Mis-targeting of the mitochondrial protein LIPT2 leads to apoptotic cell death

Abstract

Lipoyl(Octanoyl) Transferase 2 (LIPT2) is a protein involved in the post-translational modification of key energy metabolism enzymes in humans. Defects of lipoic acid synthesis and transfer start to emerge as causes of fatal or severe early-onset disease. We show that the first 31 amino acids of the N-terminus of LIPT2 represent a mitochondrial targeting sequence and inhibition of the transit of LIPT2 to the mitochondrion results in apoptotic cell death associated with activation of the apoptotic volume decrease (AVD) current in normotonic conditions, as well as over-activation of the swelling-activated chloride current (IClswell), mitochondrial membrane potential collapse, caspase-3 cleavage and nuclear DNA fragmentation. The findings presented here may help elucidate the molecular mechanisms underlying derangements of lipoic acid biosynthesis.

Protein moonlighting elucidates the essential human pathway catalyzing lipoic acid assembly on its cognate enzymes

“…7A) is expected from the phenotype of the LIPT1 disorder. If octanoylation of the 2-oxoacid dehydrogenase E2 subunits did occur, this could provide a substrate for LIAS-catalyzed sulfur insertion as suggested (26). However, if this were the case, loss of 8.…”

“…Indeed, LIPT2 is not processed. Several mass-spectral analyses of the human proteome report detection of LIPT2 peptides corresponding to the N-terminal seven residues plus residues 14-24 and 29-40 of the primary translation product (37-39) (the peptide data are collected at www.proteomicsdb.org), which would be lacking in the putative mature form (26). Note that, consistent with human LIPT2, loss of the 22 N-terminal E. coli LipB residues inactivated the protein (29).…”

“…It would follow that the lipoyl-adenylate activity would be an evolutionary remnant, as often seen in moonlighting proteins (23)(24)(25). It should be noted that recent models of the human disorders include a step in which LIPT1 somehow transfers lipoyl groups from GCSH to the 2-oxoacid dehydrogenase E2 subunits without invoking a mechanism for this transfer (15,26,27). We report biochemical evidence obtained by reconstruction of the human assembly pathway in E. coli plus direct biochemical assays with purified enzymes and acceptor proteins that demonstrate LIPT1 to be a lipoyl amidotransferase that catalyzes transfer of lipoyl moieties from GCSH to a 2-oxoacid dehydrogenase domain.…”

Lipoate protein ligase B primarily recognizes the C8-phosphopantetheine arm of its donor substrate, and weakly binds the acyl carrier protein

“…The three enzymes work in concert, performing non-redundant functions in the mitochondria. Genetic mutation of any one, or the upstream type II fatty acid synthesis enzymes causes metabolic dysfunction (36,37). The disorders due to malfunction of lipoic acid synthesis pathway cannot be treated by supplementation with exogenous lipoic acid, as none of the three enzymes can uptake free lipoic acid.…”

Protein–protein interaction based substrate control in the E. coli octanoic acid transferase, LipB

“…De novo lipoic acid biosynthesis occurs in all organisms as a branch point from type II fatty acid biosynthesis (FAB), and lipoic acid is the only known essential product of human mitochondrial FAB [1][2][3][4][5] . Octanoic acid is transferred from within the FAB onto a lipoylated target protein, whereupon thiol moieties are subsequently added through the activity of iron-sulfur cluster enzymes 1,[6][7][8] .…”