- Email: [email protected]
Characterization of secreted aspartic proteases of Candida albicans
S110
Abstracts / Journal of Bioscience and Bioengineering 108 (2009) S96–S113
Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, Japan 1 and Department of Bioengineering, Graduate School of Engineering, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, Japan 2 With the success of the human genome project, the ability to rapidly convert gene information into the molecular function of their corresponding proteins is a significant advantage for both protein science and protein engineering (1). To obtain the information about the molecular function of target proteins in a high through-put manner, both the technologies to efficiently obtain target proteins in their native form and the methodologies to rapidly determine their function are required. In these proteomics studies, one of the most fundamental problems is the fragility of the protein structure, which is easy to unfold. Furthermore, unfolded proteins irreversibly form aggregates and permanently loss their function. Therefore, in the many process of proteomics studies such as protein production, protein separation, protein isolation and so on, irreversible unfolding and aggregation have been major bottlenecks. Thus, controlling the aggregation and folding of proteins in vitro is crucial for further progress in proteomic research area. In this presentation, we reports two topics about refolding/reactivation of protein by using small molecular additives: (1) successful development of small molecular additives for protein refolding by combinatorial approach (2), (2) a technology for reactivation of denatured enzymes in SDS-PAGE gel (3).
storage. To improve the stability of the enzymes, NP and XOD were freeze-dried with various glass-forming additives i.e., sucrose, sucrose-bovine serum albumin (BSA), sucrose-dextran (MW: 10400 Da), sucrose-polyethylene glycol (PEG: MW 600 Da), and sucrose-polyvinylpyrrolidone k30 (PVP). The samples were stored at 25 °C for up to 90 days, and the remaining activity of the enzymes was determined by using a UV-VIS spectrophotometer. Glass transition temperature (Tg) and moisture content of the samples were also investigated using differential scanning calorimetry and Karl Fisher coulometer, respectively. All additive-containing samples were amorphous solid. The sucrose– polymer formulations had higher Tg than that of the sucrose alone formulation. Furthermore, the crystallization temperatures of sucrose-polymer samples were higher than those of the sucrose samples. This means that the physical stability of amorphous sucrose is improved with the addition of polymer. The activity of NP and XOD was greatly protected by sucrose, however, the combination of sucrose and BSA improved the enzymes stability more effectively. On the other hand, sucrose–PEG, sucrose–dextran and sucrose–PVP did not have synergistic stabilizing effects. Sucrose-PEG protected the enzymes in the same degree with the sucrose alone formulation. Dextran and PVP seem to lower the stabilizing ability of sucrose on the dried enzymes. Reference 1. Wang, W.: Lyophilization and development of solid protein pharmaceuticals. Int. J. Pharm., 203, 1-60 (2000).
References doi:10.1016/j.jbiosc.2009.08.322 1. Middelberg A. P. J.: Preparative protein refolding. Trends Biotechnol, 2002;20: 437-443. 2. Yamaguchi S., Yamamoto E., Tsukiji S., and Nagamune T.: Successful control of aggregation and folding rates during refolding of denatured lysozyme by adding N-methylimidazolium cations with various N'-substituents. Biotechnol. Prog. 2008;24:402-408. 3. Yamamoto E., Yamaguchi S., and Nagamune T.: Effect of beta-cyclodextrin on the renaturation of enzymes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Anal. Biochem. 2008.
EP-P35 Characterization of secreted aspartic proteases of Candida albicans Wataru Aoki, Nao Kitahara, Hironobu Morisaka, Natsuko Miura, Kouichi Kuroda, and Mitsuyoshi Ueda
doi:10.1016/j.jbiosc.2009.08.321
Kyoto University, Kyoto, Japan EP-P34 Stabilization of xanthine oxidase and nucleoside phosphorylase enzyme mixture in dried glassy matrices Paveena Srirangsan,1 Kiyoshi Kawai,2 Naoko Hamada-Sato,3 and Toru Suzuki1 Department of Food Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan 1 Department of Biofunctional science and Technology, Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8528, Japan 2 and Course of Safety Management in Food Supply Chain, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan 3 Most of enzymes are sensitive to chemical and physical stresses, which cause them to lose their activity during processing and storage (1). Nucleoside phosphorylase (NP) and xanthine oxidase (XOD) are the enzymes, which have been widely used in biotechnology. Particularly, mixtures of them have been used in freshness testing sensor to evaluate fish freshness based on the change of nucleotides and nucleosides contents in fish meat. The enzymes, however, are unstable thermally and lose their activities during freeze-drying and
Recently, virulent fungi are running rampant in human beings, especially in immunocompromised patients. A representative of virulent fungi is Candida albicans. C. albicans is a dimorphic, opportunistic fungal pathogen and does not cause diseases in healthy people. However, C. albicans causes various diseases in people whose immuno-system has been damaged by AIDS, immune-suppressing drugs, or cancers. Notably, systemic infection of C. albicans often causes multiple organ failures, and is almost lethal. It is said that the mortality rate reaches around 70%. Patients have very limited remedies available, because there are a few effective drugs against candidiasis. Amphotericin B and fluconazole are the representative anti-fungal drugs, but these drugs have severe side effects such as renal dysfunction. Furthermore, drug-resistance fungi gradually are emerging. The pathogenicity of C. albicans largely depends on adhesion to epithelial cells, yeast-hypha transition, and extracellular proteases. In this study, we pay attention to extracellular proteases of C. albicans. C. albicans has ten secreted aspartic proteases (Saps) and each of them is an important pathogenic factor. Saccharomyces cerevisiae, a nonpathogenic close relative of C. albicans, does not have Sap homolog, and Sap deletion mutants of C. albicans shows less virulence. So Saps are considered as important virulent factors. It is known that Saps degrade epithelial adhesion molecules to penetrate into the interior, and disintegrate immunoproteins such as complements and IgG to
Abstracts / Journal of Bioscience and Bioengineering 108 (2009) S96–S113 escape from immune responses. However, substrate specificity of Saps has not been investigated in detail. In this study, we have clarified characteristics of Saps. Disclosure of the properties of these proteases will enable us to understand the pathogenicity of C. albicans. doi:10.1016/j.jbiosc.2009.08.323
EP-P36 Structural and functional characterization of glycerol kinase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 Ryuta Hokao,1 Yuichi Koga,1 Kazufumi Takano,1,2 and Shigenori Kanaya1 Osaka University, Suita city, Osaka, Japan 1 and CREST-JST, Suita city, Osaka, Japan 2 The purpose of this study is the analysis of structural and functional characters of glycerol kinase (GK) from the hyperthermophilic archaeon Thermococcus kodakaraensis (Tk-GK). Glycerol kinase catalyses the Mg,ATP-dependent phosphorylation of glycerol to produce glycerol 3-phosphate, which is a key enzyme of glycerol metabolism. In E. coli, GK (Ec-GK) exists in a dimer-tetramer equilibrium. Fructose 1,6-bisphosphate (FBP) selectively binds to the tetramer and inhibits Ec-GK activity by preventing interdomain motion. Unlike Ec-GK, Tk-GK does not exist as a tetramer in solution state, and it is not sensitive to FBP. According to the gel filtration chromatography, it was shown that Tk-GK exists as a dimer (110 kDa) in the absence of glycerol or presence of ATP, and both as a dimer and as a hexamer (330 kDa) in the presence of glycerol (mainly as a hexamer). This is the first report of the hexameric structure among GKs from various organisms. It may indicate that hexameric structure is induced by the conformational change with glycerol binding. In addition, we analyzed the interface of this hexameric structure by the mutational method. We will report the role of hexameric structure which affects the functions of Tk-GK. References 1. Koga Y., Katsumi R., You D.J., Matsumura H., Takano K., and Kanaya S.: Crystal structure of highly thermostable glycerol kinase from a hyperthermophilic archaeon in a dimeric form. FEBS J., 275, 2632-2643 (2008). 2. Katsumi R., Koga Y., You D.J., Matsumura H., Takano K., and Kanaya S.: Crystallization and preliminary X-ray diffraction study of glycerol kinase from the hyperthermophilic archaeon Thermococcus kodakaraensis. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun., 63(Pt 2),126-129 (2007).
doi:10.1016/j.jbiosc.2009.08.324
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a redox-based mechanism of action. However, specific cell surface proteins and pathways coupling extracellular TRX redox activity to cellular responses have not been identified so far. We first found that exogenous TRX not only scavenger but also effected intracellular proteins using proteomic approaches. Therefore, the effect of a thiolantioxidant protein TRX have appropriated on regulation in a human skin malignant melanoma cell line (A375). Mechanism-based His pulldown and immunoprecipitation technique used to identify TRX binding partner proteins. We found that up/downregulated intracellular proteins were identified under the condition of LPS-treated A375 cells and co-treat with exogenous TRX using in vitro 2-DE proteomic approaches. As well as, confirmed in vivo mouse inflammation models used to quantitatively measure IL-6, IL-10, MCP-1 and TNF protein levels in serum samples. Our studies demonstrate that exogenous TRX has anti-inflammatory properties and intracellular regulator activity in vivo and in vitro. These results have a therapeutic role in skin inflammation therapy. doi:10.1016/j.jbiosc.2009.08.325
EP-P39 Triose phosphate Isomerase enzyme is protected by Tau through its binding in a brain cell Seung-Ah Park, Nam-Hee Kim, Yoon-Ha Kim, Min-Jeong Kwak, Jun-Seop Shin, Hae-Won Park, and Chan-Wha Kim Korea University, Seoul, Republic of Korea Tau, a microtubule associated protein, is the main component of the aberrant paired helical filaments (PHF) found in Alzheimer's Disease (AD). Otherwise, the basic role of Tau is known for the stabilization of microtubules. These opposing roles of Tau have been previously reported; Tau enhances oxidative stress [1] or antagonizes apoptosis [2]. Here, we show the controversial effect of Tau on the activity and quantity of TPI in two different conditions. Lower levels of TPI (N = 6, p b 0.05) were detected, by TPI enzyme assay in cortex of Tau over-expressed Transgenic mice (Tg) group compared with agematched healthy group (NTg), and the shortage quantity of TPI was identified on the Paired helical filament (PHF) in Tg group. However, interaction of Tau and TPI was detected more in NTg group than Tg group by immunoprecipitation in vitro. Under the oxidative stress condition (500 μM H2O2), TPI activity show the higher level (N = 5, p b 0.5) when Tau binds to TPI. Consequently, in the Tg group, some TPI are in PHF, which is the reason of the TPI deficiency and advanced glycation end products (AGEs) accumulation. On the other side, in the NTg group, soluble Tau protects TPI against oxidative stress. Our results provide new insights into the understanding complication of tau's role and into the treatment of tauopathies such as TPI protection of soluble Tau.
EP-P38 References Regulation of bacterial LPS-stimulated inflammation in vitro and in vivo lipid accumulation by the thiol-antioxidant thioredoxin Gi-Yeon Han, Eun-Kyung Lee, and Chan-Wha Kim Korea University, Seoul, Republic of Korea The thiol-disulfide oxidoreductase thioredoxin-1 (TRX) is known to be secreted by leukocytes and to exhibit cytokine-like properties. Extracellular effects of TRX require a functional active site, suggesting
1. Stamer, K., Vogel, E. T, Mandelkow E., and Mandelkow. E.M.: Tau blocks traffic of organelles, neurofilaments, and APP vesicles in neurons and enhances oxidative stress. J. Cell Biology. 156, 1051-1063 (2002). 2. Hong-Lian, L., Hai-Hong, W., Yong-Jie, Z., Qing, T., Xial-Chuan, Wang., and XialQian, C.,: Phosphorylation of tau antagonizes apoptosis by stabilizing β-catenin, a mechanism involved in Alzheimer's neurodegeneration. Proc. Natl. Acad. Sci. USA,. 104, 3591-3596 (2007).
doi:10.1016/j.jbiosc.2009.08.326
Abstracts / Journal of Bioscience and Bioengineering 108 (2009) S96–S113
Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, Japan 1 and Department of Bioengineering, Graduate School of Engineering, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, Japan 2 With the success of the human genome project, the ability to rapidly convert gene information into the molecular function of their corresponding proteins is a significant advantage for both protein science and protein engineering (1). To obtain the information about the molecular function of target proteins in a high through-put manner, both the technologies to efficiently obtain target proteins in their native form and the methodologies to rapidly determine their function are required. In these proteomics studies, one of the most fundamental problems is the fragility of the protein structure, which is easy to unfold. Furthermore, unfolded proteins irreversibly form aggregates and permanently loss their function. Therefore, in the many process of proteomics studies such as protein production, protein separation, protein isolation and so on, irreversible unfolding and aggregation have been major bottlenecks. Thus, controlling the aggregation and folding of proteins in vitro is crucial for further progress in proteomic research area. In this presentation, we reports two topics about refolding/reactivation of protein by using small molecular additives: (1) successful development of small molecular additives for protein refolding by combinatorial approach (2), (2) a technology for reactivation of denatured enzymes in SDS-PAGE gel (3).
storage. To improve the stability of the enzymes, NP and XOD were freeze-dried with various glass-forming additives i.e., sucrose, sucrose-bovine serum albumin (BSA), sucrose-dextran (MW: 10400 Da), sucrose-polyethylene glycol (PEG: MW 600 Da), and sucrose-polyvinylpyrrolidone k30 (PVP). The samples were stored at 25 °C for up to 90 days, and the remaining activity of the enzymes was determined by using a UV-VIS spectrophotometer. Glass transition temperature (Tg) and moisture content of the samples were also investigated using differential scanning calorimetry and Karl Fisher coulometer, respectively. All additive-containing samples were amorphous solid. The sucrose– polymer formulations had higher Tg than that of the sucrose alone formulation. Furthermore, the crystallization temperatures of sucrose-polymer samples were higher than those of the sucrose samples. This means that the physical stability of amorphous sucrose is improved with the addition of polymer. The activity of NP and XOD was greatly protected by sucrose, however, the combination of sucrose and BSA improved the enzymes stability more effectively. On the other hand, sucrose–PEG, sucrose–dextran and sucrose–PVP did not have synergistic stabilizing effects. Sucrose-PEG protected the enzymes in the same degree with the sucrose alone formulation. Dextran and PVP seem to lower the stabilizing ability of sucrose on the dried enzymes. Reference 1. Wang, W.: Lyophilization and development of solid protein pharmaceuticals. Int. J. Pharm., 203, 1-60 (2000).
References doi:10.1016/j.jbiosc.2009.08.322 1. Middelberg A. P. J.: Preparative protein refolding. Trends Biotechnol, 2002;20: 437-443. 2. Yamaguchi S., Yamamoto E., Tsukiji S., and Nagamune T.: Successful control of aggregation and folding rates during refolding of denatured lysozyme by adding N-methylimidazolium cations with various N'-substituents. Biotechnol. Prog. 2008;24:402-408. 3. Yamamoto E., Yamaguchi S., and Nagamune T.: Effect of beta-cyclodextrin on the renaturation of enzymes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Anal. Biochem. 2008.
EP-P35 Characterization of secreted aspartic proteases of Candida albicans Wataru Aoki, Nao Kitahara, Hironobu Morisaka, Natsuko Miura, Kouichi Kuroda, and Mitsuyoshi Ueda
doi:10.1016/j.jbiosc.2009.08.321
Kyoto University, Kyoto, Japan EP-P34 Stabilization of xanthine oxidase and nucleoside phosphorylase enzyme mixture in dried glassy matrices Paveena Srirangsan,1 Kiyoshi Kawai,2 Naoko Hamada-Sato,3 and Toru Suzuki1 Department of Food Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan 1 Department of Biofunctional science and Technology, Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8528, Japan 2 and Course of Safety Management in Food Supply Chain, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan 3 Most of enzymes are sensitive to chemical and physical stresses, which cause them to lose their activity during processing and storage (1). Nucleoside phosphorylase (NP) and xanthine oxidase (XOD) are the enzymes, which have been widely used in biotechnology. Particularly, mixtures of them have been used in freshness testing sensor to evaluate fish freshness based on the change of nucleotides and nucleosides contents in fish meat. The enzymes, however, are unstable thermally and lose their activities during freeze-drying and
Recently, virulent fungi are running rampant in human beings, especially in immunocompromised patients. A representative of virulent fungi is Candida albicans. C. albicans is a dimorphic, opportunistic fungal pathogen and does not cause diseases in healthy people. However, C. albicans causes various diseases in people whose immuno-system has been damaged by AIDS, immune-suppressing drugs, or cancers. Notably, systemic infection of C. albicans often causes multiple organ failures, and is almost lethal. It is said that the mortality rate reaches around 70%. Patients have very limited remedies available, because there are a few effective drugs against candidiasis. Amphotericin B and fluconazole are the representative anti-fungal drugs, but these drugs have severe side effects such as renal dysfunction. Furthermore, drug-resistance fungi gradually are emerging. The pathogenicity of C. albicans largely depends on adhesion to epithelial cells, yeast-hypha transition, and extracellular proteases. In this study, we pay attention to extracellular proteases of C. albicans. C. albicans has ten secreted aspartic proteases (Saps) and each of them is an important pathogenic factor. Saccharomyces cerevisiae, a nonpathogenic close relative of C. albicans, does not have Sap homolog, and Sap deletion mutants of C. albicans shows less virulence. So Saps are considered as important virulent factors. It is known that Saps degrade epithelial adhesion molecules to penetrate into the interior, and disintegrate immunoproteins such as complements and IgG to
Abstracts / Journal of Bioscience and Bioengineering 108 (2009) S96–S113 escape from immune responses. However, substrate specificity of Saps has not been investigated in detail. In this study, we have clarified characteristics of Saps. Disclosure of the properties of these proteases will enable us to understand the pathogenicity of C. albicans. doi:10.1016/j.jbiosc.2009.08.323
EP-P36 Structural and functional characterization of glycerol kinase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 Ryuta Hokao,1 Yuichi Koga,1 Kazufumi Takano,1,2 and Shigenori Kanaya1 Osaka University, Suita city, Osaka, Japan 1 and CREST-JST, Suita city, Osaka, Japan 2 The purpose of this study is the analysis of structural and functional characters of glycerol kinase (GK) from the hyperthermophilic archaeon Thermococcus kodakaraensis (Tk-GK). Glycerol kinase catalyses the Mg,ATP-dependent phosphorylation of glycerol to produce glycerol 3-phosphate, which is a key enzyme of glycerol metabolism. In E. coli, GK (Ec-GK) exists in a dimer-tetramer equilibrium. Fructose 1,6-bisphosphate (FBP) selectively binds to the tetramer and inhibits Ec-GK activity by preventing interdomain motion. Unlike Ec-GK, Tk-GK does not exist as a tetramer in solution state, and it is not sensitive to FBP. According to the gel filtration chromatography, it was shown that Tk-GK exists as a dimer (110 kDa) in the absence of glycerol or presence of ATP, and both as a dimer and as a hexamer (330 kDa) in the presence of glycerol (mainly as a hexamer). This is the first report of the hexameric structure among GKs from various organisms. It may indicate that hexameric structure is induced by the conformational change with glycerol binding. In addition, we analyzed the interface of this hexameric structure by the mutational method. We will report the role of hexameric structure which affects the functions of Tk-GK. References 1. Koga Y., Katsumi R., You D.J., Matsumura H., Takano K., and Kanaya S.: Crystal structure of highly thermostable glycerol kinase from a hyperthermophilic archaeon in a dimeric form. FEBS J., 275, 2632-2643 (2008). 2. Katsumi R., Koga Y., You D.J., Matsumura H., Takano K., and Kanaya S.: Crystallization and preliminary X-ray diffraction study of glycerol kinase from the hyperthermophilic archaeon Thermococcus kodakaraensis. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun., 63(Pt 2),126-129 (2007).
doi:10.1016/j.jbiosc.2009.08.324
S111
a redox-based mechanism of action. However, specific cell surface proteins and pathways coupling extracellular TRX redox activity to cellular responses have not been identified so far. We first found that exogenous TRX not only scavenger but also effected intracellular proteins using proteomic approaches. Therefore, the effect of a thiolantioxidant protein TRX have appropriated on regulation in a human skin malignant melanoma cell line (A375). Mechanism-based His pulldown and immunoprecipitation technique used to identify TRX binding partner proteins. We found that up/downregulated intracellular proteins were identified under the condition of LPS-treated A375 cells and co-treat with exogenous TRX using in vitro 2-DE proteomic approaches. As well as, confirmed in vivo mouse inflammation models used to quantitatively measure IL-6, IL-10, MCP-1 and TNF protein levels in serum samples. Our studies demonstrate that exogenous TRX has anti-inflammatory properties and intracellular regulator activity in vivo and in vitro. These results have a therapeutic role in skin inflammation therapy. doi:10.1016/j.jbiosc.2009.08.325
EP-P39 Triose phosphate Isomerase enzyme is protected by Tau through its binding in a brain cell Seung-Ah Park, Nam-Hee Kim, Yoon-Ha Kim, Min-Jeong Kwak, Jun-Seop Shin, Hae-Won Park, and Chan-Wha Kim Korea University, Seoul, Republic of Korea Tau, a microtubule associated protein, is the main component of the aberrant paired helical filaments (PHF) found in Alzheimer's Disease (AD). Otherwise, the basic role of Tau is known for the stabilization of microtubules. These opposing roles of Tau have been previously reported; Tau enhances oxidative stress [1] or antagonizes apoptosis [2]. Here, we show the controversial effect of Tau on the activity and quantity of TPI in two different conditions. Lower levels of TPI (N = 6, p b 0.05) were detected, by TPI enzyme assay in cortex of Tau over-expressed Transgenic mice (Tg) group compared with agematched healthy group (NTg), and the shortage quantity of TPI was identified on the Paired helical filament (PHF) in Tg group. However, interaction of Tau and TPI was detected more in NTg group than Tg group by immunoprecipitation in vitro. Under the oxidative stress condition (500 μM H2O2), TPI activity show the higher level (N = 5, p b 0.5) when Tau binds to TPI. Consequently, in the Tg group, some TPI are in PHF, which is the reason of the TPI deficiency and advanced glycation end products (AGEs) accumulation. On the other side, in the NTg group, soluble Tau protects TPI against oxidative stress. Our results provide new insights into the understanding complication of tau's role and into the treatment of tauopathies such as TPI protection of soluble Tau.
EP-P38 References Regulation of bacterial LPS-stimulated inflammation in vitro and in vivo lipid accumulation by the thiol-antioxidant thioredoxin Gi-Yeon Han, Eun-Kyung Lee, and Chan-Wha Kim Korea University, Seoul, Republic of Korea The thiol-disulfide oxidoreductase thioredoxin-1 (TRX) is known to be secreted by leukocytes and to exhibit cytokine-like properties. Extracellular effects of TRX require a functional active site, suggesting
1. Stamer, K., Vogel, E. T, Mandelkow E., and Mandelkow. E.M.: Tau blocks traffic of organelles, neurofilaments, and APP vesicles in neurons and enhances oxidative stress. J. Cell Biology. 156, 1051-1063 (2002). 2. Hong-Lian, L., Hai-Hong, W., Yong-Jie, Z., Qing, T., Xial-Chuan, Wang., and XialQian, C.,: Phosphorylation of tau antagonizes apoptosis by stabilizing β-catenin, a mechanism involved in Alzheimer's neurodegeneration. Proc. Natl. Acad. Sci. USA,. 104, 3591-3596 (2007).
doi:10.1016/j.jbiosc.2009.08.326