Celular , Línea Celular , Regulación del Desarrollo de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/trasplante , Riñón/metabolismo , Trasplante de Riñón , Ratones , Organoides/metabolismo , Organoides/trasplante , Reproducibilidad de los Resultados , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Trasplante HeterólogoRESUMEN
Progressive kidney diseases are often associated with scarring of the kidney's filtration unit, a condition called focal segmental glomerulosclerosis (FSGS). This scarring is due to loss of podocytes, cells critical for glomerular filtration, and leads to proteinuria and kidney failure. Inherited forms of FSGS are caused by Rac1-activating mutations, and Rac1 induces TRPC5 ion channel activity and cytoskeletal remodeling in podocytes. Whether TRPC5 activity mediates FSGS onset and progression is unknown. We identified a small molecule, AC1903, that specifically blocks TRPC5 channel activity in glomeruli of proteinuric rats. Chronic administration of AC1903 suppressed severe proteinuria and prevented podocyte loss in a transgenic rat model of FSGS. AC1903 also provided therapeutic benefit in a rat model of hypertensive proteinuric kidney disease. These data indicate that TRPC5 activity drives disease and that TRPC5 inhibitors may be valuable for the treatment of progressive kidney diseases.
Asunto(s)
Glomeruloesclerosis Focal y Segmentaria/tratamiento farmacológico , Hipertensión Renal/tratamiento farmacológico , Indazoles/farmacología , Proteinuria/tratamiento farmacológico , Canales Catiónicos TRPC/antagonistas & inhibidores , Animales , Modelos Animales de Enfermedad , Glomeruloesclerosis Focal y Segmentaria/genética , Mutación , Podocitos/efectos de los fármacos , Ratas , Ratas Endogámicas Dahl , Ratas Transgénicas , Bibliotecas de Moléculas Pequeñas , Canales Catiónicos TRPC/farmacología , Proteína de Unión al GTP rac1/genéticaRESUMEN
The microtubule-targeting maytansinoids accumulate in cells and induce mitotic arrest at 250- to 1000-fold lower concentrations than those required for their association with tubulin or microtubules. To identify the mechanisms of this intracellular accumulation and exceptional cytotoxicity of maytansinoids we studied interaction of a highly cytotoxic maytansinoid, S-methyl DM1 and several other maytansinoids with cells. S-methyl DM1 accumulated inside the cells with a markedly higher apparent affinity than to tubulin or microtubules. The apparent affinities of maytansinoids correlated with their cytotoxicities. The number of intracellular binding sites for S-methyl DM1 in MCF7 cells was comparable to the number of tubulin molecules per cell (~ 4-6 × 10(7) copies). Efflux of 3[H]-S-methyl DM1 from cells was enhanced in the presence of an excess of non-labeled S-methyl DM1, indicating that re-binding of 3 [H]-S-methyl DM1 to intracellular binding sites contributed to its intracellular retention. Liposomes loaded with non-polymerized tubulin recapitulated the apparent high-affinity association of S-methyl DM1 to cells. We propose a model for the intracellular accumulation of maytansinoids in which molecules of the compounds diffuse into a cell and associate with tubulin. Affinities of maytansinoids for individual tubulin molecules are weak, but the high intracellular concentration of tubulin favors, after dissociation of a compound-tubulin complex, their re-binding to a tubulin molecule, or to a tip of a microtubule in the same cell, over their efflux. As a result, a significant fraction of microtubule tips is occupied with a maytansinoid when added to cells at sub-nanomolar concentrations, inducing mitotic arrest and cell death.