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Luetzen MA, Chakraborty R, Moreno-Ramos OA, Echeverri-Peña OY, Satta Y, Montaño AM. Purifying selection of the lysosomal enzymes arylsulfatase A and beta-galactocerebrosidase and their evolutionary impact on myelin integrity. J Lipid Res 2025; 66:100769. [PMID: 40054667 PMCID: PMC12008523 DOI: 10.1016/j.jlr.2025.100769] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2025] [Revised: 03/02/2025] [Accepted: 03/03/2025] [Indexed: 04/10/2025] Open
Abstract
The myelin is responsible for providing stability to the axons of the nerve cells, but above all, to improve transmission speed of the nerve impulse in vertebrates. Over 70% of the myelin sheath is composed of lipids and the remaining portion by approximately 2,000 proteins. The myelin sheath has been constantly evolving, and it is known that unusually high concentrations of galactosylceramide (GalCer) and its sulfated form play a major role in the biophysical properties of the myelin. To gain insights of the evolutionary role of GalCer, we have studied two lysosomal enzymes involved in GalCer degradation, arylsulfatase A (ARSA) and galactocerebrosidase (GALC). Deficiency of ARSA or GALC causes demyelinating disorders. We conducted phylogenetic analyses of 105 ARSA and 110 GALC orthologs representing more than 600 million years ago of evolution. We examined i) low values of the ratio of nonsynonymous to synonymous nucleotide-substitution rates (dN/dS) indicating purifying selection and ii) negative selection of amino acids located in the active site preventing pathogenic mutations. Gene structure analyses showed evidence of rearrangement with gain and loss of exons while there were conserved regions mainly located around the active site. We also found a limited number of sites under positive selection pressure that do not cause alterations to the overall protein structure. Our results indicate that ARSA and GALC have been highly conserved during the evolutionary process to maintain the metabolism of GalCer, which is essential for the integrity of the white matter in vertebrate species.
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Affiliation(s)
- Matthew A Luetzen
- Department of Biochemistry and Molecular Biology, School of Medicine, Saint Louis University, St. Louis, MO, USA
| | - Richik Chakraborty
- School of Medicine, Saint Louis University, St. Louis, MO, USA; Clinical Trials Office, Georgetown Lombardi Comprehensive Cancer Center, Washington D.C., USA
| | - Oscar Andrés Moreno-Ramos
- Department of Pediatrics, School of Medicine, Saint Louis University, St Louis, MO, USA; Facultad de Ciencias, Departamento de Ciencias Biológicas, Centro de Investigaciones Genéticas en Enfermedades Humanas (CIGEN), Universidad de los Andes, Bogotá, Colombia
| | | | - Yoko Satta
- Department of Evolutionary Studies of Biosystems, SOKENDAI (The Graduate University for Advanced Studies), Hayama, Kanagawa, Japan
| | - Adriana M Montaño
- Department of Biochemistry and Molecular Biology, School of Medicine, Saint Louis University, St. Louis, MO, USA; Department of Pediatrics, School of Medicine, Saint Louis University, St Louis, MO, USA.
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Kanbay M, Copur S, Ozbek L, Mutlu A, Cejka D, Ciceri P, Cozzolino M, Haarhaus ML. Klotho: a potential therapeutic target in aging and neurodegeneration beyond chronic kidney disease-a comprehensive review from the ERA CKD-MBD working group. Clin Kidney J 2024; 17:sfad276. [PMID: 38213484 PMCID: PMC10783249 DOI: 10.1093/ckj/sfad276] [Citation(s) in RCA: 13] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2023] [Indexed: 01/13/2024] Open
Abstract
Klotho, a multifunctional protein, acts as a co-receptor in fibroblast growth factor 23 and exerts its impact through various molecular pathways, including Wnt, hypoxia-inducible factor and insulin-like growth factor 1 pathways. The physiological significance of Klotho is the regulation of vitamin D and phosphate metabolism as well as serving as a vital component in aging and neurodegeneration. The role of Klotho in aging and neurodegeneration in particular has gained considerable attention. In this narrative review we highlight several key insights into the molecular basis and physiological function of Klotho and synthesize current research on the role of Klotho in neurodegeneration and aging. Klotho deficiency was associated with cognitive impairment, reduced growth, diminished longevity and the development of age-related diseases in vivo. Serum Klotho levels showed a decline in individuals with advanced age and those affected by chronic kidney disease, establishing its potential diagnostic significance. Additionally, multiple medications have been demonstrated to influence Klotho levels. Therefore, this comprehensive review suggests that Klotho could open the door to novel interventions aimed at addressing the challenges of aging and neurodegenerative disorders.
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Affiliation(s)
- Mehmet Kanbay
- Department of Medicine, Nephrology, Koc University School of Medicine, Istanbul, Turkey
| | - Sidar Copur
- Department of Medicine, Koc University School of Medicine, Istanbul, Turkey
| | - Lasin Ozbek
- Department of Medicine, Koc University School of Medicine, Istanbul, Turkey
| | - Ali Mutlu
- Department of Medicine, Koc University School of Medicine, Istanbul, Turkey
| | - Daniel Cejka
- Department of Medicine III – Nephrology, Hypertension, Transplantation, Rheumatology, Geriatrics, Ordensklinikum Linz – Elisabethinen Hospital, Linz, Austria
| | - Paola Ciceri
- Department of Health Sciences, Renal Division, University of Milan, Milan, Italy
| | - Mario Cozzolino
- Department of Health Sciences, Renal Division, University of Milan, Milan, Italy
| | - Mathias Loberg Haarhaus
- Division of Renal Medicine, Department of Clinical Science, Intervention and Technology, Karolinska University Hospital, Karolinska Institutet, Stockholm, Sweden
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Sun F, Liang P, Wang B, Liu W. The fibroblast growth factor-Klotho axis at molecular level. Open Life Sci 2023; 18:20220655. [PMID: 37941788 PMCID: PMC10628560 DOI: 10.1515/biol-2022-0655] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2022] [Revised: 06/03/2023] [Accepted: 06/10/2023] [Indexed: 11/10/2023] Open
Abstract
Klotho is a recently discovered protein that has positive effects on all systems of the body, for example, regulating calcium and phosphorus metabolism, protecting nerves, delaying aging and so on. Fibroblast growth factors (FGFs) are a group of polypeptides that function throughout the body by binding with cell surface FGF receptors (FGFRs). Endocrine FGFs require Klotho as a co-receptor for FGFRs. There is increasing evidence that Klotho participates in calcium and phosphorus regulation and metabolic regulation via the FGF-Klotho axis. Moreover, soluble Klotho can function as a separate hormone to regulate homeostasis on various ion channels and carrier channels on the cell surface. This review mainly explains the molecular basis of the membrane signaling mechanism of Klotho.
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Affiliation(s)
- Fuqiang Sun
- School of Anesthesiology, Weifang Medical University, Shandong Provincial Medicine and Health Key Laboratory of Clinical Anesthesia, Weifang261053, Shandong, China
| | - Panpan Liang
- School of Basic Medical Sciences, Air Force Medical University, Xi'an, 710032, Shaanxi, China
| | - Bo Wang
- School of Anesthesiology, Weifang Medical University, Shandong Provincial Medicine and Health Key Laboratory of Clinical Anesthesia, Weifang261053, Shandong, China
| | - Wenbo Liu
- Central Laboratory of the First Affiliated Hospital, Weifang Medical University, Weifang261000, Shandong, China
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Yu T, Dou C, Lu Y, Duan L, Tan J, Li J, Kang F, Dong S, Bai Y, Xu J. Klotho upregulates the interaction between RANK and TRAF6 to facilitate RANKL-induced osteoclastogenesis via the NF-κB signaling pathway. ANNALS OF TRANSLATIONAL MEDICINE 2021; 9:1499. [PMID: 34805361 PMCID: PMC8573428 DOI: 10.21037/atm-21-4332] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/30/2021] [Accepted: 09/16/2021] [Indexed: 11/06/2022]
Abstract
Background α-Klotho (Klotho) plays a wide range of roles in pathophysiological processes, such as low-turnover osteoporosis observed in klotho mutant mice (kl/kl mice). However, the precise function and underlying mechanism of klotho during osteoclastogenesis are not fully understood. Here, we investigated the effects of klotho on osteoclastogenesis induced by receptor activator of nuclear factor kappa-B ligand (RANKL). Methods The effects of klotho deficiency on osteoclastogenesis were explored using kl/kl mice both in vivo and in vitro. In in vitro experiments, lentivirus transfection, real-time quantitative PCR (RT-qPCR) analysis, western blot analysis, immunostaining, RNA-seq analysis, differential pathway analysis, Energy-based protein docking analysis and co-immunoprecipitation were used for deeply investigating the effects of klotho on RANKL-induced Osteoclastogenesis and the underlying mechanism. Results We found that klotho deficiency impaired osteoclastogenesis. Furthermore, in vitro studies revealed that klotho facilitated osteoclastogenesis and upregulated the expression of c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) during osteoclastogenesis. Mechanistically, we confirmed that klotho co-localized with nuclear factor kappa B (RANK) and facilitated the interaction between activated RANK and TNFR-associated factor 6 (TRAF6), thus klotho exerts its function in osteoclastogenesis through the activation of the NF-κB signaling pathway. Conclusions Klotho promotes RANKL-induced osteoclastogenesis through upregulating the interaction between RANK and TARF6, Targeting on klotho may be an attractive therapeutic method for osteopenic diseases.
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Affiliation(s)
- Tao Yu
- Department of Orthopedics, Southwest Hospital, Third Military Medical University, Chongqing, China
| | - Ce Dou
- Department of Orthopedics, Southwest Hospital, Third Military Medical University, Chongqing, China
| | - Yanzhu Lu
- Department of Orthopedics, Southwest Hospital, Third Military Medical University, Chongqing, China
| | - Lianli Duan
- Department of Orthopedics, Southwest Hospital, Third Military Medical University, Chongqing, China
| | - Jiulin Tan
- Department of Orthopedics, Southwest Hospital, Third Military Medical University, Chongqing, China
| | - Jianmei Li
- Department of Biomedical Materials Science, School of Biomedical Engineering, Third Military Medical University, Chongqing, China
| | - Fei Kang
- Department of Biomedical Materials Science, School of Biomedical Engineering, Third Military Medical University, Chongqing, China
| | - Shiwu Dong
- Department of Biomedical Materials Science, School of Biomedical Engineering, Third Military Medical University, Chongqing, China
| | - Yun Bai
- Department of Orthopedics, Southwest Hospital, Third Military Medical University, Chongqing, China
| | - Jianzhong Xu
- Department of Orthopedics, Southwest Hospital, Third Military Medical University, Chongqing, China
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Erkus E, Buyukterzı Z, Karakose S, Kurku H, Kurtgoz PO, Topal M, Guney I. The relationship of soluble klotho level with uremic cardiomyopathy and ecocardiographic parameters in hemodialysis patients. Semin Dial 2020; 34:157-162. [PMID: 33252840 DOI: 10.1111/sdi.12942] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
There are studies reporting that soluble kltho (sKlotho) deficiency plays a role in cardiovascular disease in addition to traditional risk factors such as diabetes, hypertension, anemia, smoking, and excessive volume burden. Our aim in this study was to investigate the relationship of sKlotho with uremic cardiomyopathy and echocardiographic parameters in patients receiving hemodialysis treatment. According to the median value, the sKlotho value was divided into two groups as ≥1.24 and <1.24 ng/ml. Ventricular wall thicknesses, ejection fractions, left atrium, M mode aorta systole, and diastole diameter measurements were taken. The left ventricular mass (LVM) was calculated using the Devereux formula. There were significant differences between the two groups in terms of age, number of patients with diabetes mellitus, comorbidity, dialysis time, sKlotho, phosphorus, parathormone, and albumin parameters. No significant difference was found between the two groups that were separated according to the median sKlotho value, when the echocardiographic parameters of interventricular septum thickness, left ventricular posterior wall thickness, left atrial diameter, left ventricular ejection fraction, and LVM index were compared. In conclusion, sKlotho is not a marker for showing and predicting uremic cardiomyopathy in hemodialysis patients.
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Affiliation(s)
- Edip Erkus
- Nephrology Department, University of Health Sciences, Konya Research and Training Hospital, Konya, Turkey
| | - Zafer Buyukterzı
- Cardiology Department, University of Health Sciences, Konya Research and Training Hospital, Konya, Turkey
| | - Suleyman Karakose
- Nephrology Department, University of Health Sciences, Konya Research and Training Hospital, Konya, Turkey
| | - Huseyın Kurku
- Bıochemıstry Department, University of Health Sciences, Konya Research and Training Hospital, Konya, Turkey
| | - Pervın O Kurtgoz
- Nephrology Department, University of Health Sciences, Konya Research and Training Hospital, Konya, Turkey
| | - Mustafa Topal
- Nephrology Department, University of Health Sciences, Konya Research and Training Hospital, Konya, Turkey
| | - Ibrahim Guney
- Nephrology Department, University of Health Sciences, Konya Research and Training Hospital, Konya, Turkey
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Shen Y, Li Z, Huo YY, Bao L, Gao B, Xiao P, Hu X, Xu XW, Li J. Structural and Functional Insights Into CmGH1, a Novel GH39 Family β-Glucosidase From Deep-Sea Bacterium. Front Microbiol 2019; 10:2922. [PMID: 31921083 PMCID: PMC6933502 DOI: 10.3389/fmicb.2019.02922] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2019] [Accepted: 12/04/2019] [Indexed: 01/07/2023] Open
Abstract
Glucosidases play key roles in many diseases and are limiting enzymes during cellulose degradation, which is an important part of global carbon cycle. Here, we identified a novel β-glucosidase, CmGH1, isolated from marine bacterium Croceicoccus marinus E4A9T. In spite of its high sequence and structural similarity with β-xylosidase family members, CmGH1 had enzymatic activity toward p-nitrophenyl-β-D-glucopyranoside (p-NPG) and cellobiose. The Km and Kcat values of CmGH1 toward p-NPG were 0.332 ± 0.038 mM and 2.15 ± 0.081 min–1, respectively. CmGH1 was tolerant to high concentration salts, detergents, as well as many kinds of organic solvents. The crystal structure of CmGH1 was resolved with a 1.8 Å resolution, which showed that CmGH1 was composed of a canonical (α/β)8-barrel catalytic domain and an auxiliary β-sandwich domain. Although no canonical catalytic triad residues were found in CmGH1, structural comparison and mutagenesis analysis suggested that residues Gln157 and Tyr264 of CmGH1 were the active sites. Mutant Q157E significantly increased its hydrolase activity up to 15-fold, whereas Y264E totally abolished its enzymatic activity. These results might provide new insights into understanding the different catalytic mechanism during evolution for β-glucosidases and β-xylosidases.
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Affiliation(s)
- Yanfang Shen
- State Key Laboratory of Genetic Engineering, Department of Neurology, School of Life Sciences, Huashan Hospital, Shanghai Engineering Research Center of Industrial Microorganisms, Fudan University, Shanghai, China
| | - Zhengyang Li
- State Key Laboratory of Genetic Engineering, Department of Neurology, School of Life Sciences, Huashan Hospital, Shanghai Engineering Research Center of Industrial Microorganisms, Fudan University, Shanghai, China
| | - Ying-Yi Huo
- Key Laboratory of Marine Ecosystem and Biogeochemistry, Second Institute of Oceanography, State Oceanic Administration, Hangzhou, China
| | - Luyao Bao
- State Key Laboratory of Genetic Engineering, Department of Neurology, School of Life Sciences, Huashan Hospital, Shanghai Engineering Research Center of Industrial Microorganisms, Fudan University, Shanghai, China
| | - Baocai Gao
- State Key Laboratory of Genetic Engineering, Department of Neurology, School of Life Sciences, Huashan Hospital, Shanghai Engineering Research Center of Industrial Microorganisms, Fudan University, Shanghai, China
| | - Peng Xiao
- State Key Laboratory of Genetic Engineering, Department of Neurology, School of Life Sciences, Huashan Hospital, Shanghai Engineering Research Center of Industrial Microorganisms, Fudan University, Shanghai, China
| | - Xiaojian Hu
- State Key Laboratory of Genetic Engineering, Department of Neurology, School of Life Sciences, Huashan Hospital, Shanghai Engineering Research Center of Industrial Microorganisms, Fudan University, Shanghai, China
| | - Xue-Wei Xu
- Key Laboratory of Marine Ecosystem and Biogeochemistry, Second Institute of Oceanography, State Oceanic Administration, Hangzhou, China
| | - Jixi Li
- State Key Laboratory of Genetic Engineering, Department of Neurology, School of Life Sciences, Huashan Hospital, Shanghai Engineering Research Center of Industrial Microorganisms, Fudan University, Shanghai, China
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Wu Y, Shen Y, Zhu Y, Mupunga J, Zou L, Liu C, Liu S, Mao J. Broccoli ingestion increases the glucosinolate hydrolysis activity of microbiota in the mouse gut. Int J Food Sci Nutr 2019; 70:585-594. [DOI: 10.1080/09637486.2018.1554624] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Affiliation(s)
- Yuanfeng Wu
- School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Zhejiang, Hangzhou, China
- Zhejiang Provincial Key Lab for Chem & Bio Processing Technology of Farm Produces, Zhejiang, Hangzhou, China
| | - Yuke Shen
- School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Zhejiang, Hangzhou, China
- Zhejiang Provincial Key Lab for Chem & Bio Processing Technology of Farm Produces, Zhejiang, Hangzhou, China
| | - Ye Zhu
- School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Zhejiang, Hangzhou, China
- Zhejiang Provincial Key Lab for Chem & Bio Processing Technology of Farm Produces, Zhejiang, Hangzhou, China
| | - Jothame Mupunga
- School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Zhejiang, Hangzhou, China
- Zhejiang Provincial Key Lab for Chem & Bio Processing Technology of Farm Produces, Zhejiang, Hangzhou, China
| | - Ligen Zou
- Hangzhou Academy of Agricultural Sciences, Zhejiang, Hangzhou, China
| | - Chao Liu
- Hangzhou Academy of Agricultural Sciences, Zhejiang, Hangzhou, China
| | - Shiwang Liu
- School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Zhejiang, Hangzhou, China
- Zhejiang Provincial Key Lab for Chem & Bio Processing Technology of Farm Produces, Zhejiang, Hangzhou, China
| | - Jianwei Mao
- School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Zhejiang, Hangzhou, China
- Zhejiang Provincial Key Lab for Chem & Bio Processing Technology of Farm Produces, Zhejiang, Hangzhou, China
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Chen G, Tao Q. Expression of the hormonal FGF co-receptor Klotho beta in the Xenopus laevis model. Cell Biol Int 2018; 43:207-213. [PMID: 30259590 DOI: 10.1002/cbin.11059] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2018] [Accepted: 09/25/2018] [Indexed: 11/11/2022]
Abstract
Klotho beta (Klb), a single-pass transmembrane protein, has been described as a co-receptor for endocrine FGFs, such as FGF15/19 and FGF21, to regulate critical metabolic processes in multiple organs and tissues in adult mice. However, its function during early embryonic development remains largely unknown. In this paper, we evaluated for the first time the expression of klb mRNA during early development of Xenopus laevis by RT-PCR and whole mount in situ hybridization. RT-PCR experiments showed that the expression of klb was initially detected at late gastrula stage followed by a quick increasing and continued expression throughout embryonic development. Whole mount in situ hybridization detected specific expression of klb in many primordial organs at tailbud stage such as liver primordium and pancreatic buds, implying that the hormonal FGF signaling may play a role in the foregut development. The dynamic and specific expression patterns of klb also suggest that Xenopus laevis can serve a convenient model for the function of the hormonal FGF signaling in organogenesis and metabolism regulation during embryonic development.
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Affiliation(s)
- Geng Chen
- MOE Key Laboratory of Protein Sciences, Tsinghua University School of Life Sciences, Beijing, 100084, China
| | - Qinghua Tao
- MOE Key Laboratory of Protein Sciences, Tsinghua University School of Life Sciences, Beijing, 100084, China
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Kaludjerovic J, Komaba H, Lanske B. Effects of klotho deletion from bone during chronic kidney disease. Bone 2017; 100:50-55. [PMID: 28232146 PMCID: PMC5474158 DOI: 10.1016/j.bone.2017.02.006] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/14/2017] [Revised: 02/17/2017] [Accepted: 02/18/2017] [Indexed: 12/24/2022]
Abstract
Klotho is a type I transmembrane protein that acts as a permissive co-receptor for FGF23 and helps to maintain proper mineral metabolism. Mice carrying a loss-of-function mutation in either the Klotho or Fgf23 gene develop many similar phenotypes including osteoporosis. Based on these observations it was hypothesized that the bone phenotypes in Klotho- and Fgf23-null mice may be mediated through a common signaling pathway. Recent improvements in antibody specificity have shown that osteoblasts and osteocytes, which produce FGF23, also express low amount of membrane Klotho. But, the role of Klotho in bone is still largely unclear. In this review we summarize the literature and show that Klotho has an FGF23 dependent and independent effect in bone.
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Affiliation(s)
- Jovana Kaludjerovic
- Division of Bone and Mineral Research, Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, Boston, MA, USA
| | - Hirotaka Komaba
- Division of Bone and Mineral Research, Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, Boston, MA, USA
| | - Beate Lanske
- Division of Bone and Mineral Research, Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, Boston, MA, USA; Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA.
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Kunert SK, Hartmann H, Haffner D, Leifheit-Nestler M. Klotho and fibroblast growth factor 23 in cerebrospinal fluid in children. J Bone Miner Metab 2017; 35:215-226. [PMID: 27017221 DOI: 10.1007/s00774-016-0746-y] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/01/2015] [Accepted: 02/15/2016] [Indexed: 12/13/2022]
Abstract
The fibroblast growth factor (FGF) 23/Klotho axis is a principal regulator of phosphate hemostasis and vitamin D metabolism, but limited data is available on its role in the central nervous system. Here, we investigate soluble α-Klotho (sKlotho) and C-terminal as well as intact FGF23 in cerebrospinal fluid (CSF) and plasma and their relationship to mineral metabolism parameters in humans. In 39 children aged 0.3-16.8 years undergoing lumbar puncture for the exclusion of inflammatory neurological disease, sKlotho and FGF23 were investigated by Western blot analysis, followed by ELISA quantification in CSF and plasma. The percentage of intrathecal synthesis of both proteins was calculated by measuring both the expected and observed CSF/plasma ratios of sKlotho and FGF23. The secreted (KL1) and cleaved (KL1+KL2) isoforms of sKlotho, and FGF23 were clearly detected in CSF in all subjects, although protein levels were lower compared to those of plasma samples (each p < 0.01). The intrathecal percentage of CSF sKlotho and FGF23 synthesis amounted to 98 and 99 %, respectively. CSF sKlotho levels were higher in boys than in girls (p < 0.01), and correlated positively with plasma C-terminal FGF23 concentrations (p < 0.05) and standardized height (p < 0.01). Importantly, there were no significant correlations between plasma and CSF levels of sKlotho or FGF23. Plasma sKlotho as well as C-terminal and intact FGF23, respectively, were associated with parameters of mineral metabolism These results provide evidence that cleaved and secreted sKlotho and FGF23 are present in CSF, mainly derived from brain and affected by sex, height, and mineral metabolism parameters in children. Nevertheless, the absence of significant associations between plasma and CSF levels of Klotho and FGF23, respectively, suggest that the regulation of Klotho and FGF23 may be different between organs secreting these hormones into blood and CSF.
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Affiliation(s)
- Svenja Kristin Kunert
- Department of Pediatric Kidney, Liver and Metabolic Diseases, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany
| | - Hans Hartmann
- Department of Pediatric Kidney, Liver and Metabolic Diseases, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany
| | - Dieter Haffner
- Department of Pediatric Kidney, Liver and Metabolic Diseases, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany
| | - Maren Leifheit-Nestler
- Department of Pediatric Kidney, Liver and Metabolic Diseases, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany.
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Abdallah E, Mosbah O, Khalifa G, Metwaly A, El-Bendary O. Assessment of the relationship between serum soluble Klotho and carotid intima-media thickness and left ventricular dysfunction in hemodialysis patients. Kidney Res Clin Pract 2016; 35:42-9. [PMID: 27069857 PMCID: PMC4811978 DOI: 10.1016/j.krcp.2015.12.006] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2015] [Revised: 11/27/2015] [Accepted: 12/16/2015] [Indexed: 12/27/2022] Open
Abstract
BACKGROUND The aim of our study was to assess the relationship between soluble Klotho (s-Klotho) and carotid intima-media thickness (CIMT) and left ventricular (LV) dysfunction in hemodialysis (HD) patients. METHODS This is a cross-sectional study conducted on 88 patients with end-stage renal disease on regular HD. Serum levels of calcium, phosphorus, parathyroid hormone, and C-reactive protein were measured. The serum levels of s-Klotho and fibroblast growth factor-23 (FGF-23) were measured using an Enzyme linked immunosorbent assay (ELISA) kit. Echocardiography and measurement of CIMT were also conducted. The studied patients were divided according to the median s-Klotho level into 2 groups: patients with low s-Klotho (Group I) and patients with high s-Klotho (Group II). RESULTS Mean value of s-Klotho was significantly low in HD patients compared to controls (P = 0.001), and mean value of FGF-23 was significantly high in HD patients compared to controls (P = 0.001). The mean values of parathyroid hormone, FGF-23, and phosphorus were significantly high in Group I compared to Group II, whereas the mean value of serum calcium was significantly low in Group I compared to Group II. The mean values of CIMT, LV mass (LVM), LVM index, and LV ejection fraction (LVEF) were high in Group I compared to Group II. Patients with low s-Klotho had significantly more coronary artery disease (CAD). In a regression analysis of s-Klotho with different markers of cardiovascular diseases, s-Klotho showed significant association with CIMT, LVEF, and CAD, but not with LVM and LVM index. CONCLUSION The present study showed that patients with a low s-Klotho were more often associated with increased CIMT, LV dysfunction, and CAD, and it seems that there was independent association between s-Klotho and CIMT, LVEF, and CAD.
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Affiliation(s)
- Emad Abdallah
- Department of Nephrology, Theodor Bilharz Research Institute, Giza, Egypt
| | - Osama Mosbah
- Department of Nephrology, Theodor Bilharz Research Institute, Giza, Egypt
| | - Ghada Khalifa
- Department of Nephrology, Theodor Bilharz Research Institute, Giza, Egypt
| | - Amna Metwaly
- Department of Intensive Care Unit, Theodor Bilharz Research Institute, Giza, Egypt
| | - Omnia El-Bendary
- Department of Clinical Chemistry, Theodor Bilharz Research Institute, Giza, Egypt
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Ligumsky H, Rubinek T, Merenbakh-Lamin K, Yeheskel A, Sertchook R, Shahmoon S, Aviel-Ronen S, Wolf I. Tumor Suppressor Activity of Klotho in Breast Cancer Is Revealed by Structure–Function Analysis. Mol Cancer Res 2015; 13:1398-407. [DOI: 10.1158/1541-7786.mcr-15-0141] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2015] [Accepted: 06/10/2015] [Indexed: 11/16/2022]
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Zhou X, Wang X. Klotho: a novel biomarker for cancer. J Cancer Res Clin Oncol 2015; 141:961-9. [PMID: 25086986 DOI: 10.1007/s00432-014-1788-y] [Citation(s) in RCA: 59] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2014] [Accepted: 07/23/2014] [Indexed: 12/15/2022]
Abstract
BACKGROUND The Klotho gene was originally identified as an anti-aging gene in 1997. Recent studies have demonstrated aberrant expression of Klotho in a number of cancers, including breast cancer, lung cancer, hepatocellular carcinoma (HCC), and so on. METHODS A literature search focusing on dysregulation of Klotho and its possible mechanisms in cancer was performed. RESULTS AND CONCLUSIONS Downregulation of Klotho was found in several cancers, such as pancreatic cancer, HCC, and other tumors. Epigenetic modulation, such as promoter methylation and histone deacetylation, also contributed to the dysregulation of Klotho in cancers. Downregulation of Klotho resulted in promoted proliferation and reduced apoptosis of cancer cells. The relevant mechanisms include the fibroblast growth factor signaling, the insulin-like growth factor 1 receptor pathway, and the Wnt/β-catenin signaling pathway. Furthermore, the Klotho protein hopefully provides new insights into cancer target treatment.
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Affiliation(s)
- Xiangxiang Zhou
- Department of Hematology, Shandong Provincial Hospital Affiliated to Shandong University, No. 324, Jingwu Road, Jinan, 250021, Shandong, People's Republic of China
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14
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Esapa CT, Hannan FM, Babinsky VN, Potter P, Thomas GP, Croucher PI, Brown MA, Brown SDM, Cox RD, Thakker RV. N-ethyl-N-Nitrosourea (ENU) induced mutations within the klotho gene lead to ectopic calcification and reduced lifespan in mouse models. PLoS One 2015; 10:e0122650. [PMID: 25860694 PMCID: PMC4393098 DOI: 10.1371/journal.pone.0122650] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2014] [Accepted: 02/11/2015] [Indexed: 11/18/2022] Open
Abstract
Ectopic calcification (EC), which is the pathological deposition of calcium and phosphate in extra-skeletal tissues, may be associated with hypercalcaemic and hyperphosphataemic disorders, or it may occur in the absence of metabolic abnormalities. In addition, EC may be inherited as part of several monogenic disorders and studies of these have provided valuable insights into the metabolic pathways regulating mineral metabolism. For example, studies of tumoural calcinosis, a disorder characterised by hyperphosphataemia and progressive EC, have revealed mutations of fibroblast growth factor 23 (FGF23), polypeptide N-acetyl galactosaminyltransferase 3 (GALNT3) and klotho (KL), which are all part of a phosphate-regulating pathway. However, such studies in humans are limited by the lack of available large families with EC, and to facilitate such studies we assessed the progeny of mice treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) for EC. This identified two mutants with autosomal recessive forms of EC, and reduced lifespan, designated Ecalc1 and Ecalc2. Genetic mapping localized the Ecalc1 and Ecalc2 loci to a 11.0 Mb region on chromosome 5 that contained the klotho gene (Kl), and DNA sequence analysis identified nonsense (Gln203Stop) and missense (Ile604Asn) Kl mutations in Ecalc1 and Ecalc2 mice, respectively. The Gln203Stop mutation, located in KL1 domain, was severely hypomorphic and led to a 17-fold reduction of renal Kl expression. The Ile604Asn mutation, located in KL2 domain, was predicted to impair klotho protein stability and in vitro expression studies in COS-7 cells revealed endoplasmic reticulum retention of the Ile604Asn mutant. Further phenotype studies undertaken in Ecalc1 (kl203X/203X) mice demonstrated elevations in plasma concentrations of phosphate, FGF23 and 1,25-dihydroxyvitamin D. Thus, two allelic variants of Kl that develop EC and represent mouse models for tumoural calcinosis have been established.
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Affiliation(s)
- Christopher T. Esapa
- Academic Endocrine Unit, Radcliffe Department of Medicine, Oxford Centre for Diabetes, Endocrinology and Metabolism (OCDEM), University of Oxford, Oxford, United Kingdom
- Medical Research Council (MRC) Mammalian Genetics Unit and Mary Lyon Centre, MRC Harwell, Harwell Science and Innovation Campus, Harwell, United Kingdom
| | - Fadil M. Hannan
- Academic Endocrine Unit, Radcliffe Department of Medicine, Oxford Centre for Diabetes, Endocrinology and Metabolism (OCDEM), University of Oxford, Oxford, United Kingdom
- Department of Musculoskeletal Biology, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, United Kingdom
| | - Valerie N. Babinsky
- Academic Endocrine Unit, Radcliffe Department of Medicine, Oxford Centre for Diabetes, Endocrinology and Metabolism (OCDEM), University of Oxford, Oxford, United Kingdom
| | - Paul Potter
- Medical Research Council (MRC) Mammalian Genetics Unit and Mary Lyon Centre, MRC Harwell, Harwell Science and Innovation Campus, Harwell, United Kingdom
| | - Gethin P. Thomas
- University of Queensland Diamantina Institute, Translational Research Institute, Princess Alexandra Hospital, University of Queensland, Brisbane, Australia
| | | | - Matthew A. Brown
- University of Queensland Diamantina Institute, Translational Research Institute, Princess Alexandra Hospital, University of Queensland, Brisbane, Australia
| | - Steve D. M. Brown
- Medical Research Council (MRC) Mammalian Genetics Unit and Mary Lyon Centre, MRC Harwell, Harwell Science and Innovation Campus, Harwell, United Kingdom
| | - Roger D. Cox
- Medical Research Council (MRC) Mammalian Genetics Unit and Mary Lyon Centre, MRC Harwell, Harwell Science and Innovation Campus, Harwell, United Kingdom
| | - Rajesh V. Thakker
- Academic Endocrine Unit, Radcliffe Department of Medicine, Oxford Centre for Diabetes, Endocrinology and Metabolism (OCDEM), University of Oxford, Oxford, United Kingdom
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15
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Cordeiro RP, Doria JH, Zhanel GG, Sparling R, Holley RA. Role of glycoside hydrolase genes in sinigrin degradation by E. coli O157:H7. Int J Food Microbiol 2015; 205:105-11. [PMID: 25897994 DOI: 10.1016/j.ijfoodmicro.2015.04.008] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2015] [Revised: 04/01/2015] [Accepted: 04/04/2015] [Indexed: 11/24/2022]
Abstract
This work examined Escherichia coli O157:H7 strain 02-0304 for putative genes responsible for sinigrin hydrolysis. Sinigrin is a glucosinolate present in Oriental mustard (Brassica juncea), and its hydrolysis is mediated in plants by the enzyme myrosinase. Sinigrin hydrolysis by plant or bacterial myrosinase yields allyl isothiocyanate (AITC) which is bactericidal. In silico analysis using public databases found sequence similarity between plant myrosinase and enzymes encoded by genes from β-glucosidase families in E. coli O157:H7. Specifically, 6-phospho-β-glucosidase encoded by the genes bglA and ascB (family 1), and chbF (family 4) present in E. coli O157:H7 showed the highest similarity. Polymerase chain reaction (PCR) confirmed the presence of bglA, ascB, and chbF in the clinical E. coli strain tested. Disruption of these genes in wild-type E. coli O157:H7 strain 02-0304 using lambda-red replacement created single and double mutants. The relative importance of each gene in the hydrolysis of sinigrin by E. coli O157:H7 was also assessed by comparing gene expression and sinigrin degradation rates among the E. coli O157:H7 wild-type strain and its mutants. The results suggested that the genes bglA and ascB play a substantial role in the degradation of sinigrin by E. coli O157:H7 strain 02-0304.
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Affiliation(s)
- Roniele P Cordeiro
- Department of Food Science, Faculty of Agriculture and Food Sciences, University of Manitoba, Winnipeg, MB R3T 2N2, Canada
| | - Juan H Doria
- Department of Animal Science, University of Manitoba, Winnipeg, MB R3T 2N2, Canada
| | - George G Zhanel
- Department of Medical Microbiology, Faculty of Medicine, University of Manitoba, Winnipeg, MB R3E 3P5, Canada
| | - Richard Sparling
- Department of Microbiology, Faculty of Science, University of Manitoba, Winnipeg, MB R3T 2N2, Canada
| | - Richard A Holley
- Department of Food Science, Faculty of Agriculture and Food Sciences, University of Manitoba, Winnipeg, MB R3T 2N2, Canada.
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16
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Kim JH, Hwang KH, Park KS, Kong ID, Cha SK. Biological Role of Anti-aging Protein Klotho. J Lifestyle Med 2015; 5:1-6. [PMID: 26528423 PMCID: PMC4608225 DOI: 10.15280/jlm.2015.5.1.1] [Citation(s) in RCA: 151] [Impact Index Per Article: 15.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2014] [Accepted: 02/16/2015] [Indexed: 12/15/2022] Open
Abstract
Klotho-deficient mice have accelerated aging phenotypes, whereas overexpression of Klotho in mice extends lifespan. Klotho is an anti-aging single-pass membrane protein predominantly produced in the kidney, with shedding of the amino-terminal extracellular domain into the systemic circulation. Circulating levels of soluble Klotho decrease with age, and the klotho gene is associated with increased risk of age-related diseases. The three forms of Klotho protein have distinct functions. Membrane Klotho forms a complex with fibroblast growth factor (FGF) receptors, functions as an obligatory co-receptor for FGF23, which is involved in aging and the development of chronic diseases via regulation of Pi and vitamin D metabolism. Secreted Klotho functions as a humoral factor with pleiotropic activities including regulation of oxidative stress, growth factor signaling, and ion homeostasis. Secreted Klotho is also involved in organ protection. The intracellular form of Klotho suppresses inflammation-mediated cellular senescence and mineral metabolism. Herein we provide a brief overview of the structure and function and recent research about Klotho.
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Affiliation(s)
- Ji-Hee Kim
- Departments of Physiology and Global Medical Science, Yonsei University Wonju College of Medicine, Wonju, Korea
| | - Kyu-Hee Hwang
- Departments of Physiology and Global Medical Science, Yonsei University Wonju College of Medicine, Wonju, Korea
| | - Kyu-Sang Park
- Departments of Physiology and Global Medical Science, Yonsei University Wonju College of Medicine, Wonju, Korea ; Institute of Lifestyle Medicine, Yonsei University Wonju College of Medicine, Wonju, Korea
| | - In Deok Kong
- Departments of Physiology and Global Medical Science, Yonsei University Wonju College of Medicine, Wonju, Korea ; Institute of Lifestyle Medicine, Yonsei University Wonju College of Medicine, Wonju, Korea
| | - Seung-Kuy Cha
- Departments of Physiology and Global Medical Science, Yonsei University Wonju College of Medicine, Wonju, Korea ; Institute of Lifestyle Medicine, Yonsei University Wonju College of Medicine, Wonju, Korea ; Nuclear Receptor Research Consortium, Yonsei University Wonju College of Medicine, Wonju, Korea
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17
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Rotondi S, Pasquali M, Tartaglione L, Muci ML, Mandanici G, Leonangeli C, Sales S, Farcomeni A, Mazzaferro S. Soluble α -Klotho Serum Levels in Chronic Kidney Disease. Int J Endocrinol 2015; 2015:872193. [PMID: 25873958 PMCID: PMC4383388 DOI: 10.1155/2015/872193] [Citation(s) in RCA: 72] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/08/2014] [Accepted: 11/17/2014] [Indexed: 02/08/2023] Open
Abstract
Transmembrane α-Klotho (TM-Klotho), expressed in renal tubules, is a cofactor for FGF23-receptor. Circulating soluble-α-Klotho (s-Klotho) results from TM-Klotho shedding and acts on Phosphate (P) and Calcium (Ca) tubular transport. Decreased TM-Klotho, described in experimental chronic kidney disease (CKD), prevents actions of FGF23 and lessens circulating s-Klotho. Thus, levels of s-Klotho could represent a marker of CKD-MBD. To evaluate the clinical significance of s-Klotho in CKD we assayed serum s-Klotho and serum FGF23 in 68 patients (age 58 ± 15; eGFR 45 ± 21 mL/min). s-Klotho was lower than normal (519 ± 183 versus 845 ± 330 pg/mL, P < .0001) in renal patients and its reduction was detectable since CKD stage 2 (P < .01). s-Klotho correlated positively with eGFR and serum calcium (Cas) and negatively with serum phosphate (Ps), PTH and FGF23. FGF23 was higher than normal (73 ± 51 versus 36 ± 11, P < .0002) with significantly increased levels since CKD stage 2 (P < .001). Our data indicate a negative effect of renal disease on circulating s-Klotho starting very early in CKD. Assuming that s-Klotho mirrors TM-Klotho synthesis, low circulating s-Klotho seems to reflect the ensuing of tubular resistance to FGF23, which, accordingly, is increased. We endorse s-Klotho as an early marker of CKD-MBD.
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Affiliation(s)
- Silverio Rotondi
- Department of Cardiovascular, Respiratory, Nephrology, Geriatric, and Anesthetic Sciences, “Sapienza” University, 5 Piazzale Aldo Moro, 00185 Rome, Italy
| | - Marzia Pasquali
- Department of Cardiovascular, Respiratory, Nephrology, Geriatric, and Anesthetic Sciences, “Sapienza” University, 5 Piazzale Aldo Moro, 00185 Rome, Italy
| | - Lida Tartaglione
- Department of Cardiovascular, Respiratory, Nephrology, Geriatric, and Anesthetic Sciences, “Sapienza” University, 5 Piazzale Aldo Moro, 00185 Rome, Italy
| | - Maria Luisa Muci
- Department of Cardiovascular, Respiratory, Nephrology, Geriatric, and Anesthetic Sciences, “Sapienza” University, 5 Piazzale Aldo Moro, 00185 Rome, Italy
| | - Giusy Mandanici
- Department of Cardiovascular, Respiratory, Nephrology, Geriatric, and Anesthetic Sciences, “Sapienza” University, 5 Piazzale Aldo Moro, 00185 Rome, Italy
| | - Cristiana Leonangeli
- Department of Cardiovascular, Respiratory, Nephrology, Geriatric, and Anesthetic Sciences, “Sapienza” University, 5 Piazzale Aldo Moro, 00185 Rome, Italy
| | - Silvia Sales
- Department of Cardiovascular, Respiratory, Nephrology, Geriatric, and Anesthetic Sciences, “Sapienza” University, 5 Piazzale Aldo Moro, 00185 Rome, Italy
| | - Alessio Farcomeni
- Department of Public Health and Infectious Diseases, Section of Statistics, “Sapienza” University, 5 Piazzale Aldo Moro, 00185 Rome, Italy
| | - Sandro Mazzaferro
- Department of Cardiovascular, Respiratory, Nephrology, Geriatric, and Anesthetic Sciences, “Sapienza” University, 5 Piazzale Aldo Moro, 00185 Rome, Italy
- *Sandro Mazzaferro:
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Martín-Núñez E, Donate-Correa J, Muros-de-Fuentes M, Mora-Fernández C, Navarro-González JF. Implications of Klotho in vascular health and disease. World J Cardiol 2014; 6:1262-1269. [PMID: 25548616 PMCID: PMC4278161 DOI: 10.4330/wjc.v6.i12.1262] [Citation(s) in RCA: 47] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/25/2014] [Revised: 09/13/2014] [Accepted: 10/10/2014] [Indexed: 02/06/2023] Open
Abstract
Cardiovascular disease (CVD) is a prevalent condition in general population and the first cause of death overall. Klotho, a pleiotropic protein related to longevity that acts as a co-receptor of the fibroblast growth factor 23, has been proposed as a key regulator of the development of CVD. In the few clinical studies made, it has been observed a relationship between low levels of soluble Klotho and the occurrence and severity of CVD, as well as a reduction of cardiovascular risk when they are high. Also, different polymorphisms of human Klotho gene have been related to the incidence of cardiovascular events. Moreover, several experimental studies indicate that this protein acts in the maintenance of vascular homeostasis. Klotho improves endothelial dysfunction through promotion of NO production and mediates anti-inflammatory and anti-aging effects such as suppression of adhesion molecules expression, attenuation of nuclear factor-kappa B or inhibition of Wnt signaling. Furthermore, this protein is related to the attenuation of vascular calcification as well as prevention of cardiac hypertrophy. The expression of this protein in the vascular wall implies a new scenario for the treatment of vascular disorders. The purpose of this review is to provide an overview of the relationship between the Klotho protein and CVD, in addition to its role in the maintenance of functional vascular integrity.
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19
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Identification of Five Phospho-β-glycosidases fromLactobacillus gasseriATCC33323TCultured in Lactose Medium. Biosci Biotechnol Biochem 2014; 72:1954-7. [DOI: 10.1271/bbb.80089] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
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20
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Molecular evolution of glycoside hydrolase genes in the Western corn rootworm (Diabrotica virgifera virgifera). PLoS One 2014; 9:e94052. [PMID: 24718603 PMCID: PMC3981738 DOI: 10.1371/journal.pone.0094052] [Citation(s) in RCA: 52] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2013] [Accepted: 03/11/2014] [Indexed: 12/20/2022] Open
Abstract
Cellulose is an important nutritional resource for a number of insect herbivores. Digestion of cellulose and other polysaccharides in plant-based diets requires several types of enzymes including a number of glycoside hydrolase (GH) families. In a previous study, we showed that a single GH45 gene is present in the midgut tissue of the western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae). However, the presence of multiple enzymes was also suggested by the lack of a significant biological response when the expression of the gene was silenced by RNA interference. In order to clarify the repertoire of cellulose-degrading enzymes and related GH family proteins in D. v. virgifera, we performed next-generation sequencing and assembled transcriptomes from the tissue of three different developmental stages (eggs, neonates, and third instar larvae). Results of this study revealed the presence of seventy-eight genes that potentially encode GH enzymes belonging to eight families (GH45, GH48, GH28, GH16, GH31, GH27, GH5, and GH1). The numbers of GH45 and GH28 genes identified in D. v. virgifera are among the largest in insects where these genes have been identified. Three GH family genes (GH45, GH48, and GH28) are found almost exclusively in two coleopteran superfamilies (Chrysomeloidea and Curculionoidea) among insects, indicating the possibility of their acquisitions by horizontal gene transfer rather than simple vertical transmission from ancestral lineages of insects. Acquisition of GH genes by horizontal gene transfers and subsequent lineage-specific GH gene expansion appear to have played important roles for phytophagous beetles in specializing on particular groups of host plants and in the case of D. v. virgifera, its close association with maize.
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Siomou E, Stefanidis CJ. FGF-23 in children with CKD: a new player in the development of CKD-mineral and bone disorder. Nephrol Dial Transplant 2012; 27:4259-62. [PMID: 22848110 DOI: 10.1093/ndt/gfs315] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
Disturbances in mineral and bone metabolism in children with chronic kidney disease (CKD) lead to specific abnormalities of skeletal homeostasis called CKD-mineral and bone disorder (CKD-MBD). These disturbances should be diagnosed and managed appropriately to prevent bone deformities and disturbed growth. Changes in the vitamin D and parathyroid hormone (PTH), and the subsequent alterations in calcium (Ca) and phosphate (P) homeostasis are considered responsible for the development of CKD-MBD. Recently, a phosphaturic hormone, the fibroblast growth factor-23 (FGF-23), has been reported as a key regulator of P and vitamin D metabolism. A number of recent studies in paediatric populations have documented that the FGF-23 levels are increased early in CKD, before any abnormalities in serum Ca, P or PTH are apparent. The elevated FGF-23 levels result in a negative P balance to maintain P homeostasis, inducing phosphaturia, independently of PTH, and suppressing vitamin D synthesis. Therefore, the bone-kidney-parathyroid endocrine axis mediated by FGF-23 should be a novel therapeutic target in clinical practice, even in early stages of CKD in children.
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Affiliation(s)
- Ekaterini Siomou
- Department of Child Health, Medical School, University of Ioannina, Ioannina, Greece.
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Chang B, Kim J, Jeong D, Jeong Y, Jeon S, Jung SI, Yang Y, Kim KI, Lim JS, Kim C, Lee MS. Klotho inhibits the capacity of cell migration and invasion in cervical cancer. Oncol Rep 2012; 28:1022-8. [PMID: 22710352 DOI: 10.3892/or.2012.1865] [Citation(s) in RCA: 58] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2012] [Accepted: 05/22/2012] [Indexed: 11/05/2022] Open
Abstract
Aberrant activation of the Wnt/β-catenin signaling pathway is common in human cervical cancers. However, the mechanisms of Wnt activation in cervical cancer remain largely unknown. In the present study, we demonstrate that Klotho, a Wnt antagonist, is downregulated in invasive human cervical tumors and in a cell line we analyzed. Our data demonstrated that in vivo Klotho expression was not observed in invasive cervical carcinoma. In vitro restoration of Klotho expression in SiHa cells resulted in a decreased cell motility and invasiveness through upregulation of E-cadherin, downregulation of N-cadherin and reduced expression of MMP7 and -9. Ectopic expression of Klotho also reduced the expression of the epithelial-to-mesenchymal transition (EMT) transcription factors Slug and Twist. Furthermore, Klotho causes a significant inhibition of the Wnt/β-catenin pathway in cervical cancer cells, as supported by the expression of Wnt/β-catenin transcriptional target genes such as c-Myc and cyclin D1. Consequently, our findings demonstrate for the first time that Klotho regulates tumor invasion through the EMT process and provide novel mechanistic insights into the role of Klotho in cervical cancer progression and contribute to treatment for metastatic cervical cancer patients.
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Affiliation(s)
- Boogi Chang
- Research Center for Women's Diseases and Department of Biological Sciences, Sookmyung Women's University, Seoul, Republic of Korea
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Abstract
Endocrine fibroblast growth factors (FGFs) have been recognized as hormones that regulate a variety of metabolic processes. FGF19 is secreted from intestine upon feeding and acts on liver to suppress bile acid synthesis. FGF21 is secreted from liver upon fasting and acts on adipose tissue to promote lipolysis and responses to fasting. FGF23 is secreted from bone and acts on kidney to inhibit phosphate reabsorption and vitamin D synthesis. One critical feature of endocrine FGFs is that they require the Klotho gene family of transmembrane proteins as coreceptors to bind their cognate FGF receptors and exert their biological activities. This chapter overviews function of Klotho family proteins as obligate coreceptors for endocrine FGFs and discusses potential link between Klothos and age-related diseases.
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Li F, Hullar MAJ, Beresford SAA, Lampe JW. Variation of glucoraphanin metabolism in vivo and ex vivo by human gut bacteria. Br J Nutr 2011; 106:408-16. [PMID: 21342607 PMCID: PMC3137642 DOI: 10.1017/s0007114511000274] [Citation(s) in RCA: 88] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Glucosinolates, phytochemicals found in cruciferous vegetables, are metabolised to bioactive isothiocyanates (ITC) by certain bacteria in the human gut. Substantial individual variation in urinary ITC excretion has been observed in previous cruciferous vegetable-feeding studies. We hypothesised that individual differences in gut microbial community contribute to the observed variation in glucosinolate metabolism, i.e. gut microbiota composition between high- and low-ITC excreters differs. We recruited twenty-three healthy individuals and fed them a standardised meal containing 200 g of cooked broccoli. After the meal, 24 h urinary ITC excretion was measured. Study participants with the highest (n 5) and lowest (n 5) ITC excretion provided faecal samples for ex vivo bacterial cultivation with 50 μm-glucoraphanin, the major glucosinolate found in broccoli. When grown ex vivo, faecal bacteria from the selected high-ITC excreters were able to degrade more glucoraphanin than those from the low-ITC excreters (P = 0·05). However, bacterial fingerprints of faecal and ex vivo culture microbiota revealed no statistically significant differences between the high- and low-ITC excreters in terminal restriction fragment length polymorphism analysis of the bacterial 16S ribosomal RNA gene. In conclusion, glucosinolate degradation by faecal bacteria ex vivo may be associated with in vivo bacterial glucosinolate metabolism capacity, but no direct link to specific bacterial species could be established, possibly due to the complexity and functional redundancy of the gut microbiota.
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Affiliation(s)
- Fei Li
- Fred Hutchinson Cancer Research Center, Seattle, WA USA 98109
- Interdisplinary Graduate Program in Nutritional Sciences, University of Washington, Seattle, WA USA 98195
| | | | - Shirley AA Beresford
- Fred Hutchinson Cancer Research Center, Seattle, WA USA 98109
- Interdisplinary Graduate Program in Nutritional Sciences, University of Washington, Seattle, WA USA 98195
| | - Johanna W Lampe
- Fred Hutchinson Cancer Research Center, Seattle, WA USA 98109
- Interdisplinary Graduate Program in Nutritional Sciences, University of Washington, Seattle, WA USA 98195
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Chen B, Wang X, Zhao W, Wu J. Klotho inhibits growth and promotes apoptosis in human lung cancer cell line A549. JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH : CR 2010; 29:99. [PMID: 20642846 PMCID: PMC2912837 DOI: 10.1186/1756-9966-29-99] [Citation(s) in RCA: 89] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/15/2010] [Accepted: 07/19/2010] [Indexed: 01/02/2023]
Abstract
Background Klotho, as a new anti-aging gene, can shed into circulation and act as a multi-functional humoral factor that influences multiple biological processes. Recently, published studies suggest that klotho can also serve as a potential tumor suppressor. The aim of this study is to investigate the effects and possible mechanisms of action of klotho in human lung cancer cell line A549. Methods In this study, plasmids encoding klotho or klotho specific shRNAs were constructed to overexpress or knockdown klotho in vitro. A549 cells were respectively treated with pCMV6-MYC-KL or klotho specific shRNAs. The MTT assay was used to evaluate the cytotoxic effects of klotho and flow cytometry was utilized to observe and detect the apoptosis of A549 cells induced by klotho. The activation of IGF-1/insulin signal pathways in A549 cells treated by pCMV6-MYC-KL or shRNAs were evaluated by western blotting. The expression levels of bcl-2 and bax transcripts were evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Results Overexpression of klotho reduced the proliferation of lung cancer A549 cells, whereas klotho silencing in A549 cells enhanced proliferation. Klotho did not show any effects on HEK-293 cells. Klotho overexpression in A549 cells was associated with reduced IGF-1/insulin-induced phosphorylation of IGF-1R (IGF-1 receptor)/IR (insulin receptor) (P < 0.01). Overexpression of klotho can promote the apoptosis of A549 cells (P < 0.01). Overexpression of klotho, a bcl family gene bax, was found up-regulated and bcl-2, an anti-apoptosis gene, was found down-regulated (P < 0.01). In contrast, bax and bcl-2 were found down-regulated (P < 0.05) and up-regulated (P < 0.01), respectively when silencing klotho using shRNAs. Conclusions Klotho can inhibit proliferation and increase apoptosis of A549 cells, this may be partly due to the inhibition of IGF-1/insulin pathways and involving regulating the expression of the apoptosis-related genes bax/bcl-2. Thus, klotho can serve as a potential tumor suppressor in A549 cells.
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Affiliation(s)
- Bo Chen
- Department of Geriatrics, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu, China
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Overview of the FGF23-Klotho axis. Pediatr Nephrol 2010; 25:583-90. [PMID: 19626341 DOI: 10.1007/s00467-009-1260-4] [Citation(s) in RCA: 111] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/15/2009] [Revised: 06/15/2009] [Accepted: 06/16/2009] [Indexed: 01/31/2023]
Abstract
Recent studies have identified a novel bone-kidney endocrine axis that maintains phosphate homeostasis. When phosphate is in excess, fibroblast growth factor-23 (FGF23) is secreted from bone and acts on the kidney to promote phosphate excretion into urine and suppress vitamin D synthesis, thereby inducing negative phosphate balance. One critical feature of FGF23 is that it requires Klotho, a single-pass transmembrane protein expressed in renal tubules, as an obligate coreceptor to bind and activate FGF receptors. Several hereditary disorders that exhibit inappropriately high serum FGF23 levels are associated with phosphate wasting and impaired bone mineralization. In contrast, defects in either FGF23 or Klotho are associated with phosphate retention and a premature-aging syndrome. The aging-like phenotypes in Klotho-deficient or FGF23-deficient mice can be rescued by resolving hyperphosphatemia with dietary or genetic manipulation, suggesting a novel concept that phosphate retention accelerates aging. Phosphate retention is universally observed in patients with chronic kidney disease (CKD) and identified as a potent risk of death in epidemiological studies. Thus, the bone-kidney endocrine axis mediated by FGF23 and Klotho has emerged as a novel target of therapeutic interventions in CKD.
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Abstract
The klotho gene was identified as an "aging-suppressor" gene in mice that accelerates aging when disrupted and extends life span when overexpressed. It encodes a single-pass transmembrane protein and is expressed primarily in renal tubules. The extracellular domain of Klotho protein is secreted into blood and urine by ectodomain shedding. The two forms of Klotho protein, membrane Klotho and secreted Klotho, exert distinct functions. Membrane Klotho forms a complex with fibroblast growth factor (FGF) receptors and functions as an obligate co-receptor for FGF23, a bone-derived hormone that induces phosphate excretion into urine. Mice lacking Klotho or FGF23 not only exhibit phosphate retention but also display a premature-aging syndrome, revealing an unexpected link between phosphate metabolism and aging. Secreted Klotho functions as a humoral factor that regulates activity of multiple glycoproteins on the cell surface, including ion channels and growth factor receptors such as insulin/insulin-like growth factor-1 receptors. Potential contribution of these multiple activities of Klotho protein to aging processes is discussed.
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Kuro-o M. Klotho and aging. BIOCHIMICA ET BIOPHYSICA ACTA 2009; 1790:1049-58. [PMID: 19230844 PMCID: PMC2743784 DOI: 10.1016/j.bbagen.2009.02.005] [Citation(s) in RCA: 246] [Impact Index Per Article: 15.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Received: 12/19/2008] [Revised: 02/03/2009] [Accepted: 02/07/2009] [Indexed: 01/12/2023]
Abstract
The klotho gene encodes a single-pass transmembrane protein that forms a complex with multiple fibroblast growth factor (FGF) receptors and functions as an obligatory co-receptor for FGF23, a bone-derived hormone that induces negative phosphate balance. Defects in either Klotho or Fgf23 gene expression cause not only phosphate retention but also a premature-aging syndrome in mice, unveiling a potential link between phosphate metabolism and aging. In addition, the extracellular domain of Klotho protein is clipped on the cell surface and secreted into blood stream, potentially functioning as an endocrine factor. The secreted Klotho protein has a putative sialidase activity that modifies glycans on the cell surface, which may explain the ability of secreted Klotho protein to regulate activity of multiple ion channels and growth factors including insulin, IGF-1, and Wnt. Secreted Klotho protein also protects cells and tissues from oxidative stress through a mechanism yet to be identified. Thus, the transmembrane and secreted forms of Klotho protein have distinct functions, which may collectively affect aging processes in mammals.
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Affiliation(s)
- Makoto Kuro-o
- Department of Pathology, The University of Texas Southwestern Medical Center at Dallas, 6000 Harry Hines Blvd., Dallas, TX 75390-9072, USA.
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Li F, Hullar MAJ, Schwarz Y, Lampe JW. Human gut bacterial communities are altered by addition of cruciferous vegetables to a controlled fruit- and vegetable-free diet. J Nutr 2009; 139:1685-91. [PMID: 19640972 PMCID: PMC2728691 DOI: 10.3945/jn.109.108191] [Citation(s) in RCA: 132] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
In the human gut, commensal bacteria metabolize food components that typically serve as energy sources. These components have the potential to influence gut bacterial community composition. Cruciferous vegetables, such as broccoli and cabbage, contain distinctive compounds that can be utilized by gut bacteria. For example, glucosinolates can be hydrolyzed by certain bacteria, and dietary fibers can be fermented by a range of species. We hypothesized that cruciferous vegetable consumption would alter growth of certain bacteria, thereby altering bacterial community composition. We tested this hypothesis in a randomized, crossover, controlled feeding study. Fecal samples were collected from 17 participants at the end of 2 14-d intake periods: a low-phytochemical, low-fiber basal diet (i.e. refined grains without fruits or vegetables) and a high ("double") cruciferous vegetable diet [basal diet + 14 g cruciferous vegetables/(kg body weightd)]. Fecal bacterial composition was analyzed by the terminal restriction fragment length polymorphism (tRFLP) method using the bacterial 16S ribosomal RNA gene and nucleotide sequencing. Using blocked multi-response permutation procedures analysis, we found that overall bacterial community composition differed between the 2 consumption periods (delta = 0.603; P = 0.011). The bacterial community response to cruciferous vegetables was individual-specific, as revealed by nonmetric multidimensional scaling ordination analysis. Specific tRFLP fragments that characterized each of the diets were identified using indicator species analysis. Putative species corresponding to these fragments were identified through gene sequencing as Eubacterium hallii, Phascolarctobacterium faecium, Burkholderiales spp., Alistipes putredinis, and Eggerthella spp. In conclusion, human gut bacterial community composition was altered by cruciferous vegetable consumption, which could ultimately influence gut metabolism of bioactive food components and host exposure to these compounds.
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Affiliation(s)
- Fei Li
- Fred Hutchinson Cancer Research Center, Seattle, WA 98109 and Interdisplinary Graduate Program in Nutritional Sciences, University of Washington, Seattle, WA 98195
| | - Meredith A. J. Hullar
- Fred Hutchinson Cancer Research Center, Seattle, WA 98109 and Interdisplinary Graduate Program in Nutritional Sciences, University of Washington, Seattle, WA 98195
| | - Yvonne Schwarz
- Fred Hutchinson Cancer Research Center, Seattle, WA 98109 and Interdisplinary Graduate Program in Nutritional Sciences, University of Washington, Seattle, WA 98195
| | - Johanna W. Lampe
- Fred Hutchinson Cancer Research Center, Seattle, WA 98109 and Interdisplinary Graduate Program in Nutritional Sciences, University of Washington, Seattle, WA 98195
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31
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Abstract
Klotho is a new anti-aging gene. Genetic mutation of klotho causes multiple premature aging-like phenotypes and strikingly shortens lifespan. Overexpression of the klotho gene in mice suppresses aging and extends lifespan which may involve the mechanism of suppression of insulin signaling and oxidant stress. Klotho functions as a cofactor/coreceptor regulating fibroblast growth factor (FGF) 23 signaling. Klotho acts as a glucuronidase and activates ion channel TRPV5. Klotho protects against endothelial dysfunction and regulates the production of nitric oxide. Klotho also influences intracellular signaling pathways including p53/p21, cAMP, protein kinase C (PKC) and Wnt signaling pathways. The discovery of klotho has a great impact on aging research. The purpose of this review is to provide the recent progress and future directions of klotho research. Specifically, this review will cover: klotho and aging, structure and expression of the klotho gene, localization of klotho expression, source of circulating klotho, current understanding of klotho functions, and signaling pathways of klotho.
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Affiliation(s)
- Yuhong Wang
- Department of Physiology, College of Medicine, University of Oklahoma Health Sciences Center (OUHSC), Oklahoma City, OK 73104-0901, USA
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Sterner B, Singh R, Berger B. Predicting and annotating catalytic residues: an information theoretic approach. J Comput Biol 2007; 14:1058-73. [PMID: 17887954 DOI: 10.1089/cmb.2007.0042] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
We introduce a computational method to predict and annotate the catalytic residues of a protein using only its sequence information, so that we describe both the residues' sequence locations (prediction) and their specific biochemical roles in the catalyzed reaction (annotation). While knowing the chemistry of an enzyme's catalytic residues is essential to understanding its function, the challenges of prediction and annotation have remained difficult, especially when only the enzyme's sequence and no homologous structures are available. Our sequence-based approach follows the guiding principle that catalytic residues performing the same biochemical function should have similar chemical environments; it detects specific conservation patterns near in sequence to known catalytic residues and accordingly constrains what combination of amino acids can be present near a predicted catalytic residue. We associate with each catalytic residue a short sequence profile and define a Kullback-Leibler (KL) distance measure between these profiles, which, as we show, effectively captures even subtle biochemical variations. We apply the method to the class of glycohydrolase enzymes. This class includes proteins from 96 families with very different sequences and folds, many of which perform important functions. In a cross-validation test, our approach correctly predicts the location of the enzymes' catalytic residues with a sensitivity of 80% at a specificity of 99.4%, and in a separate cross-validation we also correctly annotate the biochemical role of 80% of the catalytic residues. Our results compare favorably to existing methods. Moreover, our method is more broadly applicable because it relies on sequence and not structure information; it may, furthermore, be used in conjunction with structure-based methods.
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Affiliation(s)
- Beckett Sterner
- Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
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Torres PU, Prié D, Molina-Blétry V, Beck L, Silve C, Friedlander G. Klotho: an antiaging protein involved in mineral and vitamin D metabolism. Kidney Int 2007; 71:730-7. [PMID: 17332731 DOI: 10.1038/sj.ki.5002163] [Citation(s) in RCA: 102] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Klotho gene mutation leads to a syndrome strangely resembling chronic kidney disease patients undergoing dialysis with multiple accelerated age-related disorders, including hypoactivity, sterility, skin thinning, muscle atrophy, osteoporosis, vascular calcifications, soft-tissue calcifications, defective hearing, thymus atrophy, pulmonary emphysema, ataxia, and abnormalities of the pituitary gland, as well as hypoglycemia, hyperphosphatemia, and paradoxically high-plasma calcitriol levels. Conversely, mice overexpressing klotho show an extended existence and a slow aging process through a mechanism that may involve the induction of a state of insulin and oxidant stress resistance. Two molecules are produced by the klotho gene, a membrane bound form and a circulating form. However, their precise biological roles and molecular functions have been only partly deciphered. Klotho can act as a circulating factor or hormone, which binds to a not yet identified high-affinity receptor and inhibits the intracellular insulin/insulin-like growth factor-1 (IGF-1) signaling cascade; klotho can function as a novel beta-glucuronidase, which deglycosylates steroid beta-glucuronides and the calcium channel transient receptor potential vallinoid-5 (TRPV5); as a cofactor essential for the stimulation of fibroblast growth factor (FGF) receptor by FGF23. The two last functions have propelled klotho to the group of key factors regulating mineral and vitamin D metabolism, and have also stimulated the interest of the nephrology community. The purpose of this review is to provide a nephrology-oriented overview of klotho and its potential implications in normal and altered renal function states.
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Affiliation(s)
- P-Ureña Torres
- Service de Néphrologie et Dialyse, Clinique de l'Orangerie, Aubervilliers, France.
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Mian IS, Worthey EA, Salavati R. Taking U out, with two nucleases? BMC Bioinformatics 2006; 7:305. [PMID: 16780580 PMCID: PMC1525001 DOI: 10.1186/1471-2105-7-305] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2006] [Accepted: 06/16/2006] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND REX1 and REX2 are protein components of the RNA editing complex (the editosome) and function as exouridylylases. The exact roles of REX1 and REX2 in the editosome are unclear and the consequences of the presence of two related proteins are not fully understood. Here, a variety of computational studies were performed to enhance understanding of the structure and function of REX proteins in Trypanosoma and Leishmania species. RESULTS Sequence analysis and homology modeling of the Endonuclease/Exonuclease/Phosphatase (EEP) domain at the C-terminus of REX1 and REX2 highlights a common active site shared by all EEP domains. Phylogenetic analysis indicates that REX proteins contain a distinct subfamily of EEP domains. Inspection of three-dimensional models of the EEP domain in Trypanosoma brucei REX1 and REX2, and Leishmania major REX1 suggests variations of previously characterized key residues likely to be important in catalysis and determining substrate specificity. CONCLUSION We have identified features of the REX EEP domain that distinguish it from other family members and hence subfamily specific determinants of catalysis and substrate binding. The results provide specific guidance for experimental investigations about the role(s) of REX proteins in RNA editing.
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Affiliation(s)
- I Saira Mian
- Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720-8265, USA
| | | | - Reza Salavati
- Seattle Biomedical Research Institute, Seattle, Washington, 98109, USA
- McGill University, Institute of Parasitology, Ste.-Anne-De-Bellevue, Quebec, H9X 3V9, Canada
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Marques AR, Coutinho PM, Videira P, Fialho AM, Sá-Correia I. Sphingomonas paucimobilis beta-glucosidase Bgl1: a member of a new bacterial subfamily in glycoside hydrolase family 1. Biochem J 2003; 370:793-804. [PMID: 12444924 PMCID: PMC1223213 DOI: 10.1042/bj20021249] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2002] [Revised: 11/13/2002] [Accepted: 11/22/2002] [Indexed: 01/08/2023]
Abstract
The Sphingomonas paucimobilis beta-glucosidase Bgl1 is encoded by the bgl1 gene, associated with an 1308 bp open reading frame. The deduced protein has a potential signal peptide of 24 amino acids in the N-terminal region, and experimental evidence is consistent with the processing and export of the Bgl1 protein through the inner membrane to the periplasmic space. A His(6)-tagged 44.3 kDa protein was over-produced in the cytosol of Escherichia coli from a recombinant plasmid, which contained the S. paucimobilis bgl1 gene lacking the region encoding the putative signal peptide. Mature beta-glucosidase Bgl1 is specific for aryl-beta-glucosides and has no apparent activity with oligosaccharides derived from cellulose hydrolysis and other saccharides. A structure-based alignment established structural relations between S. paucimobilis Bgl1 and other members of the glycoside hydrolase (GH) family 1 enzymes. At subsite -1, the conserved residues required for catalysis by GH1 enzymes are present in Bgl1 with only minor differences. Major differences are found at subsite +1, the aglycone binding site. This alignment seeded a sequence-based phylogenetic analysis of GH1 enzymes, revealing an absence of horizontal transfer between phyla. Bootstrap analysis supported the definition of subfamilies and revealed that Bgl1, the first characterized beta-glucosidase from the genus Sphingomonas, represents a very divergent bacterial subfamily, closer to archaeal subfamilies than to others of bacterial origin.
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Affiliation(s)
- Ana Rita Marques
- Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal
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Grate LR, Bhattacharyya C, Jordan MI, Mian IS. Integrated analysis of transcript profiling and protein sequence data. Mech Ageing Dev 2003; 124:109-14. [PMID: 12618013 DOI: 10.1016/s0047-6374(02)00174-4] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
Transcript profiling can be used to elucidate the molecular and cellular mechanisms involved in ageing and cancer. A recent study of human gastrointestinal stromal tumours (GISTs) with mutations in the KIT gene, Cancer Res. 61 (2001) 8624 exemplifies a common type of investigation. cDNA microarrays were used to generate measurements for 1987 clones in two types of tissues: 13 KIT mutation-positive GISTs and 6 spindle cell tumours from locations outside the gastrointestinal tract. Statistical problems associated with such two-class, high-dimensional profiling data include simultaneous classification and relevant feature identification, probabilistic clustering and protein sequence family modelling. Here, the GIST data were reexamined using specific solutions to these problems, namely sparse hyperplanes, nai;ve Bayes models and profile hidden Markov models respectively. The integrated analysis of molecular profiling and sequence data highlighted 6 clones that may be of clinical and experimental interest. The protein encoded by one of these putative biomarkers defined a novel protein family present in diverse eucarya. The family may be involved in chromosome segregation and/or stability. One family member is a potential biomarker identified recently from a retrospective analysis of transcript profiles for sporadic breast cancer samples from patients with poor and good prognosis, Signal Process. (in press).
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Affiliation(s)
- L R Grate
- Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA
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37
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Sanchez JM, Perillo MA. Membrane topology modulates beta-galactosidase activity against soluble substrates. Biophys Chem 2002; 99:281-95. [PMID: 12408942 DOI: 10.1016/s0301-4622(02)00229-6] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
The effect of bio-surfaces of contrasting curvature, on the kinetic parameters of ortho-nitrophenyl-beta-D-galactopiranoside hydrolysis catalyzed by E. coli beta-galactosidase, was investigated. The self-aggregating state and structure of the amphiphiles (Phosphatidylcholine, Lubrol-PX, Triton X-100, DocNa, SDS and CTAB) were inferred from their c.m.c. values and light-scattering measurements. Low curvature phosphatidylcholine or mixed phosphatidylcholine-detergent vesicles increased V(max) without affecting K(M). High curvature micellar structures containing ionic detergents modulated negatively the enzyme activity (decreased or abolished V(max) and increased K(M)). Neither micelles containing non-ionic detergents nor the amphiphiles in a monomeric form, affected enzyme activity. CTAB at a concentration below its c.m.c but incorporated into a bilayer, became an activator (K(M) decreased respect to the control). Non-enzymatic interfacial hydrolysis of the substrate was discarded. Enzyme-membrane interaction and membrane elasticity, were evaluated using monomolecular layers at the air-water interface. Beyond particular molecular structures, topology affected the direction of the modulatory effects exerted by these amphiphiles on beta-galactosidase activity.
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Affiliation(s)
- Julieta M Sanchez
- Departamento de Química, Facultad de Ciencias Exactas, Fisicas y Naturales, Universidad Nacional de Cordoba, Av. Velez Sarsfield 299, 5000 Cordoba, Argentina
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38
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Ito S, Fujimori T, Hayashizaki Y, Nabeshima YI. Identification of a novel mouse membrane-bound family 1 glycosidase-like protein, which carries an atypical active site structure. BIOCHIMICA ET BIOPHYSICA ACTA 2002; 1576:341-5. [PMID: 12084582 DOI: 10.1016/s0167-4781(02)00281-6] [Citation(s) in RCA: 88] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
We have identified a novel mouse gene klph (Klotho-LPH related protein; where LPH stands for lactase-phlorizin hydrolase) that encodes a novel mammalian family 1 glycosidase-like protein. KLPH was a putative type I membrane protein that consists of N-terminal signal sequence, glycosidase domain, transmembrane region and short cytoplasmic tail. Despite its overall structural similarity to other family 1 glycosidases, the glutamic acid for the acid-base catalyst was not conserved in this protein. klph mRNA was predominantly expressed in the kidney and skin. Epitope-tagged KLPH was localized to the perinuclear tubular network structure of the endoplasmic reticulum in cultured cells.
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Affiliation(s)
- Shinji Ito
- Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Yoshida Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
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39
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Arking DE, Krebsova A, Macek M, Macek M, Arking A, Mian IS, Fried L, Hamosh A, Dey S, McIntosh I, Dietz HC. Association of human aging with a functional variant of klotho. Proc Natl Acad Sci U S A 2002; 99:856-61. [PMID: 11792841 PMCID: PMC117395 DOI: 10.1073/pnas.022484299] [Citation(s) in RCA: 357] [Impact Index Per Article: 15.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Mice deficient in Klotho gene expression exhibit a syndrome resembling premature human aging. To determine whether variation in the human KLOTHO locus contributes to survival, we applied two newly characterized polymorphic microsatellite markers flanking the gene in a population-based association study. In a cohort chosen for its homogeneity, Bohemian Czechs, we demonstrated significant differences in selected marker allele frequencies between newborn and elderly individuals (P < 0.05). These results precipitated a search for functional variants of klotho. We identified an allele, termed KL-VS, containing six sequence variants in complete linkage disequilibrium, two of which result in amino acid substitutions F352V and C370S. Homozygous elderly individuals were underrepresented in three distinct populations: Bohemian Czechs, Baltimore Caucasians, and Baltimore African-Americans [combined odds ratio (OR) = 2.59, P < 0.0023]. In a transient transfection assay, secreted levels of klotho harboring V352 are reduced 6-fold, whereas extracellular levels of the S370 form are increased 2.9-fold. The V352/S370 double mutant exhibits an intermediate phenotype (1.6-fold increase), providing a rare example of intragenic complementation in cis by human single nucleotide polymorphisms. The remarkable conservation of F352 among homologous proteins suggests that it is functionally important. The corresponding substitution, F289V, in the closest human klotho paralog with a known substrate, cBGL1, completely eliminates its ability to cleave p-nitrophenyl-beta-D-glucoside. These results suggest that the KL-VS allele influences the trafficking and catalytic activity of klotho, and that variation in klotho function contributes to heterogeneity in the onset and severity of human age-related phenotypes.
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Affiliation(s)
- Dan E Arking
- Institute of Genetic Medicine, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA
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de Graaf M, van Veen IC, van der Meulen-Muileman IH, Gerritsen WR, Pinedo HM, Haisma HJ. Cloning and characterization of human liver cytosolic beta-glycosidase. Biochem J 2001; 356:907-10. [PMID: 11389701 PMCID: PMC1221920 DOI: 10.1042/0264-6021:3560907] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
Cytosolic beta-glucosidase (EC 3.2.1.21) from mammalian liver is a member of the family 1 glycoside hydrolases and is known for its ability to hydrolyse a range of beta-D-glycosides, including beta-D-glucoside and beta-D-galactoside. We therefore refer to this enzyme as cytosolic beta-glycosidase. We cloned the cDNA encoding the human cytosolic beta-glycosidase by performing PCR on cDNA prepared from total human liver RNA. Specific primers were based on human expressed sequence tags found in the expressed sequence tag database. The cloned cDNA contained 1407 nt with an open reading frame encoding 469 amino acid residues. Amino acid sequence analysis indicates that human cytosolic beta-glycosidase is most closely related to lactase phlorizin hydrolase and klotho protein. The enzyme was characterized by using cell lysates of COS-7 cells transfected with a eukaryotic expression vector containing the cDNA. The biochemical, kinetic and inhibition properties of the cloned enzyme were found to be identical with those reported for the enzyme purified from human liver.
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Affiliation(s)
- M de Graaf
- Department of Medical Oncology, Division of Gene Therapy, University Hospital Vrije Universiteit, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands.
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