Olga Charlat - Academia.edu (original) (raw)
Papers by Olga Charlat
Genomics, 1997
During the recent cloning of the mouseLystgene we developed both a high-resolution genetic map an... more During the recent cloning of the mouseLystgene we developed both a high-resolution genetic map and a complete YAC and BAC contig of theLystcritical region on mouse Chromosome 13. We also report the mapping of the human homologue of the mouseLystgene (LYST) to 1q43. These data are consistent withLYSTbeing the gene for the human Chediak–Higashi Syndrome and strengthen the synteny relationship between MMU13 and human 1q43.
Molecular Cell, 2015
Tumor suppressors ZNRF3 and RNF43 inhibit Wnt signaling through promoting degradation of Wnt core... more Tumor suppressors ZNRF3 and RNF43 inhibit Wnt signaling through promoting degradation of Wnt coreceptors Frizzled (FZD) and LRP6, and this activity is counteracted by stem cell growth factor R-spondin. The mechanism by which ZNRF3 and RNF43 recognize Wnt receptors remains unclear. Here we uncover an unexpected role of Dishevelled (DVL), a positive Wnt regulator, in promoting Wnt receptor degradation. DVL knockout cells have significantly increased cell surface levels of FZD and LRP6. DVL is required for ZNRF3/RNF43-mediated ubiquitination and degradation of FZD. Physical interaction with DVL is essential for the Wnt inhibitory activity of ZNRF3/RNF43. Binding of FZD through the DEP domain of DVL is required for DVL-mediated downregulation of FZD. Fusion of the DEP domain to ZNRF3/RNF43 overcomes their DVL dependency to downregulate FZD. Our study reveals DVL as a dual function adaptor to recruit negative regulators ZNRF3/RNF43 to Wnt receptors to ensure proper control of pathway activity.
PloS one, 2012
The Wnt/β-catenin signaling pathbway controls many important biological processes. R-Spondin (RSP... more The Wnt/β-catenin signaling pathbway controls many important biological processes. R-Spondin (RSPO) proteins are a family of secreted molecules that strongly potentiate Wnt/β-catenin signaling, however, the molecular mechanism of RSPO action is not yet fully understood. We performed an unbiased siRNA screen to identify molecules specifically required for RSPO, but not Wnt, induced β-catenin signaling. From this screen, we identified LGR4, then an orphan G protein-coupled receptor (GPCR), as the cognate receptor of RSPO. Depletion of LGR4 completely abolished RSPO-induced β-catenin signaling. The loss of LGR4 could be compensated by overexpression of LGR5, suggesting that LGR4 and LGR5 are functional homologs. We further demonstrated that RSPO binds to the extracellular domain of LGR4 and LGR5, and that overexpression of LGR4 strongly sensitizes cells to RSPO-activated β-catenin signaling. Supporting the physiological significance of RSPO-LGR4 interaction, Lgr4-/- crypt cultures fail...
Proceedings of the National Academy of Sciences, 2013
A growing number of agents targeting ligand-induced Wnt/β-catenin signaling are being developed f... more A growing number of agents targeting ligand-induced Wnt/β-catenin signaling are being developed for cancer therapy. However, clinical development of these molecules is challenging because of the lack of a genetic strategy to identify human tumors dependent on ligand-induced Wnt/β-catenin signaling. Ubiquitin E3 ligase ring finger 43 (RNF43) has been suggested as a negative regulator of Wnt signaling, and mutations of RNF43 have been identified in various tumors, including cystic pancreatic tumors. However, loss of function study of RNF43 in cell culture has not been conducted, and the functional significance of RNF43 mutations in cancer is unknown. Here, we show that RNF43 inhibits Wnt/β-catenin signaling by reducing the membrane level of Frizzled in pancreatic cancer cells, serving as a negative feedback mechanism. Inhibition of endogenous Wnt/β-catenin signaling increased the cell surface level of Frizzled. A panel of 39 pancreatic cancer cell lines was tested for Wnt dependency using LGK974, a selective Porcupine inhibitor being examined in a phase 1 clinical trial. Strikingly, all LGK974-sensitive lines carried inactivating mutations of RNF43. Inhibition of Wnt secretion, depletion of β-catenin, or expression of wild-type RNF43 blocked proliferation of RNF43 mutant but not RNF43-wild-type pancreatic cancer cells.
Proceedings of the National Academy of Sciences, 2010
Disregulated Wnt/β-catenin signaling has been linked to various human diseases, including cancers... more Disregulated Wnt/β-catenin signaling has been linked to various human diseases, including cancers. Inhibitors of oncogenic Wnt signaling are likely to have a therapeutic effect in cancers. LRP5 and LRP6 are closely related membrane coreceptors for Wnt proteins. Using a phage-display library, we identified anti-LRP6 antibodies that either inhibit or enhance Wnt signaling. Two classes of LRP6 antagonistic antibodies were discovered: one class specifically inhibits Wnt proteins represented by Wnt1, whereas the second class specifically inhibits Wnt proteins represented by Wnt3a. Epitope-mapping experiments indicated that Wnt1 class-specific antibodies bind to the first propeller and Wnt3a class-specific antibodies bind to the third propeller of LRP6, suggesting that Wnt1and Wnt3a-class proteins interact with distinct LRP6 propeller domains. This conclusion is further supported by the structural functional analysis of LRP5/6 and the finding that the Wnt antagonist Sclerostin interacts with the first propeller of LRP5/6 and preferentially inhibits the Wnt1-class proteins. We also show that Wnt1 or Wnt3a class-specific anti-LRP6 antibodies specifically block growth of MMTV-Wnt1 or MMTV-Wnt3 xenografts in vivo. Therapeutic application of these antibodies could be limited without knowing the type of Wnt proteins expressed in cancers. This is further complicated by our finding that bivalent LRP6 antibodies sensitize cells to the nonblocked class of Wnt proteins. The generation of a biparatopic LRP6 antibody blocks both Wnt1-and Wnt3a-mediated signaling without showing agonistic activity. Our studies provide insights into Wnt-induced LRP5/6 activation and show the potential utility of LRP6 antibodies in Wntdriven cancer.
Proceedings of the National Academy of Sciences, 2001
We have searched for genes predisposing to bipolar disorder (BP) by studying individuals with the... more We have searched for genes predisposing to bipolar disorder (BP) by studying individuals with the most extreme form of the affected phenotype, BP-I, ascertained from the genetically isolated population of the Central Valley of Costa Rica (CVCR). The results of a previous linkage analysis on two extended CVCR BP-I pedigrees, CR001 and CR004, and of linkage disequilibrium (LD) analyses of a CVCR population sample of BP-I patients implicated a candidate region on 18p11.3. We further investigated this region by creating a physical map and developing 4 new microsatellite and 26 single-nucleotide polymorphism markers for typing in the pedigree and population samples. We report the results of fine-scale association analyses in the population sample, as well as evaluation of haplotypes in pedigree CR001. Our results suggest a candidate region containing six genes but also highlight the complexities of LD mapping of common disorders.
Nature Cell Biology, 2011
The Wnt/β-catenin signalling pathway plays essential roles in embryonic development and adult tis... more The Wnt/β-catenin signalling pathway plays essential roles in embryonic development and adult tissue homeostasis, and deregulation of this pathway has been linked to cancer. Axin is a concentration-limiting component of the β-catenin destruction complex, and its stability is regulated by tankyrase. However, the molecular mechanism by which tankyrase-dependent poly(ADP-ribosyl)ation (PARsylation) is coupled to ubiquitylation and degradation of axin remains undefined. Here, we identify RNF146, a RING-domain E3 ubiquitin ligase, as a positive regulator of Wnt signalling. RNF146 promotes Wnt signalling by mediating tankyrase-dependent degradation of axin. Mechanistically, RNF146 directly interacts with poly(ADP-ribose) through its WWE domain, and promotes degradation of PARsylated proteins. Using proteomics approaches, we have identified BLZF1 and CASC3 as further substrates targeted by tankyrase and RNF146 for degradation. Thus, identification of RNF146 as a PARsylation-directed E3 ligase establishes a molecular paradigm that links tankyrase-dependent PARsylation to ubiquitylation. RNF146-dependent protein degradation may emerge as a major mechanism by which tankyrase exerts its function.
Nature, 2012
R-spondin proteins strongly potentiate Wnt signalling and function as stem-cell growth factors. D... more R-spondin proteins strongly potentiate Wnt signalling and function as stem-cell growth factors. Despite the biological and therapeutic significance, the molecular mechanism of R-spondin action remains unclear. Here we show that the cell-surface transmembrane E3 ubiquitin ligase zinc and ring finger 3 (ZNRF3) and its homologue ring finger 43 (RNF43) are negative feedback regulators of Wnt signalling. ZNRF3 is associated with the Wnt receptor complex, and inhibits Wnt signalling by promoting the turnover of frizzled and LRP6. Inhibition of ZNRF3 enhances Wnt/b-catenin signalling and disrupts Wnt/planar cell polarity signalling in vivo. Notably, R-spondin mimics ZNRF3 inhibition by increasing the membrane level of Wnt receptors. Mechanistically, R-spondin interacts with the extracellular domain of ZNRF3 and induces the association between ZNRF3 and LGR4, which results in membrane clearance of ZNRF3. These data suggest that R-spondin enhances Wnt signalling by inhibiting ZNRF3. Our study provides new mechanistic insights into the regulation of Wnt receptor turnover, and reveals ZNRF3 as a tractable target for therapeutic exploration.
Nature, 2009
The stability of the Wnt pathway transcription factor beta-catenin is tightly regulated by the mu... more The stability of the Wnt pathway transcription factor beta-catenin is tightly regulated by the multi-subunit destruction complex. Deregulated Wnt pathway activity has been implicated in many cancers, making this pathway an attractive target for anticancer therapies. However, the development of targeted Wnt pathway inhibitors has been hampered by the limited number of pathway components that are amenable to small molecule inhibition. Here, we used a chemical genetic screen to identify a small molecule, XAV939, which selectively inhibits beta-catenin-mediated transcription. XAV939 stimulates beta-catenin degradation by stabilizing axin, the concentration-limiting component of the destruction complex. Using a quantitative chemical proteomic approach, we discovered that XAV939 stabilizes axin by inhibiting the poly-ADP-ribosylating enzymes tankyrase 1 and tankyrase 2. Both tankyrase isoforms interact with a highly conserved domain of axin and stimulate its degradation through the ubiquitin-proteasome pathway. Thus, our study provides new mechanistic insights into the regulation of axin protein homeostasis and presents new avenues for targeted Wnt pathway therapies.
Molecular and Cellular Biology, 2000
The mouse tubby phenotype is characterized by maturity-onset obesity accompanied by retinal and c... more The mouse tubby phenotype is characterized by maturity-onset obesity accompanied by retinal and cochlear degeneration. A positional cloning effort to find the gene responsible for this phenotype led to the identification of tub, a member of a novel gene family of unknown function. A splice defect mutation in the 3 end of the tub gene, predicted to disrupt the C terminus of the Tub protein, has been implicated in the genesis of the tubby phenotype. It is not clear, however, whether the Tub mutant protein retains any biological activity, or perhaps has some dominant function, nor is it established that the tubby mutation is itself responsible for all of the observed tubby phenotypes. To address these questions, we generated tub-deficient mice and compared their phenotype to that of tubby mice. Our results demonstrate that tubby is a loss-of-function mutation of the tub gene and that loss of the tub gene is sufficient to give rise to the full spectrum of tubby phenotypes. We also demonstrate that loss of photoreceptors in the retina of tubby and tub-deficient mice occurs by apoptosis. In addition, we show that Tub protein expression is not significantly altered in the ob, db, or melanocortin 4 receptor-deficient mouse model of obesity.
Cell, 1996
The mutated gene responsible for the tubby obesity phenotype has been identified by positional cl... more The mutated gene responsible for the tubby obesity phenotype has been identified by positional cloning. A single base change within a splice donor site results in the incorrect retention of a single intron in the mature tub mRNA transcript. The consequence of this mutation is the substitution of the carboxy-terminal 44 amino acids with 24 intron-encoded amino acids. The normal transcript appears to be abundantly expressed in the hypothalamus, a region of the brain involved in body weight regulation. Variation in the relative abundance of alternative splice products is observed between inbred mouse strains and appears to correlate with an intron length polymorphism. This allele of tub is a candidate for a previously reported diet-induced obesity quantitative trait locus on mouse chromosome 7.
Genomics, 1997
Lyst bg8J 1 CAST/Ei) 1 DBA/2 Co-Lyst bg8J ]. The off-During the recent cloning of the mouse Lyst ... more Lyst bg8J 1 CAST/Ei) 1 DBA/2 Co-Lyst bg8J ]. The off-During the recent cloning of the mouse Lyst gene we spring from each of these backcrosses, which totaled developed both a high-resolution genetic map and a 1496, were analyzed by coat color to determine their complete YAC and BAC contig of the Lyst critical regenotype at the Lyst locus. Genomic DNA was made gion on mouse Chromosome 13. We also report the from a tail biopsy of each mouse and analyzed for mapping of the human homologue of the mouse Lyst multiple simple sequence length repeat polymorphisms gene (LYST) to 1q43. These data are consistent with (SSLP). Not all strain combinations were polymorphic LYST being the gene for the human Chediak-Higashi for all markers. The loci, nidogen (Nid) and Ras-like Syndrome and strengthen the synteny relationship beprotein 1 (Rasl1), were also genotyped in mice from
EMBO reports, 2013
R-spondin proteins sensitize cells to Wnt signalling and act as potent stem cell growth factors. ... more R-spondin proteins sensitize cells to Wnt signalling and act as potent stem cell growth factors. Various membrane proteins have been proposed as potential receptors of R-spondin, including LGR4/5, membrane E3 ubiquitin ligases ZNRF3/RNF43 and several others proteins. Here, we show that R-spondin interacts with ZNRF3/RNF43 and LGR4 through distinct motifs. Both LGR4 and ZNRF3 binding motifs are required for R-spondin-induced LGR4/ZNRF3 interaction, membrane clearance of ZNRF3 and activation of Wnt signalling. Importantly, Wnt-inhibitory activity of ZNRF3, but not of a ZNRF3 mutant with reduced affinity to R-spondin, can be strongly suppressed by R-spondin, suggesting that R-spondin primarily functions by binding and inhibiting ZNRF3. Together, our results support a dual receptor model of R-spondin action, where LGR4/5 serve as the engagement receptor whereas ZNRF3/RNF43 function as the effector receptor.
Cell, 1996
However, db/db mice fail to respond to recombinant Elizabeth A. Woolf, Xun Weng, Stephen J. Ellis... more However, db/db mice fail to respond to recombinant Elizabeth A. Woolf, Xun Weng, Stephen J. Ellis, leptin, again suggesting that db/db mice are defective Nathan D. Lakey, Janice Culpepper, Karen J. More, in leptin signal reception (Campfield et al., 1995; Halaas Roger E. Breitbart, Geoffrey M. Duyk, Robert I. Tepper, et al., 1995; Pelleymounter et al., 1995; Stephens et al., and Jay P. Morgenstern 1995). Millennium Pharmaceuticals, Incorporated Recently, we have identified a cDNA from mouse cho-640 Memorial Drive roid plexus that encodes a high affinity receptor for Cambridge, Massachusetts 02139 leptin, OB-R (Tartaglia et al., 1995). Genetic mapping of the gene encoding OB-R localized it within the 5.1 cM interval of mouse chromosome 4 that contains the db locus, suggesting it as an excellent candidate for the Summary db gene. However, sequencing within the coding region OB-R is a high affinity receptor for leptin, an important of OB-R cDNAs from db/db (the original allele on C57BL/ circulating signal for the regulation of body weight.
Cancer Chemotherapy and Pharmacology, 2003
Dihydropyrimidine dehydrogenase (DPD) and thymidylate synthase (TS) are key enzymes for predictin... more Dihydropyrimidine dehydrogenase (DPD) and thymidylate synthase (TS) are key enzymes for predicting the efficacy of 5-FU in the treatment of malignant tumors. However, 5-FU is not commonly commonly chosen for chemotherapeutic treatment of hepatocellular carcinoma (HCC) in practice. The aim of this study was to determine the activities of both DPD and TS in HCCs and corresponding liver parenchyma and to assess the correlation between the activities of these enzymes and clinicopathological features. The possibility of using 5-FU as a first-choice chemotherapeutic agent for HCC was also evaluated. The study material comprised 33 pairs of hepatocellular carcinoma and noncancerous liver samples. The DPD and TS activities were quantified by a radiometric enzymatic assay and a 5-fluoro-2'-deoxyuridine-5'- monophosphate (FdUMP) ligand-binding assay, respectively. Pathologically invasive HCCs tended to show higher DPD activity and lower TS activity with some exceptions. DPD activity was lower in the HCCs regardless of their clinical features than in the noncancerous liver parenchyma, whereas TS activity was generally lower in HCCs except for those with certain clinical features. HCCs with multiple nodules showed lower DPD activity and those with a diameter of more than 5 cm showed lower TS activity. In the noncancerous liver parenchyma, a gradual decrease in DPD activity and an increase in TS activity were associated with the age of the patient, liver damage and z-factor. Of 30 HCC samples, 10 exhibited comparatively low DPD and TS activity, and these could be considered 5-FU-sensitive HCC. DPD and TS activity may be affected by the clinicopathological status in both the HCC and the corresponding liver parenchyma. However, further investigation is necessary. Some HCC patients may be good candidates for 5-FU-based chemotherapy based on measurements of tumor levels of DPD and TS.
Journal of Medicinal Chemistry, 2012
The Wnt signaling pathway is critical to the regulation of key cellular processes. When deregulat... more The Wnt signaling pathway is critical to the regulation of key cellular processes. When deregulated, it has been shown to play a crucial role in the growth and progression of multiple human cancers. The identification of small molecule modulators of Wnt signaling has proven challenging, largely due to the relative paucity of druggable nodes in this pathway. Several recent publications have identified small molecule inhibitors of the Wnt pathway, and tankyrase (TNKS) inhibition has been demonstrated to antagonize Wnt signaling via axin stabilization. Herein, we report the early hit assessment of a series of compounds previously reported to antagonize Wnt signaling. We report the biophysical, computational characterization, structure-activity relationship, and physicochemical properties of a novel series of [1,2,4]triazol-3-ylsulfanylmethyl)-3-phenyl-[1,2,4]oxadiazole inhibitors of TNKS1 and 2. Furthermore, a cocrystal structure of compound 24 complexed to TNKS1 demonstrates an alternate binding mode for PARP family member proteins that does not involve interactions with the nicotinamide binding pocket.
Genomics, 1997
During the recent cloning of the mouseLystgene we developed both a high-resolution genetic map an... more During the recent cloning of the mouseLystgene we developed both a high-resolution genetic map and a complete YAC and BAC contig of theLystcritical region on mouse Chromosome 13. We also report the mapping of the human homologue of the mouseLystgene (LYST) to 1q43. These data are consistent withLYSTbeing the gene for the human Chediak–Higashi Syndrome and strengthen the synteny relationship between MMU13 and human 1q43.
Molecular Cell, 2015
Tumor suppressors ZNRF3 and RNF43 inhibit Wnt signaling through promoting degradation of Wnt core... more Tumor suppressors ZNRF3 and RNF43 inhibit Wnt signaling through promoting degradation of Wnt coreceptors Frizzled (FZD) and LRP6, and this activity is counteracted by stem cell growth factor R-spondin. The mechanism by which ZNRF3 and RNF43 recognize Wnt receptors remains unclear. Here we uncover an unexpected role of Dishevelled (DVL), a positive Wnt regulator, in promoting Wnt receptor degradation. DVL knockout cells have significantly increased cell surface levels of FZD and LRP6. DVL is required for ZNRF3/RNF43-mediated ubiquitination and degradation of FZD. Physical interaction with DVL is essential for the Wnt inhibitory activity of ZNRF3/RNF43. Binding of FZD through the DEP domain of DVL is required for DVL-mediated downregulation of FZD. Fusion of the DEP domain to ZNRF3/RNF43 overcomes their DVL dependency to downregulate FZD. Our study reveals DVL as a dual function adaptor to recruit negative regulators ZNRF3/RNF43 to Wnt receptors to ensure proper control of pathway activity.
PloS one, 2012
The Wnt/β-catenin signaling pathbway controls many important biological processes. R-Spondin (RSP... more The Wnt/β-catenin signaling pathbway controls many important biological processes. R-Spondin (RSPO) proteins are a family of secreted molecules that strongly potentiate Wnt/β-catenin signaling, however, the molecular mechanism of RSPO action is not yet fully understood. We performed an unbiased siRNA screen to identify molecules specifically required for RSPO, but not Wnt, induced β-catenin signaling. From this screen, we identified LGR4, then an orphan G protein-coupled receptor (GPCR), as the cognate receptor of RSPO. Depletion of LGR4 completely abolished RSPO-induced β-catenin signaling. The loss of LGR4 could be compensated by overexpression of LGR5, suggesting that LGR4 and LGR5 are functional homologs. We further demonstrated that RSPO binds to the extracellular domain of LGR4 and LGR5, and that overexpression of LGR4 strongly sensitizes cells to RSPO-activated β-catenin signaling. Supporting the physiological significance of RSPO-LGR4 interaction, Lgr4-/- crypt cultures fail...
Proceedings of the National Academy of Sciences, 2013
A growing number of agents targeting ligand-induced Wnt/β-catenin signaling are being developed f... more A growing number of agents targeting ligand-induced Wnt/β-catenin signaling are being developed for cancer therapy. However, clinical development of these molecules is challenging because of the lack of a genetic strategy to identify human tumors dependent on ligand-induced Wnt/β-catenin signaling. Ubiquitin E3 ligase ring finger 43 (RNF43) has been suggested as a negative regulator of Wnt signaling, and mutations of RNF43 have been identified in various tumors, including cystic pancreatic tumors. However, loss of function study of RNF43 in cell culture has not been conducted, and the functional significance of RNF43 mutations in cancer is unknown. Here, we show that RNF43 inhibits Wnt/β-catenin signaling by reducing the membrane level of Frizzled in pancreatic cancer cells, serving as a negative feedback mechanism. Inhibition of endogenous Wnt/β-catenin signaling increased the cell surface level of Frizzled. A panel of 39 pancreatic cancer cell lines was tested for Wnt dependency using LGK974, a selective Porcupine inhibitor being examined in a phase 1 clinical trial. Strikingly, all LGK974-sensitive lines carried inactivating mutations of RNF43. Inhibition of Wnt secretion, depletion of β-catenin, or expression of wild-type RNF43 blocked proliferation of RNF43 mutant but not RNF43-wild-type pancreatic cancer cells.
Proceedings of the National Academy of Sciences, 2010
Disregulated Wnt/β-catenin signaling has been linked to various human diseases, including cancers... more Disregulated Wnt/β-catenin signaling has been linked to various human diseases, including cancers. Inhibitors of oncogenic Wnt signaling are likely to have a therapeutic effect in cancers. LRP5 and LRP6 are closely related membrane coreceptors for Wnt proteins. Using a phage-display library, we identified anti-LRP6 antibodies that either inhibit or enhance Wnt signaling. Two classes of LRP6 antagonistic antibodies were discovered: one class specifically inhibits Wnt proteins represented by Wnt1, whereas the second class specifically inhibits Wnt proteins represented by Wnt3a. Epitope-mapping experiments indicated that Wnt1 class-specific antibodies bind to the first propeller and Wnt3a class-specific antibodies bind to the third propeller of LRP6, suggesting that Wnt1and Wnt3a-class proteins interact with distinct LRP6 propeller domains. This conclusion is further supported by the structural functional analysis of LRP5/6 and the finding that the Wnt antagonist Sclerostin interacts with the first propeller of LRP5/6 and preferentially inhibits the Wnt1-class proteins. We also show that Wnt1 or Wnt3a class-specific anti-LRP6 antibodies specifically block growth of MMTV-Wnt1 or MMTV-Wnt3 xenografts in vivo. Therapeutic application of these antibodies could be limited without knowing the type of Wnt proteins expressed in cancers. This is further complicated by our finding that bivalent LRP6 antibodies sensitize cells to the nonblocked class of Wnt proteins. The generation of a biparatopic LRP6 antibody blocks both Wnt1-and Wnt3a-mediated signaling without showing agonistic activity. Our studies provide insights into Wnt-induced LRP5/6 activation and show the potential utility of LRP6 antibodies in Wntdriven cancer.
Proceedings of the National Academy of Sciences, 2001
We have searched for genes predisposing to bipolar disorder (BP) by studying individuals with the... more We have searched for genes predisposing to bipolar disorder (BP) by studying individuals with the most extreme form of the affected phenotype, BP-I, ascertained from the genetically isolated population of the Central Valley of Costa Rica (CVCR). The results of a previous linkage analysis on two extended CVCR BP-I pedigrees, CR001 and CR004, and of linkage disequilibrium (LD) analyses of a CVCR population sample of BP-I patients implicated a candidate region on 18p11.3. We further investigated this region by creating a physical map and developing 4 new microsatellite and 26 single-nucleotide polymorphism markers for typing in the pedigree and population samples. We report the results of fine-scale association analyses in the population sample, as well as evaluation of haplotypes in pedigree CR001. Our results suggest a candidate region containing six genes but also highlight the complexities of LD mapping of common disorders.
Nature Cell Biology, 2011
The Wnt/β-catenin signalling pathway plays essential roles in embryonic development and adult tis... more The Wnt/β-catenin signalling pathway plays essential roles in embryonic development and adult tissue homeostasis, and deregulation of this pathway has been linked to cancer. Axin is a concentration-limiting component of the β-catenin destruction complex, and its stability is regulated by tankyrase. However, the molecular mechanism by which tankyrase-dependent poly(ADP-ribosyl)ation (PARsylation) is coupled to ubiquitylation and degradation of axin remains undefined. Here, we identify RNF146, a RING-domain E3 ubiquitin ligase, as a positive regulator of Wnt signalling. RNF146 promotes Wnt signalling by mediating tankyrase-dependent degradation of axin. Mechanistically, RNF146 directly interacts with poly(ADP-ribose) through its WWE domain, and promotes degradation of PARsylated proteins. Using proteomics approaches, we have identified BLZF1 and CASC3 as further substrates targeted by tankyrase and RNF146 for degradation. Thus, identification of RNF146 as a PARsylation-directed E3 ligase establishes a molecular paradigm that links tankyrase-dependent PARsylation to ubiquitylation. RNF146-dependent protein degradation may emerge as a major mechanism by which tankyrase exerts its function.
Nature, 2012
R-spondin proteins strongly potentiate Wnt signalling and function as stem-cell growth factors. D... more R-spondin proteins strongly potentiate Wnt signalling and function as stem-cell growth factors. Despite the biological and therapeutic significance, the molecular mechanism of R-spondin action remains unclear. Here we show that the cell-surface transmembrane E3 ubiquitin ligase zinc and ring finger 3 (ZNRF3) and its homologue ring finger 43 (RNF43) are negative feedback regulators of Wnt signalling. ZNRF3 is associated with the Wnt receptor complex, and inhibits Wnt signalling by promoting the turnover of frizzled and LRP6. Inhibition of ZNRF3 enhances Wnt/b-catenin signalling and disrupts Wnt/planar cell polarity signalling in vivo. Notably, R-spondin mimics ZNRF3 inhibition by increasing the membrane level of Wnt receptors. Mechanistically, R-spondin interacts with the extracellular domain of ZNRF3 and induces the association between ZNRF3 and LGR4, which results in membrane clearance of ZNRF3. These data suggest that R-spondin enhances Wnt signalling by inhibiting ZNRF3. Our study provides new mechanistic insights into the regulation of Wnt receptor turnover, and reveals ZNRF3 as a tractable target for therapeutic exploration.
Nature, 2009
The stability of the Wnt pathway transcription factor beta-catenin is tightly regulated by the mu... more The stability of the Wnt pathway transcription factor beta-catenin is tightly regulated by the multi-subunit destruction complex. Deregulated Wnt pathway activity has been implicated in many cancers, making this pathway an attractive target for anticancer therapies. However, the development of targeted Wnt pathway inhibitors has been hampered by the limited number of pathway components that are amenable to small molecule inhibition. Here, we used a chemical genetic screen to identify a small molecule, XAV939, which selectively inhibits beta-catenin-mediated transcription. XAV939 stimulates beta-catenin degradation by stabilizing axin, the concentration-limiting component of the destruction complex. Using a quantitative chemical proteomic approach, we discovered that XAV939 stabilizes axin by inhibiting the poly-ADP-ribosylating enzymes tankyrase 1 and tankyrase 2. Both tankyrase isoforms interact with a highly conserved domain of axin and stimulate its degradation through the ubiquitin-proteasome pathway. Thus, our study provides new mechanistic insights into the regulation of axin protein homeostasis and presents new avenues for targeted Wnt pathway therapies.
Molecular and Cellular Biology, 2000
The mouse tubby phenotype is characterized by maturity-onset obesity accompanied by retinal and c... more The mouse tubby phenotype is characterized by maturity-onset obesity accompanied by retinal and cochlear degeneration. A positional cloning effort to find the gene responsible for this phenotype led to the identification of tub, a member of a novel gene family of unknown function. A splice defect mutation in the 3 end of the tub gene, predicted to disrupt the C terminus of the Tub protein, has been implicated in the genesis of the tubby phenotype. It is not clear, however, whether the Tub mutant protein retains any biological activity, or perhaps has some dominant function, nor is it established that the tubby mutation is itself responsible for all of the observed tubby phenotypes. To address these questions, we generated tub-deficient mice and compared their phenotype to that of tubby mice. Our results demonstrate that tubby is a loss-of-function mutation of the tub gene and that loss of the tub gene is sufficient to give rise to the full spectrum of tubby phenotypes. We also demonstrate that loss of photoreceptors in the retina of tubby and tub-deficient mice occurs by apoptosis. In addition, we show that Tub protein expression is not significantly altered in the ob, db, or melanocortin 4 receptor-deficient mouse model of obesity.
Cell, 1996
The mutated gene responsible for the tubby obesity phenotype has been identified by positional cl... more The mutated gene responsible for the tubby obesity phenotype has been identified by positional cloning. A single base change within a splice donor site results in the incorrect retention of a single intron in the mature tub mRNA transcript. The consequence of this mutation is the substitution of the carboxy-terminal 44 amino acids with 24 intron-encoded amino acids. The normal transcript appears to be abundantly expressed in the hypothalamus, a region of the brain involved in body weight regulation. Variation in the relative abundance of alternative splice products is observed between inbred mouse strains and appears to correlate with an intron length polymorphism. This allele of tub is a candidate for a previously reported diet-induced obesity quantitative trait locus on mouse chromosome 7.
Genomics, 1997
Lyst bg8J 1 CAST/Ei) 1 DBA/2 Co-Lyst bg8J ]. The off-During the recent cloning of the mouse Lyst ... more Lyst bg8J 1 CAST/Ei) 1 DBA/2 Co-Lyst bg8J ]. The off-During the recent cloning of the mouse Lyst gene we spring from each of these backcrosses, which totaled developed both a high-resolution genetic map and a 1496, were analyzed by coat color to determine their complete YAC and BAC contig of the Lyst critical regenotype at the Lyst locus. Genomic DNA was made gion on mouse Chromosome 13. We also report the from a tail biopsy of each mouse and analyzed for mapping of the human homologue of the mouse Lyst multiple simple sequence length repeat polymorphisms gene (LYST) to 1q43. These data are consistent with (SSLP). Not all strain combinations were polymorphic LYST being the gene for the human Chediak-Higashi for all markers. The loci, nidogen (Nid) and Ras-like Syndrome and strengthen the synteny relationship beprotein 1 (Rasl1), were also genotyped in mice from
EMBO reports, 2013
R-spondin proteins sensitize cells to Wnt signalling and act as potent stem cell growth factors. ... more R-spondin proteins sensitize cells to Wnt signalling and act as potent stem cell growth factors. Various membrane proteins have been proposed as potential receptors of R-spondin, including LGR4/5, membrane E3 ubiquitin ligases ZNRF3/RNF43 and several others proteins. Here, we show that R-spondin interacts with ZNRF3/RNF43 and LGR4 through distinct motifs. Both LGR4 and ZNRF3 binding motifs are required for R-spondin-induced LGR4/ZNRF3 interaction, membrane clearance of ZNRF3 and activation of Wnt signalling. Importantly, Wnt-inhibitory activity of ZNRF3, but not of a ZNRF3 mutant with reduced affinity to R-spondin, can be strongly suppressed by R-spondin, suggesting that R-spondin primarily functions by binding and inhibiting ZNRF3. Together, our results support a dual receptor model of R-spondin action, where LGR4/5 serve as the engagement receptor whereas ZNRF3/RNF43 function as the effector receptor.
Cell, 1996
However, db/db mice fail to respond to recombinant Elizabeth A. Woolf, Xun Weng, Stephen J. Ellis... more However, db/db mice fail to respond to recombinant Elizabeth A. Woolf, Xun Weng, Stephen J. Ellis, leptin, again suggesting that db/db mice are defective Nathan D. Lakey, Janice Culpepper, Karen J. More, in leptin signal reception (Campfield et al., 1995; Halaas Roger E. Breitbart, Geoffrey M. Duyk, Robert I. Tepper, et al., 1995; Pelleymounter et al., 1995; Stephens et al., and Jay P. Morgenstern 1995). Millennium Pharmaceuticals, Incorporated Recently, we have identified a cDNA from mouse cho-640 Memorial Drive roid plexus that encodes a high affinity receptor for Cambridge, Massachusetts 02139 leptin, OB-R (Tartaglia et al., 1995). Genetic mapping of the gene encoding OB-R localized it within the 5.1 cM interval of mouse chromosome 4 that contains the db locus, suggesting it as an excellent candidate for the Summary db gene. However, sequencing within the coding region OB-R is a high affinity receptor for leptin, an important of OB-R cDNAs from db/db (the original allele on C57BL/ circulating signal for the regulation of body weight.
Cancer Chemotherapy and Pharmacology, 2003
Dihydropyrimidine dehydrogenase (DPD) and thymidylate synthase (TS) are key enzymes for predictin... more Dihydropyrimidine dehydrogenase (DPD) and thymidylate synthase (TS) are key enzymes for predicting the efficacy of 5-FU in the treatment of malignant tumors. However, 5-FU is not commonly commonly chosen for chemotherapeutic treatment of hepatocellular carcinoma (HCC) in practice. The aim of this study was to determine the activities of both DPD and TS in HCCs and corresponding liver parenchyma and to assess the correlation between the activities of these enzymes and clinicopathological features. The possibility of using 5-FU as a first-choice chemotherapeutic agent for HCC was also evaluated. The study material comprised 33 pairs of hepatocellular carcinoma and noncancerous liver samples. The DPD and TS activities were quantified by a radiometric enzymatic assay and a 5-fluoro-2'-deoxyuridine-5'- monophosphate (FdUMP) ligand-binding assay, respectively. Pathologically invasive HCCs tended to show higher DPD activity and lower TS activity with some exceptions. DPD activity was lower in the HCCs regardless of their clinical features than in the noncancerous liver parenchyma, whereas TS activity was generally lower in HCCs except for those with certain clinical features. HCCs with multiple nodules showed lower DPD activity and those with a diameter of more than 5 cm showed lower TS activity. In the noncancerous liver parenchyma, a gradual decrease in DPD activity and an increase in TS activity were associated with the age of the patient, liver damage and z-factor. Of 30 HCC samples, 10 exhibited comparatively low DPD and TS activity, and these could be considered 5-FU-sensitive HCC. DPD and TS activity may be affected by the clinicopathological status in both the HCC and the corresponding liver parenchyma. However, further investigation is necessary. Some HCC patients may be good candidates for 5-FU-based chemotherapy based on measurements of tumor levels of DPD and TS.
Journal of Medicinal Chemistry, 2012
The Wnt signaling pathway is critical to the regulation of key cellular processes. When deregulat... more The Wnt signaling pathway is critical to the regulation of key cellular processes. When deregulated, it has been shown to play a crucial role in the growth and progression of multiple human cancers. The identification of small molecule modulators of Wnt signaling has proven challenging, largely due to the relative paucity of druggable nodes in this pathway. Several recent publications have identified small molecule inhibitors of the Wnt pathway, and tankyrase (TNKS) inhibition has been demonstrated to antagonize Wnt signaling via axin stabilization. Herein, we report the early hit assessment of a series of compounds previously reported to antagonize Wnt signaling. We report the biophysical, computational characterization, structure-activity relationship, and physicochemical properties of a novel series of [1,2,4]triazol-3-ylsulfanylmethyl)-3-phenyl-[1,2,4]oxadiazole inhibitors of TNKS1 and 2. Furthermore, a cocrystal structure of compound 24 complexed to TNKS1 demonstrates an alternate binding mode for PARP family member proteins that does not involve interactions with the nicotinamide binding pocket.