Analysis of the peroxisomal malate/aspartate and lactate/pyruvate shuttle components and their metabolic function

by Ignacio Felipe Lobos Matthei
Doctoral thesis
Date of Examination:2022-01-28
Date of issue:2022-05-19
Advisor:Prof. Dr. Sven Thoms
Referee:Prof. Dr. Ralph Kehlenbach
Referee:Prof. Dr. Michael Müller
Persistent Address: http://dx.doi.org/10.53846/goediss-9254

 

 

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Abstract

English

Peroxisomes are responsible for several metabolic processes. Among the best known functions are the breakdown of peroxides like H2O2 and the catabolism of fatty acids by α- and β-oxidation. The proper function of these pathways relies on the transport of not only the enzymes responsible of these processes, but also of the metabolites in play. Small molecules, up to 400 Da, can freely move through the peroxisomal membrane, but bulkier molecules like ATP, NAD+, NADH, NADP+ NADPH, and CoA, require of transporters to cross the peroxisomal membrane. So far, no protein responsible for the transport of NAD+, nor NADH has been found. In the last decade, two human proteins have been found to acquire a peroxisomal targeting signal by translational readthrough: malate dehydrogenase 1 (MDH1) and lactate dehydrogenase B (LDBH). Both these proteins are hypothesized to be part of independent shuttling systems, responsible of oxidizing NADH within peroxisomes, MDH1 as part of the malate/aspartate shuttle (MAS), and LDHB as part of the lactate/pyruvate shuttle (LS). In this work, I showed evidence of the presence of both these shuttling systems within peroxisomes. First, by demonstrating an increase in peroxisomal colocalization of MDH1 and LDHB when readthrough levels are induced with antibiotics. Then, showing a reduction in the peroxisomal levels of MDH1 in cells lacking the extended MDH1 (MDH1x), which contains a PTS1 at the very C-termini. For LDHB, no reduction in the peroxisomal levels of LDHB were found in cells lacking LDHBx. Nevertheless, cells lacking both MDH1x and LDHBx showed metabolic differences than the wild type, with higher glutathione (GSH) levels and higher catalase activity levels. Additionally, the presence of the carriers AGC1, AGC2 and OGC, responsible for the metabolite transport through the peroxisomal membrane in the MAS, was confirmed in HeLa cells, whereas in differentiated cardiomyocytes (iPSC-CM) only AGC2 was detected in peroxisomes. Altogether, the evidence gathered in this work supports the hypothesis of a MAS shuttle in human peroxisomes, suggesting a mechanism for the reoxidation of NADH within peroxisomes.
Keywords: Peroxisomes; MDH1; LDHB; Malate-aspartate shuttle; Lactate-pyruvate shuttle
 
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