Flavonoids as Potential Drugs for VPS13-Dependent Rare Neurodegenerative Diseases.

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.

Molecular Basis of the Pathogenic Mechanism Induced by the m.9191T>C Mutation in Mitochondrial ATP6 Gene.

Probing the pathogenicity and functional consequences of mitochondrial DNA (mtDNA) mutations from patient's cells and tissues is difficult due to genetic heteroplasmy (co-existence of wild type and mutated mtDNA in cells), occurrence of numerous mtDNA polymorphisms, and absence of methods for genetically transforming human mitochondria. Owing to its good fermenting capacity that enables survival to loss-of-function mtDNA mutations, its amenability to mitochondrial genome manipulation, and lack of heteroplasmy, Saccharomyces cerevisiae is an excellent model for studying and resolving the molecular bases of human diseases linked to mtDNA in a controlled genetic background. Using this model, we previously showed that a pathogenic mutation in mitochondrial ATP6 gene (m.9191T>C), that converts a highly conserved leucine residue into proline in human ATP synthase subunit a (aL222P), severely compromises the assembly of yeast ATP synthase and reduces by 90% the rate of mitochondrial ATP synthesis. Herein, we report the isolation of intragenic suppressors of this mutation. In light of recently described high resolution structures of ATP synthase, the results indicate that the m.9191T>C mutation disrupts a four α-helix bundle in subunit a and that the leucine residue it targets indirectly optimizes proton conduction through the membrane domain of ATP synthase.

The binding of the APT1 domains to phosphoinositides is regulated by metal ions in vitro.

Chorein is a protein of the Vps13 family, and defects in this protein cause the rare neurodegenerative disorder chorea-acanthocytosis (ChAc). Chorein is involved in the actin cytoskeleton organization, calcium ion flux, neuronal cell excitability, exocytosis and autophagy. The function of this protein is poorly understood, and obtaining this knowledge is a key to finding a cure for ChAc. Chorein, as well as the Vps13 protein from yeast, contains the APT1 domain. Our previous research has shown that the APT1 domain from yeast Vps13 (yAPT1v) binds phosphatidylinositol 3-phosphate (PI3P) in vitro. In this study, we showed that although the APT1 domain from chorein (hAPT1) binds to PI3P it could not functionally replace yAPT1v. The hAPT1 domain binds, in addition to PI3P, to phosphatidylinositol 5-phosphate (PI5P). The binding of hAPT1 to PI3P, unlike the binding of yAPT1v to PI3P, is regulated by the bivalent ions, calcium and magnesium. Regulation of PI3P binding via calcium is also observed for the APT1 domain of yeast autophagy protein Atg2. The substitution I2771R, found in chorein of patient suffering from ChAc, reduces the binding of the hAPT1 domain to PI3P and PI5P. These results suggest that the ability of APT1 domains to bind phosphoinositides is regulated differently in yeast and human protein and that this regulation is important for chorein function.

Yeast-model-based study identified myosin- and calcium-dependent calmodulin signalling as a potential target for drug intervention in chorea-acanthocytosis.

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.

Amino acid substitution equivalent to human chorea-acanthocytosis I2771R in yeast Vps13 protein affects its binding to phosphatidylinositol 3-phosphate.

The rare human disorder chorea-acanthocytosis (ChAc) is caused by mutations in hVPS13A gene. The hVps13A protein interacts with actin and regulates the level of phosphatidylinositol 4-phosphate (PI4P) in the membranes of neuronal cells. Yeast Vps13 is involved in vacuolar protein transport and, like hVps13A, participates in PI4P metabolism. Vps13 proteins are conserved in eukaryotes, but their molecular function remains unknown. One of the mutations found in ChAc patients causes amino acids substitution I2771R which affects the localization of hVps13A in skeletal muscles. To dissect the mechanism of pathogenesis of I2771R, we created and analyzed a yeast strain carrying the equivalent mutation. Here we show that in yeast, substitution I2749R causes dysfunction of Vps13 protein in endocytosis and vacuolar transport, although the level of the protein is not affected, suggesting loss of function. We also show that Vps13, like hVps13A, influences actin cytoskeleton organization and binds actin in immunoprecipitation experiments. Vps13-I2749R binds actin, but does not function in the actin cytoskeleton organization. Moreover, we show that Vps13 binds phospholipids, especially phosphatidylinositol 3-phosphate (PI3P), via its SHR_BD and APT1 domains. Substitution I2749R attenuates this ability. Finally, the localization of Vps13-GFP is altered when cellular levels of PI3P are decreased indicating its trafficking within the endosomal membrane system. These results suggest that PI3P regulates the functioning of Vps13, both in protein trafficking and actin cytoskeleton organization. Attenuation of PI3P-binding ability in the mutant hVps13A protein may be one of the reasons for its mislocalization and disrupted function in cells of patients suffering from ChAc.

Phosphatidylinositol-3-phosphate regulates response of cells to proteotoxic stress.

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.

Mimicking the phosphorylation of Rsp5 in PKA site T761 affects its function and cellular localization.

Rsp5 ubiquitin ligase belongs to the Nedd4 family of proteins, which affect a wide variety of processes in the cell. Here we document that Rsp5 shows several phosphorylated variants of different mobility and the migration of the phosphorylated forms of Rsp5 was faster for the tpk1Δ tpk3Δ mutant devoid of two alternative catalytic subunits of protein kinase A (PKA), indicating that PKA possibly phosphorylates Rsp5 in vivo. We demonstrated by immunoprecipitation and Western blot analysis of GFP-HA-Rsp5 protein using the anti-phospho PKA substrate antibody that Rsp5 is phosphorylated in PKA sites. Rsp5 contains the sequence 758-RRFTIE-763 with consensus RRXS/T in the catalytic HECT domain and four other sites with consensus RXXS/T, which might be phosphorylated by PKA. The strain bearing the T761D substitution in Rsp5 which mimics phosphorylation grew more slowly at 28°C and did not grow at 37°C, and showed defects in pre-tRNA processing and protein sorting. The rsp5-T761D strain also demonstrated a reduced ability to form colonies, an increase in the level of reactive oxygen species (ROS) and hypersensitivity to ROS-generating agents. These results indicate that PKA may downregulate many functions of Rsp5, possibly affecting its activity. Rsp5 is found in the cytoplasm, nucleus, multivesicular body and cortical patches. The rsp5-T761D mutation led to a strongly increased cortical localization while rsp5-T761A caused mutant Rsp5 to locate more efficiently in internal spots. Rsp5-T761A protein was phosphorylated less efficiently in PKA sites under specific growth conditions. Our data suggests that Rsp5 may be phosphorylated by PKA at position T761 and that this regulation is important for its localization and function.

Nedd4, a human ubiquitin ligase, affects actin cytoskeleton in yeast cells.

Human Nedd4 ubiquitin ligase is involved in protein trafficking, signal transduction and oncogenesis. Nedd4 with an inactive WW4 domain is toxic to yeast cells. We report here that actin cytoskeleton is abnormal in yeast cells expressing the NEDD4 or NEDD4w4 gene and these cells are more sensitive to Latrunculin A, an actin-depolymerizing drug. These phenotypes are less pronounced when a mutation inactivating the catalytic domain of the ligase has been introduced. In contrast, overexpression of the LAS17 gene, encoding an activator of the Arp2/3 actin nucleating complex, is detrimental to NEDD4w4-expressing cells. The level of Las17p is increased in cells overproducing Nedd4w4 and this depends partially on its catalytic domain. Expression of genes encoding Nedd4 variants, like overexpression of LAS17, suppresses the growth defect of the arp2-1 strain. Our results suggest that human Nedd4 ligase inhibits yeast cell growth by disturbing the actin cytoskeleton, in part by increasing Las17p level, and that Nedd4 ubiquitination targets may include actin cytoskeleton-associated proteins conserved in evolution.

Physiological effects of unassembled chaperonin Cct subunits in the yeast Saccharomyces cerevisiae.

Eukaryotic chaperonins, the Cct complexes, are assembled into two rings, each of which is composed of a stoichiometric array of eight different subunits, which are denoted Cct1p-Cct8p. Overexpression of a single CCT gene in Saccharomyces cerevisiae causes an increase of the corresponding Cct subunit, but not of the Cct complex. Nevertheless, overexpression of certain Cct subunits, especially CCT6, suppresses a wide range of abnormal phenotypes, including those caused by the diverse types of conditional mutations tor2-21, lst8-2 and rsp5-9 and those caused by the concomitant overexpression of Sit4p and Sap155p. The examination of 73 altered forms of Cct6p revealed that the cct6-24 mutation, containing GDGTT --> AAAAA replacements of the conserved ATP-binding motif, was unable to suppress any of these traits, although the cct6-24 allele was completely functional for growth. These results provide evidence for functional differences among Cct subunits and for physiological properties of unassembled subunits. We suggest that the suppression is due to the competition of specific Cct subunits for activities that normally modify various cellular components. Furthermore, we also suggest that the Cct subunits can act as suppressors only in certain states, such as when associated with ATP.

Rsp5p is required for ER bound Mga2p120 polyubiquitination and release of the processed/tethered transactivator Mga2p90.

A number of eukaryotic transcription factors are held in a latent state by being embedded in, or tethered to, cellular membranes. Mga2p of Saccharomyces cerevisiae is an endoplasmic reticulum (ER)-localized transcription factor that plays an overlapping role with homologous Spt23p in upregulating expression of OLE1, a gene required for the synthesis of essential oleic acid. Previous studies have documented that proteasome-dependent processing of ER bound 120 kDa Mga2p and Spt23p proteins generates transcriptionally competent 90 kDa polypeptides. In the case of Spt23p90, it is held at the membrane prior to release via a self-interaction with the unprocessed Spt23p120 anchor. It is currently thought that the highly conserved Rsp5p ubiquitin ligase provides the signal for partial degradation of both proteins. Cells lacking Rsp5p function require oleic acid for growth, and Spt23p processing is suppressed in rsp5 Delta cells and in wild-type RSP5 cells upon expression of Rsp5p dominant-negative mutants. We report here that Rsp5p is dispensable for Mga2p90 generation but not for release of the processed product from the ER. In addition, we demonstrate that polyubiquitinated Mga2p120 accumulates in cells lacking Npl4p or proteasome function and Rsp5p is required for Mga2p120 polyubiquitination. Finally, we provide evidence that Mga2p90 and Mga2p120 dimerize and that Rsp5p binds heterodimeric Mga2p complexes both in vitro and in vivo. In light of these experiments, we propose that Rsp5p facilitates Mga2p90 release from the ER by promoting polyubiquitination and Npl4p-proteasome-mediated degradation of the interacting Mga2p120 ER bound anchor.