Publications by authors named "Katharina Iwan"

8 Publications

  • Page 1 of 1

Urine proteomics analysis of patients with neuronal ceroid lipofuscinoses.

iScience 2021 Feb 31;24(2):102020. Epub 2020 Dec 31.

Inborn Errors of Metabolism Section, Genetics & Genomic Medicine Unit, Great Ormond Street Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK.

The neuronal ceroid lipofuscinoses (NCL) are a group of 13 rare neurodegenerative disorders characterized by accumulation of cellular storage bodies. There are few therapeutic options, and existing tests do not monitor disease progression and treatment response. However, urine biomarkers could address this need. Proteomic analysis of CLN2 patient urine revealed activation of immune response pathways and pathways associated with the unfolded protein response. Analysis of CLN5 and CLN6 sheep model urine showed subtle changes. To confirm and investigate the relevance of candidate biomarkers a targeted LC-MS/MS proteomic assay was created. We applied this assay to additional CLN2 samples as well as other patients with NCL (CLN1, CLN3, CLN5, CLN6, and CLN7) and demonstrated that hexosaminidase-A, aspartate aminotransferase-1, and LAMP1 are increased in NCL samples and betaine-homocysteine S-methyltransferase-1 was specifically increased in patients with CLN2. These proteins could be used to monitor the effectiveness of future therapies aimed at treating systemic NCL disease.
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http://dx.doi.org/10.1016/j.isci.2020.102020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822952PMC
February 2021

Unified prebiotically plausible synthesis of pyrimidine and purine RNA ribonucleotides.

Science 2019 10;366(6461):76-82

Center for Integrated Protein Science, Department of Chemistry, LMU München, Butenandtstrasse 5-13, 81377 München, Germany.

Theories about the origin of life require chemical pathways that allow formation of life's key building blocks under prebiotically plausible conditions. Complex molecules like RNA must have originated from small molecules whose reactivity was guided by physico-chemical processes. RNA is constructed from purine and pyrimidine nucleosides, both of which are required for accurate information transfer, and thus Darwinian evolution. Separate pathways to purines and pyrimidines have been reported, but their concurrent syntheses remain a challenge. We report the synthesis of the pyrimidine nucleosides from small molecules and ribose, driven solely by wet-dry cycles. In the presence of phosphate-containing minerals, 5'-mono- and diphosphates also form selectively in one-pot reactions. The pathway is compatible with purine synthesis, allowing the concurrent formation of all Watson-Crick bases.
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http://dx.doi.org/10.1126/science.aax2747DOI Listing
October 2019

Isotope-dilution mass spectrometry for exact quantification of noncanonical DNA nucleosides.

Nat Protoc 2019 01;14(1):283-312

Center for Integrated Protein Science Munich (CiPSM), Department of Chemistry, Ludwig-Maximilians-Universität München, Munich, Germany.

DNA contains not only canonical nucleotides but also a variety of modifications of the bases. In particular, cytosine and adenine are frequently modified. Determination of the exact quantity of these noncanonical bases can contribute to the characterization of the state of a biological system, e.g., determination of disease or developmental processes, and is therefore extremely important. Here, we present a workflow that includes detailed description of critical sample preparation steps and important aspects of mass spectrometry analysis and validation. In this protocol, extraction and digestion of DNA by an optimized spin-column and enzyme-based method are described. Isotopically labeled standards are added in the course of DNA digestion, which allows exact quantification by isotope dilution mass spectrometry. To overcome the major bottleneck of such analyses, we developed a short (~14-min-per-sample) ultra-HPLC (UHPLC) and triple quadrupole mass spectrometric (QQQ-MS) method. Easy calculation of the modification abundance in the genome is possible with the provided evaluation sheets. Compared to alternative methods, the quantification procedure presented here allows rapid, ultrasensitive (low femtomole range) and highly reproducible quantification of different nucleosides in parallel. Including sample preparation and evaluation, quantification of DNA modifications can be achieved in less than a week.
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http://dx.doi.org/10.1038/s41596-018-0094-6DOI Listing
January 2019

ALKBH5-induced demethylation of mono- and dimethylated adenosine.

Chem Commun (Camb) 2018 Aug 16;54(62):8591-8593. Epub 2018 Jul 16.

Center for Integrated Protein Science, Department of Chemistry, Ludwig-Maximilians-Universität München, Butenandtstraße 5-13, 81377 Munich, Germany.

RNA contains methylated A-base derivatives. A methylation to mA and then demethylation regulate homeostasis in mRNA. It is assumed that mA is mainly demethylated by the α-ketoglutarate dependent oxidase ALKBH5. Here we show that ALKBH5 also demethylates the dimethylated adenosine mA, which is a non-canonical base present in ribosomal RNA.
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http://dx.doi.org/10.1039/c8cc03980aDOI Listing
August 2018

A Predictive Approach for the Optical Control of Carbonic Anhydrase II Activity.

ACS Chem Biol 2018 03 9;13(3):793-800. Epub 2018 Feb 9.

Department of Chemistry , Ludwig-Maximilian-University Munich and Munich Center for Integrated Protein Science (CIPSM) , Butenandtstrasse 5-13 , 83177 Munich , Germany.

Optogenetics and photopharmacology are powerful approaches to investigating biochemical systems. While the former is based on genetically encoded photoreceptors that utilize abundant chromophores, the latter relies on synthetic photoswitches that are either freely diffusible or covalently attached to specific bioconjugation sites, which are often native or engineered cysteines. The identification of suitable cysteine sites and appropriate linkers for attachment is generally a lengthy and cumbersome process. Herein, we describe an in silico screening approach that is designed to propose a small number of optimal combinations. By applying this computational approach to human carbonic anhydrase and a set of three photochromic tethered ligands, the number of potential site-ligand combinations was narrowed from over 750 down to 6, which we then evaluated experimentally. Two of these six combinations resulted in light-responsive human Carbonic Anhydrases (LihCAs), which were characterized with enzymatic activity assays, mass spectrometry, and X-ray crystallography. Our study also provides insights into the reactivity of cysteines toward maleimides and the hydrolytic stability of the adducts obtained.
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http://dx.doi.org/10.1021/acschembio.7b00862DOI Listing
March 2018

5-Formylcytosine to cytosine conversion by C-C bond cleavage in vivo.

Nat Chem Biol 2018 Jan 27;14(1):72-78. Epub 2017 Nov 27.

Center for Integrated Protein Science Munich CiPSM at the Department of Chemistry, Ludwig-Maximilians-Universität München, Munich, Germany.

Tet enzymes oxidize 5-methyl-deoxycytidine (mdC) to 5-hydroxymethyl-dC (hmdC), 5-formyl-dC (fdC) and 5-carboxy-dC (cadC) in DNA. It was proposed that fdC and cadC deformylate and decarboxylate, respectively, to dC over the course of an active demethylation process. This would re-install canonical dC bases at previously methylated sites. However, whether such direct C-C bond cleavage reactions at fdC and cadC occur in vivo remains an unanswered question. Here we report the incorporation of synthetic isotope- and (R)-2'-fluorine-labeled dC and fdC derivatives into the genome of cultured mammalian cells. Following the fate of these probe molecules using UHPLC-MS/MS provided quantitative data about the formed reaction products. The data show that the labeled fdC probe is efficiently converted into the corresponding labeled dC, most likely after its incorporation into the genome. Therefore, we conclude that fdC undergoes C-C bond cleavage in stem cells, leading to the direct re-installation of unmodified dC.
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http://dx.doi.org/10.1038/nchembio.2531DOI Listing
January 2018

Synthesis of (R)-Configured 2'-Fluorinated mC, hmC, fC, and caC Phosphoramidites and Oligonucleotides.

Org Lett 2016 09 19;18(17):4368-71. Epub 2016 Aug 19.

Center for Integrated Protein Science, Department of Chemistry, Ludwig-Maximilians-Universität München , Butenandtstraße 5-13, 81377 Munich, Germany.

Investigation of the function of the new epigenetic bases requires the development of stabilized analogues that are stable during base excision repair (BER). Here we report the synthesis of 2'-(R)-fluorinated versions of the phosphoramidites of 5-methylcytosine (mC), 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxycytosine (caC). For oligonucleotides containing 2'-(R)-F-fdC, we show that these compounds cannot be cleaved by the main BER enzyme thymine-DNA glycosylase (TDG).
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http://dx.doi.org/10.1021/acs.orglett.6b02110DOI Listing
September 2016

Optical control of acetylcholinesterase with a tacrine switch.

Angew Chem Int Ed Engl 2014 Jul 4;53(29):7657-60. Epub 2014 Jun 4.

Department Chemie, Ludwig-Maximilians-Universität München und Center of Integrated Protein Science Munich, Butenandtstrasse 5-13, 81377 München (Germany).

Photochromic ligands have been used to control a variety of biological functions, especially in neural systems. Recently, much effort has been invested in the photocontrol of ion channels and G-protein coupled receptors found in the synapse. Herein, we describe the expansion of our photopharmacological approach toward the remote control of an enzyme. Building on hallmark studies dating from the late 1960s, we evaluated photochromic inhibitors of one of the most important enzymes in synaptic transmission, acetylcholinesterase (AChE). Using structure-based design, we synthesized several azobenzene analogues of the well-known AChE inhibitor tacrine (THA) and determined their effects on enzymatic activity. One of our compounds, AzoTHA, is a reversible photochromic blocker of AChE in vitro and ex vivo with high affinity and fast kinetics. As such, AzoTHA can be used to control synaptic transmission on the neuromuscular endplate based on the light-dependent clearance of a neurotransmitter.
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http://dx.doi.org/10.1002/anie.201403666DOI Listing
July 2014