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| Author(s): |
Izabella Agostinho Pena
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Campinas, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia |
| Defense date: | 2015-06-08 |
| Examining board members: |
Paulo Arruda;
Opher Gileadi;
Wyatt Yue;
Mário Henrique Bengtson;
Pedro Otavio de Campos Lima
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| Advisor: | Paulo Arruda; José Andrés Yunes |
| Abstract | |
The saccharopine pathway is considered the main route for lysine catabolism in higher eukaryotes. In this pathway, the enzyme aminoadipate-semialdehyde synthase (AASS) catalyzes the oxidation of lysine into aminoadipic semialdehyde (AASA). The latter is immediately oxidized to aminoadipic acid (AAA) by aminoadipic semialdehyde dehydrogenase (AASADH). In humans, mutations affecting AASS activity lead to hyperlysinemia, a benign inborn error of metabolism. In contrast, mutations affecting AASADH activity cause pyridoxine dependent epilepsy (PDE), in which the accumulation of AASA and its cyclic form piperideine-6-carboxylate (P6C) are considered the main pathogenic drivers of this disease as it depletes pyridoxal 5?-phosphate (PLP) by formation of Knoevenagel condensation products. Although there exist another lysine catabolic route known as the pipecolate pathway, we hypothesize that the elevated plasma and urine levels of AASA/P6C observed in PDE patients arises predominantly from the saccharopine pathway. In the work comprising this thesis, a series of experiments were carried out employing diverse bilological models to study lysine catabolism to AAA. In addition, a new quantitative LC-MS/MS method was developed, which allowed the analysis of lysine catabolism products in mouse plasma and tissues. The quantification of lysine metabolites and the tracking of N15-labelled lysine suggest that AASS is the main enzyme of lysine degradation. We suggest that the primary source of pathogenic accumulation of AASA/P6C is the liver and the kidney, since they present high levels of AASS activity. In addition, we demonstrate that the pipecolate pathway plays only a minor role in the overall figure of lysine oxidation to AAA. The idea of the predominant role of the sacharopine pathway in lysine degradation was reinforced by studies performed in bacteria and plants and also from the observation that a significant proportion of the circulating pipecolate is actually produced from the saccharopine pathway. Taken together these results may support the hypothesis that targeting AASS inhibition or AASS down-regulation at transcription level would reduce the toxic levels of AASA/P6C and this may result in a better outcome for PDE. We then knocked-down AASS in HEK293T cells and observed that this does not imply in any detrimental phenotype. Then we used primary skin fibroblasts from PDE patients and observed that these cells display reduced viability in the presence of high lysine levels when compared to skin fibroblasts derived from normal children. As proof-of-concept, we recovered the normal cell survival in the presence of high lysine levels by knocking-down AASS expression in skin fibroblasts obtained from PDE patients, which suggest that inhibition of AASS prevents accumulation of AASA/P6C and thus improves cell performance. This may potentially be a new method to treat PDE. In order to understand the biochemistry and structural biology of human AASS as a tool for the design of an enzyme inhibitor, we expressed, purified and obtained the crystallographic structure by X-ray diffraction at 2.6 Å of the saccharopine dehydrogenase (SDH) domain of human AASS. The solved structure consists on a homodimer bound to the cofactor NAD+. Structure-based drug design approaches are being used to design potential ligands that may act as competitive inhibitors to saccharopine binding site (AU) | |
| FAPESP's process: | 12/00235-5 - Mechanisms of saccharopine pathway induction in human cells |
| Grantee: | Izabella Agostinho Pena |
| Support Opportunities: | Scholarships in Brazil - Doctorate (Direct) |