Publications by authors named "Reiko Sakaguchi"

35 Publications

Safety and Effectiveness of Lurasidone in Patients with Schizophrenia: A 12-Week, Open-Label Extension Study.

Neuropsychiatr Dis Treat 2021 16;17:2683-2695. Epub 2021 Aug 16.

Japan Depression Center, Tokyo, Japan.

Purpose: The goal of this study was to evaluate the safety and effectiveness of lurasidone among patients with schizophrenia in a 12-week open-label extension study.

Patients And Methods: Patients who completed a 6-week, double-blind, placebo-controlled study were enrolled in a 12-week open-label extension study with flexible dosing of lurasidone at 40 or 80 mg/day. Safety assessments included adverse events, vital signs, laboratory tests, and electrocardiogram (ECG) parameters. Effectiveness measures included the Positive and Negative Syndrome Scale (PANSS) total score, Clinical Global Impression-Severity Scale (CGI-S), Calgary Depression Scale for Schizophrenia (CDSS) and quality of life measure.

Results: A total of 289 patients were enrolled in the open-label extension study. Rates of treatment-emergent adverse events (TEAEs) were low; akathisia was the most common TEAE with an incidence of 6.6%. There were 54 patients (18.7%) who discontinued the extension study, with 17 (5.9%) discontinuing due to adverse events. Minimal or no effects of lurasidone on weight, body mass index, metabolic parameters, prolactin, and ECG parameters were evident. There was continued improvement to week 12 in PANSS and CGI-S scores beyond the initial gains made during the prior 6-week double-blind study. Non-responders to lurasidone 40 mg/day in the prior 6-week study showed a mean (standard deviation) improvement from open-label baseline of 10.7 (13.8) points on the PANSS total score after lurasidone dose was increased to a modal dose of 80 mg/day during the extension study. Changes from double-blind baseline in CDSS and quality of life were maintained in the extension study.

Conclusion: Treatment with lurasidone 40 or 80 mg once daily (flexibly dosed) continued to be well tolerated with patients demonstrating further improvement in symptoms over the course of a 12-week open-label extension study in patients with schizophrenia.
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http://dx.doi.org/10.2147/NDT.S320021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8379682PMC
August 2021

Efficacy and safety of lurasidone in acutely psychotic patients with schizophrenia: A 6-week, randomized, double-blind, placebo-controlled study.

Psychiatry Clin Neurosci 2021 Apr 23. Epub 2021 Apr 23.

Japan Depression Center, Tokyo, Japan.

Aim: The aim of this study was to evaluate the efficacy of lurasidone in acute schizophrenia in Japan and other countries.

Methods: Subjects (aged 18-74 years) diagnosed with schizophrenia were randomized to lurasidone 40 mg/day or placebo. The primary efficacy endpoint was change from baseline on the Positive and Negative Syndrome Scale (PANSS) total score at Week 6. Secondary efficacy assessments included the Clinical Global Impression-Severity Scale (CGI-S). Safety endpoints included adverse events, and laboratory and electrocardiogram parameters.

Results: A total of 483 subjects were randomized to lurasidone or placebo; 107 subjects were from Japan. Mean changes from baseline at Week 6 endpoint in PANSS total scores were -19.3 in the lurasidone group and -12.7 in the placebo group (treatment difference: P < 0.001, effect size = 0.41). Changes from baseline for Week 6 CGI-S scores were -1.0 for lurasidone and -0.7 for placebo (treatment difference: P < 0.001, effect size = 0.41). All-cause discontinuation during the 6-week, double-blind period was 19.4% for lurasidone and 25.4% for placebo, and discontinuation rates due to adverse event were 5.7% for lurasidone and 6.4% for placebo. The following common treatment-emergent adverse events occurred in more than 2% on lurasidone and at a rate at least twice that of the placebo group: akathisia (4.0%), dizziness (2.8%), somnolence (2.8%), abdominal discomfort (2.0%) and asthenia (2.0%). No significant changes in bodyweight or metabolic parameters were observed.

Conclusion: Lurasidone 40 mg once daily dosing demonstrated efficacy in a patient population with acute schizophrenia, including subjects from Japan, and was generally safe and well-tolerated.
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http://dx.doi.org/10.1111/pcn.13221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8361730PMC
April 2021

Inositol 1,4,5-trisphosphate 3-kinase B promotes Ca mobilization and the inflammatory activity of dendritic cells.

Sci Signal 2021 Mar 30;14(676). Epub 2021 Mar 30.

Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126 Milan, Italy.

Innate immune responses to Gram-negative bacteria depend on the recognition of lipopolysaccharide (LPS) by a receptor complex that includes CD14 and TLR4. In dendritic cells (DCs), CD14 enhances the activation not only of TLR4 but also that of the NFAT family of transcription factors, which suppresses cell survival and promotes the production of inflammatory mediators. NFAT activation requires Ca mobilization. In DCs, Ca mobilization in response to LPS depends on phospholipase C γ2 (PLCγ2), which produces inositol 1,4,5-trisphosphate (IP). Here, we showed that the IP receptor 3 (IPR3) and ITPKB, a kinase that converts IP to inositol 1,3,4,5-tetrakisphosphate (IP), were both necessary for Ca mobilization and NFAT activation in mouse and human DCs. A pool of IPR3 was located on the plasma membrane of DCs, where it colocalized with CD14 and ITPKB. Upon LPS binding to CD14, ITPKB was required for Ca mobilization through plasma membrane-localized IPR3 and for NFAT nuclear translocation. Pharmacological inhibition of ITPKB in mice reduced both LPS-induced tissue swelling and the severity of inflammatory arthritis to a similar extent as that induced by the inhibition of NFAT using nanoparticles that delivered an NFAT-inhibiting peptide specifically to phagocytic cells. Our results suggest that ITPKB may represent a promising target for anti-inflammatory therapies that aim to inhibit specific DC functions.
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http://dx.doi.org/10.1126/scisignal.aaz2120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8091591PMC
March 2021

Terahertz pulse-altered gene networks in human induced pluripotent stem cells.

Opt Lett 2020 Nov;45(21):6078-6081

Terahertz (THz) irradiation has been exploited in biomedical applications involving non-invasive manipulation of living cells. We developed an apparatus for studying the effects of THz pulse irradiation on living human induced pluripotent stem cells. The THz pulse of the maximum electric field reached 0.5 MV/cm and was applied for one hour with 1 kHz repetition to the entire cell-culture area, a diameter of 1 mm. RNA sequencing of global gene-expression revealed that many THz-regulated genes were driven by zinc-finger transcription factors. Combined with a consideration of the interactions of metal ions and a THz electric field, these results imply that the local intracellular concentration of metal ions, such as , was changed by the effective electrical force of our THz pulse.
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November 2020

O-Dependent Protein Internalization Underlies Astrocytic Sensing of Acute Hypoxia by Restricting Multimodal TRPA1 Channel Responses.

Curr Biol 2020 09 16;30(17):3378-3396.e7. Epub 2020 Jul 16.

Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan. Electronic address:

Hypoxia sensors are essential for regulating local oxygen (O) homeostasis within the body. This is especially pertinent within the CNS, which is particularly vulnerable to O deprivation due to high energetic demand. Here, we reveal hypoxia-monitoring function exerted by astrocytes through an O-regulated protein trafficking mechanism within the CNS. Strikingly, cultured mouse astrocytes isolated from the parafacial respiratory group (pFRG) and retrotrapezoid nucleus (RTN) region are capable of rapidly responding to moderate hypoxia via the sensor cation channel transient receptor potential (TRP) A1 but, unlike multimodal sensory neurons, are inert to hyperoxia and other TRPA1 activators (carbon dioxide, electrophiles, and oxidants) in normoxia. Mechanistically, O suppresses TRPA1 channel activity by protein internalization via O-dependent proline hydroxylation and subsequent ubiquitination by an E3 ubiquitin ligase, NEDD4-1 (neural precursor cell-expressed developmentally down-regulated protein 4). Hypoxia inhibits this process and instantly accumulates TRPA1 proteins at the plasma membrane, inducing TRPA1-mediated Ca influx that triggers ATP release from pFRG/RTN astrocytes, potentiating respiratory center activity. Furthermore, astrocyte-specific Trpa1 disruption in a mouse brainstem-spinal cord preparation impedes the amplitude augmentation of the central autonomic respiratory output during hypoxia. Thus, reversible coupling of the TRPA1 channels with O-dependent protein translocation allows astrocytes to act as acute hypoxia sensors in the medullary respiratory center.
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http://dx.doi.org/10.1016/j.cub.2020.06.047DOI Listing
September 2020

Fluorescence detection of the nitric oxide-induced structural change at the putative nitric oxide sensing segment of TRPC5.

Bioorg Med Chem 2020 04 17;28(8):115430. Epub 2020 Mar 17.

Institute of Advanced Energy, Kyoto University, Uji, Kyoto 611-0011, Japan. Electronic address:

The plausible nitric oxide (NO)-sensing module of TRPC5 was incorporated in a enhanced green fluorescent protein (EGFP) to evaluate its conformational change as an optical response upon the reaction with NO. Two cysteine residues located in the NO-sensing module have been proposed to form a disulfide bond through S-nitrosylation of the thiol group by NO. Modification of the cysteine residues by NO resulted a ratiometric change of EGFP emission through transducing the conformational change of NO-sensing module to the EGFP chromophore. The oxidized form of NO-sensing module fused EGFP changed the intensity of emission spectra upon reduction of the disulfide bond at the NO-reactive module. The NO-sensing module fused EGFP in its reduced form avidly reacted with NO and realized the ratiometric fluorescence intensity changes depending on the formation of disulfide bond. These results support the notion that NO induces a conformational change at the putative NO-sensing segment of TRPC5, and provide a prototype for the genetically encoded cellular NO sensors.
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http://dx.doi.org/10.1016/j.bmc.2020.115430DOI Listing
April 2020

Variants That Affect Function of Calcium Channel TRPV6 Are Associated With Early-Onset Chronic Pancreatitis.

Gastroenterology 2020 05 10;158(6):1626-1641.e8. Epub 2020 Jan 10.

Department of Gastroenterology and Hepatology, Tokyo Medical University, Tokyo, Japan.

Background & Aims: Changes in pancreatic calcium levels affect secretion and might be involved in development of chronic pancreatitis (CP). We investigated the association of CP with the transient receptor potential cation channel subfamily V member 6 gene (TRPV6), which encodes a Ca-selective ion channel, in an international cohort of patients and in mice.

Methods: We performed whole-exome DNA sequencing from a patient with idiopathic CP and from his parents, who did not have CP. We validated our findings by sequencing DNA from 300 patients with CP (not associated with alcohol consumption) and 1070 persons from the general population in Japan (control individuals). In replication studies, we sequenced DNA from patients with early-onset CP (20 years or younger) not associated with alcohol consumption from France (n = 470) and Germany (n = 410). We expressed TRPV6 variants in HEK293 cells and measured their activity using Ca imaging assays. CP was induced by repeated injections of cerulein in TRPV6 mice.

Results: We identified the variants c.629C>T (p.A210V) and c.970G>A (p.D324N) in TRPV6 in the index patient. Variants that affected function of the TRPV6 product were found in 13 of 300 patients (4.3%) and 1 of 1070 control individuals (0.1%) from Japan (odds ratio [OR], 48.4; 95% confidence interval [CI], 6.3-371.7; P = 2.4 × 10). Twelve of 124 patients (9.7%) with early-onset CP had such variants. In the replication set from Europe, 18 patients with CP (2.0%) carried variants that affected the function of the TRPV6 product compared with 0 control individuals (P = 6.2 × 10). Variants that did not affect the function of the TRPV6 product (p.I223T and p.D324N) were overrepresented in Japanese patients vs control individuals (OR, 10.9; 95% CI, 4.5-25.9; P = 7.4 × 10 for p.I223T and P = .01 for p.D324N), whereas the p.L299Q was overrepresented in European patients vs control individuals (OR, 3.0; 95% CI, 1.9-4.8; P = 1.2 × 10). TRPV6 mice given cerulein developed more severe pancreatitis than control mice, as shown by increased levels of pancreatic enzymes, histologic alterations, and pancreatic fibrosis.

Conclusions: We found that patients with early-onset CP not associated with alcohol consumption carry variants in TRPV6 that affect the function of its product, perhaps by altering Ca balance in pancreatic cells. TRPV6 regulates Ca homeostasis and pancreatic inflammation.
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http://dx.doi.org/10.1053/j.gastro.2020.01.005DOI Listing
May 2020

Detection of Inositol Phosphates by Split PH Domains.

Methods Mol Biol 2020 ;2091:47-57

Institute of Advanced Energy, Kyoto University, Uji, Kyoto, Japan.

The pleckstrin homology (PH) domain is a family of structurally conserved proteins which can bind inositol phosphate derivatives. Some proteins involved in cellular signaling and cytoskeletal organization possess split PH domains that assemble into a structure which can bind specific inositol phosphates. Here we describe the design of split PH domain from a structurally well-characterized PH domain of phospholipase C (PLC) δ and Bruton's tyrosine kinase (Btk), which selectively bind Ins(1,4,5)P and Ins(1,3,4,5)P, respectively. The PH domains fold into a functional structure when the split halves are brought to close proximity, and can be utilized to detect specific inositol phosphate of interest.
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http://dx.doi.org/10.1007/978-1-0716-0167-9_4DOI Listing
January 2021

Transient receptor potential (TRP) channels: Biosensors for redox environmental stimuli and cellular status.

Free Radic Biol Med 2020 01 2;146:36-44. Epub 2019 Nov 2.

Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, 615-8510, Japan; The World Premier International Research Initiative-Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto, 615-8510, Japan. Electronic address:

Transient receptor potential (TRP) channels are a family of cation channels that depolarizes the membrane potential and regulates intracellular concentrations of cations such as Ca. TRP channels are also known to function as "biosensors" to detect changes of the surrounding environment and cellular status. Lines of evidence have unveiled that numerous proteins are subject to redox modification and subsequent signaling. For example, TRPM2, TRPC5, TRPV1, and TRPA1 are known as redox sensors activated by hydrogen peroxide (HO), nitric oxide (NO), and electrophiles. Thus, these channels facilitate the influx of cations which in turn triggers the appropriate cellular responses against environmental redox stimuli and cellular redox status. In this review, we focus on the recent findings regarding the functions of TRP channels in relation to other ion channels, and other proteins which also go through redox modification of cysteine (Cys) residues. We aim to understand the structural and molecular basis of the redox-sensing mechanisms of TRP channels in exerting various functions under physiological conditions as well as pathological conditions such as cancer malignancy. Their future potential as drug targets will also be discussed.
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http://dx.doi.org/10.1016/j.freeradbiomed.2019.10.415DOI Listing
January 2020

Contribution of Coiled-Coil Assembly to Ca/Calmodulin-Dependent Inactivation of TRPC6 Channel and its Impacts on FSGS-Associated Phenotypes.

J Am Soc Nephrol 2019 09 2;30(9):1587-1603. Epub 2019 Jul 2.

Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering,

Background: TRPC6 is a nonselective cation channel, and mutations of this gene are associated with FSGS. These mutations are associated with TRPC6 current amplitude amplification and/or delay of the channel inactivation (gain-of-function phenotype). However, the mechanism of the gain-of-function in TRPC6 activity has not yet been clearly solved.

Methods: We performed electrophysiologic, biochemical, and biophysical experiments to elucidate the molecular mechanism underlying calmodulin (CaM)-mediated Ca-dependent inactivation (CDI) of TRPC6. To address the pathophysiologic contribution of CDI, we assessed the actin filament organization in cultured mouse podocytes.

Results: Both lobes of CaM helped induce CDI. Moreover, CaM binding to the TRPC6 CaM-binding domain (CBD) was Ca-dependent and exhibited a 1:2 (CaM/CBD) stoichiometry. The TRPC6 coiled-coil assembly, which brought two CBDs into adequate proximity, was essential for CDI. Deletion of the coiled-coil slowed CDI of TRPC6, indicating that the coiled-coil assembly configures both lobes of CaM binding on two CBDs to induce normal CDI. The FSGS-associated TRPC6 mutations within the coiled-coil severely delayed CDI and often increased TRPC6 current amplitudes. In cultured mouse podocytes, FSGS-associated channels and CaM mutations led to sustained Ca elevations and a disorganized cytoskeleton.

Conclusions: The gain-of-function mechanism found in FSGS-causing mutations in TRPC6 can be explained by impairments of the CDI, caused by disruptions of TRPC's coiled-coil assembly which is essential for CaM binding. The resulting excess Ca may contribute to structural damage in the podocytes.
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http://dx.doi.org/10.1681/ASN.2018070756DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6727271PMC
September 2019

TRPM7 channels mediate spontaneous Ca fluctuations in growth plate chondrocytes that promote bone development.

Sci Signal 2019 04 9;12(576). Epub 2019 Apr 9.

Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 615-8501, Japan.

During endochondral ossification of long bones, the proliferation and differentiation of chondrocytes cause them to be arranged into layered structures constituting the epiphyseal growth plate, where they secrete the cartilage matrix that is subsequently converted into trabecular bone. Ca signaling has been implicated in chondrogenesis in vitro. Through fluorometric imaging of bone slices from embryonic mice, we demonstrated that live growth plate chondrocytes generated small, cell-autonomous Ca fluctuations that were associated with weak and intermittent Ca influx. Several genes encoding Ca-permeable channels were expressed in growth plate chondrocytes, but only pharmacological inhibitors of transient receptor potential cation channel subfamily M member 7 (TRPM7) reduced the spontaneous Ca fluctuations. The TRPM7-mediated Ca influx was likely activated downstream of basal phospholipase C activity and was potentiated upon cell hyperpolarization induced by big-conductance Ca-dependent K channels. Bones from embryos in which was conditionally knocked out during ex vivo culture exhibited reduced outgrowth and displayed histological abnormalities accompanied by insufficient autophosphorylation of Ca/calmodulin-dependent protein kinase II (CaMKII) in the growth plate. The link between TRPM7-mediated Ca fluctuations and CaMKII-dependent chondrogenesis was further supported by experiments with chondrocyte-specific knockout mice. Thus, growth plate chondrocytes generate spontaneous, TRPM7-mediated Ca fluctuations that promote self-maturation and bone development.
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http://dx.doi.org/10.1126/scisignal.aaw4847DOI Listing
April 2019

Intracellular thermometry with fluorescent sensors for thermal biology.

Pflugers Arch 2018 05 4;470(5):717-731. Epub 2018 Feb 4.

Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, 615-8510, Japan.

Temperature influences the activities of living organisms at various levels. Cells not only detect environmental temperature changes through their unique temperature-sensitive molecular machineries but also muster an appropriate response to the temperature change to maintain their inherent functions. Despite the fundamental involvement of temperature in physiological phenomena, the mechanism by which cells produce and use heat is largely unknown. Recently, fluorescent thermosensors that function as thermometers in live cells have attracted much attention in biology. These new tools, made of various temperature-sensitive molecules, have allowed for intracellular thermometry at the single-cell level. Intriguing spatiotemporal temperature variations, including organelle-specific thermogenesis, have been revealed with these fluorescent thermosensors, which suggest an intrinsic connection between temperature and cell functions. Moreover, fluorescent thermosensors have shown that intracellular temperature changes at the microscopic level are largely different from those assumed for a water environment at the macroscopic level. Thus, the employment of fluorescent thermosensors will uncover novel mechanisms of intracellular temperature-assisted physiological functions.
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http://dx.doi.org/10.1007/s00424-018-2113-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5942359PMC
May 2018

A genetically encoded fluorescent tRNA is active in live-cell protein synthesis.

Nucleic Acids Res 2017 04;45(7):4081-4093

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107, USA.

Transfer RNAs (tRNAs) perform essential tasks for all living cells. They are major components of the ribosomal machinery for protein synthesis and they also serve in non-ribosomal pathways for regulation and signaling metabolism. We describe the development of a genetically encoded fluorescent tRNA fusion with the potential for imaging in live Escherichia coli cells. This tRNA fusion carries a Spinach aptamer that becomes fluorescent upon binding of a cell-permeable and non-toxic fluorophore. We show that, despite having a structural framework significantly larger than any natural tRNA species, this fusion is a viable probe for monitoring tRNA stability in a cellular quality control mechanism that degrades structurally damaged tRNA. Importantly, this fusion is active in E. coli live-cell protein synthesis allowing peptidyl transfer at a rate sufficient to support cell growth, indicating that it is accommodated by translating ribosomes. Imaging analysis shows that this fusion and ribosomes are both excluded from the nucleoid, indicating that the fusion and ribosomes are in the cytosol together possibly engaged in protein synthesis. This fusion methodology has the potential for developing new tools for live-cell imaging of tRNA with the unique advantage of both stoichiometric labeling and broader application to all cells amenable to genetic engineering.
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http://dx.doi.org/10.1093/nar/gkw1229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5397188PMC
April 2017

Methyl transfer by substrate signaling from a knotted protein fold.

Nat Struct Mol Biol 2016 Oct 29;23(10):941-948. Epub 2016 Aug 29.

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.

Proteins with knotted configurations, in comparison with unknotted proteins, are restricted in conformational space. Little is known regarding whether knotted proteins have sufficient dynamics to communicate between spatially separated substrate-binding sites. TrmD is a bacterial methyltransferase that uses a knotted protein fold to catalyze methyl transfer from S-adenosyl methionine (AdoMet) to G37-tRNA. The product, mG37-tRNA, is essential for life and maintains protein-synthesis reading frames. Using an integrated approach of structural, kinetic, and computational analysis, we show that the structurally constrained TrmD knot is required for its catalytic activity. Unexpectedly, the TrmD knot undergoes complex internal movements that respond to AdoMet binding and signaling. Most of the signaling propagates the free energy of AdoMet binding, thereby stabilizing tRNA binding and allowing assembly of the active site. This work demonstrates new principles of knots as organized structures that capture the free energies of substrate binding and facilitate catalysis.
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http://dx.doi.org/10.1038/nsmb.3282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5429141PMC
October 2016

Different Contribution of Redox-Sensitive Transient Receptor Potential Channels to Acetaminophen-Induced Death of Human Hepatoma Cell Line.

Front Pharmacol 2016 9;7:19. Epub 2016 Feb 9.

Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto UniversityKyoto, Japan; World Premier International Research Initiative-Institute for Integrated Cell-Material Sciences, Kyoto UniversityKyoto, Japan; Laboratory of Environmental Systems Biology, Department of Technology and Ecology, Hall of Global Environmental Studies, Kyoto UniversityKyoto, Japan.

Acetaminophen (APAP) is a safe analgesic antipyretic drug at prescribed doses. Its overdose, however, can cause life-threatening liver damage. Though, involvement of oxidative stress is widely acknowledged in APAP-induced hepatocellular death, the mechanism of this increased oxidative stress and the associated alterations in Ca(2+) homeostasis are still unclear. Among members of transient receptor potential (TRP) channels activated in response to oxidative stress, we here identify that redox-sensitive TRPV1, TRPC1, TRPM2, and TRPM7 channels underlie Ca(2+) entry and downstream cellular damages induced by APAP in human hepatoma (HepG2) cells. Our data indicate that APAP treatment of HepG2 cells resulted in increased reactive oxygen species (ROS) production, glutathione (GSH) depletion, and Ca(2+) entry leading to increased apoptotic cell death. These responses were significantly suppressed by pretreatment with the ROS scavengers N-acetyl-L-cysteine (NAC) and 4,5-dihydroxy-1,3-benzene disulfonic acid disodium salt monohydrate (Tiron), and also by preincubation of cells with the glutathione inducer Dimethylfumarate (DMF). TRP subtype-targeted pharmacological blockers and siRNAs strategy revealed that suppression of either TRPV1, TRPC1, TRPM2, or TRPM7 reduced APAP-induced ROS formation, Ca(2+) influx, and cell death; the effects of suppression of TRPV1 or TRPC1, known to be activated by oxidative cysteine modifications, were stronger than those of TRPM2 or TRPM7. Interestingly, TRPV1 and TRPC1 were labeled by the cysteine-selective modification reagent, 5,5'-dithiobis (2-nitrobenzoic acid)-2biotin (DTNB-2Bio), and this was attenuated by pretreatment with APAP, suggesting that APAP and/or its oxidized metabolites act directly on the modification target cysteine residues of TRPV1 and TRPC1 proteins. In human liver tissue, TRPV1, TRPC1, TRPM2, and TRPM7 channels transcripts were localized mainly to hepatocytes and Kupffer cells. Our findings strongly suggest that APAP-induced Ca(2+) entry and subsequent hepatocellular death are regulated by multiple redox-activated cation channels, among which TRPV1 and TRPC1 play a prominent role.
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http://dx.doi.org/10.3389/fphar.2016.00019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4746322PMC
February 2016

Deciphering Subtype-Selective Modulations in TRPA1 Biosensor Channels.

Curr Neuropharmacol 2015 ;13(2):266-78

Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura Campus, Nishikyoku, Kyoto 615-8510, Japan.

The transient receptor potential (TRP) proteins are a family of ion channels that act as cellular sensors. Several members of the TRP family are sensitive to oxidative stress mediators. Among them, TRPA1 is remarkably susceptible to various oxidants, and is known to mediate neuropathic pain and respiratory, vascular and gastrointestinal functions, making TRPA1 an attractive therapeutic target. Recent studies have revealed a number of modulators (both activators and inhibitors) that act on TRPA1. Endogenous mediators of oxidative stress and exogenous electrophiles activate TRPA1 through oxidative modification of cysteine residues. Non-electrophilic compounds also activate TRPA1. Certain non-electrophilic modulators may act on critical non-cysteine sites in TRPA1. However, a method to achieve selective modulation of TRPA1 by small molecules has not yet been established. More recently, we found that a novel N-nitrosamine compound activates TRPA1 by S-nitrosylation (the addition of a nitric oxide (NO) group to cysteine thiol), and does so with significant selectivity over other NO-sensitive TRP channels. It is proposed that this subtype selectivity is conferred through synergistic effects of electrophilic cysteine transnitrosylation and molecular recognition of the non-electrophilic moiety on the N-nitrosamine. In this review, we describe the molecular pharmacology of these TRPA1 modulators and discuss their modulatory mechanisms.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4598439PMC
http://dx.doi.org/10.2174/1570159x1302150525122020DOI Listing
July 2016

Validating subcellular thermal changes revealed by fluorescent thermosensors.

Nat Methods 2015 Sep;12(9):801-2

Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan.

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http://dx.doi.org/10.1038/nmeth.3548DOI Listing
September 2015

A divalent metal ion-dependent N(1)-methyl transfer to G37-tRNA.

Chem Biol 2014 Oct 11;21(10):1351-1360. Epub 2014 Sep 11.

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 South 10(th) Street, BLSB 220, Philadelphia, PA 19107, USA. Electronic address:

The catalytic mechanism of the majority of S-adenosyl methionine (AdoMet)-dependent methyl transferases requires no divalent metal ions. Here we report that methyl transfer from AdoMet to N(1) of G37-tRNA, catalyzed by the bacterial TrmD enzyme, is strongly dependent on divalent metal ions and that Mg(2+) is the most physiologically relevant. Kinetic isotope analysis, metal rescue, and spectroscopic measurements indicate that Mg(2+) is not involved in substrate binding, but in promoting methyl transfer. On the basis of the pH-activity profile indicating one proton transfer during the TrmD reaction, we propose a catalytic mechanism in which the role of Mg(2+) is to help to increase the nucleophilicity of N(1) of G37 and stabilize the negative developing charge on O(6) during attack on the methyl sulfonium of AdoMet. This work demonstrates how Mg(2+) contributes to the catalysis of AdoMet-dependent methyl transfer in one of the most crucial posttranscriptional modifications to tRNA.
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http://dx.doi.org/10.1016/j.chembiol.2014.07.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4224600PMC
October 2014

Fluorescent sensors reveal subcellular thermal changes.

Curr Opin Biotechnol 2015 Feb 29;31:57-64. Epub 2014 Aug 29.

World Premier International Research Initiative-Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto 606-8501, Japan; Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan; Department of Technology and Ecology, Hall of Global Environmental Studies, Kyoto University, Kyoto 615-8510, Japan. Electronic address:

In mammals and birds, thermoregulation to conserve body temperature is vital to life. Multiple mechanisms of thermogeneration have been proposed, localized in different subcellular organelles. However, studying thermogenesis directly in intact organelles has been challenging. Visualizing patterns of thermal changes at subcellular resolution would reveal physiologically relevant spatio-temporal information, especially if this could be done in the native cellular configuration of the cell. Here we review and compare the wide variety of intracellular thermosensors currently identified. This review focuses particularly on genetically encoded sensors. It also explores the notable physiological discoveries made using these imaging methods, which are rapidly becoming indispensible to the study of thermal biology.
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http://dx.doi.org/10.1016/j.copbio.2014.07.013DOI Listing
February 2015

Impaired function is a common feature of neuropathy-associated glycyl-tRNA synthetase mutations.

Hum Mutat 2014 Nov;35(11):1363-71

Cellular and Molecular Biology Program, University of Michigan Medical School, Ann Arbor, Michigan; Medical Scientist Training Program, University of Michigan Medical School, Ann Arbor, Michigan.

Charcot-Marie-Tooth disease type 2D (CMT2D) is an autosomal-dominant axonal peripheral neuropathy characterized by impaired motor and sensory function in the distal extremities. Mutations in the glycyl-tRNA synthetase (GARS) gene cause CMT2D. GARS is a member of the ubiquitously expressed aminoacyl-tRNA synthetase (ARS) family and is responsible for charging tRNA with glycine. To date, 13 GARS mutations have been identified in patients with CMT disease. While functional studies have revealed loss-of-function characteristics, only four GARS mutations have been rigorously studied. Here, we report the functional evaluation of nine CMT-associated GARS mutations in tRNA charging, yeast complementation, and subcellular localization assays. Our results demonstrate that impaired function is a common characteristic of CMT-associated GARS mutations. Additionally, one mutation previously associated with CMT disease (p.Ser581Leu) does not demonstrate impaired function, was identified in the general population, and failed to segregate with disease in two newly identified families with CMT disease. Thus, we propose that this variant is not a disease-causing mutation. Together, our data indicate that impaired function is a key component of GARS-mediated CMT disease and emphasize the need for careful genetic and functional evaluation before implicating a variant in disease onset.
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http://dx.doi.org/10.1002/humu.22681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4213347PMC
November 2014

A dual-targeted aminoacyl-tRNA synthetase in Plasmodium falciparum charges cytosolic and apicoplast tRNACys.

Biochem J 2014 Mar;458(3):513-23

*Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, VIC 3010, Australia.

Plasmodium parasites possess two endosymbiotic organelles: a mitochondrion and a relict plastid called the apicoplast. To accommodate the translational requirements of these organelles in addition to its cytosolic translation apparatus, the parasite must maintain a supply of charged tRNA molecules in each of these compartments. In the present study we investigate how the parasite manages these translational requirements for charged tRNACys with only a single gene for CysRS (cysteinyl-tRNA synthetase). We demonstrate that the single PfCysRS (Plasmodium falciparum CysRS) transcript is alternatively spliced, and, using a combination of endogenous and heterologous tagging experiments in both P. falciparum and Toxoplasma gondii, we show that CysRS isoforms traffic to the cytosol and apicoplast. PfCysRS can recognize and charge the eukaryotic tRNACys encoded by the Plasmodium nucleus as well as the bacterial-type tRNA encoded by the apicoplast genome, albeit with a preference for the eukaryotic type cytosolic tRNA. The results of the present study indicate that apicomplexan parasites have lost their original plastidic cysteinyl-tRNA synthetase, and have replaced it with a dual-targeted eukaryotic type CysRS that recognizes plastid and nuclear tRNACys. Inhibitors of the Plasmodium dual-targeted CysRS would potentially offer a therapy capable of the desirable immediate effects on parasite growth as well as the irreversibility of inhibitors that disrupt apicoplast inheritance.
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http://dx.doi.org/10.1042/BJ20131451DOI Listing
March 2014

Genetically encoded fluorescent thermosensors visualize subcellular thermoregulation in living cells.

Nat Methods 2013 Dec 13;10(12):1232-8. Epub 2013 Oct 13.

1] Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan. [2] Department of Technology and Ecology, Hall of Global Environmental Studies, Kyoto University, Kyoto, Japan. [3] Core Research for Evolution Science and Technology, Japan Science and Technology Agency, Tokyo, Japan.

In mammals and birds, thermoregulation to conserve body temperature is vital to life. Multiple mechanisms of thermogeneration have been proposed, localized in different subcellular organelles. However, visualizing thermogenesis directly in intact organelles has been challenging. Here we have developed genetically encoded, GFP-based thermosensors (tsGFPs) that enable visualization of thermogenesis in discrete organelles in living cells. In tsGFPs, a tandem formation of coiled-coil structures of the Salmonella thermosensing protein TlpA transmits conformational changes to GFP to convert temperature changes into visible and quantifiable fluorescence changes. Specific targeting of tsGFPs enables visualization of thermogenesis in the mitochondria of brown adipocytes and the endoplasmic reticulum of myotubes. In HeLa cells, tsGFP targeted to mitochondria reveals heterogeneity in thermogenesis that correlates with the electrochemical gradient. Thus, tsGFPs are powerful tools to noninvasively assess thermogenesis in living cells.
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http://dx.doi.org/10.1038/nmeth.2690DOI Listing
December 2013

The temperature sensitivity of a mutation in the essential tRNA modification enzyme tRNA methyltransferase D (TrmD).

J Biol Chem 2013 Oct 28;288(40):28987-96. Epub 2013 Aug 28.

From the Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.

Conditional temperature-sensitive (ts) mutations are important reagents to study essential genes. Although it is commonly assumed that the ts phenotype of a specific mutation arises from thermal denaturation of the mutant enzyme, the possibility also exists that the mutation decreases the enzyme activity to a certain level at the permissive temperature and aggravates the negative effect further upon temperature upshifts. Resolving these possibilities is important for exploiting the ts mutation for studying the essential gene. The trmD gene is essential for growth in bacteria, encoding the enzyme for converting G37 to m(1)G37 on the 3' side of the tRNA anticodon. This conversion involves methyl transfer from S-adenosyl methionine and is critical to minimize tRNA frameshift errors on the ribosome. Using the ts-S88L mutation of Escherichia coli trmD as an example, we show that although the mutation confers thermal lability to the enzyme, the effect is relatively minor. In contrast, the mutation decreases the catalytic efficiency of the enzyme to 1% at the permissive temperature, and at the nonpermissive temperature, it renders further deterioration of activity to 0.1%. These changes are accompanied by losses of both the quantity and quality of tRNA methylation, leading to the potential of cellular pleiotropic effects. This work illustrates the principle that the ts phenotype of an essential gene mutation can be closely linked to the catalytic defect of the gene product and that such a mutation can provide a useful tool to study the mechanism of catalytic inactivation.
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http://dx.doi.org/10.1074/jbc.M113.485797DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3789996PMC
October 2013

The catalytic domain of topological knot tRNA methyltransferase (TrmH) discriminates between substrate tRNA and nonsubstrate tRNA via an induced-fit process.

J Biol Chem 2013 Aug 18;288(35):25562-25574. Epub 2013 Jul 18.

From the Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime University, 3 Bunkyo, Matsuyama, Ehime 790-8577, Japan,; the Venture Business Laboratory, Ehime University, 3 Bunkyo, Matsuyama, Ehime 790-8577, Japan. Electronic address:

A conserved guanosine at position 18 (G18) in the D-loop of tRNAs is often modified to 2'-O-methylguanosine (Gm). Formation of Gm18 in eubacterial tRNA is catalyzed by tRNA (Gm18) methyltransferase (TrmH). TrmH enzymes can be divided into two types based on their substrate tRNA specificity. Type I TrmH, including Thermus thermophilus TrmH, can modify all tRNA species, whereas type II TrmH, for example Escherichia coli TrmH, modifies only a subset of tRNA species. Our previous crystal study showed that T. thermophilus TrmH is a class IV S-adenosyl-l-methionine-dependent methyltransferase, which maintains a topological knot structure in the catalytic domain. Because TrmH enzymes have short stretches at the N and C termini instead of a clear RNA binding domain, these stretches are believed to be involved in tRNA recognition. In this study, we demonstrate by site-directed mutagenesis that both N- and C-terminal regions function in tRNA binding. However, in vitro and in vivo chimera protein studies, in which four chimeric proteins of type I and II TrmHs were used, demonstrated that the catalytic domain discriminates substrate tRNAs from nonsubstrate tRNAs. Thus, the N- and C-terminal regions do not function in the substrate tRNA discrimination process. Pre-steady state analysis of complex formation between mutant TrmH proteins and tRNA by stopped-flow fluorescence measurement revealed that the C-terminal region works in the initial binding process, in which nonsubstrate tRNA is not excluded, and that structural movement of the motif 2 region of the catalytic domain in an induced-fit process is involved in substrate tRNA discrimination.
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http://dx.doi.org/10.1074/jbc.M113.485128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3757217PMC
August 2013

Simultaneous detection of ATP and GTP by covalently linked fluorescent ribonucleopeptide sensors.

J Am Chem Soc 2013 Mar 22;135(9):3465-73. Epub 2013 Feb 22.

Institute of Advanced Energy, Kyoto University, Uji, Kyoto 611-0011, Japan.

A noncovalent RNA complex embedding an aptamer function and a fluorophore-labeled peptide affords a fluorescent ribonucleopeptide (RNP) framework for constructing fluorescent sensors. By taking an advantage of the noncovalent properties of the RNP complex, the ligand-binding and fluorescence characteristics of the fluorescent RNP can be independently tuned by taking advantage of the nature of the RNA and peptide subunits, respectively. Fluorescent sensors tailored for given measurement conditions, such as a detection wavelength and a detection concentration range for a ligand of interest can be easily identified by screening of fluorescent RNP libraries. The noncovalent configuration of a RNP becomes a disadvantage when the sensor is to be utilized at very low concentrations or when multiple sensors are applied to the same solution. Here, we report a strategy to convert a fluorescent RNP sensor in the noncovalent configuration into a covalently linked stable fluorescent RNP sensor. This covalently linked fluorescent RNP sensor enabled ligand detection at a low sensor concentration, even in cell extracts. Furthermore, application of both ATP and GTP sensors enabled simultaneous detection of ATP and GTP by monitoring each wavelength corresponding to the respective sensor. Importantly, when a fluorescein-modified ATP sensor and a pyrene-modified GTP sensor were co-incubated in the same solution, the ATP sensor responded at 535 nm only to changes in the concentration of ATP, whereas the GTP sensor detected GTP at 390 nm without any effect on the ATP sensor. Finally, simultaneous monitoring by these sensors enabled real-time measurement of adenosine deaminase enzyme reactions.
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http://dx.doi.org/10.1021/ja3097652DOI Listing
March 2013

Recognition of guanosine by dissimilar tRNA methyltransferases.

RNA 2012 Sep 30;18(9):1687-701. Epub 2012 Jul 30.

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.

Guanosines are important for biological activities through their specific functional groups that are recognized for RNA or protein interactions. One example is recognition of N(1) of G37 in tRNA by S-adenosyl-methionine (AdoMet)-dependent tRNA methyltransferases to synthesize m(1)G37-tRNA, which is essential for translational fidelity in all biological domains. Synthesis of m(1)G37-tRNA is catalyzed by TrmD in bacteria and by Trm5 in eukarya and archaea, using unrelated and dissimilar structural folds. This raises the question of how dissimilar proteins recognize the same guanosine. Here we probe the mechanism of discrimination among functional groups of guanosine by TrmD and Trm5. Guanosine analogs were systematically introduced into tRNA through a combination of chemical and enzymatic synthesis. Single turnover kinetic assays and thermodynamic analysis of the effect of each analog on m(1)G37-tRNA synthesis reveal that TrmD and Trm5 discriminate functional groups differently. While both recognize N(1) and O(6) of G37, TrmD places a much stronger emphasis on these functional groups than Trm5. While the exocyclic 2-amino group of G37 is important for TrmD, it is dispensable for Trm5. In addition, while an adjacent G36 is obligatory for TrmD, it is nonessential for Trm5. These results depict a more rigid requirement of guanosine functional groups for TrmD than for Trm5. However, the sensitivity of both enzymes to analog substitutions, together with an experimental revelation of their low cellular concentrations relative to tRNA substrates, suggests a model in which these enzymes rapidly screen tRNA by direct recognition of G37 in order to monitor the global state of m(1)G37-tRNA.
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http://dx.doi.org/10.1261/rna.032029.111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3425783PMC
September 2012

A recurrent loss-of-function alanyl-tRNA synthetase (AARS) mutation in patients with Charcot-Marie-Tooth disease type 2N (CMT2N).

Hum Mutat 2012 Jan 9;33(1):244-53. Epub 2011 Nov 9.

Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI 48109-5618, USA.

Charcot-Marie-Tooth (CMT) disease comprises a heterogeneous group of peripheral neuropathies characterized by muscle weakness and wasting, and impaired sensation in the extremities. Four genes encoding an aminoacyl-tRNA synthetase (ARS) have been implicated in CMT disease. ARSs are ubiquitously expressed, essential enzymes that ligate amino acids to cognate tRNA molecules. Recently, a p.Arg329His variant in the alanyl-tRNA synthetase (AARS) gene was found to segregate with dominant axonal CMT type 2N (CMT2N) in two French families; however, the functional consequence of this mutation has not been determined. To investigate the role of AARS in CMT, we performed a mutation screen of the AARS gene in patients with peripheral neuropathy. Our results showed that p.Arg329His AARS also segregated with CMT disease in a large Australian family. Aminoacylation and yeast viability assays showed that p.Arg329His AARS severely reduces enzyme activity. Genotyping analysis indicated that this mutation arose on three distinct haplotypes, and the results of bisulfite sequencing suggested that methylation-mediated deamination of a CpG dinucleotide gives rise to the recurrent p.Arg329His AARS mutation. Together, our data suggest that impaired tRNA charging plays a role in the molecular pathology of CMT2N, and that patients with CMT should be directly tested for the p.Arg329His AARS mutation.
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http://dx.doi.org/10.1002/humu.21635DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3240693PMC
January 2012

Allosteric communication in cysteinyl tRNA synthetase: a network of direct and indirect readout.

J Biol Chem 2011 Oct 2;286(43):37721-31. Epub 2011 Sep 2.

Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India.

Protein structure networks are constructed for the identification of long-range signaling pathways in cysteinyl tRNA synthetase (CysRS). Molecular dynamics simulation trajectory of CysRS-ligand complexes were used to determine conformational ensembles in order to gain insight into the allosteric signaling paths. Communication paths between the anticodon binding region and the aminoacylation region have been identified. Extensive interaction between the helix bundle domain and the anticodon binding domain, resulting in structural rigidity in the presence of tRNA, has been detected. Based on the predicted model, six residues along the communication paths have been examined by mutations (single and double) and shown to mediate a coordinated coupling between anticodon recognition and activation of amino acid at the active site. This study on CysRS clearly shows that specific key residues, which are involved in communication between distal sites in allosteric proteins but may be elusive in direct structure analysis, can be identified from dynamics of protein structure networks.
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http://dx.doi.org/10.1074/jbc.M111.246702DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3199515PMC
October 2011

Compound heterozygosity for loss-of-function lysyl-tRNA synthetase mutations in a patient with peripheral neuropathy.

Am J Hum Genet 2010 Oct;87(4):560-6

Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI 48109, USA.

Charcot-Marie-Tooth (CMT) disease comprises a genetically and clinically heterogeneous group of peripheral nerve disorders characterized by impaired distal motor and sensory function. Mutations in three genes encoding aminoacyl-tRNA synthetases (ARSs) have been implicated in CMT disease primarily associated with an axonal pathology. ARSs are ubiquitously expressed, essential enzymes responsible for charging tRNA molecules with their cognate amino acids. To further explore the role of ARSs in CMT disease, we performed a large-scale mutation screen of the 37 human ARS genes in a cohort of 355 patients with a phenotype consistent with CMT. Here we describe three variants (p.Leu133His, p.Tyr173SerfsX7, and p.Ile302Met) in the lysyl-tRNA synthetase (KARS) gene in two patients from this cohort. Functional analyses revealed that two of these mutations (p.Leu133His and p.Tyr173SerfsX7) severely affect enzyme activity. Interestingly, both functional variants were found in a single patient with CMT disease and additional neurological and non-neurological sequelae. Based on these data, KARS becomes the fourth ARS gene associated with CMT disease, indicating that this family of enzymes is specifically critical for axon function.
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http://dx.doi.org/10.1016/j.ajhg.2010.09.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2948804PMC
October 2010
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