Publications by authors named "Katrin Watschinger"

32 Publications

Sapropterin (BH4) Aggravates Autoimmune Encephalomyelitis in Mice.

Neurotherapeutics 2021 Apr 12. Epub 2021 Apr 12.

Institute of Clinical Pharmacology, Medical Faculty, Goethe-University, Frankfurt, Germany.

Depletion of the enzyme cofactor, tetrahydrobiopterin (BH4), in T-cells was shown to prevent their proliferation upon receptor stimulation in models of allergic inflammation in mice, suggesting that BH4 drives autoimmunity. Hence, the clinically available BH4 drug (sapropterin) might increase the risk of autoimmune diseases. The present study assessed the implications for multiple sclerosis (MS) as an exemplary CNS autoimmune disease. Plasma levels of biopterin were persistently low in MS patients and tended to be lower with high Expanded Disability Status Scale (EDSS). Instead, the bypass product, neopterin, was increased. The deregulation suggested that BH4 replenishment might further drive the immune response or beneficially restore the BH4 balances. To answer this question, mice were treated with sapropterin in immunization-evoked autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. Sapropterin-treated mice had higher EAE disease scores associated with higher numbers of T-cells infiltrating the spinal cord, but normal T-cell subpopulations in spleen and blood. Mechanistically, sapropterin treatment was associated with increased plasma levels of long-chain ceramides and low levels of the poly-unsaturated fatty acid, linolenic acid (FA18:3). These lipid changes are known to contribute to disruptions of the blood-brain barrier in EAE mice. Indeed, RNA data analyses revealed upregulations of genes involved in ceramide synthesis in brain endothelial cells of EAE mice (LASS6/CERS6, LASS3/CERS3, UGCG, ELOVL6, and ELOVL4). The results support the view that BH4 fortifies autoimmune CNS disease, mechanistically involving lipid deregulations that are known to contribute to the EAE pathology.
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http://dx.doi.org/10.1007/s13311-021-01043-4DOI Listing
April 2021

When the genome bluffs: a tandem duplication event during generation of a novel Agmo knockout mouse model fools routine genotyping.

Cell Biosci 2021 Mar 16;11(1):54. Epub 2021 Mar 16.

Institute of Biological Chemistry, Biocenter, Medical University of Innsbruck, Innsbruck, Austria.

Background: Genome editing in mice using either classical approaches like homologous recombination or CRISPR/Cas9 has been reported to harbor off target effects (insertion/deletion, frame shifts or gene segment duplications) that lead to mutations not only in close proximity to the target site but also outside. Only the genomes of few engineered mouse strains have been sequenced. Since the role of the ether-lipid cleaving enzyme alkylglycerol monooxygenase (AGMO) in physiology and pathophysiology remains enigmatic, we created a knockout mouse model for AGMO using EUCOMM stem cells but unforeseen genotyping issues that did not agree with Mendelian distribution and enzyme activity data prompted an in-depth genomic validation of the mouse model.

Results: We report a gene segment tandem duplication event that occurred during the generation of an Agmo knockout-first allele by homologous recombination. Only low homology was seen between the breakpoints. While a single copy of the recombinant 18 kb cassette was integrated correctly around exon 2 of the Agmo gene, whole genome nanopore sequencing revealed a 94 kb duplication in the Agmo locus that contains Agmo wild-type exons 1-3. The duplication fooled genotyping by routine PCR, but could be resolved using qPCR-based genotyping, targeted locus amplification sequencing and nanopore sequencing. Despite this event, this Agmo knockout mouse model lacks AGMO enzyme activity and can therefore be used to study its physiological role.

Conclusions: A duplication event occurred at the exact locus of the homologous recombination and was not detected by conventional quality control filters such as FISH or long-range PCR over the recombination sites. Nanopore sequencing provides a cost convenient method to detect such underrated off-target effects, suggesting its use for additional quality assessment of gene editing in mice and also other model organisms.
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http://dx.doi.org/10.1186/s13578-021-00566-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7962373PMC
March 2021

The Emerging Physiological Role of AGMO 10 Years after Its Gene Identification.

Life (Basel) 2021 Jan 26;11(2). Epub 2021 Jan 26.

Biocenter, Institute of Biological Chemistry, Medical University of Innsbruck, 6020 Innsbruck, Austria.

The gene encoding alkylglycerol monooxygenase (AGMO) was assigned 10 years ago. So far, AGMO is the only known enzyme capable of catalysing the breakdown of alkylglycerols and lyso-alkylglycerophospholipids. With the knowledge of the genetic information, it was possible to relate a potential contribution for mutations in the AGMO locus to human diseases by genome-wide association studies. A possible role for AGMO was implicated by genetic analyses in a variety of human pathologies such as type 2 diabetes, neurodevelopmental disorders, cancer, and immune defence. Deficient catabolism of stored lipids carrying an alkyl bond by an absence of AGMO was shown to impact on the overall lipid composition also outside the ether lipid pool. This review focuses on the current evidence of AGMO in human diseases and summarises experimental evidence for its role in immunity, energy homeostasis, and development in humans and several model organisms. With the progress in lipidomics platform and genetic identification of enzymes involved in ether lipid metabolism such as AGMO, it is now possible to study the consequence of gene ablation on the global lipid pool and further on certain signalling cascades in a variety of model organisms in more detail.
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http://dx.doi.org/10.3390/life11020088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7911779PMC
January 2021

Unequivocal Mapping of Molecular Ether Lipid Species by LC-MS/MS in Plasmalogen-Deficient Mice.

Anal Chem 2020 08 7;92(16):11268-11276. Epub 2020 Aug 7.

Institute of Human Genetics, Medical University of Innsbruck, 6020 Innsbruck, Austria.

Deficient ether lipid biosynthesis in rhizomelic chondrodysplasia punctata and other disorders is associated with a wide range of severe symptoms including small stature with proximal shortening of the limbs, contractures, facial dysmorphism, congenital cataracts, ichthyosis, spasticity, microcephaly, and mental disability. Mouse models are available but show less severe symptoms. In both humans and mice, it has remained elusive which of the symptoms can be attributed to lack of plasmanyl or plasmenyl ether lipids. The latter compounds, better known as plasmalogens, harbor a vinyl ether double bond conferring special chemical and physical properties. Discrimination between plasmanyl and plasmenyl ether lipids is a major analytical challenge, especially in complex lipid extracts with many isobaric species. Consequently, these lipids are often neglected also in recent lipidomic studies. Here, we present a comprehensive LC-MS/MS based approach that allows unequivocal distinction of these two lipid subclasses based on their chromatographic properties. The method was validated using a novel plasmalogen-deficient mouse model, which lacks plasmanylethanolamine desaturase and therefore cannot form plasmenyl ether lipids. We demonstrate that plasmanylethanolamine desaturase deficiency causes an accumulation of plasmanyl species, a too little studied but biologically important substance class.
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http://dx.doi.org/10.1021/acs.analchem.0c01933DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7439256PMC
August 2020

The gene encodes plasmanylethanolamine desaturase which introduces the characteristic vinyl ether double bond into plasmalogens.

Proc Natl Acad Sci U S A 2020 04 24;117(14):7792-7798. Epub 2020 Mar 24.

Institute of Biological Chemistry, Biocenter, Medical University of Innsbruck, A-6020 Innsbruck, Austria;

A significant fraction of the glycerophospholipids in the human body is composed of plasmalogens, particularly in the brain, cardiac, and immune cell membranes. A decline in these lipids has been observed in such diseases as Alzheimer's and chronic obstructive pulmonary disease. Plasmalogens contain a characteristic 1--alk-1'-enyl ether (vinyl ether) double bond that confers special biophysical, biochemical, and chemical properties to these lipids. However, the genetics of their biosynthesis is not fully understood, since no gene has been identified that encodes plasmanylethanolamine desaturase (E.C. 1.14.99.19), the enzyme introducing the crucial alk-1'-enyl ether double bond. The present work identifies this gene as transmembrane protein 189 (). Inactivation of the gene in human HAP1 cells led to a total loss of plasmanylethanolamine desaturase activity, strongly decreased plasmalogen levels, and accumulation of plasmanylethanolamine substrates and resulted in an inability of these cells to form labeled plasmalogens from labeled alkylglycerols. Transient expression of TMEM189 protein, but not of other selected desaturases, recovered this deficit. TMEM189 proteins contain a conserved protein motif (pfam10520) with eight conserved histidines that is shared by an alternative type of plant desaturase but not by other mammalian proteins. Each of these histidines is essential for plasmanylethanolamine desaturase activity. Mice homozygous for an inactivated gene lacked plasmanylethanolamine desaturase activity and had dramatically lowered plasmalogen levels in their tissues. These results assign the gene to plasmanylethanolamine desaturase and suggest that the previously characterized phenotype of -deficient mice may be caused by a lack of plasmalogens.
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http://dx.doi.org/10.1073/pnas.1917461117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7149458PMC
April 2020

Phospholipid Acyl Chain Diversity Controls the Tissue-Specific Assembly of Mitochondrial Cardiolipins.

Cell Rep 2020 03;30(12):4281-4291.e4

Institute of Human Genetics, Medical University of Innsbruck, 6020 Innsbruck, Austria. Electronic address:

Cardiolipin (CL) is a phospholipid specific for mitochondrial membranes and crucial for many core tasks of this organelle. Its acyl chain configurations are tissue specific, functionally important, and generated via post-biosynthetic remodeling. However, this process lacks the necessary specificity to explain CL diversity, which is especially evident for highly specific CL compositions in mammalian tissues. To investigate the so far elusive regulatory origin of CL homeostasis in mice, we combine lipidomics, integrative transcriptomics, and data-driven machine learning. We demonstrate that not transcriptional regulation, but cellular phospholipid compositions are closely linked to the tissue specificity of CL patterns allowing artificial neural networks to precisely predict cross-tissue CL compositions in a consistent mechanistic specificity rationale. This is especially relevant for the interpretation of disease-related perturbations of CL homeostasis, by allowing differentiation between specific aberrations in CL metabolism and changes caused by global alterations in cellular (phospho-)lipid metabolism.
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http://dx.doi.org/10.1016/j.celrep.2020.02.115DOI Listing
March 2020

Biallelic variants in AGMO with diminished enzyme activity are associated with a neurodevelopmental disorder.

Hum Genet 2019 Dec 25;138(11-12):1259-1266. Epub 2019 Sep 25.

Department of Pediatrics, Columbia University Medical Center, 1150 St. Nicholas Avenue, New York, NY, 10032, USA.

Alkylglycerol monooxygenase (AGMO) is the only enzyme known to cleave the O-alkyl bonds of ether lipids (alkylglycerols) which are essential components of cell membranes. A homozygous frameshift variant [p.(Glu324LysfsTer12)] in AGMO has recently been reported in two male siblings with syndromic microcephaly. In this study, we identified rare nonsense, in frame deletion, and missense biallelic variants in AGMO in two unrelated individuals with neurodevelopmental disabilities. We assessed the activity of seven disease associated AGMO variants including the four variants identified in our two affected individuals expressed in human embryonic kidney (HEK293T) cells. We demonstrated significantly diminished enzyme activity for all disease-associated variants, supporting the mechanism as decreased AGMO activity. Future mechanistic studies are necessary to understand how decreased AGMO activity leads to the neurologic manifestations.
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http://dx.doi.org/10.1007/s00439-019-02065-xDOI Listing
December 2019

Mast cell tetrahydrobiopterin contributes to itch in mice.

J Cell Mol Med 2019 02 18;23(2):985-1000. Epub 2018 Nov 18.

Institute of Clinical Pharmacology, Goethe-University Hospital, Frankfurt, Germany.

GTP cyclohydrolase (GCH1) governs de novo synthesis of the enzyme cofactor, tetrahydrobiopterin (BH4), which is essential for biogenic amine production, bioactive lipid metabolism and redox coupling of nitric oxide synthases. Overproduction of BH4 via upregulation of GCH1 in sensory neurons is associated with nociceptive hypersensitivity in rodents, and neuron-specific GCH1 deletion normalizes nociception. The translational relevance is revealed by protective polymorphisms of GCH1 in humans, which are associated with a reduced chronic pain. Because myeloid cells constitute a major non-neuronal source of BH4 that may contribute to BH4-dependent phenotypes, we studied here the contribution of myeloid-derived BH4 to pain and itch in lysozyme M Cre-mediated GCH1 knockout (LysM-GCH1 ) and overexpressing mice (LysM-GCH1-HA). Unexpectedly, knockout or overexpression in myeloid cells had no effect on nociceptive behaviour, but LysM-driven GCH1 knockout reduced, and its overexpression increased the scratching response in Compound 48/80 and hydroxychloroquine-evoked itch models, which involve histamine and non-histamine dependent signalling pathways. Mechanistically, GCH1 overexpression increased BH4, nitric oxide and hydrogen peroxide, and these changes were associated with increased release of histamine and serotonin and degranulation of mast cells. LysM-driven GCH1 knockout had opposite effects, and pharmacologic inhibition of GCH1 provided even stronger itch suppression. Inversely, intradermal BH4 provoked scratching behaviour in vivo and BH4 evoked an influx of calcium in sensory neurons. Together, these loss- and gain-of-function experiments suggest that itch in mice is contributed by BH4 release plus BH4-driven mediator release from myeloid immune cells, which leads to activation of itch-responsive sensory neurons.
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http://dx.doi.org/10.1111/jcmm.13999DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6349351PMC
February 2019

Molecular structural diversity of mitochondrial cardiolipins.

Proc Natl Acad Sci U S A 2018 04 4;115(16):4158-4163. Epub 2018 Apr 4.

Division of Human Genetics, Medical University of Innsbruck, 6020 Innsbruck, Austria;

Current strategies used to quantitatively describe the biological diversity of lipids by mass spectrometry are often limited in assessing the exact structural variability of individual molecular species in detail. A major challenge is represented by the extensive isobaric overlap present among lipids, hampering their accurate identification. This is especially true for cardiolipins, a mitochondria-specific class of phospholipids, which are functionally involved in many cellular functions, including energy metabolism, cristae structure, and apoptosis. Substituted with four fatty acyl side chains, cardiolipins offer a particularly high potential to achieve complex mixtures of molecular species. Here, we demonstrate how systematically generated high-performance liquid chromatography-mass spectral data can be utilized in a mathematical structural modeling approach, to comprehensively analyze and characterize the molecular diversity of mitochondrial cardiolipin compositions in cell culture and disease models, cardiolipin modulation experiments, and a broad variety of frequently studied model organisms.
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http://dx.doi.org/10.1073/pnas.1719407115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5910844PMC
April 2018

A novel assay for the introduction of the vinyl ether double bond into plasmalogens using pyrene-labeled substrates.

J Lipid Res 2018 05 14;59(5):901-909. Epub 2018 Mar 14.

Division of Biological Chemistry, Biocenter Medical University of Innsbruck, Innsbruck, Austria;

Plasmanylethanolamine desaturase (PEDS) (EC 1.14.99.19) introduces the 1-prime double bond into plasmalogens, one of the most abundant phospholipids in the human body. This labile membrane enzyme has not been purified and its coding sequence is unknown. Previous assays for this enzyme used radiolabeled substrates followed by multistep processing. We describe here a straight-forward method for the quantification of PEDS in enzyme incubation mixtures using pyrene-labeled substrates and reversed-phase HPLC with fluorescence detection. After stopping the reaction with hydrochloric acid in acetonitrile, the mixture was directly injected into the HPLC system without the need of lipid extraction. The substrate, 1--pyrenedecyl-2-acyl--glycero-3-phosphoethanolamine, and the lyso-substrate, 1--pyrenedecyl--glycero-3-phosphoethanolamine, were prepared from RAW-12 cells deficient in PEDS activity and were compared for their performance in the assay. Plasmalogen levels in mouse tissues and in cultured cells did not correlate with PEDS levels, indicating that the desaturase might not be the rate limiting step for plasmalogen biosynthesis. Among selected mouse organs, the highest activities were found in kidney and in spleen. Incubation of intact cultivated mammalian cells with 1--pyrenedecyl--glycerol, extraction of lipids, and treatment with hydrochloric or acetic acid in acetonitrile allowed sensitive monitoring of PEDS activity in intact cells.
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http://dx.doi.org/10.1194/jlr.D080283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5928432PMC
May 2018

Impaired Endothelial Nitric Oxide Synthase Homodimer Formation Triggers Development of Transplant Vasculopathy - Insights from a Murine Aortic Transplantation Model.

Sci Rep 2016 11 24;6:37917. Epub 2016 Nov 24.

Centre of Operative Medicine, Department of Visceral, Transplant and Thoracic Surgery, Medical University Innsbruck, Innsbruck, Austria.

Transplant vasculopathy (TV) represents a major obstacle to long-term graft survival and correlates with severity of ischemia reperfusion injury (IRI). Donor administration of the nitric oxide synthases (NOS) co-factor tetrahydrobiopterin has been shown to prevent IRI. Herein, we analysed whether tetrahydrobiopterin is also involved in TV development. Using a fully allogeneic mismatched (BALB/c to C57BL/6) murine aortic transplantation model grafts subjected to long cold ischemia time developed severe TV with intimal hyperplasia (α-smooth muscle actin positive cells in the neointima) and endothelial activation (increased P-selectin expression). Donor pretreatment with tetrahydrobiopterin significantly minimised these changes resulting in only marginal TV development. Severe TV observed in the non-treated group was associated with increased protein oxidation and increased occurrence of endothelial NOS monomers in the aortic grafts already during graft procurement. Tetrahydrobiopterin supplementation of the donor prevented all these early oxidative changes in the graft. Non-treated allogeneic grafts without cold ischemia time and syngeneic grafts did not develop any TV. We identified early protein oxidation and impaired endothelial NOS homodimer formation as plausible mechanistic explanation for the crucial role of IRI in triggering TV in transplanted aortic grafts. Therefore, targeting endothelial NOS in the donor represents a promising strategy to minimise TV.
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http://dx.doi.org/10.1038/srep37917DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5121662PMC
November 2016

Cuticle integrity and biogenic amine synthesis in Caenorhabditis elegans require the cofactor tetrahydrobiopterin (BH4).

Genetics 2015 May 24;200(1):237-53. Epub 2015 Mar 24.

Department of Biomedicine, University of Bergen, 5009 Bergen, Norway.

Tetrahydrobiopterin (BH4) is the natural cofactor of several enzymes widely distributed among eukaryotes, including aromatic amino acid hydroxylases (AAAHs), nitric oxide synthases (NOSs), and alkylglycerol monooxygenase (AGMO). We show here that the nematode Caenorhabditis elegans, which has three AAAH genes and one AGMO gene, contains BH4 and has genes that function in BH4 synthesis and regeneration. Knockout mutants for putative BH4 synthetic enzyme genes lack the predicted enzymatic activities, synthesize no BH4, and have indistinguishable behavioral and neurotransmitter phenotypes, including serotonin and dopamine deficiency. The BH4 regeneration enzymes are not required for steady-state levels of biogenic amines, but become rate limiting in conditions of reduced BH4 synthesis. BH4-deficient mutants also have a fragile cuticle and are generally hypersensitive to exogenous agents, a phenotype that is not due to AAAH deficiency, but rather to dysfunction in the lipid metabolic enzyme AGMO, which is expressed in the epidermis. Loss of AGMO or BH4 synthesis also specifically alters the sensitivity of C. elegans to bacterial pathogens, revealing a cuticular function for AGMO-dependent lipid metabolism in host-pathogen interactions.
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http://dx.doi.org/10.1534/genetics.114.174110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4423366PMC
May 2015

Tetrahydrobiopterin and alkylglycerol monooxygenase substantially alter the murine macrophage lipidome.

Proc Natl Acad Sci U S A 2015 Feb 9;112(8):2431-6. Epub 2015 Feb 9.

Division of Biological Chemistry, Biocenter, Medical University of Innsbruck, 6020 Innsbruck, Austria;

Tetrahydrobiopterin is a cofactor synthesized from GTP with well-known roles in enzymatic nitric oxide synthesis and aromatic amino acid hydroxylation. It is used to treat mild forms of phenylketonuria. Less is known about the role of tetrahydrobiopterin in lipid metabolism, although it is essential for irreversible ether lipid cleavage by alkylglycerol monooxygenase. Here we found intracellular alkylglycerol monooxygenase activity to be an important regulator of alkylglycerol metabolism in intact murine RAW264.7 macrophage-like cells. Alkylglycerol monooxygenase was expressed and active also in primary mouse bone marrow-derived monocytes and "alternatively activated" M2 macrophages obtained by interleukin 4 treatment, but almost missing in M1 macrophages obtained by IFN-γ and lipopolysaccharide treatment. The cellular lipidome of RAW264.7 was markedly changed in a parallel way by modulation of alkylglycerol monooxygenase expression and of tetrahydrobiopterin biosynthesis affecting not only various ether lipid species upstream of alkylglycerol monooxygenase but also other more complex lipids including glycosylated ceramides and cardiolipins, which have no direct connection to ether lipid pathways. Alkylglycerol monooxygenase activity manipulation modulated the IFN-γ/lipopolysaccharide-induced expression of inducible nitric oxide synthase, interleukin-1β, and interleukin 1 receptor antagonist but not transforming growth factor β1, suggesting that alkylglycerol monooxygenase activity affects IFN-γ/lipopolysaccharide signaling. Our results demonstrate a central role of tetrahydrobiopterin and alkylglycerol monooxygenase in ether lipid metabolism of murine macrophages and reveal that alteration of alkylglycerol monooxygenase activity has a profound impact on the lipidome also beyond the class of ether lipids.
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http://dx.doi.org/10.1073/pnas.1414887112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4345615PMC
February 2015

A requirement for Gch1 and tetrahydrobiopterin in embryonic development.

Dev Biol 2015 Mar 31;399(1):129-138. Epub 2014 Dec 31.

BHF Centre of Research Excellence, Division of Cardiovascular Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, UK; Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, UK.

Introduction: GTP cyclohydrolase I (GTPCH) catalyses the first and rate-limiting reaction in the synthesis of the enzymatic cofactor, tetrahydrobiopterin (BH4). Loss of function mutations in the GCH1 gene lead to congenital neurological diseases such as DOPA-responsive dystonia and hyperphenylalaninemia. However, little is known about how GTPCH and BH4 affects embryonic development in utero, and in particular whether metabolic replacement or supplementation in pregnancy is sufficient to rescue genetic GTPCH deficiency in the developing embryo.

Methods And Results: Gch1 deficient mice were generated by the insertion of loxP sites flanking exons 2-3 of the Gch1 gene. Gch1(fl/fl) mice were bred with Sox2cre mice to generate mice with global Gch1 deficiency. Genetic ablation of Gch1 caused embryonic lethality by E13.5. Despite loss of Gch1 mRNA and GTPCH enzymatic activity, whole embryo BH4 levels were maintained until E11.5, indicating sufficient maternal transfer of BH4 to reach this stage of development. After E11.5, Gch1(-/-) embryos were deficient in BH4, but an unbiased metabolomic screen indicated that the lethality was not due to a gross disturbance in metabolic profile. Embryonic lethality in Gch1(-/-) embryos was not caused by structural abnormalities, but was associated with significant bradycardia at E11.5. Embryonic lethality was not rescued by maternal supplementation of BH4, but was partially rescued, up to E15.5, by maternal supplementation of BH4 and l-DOPA.

Conclusion: These findings demonstrate a requirement for Gch1 in embryonic development and have important implications for the understanding of pathogenesis and treatment of genetic BH4 deficiencies, as well as the identification of new potential roles for BH4.
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http://dx.doi.org/10.1016/j.ydbio.2014.12.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4347993PMC
March 2015

Crucial role for neuronal nitric oxide synthase in early microcirculatory derangement and recipient survival following murine pancreas transplantation.

PLoS One 2014 12;9(11):e112570. Epub 2014 Nov 12.

Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Innsbruck, Austria.

Objective: Aim of this study was to identify the nitric oxide synthase (NOS) isoform involved in early microcirculatory derangements following solid organ transplantation.

Background: Tetrahydrobiopterin donor treatment has been shown to specifically attenuate these derangements following pancreas transplantation, and tetrahydrobiopterin-mediated protective effects to rely on its NOS-cofactor activity, rather than on its antioxidant capacity. However, the NOS-isoform mainly involved in this process has still to be defined.

Methods: Using a murine pancreas transplantation model, grafts lacking one of the three NOS-isoforms were compared to grafts from wild-type controls. Donors were treated with either tetrahydrobiopterin or remained untreated. All grafts were subjected to 16 h cold ischemia time and transplanted into wild-type recipients. Following 4 h graft reperfusion, microcirculation was analysed by confocal intravital fluorescence microscopy. Recipient survival was monitored for 50 days.

Results: Transplantation of the pancreas from untreated wild-type donor mice resulted in microcirculatory damage of the transplanted graft and no recipient survived more than 72 h. Transplanting grafts from untreated donor mice lacking either endothelial or inducible NOS led to similar outcomes. In contrast, donor treatment with tetrahydrobiopterin prevented microcirculatory breakdown enabling long-term survival. Sole exception was transplantation of grafts from untreated donor mice lacking neuronal NOS. It resulted in intact microvascular structure and long-term recipient survival, either if donor mice were untreated or treated with tetrahydrobiopterin.

Conclusion: We demonstrate for the first time the crucial involvement of neuronal NOS in early microcirculatory derangements following solid organ transplantation. In this model, protective effects of tetrahydrobiopterin are mediated by targeting this isoform.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0112570PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4229216PMC
July 2015

A gatekeeper helix determines the substrate specificity of Sjögren-Larsson Syndrome enzyme fatty aldehyde dehydrogenase.

Nat Commun 2014 Jul 22;5:4439. Epub 2014 Jul 22.

1] European Molecular Biology Laboratory, Grenoble Outstation, 6 rue Jules Horowitz, 38042 Grenoble, France [2] Unit of Virus Host-Cell Interactions, University of Grenoble Alpes-EMBL-CNRS, 6 rue Jules Horowitz, 38042 Grenoble, France [3].

Mutations in the gene coding for membrane-bound fatty aldehyde dehydrogenase (FALDH) lead to toxic accumulation of lipid species and development of the Sjögren-Larsson Syndrome (SLS), a rare disorder characterized by skin defects and mental retardation. Here, we present the crystallographic structure of human FALDH, the first model of a membrane-associated aldehyde dehydrogenase. The dimeric FALDH displays a previously unrecognized element in its C-terminal region, a 'gatekeeper' helix, which extends over the adjacent subunit, controlling the access to the substrate cavity and helping orientate both substrate cavities towards the membrane surface for efficient substrate transit between membranes and catalytic site. Activity assays demonstrate that the gatekeeper helix is important for directing the substrate specificity of FALDH towards long-chain fatty aldehydes. The gatekeeper feature is conserved across membrane-associated aldehyde dehydrogenases. Finally, we provide insight into the previously elusive molecular basis of SLS-causing mutations.
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http://dx.doi.org/10.1038/ncomms5439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4109017PMC
July 2014

Alkylglycerol monooxygenase.

IUBMB Life 2013 Apr 25;65(4):366-72. Epub 2013 Feb 25.

Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Innsbruck, Austria.

Alkylglycerol monooxygenase (E.C. 1.14.16.5), also called glyceryl ether monooxygenase, is a tetrahydrobiopterin-dependent enzyme. It is the only enzyme known to cleave the ether bond of alkylglycerols and lyso-alkylglycerol phospholipids, including lyso-platelet activating factor. Although it has been first described already in 1964, it was not possible so far to purify the protein. It took until 2010 to assign a sequence to this labile integral membrane enzyme by bioinformatic selection of candidate genes, recombinant expression of these, and sensitive monitoring of the enzymatic activity by a fluorescence-based assay. The sequence shows no significant similarity with the other known tetrahydrobiopterin-dependent enzymes but contains the fatty acid hydroxylase protein motif signature. Proteins containing this signature are all labile and catalyze reactions similar to the alkylglycerol monooxygenase reaction. They are thought to use a di-iron centre for catalysis. Site directed mutagenesis of alkylglycerol monooxygenase defined a region of the active site and a conserved glutamate residue important for tetrahydrobiopterin interaction. Current research now focuses on defining a physiological role of this enzyme which occurs not only in mammals but also in commonly used model organisms such as zebrafish and the nematode Caenorhabditis elegans.
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http://dx.doi.org/10.1002/iub.1143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3617469PMC
April 2013

Prevention of lethal murine pancreas ischemia reperfusion injury is specific for tetrahydrobiopterin.

Transpl Int 2012 Oct 17;25(10):1084-95. Epub 2012 Jul 17.

Center of Operative Medicine, Department of Visceral, Transplant and Thoracic Surgery, Innsbruck Medical University, Innsbruck, Austria.

Tetrahydrobiopterin has been shown to efficiently abrogate ischemia reperfusion injury (IRI). However, it is unclear, whether its beneficial action relies on cofactor activity of one of the five known tetrahydrobiopterin-dependent reactions or on its antioxidative capacity. We therefore compared tetrahydrobiopterin with the pterin derivate tetrahydroneopterin (similar biochemical properties, but no nitric oxide synthase cofactor activity) and the antioxidants vitamin C and 5-methyltetrahydrofolate. Donor mice were pretreated with tetrahydrobiopterin, tetrahydroneopterin, vitamin C, or 5-methyltetrahydrofolate. Pancreatic grafts were subjected to 16-h cold ischemia time and implanted in syngeneic recipients. Untreated and nontransplanted animals served as controls. Following 2-h reperfusion, microcirculation was analyzed by intravital fluorescence microscopy. Graft damage was assessed by histology and nitrotyrosine immunostaining, and tetrahydrobiopterin levels were determined by HPLC. Recipient survival served as ultimate readout. Prolonged cold ischemia time resulted in microcirculatory breakdown. Only tetrahydrobiopterin pretreatment succeeded to preserve the capillary net, whereas all other compounds showed no beneficial effects. Along with increased intragraft tetrahydrobiopterin levels during recovery and implantation, only tetrahydrobiopterin pretreatment led to significant reduction of IRI-related parenchymal damage enabling recipient survival. These results show a striking superiority of tetrahydrobiopterin in preventing lethal IRI compared with related compounds and suggest nitric oxide synthases as treatment target.
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http://dx.doi.org/10.1111/j.1432-2277.2012.01530.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499685PMC
October 2012

Orphan enzymes in ether lipid metabolism.

Biochimie 2013 Jan 4;95(1):59-65. Epub 2012 Jul 4.

Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Innrain, Innsbruck, Austria.

Ether lipids are an emerging class of lipids which have so far not been investigated and understood in every detail. They have important roles as membrane components of e.g. lens, brain and testis, and as mediators such as platelet-activating factor. The metabolic enzymes for biosynthesis and degradation have been investigated to some extent. As most involved enzymes are integral membrane proteins they are tricky to handle in biochemical protocols. The sequence of some ether lipid metabolising enzymes has only recently been reported and other sequences still remain obscure. Defined enzymes without assigned sequence are known as orphan enzymes. One of these enzymes with uncharacterised sequence is plasmanylethanolamine desaturase, a key enzyme for the biosynthesis of one of the most abundant phospholipids in our body, the plasmalogens. This review aims to briefly summarise known functions of ether lipids, give an overview on their metabolism including the most prominent members, platelet-activating factor and the plasmalogens. A special focus is set on the description of orphan enzymes in ether lipid metabolism and on the successful strategies how four previous orphans have recently been assigned a sequence. Only one of these four was characterised by classical protein purification and sequencing, whereas the other three required alternative strategies such as bioinformatic candidate gene selection and recombinant expression or development of an inhibitor and multidimensional metabolic profiling.
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http://dx.doi.org/10.1016/j.biochi.2012.06.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3520006PMC
January 2013

Studying fatty aldehyde metabolism in living cells with pyrene-labeled compounds.

J Lipid Res 2012 Jul 16;53(7):1410-6. Epub 2012 Apr 16.

Division of Biological Chemistry, Biocenter, Innsbruck Medical University, 6020 Innsbruck, Austria.

The lack of fatty aldehyde dehydrogenase function in Sjögren Larsson Syndrome (SLS) patient cells not only impairs the conversion of fatty aldehydes into their corresponding fatty acid but also has an effect on connected pathways. Alteration of the lipid profile in these cells is thought to be responsible for severe symptoms such as ichtyosis, mental retardation, and spasticity. Here we present a novel approach to examine fatty aldehyde metabolism in a time-dependent manner by measuring pyrene-labeled fatty aldehyde, fatty alcohol, fatty acid, and alkylglycerol in the culture medium of living cells using HPLC separation and fluorescence detection. Our results show that in fibroblasts from SLS patients, fatty aldehyde is not accumulating but is converted readily into fatty alcohol. In control cells, in contrast, exclusively the corresponding fatty acid is formed. SLS patient cells did not display a hypersensitivity toward hexadecanal or hexadecanol, but 3-fold lower concentrations of the fatty alcohol than the corresponding fatty aldehyde were needed to induce toxicity in SLS patient and in control cells.
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http://dx.doi.org/10.1194/jlr.D025650DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3371253PMC
July 2012

Catalytic residues and a predicted structure of tetrahydrobiopterin-dependent alkylglycerol mono-oxygenase.

Biochem J 2012 Apr;443(1):279-86

Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Fritz-Pregl-Strasse 3/VI, A-6020 Innsbruck, Austria.

Alkylglycerol mono-oxygenase (EC 1.14.16.5) forms a third, distinct, class among tetrahydrobiopterin-dependent enzymes in addition to aromatic amino acid hydroxylases and nitric oxide synthases. Its protein sequence contains the fatty acid hydroxylase motif, a signature indicative of a di-iron centre, which contains eight conserved histidine residues. Membrane enzymes containing this motif, including alkylglycerol mono-oxygenase, are especially labile and so far have not been purified to homogeneity in active form. To obtain a first insight into structure-function relationships of this enzyme, we performed site-directed mutagenesis of 26 selected amino acid residues and expressed wild-type and mutant proteins containing a C-terminal Myc tag together with fatty aldehyde dehydrogenase in Chinese-hamster ovary cells. Among all of the acidic residues within the eight-histidine motif, only mutation of Glu137 to alanine led to an 18-fold increase in the Michaelis-Menten constant for tetrahydrobiopterin, suggesting a role in tetrahydrobiopterin interaction. A ninth additional histidine residue essential for activity was also identified. Nine membrane domains were predicted by four programs: ESKW, TMHMM, MEMSAT and Phobius. Prediction of a part of the structure using the Rosetta membrane ab initio method led to a plausible suggestion for a structure of the catalytic site of alkylglycerol mono-oxygenase.
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http://dx.doi.org/10.1042/BJ20111509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3304488PMC
April 2012

IDO and regulatory T cell support are critical for cytotoxic T lymphocyte-associated Ag-4 Ig-mediated long-term solid organ allograft survival.

J Immunol 2012 Jan 30;188(1):37-46. Epub 2011 Nov 30.

Department of Visceral, Transplant and Thoracic Surgery, Innsbruck Medical University, A-6020 Innsbruck, Austria.

Costimulatory blockade of CD28-B7 interaction with CTLA4Ig is a well-established strategy to induce transplantation tolerance. Although previous in vitro studies suggest that CTLA4Ig upregulates expression of the immunoregulatory enzyme IDO in dendritic cells, the relationship of CTLA4Ig and IDO in in vivo organ transplantation remains unclear. In this study, we studied whether concerted immunomodulation in vivo by CTLA4Ig depends on IDO. C57BL/6 recipients receiving a fully MHC-mismatched BALB/c heart graft treated with CTLA4Ig + donor-specific transfusion showed indefinite graft survival (>100 d) without signs of chronic rejection or donor specific Ab formation. Recipients with long-term surviving grafts had significantly higher systemic IDO activity as compared with rejectors, which markedly correlated with intragraft IDO and Foxp3 levels. IDO inhibition with 1-methyl-dl-tryptophan, either at transplant or at postoperative day 50, abrogated CTLA4Ig + DST-induced long-term graft survival. Importantly, IDO1 knockout recipients experienced acute rejection and graft survival comparable to controls. In addition, αCD25 mAb-mediated depletion of regulatory T cells (Tregs) resulted in decreased IDO activity and again prevented CTLA4Ig + DST induced indefinite graft survival. Our results suggest that CTLA4Ig-induced tolerance to murine cardiac allografts is critically dependent on synergistic cross-linked interplay of IDO and Tregs. These results have important implications for the clinical development of this costimulatory blocker.
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http://dx.doi.org/10.4049/jimmunol.1002777DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3249458PMC
January 2012

Cryoflotation: densities of amorphous and crystalline ices.

J Phys Chem B 2011 Dec 31;115(48):14167-75. Epub 2011 Aug 31.

Institute of Physical Chemistry, University of Innsbruck, Innrain 52a, A-6020 Innsbruck, Austria.

We present an experimental method aimed at measuring mass densities of solids at ambient pressure. The principle of the method is flotation in a mixture of liquid nitrogen and liquid argon, where the mixing ratio is varied until the solid hovers in the liquid mixture. The temperature of such mixtures is in the range of 77-87 K, and therefore, the main advantage of the method is the possibility of determining densities of solid samples, which are instable above 90 K. The accessible density range (~0.81-1.40 g cm(-3)) is perfectly suitable for the study of crystalline ice polymorphs and amorphous ices. As a benchmark, we here determine densities of crystalline polymorphs (ices I(h), I(c), II, IV, V, VI, IX, and XII) by flotation and compare them with crystallographic densities. The reproducibility of the method is about ±0.005 g cm(-3), and in general, the agreement with crystallographic densities is very good. Furthermore, we show measurements on a range of amorphous ice samples and correlate the density with the d spacing of the first broad halo peak in diffraction experiments. Finally, we discuss the influence of microstructure, in particular voids, on the density for the case of hyperquenched glassy water and cubic ice samples prepared by deposition of micrometer-sized liquid droplets.
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http://dx.doi.org/10.1021/jp204752wDOI Listing
December 2011

Identification of the gene encoding alkylglycerol monooxygenase defines a third class of tetrahydrobiopterin-dependent enzymes.

Proc Natl Acad Sci U S A 2010 Aug 19;107(31):13672-7. Epub 2010 Jul 19.

Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Fritz-Pregl-Strasse 3, A-6020 Innsbruck, Austria.

Alkylglycerol monooxygenase (glyceryl-ether monooxygenase, EC 1.14.16.5) is the only enzyme known to cleave the O-alkyl bond of ether lipids which are essential components of brain membranes, protect the eye from cataract, interfere or mediate signalling processes, and are required for spermatogenesis. Along with phenylalanine hydroxylase, tyrosine hydroxylase, tryptophan hydroxylase, and nitric oxide synthase, alkylglycerol monooxygenase is one of five known enzymatic reactions which depend on tetrahydrobiopterin. Although first described in 1964, no sequence had been assigned to this enzyme so far since it lost activity upon protein purification attempts. A functional library screen using pools of plasmids of a rat liver expression library transfected to CHO cells was also unsuccessful. We therefore selected human candidate genes by bioinformatic approaches and by proteomic analysis of partially purified enzyme and tested alkylglycerol monooxygenase activity in CHO cells transfected with expression plasmids. Transmembrane protein 195, a predicted membrane protein with unassigned function which occurs in bilateral animals, was found to encode for tetrahydrobiopterin-dependent alkylglycerol monooxygenase. This sequence assignment was confirmed by injection of transmembrane protein 195 cRNA into Xenopus laevis oocytes. Transmembrane protein 195 shows no sequence homology to aromatic amino acid hydroxylases or nitric oxide synthases, but contains the fatty acid hydroxylase motif. This motif is found in enzymes which contain a diiron center and which carry out hydroxylations of lipids at aliphatic carbon atoms like alkylglycerol monooxygenase. This sequence assignment suggests that alkylglycerol monooxygenase forms a distinct third group among tetrahydrobiopterin-dependent enzymes.
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http://dx.doi.org/10.1073/pnas.1002404107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2922233PMC
August 2010

Monitoring of fatty aldehyde dehydrogenase by formation of pyrenedecanoic acid from pyrenedecanal.

J Lipid Res 2010 Jun 5;51(6):1554-9. Epub 2009 Nov 5.

Division of Biological Chemistry, Biocenter, Innsbruck Medical University, A-6020 Innsbruck, Austria.

Fatty aldehyde dehydrogenase (EC 1.2.1.48) converts long-chain fatty aldehydes to the corresponding acids. Deficiency in this enzyme causes the Sjogren Larsson Syndrome, a rare inherited disorder characterized by ichthyosis, spasticity, and mental retardation. Using a fluorescent aldehyde, pyrenedecanal, and HPLC with fluorescence detection, we developed a novel method to monitor fatty aldehyde dehydrogenase activity by quantification of the product pyrenedecanoic acid together with the substrate pyrenedecanal and possible side products, such as aldehyde adducts. As shown with recombinant enzymes, pyrenedecanal showed a high preference for fatty aldehyde dehydrogenase compared with other aldehyde dehydrogenases. The method allowed detection of fatty aldehyde dehydrogenase activity in nanogram amounts of microsomal or tissue protein and microgram amounts of Sjogren Larsson syndrome patients' skin fibroblast protein. It could successfully be adapted for the analysis of fatty aldehyde dehydrogenase activity in gel slices derived from low-temperature SDS-PAGE, showing that fatty aldehyde dehydrogenase activity from solubilized rat liver microsomes migrates as a dimer. Thus, monitoring of pyrenedecanoic acid formation from pyrenedecanal by HPLC with fluorescence detection provides a robust and sensitive method for determination of fatty aldehyde dehydrogenase activity.
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http://dx.doi.org/10.1194/jlr.D002220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3035519PMC
June 2010

Glyceryl ether monooxygenase resembles aromatic amino acid hydroxylases in metal ion and tetrahydrobiopterin dependence.

Biol Chem 2009 Jan;390(1):3-10

Division of Biological Chemistry, Biocentre, Innsbruck Medical University, Fritz-Pregl-Str. 3/VI, A-6020 Innsbruck, Austria.

Glyceryl ether monooxygenase is a tetrahydrobiopterin-dependent membrane-bound enzyme which catalyses the cleavage of lipid ethers into glycerol and the corresponding aldehyde. Despite many different characterisation and purification attempts, so far no gene and primary sequence have been assigned to this enzyme. The seven other tetrahydrobiopterin-dependent enzymes can be divided in the family of aromatic amino acid hydroxylases - comprising phenylalanine hydroxylase, tyrosine hydroxylase and the two tryptophan hydroxylases - and into the three nitric oxide synthases. We tested the influences of different metal ions and metal ion chelators on glyceryl ether monooxygenase, phenylalanine hydroxylase and nitric oxide synthase activity to elucidate the relationship of glyceryl ether monooxygenase to these two families. 1,10-Phenanthroline, an inhibitor of non-heme iron-dependent enzymes, was able to potently block glyceryl ether monooxygenase as well as phenylalanine hydroxylase, but had no effect on inducible nitric oxide synthase. Two tetrahydrobiopterin analogues, N(5)-methyltetrahydrobiopterin and 4-aminotetrahydrobiopterin, had a similar impact on glyceryl ether monooxygenase activity, as has already been shown for phenylalanine hydroxylase. These observations point to a close analogy of the role of tetrahydrobiopterin in glyceryl ether monooxygenase and in aromatic amino acid hydroxylases and suggest that glyceryl ether monooxygenase may require a non-heme iron for catalysis.
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http://dx.doi.org/10.1515/BC.2009.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2847825PMC
January 2009

Functional properties and modulation of extracellular epitope-tagged Ca(V)2.1 voltage-gated calcium channels.

Channels (Austin) 2008 Nov-Dec;2(6):461-73. Epub 2008 Nov 15.

Pharmacology and Toxicology Unit, Institute of Pharmacy and Centre for Molecular Biosciences Innsbruck, University of Innsbruck, Innsbruck, Austria.

Depolarisation-induced Ca2+ influx into electrically excitable cells is determined by the density of voltage-gated Ca2+ channels at the cell surface. Surface expression is modulated by physiological stimuli as well as by drugs and can be altered under pathological conditions. Extracellular epitope-tagging of channel subunits allows to quantify their surface expression and to distinguish surface channels from those in intracellular compartments. Here we report the first systematic characterisation of extracellularly epitope-tagged Ca(V)2.1 channels. We identified a permissive region in the pore-loop of repeat IV within the Ca(V)2.1 alpha(1) subunit, which allowed integration of several different tags (hemagluttinine [HA], double HA; 6-histidine tag [His], 9-His, bungarotoxin-binding site) without compromising alpha(1) subunit protein expression (in transfected tsA-201 cells) and function (after expression in X. laevis oocytes). Immunofluorescence studies revealed that the double-HA tagged construct (1722-HAGHA) was targeted to presynaptic sites in transfected cultured hippocampal neurons as expected for Ca(V)2.1 channels. We also demonstrate that introduction of tags into this permissive position creates artificial sites for channel modulation. This was demonstrated by partial inhibition of 1722-HA channel currents with anti-HA antibodies and the concentration-dependent stimulation or partial inhibition by Ni-nitrilo triacetic acid (NTA) and novel bulkier derivatives (Ni-trisNTA, Ni-tetrakisNTA, Ni-nitro-o-phenyl-bisNTA, Ni-nitro-p-phenyl-bisNTA). Therefore our data also provide evidence for the concept that artificial modulatory sites for small ligands can be introduced into voltage-gated Ca2+ channel for their selective modulation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3942855PMC
http://dx.doi.org/10.4161/chan.2.6.6793DOI Listing
April 2009

Time course and specificity of the pharmacological disruption of the trafficking of voltage-gated calcium channels by gabapentin.

Channels (Austin) 2008 Jan-Feb;2(1):4-9. Epub 2008 Apr 4.

Laboratory of Cellular and Molecular Neuroscience, Department of Pharmacology, University College London, London, United Kingdom.

The mechanism of action of gabapentin is still not well understood. It binds to the alpha(2)delta-1 and alpha(2)delta-2 subunits of voltage-gated calcium channels but has little acute effect on calcium currents in several systems. However, our recent results conclusively demonstrated that gabapentin inhibited calcium currents when applied chronically but not acutely, both in heterologous expression systems and in dorsal root ganglion neurons.(1) In that study we only examined a 40-hour time point of incubation with gabapentin, and here we have extended these results to include the effect of up to 6 and 20 hours incubation with gabapentin on calcium channel currents formed from Ca(V)2.1/beta(4)/alpha(2)delta-2 subunits. Gabapentin was significantly effective to inhibit the currents if included for 17-20 hours prior to recording, but it did not produce a significant inhibition if included for 3-6 hours. We previously concluded that gabapentin acts primarily at an intracellular location, requiring uptake into cells. However, this effect is mediated by alpha(2)delta subunits, being prevented by mutations in either alpha(2)delta-1 or alpha(2)delta-2 that abolish gabapentin binding.(1) Furthermore, we also showed that the trafficking of alpha(2)delta-2 and Ca(V)2 channels was disrupted by gabapentin. Here we have also extended that study, to show that the cell-surface expression of Ca(V)2.1 is not reduced by chronic gabapentin if it is co-expressed with alpha(2)delta-2 containing a point mutation (R282A) that prevents gabapentin binding.
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http://dx.doi.org/10.4161/chan.2.1.6045DOI Listing
November 2008

A destructive interaction mechanism accounts for dominant-negative effects of misfolded mutants of voltage-gated calcium channels.

J Neurosci 2008 Apr;28(17):4501-11

Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5203, Institut de Génomique Fonctionnelle, Institut National de la Santé et de la Recherche Médicale, 34094 Montpellier, France.

Channelopathies are often linked to defective protein folding and trafficking. Among them, the calcium channelopathy episodic ataxia type-2 (EA2) is an autosomal dominant disorder related to mutations in the pore-forming Ca(v)2.1 subunit of P/Q-type calcium channels. Although EA2 is linked to loss of Ca(v)2.1 channel activity, the molecular mechanism underlying dominant inheritance remains unclear. Here, we show that EA2 mutants as well as a truncated form (D(I-II)) of the Ca(v)3.2 subunit of T-type calcium channel are misfolded, retained in the endoplasmic reticulum, and subject to proteasomal degradation. Pulse-chase experiments revealed that misfolded mutants bind to nascent wild-type Ca(v) subunits and induce their subsequent degradation, thereby abolishing channel activity. We conclude that this destructive interaction mechanism promoted by Ca(v) mutants is likely to occur in EA2 and in other inherited dominant channelopathies.
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http://dx.doi.org/10.1523/JNEUROSCI.2844-07.2008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6670939PMC
April 2008