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Unveiling the Multifaceted Antimicrobial Mechanism of Action of Human Host Defence Rnases

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Unveiling the Multifaceted Antimicrobial Mechanism of Action of Human Host Defence Rnases ADVERTIMENT. Lʼaccés als continguts dʼaquesta tesi queda condicionat a lʼacceptació de les condicions dʼús establertes per la següent llicència Creative Commons: http://cat.creativecommons.org/?page_id=184 ADVERTENCIA. El acceso a los contenidos de esta tesis queda condicionado a la aceptación de las condiciones de uso establecidas por la siguiente licencia Creative Commons: http://es.creativecommons.org/blog/licencias/ WARNING. The access to the contents of this doctoral thesis it is limited to the acceptance of the use conditions set by the following Creative Commons license: https://creativecommons.org/licenses/?lang=en Departament de Bioquímica, Biología Molecular i Biomedicina Unveiling the multifaceted antimicrobial mechanism of action of human host defence RNases Tesis presentada por Javier Arranz Trullén para optar al grado de Doctor en Bioquímica, Biología Molecular y Biomedicina bajo la dirección de la Dra. Ester Boix Borràs y el Dr. David Pulido Gómez Unitat de Biociències del Departament de Bioquímica i Biología Molecular. Universitat Autònoma de Barcelona Dra. Ester Boix Borràs Dr. David Pulido Gómez Javier Arranz Trullén Campus de Bellaterra, Septiembre 2016 1 2 Biochemistry, Molecular Biology and Biomedicine Department Unveiling the multifaceted antimicrobial mechanism of action of human host defence RNases Thesis presented by Javier Arranz Trullén to obtain the PhD in Biochemistry, Molecular Biology and Biomedicine directed by Dr. Ester Boix Borràs and Dr. David Pulido Gómez Biosciences Unit of the Biochemistry and Molecular Biology Department Universitat Autònoma de Barcelona Dra. Ester Boix Borràs Dr. David Pulido Gómez Javier Arranz Trullén Campus de Bellaterra, September 2016 3 4 Content Summary/Resumen ..................................................................................................... 7 List of publications ....................................................................................................... 11 Abbreviations .............................................................................................................. 13 1. General introduction ................................................................................................. 17 1.1 Structural variety establishment and classification of antimicrobial peptides ... 17 1.1.1 Short antimicrobial peptides .................................................................. 17 1.1.1.1 α-helical amino acid structures ............................................................ 19 1.1.1.2 β-sheet amino acid structures .............................................................. 21 1.1.1.3 α-helical and β-sheet mixed structures ................................................ 22 1.1.1.4 Non-αβ peptides (extended structures) ............................................... 23 1.1.2 Antimicrobial polypeptides/proteins ...................................................... 24 1.2 Bio-structural features for antimicrobial activity ............................................... 24 1.2.1 Secondary structure and length .............................................................. 25 1.2.2 Amphipathicity and polar angle .............................................................. 25 1.2.3 Cyclic structures ...................................................................................... 26 1.2.4 Hydrophobicity ........................................................................................ 27 1.2.5 Conserved salt bridges ............................................................................ 27 1.2.6 Cationicity ............................................................................................... 28 1.2.7 Antimicrobial activity hallmarKs: specific amino acid residues ............... 28 1.3 Modes of action of AMPs .................................................................................... 30 1.4 Immune system and AMPs: an immunomodulatory linK ................................... 34 1.4.1 Neutralization of LPS ............................................................................... 34 1.4.2 Chemotaxis activity ................................................................................. 35 1.4.3 Epidermal growth factor interactions and wound healing ..................... 35 1.4.4 Adaptive immune response cooperation ............................................... 35 1.5 Human antimicrobial RNases .............................................................................. 36 1.5.1 RNase 3/Eosinophil Cationic Protein ...................................................... 38 1.5.2 RNase 6 ................................................................................................... 38 1.5.3 RNase 7 .................................................................................................... 39 1.5.4 N-terminal RNase derived peptides ........................................................ 40 2. Aims of the thesis ..................................................................................................... 45 3. Results ....................................................................................................................... 49 Paper I. Insights into the antimicrobial mechanism of action of Human RNase 6: Structural determinants for bacterial cell agglutination and membrane permeation ........................................................................................................... 53 5 Paper II. The first crystal structure of human RNase 6 reveals a novel substrate binding and cleavage site arrangement ............................................................... 75 Paper III. Exploring the mechanisms of action of human secretory RNase 3 and RNase 7 against Candida albicans ........................................................................ 91 Paper IV. Antifungal mechanism of action at the N-terminus domain of the human RNases .................................................................................................................. 109 Paper V. Endogenous host antimicrobial peptides (AMPs) to tackle antimicrobial resistance in tuberculosis ..................................................................................... 127 Paper VI. Unveiling the mode of action of human antimicrobial RNases against Mycobacterium tuberculosis using a surrogate macrophage infected model .... 163 4. General discussion and future Outlook ................................................................... 187 4.1 Unveiling the antimicrobial mechanism of action of human RNase 6 ............... 187 4.2 Crystal structure of human RNase 6: Structure- functional insights into its nucleotide binding mode .................................................................................... 188 4.3 Characterization of the antimicrobial mechanism of RNases and their N-terminal derived peptides using C. albicans, an eucariotic organism ............................. 188 4.4 Host immunogenic antimicrobial peptides (AMPs) to tackle antimicrobial resistance in mycobacteria: An integrated surrogate model for screening of AMPs against Mycobacterium tuberculosis .................................................................. 190 5. Conclusions ............................................................................................................... 195 6. References ................................................................................................................ 199 7. Annexes .................................................................................................................... 219 7.1 Supplementary material of Paper I ..................................................................... 221 7.2 Supplementary material of Paper II ..................................................................... 223 7.3 Supplementary material of Paper III ................................................................... 236 7.4 Supplementary material of Paper VI ................................................................... 244 6 Summary My PhD project is integrated into the large-scale study of the structure and function of human antimicrobial ribonucleases. These cationic and low molecular weight proteins are grouped into the ribonuclease A superfamily, considered one of the best characterized enzymes of the twentieth century. The RNase A superfamily is specific for vertebrates and has attracted remarkable interest due to the diversity of displayed biological properties; and represents an excellent example of a multifunctional protein´s family. Together with the main enzymatic activity we must highlight other biological properties such as the angiogenic, immunomodulatory and antimicrobial activities. The reported antimicrobial activity of distantly related family members in early vertebrates suggests that the family arouse with an ancestral function in host defence. Besides, the expression of several human RNases has been reported to be induced by infection. In particular, the RNases studied in this work, the human RNases 3, 6 and 7, are mainly expressed in eosinophils, monocytes and epithelial cells respectively. These proteins show a high cationicity due to the high proportion of basic residues and a remarkable antimicrobial activity against a wide range of human pathogens. Our research group has a consolidated experience in the study of the mechanism of action of human ribonucleases and the experimental work presented in this thesis is contributing to this overall research project. The main results achieved by
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