Structural and functional characterization of novel mitochondrial acyl-CoA thioesterase Them5/CTMP2

Thioesterases hydrolyze thioester bonds in a variety of substrates, including fatty
acid CoA esters, and palmitoylated or myristoylated proteins, and participate in lipid
metabolism, alpha- and beta-oxidation, cholesterol metabolism, and other processes.
According to their folding and the catalytic reaction mechanism, thioesterases are
subdivided into two groups: alpha/beta-hydrolases and hotdog-fold thioesterases. Whereas alpha/beta hydrolases have been well characterized in mammals, the second group of enzymes, mammalian hotdog thioesterases have been studied to a much lesser extent.
CTMP/Them4 protein has been identified in a yeast two-hybrid screening as an
interactor and negative regulator of PKB. We have recently shown that CTMP/Them4 is a mitochondrial protein which is released form mitochondria after apoptosis induction and promotes cell death. CTMP is located in the intramembrane space and is associated with the inner mitochondrial membrane. Proper processing of CTMP is important for maintaining mitochondrial morphology. In a knock-out mouse model we have shown that mitochondria from CTMP1-/- mice are more elongated and interconnected.
Human CTMP2/Them5 protein shares 38% similarity with Them4, which has
previously been shown to be an acyl-CoA thioesterase with a broad substrate range. Interestingly, while Them4 orthologs have been found in yeast and lower eukaryotes, Them5 orthologs have only been found in higher eukaryotes.
Therefore, we were keen to investigate CTMP2/Them5. We have determined
structures of both human Them4 and Them5 and confirmed that these proteins
belong to the hotdog class of thioesterases. Previous reports have shown that
mammalian hotdog proteins are organized in tetrameric or higher-order structures.
However, both CTMP/Them4 and CTMP2/Them5 form homodimers, and thus seem
to be more closely related to bacterial hotdog thioesterases. We also identified
residues which participate in the formation of active centers and are important for the
acyl-CoA hydrolysis. Although Them4 and Them5 have very similar folding, they
exhibit structural differences and different substrate specificity. For example, Them4
reacts with acetyl-CoA, whereas Them5 does not hydrolyze it.
Hotdog-fold thioesterases play very diverse biological roles, and their function in
mammals has not been fully studied. We could show that Them5-deficient mice are
viable and fertile, and show no gross developmental abnormalities. Similarly to
Them4, which has been reported to localize in mitochondria, Them5 is also a
mitochondrial protein. Them5 is located in the mitochondrial matrix and the inner
mitochondrial membrane, facing the matrix side. We therefore analyzed the
morphology of mitochondria in Them5 knock-out mice. We demonstrate that Them5-
/- mice are characterized by a highly interconnected and elongated mitochondrial
network compared to wildtype control mice. However, loss of Them5 does not affect
the biogenesis of mitochondria. More importantly, we show that overexpression of a
thioesterase-dead version of Them5 in a cell culture system leads to the appearance
of a more elongated and interconnected mitochondrial network, similar to the
phenotype observed in Them5-/-.
In order to assess the lipid profile after Them5 ablation, we performed a detailed
mass spectrometry analysis of lipid extracts from Them5-/- tissues. We found that loss of Them5 leads to a two-fold increase in major species of monolysocardiolipin (MCL), which act as upstream metabolites in the remodeling cycle of cardiolipin (CL). CL is a phospholipid localized predominantly within the inner mitochondrial membrane. On the basis of these results, we propose that Them5 has a rather specific action in vivo, namely that of regulating the initial metabolism of mitochondrial CL by maintaining, in particular, the pool of acyl groups used to re-acetylate one metabolic intermediate of cardiolipin, SP2-MCL (stearoyl-di-palmitoyl-monolysocardiolipin). More importantly, Them5 knock-out mice develop fatty livers and show deregulation of enzymes participating in lipid metabolism. Interestingly, young Them5-/- males are insulin-hypersensitive, which we have shown in both in vivo and in vitro settings. We were able to demonstrate that insulin-induced PKB phosphorylation is stronger and more sustained in knock-out tissues, and this led us to suggest that Them5/CTMP2, in addition to CTMP/Them4, also participates in regulation of PKB signaling.
This work presents a structural and functional analysis of two members of a
previously uncharacterized class of mammalian hotdog-fold enzymes. In addition,
our data indicate for the first time a connection between the loss of thioesterase
activity, mitochondrial morphology and function, and development of fatty liver
disease. Considering that very limited information is available about the biological
role of mitochondrial hotdog-fold thioesterases in mammals, our work provides a
framework for future research in this area.