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Glycosome Biogenesis in Trypanosomes : Identification And Glycosome biogenesis in trypanosomes ‐ Identification and characterization of PEX16 and inhibitors of PEX5‐PEX14 interaction Dissertation to obtain the degree Doctor Philosophiae (Doctor of Philosophy, PhD) At the Faculty of Biology and Biotechnology Ruhr‐University Bochum International Graduate School of Biosciences Ruhr‐University Bochum Institute of Biochemistry and Pathobiochemistry Department of Systems Biochemistry Faculty of Medicine Submitted by Vishal C. Kalel M.Sc. Biochemistry from Walchandnagar, India Bochum October 2015 First supervisor: Prof. Dr. Ralf Erdmann Second supervisor: PD Dr. Mathias Lübben I Biogenese von Glykosomen in Trypanosomen ‐ Identifizierung und Charakterisierung von PEX16 und Inhibitoren der PEX5‐PEX14‐Interaktion Dissertation zur Erlangung des Grades eines Doktors der Naturwissenschaften der Fakultät für Biologie und Biotechnologie an der Internationalen Graduiertenschule Biowissenschaften der Ruhr‐Universität Bochum angefertigt im Institut für Biochemie und Pathobiochemie Abteilung für Systembiochemie der Fakultät für Medizin vorgelegt von Vishal C. Kalel M.Sc. Biochemie aus Walchandnagar, Indien Bochum Oktober 2015 Referent: Prof. Dr. Ralf Erdmann Korreferent: PD Dr. Mathias Lübben II ERKLÄRUNG Hiermit erkläre ich, dass ich die Arbeit selbstständig verfasst und bei keiner anderen Fakultät eingereicht und dass ich keine anderen als die angegeben Hilfsmittel verwendet habe. Es handelt sich bei der heute von mir eingereichten Dissertation um sechs in Wort und Bild völlig übereinstimmende Exemplare. Weiterhin erkläre ich, dass digitale Abbildungen nur die originalen Daten enthalten und in keinem Fall inhaltsverändernde Bildbearbeitung vorgenommen wurde. Bochum, den (Unterschrift) III Dedicated to... The Parasitologists and The Organziations who are helping to fight the Neglected Diseases. With special mention of following scientists after whom the Trypanosomatid parasite species are named David Bruce William Leishman Charles Donovan Ostwaldo Cruz IV Table of contents I INTRODUCTION ............................................................................................................. 1 1. Trypanosomatid parasites and the diseases .............................................................. 1 2. Glycosomes .................................................................................................................. 3 3. Peroxisome and glycosome biogenesis ...................................................................... 5 3.1 Matrix protein import ...................................................................................... 5 3.1.1 PEX5‐PEX14 interaction .......................................................................... 9 3.2 Membrane protein import ............................................................................. 10 3.3 Inheritance and degradation of peroxisomes ............................................... 14 3.4 Mechanisms of peroxisome biogenesis ......................................................... 15 3.5 Comparison of PEX proteins between human and trypanosomes ............... 17 4. Aims and objectives .................................................................................................. 19 II RESULTS ............................................................................................................................ 21 Chapter I: Identification and functional characterization of Trypanosoma brucei peroxin 16 .................................................................................. 22 a) Publication .......................................................................................................... 23 b) Supplementary (unpublished) ............................................................................ 37 1. Supplementary materials and methods ................................................. 38 1.1 Cells and growth conditions.............................................................. 38 1.2 Plasmids construction ....................................................................... 38 1.3 Microscopy ........................................................................................ 39 1.4 Yeast two‐hybrid ............................................................................... 40 2. Supplementary results ............................................................................ 41 2.1 Trypanosomatid PEX16 localization in human cells ........................ 41 2.2 Parasite PEX16 proteins do not rescue human PEX16 defects ........ 42 2.3 Trypanosomatid PEX16 localization in yeast ................................... 44 2.4 Identification of Trypanosoma PEX16 binding partners .................. 45 2.5 Overexpression of GFP‐PEX16 leads to glycosome aggregation ..... 49 V Chapter II: Identification and characterization of inhibitors of PEX5‐PEX14 interaction ...... 51 1. Materials and methods ......................................................................................... 52 1.1 Anti‐trypanosomal assay ......................................................................... 52 1.2 Human cell cytotoxicity assay ................................................................. 52 1.3 Glucose dependence of inhibitor toxicity ............................................... 53 1.4 ATP Assay and microscopy ...................................................................... 53 2. Results ................................................................................................................... 54 2.1 in vitro and bioactivity assays of PEX5‐PEX14 inhibitors (schemes) ..... 54 2.2 Anti‐trypanosomal activity and human cell cytotoxicity of inhibitors .. 57 2.3 Correlation between in vitro and in vivo activities ................................ 60 2.4 Inhibitors are also active on procyclic form trypanosomes ................... 61 2.5 Glucose is toxic in presence of inhibitors ............................................... 62 2.6 Cellular ATP levels are depleted by the inhibitor ................................... 64 2.7 Inhibitor disrupts the glycosomal protein import .................................. 65 2.8 Inhibitor reduces parasite load in mouse model of trypanosomiasis ... 66 III DISCUSSION .................................................................................................................... 68 IV SUMMARY ...................................................................................................................... 89 IV ZUSAMMENFASSUNG .................................................................................................... 91 V REFERENCES .................................................................................................................... 93 VI APPENDIX ..................................................................................................................... 103 1. Abbreviation used in the study ............................................................................... 103 2. Publications .............................................................................................................. 104 2.1 Complete list of publications ............................................................................ 104 2.2 Conference contributions ................................................................................. 105 2. Curriculum vitae ....................................................................................................... 106 3. Acknowledgments .................................................................................................... 107 4. Publication (Review) ‐ Nagotu and Kalel et al., BBA Mol Bas. Dis. (2012) ............. 109 VI INTRODUCTION I INTRODUCTION 1. Trypanosomatid parasites and the diseases Trypanosomatidae family parasites Trypanosoma and Leishmania are the causative agents of infectious diseases termed Trypanosomiasis and Leishmaniasis respectively. These along with 15 other infectious diseases are regarded as Neglected Tropical Diseases (NTDs) which collectively affect 1 billion people worldwide, mostly in poor and developing countries (WHO NTD report 2015). The protozoan parasites Trypanosoma and Leishmania not only infect humans but also affect livestock animals like cattle leading to economic loss of over 1 billion US$ annually (Kristjanson et al., 1999). Trypanosoma brucei rhodesiense (east African form) and T. b. gambiense (west African form) are transmitted by the bite of Tsetse fly (Glossina species) causing Human African Trypanosomiasis (HAT). During the first stage of the disease, trypanosomes reside and proliferate in bloodstream of human host. During second stage, the parasites invade central nervous system causing disruption of normal sleep cycle and hence the disease is commonly known as Sleeping Sickness (Mogk et al., 2014). Mainly in South America, T. cruzi is the infectious agent of Chagas’ disease (also referred to as Human American Trypanosomiasis). Bloodsucking triatomine insects also known as kissing bugs transmit T. cruzi parasites to humans through their feces laid at bite wound. Although the symptoms in acute stage are mild and non‐specific, chronic stage Chagas’ cardiomyopathy is life‐threatening (Bern, 2015). Leishmania genus comprises the most diverse pathogenic species causing wide spectrum of clinical manifestations collectively termed as Leishmaniasis (Pace, 2014). Female sandflies from the Phleobotomus and Lutzomyia transmit these parasites. In South Asian sub‐continent, L. donovani is the causative agent of visceral
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