Anabelle Decottignies

Complete inventory of the yeast ABC proteins.

The complete sequence of the yeast genome predicts the existence of 29 proteins belonging to the ubiquitous ATP-binding cassette (ABC) super-family. Using binary comparison, phylogenetic classification and detection of conserved amino acid residues, the yeast ABC proteins have been classified in a total of six clusters, including ten subclusters of distinct predicted topology and presumed distinct function. Study of the yeast ABC proteins provides insight into the physiological function and biochemical mechanisms of their human homologues, such as those involved in cystic fibrosis, adrenoleukodystrophy, Zellweger syndrome, multidrug resistance and the antiviral activity of interferons.

ATPase and multidrug transport activities of the overexpressed yeast ABC protein Yor1p.

The Saccharomyces cerevisiae genome encodes 15 full-size ATPbinding cassette transporters (ABC), of whichPDR5, SNQ2, and YOR1 are known to be regulated by the transcription factors Pdr1p and Pdr3p (pleiotropic drug resistance). We have identified two new ABC transporter-encoding genes,PDR10 and PDR15, which were up-regulated by thePDR1–3 mutation. These genes, as well as four other ABC transporter-encoding genes, were deleted in order to study the properties of Yor1p. The PDR1–3 gain-of-function mutant was then used to overproduce Yor1p up to 10% of the total plasma membrane proteins. Overexpressed Yor1p was photolabeled by [γ-32P]2′,3′-O-(2,4,6-trinitrophenyl)-8-azido-ATP (K 0.5 = 45 μm) and inhibited by ATP (K D = 0.3 mm) in plasma membranes. Solubilization and partial purification on sucrose gradient allowed to detect significant Yor1p ATP hydrolysis activity (∼100 nmol of Pi·min−1·mg−1). This activity was phospholipid-dependent and sensitive to low concentrations of vanadate (I50 = 0.3 μm) and oligomycin (I50 = 8.5 μg/ml). In vivo, we observed a correlation between the amount of Yor1p in the plasma membrane and the level of resistance to oligomycin. We also demonstrated that Yor1p drives an energy-dependent, proton uncoupler-insensitive, cellular extrusion of rhodamine B. Furthermore, cells lacking both Yor1p and Pdr5p (but not Snq2p) showed increased accumulation of the fluorescent derivative of 1-myristoyl-2-[6-(NBD)aminocaproyl]phosphatidylethanolamine. Despite their different topologies, both Yor1p and Pdr5p mediated the ATP-dependent translocation of similar drugs and phospholipids across the yeast cell membrane. Both ABC transporters exhibit ATP hydrolysis in vitro, but Pdr5p ATPase activity is about 15 times higher than that of Yor1p, which may indicate mechanistic or regulatory differences between the two enzymes.

Functional Expression of Candida albicans Drug Efflux Pump Cdr1p in a Saccharomyces cerevisiae Strain Deficient in Membrane Transporters

ABSTRACT Analysis of the transport functions of individualCandida albicans plasma membrane drug efflux pumps is hampered by the multitude of endogenous transporters. We have stably expressed C. albicans Cdr1p, the major pump implicated in multiple-drug-resistance phenotypes, from the genomicPDR5 locus in a Saccharomyces cerevisiae mutant (AD1-8u−) from which seven major transporters of the ATP-binding cassette (ABC) family have been deleted. High-level expression of Cdr1p, under the control of the S. cerevisiae PDR5 promoter and driven by S. cerevisiae Pdr1p transcriptional regulator mutation pdr1-3, was demonstrated by increased levels of mRNA transcription, increased levels of nucleoside triphosphatase activity, and immunodetection in plasma membrane fractions. S. cerevisiae AD1-8u− was hypersensitive to azole antifungals (the MICs at which 80% of cells were inhibited [MIC80s] were 0.625 μg/ml for fluconazole, <0.016 μg/ml for ketoconazole, and <0.016 μg/ml for itraconazole), whereas the strain (AD1002) that overexpressed C. albicans Cdr1p was resistant to azoles (MIC80s of fluconazole, ketoconazole, and itraconazole, 30, 0.5, and 4 μg/ml, respectively). Drug resistance correlated with energy-dependent drug efflux. AD1002 demonstrated resistance to a variety of structurally unrelated chemicals which are potential drug pump substrates. The controlled overexpression of C. albicansCdr1p in an S. cerevisiae background deficient in other pumps allows the functional analysis of pumping specificity and mechanisms of a major ABC transporter involved in drug efflux from an important human pathogen.

Telomere length regulates TERRA levels through increased trimethylation of telomeric H3K9 and HP1α

Gene silencing by the repressive telomeric chromatin environment, referred to as telomere position effect (TPE), has been well characterized in yeast and depends on telomere length. However, proof of its existence at native human chromosome ends has remained elusive, mainly owing to the paucity of genes near telomeres. The discovery of TERRAs, the telomeric noncoding RNAs transcribed from subtelomeric promoters, paved the way to probing for telomere-length impact on physiological TPE. Using cell lines of various origins, we show that telomere elongation consistently represses TERRA expression. Repression is mediated by increased trimethylated H3K9 density at telomeres and by heterochromatin protein HP1α, with no detectable spreading of the marks beyond the telomeric tract, restricting human TPE to telomere transcription. Our data further support the existence of a negative-feedback mechanism in which longer TERRA molecules repress their own transcription upon telomere elongation.

Chemosensitization of Fluconazole Resistance in Saccharomyces cerevisiae and Pathogenic Fungi by a d-Octapeptide Derivative

ABSTRACT Hyperexpression of the Saccharomyces cerevisiae multidrug ATP-binding cassette (ABC) transporter Pdr5p was driven by the pdr1-3 mutation in the Pdr1p transcriptional regulator in a strain (AD/PDR5+) with deletions of five other ABC-type multidrug efflux pumps. The strain had high-level fluconazole (FLC) resistance (MIC, 600 μg ml−1), and plasma membrane fractions showed oligomycin-sensitive ATPase activity up to fivefold higher than that shown by fractions from an isogenic PDR5-null mutant (FLC MIC, 0.94 μg ml−1). In vitro inhibition of the Pdr5p ATPase activity and chemosensitization of cells to FLC allowed the systematic screening of a 1.8-million-member designer d-octapeptide combinatorial library for surface-active Pdr5p antagonists with modest toxicity against yeast cells. Library deconvolution identified the 4-methoxy-2,3,6-trimethylbenzensulfonyl-substituted d-octapeptide KN20 as a potent Pdr5p ATPase inhibitor (concentration of drug causing 50% inhibition of enzyme activity [IC50], 4 μM) which chemosensitized AD/PDR5+ to FLC, itraconazole, and ketoconazole. It also inhibited the ATPase activity of other ABC transporters, such as Candida albicans Cdr1p (IC50, 30 μM) and Cdr2p (IC50, 2 μM), and chemosensitized clinical isolates of pathogenic Candida species and S. cerevisiae strains that heterologously hyperexpressed either ABC-type multidrug efflux pumps, the C. albicans major facilitator superfamily-type drug transporter BenRp, or the FLC drug target lanosterol 14α-demethylase (Erg11p). Although KN20 also inhibited the S. cerevisiae plasma membrane proton pump Pma1p (IC50, 1 μM), the peptide concentrations required for chemosensitization made yeast cells permeable to rhodamine 6G. KN20 therefore appears to indirectly chemosensitize cells to FLC by a nonlethal permeabilization of the fungal plasma membrane.

Binding of Cdc48p to a ubiquitin-related UBX domain from novel yeast proteins involved in intracellular proteolysis and sporulation.

The Cdc48/p97 AAA‐ATPase functions in membrane fusion and ubiquitin‐dependent protein degradation. Here, we show that, in yeast, Cdc48p interacts with three novel proteins, Cuil–3p, which contain a conserved ubiquitin‐related (UBX) domain. Cui2p and Cui3p are closely related, interact with each other, and are localized at the perinuclear membrane. Cdc48p binds directly the UBX domain of Cui3p in vitro. Multiple deletions of the CUI1, CUI2 and CUI3 genes confer deficiency in sporulation and degradation of model ubiquitin–protein fusions. The Cuil–3 proteins were also found to interact with Ufd3p, a WD repeat protein known to associate with Cdc48p. Together, these results indicate that the Cuil–3 proteins form complexes that are components of the ubiquitin–proteasome system. Copyright

Casein Kinase I-dependent Phosphorylation and Stability of the Yeast Multidrug Transporter Pdr5p

The pleiotropic drug resistance protein, Pdr5p, is an ATP-binding cassette transporter of the plasma membrane ofSaccharomyces cerevisiae. Overexpression of Pdr5p results in increased cell resistance to a variety of cytotoxic compounds, a phenotype reminiscent of the multiple drug resistance seen in tumor cells. Pdr5p and two other yeast ATP-binding cassette transporters, Snq2p and Yor1p, were found to be phosphorylated on serine residuesin vitro. Mutations in the plasma membrane-bound casein kinase I isoforms, Yck1p and Yck2p, abolished Pdr5p phosphorylation and modified the multiple drug resistance profile. We showed Pdr5p to be ubiquitylated when overexpressed. However, instability of Pdr5p was only seen in Yck1p- and Yck2p-deficient strains, in which it was degraded in the vacuole via a Pep4p-dependent mechanism. Our results suggest that casein kinase I activity is required for membrane trafficking of Pdr5p to the cell surface. In the absence of functional Yck1p and Yck2p, Pdr5p is transported to the vacuole for degradation.

Subtelomeric DNA hypomethylation is not required for telomeric sister chromatid exchanges in ALT cells.

Most human tumor cells acquire immortality by activating the expression of telomerase, a ribonucleoprotein that maintains stable telomere lengths at chromosome ends throughout cell divisions. Other tumors use an alternative mechanism of telomere lengthening (ALT), characterized by high frequencies of telomeric sister chromatid exchanges (T-SCEs). Mechanisms of ALT activation are still poorly understood, but recent studies suggest that DNA hypomethylation of chromosome ends might contribute to the process by facilitating T-SCEs. Here, we show that ALT/T-SCEhigh tumor cells display low DNA-methylation levels at the D4Z4 and DNF92 subtelomeric sequences. Surprisingly, however, the same sequences retained high methylation levels in ALT/T-SCEhigh SV40-immortalized fibroblasts. Moreover, T-SCE rates were efficiently reduced by ectopic expression of active telomerase in ALT tumor cells, even though subtelomeric sequences remained hypomethylated. We also show that hypomethylation of subtelomeric sequences in ALT tumor cells is correlated with genome-wide hypomethylation of Alu repeats and pericentromeric Sat2 DNA sequences. Overall, this study suggests that, although subtelomeric DNA hypomethylation is often coincident with the ALT process in human tumor cells, it is not required for T-SCE.

A novel cancer-germline transcript carrying pro-metastatic miR-105 and TET-targeting miR-767 induced by DNA hypomethylation in tumors

Genome hypomethylation is a common epigenetic alteration in human tumors, where it often leads to aberrant activation of a group of germline-specific genes, commonly referred to as “cancer-germline” genes. The cellular functions and tumor promoting potential of these genes remain, however, largely uncertain. Here, we report identification of a novel cancer-germline transcript (CT-GABRA3) displaying DNA hypomethylation-dependent activation in various tumors, including melanoma and lung carcinoma. Importantly, CT-GABRA3 harbors a microRNA (miR-105), which has recently been identified as a promoter of cancer metastasis by its ability to weaken vascular endothelial barriers following exosomal secretion. CT-GABRA3 also carries a microRNA (miR-767) with predicted target sites in TET1 and TET3, two members of the ten-eleven-translocation family of tumor suppressor genes, which are involved in the conversion of 5-methylcytosines to 5-hydroxymethylcytosines (5hmC) in DNA. Decreased TET activity is a hallmark of cancer; here, we provide evidence that aberrant activation of miR-767 contributes to this phenomenon. We demonstrate that miR-767 represses TET1/3 mRNA and protein expression and regulates genomic 5hmC levels. Additionally, we show that high CT-GABRA3 transcription correlates with reduced TET1 mRNA levels in vivo in lung tumors. Together, our study identified a cancer-germline gene that produces microRNAs with oncogenic potential. Moreover, our data indicate that DNA hypomethylation in tumors can contribute to reduced 5hmC levels via activation of a TET-targeting microRNA.

ABCB1 1199G>A genetic polymorphism (Rs2229109) influences the intracellular accumulation of tacrolimus in HEK293 and K562 recombinant cell lines.

Objective ATP-binding cassette, subfamily B, member 1 (ABCB1) transporter, or P-glycoprotein, is an efflux protein implicated in the absorption and the distribution of various compounds, including tacrolimus and cyclosporine A. In vivo studies suggest an association between the ABCB1 1199G>A single nucleotide polymorphism (SNP) and tacrolimus intracellular accumulation. The aim of the present experimental study was to clarify in vitro the impact of the coding ABCB1 1199G>A SNP on ABCB1 transport activity towards both immunosuppressive drugs. Method Two recombinant cell lines, i.e. Human Embryonic Kidney (HEK293) and Human Myelogenous Leukemia (K562) cells, overexpressing ABCB1 carrying either the wild-type allele (1199G) or its mutated counterpart (1199A), were generated. The impact of the 1199G>A SNP on ABCB1 activity towards rhodamine (Rh123), doxorubicin, vinblastine, tacrolimus and cyclosporine A was assessed by accumulation, cytotoxicity and/or kinetic experiments. Results Tacrolimus accumulation was strongly decreased in cells overexpressing the wild-type protein (1199G) compared to control cells, confirming the ability of ABCB1 to transport tacrolimus. By contrast, overexpression of the variant protein (1199A) had nearly no effect on tacrolimus intracellular accumulation whatever the model used and the concentration tested. Unlike tacrolimus, our results also indicate that cyclosporine A, Rh123 and doxorubicin are transported in a similar extent by the wild-type and variant ABCB1 proteins while the variant protein seems to be more efficient for the transport of vinblastine. Conclusion ABCB1 encoded by the 1199G wild-type allele transports more efficiently tacrolimus in comparison to the 1199A variant protein. This observation indicates that the amino-acid substitution (Ser400Asn) encoded by the 1199A allele drastically decreases the ability of ABCB1 to drive the efflux of tacrolimus in a substrate-specific manner, in agreement with our previously published clinical data. Our study emphasizes the importance of the ABCB1 1199G>A polymorphism for ABCB1 activity and its potential to explain differences in drug response.