Full text
| |
| Author(s): |
Danilo Bilches Medinas
Total Authors: 1
|
| Document type: | Doctoral Thesis |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ) |
| Defense date: | 2010-02-24 |
| Examining board members: |
Ohara Augusto;
Gerson Chadi;
Shaker Chuck Farah;
Ana Maria da Costa Ferreira;
Iseli Lourenço Nantes
|
| Advisor: | Ohara Augusto |
| Abstract | |
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of motors neurons that causes muscle atrophy, weakness, and death by respiratory failure. This pathology occurs in both sporadic and familiar forms that are clinically indistinguishable. Mutations in the antioxidant enzyme superoxide dismutase 1 (hSod1) respond to about 20% of the familiar cases of ALS. Besides, the autosomal dominant nature of these hSod1-associated ALS suggests that the mutants gain toxic properties to motor neurons. Currently, two hypotheses exist to explain the toxicity of hSod1 mutants but they do not exclude each other. The first one is related to the production of oxidants by the increased peroxidase activity of the ALS-linked mutants that could contribute to the oxidative stress reported in ALS. The second refers to protein aggregation as proposed in other neurodegenerative diseases. Noteworthy, the carbonate radical produced during hSod1 peroxidase activity leads to the formation of a covalent dimer of the protein similar to a hSod1 species often detected in experimental models and patients of the disease and implicated in the toxic properties of hSod1 mutants. Thus, the present work aimed to determine the mechanism of carbonate radical production by hSod1 and to characterize the covalent dimer of the protein in vitro followed by the study of covalent aggregates of hSod1 in a rat model of ALS that overexpresses the G93A mutant of the protein. The kinetic studies of the effect of bicarbonate/CO2, nitrite and formate in the peroxidase activity of hSod1 at various pH, measured by hydrogen peroxide consumption and radical production, permitted to exclude the Fenton mechanism to explain the enzyme peroxidative cycle in bicarbonate buffer in favor of other reactive intermediates. Furthermore, 13C NMR, molecular docking and stopped-flow experiments with asymmetric mixing demonstrated that the anion peroxomonocarbonate is the precursor of the carbonate radical produced by hSod1. The characterization of hSod1 covalent dimer by proteolysis with trypsin followed by HPLC/UV-vis and HPLC/ESI-MS analysis identified a peptide characteristic of the covalent dimer of the protein. The enzymatic digestion in H2 18 O irrefutably demonstrated the dimeric nature of this peptide because of the C-terminal labeling with oxygen-18 isotopes. In addition, sequencing of the dimeric peptide by MS/MS determined the primary structure ESNGPVKVW(ESNGPVKVWGSIK)GSIK, in which the polipeptide chains are crosslinked through a ditryptophan adduct formed by a covalent bond between the Trp32 residues of each subunit. So, this dimeric peptide can be employed as a biochemical marker for studying the hSod1 covalent dimer in vivo. The analysis of protein extracts from the spinal cord of the rat model of ALS by Western-blot identified fifteen candidates to hSod1 covalent dimer, but two of them were excluded by mass spectrometry analysis that identified unmodified Trp32 residues. Moreover, neither the dimeric peptide nor the Trp32 residue were observed in the remaining species. Therefore, these thirteen candidates must be reexamined in subsequent studies. In conclusion, the anion peroxomonocarbonate is the key intermediate in the production of the carbonate radical by hSod1 and the dimeric peptide constitutes a specific tool to study hSod1 covalent aggregation in ALS (AU) | |