Synthetic toxic Aß1-42 oligomers can assemble in different morphologies.

BACKGROUND: Alzheimer's disease is the most common neurodegenerative disease associated with aggregation of Aß peptides. Aß toxicity is mostly related to the capacity of intermediate oligomers to disrupt membrane integrity. We previously expressed Aß1-42 in a eukaryotic cellular system and selected synthetic variants on their sole toxicity. The most toxic mutant G37C forms stable oligomers. METHODS: Different biophysical methods (Fluorescence spectroscopy, cross-linking, mass spectrometry (MS), Small Angle X-ray Scattering (SAXS), Atomic Force Microscopy (AFM), Transmission Electron Microscopy (TEM), calcein leakage) were used. RESULTS: The oligomers are mostly populated by a 14mers resulting from the packing of homodimers. These homodimers come from the formation of a disulfide bridge between two monomers. This link stabilizes the multimers and prevents the assembly into amyloid fibrils. These oligomers affect the membrane integrity. The reduction of disulfide bonds leads to a rearrangement and redirects assembly of Aß amyloid fibrils. CONCLUSION: The toxic synthetic AßG37C mutant can assemble into an amyloid of unusual morphology through the formation of anti-parallel ß-sheets. This pathway involves the formation of oligomers resulting from the arrangement of Aß dimers linked by covalent di-sulfide link, being these oligomers harmful for the membranes. GENERAL SIGNIFICANCE: The capacity to produce large amount of stable oligomers without additional detergents or extrinsic cross-linkers allow further structural and biophysical studies to understand their capacity to assemble and disrupt the membranes, a key event in Alzheimer's disease.

The Impact of ESCRT on Aß1-42 Induced Membrane Lesions in a Yeast Model for Alzheimer's Disease.

Aß metabolism plays a pivotal role in Alzheimer's disease. Here, we used a yeast model to monitor Aß42 toxicity when entering the secretory pathway and demonstrate that processing in, and exit from the endoplasmic reticulum (ER) is required to unleash the full Aß42 toxic potential. Consistent with previously reported data, our data suggests that Aß42 interacts with mitochondria, thereby enhancing formation of reactive oxygen species and eventually leading to cell demise. We used our model to search for genes that modulate this deleterious effect, either by reducing or enhancing Aß42 toxicity, based on screening of the yeast knockout collection. This revealed a reduced Aß42 toxicity not only in strains hampered in ER-Golgi traffic and mitochondrial functioning but also in strains lacking genes connected to the cell cycle and the DNA replication stress response. On the other hand, increased Aß42 toxicity was observed in strains affected in the actin cytoskeleton organization, endocytosis and the formation of multivesicular bodies, including key factors of the ESCRT machinery. Since the latter was shown to be required for the repair of membrane lesions in mammalian systems, we studied this aspect in more detail in our yeast model. Our data demonstrated that Aß42 heavily disturbed the plasma membrane integrity in a strain lacking the ESCRT-III accessory factor Bro1, a phenotype that came along with a severe growth defect and enhanced loading of lipid droplets. Thus, it appears that also in yeast ESCRT is required for membrane repair, thereby counteracting one of the deleterious effects induced by the expression of Aß42. Combined, our studies once more validated the use of yeast as a model to investigate fundamental mechanisms underlying the etiology of neurodegenerative disorders.

Using Histone Deacetylase Inhibitors to Analyze the Relevance of HDACs for Translation.

Gene expression is regulated in part through the reversible acetylation of histones, by the action of histone acetyltransferases (HAT) and histone deacetylases (HDAC). HAT activity results in the addition of acetyl groups on the lysine residues of histone tails leading to decondensation of the chromatin, and increased gene transcription in general, whereas HDACs remove these acetyl groups, thus leading to an overall suppression of gene transcription. Recent evidence has elucidated that histones are not the only components of the proteome that are targeted by HATs and HDACs. A large number of nonhistone proteins undergo posttranslational acetylation. They include proteins involved in mRNA stability, protein localization and degradation, as well as protein-protein and protein-DNA interactions. In recent years, numerous studies have discovered increased HDAC expression and/or activity in numerous disease states, including cancer, where the upregulation of HDAC family members leads to dysregulation of genes and proteins involved in cell proliferation, cell cycle regulation, and apoptosis. These observations have pushed HDAC inhibitors (HDACi) to the forefront of therapeutic development of oncological conditions. HDACi, such as Vorinostat (Suberoylanilide hydroxamic acid (SAHA)), affect cancer cells in part by suppressing the translation of key proteins linked to tumorigenesis, such as cyclin D1 and hypoxia inducible factor 1 alpha (HIF-1α). Herein we describe methodologies to analyze the impact of the HDACi Vorinostat on HIF-1α translational regulation and downstream effectors.

Extracts from two ubiquitous Mediterranean plants ameliorate cellular and animal models of neurodegenerative proteinopathies.

A signature feature of age-related neurodegenerative proteinopathies is the misfolding and aggregation of proteins, typically amyloid-ß (Aß) in Alzheimer's disease (AD) and α-synuclein (α-syn) in Parkinson's disease (PD), into soluble oligomeric structures that are highly neurotoxic. Cellular and animal models that faithfully replicate the hallmark features of these disorders are being increasing exploited to identify disease-modifying compounds. Natural compounds have been identified as a useful source of bioactive molecules with promising neuroprotective capabilities. In the present report, we investigated whether extracts derived from two ubiquitous Mediterranean plants namely, the prickly pear Opuntia ficus-indica (EOFI) and the brown alga Padina pavonica (EPP) alleviate neurodegenerative phenotypes in yeast (Saccharomyces cerevisiae) and fly (Drosophila melanogaster) models of AD and PD. Pre-treatment with EPP or EOFI in the culture medium significantly improved the viability of yeast expressing the Arctic Aß42 (E22G) mutant. Supplementing food with EOFI or EPP dramatically ameliorated lifespan and behavioural signs of flies with brain-specific expression of wild-type Aß42 (model of late-onset AD) or the Arctic Aß42 variant (model of early-onset AD). Additionally, we show that either extract prolonged the survival of a PD fly model based on transgenic expression of the human α-syn A53T mutant. Taken together, our findings suggest that the plant-derived extracts interfere with shared mechanisms of neurodegeneration in AD and PD. This notion is strengthened by evidence demonstrating that EOFI and to a greater extent EPP, while strongly inhibiting the fibrillogenesis of both Aß42 and α-syn, accumulate remodelled oligomeric aggregates that are less effective at disrupting lipid membrane integrity. Our work therefore opens new avenues for developing therapeutic applications of these natural plant extracts in the treatment of amyloidogenic neurodegenerative disorders.

ERAD defects and the HFE-H63D variant are associated with increased risk of liver damages in Alpha 1-Antitrypsin Deficiency.

BACKGROUND: The most common and severe disease causing allele of Alpha 1-Antitrypsin Deficiency (1ATD) is Z-1AT. This protein aggregates in the endoplasmic reticulum, which is the main cause of liver disease in childhood. Based on recent evidences and on the frequency of liver disease occurrence in Z-1AT patients, it seems that liver disease progression is linked to still unknown genetic factors. METHODS: We used an innovative approach combining yeast genetic screens with next generation exome sequencing to identify and functionally characterize the genes involved in 1ATD associated liver disease. RESULTS: Using yeast genetic screens, we identified HRD1, an Endoplasmic Reticulum Associated Degradation (ERAD) associated protein, as an inducer of Z-mediated toxicity. Whole exome sequencing of 1ATD patients resulted in the identification of two variants associated with liver damages in Z-1AT homozygous cases: HFE H63D and HERPUD1 R50H. Functional characterization in Z-1AT model cell lines demonstrated that impairment of the ERAD machinery combined with the HFE H63D variant expression decreased both cell proliferation and cell viability, while Unfolded Protein Response (UPR)-mediated cell death was hyperstimulated. CONCLUSION: This powerful experimental pipeline allowed us to identify and functionally validate two genes involved in Z-1AT-mediated severe liver toxicity. This pilot study moves forward our understanding on genetic modifiers involved in 1ATD and highlights the UPR pathway as a target for the treatment of liver diseases associated with 1ATD. Finally, these findings support a larger scale screening for HERPUD1 R50H and HFE H63D variants in the sub-group of 1ATD patients developing significant chronic hepatic injuries (hepatomegaly, chronic cholestasis, elevated liver enzymes) and at risk developing liver cirrhosis.

A yeast model for amyloid-ß aggregation exemplifies the role of membrane trafficking and PICALM in cytotoxicity.

Alzheimer's disease is the most common neurodegenerative disease, associated with aggregation of amyloid-ß (Aß) peptides. The exact mechanism of neuronal cell dysfunction in Alzheimer's disease is poorly understood and numerous models have been used to decipher the mechanisms leading to cellular death. Yeast cells might be a good model to understand the intracellular toxicity triggered by Aß peptides. Indeed, yeast has been used as a model to examine protein functions or cellular pathways that mediate the secretion, aggregation and subsequent toxicity of proteins associated with human neurodegenerative disorders. In the present study, we use the yeast Saccharomyces cerevisiae as a model system to study the effects of intracellular Aß in fusion with green fluorescent protein. We sent this fusion protein into the secretory pathway and showed that intracellular traffic pathways are necessary for the generation of toxic species. Yeast PICALM orthologs are involved in cellular toxicity, indicating conservation of the mechanisms of toxicity from mammals to yeast. Finally, our model demonstrates the capacity for intracellular Aß to cross intracellular membranes and target mitochondrial organelles.

The HIV-1 Vpu protein induces apoptosis in Drosophila via activation of JNK signaling.

The genome of the human immunodeficiency virus type 1 (HIV-1) encodes the canonical retroviral proteins, as well as additional accessory proteins that enhance the expression of viral genes, the infectivity of the virus and the production of virions. The accessory Viral Protein U (Vpu), in particular, enhances viral particle production, while also promoting apoptosis of HIV-infected human T lymphocytes. Some Vpu effects rely on its interaction with the ubiquitin-proteasome protein degradation system, but the mechanisms responsible for its pro-apoptotic effects in vivo are complex and remain largely to be elucidated.We took advantage of the Drosophila model to study the effects of Vpu activity in vivo. Expression of Vpu in the developing Drosophila wing provoked tissue loss due to caspase-dependent apoptosis. Moreover, Vpu induced expression of the pro-apoptotic gene reaper, known to down-regulate Inhibitor of Apoptosis Proteins (IAPs) which are caspase-antagonizing E3 ubiquitin ligases. Indeed, Vpu also reduced accumulation of Drosophila IAP1 (DIAP1). Though our results demonstrate a physical interaction between Vpu and the proteasome-addressing SLIMB/ß-TrCP protein, as in mammals, both SLIMB/ßTrCP-dependent and -independent Vpu effects were observed in the Drosophila wing. Lastly, the pro-apoptotic effect of Vpu in this tissue was abrogated upon inactivation of the c-Jun N-terminal Kinase (JNK) pathway. Our results in the fly thus provide the first functional evidence linking Vpu pro-apoptotic effects to activation of the conserved JNK pathway.

The toxicity of an "artificial" amyloid is related to how it interacts with membranes.

Despite intensive research into how amyloid structures can impair cellular viability, the molecular nature of these toxic species and the cellular mechanisms involved are not clearly defined and may differ from one disease to another. We systematically analyzed, in Saccharomyces cerevisiae, genes that increase the toxicity of an amyloid (M8), previously selected in yeast on the sole basis of its cellular toxicity (and consequently qualified as "artificial"). This genomic screening identified the Vps-C HOPS (homotypic vacuole fusion and protein sorting) complex as a key-player in amyloid toxicity. This finding led us to analyze further the phenotype induced by M8 expression. M8-expressing cells displayed an identical phenotype to vps mutants in terms of endocytosis, vacuolar morphology and salt sensitivity. The direct and specific interaction between M8 and lipids reinforces the role of membrane formation in toxicity due to M8. Together these findings suggest a model in which amyloid toxicity results from membrane fission.

The cellular concentration of the yeast Ure2p prion protein affects its propagation as a prion.

The [URE3] yeast prion is a self-propagating inactive form of the Ure2p protein. We show here that Ure2p from the species Saccharomyces paradoxus (Ure2p(Sp)) can be efficiently converted into a prion form and propagate [URE3] when expressed in Saccharomyces cerevisiae at physiological level. We found however that Ure2p(Sp) overexpression prevents efficient prion propagation. We have compared the aggregation rate and propagon numbers of Ure2p(Sp) and of S. cerevisiae Ure2p (Ure2p(Sc)) in [URE3] cells both at different expression levels. Overexpression of both Ure2p orthologues accelerates formation of large aggregates but Ure2p(Sp) aggregates faster than Ure2p(Sc). Although the yeast cells that contain these large Ure2p aggregates do not transmit [URE3] to daughter cells, the corresponding crude extract retains the ability to induce [URE3] in wild-type [ure3-0] cells. At low expression level, propagon numbers are higher with Ure2p(Sc) than with Ure2p(Sp). Overexpression of Ure2p decreases the number of [URE3] propagons with Ure2p(Sc). Together, our results demonstrate that the concentration of a prion protein is a key factor for prion propagation. We propose a model to explain how prion protein overexpression can produce a detrimental effect on prion propagation and why Ure2p(Sp) might be more sensitive to such effects than Ure2p(Sc).

Involvement of the betaTrCP in the ubiquitination and stability of the HIV-1 Vpu protein.

The human immunodeficiency virus type 1 (HIV-1) Vpu protein binds to the CD4 receptor and targets it to the proteasome for degradation. This process requires the recruitment of human betaTrCP, a component of the Skp1-Cullin-F box (SCF) ubiquitin ligase complex, that interacts with phosphorylated Vpu molecules. Vpu, unlike other ligands of betaTrCP, has never been reported to be degraded. We provide evidence that Vpu, itself, is ubiquitinated and targeted for degradation by the proteasome. We demonstrate that the mutant Vpu2.6, which cannot interact with betaTrCP, is stable and, unlike wild-type Vpu, is not polyubiquitinated. These results suggest that betaTrCP is involved in Vpu polyubiquitination.

In vitro analysis of SpUre2p, a prion-related protein, exemplifies the relationship between amyloid and prion.

The yeast Saccharomyces cerevisiae contains in its proteome at least three prion proteins. These proteins (Ure2p, Sup35p, and Rnq1p) share a set of remarkable properties. In vivo, they form aggregates that self-perpetuate their aggregation. This aggregation is controlled by Hsp104, which plays a major role in the growth and severing of these prions. In vitro, these prion proteins form amyloid fibrils spontaneously. The introduction of such fibrils made from Ure2p or Sup35p into yeast cells leads to the prion phenotypes [URE3] and [PSI], respectively. Previous studies on evolutionary biology of yeast prions have clearly established that [URE3] is not well conserved in the hemiascomycetous yeasts and particularly in S. paradoxus. Here we demonstrated that the S. paradoxus Ure2p is able to form infectious amyloid. These fibrils are more resistant than S. cerevisiae Ure2p fibrils to shear force. The observation, in vivo, of a distinct aggregation pattern for GFP fusions confirms the higher propensity of SpUre2p to form fibrillar structures. Our in vitro and in vivo analysis of aggregation propensity of the S. paradoxus Ure2p provides an explanation for its loss of infective properties and suggests that this protein belongs to the non-prion amyloid world.

Casein kinase I controls a late step in the endocytic trafficking of yeast uracil permease.

The modification of yeast uracil permease by phosphorylation at the plasma membrane is a key mechanism for regulating transporter endocytosis. Uracil permease is phosphorylated at several serine residues within a well characterized PEST sequence. The phosphorylation of these residues facilitates the ubiquitination and internalization of the permease. Following endocytosis, the permease is targeted to the lysosome/vacuole for proteolysis. We have shown that in casein kinase 1 (CK1)-deficient cells, the permease is poorly phosphorylated, poorly ubiquitinated and that Yck activity may play a direct role in phosphorylating the permease. We show here that CK1-deficient cells accumulated permease that was subjected to endocytosis in an internal compartment on its way to the vacuole. Uracil permease, produced as a fusion protein with green fluorescent protein in CK1-deficient cells, was detected in dots adjacent to the vacuole. These dots probably correspond to the late endosome/prevacuolar compartment because they were partially colocalized with the Pep12p marker. This accumulation was abolished by mutations affecting the adaptor-related complex, AP-3. The CPY and ALP pathways to the vacuole were both unaffected in CK1-deficient cells. Our analysis provides the first evidence that CK1 is important for the delivery of proteins to the vacuole after endocytosis.

Directed expression of the HIV-1 accessory protein Vpu in Drosophila fat-body cells inhibits Toll-dependent immune responses.

Human immunodeficiency virus 1 (HIV-1) expresses several accessory proteins that manipulate various host-cell processes to achieve optimum replicative efficiency. One of them, viral protein U (Vpu), has been shown to interfere with the cellular degradation machinery through interaction with SCF(beta-TrCP) complexes. To learn more about Vpu function in vivo, we used the genetically tractable fruit fly, Drosophila melanogaster. Our results show that the directed expression of Vpu, but not the non-phosphorylated form, Vpu2/6, in fat-body cells affects Drosophila antimicrobial responses. In flies, the Toll and Imd pathways regulate antimicrobial-peptide gene expression. We show that Vpu specifically affects Toll pathway activation by inhibiting Cactus degradation. Given the conservation of the Toll/nuclear factor-kappa B (NF-kappa B) signalling pathways between flies and mammals, our results suggest a function for Vpu in the inhibition of host NF-kappa B-mediated innate immune defences and provide a powerful genetic approach for studying Vpu inhibition of NF-kappa B signalling in vivo.

HIV-1 Vpu sequesters beta-transducin repeat-containing protein (betaTrCP) in the cytoplasm and provokes the accumulation of beta-catenin and other SCFbetaTrCP substrates.

The human immunodeficiency virus type 1 Vpu protein acts as an adaptor for the proteasomal degradation of CD4 by recruiting CD4 and beta-transducin repeat-containing protein (betaTrCP), the receptor component of the multisubunit SCF-betaTrCP E3 ubiquitin ligase complex. We showed that the expression of a Vpu-green fluorescent fusion protein prevented the proteosomal degradation of betaTrCP substrates such as beta-catenin, IkappaBalpha, and ATF4, which are normally directly targeted to the proteasome for degradation. Beta-catenin was translocated into the nucleus, whereas the tumor necrosis factor-induced nuclear translocation of NFkappaB was impaired. Beta-catenin was also up-regulated in cells producing Vpu+ human immunodeficiency virus type 1 but not in cells producing Vpu-deficient viruses. The overexpression of ATF4 also provoked accumulation of beta-catenin, but to a lower level than that resulting from the expression of Vpu. Finally, the expression of Vpu induces the exclusion of betaTrCP from the nucleus. These data suggest that Vpu is a strong competitive inhibitor of betaTrCP that impairs the degradation of SCFbetaTrCP substrates as long as Vpu has an intact phosphorylation motif and can bind to betaTrCP.