Browse

Structural and Functional Studies of Enhanced Intracellular Survival Proteins from Mycobacterium tuberculosis and Mycobacterium smegmatis

DC Field Value Language
dc.contributor.advisor서세원-
dc.contributor.author김경훈-
dc.date.accessioned2017-07-14T05:52:32Z-
dc.date.available2017-07-14T05:52:32Z-
dc.date.issued2014-02-
dc.identifier.other000000016844-
dc.identifier.urihttps://hdl.handle.net/10371/125245-
dc.description학위논문 (박사)-- 서울대학교 대학원 : 화학부, 2014. 2. 서세원.-
dc.description.abstractThe intracellular pathogen Mycobacterium tuberculosis causes tuberculosis. Enhanced intracellular survival (Eis) protein, secreted by M. tuberculosis, enhances survival of Mycobacterium smegmatis in macrophages. M. tuberculosis Eis was shown to suppress host immune defenses by negatively modulating autophagy, inflammation, and cell death through c-Jun N-terminal kinase (JNK)-dependent inhibition of reactive oxygen species generation. M. tuberculosis Eis was demonstrated to contribute to drug resistance by acetylating multiple amines of aminoglycosides. However, the mechanism of enhanced intracellular survival by M. tuberculosis Eis remains unanswered. Therefore, I have characterized both M. tuberculosis and M. smegmatis Eis proteins biochemically and structurally. I have discovered that M. tuberculosis Eis is an efficient N-acetyltransferase, rapidly acetylating Lys55 of dual-specificity protein phosphatase 16 (DUSP16)/mitogen-activated protein kinase phosphatase-7 (MKP-7), a JNK-specific phosphatase. In contrast, M. smegmatis Eis is more efficient as an N-acetyltransferase. I also show that M. smegmatis Eis acetylates aminoglycosides as readily as M. tuberculosis Eis. Furthermore, M. tuberculosis Eis, but not M. smegmatis Eis, inhibits lipopolysaccharide-induced JNK phosphorylation. This functional difference against DUSP16/MKP-7 can be understood by comparing the structures of two Eis proteins. The active site of M. tuberculosis Eis with a narrow channel appears to be more suitable for sequence-specific recognition of the protein substrate than the pocket-shaped active site of M. smegmatis Eis. I propose that M. tuberculosis Eis initiates the inhibition of JNK-dependent autophagy, phagosome maturation, and reactive oxygen species generation by acetylating Lys55 of DUSP16/MKP-7. My work thus provides new insights into the mechanism of suppressing host immune responses and enhancing mycobacterial survival within macrophages by M. tuberculosis Eis.-
dc.description.tableofcontents1. Introduction
2. Material and Methods
2.1 Protein expression, purification, and mutagenesis
2.2 Crystallization and X-ray data collection
2.3 Structure solution and refinement
2.4 Spectrophotometric acetylation assay of aminoglycosides and steady-state kinetic measurements
2.5 In vitro acetylation of synthetic peptides and mass spectrometry
2.6 In vitro protein N-acetylation assay
2.7 Cell culture and Western blotting
3. Results and discussion
3.1 Overall structure and structural similarity search
3.2 Quaternary structure of Eis proteins
3.3 Ligand binding at the active site
3.4 Comparison of aminoglycosides acetyltransferase activity of Eis proteins from M. tuberculosis and M. smegmatis
3.5 Comparison of overall structure of Eis proteins from M. tuberculosis and M. smegmatis
3.6 Identification of DUSP16/MKP-7 as the N-acetylation target of M. tuberculosis Eis.
3.7 Comparison of substrate binding sites in Eis proteins
3.8 M. tuberculosis Eis, but not M. smegmatis Eis, inhibits lipopolysaccharide-induced JNK phosphorylation
3.9 Mechanism of acetylation by M. tuberculosis Eis
3.10 Mechanism of M. tuberculosis Eis protein in immune system of host cell
4. References
Abstract (in Korean)
Acknowledgements
Appendix: Printouts of the first author publications.
-
dc.formatapplication/pdf-
dc.format.extent14879092 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectEnhanced intracellular survival protein-
dc.subjecttuberculosis-
dc.subjectlysine acetylation-
dc.subject.ddc540-
dc.titleStructural and Functional Studies of Enhanced Intracellular Survival Proteins from Mycobacterium tuberculosis and Mycobacterium smegmatis-
dc.typeThesis-
dc.contributor.AlternativeAuthorKyoung Hoon, Kim-
dc.description.degreeDoctor-
dc.citation.pagesxvi, 119-
dc.contributor.affiliation자연과학대학 화학부-
dc.date.awarded2014-02-
Appears in Collections:
College of Natural Sciences (자연과학대학)Dept. of Chemistry (화학부)Theses (Ph.D. / Sc.D._화학부)
Files in This Item:
  • mendeley

Items in S-Space are protected by copyright, with all rights reserved, unless otherwise indicated.

Browse