Bypassing Mendel's First Law: Transmission Ratio Distortion in Mammals.

Mendel's law of segregation states that the two alleles at a diploid locus should be transmitted equally to the progeny. A genetic segregation distortion, also referred to as transmission ratio distortion (TRD), is a statistically significant deviation from this rule. TRD has been observed in several mammal species and may be due to different biological mechanisms occurring at diverse time points ranging from gamete formation to lethality at post-natal stages. In this review, we describe examples of TRD and their possible mechanisms in mammals based on current knowledge. We first focus on the differences between TRD in male and female gametogenesis in the house mouse, in which some of the most well studied TRD systems have been characterized. We then describe known TRD in other mammals, with a special focus on the farmed species and in the peculiar common shrew species. Finally, we discuss TRD in human diseases. Thus far, to our knowledge, this is the first time that such description is proposed. This review will help better comprehend the processes involved in TRD. A better understanding of these molecular mechanisms will imply a better comprehension of their impact on fertility and on genome evolution. In turn, this should allow for better genetic counseling and lead to better care for human families.

Identification of 8-Hydroxyquinoline Derivatives That Decrease Cystathionine Beta Synthase (CBS) Activity.

CBS encodes a pyridoxal 5'-phosphate-dependent enzyme that catalyses the condensation of homocysteine and serine to form cystathionine. Due to its implication in some cancers and in the cognitive pathophysiology of Down syndrome, the identification of pharmacological inhibitors of this enzyme is urgently required. However, thus far, attempts to identify such molecules have only led to the identification of compounds with low potency and limited selectivity. We consequently developed an original, yeast-based screening method that identified three FDA-approved drugs of the 8-hydroxyquinoline family: clioquinol, chloroxine and nitroxoline. These molecules reduce CBS enzymatic activity in different cellular models, proving that the molecular mechanisms involved in yeast phenotypic rescue are conserved in mammalian cells. A combination of genetic and chemical biology approaches also revealed the importance of copper and zinc intracellular levels in the regulation of CBS enzymatic activity-copper promoting CBS activity and zinc inhibiting its activity. Taken together, these results indicate that our effective screening approach identified three new potent CBS inhibitors and provides new findings for the regulation of CBS activity, which is crucial to develop new therapies for CBS-related human disorders.

A homozygous splice variant in ATP5PO, disrupts mitochondrial complex V function and causes Leigh syndrome in two unrelated families.

Mitochondrial complex V plays an important role in oxidative phosphorylation by catalyzing the generation of ATP. Most complex V subunits are nuclear encoded and not yet associated with recognized Mendelian disorders. Using exome sequencing, we identified a rare homozygous splice variant (c.87+3A>G) in ATP5PO, the complex V subunit which encodes the oligomycin sensitivity conferring protein, in three individuals from two unrelated families, with clinical suspicion of a mitochondrial disorder. These individuals had a similar, severe infantile and often lethal multi-systemic disorder that included hypotonia, developmental delay, hypertrophic cardiomyopathy, progressive epileptic encephalopathy, progressive cerebral atrophy, and white matter abnormalities on brain MRI consistent with Leigh syndrome. cDNA studies showed a predominant shortened transcript with skipping of exon 2 and low levels of the normal full-length transcript. Fibroblasts from the affected individuals demonstrated decreased ATP5PO protein, defective assembly of complex V with markedly reduced amounts of peripheral stalk proteins, and complex V hydrolytic activity. Further, expression of human ATP5PO cDNA without exon 2 (hATP5PO-∆ex2) in yeast cells deleted for yATP5 (ATP5PO homolog) was unable to rescue growth on media which requires oxidative phosphorylation when compared to the wild type construct (hATP5PO-WT), indicating that exon 2 deletion leads to a non-functional protein. Collectively, our findings support the pathogenicity of the ATP5PO c.87+3A>G variant, which significantly reduces but does not eliminate complex V activity. These data along with the recent report of an affected individual with ATP5PO variants, add to the evidence that rare biallelic variants in ATP5PO result in defective complex V assembly, function and are associated with Leigh syndrome.

New insights into the regulation of Cystathionine beta synthase (CBS), an enzyme involved in intellectual deficiency in Down syndrome.

Down syndrome (DS), the most frequent chromosomic aberration, results from the presence of an extra copy of chromosome 21. The identification of genes which overexpression contributes to intellectual disability (ID) in DS is important to understand the pathophysiological mechanisms involved and develop new pharmacological therapies. In particular, gene dosage of Dual specificity tyrosine phosphorylation Regulated Kinase 1A (DYRK1A) and of Cystathionine beta synthase (CBS) are crucial for cognitive function. As these two enzymes have lately been the main targets for therapeutic research on ID, we sought to decipher the genetic relationship between them. We also used a combination of genetic and drug screenings using a cellular model overexpressing CYS4, the homolog of CBS in Saccharomyces cerevisiae, to get further insights into the molecular mechanisms involved in the regulation of CBS activity. We showed that overexpression of YAK1, the homolog of DYRK1A in yeast, increased CYS4 activity whereas GSK3ß was identified as a genetic suppressor of CBS. In addition, analysis of the signaling pathways targeted by the drugs identified through the yeast-based pharmacological screening, and confirmed using human HepG2 cells, emphasized the importance of Akt/GSK3ß and NF-κB pathways into the regulation of CBS activity and expression. Taken together, these data provide further understanding into the regulation of CBS and in particular into the genetic relationship between DYRK1A and CBS through the Akt/GSK3ß and NF-κB pathways, which should help develop more effective therapies to reduce cognitive deficits in people with DS.

Anti-prion Drugs Targeting the Protein Folding Activity of the Ribosome Reduce PABPN1 Aggregation.

Prion diseases are caused by the propagation of PrPSc, the pathological conformation of the PrPC prion protein. The molecular mechanisms underlying PrPSc propagation are still unsolved and no therapeutic solution is currently available. We thus sought to identify new anti-prion molecules and found that flunarizine inhibited PrPSc propagation in cell culture and significantly prolonged survival of prion-infected mice. Using an in silico therapeutic repositioning approach based on similarities with flunarizine chemical structure, we tested azelastine, duloxetine, ebastine, loperamide and metixene and showed that they all have an anti-prion activity. Like flunarizine, these marketed drugs reduced PrPSc propagation in cell culture and in mouse cerebellum organotypic slice culture, and inhibited the protein folding activity of the ribosome (PFAR). Strikingly, some of these drugs were also able to alleviate phenotypes due to PABPN1 nuclear aggregation in cell and Drosophila models of oculopharyngeal muscular dystrophy (OPMD). These data emphasize the therapeutic potential of anti-PFAR drugs for neurodegenerative and neuromuscular proteinopathies.

p53, A Victim of the Prion Fashion.

Identified in the late 1970s as an oncogene, a driving force leading to tumor development, p53 turned out to be a key tumor suppressor gene. Now p53 is considered a master gene regulating the transcription of over 3000 target genes and controlling a remarkable number of cellular functions. The elevated prevalence of p53 mutations in human cancers has led to a recurring questioning about the roles of mutant p53 proteins and their functional consequences. Both mutants and isoforms of p53 have been attributed dominant-negative and gain of function properties among which is the ability to form amyloid aggregates and behave in a prion-like manner. This report challenges the ongoing "prion p53" hypothesis by reviewing evidence of p53 behavior in light of our current knowledge regarding amyloid proteins, prionoids and prions.

Novel cationic bis(acylhydrazones) as modulators of Epstein-Barr virus immune evasion acting through disruption of interaction between nucleolin and G-quadruplexes of EBNA1 mRNA.

The oncogenic Epstein-Barr virus (EBV) evades the immune system through limiting the expression of its highly antigenic and essential genome maintenance protein, EBNA1, to the minimal level to ensure viral genome replication, thereby also minimizing the production of EBNA1-derived antigenic peptides. This regulation is based on inhibition of translation of the virally-encoded EBNA1 mRNA, and involves the interaction of host protein nucleolin (NCL) with G-quadruplex (G4) structures that form in the glycine-alanine repeat (GAr)-encoding sequence of the EBNA1 mRNA. Ligands that bind to these G4-RNA can prevent their interaction with NCL, leading to disinhibition of EBNA1 expression and antigen presentation, thereby interfering with the immune evasion of EBNA1 and therefore of EBV (M.J. Lista et al., Nature Commun., 2017, 8, 16043). In this work, we synthesized and studied a series of 20 cationic bis(acylhydrazone) derivatives designed as G4 ligands. The in vitro evaluation showed that most derivatives based on central pyridine (Py), naphthyridine (Naph) or phenanthroline (Phen) units were efficient G4 binders, in contrast to their pyrimidine (Pym) counterparts, which were poor G4 binders due to a significantly different molecular geometry. The influence of lateral heterocyclic units (N-substituted pyridinium or quinolinium residues) on G4-binding properties was also investigated. Two novel compounds, namely PyDH2 and PhenDH2, used at a 5 µM concentration, were able to significantly enhance EBNA1 expression in H1299 cells in a GAr-dependent manner, while being significantly less toxic than the prototype drug PhenDC3 (GI50 > 50 µM). Antigen presentation, RNA pull-down and proximity ligation assays confirmed that the effect of both drugs was related to the disruption of NCL-EBNA1 mRNA interaction and the subsequent promotion of GAr-restricted antigen presentation. Our work provides a novel modular scaffold for the development of G-quadruplex-targeting drugs acting through interference with G4-protein interaction.

Pharmacological modulation of the ER stress response ameliorates oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is a rare late onset genetic disease leading to ptosis, dysphagia and proximal limb muscles at later stages. A short abnormal (GCN) triplet expansion in the polyA-binding protein nuclear 1 (PABPN1) gene leads to PABPN1-containing aggregates in the muscles of OPMD patients. Here we demonstrate that treating mice with guanabenz acetate (GA), an FDA-approved antihypertensive drug, reduces the size and number of nuclear aggregates, improves muscle force, protects myofibers from the pathology-derived turnover and decreases fibrosis. GA targets various cell processes, including the unfolded protein response (UPR), which acts to attenuate endoplasmic reticulum (ER) stress. We demonstrate that GA increases both the phosphorylation of the eukaryotic translation initiation factor 2α subunit and the splicing of Xbp1, key components of the UPR. Altogether these data show that modulation of protein folding regulation is beneficial for OPMD and promote the further development of GA or its derivatives for treatment of OPMD in humans. Furthermore, they support the recent evidences that treating ER stress could be therapeutically relevant in other more common proteinopathies.

A yeast model for the mechanism of the Epstein-Barr virus immune evasion identifies a new therapeutic target to interfere with the virus stealthiness.

The oncogenic Epstein-Barr virus (EBV) evades the immune system but has an Achilles heel: its genome maintenance protein EBNA1. Indeed, EBNA1 is essential for viral genome replication and maintenance but also highly antigenic. Hence, EBV evolved a system in which the glycine-alanine repeat (GAr) of EBNA1 limits the translation of its own mRNA at a minimal level to ensure its essential function thereby, at the same time, minimizing immune recognition. Defining intervention points where to interfere with EBNA1 immune evasion is an important step to trigger an immune response against EBV-carrying cancers. Thanks to a yeast-based assay that recapitulates all the aspects of EBNA1 self-limitation of expression, a recent study by Lista et al. [Nature Communications (2017) 7, 435-444] has uncovered the role of the host cell nucleolin (NCL) in this process via a direct interaction of this protein with G-quadruplexes (G4) formed in GAr-encoding sequence of EBNA1 mRNA. In addition, the G4 ligand PhenDC3 prevents NCL binding on EBNA1 mRNA and reverses GAr-mediated repression of translation and antigen presentation. This shows that the NCL-EBNA1 mRNA interaction is a relevant therapeutic target to unveil EBV-carrying cancers to the immune system and that the yeast model can be successfully used for uncovering drugs and host factors that interfere with EBV stealthiness.

p53, p63 and p73 in the wonderland of S. cerevisiae.

Since its discovery in 1979, p53 has been on the forefront of cancer research. It is considered a master gene of cancer suppression and is found mutated in around 50% of all human tumors. In addition, the progressive identification of p53-related transcription factors p63 and p73 as well as their multiple isoforms have added further layers of complexity to an already dense network. Among the numerous models used to unravel the p53 family mysteries, S. cerevisiae has been particularly useful. This seemingly naive model allows the expression of a functional human p53 and thus the assessment of p53 intrinsic transcriptional activity. The aim of this article is to review the various contributions that the budding yeast has made to the understanding of p53, p63 and p73 biology and to envision new possible directions for yeast-based assays in the field of cancer as well as other p53-family-related diseases.

Nucleolin directly mediates Epstein-Barr virus immune evasion through binding to G-quadruplexes of EBNA1 mRNA.

The oncogenic Epstein-Barr virus (EBV) evades the immune system but has an Achilles heel: its genome maintenance protein EBNA1, which is essential for viral genome maintenance but highly antigenic. EBV has seemingly evolved a system in which the mRNA sequence encoding the glycine-alanine repeats (GAr) of the EBNA1 protein limits its expression to the minimal level necessary for function while minimizing immune recognition. Here, we identify nucleolin (NCL) as a host factor required for this process via a direct interaction with G-quadruplexes formed in GAr-encoding mRNA sequence. Overexpression of NCL enhances GAr-based inhibition of EBNA1 protein expression, whereas its downregulation relieves the suppression of both expression and antigen presentation. Moreover, the G-quadruplex ligand PhenDC3 prevents NCL binding to EBNA1 mRNA and reverses GAr-mediated repression of EBNA1 expression and antigen presentation. Hence the NCL-EBNA1 mRNA interaction is a relevant therapeutic target to trigger an immune response against EBV-carrying cancers.

The double life of the ribosome: When its protein folding activity supports prion propagation.

It is no longer necessary to demonstrate that ribosome is the central machinery of protein synthesis. But it is less known that it is also key player of the protein folding process through another conserved function: the protein folding activity of the ribosome (PFAR). This ribozyme activity, discovered more than 2 decades ago, depends upon the domain V of the large rRNA within the large subunit of the ribosome. Surprisingly, we discovered that anti-prion compounds are also potent PFAR inhibitors, highlighting an unexpected link between PFAR and prion propagation. In this review, we discuss the ancestral origin of PFAR in the light of the ancient RNA world hypothesis. We also consider how this ribosomal activity fits into the landscape of cellular protein chaperones involved in the appearance and propagation of prions and other amyloids in mammals. Finally, we examine how drugs targeting the protein folding activity of the ribosome could be active against mammalian prion and other protein aggregation-based diseases, making PFAR a promising therapeutic target for various human protein misfolding diseases.

The dominant-negative interplay between p53, p63 and p73: A family affair.

The tumor suppression activity of p53 is frequently impaired in cancers even when a wild-type copy of the gene is still present, suggesting that a dominant-negative effect is exerted by some of p53 mutants and isoforms. p63 and p73, which are related to p53, have also been reported to be subjected to a similar loss of function, suggesting that a dominant-negative interplay might happen between p53, p63 and p73. However, to which extent p53 hotspot mutants and isoforms of p53, p63 and p73 are able to interfere with the tumor suppressive activity of their siblings as well as the underlying mechanisms remain undeciphered. Using yeast, we showed that a dominant-negative effect is widely spread within the p53/p63/p73 family as all p53 loss-of-function hotspot mutants and several of the isoforms of p53 and p73 tested exhibit a dominant-negative potential. In addition, we found that this dominant-negative effect over p53 wild-type is based on tetramer poisoning through the formation of inactive hetero-tetramers and does not rely on a prion-like mechanism contrary to what has been previously suggested. We also showed that mutant p53-R175H gains the ability to inhibit p63 and p73 activity by a mechanism that is only partially based on tetramerization.

Protein Folding Activity of the Ribosome is involved in Yeast Prion Propagation.

6AP and GA are potent inhibitors of yeast and mammalian prions and also specific inhibitors of PFAR, the protein-folding activity borne by domain V of the large rRNA of the large subunit of the ribosome. We therefore explored the link between PFAR and yeast prion [PSI(+)] using both PFAR-enriched mutants and site-directed methylation. We demonstrate that PFAR is involved in propagation and de novo formation of [PSI(+)]. PFAR and the yeast heat-shock protein Hsp104 partially compensate each other for [PSI(+)] propagation. Our data also provide insight into new functions for the ribosome in basal thermotolerance and heat-shocked protein refolding. PFAR is thus an evolutionarily conserved cell component implicated in the prion life cycle, and we propose that it could be a potential therapeutic target for human protein misfolding diseases.

The long-lasting love affair between the budding yeast Saccharomyces cerevisiae and the Epstein-Barr virus.

The Epstein-Barr gammaherpesvirus (EBV) is the first oncogenic virus discovered in human. Indeed, EBV has been known for more than 50 years to be tightly associated with certain human cancers. As such, EBV has been the subject of extensive studies aiming at deciphering various aspects of its biological cycle, ranging from the regulation of its genome replication and maintenance to the induction of its lytic cycle, including the mechanisms that allow its immune evasion or that are related to its tumorogenicity. For more than 30 years the budding yeast Saccharomyces cerevisiae has fruitfully contributed to a number of these studies. The aim of this article is to review the various aspects of EBV biology for which yeast has been instrumental, and to propose new possible applications for these yeast-based assays, as well as the creation of further yeast models dedicated to EBV. This review article illustrates the tremendous potential of S. cerevisiae in integrated chemobiological approaches for the biomedical research.

Epstein-Barr virus-encoded EBNA1 and ZEBRA: targets for therapeutic strategies against EBV-carrying cancers.

The EBV-encoded EBNA1 was first discovered 40 years ago, approximately 10 years after the presence of EBV had been demonstrated in Burkitt's lymphoma cells. It took another 10 years before the functions of EBNA1 in maintaining the viral genome were revealed, and it has since been shown to be an essential viral factor expressed in all EBV-carrying cells. Apart from serving to maintain the viral episome and to control viral replication and gene expression, EBNA1 also harbours a cis-acting mechanism that allows virus-carrying host cells to evade the immune system. This relates to a particular glycine-alanine repeat (GAr) within EBNA1 that has the capacity to suppress antigen presentation to the major histocompatibility complex (MHC) class I pathway. We discuss the role of the GAr sequence at the level of mRNA translation initiation, rather than at the protein level, as at least part of the mechanism to avoid MHC presentation. Interfering with this mechanism has become the focus of the development of immune-based therapies against EBV-carrying cancers, and some lead compounds that affect translation of GAr-carrying mRNAs have been identified. In addition, we describe the EBV-encoded ZEBRA factor and the switch from the latent to the lytic cycle as an alternative virus-specific target for treating EBV-carrying cancers. Understanding the molecular mechanisms of how EBNA1 and ZEBRA interfere with cellular pathways not only opens new therapeutic approaches but continues to reveal new cell-biological insights on the interplay between host and virus. This review is a tale of discoveries relating to how EBNA1 and ZEBRA have emerged as targets for specific cancer therapies against EBV-carrying diseases, and serves as an illustration of how mRNA translation can play roles in future immune-based strategies to target viral disease.

[Chemobiology at happy hour: yeast as a model for pharmacological screening]. / Chémobiologie à l'happy hour - La levure comme modèle de criblage pharmacologique.

Since its discovery and description by Louis Pasteur, the budding yeast Saccharomyces cerevisiae, which was used for thousands of years for alcoholic fermentation and as a leavening agent, has become a popular model system in biology. One of the reasons for this popularity is the strong conservation from yeast to human of most of the pathways controlling cell growth and fate. In addition, at least 30 % of human genes involved in diseases have a functional homolog in yeast. Hence, yeast is now widely used for modelling and deciphering physiopathological mechanisms as well as for developing pharmacological approaches like phenotype-based drug screening. Three examples of such yeast-based chemobiological studies are presented.

Mitochondrial protein sorting as a therapeutic target for ATP synthase disorders.

Mitochondrial diseases are systemic, prevalent and often fatal; yet treatments remain scarce. Identifying molecular intervention points that can be therapeutically targeted remains a major challenge, which we confronted via a screening assay we developed. Using yeast models of mitochondrial ATP synthase disorders, we screened a drug repurposing library, and applied genomic and biochemical techniques to identify pathways of interest. Here we demonstrate that modulating the sorting of nuclear-encoded proteins into mitochondria, mediated by the TIM23 complex, proves therapeutic in both yeast and patient-derived cells exhibiting ATP synthase deficiency. Targeting TIM23-dependent protein sorting improves an array of phenotypes associated with ATP synthase disorders, including biogenesis and activity of the oxidative phosphorylation machinery. Our study establishes mitochondrial protein sorting as an intervention point for ATP synthase disorders, and because of the central role of this pathway in mitochondrial biogenesis, it holds broad value for the treatment of mitochondrial diseases.

Structure-activity relationship study around guanabenz identifies two derivatives retaining antiprion activity but having lost α2-adrenergic receptor agonistic activity.

Guanabenz (GA) is an orally active α2-adrenergic agonist that has been used for many years for the treatment of hypertension. We recently described that GA is also active against both yeast and mammalian prions in an α2-adrenergic receptor-independent manner. These data suggest that this side-activity of GA could be explored for the treatment of prion-based diseases and other amyloid-based disorders. In this perspective, the potent antihypertensive activity of GA happens to be an annoying side-effect that could limit its use. In order to get rid of GA agonist activity at α2-adrenergic receptors, we performed a structure-activity relationship study around GA based on changes of the chlorine positions on the benzene moiety and then on the modifications of the guanidine group. Hence, we identified the two derivatives 6 and 7 that still possess a potent antiprion activity but were totally devoid of any agonist activity at α2-adrenergic receptors. Similarly to GA, 6 and 7 were also able to inhibit the protein folding activity of the ribosome (PFAR) which has been suggested to be involved in prion appearance/maintenance. Therefore, these two GA derivatives are worth being considered as drug candidates.

Evaluation of the antiprion activity of 6-aminophenanthridines and related heterocycles.

Series of 6-aminophenanthridines and related heterocyclic compounds such as benzonaphtyridines were prepared. Reduction of one of the three aromatic rings was also performed. The compounds were first tested for their antiprion activity in a previously described yeast-based colourimetric prion assay. The most potent derivatives were then assayed ex vivo against the mammalian prion PrP(Sc) in a cell-based assay. Several of the new compounds were found more potent than the parent lead 6-aminophenanthridine. The most promising compounds against yeast and mammalian prions were 8-azido-6-aminophenanthridine (3m), and 7,10-dihydrophenanthridin-6-amine (14). In the mammalian cell-based assay, the IC50 of these two compounds were around 5 µM and 1.8 µM, respectively.