The enzyme 4-hydroxy-2-oxoglutarate aldolase and its role in mitochondrial metabolism

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dc.contributor.advisor Loomes, K en
dc.contributor.advisor Hickey, A en
dc.contributor.advisor Baker, T en
dc.contributor.advisor Phillips, A en
dc.contributor.author MacDonald, Julia en
dc.date.accessioned 2016-02-24T20:23:07Z en
dc.date.issued 2015 en
dc.identifier.citation 2015 en
dc.identifier.uri http://hdl.handle.net/2292/28287 en
dc.description.abstract 4-Hydroxy-2-oxoglutarate aldolase (HOGA) is a Class I aldolase implicated in the rare autosomal recessive disease Primary Hyperoxaluria Type 3 (PH3), characterized by excessive oxalate production and calcium oxalate stone formation. HOGA is expressed primarily in animal liver and kidney mitochondria, and has dual activities; (1) HOG aldolase activity involved in hydroxyproline catabolism; and (2) oxaloacetate decarboxylase (OAD) activity putatively influencing the tricarboxylic acid (TCA) cycle. The mechanism whereby PH3 HOGA mutations lead to hyperoxaluria is uncharacterized. There are 26 HOGA mutations identified in PH3 patients to date, and this thesis investigates the two most common mutations, the deletion of glutamate residue 315 (315) and the intronic in-frame insertion of 17 amino acids between exons 5 and 6 (c.700+5G>T). Following their recombinant expression in Escherichia coli, the mutant proteins 315 and c.700+5G>T HOGA aggregated, were thermally unstable and kinetically inactive. To investigate intracellular processing of PH3 HOGA mutants, 315 HOGA was transfected in Flp-In® HEK-293 T-REx (F-293) cells; this allowed tetracycline-inducible expression of HOGA. mRNA was upregulated similarly in wild-type and 315 HOGA F-293 cells following induction of transcription, however HOGA protein was not detected in 315 HOGA cell lysate, in contrast to wild-type HOGA F-293 cells. Intracellular degradation of the 315 HOGA protein was inhibited using the proteasome inhibitor PS-341, which permitted the detection of 315 HOGA protein by western blot. It was hypothesized that PH3 mutations altered the quaternary structure of HOGA; however, this theory was invalid following the finding of misfolded recombinant PH3 HOGA mutant protein. The quaternary structure of wild-type HOGA formed a tetramer across a wide-concentration range. In a kinetic investigation, HOG aldolase activity was 4.8-fold more efficient compared to OAD activity of wild-type recombinant human HOGA (hHOGA), due to a lower KM (56 μM and 130 μM for HOG and oxaloacetate, respectively), and 2-fold greater turnover rate (kcat 0.5 s-1 vs 1 s-1 for HOG and oxaloacetate, respectively). HOG and oxaloacetate cleavage utilized the same kinetic mechanism, and the TCA cycle intermediate α-ketoglutarate (αKG) competitively inhibited OAD activity (Ki = 2.8 mM), and HOG aldolase activity (Ki = 22 mM). As HOGA has OAD activity, it was hypothesised that it may contribute to TCA cycle regulation in malate-fuelled mitochondria, through turnover of malate to oxaloacetate by malate dehydrogenase and the subsequent oxaloacetate decarboxylase to form pyruvate. As the net reactions of HOGA and malate dehydrogenase are similar to malic enzyme (ME), the contribution of HOGA and ME to TCA cycle turnover was investigated in mitochondria isolated from: (1) F-293 cells vs F-293 cells overexpressing hHOGA, and (2) metabolically diverse rat organs with differing expression of HOGA and ME. Malate supported respiration was high in F-293 mitochondria, and not altered by HOGA overexpression. During blockage of the ME2 pathway with the novel inhibitor NPD-389, 80 % of malate respiration was inhibited in F-293 cells, and this revealed 16 % (p<0.01) higher malate respiration in F-293 cell mitochondria overexpressing HOGA. Malate respiration varied across metabolically diverse rat organs, however rat tissue was insensitive to the ME2 inhibitor, thus the contribution of ME2 and HOGA could not be determined in rat tissues. In conclusion, the two most common PH3 HOGA mutations are confirmed to be loss of function mutations, and 315 HOGA protein is proteasomally degraded in vitro. The two activities of HOGA, HOG aldolase and OAD activity, proceed via the same catalytic mechanism, and HOG aldolase activity is 4-fold more efficient. Both activities are regulated by the TCA intermediate αKG, and preliminary evidence shows that HOGA may be involved in TCA cycle regulation. en
dc.publisher ResearchSpace@Auckland en
dc.relation.ispartof PhD Thesis - University of Auckland en
dc.relation.isreferencedby UoA99264865397302091 en
dc.rights Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. Previously published items are made available in accordance with the copyright policy of the publisher. en
dc.rights.uri https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm en
dc.title The enzyme 4-hydroxy-2-oxoglutarate aldolase and its role in mitochondrial metabolism en
dc.type Thesis en
thesis.degree.discipline Biological Sciences en
thesis.degree.grantor The University of Auckland en
thesis.degree.level Doctoral en
thesis.degree.name PhD en
dc.rights.holder Copyright: The Author en
dc.rights.accessrights http://purl.org/eprint/accessRights/OpenAccess en
pubs.elements-id 523466 en
pubs.org-id Science en
pubs.org-id Biological Sciences en
pubs.org-id Science Research en
pubs.org-id Maurice Wilkins Centre (2010-2014) en
pubs.record-created-at-source-date 2016-02-25 en


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