An allosteric inhibitor of the human Cdc34 ubiquitin-conjugating enzyme.

In the ubiquitin-proteasome system (UPS), E2 enzymes mediate the conjugation of ubiquitin to substrates and thereby control protein stability and interactions. The E2 enzyme hCdc34 catalyzes the ubiquitination of hundreds of proteins in conjunction with the cullin-RING (CRL) superfamily of E3 enzymes. We identified a small molecule termed CC0651 that selectively inhibits hCdc34. Structure determination revealed that CC0651 inserts into a cryptic binding pocket on hCdc34 distant from the catalytic site, causing subtle but wholesale displacement of E2 secondary structural elements. CC0651 analogs inhibited proliferation of human cancer cell lines and caused accumulation of the SCF(Skp2) substrate p27(Kip1). CC0651 does not affect hCdc34 interactions with E1 or E3 enzymes or the formation of the ubiquitin thioester but instead interferes with the discharge of ubiquitin to acceptor lysine residues. E2 enzymes are thus susceptible to noncatalytic site inhibition and may represent a viable class of drug target in the UPS.

Documents d'information et outils d'aide à la décision pour le dépistage du cancer du sein.

CONTEXT: In France, breast cancer has been the most commonly diagnosed and the most lethal cancer in women. The risk-benefit ratio of organized breast cancer screening has been the focus of much scientific controversy, especially about overdiagnosis. A citizen conference and some scientific organizations have asked for a better education for women. The aim of this study was to analyze the numerous decision support tools and other information media, both in form and in substance. METHODS: A review of the existing publications has been done by 4 researchers between 2006 and 2016. Original articles included, in French or in English, focused on information media or decision aid tools for organized screening without any restriction on the type of studies. RESULTS: The 69 articles included in this review have shown that a better education of the patients was not related to an increase in the will to take part or in the actual participation in screening. The information media (pamphlet, video…) does not appear to have any influence on those ratios. The participants like having a discussion with a trained interviewer. In a third of the studies, the population was included on ethnic and social criteria. 8 studies focused on overdiagnosis. CONCLUSION: This review underlines the importance of repeated interviews, as a support for shared decision making. Specific interactions (such as patient navigator) could help to reduce social health inequalities. The patients should be informed about the current uncertainty regarding the ratio of overdiagnosis.

[Information media and decision support tools for cancer screening.] / Documents d'information et outils d'aide à la décision pour le dépistage du cancer du sein.

CONTEXT: In France, breast cancer has been the most commonly diagnosed and the most lethal cancer in women. The risk-benefit ratio of organized breast cancer screening has been the focus of much scientific controversy, especially about overdiagnosis. A citizen conference and some scientific organizations have asked for a better education for women. The aim of this study was to analyze the numerous decision support tools and other information media, both in form and in substance. METHODS: A review of the existing publications has been done by 4 researchers between 2006 and 2016. Original articles included, in French or in English, focused on information media or decision aid tools for organized screening without any restriction on the type of studies. RESULTS: The 69 articles included in this review have shown that a better education of the patients was not related to an increase in the will to take part or in the actual participation in screening. The information media (pamphlet, video…) does not appear to have any influence on those ratios. The participants like having a discussion with a trained interviewer. In a third of the studies, the population was included on ethnic and social criteria. 8 studies focused on overdiagnosis. CONCLUSION: This review underlines the importance of repeated interviews, as a support for shared decision making. Specific interactions (such as patient navigator) could help to reduce social health inequalities. The patients should be informed about the current uncertainty regarding the ratio of overdiagnosis.

Discovery of Benzodiazepine-Based Inhibitors of the E2 Enzyme UBCH10 from a Cell-Based p21 Degradation Screen.

p21Cip1 (p21) is a universal cyclin-dependent kinase (CDK) inhibitor that halts cell proliferation and tumor growth by multiple mechanisms. The expression of p21 is often downregulated in cancer cells as a result of the loss of function of transcriptional activators, such as p53, or the increased degradation rate of the protein. To identify small molecules that block the ubiquitin-mediated degradation of p21 as a future avenue for cancer drug discovery, we have screened a compound library using a cell-based reporter assay of p21 degradation. This led to the identification of a benzodiazepine series of molecules that induce the accumulation of p21 in cells. Using a chemical proteomic strategy, we identified the ubiquitin-conjugating enzyme UBCH10 as a cellular target of this benzodiazepine series. We show that an optimized benzodiazepine analogue inhibits UBCH10 ubiquitin-conjugating activity and substrate proteolysis by the anaphase-promoting complex.

Iron activates in vivo DNA binding of Schizosaccharomyces pombe transcription factor Fep1 through its amino-terminal region.

In Schizosaccharomyces pombe, the iron sensor Fep1 mediates the transcriptional repression of iron transport genes in response to high concentrations of iron. On the other hand, fep1(+) expression is downregulated under conditions of iron starvation by the CCAAT-binding factor Php4. In this study, we created a fep1Delta php4Delta double mutant strain where expression of fep1(+) was disengaged from its iron limitation-dependent repression by Php4 to examine the effects of iron on constitutively expressed functional fep1(+)-GFP and TAP-fep1(+) alleles and their gene products. In these cells, Fep1-green fluorescent protein was invariably localized in the nucleus under both iron-limiting and iron-replete conditions. Using chromatin immunoprecipitation assays, we found that Fep1 is associated with iron-responsive promoters in vivo. Chromatin binding was iron dependent, with a loss of binding observed in the presence of low iron. Functional dissection of the protein revealed that the N-terminal 241-residue segment that includes two consensus Cys(2)/Cys(2)-type zinc finger motifs and a Cys-rich region is required for optimal promoter occupancy by Fep1. Within this segment, a minimal module encompassing amino acids 60 to 241 is sufficient for iron-dependent chromatin binding. Using yeast one-hybrid analysis, we showed that the replacement of the repression domain of Fep1 by fusing the activation domain of VP16 to the chromatin-binding fragment of amino acids 1 to 241 of Fep1 converts the protein from an iron-dependent repressor into an iron-dependent transcriptional activator. Thus, the repression function of Fep1 can be replaced with that of a transcriptional activation function without the loss of its iron-dependent DNA-binding activity.

Fep1 represses expression of the fission yeast Schizosaccharomyces pombe siderophore-iron transport system.

When iron repletes, Schizosaccharomyces pombe cells repress transcription of genes encoding components involved in the reductive iron transport system. Fep1 mediates this transcriptional control by interacting specifically with GATA-type cis-acting elements. To further investigate the role that Fep1 plays in iron homeostasis, we searched for additional Fep1-regulated genes. We found that str1+ is subject to negative transcriptional regulation, which is exerted through binding of Fep1 to a single GATA element in the str1+ promoter. Introduction of str1+ into a Saccharomyces cerevisiae fet3Delta arn1-4Delta strain led to assimilation of iron from ferrichrome, revealing that Str1 functions as a siderophore-iron transporter in S.pombe. We also identified two additional target genes of Fep1, named str2+ and str3+. We demonstrate that the str1+, str2+ and str3+ genes share a common promoter element, 5'-(A/T)GATAA-3'. We found that the N-terminal 241 residue segment of Fep1 expressed in Escherichia coli specifically interacts with the 5'-(A/T)GATAA-3' element present in each of these promoters. Consistent with this, constitutive high level str1+, str2+ and str3+ gene expression was observed in a fep1Delta mutant strain. Taken together, these results demonstrate that Fep1 occupies a central role in coordinating transcriptional regulation of genes encoding components of the reductive and non-reductive iron transport systems in fission yeast.

Iron homeostasis in the fission yeast Schizosaccharomyces pombe.

Schizosaccharomyces pombe has acquisition processes for iron, an essential nutrient. One pathway consists to produce, excrete, and capture siderophore-iron complexes. A second pathway requires enzymatic reduction of ferric iron at the cell surface prior to uptake by a permease-oxidase complex. Genes encoding proteins involved in iron assimilation are transcriptionally regulated as a function of iron availability. Under high iron conditions, the GATA-type regulator Fep1 represses the expression of iron uptake genes. The repressor function of Fep1 requires the presence of the Tup11 or Tup12 transcriptional co-repressor. Under low iron conditions, two regulatory mechanisms occur. First, the iron transport genes are highly induced. Second, there is a transcription factor cascade implicating the heteromeric CCAAT-binding complex that turns off a set of genes encoding iron-utilizing proteins, presumably to avoid a futile expenditure of energy in producing iron-using proteins that lack the necessary cofactor to function. Thus, collectively, these regulatory responses to variations in iron concentrations ensure that iron is present within cells for essential biochemical reactions, yet prevent the accumulation of iron or iron-using proteins to deleterious levels.

Expression of Candida albicans Sfu1 in fission yeast complements the loss of the iron-regulatory transcription factor Fep1 and requires Tup co-repressors.

The opportunistic pathogenic yeast Candida albicans contains a gene which encodes a putative member of the iron-regulatory GATA factor protein family. This protein, referred to as suppressor of ferric uptake (Sfu1), has two Cys(2)/Cys(2)-type zinc finger domains separated by a conserved Cys-rich region. In Schizosaccharomyces pombe, the GATA-type transcription factor Fe protein 1 (Fep1) represses target gene expression when iron levels exceed those needed by the cell. To ascertain the functional similarity between Sfu1 and Fep1, the C. albicans Sfu1 was expressed in Sz. pombe cells lacking the endogenous fep1(+) gene. We determined that Sfu1 is capable of suppressing iron-related phenotypes of fep1Delta mutant cells. Using a functional SFU1-GFP fusion allele, the Sfu1 protein was localized to the nucleus under both iron-replete and iron-starved conditions. Sfu1 effectively regulated the expression of genes encoding components of the reductive and non-reductive iron transport systems. Furthermore, the iron-responsive regulation mediated by Sfu1 was GATA-dependent. The N-terminal 250 amino acid segment of Sfu1 expressed in and purified from Escherichia coli specifically associated with the hexanucleotide sequence AGATAA in an iron-dependent manner. On the other hand, expression of the full-length C. albicans Sfu1 in Sz. pombe fep1Delta tup11Delta tup12Delta triple mutant cells failed to repress target gene expression under conditions of high iron concentration. Using two-hybrid analysis, we demonstrated that Tup11 and Tup12 physically interacted with Sfu1. Taken together, these results reveal a remarkable functional conservation between Sfu1 from C. albicans and Fep1 from Sz. pombe in their ability to sense excess iron and respond by repressing target gene transcription.

A transcription factor cascade involving Fep1 and the CCAAT-binding factor Php4 regulates gene expression in response to iron deficiency in the fission yeast Schizosaccharomyces pombe.

We have identified genes encoding candidate proteins involved in iron storage (pcl1+), the tricarboxylic acid cycle (sdh4+), and iron-sulfur cluster assembly (isa1+) that are negatively regulated in response to iron deprivation. Promoter deletion and site-directed mutagenesis permitted identification of a new cis-regulatory element in the promoter region of the pcl1+ gene. This cis-acting regulatory sequence containing the pentanucleotide sequence CCAAT is responsible for transcriptional repression of pcl1+ under low iron supply conditions. In Schizosaccharomyces pombe, the CCAAT-binding factor is a heteromeric DNA-binding complex that contains three subunits, designated Php2, Php3, and Php5. Inactivation of the php2+ locus negatively affects the transcriptional competency of pcl1+. A fourth subunit, designated Php4, is not essential for the transcriptional activation of target genes under basal and iron-replete conditions. We demonstrate that, in response to iron-limiting conditions, Php4 is required for down-regulation of pcl1+, sdh4+, and isa1+ mRNA levels. In vivo RNase protection studies reveal that the expression of php4+ is negatively regulated by iron and that this regulated expression requires a functional fep1+ gene. The results of these studies reveal that Fep1 represses php4+ expression in response to iron. In contrast, when iron is scarce, Fep1 becomes inactive and php4+ is expressed to act as a regulatory subunit of the CCAAT-binding factor that is required to block pcl1+, sdh4+, and isa1+ gene transcription.

Functional characterization of the iron-regulatory transcription factor Fep1 from Schizosaccharomyces pombe.

In response to excess iron, Schizosaccharomyces pombe cells repress transcription of genes encoding components involved in iron uptake through the Fep1 transcription factor. Fep1 mediates this control by interacting with the consensus sequence 5'-(A/T)GATAA-3', found in iron-dependent promoters. In this report, we show that Fep1 localizes to the nucleus under both iron-replete and iron-starved conditions. The Fep1 DNA binding domain (amino acids 1-241) contains two GATA-type zinc finger motifs. Although we determine that the Fep1 C-terminal zinc finger (ZF2) is essential for DNA binding, we show that the N-terminal zinc finger (ZF1) enhances DNA binding affinity approximately 5-fold. Between the two zinc finger motifs of Fep1 resides an invariant amino acid sequence, denoted the Cys-rich region (amino acids 68-94), in which four highly conserved Cys residues are found. Cells harboring mutant alleles in which two or more of the conserved Cys residues were substituted by alanine exhibited elevated fio1(+) mRNA levels. We determine that the dissociation constant for the resulting complex between each of the Cys mutants and the sequence 5'-(A/T)GATAA-3' reflects a much lower affinity that correlates with failure to repress fio1(+) gene expression. Deletion analysis identified two heptad repeats (amino acids 522-536) within the C-terminal region of Fep1 that are necessary and sufficient to mediate Fep1 dimerization. Moreover, mutations that impair dimerization also negatively affect transcriptional repression. Together these findings reveal several novel features of Fep1, a non-canonical GATA factor required for iron homeostasis.

The Schizosaccharomyces pombe corepressor Tup11 interacts with the iron-responsive transcription factor Fep1.

The Schizosaccharomyces pombe fep1(+) gene encodes a GATA transcription factor that represses the expression of iron transport genes in response to elevated iron concentrations. This transcriptional response is altered only in strains harboring a combined deletion of both tup11(+) and tup12(+) genes. This suggests that Tup11 is capable of negatively regulating iron transport gene expression in the absence of Tup12 and vice versa. The tup11(+)- and tup12(+)-encoded proteins resemble the Saccharomyces cerevisiae Tup1 corepressor. Using yeast two-hybrid analysis we show that Tup11 and Fep1 physically interact with each other. The C-terminal region from amino acids 242 to 564 of Fep1 is required for interaction with Tup11. Within this region, a minimal domain encompassing amino acids 405-541 was sufficient for Tup11-Fep1 association. Deletion mapping analysis revealed that the WD40-repeat sequence motifs of Tup11 are necessary for its interaction with Fep1. Analysis of Tup11 mutants with single amino acid substitutions in the WD40 repeats suggested that the Fep1 transcription factor interacts with a putative flat upper surface on the predicted beta-propeller structure of this motif. Further analysis by in vivo coimmunoprecipitation showed that Tup11 and Fep1 are physically associated. In vitro pull-down experiments further verified a direct interaction between the Fep1 C terminus and the Tup11 C-terminal WD40 repeat domain. Taken together, these results describe the first example of a physical interaction between a corepressor and an iron-sensing factor controlling the expression of iron uptake genes.

Fep1, an iron sensor regulating iron transporter gene expression in Schizosaccharomyces pombe.

Schizosaccharomyces pombe cells acquire iron under high affinity conditions through the action of a cell surface ferric reductase encoded by the frp1(+) gene and a two-component iron-transporting complex encoded by the fip1(+) and fio1(+) genes. When cells are grown in the presence of iron, transcription of all three genes is blocked. A conserved regulatory element, 5'-(A/T)GATAA-3', located upstream of the frp1(+), fip1(+), and fio1(+) genes, is necessary for iron repression. We have cloned a novel gene, termed fep1(+), which encodes an iron-sensing transcription factor. Binding studies reveal that the putative DNA binding domain of Fep1 expressed as a fusion protein in Escherichia coli specifically interacts with the 5'-(A/T)GATAA-3' sequence in an iron-dependent manner. In a fep1 Delta mutant strain, the fio1(+) gene is highly expressed and is unregulated by iron. Furthermore, the fep1 Delta mutation increases activity of the cell surface iron reductase and renders cells hypersensitive to the iron-dependent free radical generator phleomycin. Mutations in the transcriptional co-repressors tup11(+) and tup12(+) are phenocopies to fep1(+). Indeed, strains with both tup11 Delta and tup12 Delta deletions fail to sense iron. This suggests that in the presence of iron and Fep1, the Tup11 and Tup12 proteins may act as co-repressors for down-regulation of genes encoding components of the reductive iron transport machinery.