RESUMEN
Mutations in human VPS13A-D genes result in rare neurological diseases, including chorea-acanthocytosis. The pathogenesis of these diseases is poorly understood, and no effective treatment is available. As VPS13 genes are evolutionarily conserved, the effects of the pathogenic mutations could be studied in model organisms, including yeast, where one VPS13 gene is present. In this review, we summarize advancements obtained using yeast. In recent studies, vps13Δ and vps13-I2749 yeast mutants, which are models of chorea-acanthocytosis, were used to screen for multicopy and chemical suppressors. Two of the suppressors, a fragment of the MYO3 and RCN2 genes, act by downregulating calcineurin activity. In addition, vps13Δ suppression was achieved by using calcineurin inhibitors. The other group of multicopy suppressors were genes: FET4, encoding iron transporter, and CTR1, CTR3 and CCC2, encoding copper transporters. Mechanisms of their suppression rely on causing an increase in the intracellular iron content. Moreover, among the identified chemical suppressors were copper ionophores, which require a functional iron uptake system for activity, and flavonoids, which bind iron. These findings point at areas for further investigation in a higher eukaryotic model of VPS13-related diseases and to new therapeutic targets: calcium signalling and copper and iron homeostasis. Furthermore, the identified drugs are interesting candidates for drug repurposing for these diseases.
Asunto(s)
Neuroacantocitosis , Enfermedades Neurodegenerativas , Proteínas de Saccharomyces cerevisiae , Señalización del Calcio , Proteínas de Unión al Calcio/metabolismo , Cobre/metabolismo , Proteínas Transportadoras de Cobre , Humanos , Hierro/metabolismo , Proteínas de Unión a Hierro/metabolismo , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Ion homeostasis is crucial for organism functioning, and its alterations may cause diseases. For example, copper insufficiency and overload are associated with Menkes and Wilson's diseases, respectively, and iron imbalance is observed in Parkinson's and Alzheimer's diseases. To better understand human diseases, Saccharomyces cerevisiae yeast are used as a model organism. In our studies, we used the vps13Δ yeast strain as a model of rare neurological diseases caused by mutations in VPS13A-D genes. In this work, we show that overexpression of genes encoding copper transporters, CTR1, CTR3, and CCC2, or the addition of copper salt to the medium, improved functioning of the vps13Δ mutant. We show that their mechanism of action, at least partially, depends on increasing iron content in the cells by the copper-dependent iron uptake system. Finally, we present that treatment with copper ionophores, disulfiram, elesclomol, and sodium pyrithione, also resulted in alleviation of the defects observed in vps13Δ cells. Our study points at copper and iron homeostasis as a potential therapeutic target for further investigation in higher eukaryotic models of VPS13-related diseases.
Asunto(s)
Proteínas Transportadoras de Cobre/metabolismo , Cobre/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Homeostasis , Mutación , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas Transportadoras de Cobre/genética , Humanos , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Regulation of calcineurin, a Ca2+/calmodulin-regulated phosphatase, is important for the nervous system, and its abnormal activity is associated with various pathologies, including neurodegenerative disorders. In yeast cells lacking the VPS13 gene (vps13Δ), a model of VPS13-linked neurological diseases, we recently demonstrated that calcineurin is activated, and its downregulation reduces the negative effects associated with vps13Δ mutation. Here, we show that overexpression of the RCN2 gene, which encodes a negative regulator of calcineurin, is beneficial for vps13Δ cells. We studied the molecular mechanism underlying this effect through site-directed mutagenesis of RCN2. The interaction of the resulting Rcn2 variants with a MAPK kinase, Slt2, and subunits of calcineurin was tested. We show that Rcn2 binds preferentially to Cmp2, one of two alternative catalytic subunits of calcineurin, and partially inhibits calcineurin. Rcn2 ability to bind to and reduce the activity of calcineurin was important for the suppression. The binding of Rcn2 to Cmp2 requires two motifs in Rcn2: the previously characterized C-terminal motif and a new N-terminal motif that was discovered in this study. Altogether, our findings can help to better understand calcineurin regulation and to develop new therapeutic strategies against neurodegenerative diseases based on modulation of the activity of selected calcineurin isoforms.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Calcineurina/metabolismo , Proteínas de Transporte Vesicular/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Sitios de Unión , Dosificación de Gen , Regulación de la Expresión Génica , Mutación , Unión Proteica , Dominios y Motivos de Interacción de ProteínasRESUMEN
Several rare neurodegenerative diseases, including chorea acanthocytosis, are caused by mutations in the VPS13A-D genes. Only symptomatic treatments for these diseases are available. Saccharomyces cerevisiae contains a unique VPS13 gene and the yeast vps13Δ mutant has been proven as a suitable model for drug tests. A library of drugs and an in-house library of natural compounds and their derivatives were screened for molecules preventing the growth defect of vps13Δ cells on medium with sodium dodecyl sulfate (SDS). Seven polyphenols, including the iron-binding flavone luteolin, were identified. The structure-activity relationship and molecular mechanisms underlying the action of luteolin were characterized. The FET4 gene, which encodes an iron transporter, was found to be a multicopy suppressor of vps13Δ, pointing out the importance of iron in response to SDS stress. The growth defect of vps13Δ in SDS-supplemented medium was also alleviated by the addition of iron salts. Suppression did not involve cell antioxidant responses, as chemical antioxidants were not active. Our findings support that luteolin and iron may target the same cellular process, possibly the synthesis of sphingolipids. Unveiling the mechanisms of action of chemical and genetic suppressors of vps13Δ may help to better understand VPS13A-D-dependent pathogenesis and to develop novel therapeutic strategies.
Asunto(s)
Productos Biológicos/farmacología , Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Luteolina/farmacología , Fármacos Neuroprotectores/farmacología , Proteínas de Saccharomyces cerevisiae/metabolismo , Productos Biológicos/química , Proliferación Celular/efectos de los fármacos , Proteínas Transportadoras de Cobre/genética , Proteínas Transportadoras de Cobre/metabolismo , Hierro/metabolismo , Proteínas de Unión a Hierro/genética , Proteínas de Unión a Hierro/metabolismo , Luteolina/química , Fármacos Neuroprotectores/química , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad , Supresión GenéticaRESUMEN
Chorea-acanthocytosis (ChAc) is a rare neurodegenerative disease associated with mutations in the human VPS13A gene. The mechanism of ChAc pathogenesis is unclear. A simple yeast model was used to investigate the function of the single yeast VSP13 orthologue, Vps13. Vps13, like human VPS13A, is involved in vesicular protein transport, actin cytoskeleton organisation and phospholipid metabolism. A newly identified phenotype of the vps13Δ mutant, sodium dodecyl sulphate (SDS) hypersensitivity, was used to screen a yeast genomic library for multicopy suppressors. A fragment of the MYO3 gene, encoding Myo3-N (the N-terminal part of myosin, a protein involved in the actin cytoskeleton and in endocytosis), was isolated. Myo3-N protein contains a motor head domain and a linker. The linker contains IQ motifs that mediate the binding of calmodulin, a negative regulator of myosin function. Amino acid substitutions that disrupt the interaction of Myo3-N with calmodulin resulted in the loss of vps13Δ suppression. Production of Myo3-N downregulated the activity of calcineurin, a protein phosphatase regulated by calmodulin, and alleviated some defects in early endocytosis events. Importantly, ethylene glycol tetraacetic acid (EGTA), which sequesters calcium and thus downregulates calmodulin and calcineurin, was a potent suppressor of vps13Δ. We propose that Myo3-N acts by sequestering calmodulin, downregulating calcineurin and increasing activity of Myo3, which is involved in endocytosis and, together with Osh2/3 proteins, functions in endoplasmic reticulum-plasma membrane contact sites. These results show that defects associated with vps13Δ could be overcome, and point to a functional connection between Vps13 and calcium signalling as a possible target for chemical intervention in ChAc. Yeast ChAc models may uncover the underlying pathological mechanisms, and may also serve as a platform for drug testing.This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Calmodulina/metabolismo , Modelos Biológicos , Miosinas/metabolismo , Neuroacantocitosis/tratamiento farmacológico , Neuroacantocitosis/metabolismo , Saccharomyces cerevisiae/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Alelos , Sustitución de Aminoácidos , Calcineurina/metabolismo , Señalización del Calcio/efectos de los fármacos , Canavanina/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Endocitosis/efectos de los fármacos , Genes Supresores , Mutación/genética , Dominios Proteicos , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/metabolismo , Dodecil Sulfato de Sodio , Transcripción Genética/efectos de los fármacos , Vacuolas/metabolismoRESUMEN
Human Nedd4 ubiquitin ligase, or its variants, inhibit yeast cell growth by disturbing the actin cytoskeleton organization and dynamics, and lead to an increase in levels of ubiquitinated proteins. In a screen for multicopy suppressors which rescue growth of yeast cells producing Nedd4 ligase with an inactive WW4 domain (Nedd4w4), we identified a fragment of ATG2 gene encoding part of the Atg2 core autophagy protein. Expression of the Atg2-C1 fragment (aa 1074-1447) improved growth, actin cytoskeleton organization, but did not significantly change the levels of ubiquitinated proteins in these cells. The GFP-Atg2-C1 protein in Nedd4w4-producing cells primarily localized to a single defined structure adjacent to the vacuole, surrounded by an actin filament ring, containing Hsp42 and Hsp104 chaperones. This localization was not affected in several atg deletion mutants, suggesting that it might be distinct from the phagophore assembly site (PAS). However, deletion of ATG18 encoding a phosphatidylinositol-3-phosphate (PI3P)-binding protein affected the morphology of the GFP-Atg2-C1 structure while deletion of ATG14 encoding a subunit of PI3 kinase suppressed toxicity of Nedd4w4 independently of GFP-Atg2-C1. Further analysis of the Atg2-C1 revealed that it contains an APT1 domain of previously uncharacterized function. Most importantly, we showed that this domain is able to bind phosphatidylinositol phosphates, especially PI3P, which is abundant in the PAS and endosomes. Together our results suggest that human Nedd4 ubiquitinates proteins in yeast and causes proteotoxic stress and, with some Atg proteins, leads to formation of a perivacuolar structure, which may be involved in sequestration, aggregation or degradation of proteins.