Antimicrobial and immune-modulation action within the rnase a superfamily using macrophage infection models
- Autores: Lu Lu
- Directores de la Tesis: Ester Boix i Borràs (dir. tes.)
- Lectura: En la Universitat Autònoma de Barcelona ( España ) en 2020
- Idioma: español
- Tribunal Calificador de la Tesis: Maria Plana i Coll (presid.), Xavier Fernández Busquets (secret.), Anna Aris Giralt (voc.)
- Programa de doctorado: Programa de Doctorado en Bioquímica, Biología Molecular y Biomedicina por la Universidad Autónoma de Barcelona
- Materias:
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- Resumen
The thesis focused on the biological function analysis of human RNases, emphasizing the immune-regulation action within human macrophages to fight pathogen infection and the potential molecular mechanisms involved. Our laboratory has extensively studied human RNases and found that RNases have a direct antimicrobial activity against diverse bacterial species in vitro, suggesting a physiological host-defense function. In this doctoral thesis, the main goal is to understand how human RNases function in human innate cells against pathogen infection. The majority of the research presented in this thesis is taken from 4 published papers, 1 manuscript submitted and 1 in preparation. Below is a description of each chapter within this thesis: The first chapter mainly summarized the antipathogenic mechanism of human antimicrobial peptides and proteins in mycobacterial infections and their applied therapies against tuberculosis.
The second chapter reviewed the immune-regulation functions of human RNase superfamily. 8 human RNase A family members were summarized based on their contribution to innate immunity at the extracellular space.
This third chapter focused on screening the antimicrobial profiles of human RNases against M. aurum and identifying their ability to induce autophagy in infected macrophages. Moreover, we found that the antimicrobial and autophagy activities were not dependent on the RNase enzymatic activity.
In the fourth chapter, the transcriptome of macrophages treated with RNase3 and RNase3-H15A (a catalytic defective mutant) were compared to identify the molecular mechanism of the immune-regulation action of RNase3. Moreover, we overexpressed the endogenous RNase3 in THP1 derived-macrophage cells and analyzed its effect on the infectivity by Mycobacterium aurum and the Respiratory Syncytial Virus. We identified RNase3 immune-regulation activities on macrophages, either in a ribonucleolytic-dependent or independent manner. We propose that RNase3 directly targets the EGFR receptor. On the other hand, the RNA products cleaved by RNase3 may be responsible for the ribonucleolytic-dependent macrophage immune response.
In the fifth chapter, CRISPR/Cas9 gene editing tool was applied to knock out RNase2 in macrophage THP1 cells. Then, RSV infectivity, transcriptome change, and tRNA population were compared in cells with and without endogenous RNase2. Our results indicated that RNase2 is crucial in controlling the macrophage intracellular RSV infection. Transcriptome analysis suggests a direct interaction of RNase2 with the EGFR receptor. In addition, by screening a library of tRNA fragments we identified the tRNA fragments produced by RNase2 that might participate in the virus infection inhibition.
In the sixth chapter, we analyzed the enzyme substrate specificity at the secondary nucleotide base binding site and performed a kinetic characterization of the canonical RNase types together with a molecular dynamic simulation of the selected representative family members. The results highlight an evolutionary trend from lower to higher order vertebrates towards an enhanced discrimination for adenine respect to guanine preference, The RNase binding involves specific bidentate polar and electrostatic interactions: Asn to N1/N6 and N6/N7 adenine groups in mammals versus Glu/Asp and Arg to N1/N2, N1/O6 and O6/N7 guanine groups in non-mammals. The study aims to identify the structural basis for the specific recognition of cellular RNA substrates.
In conclusion, the results presented in this thesis contribute to understand the role of RNases in innate immune cells and how they facilitate the host clearance of pathogens, which is crucial for designing and developing new therapeutic agents.
