Mitochondrial Thioesterases: Localization, Regulation and Functional Relevance of Acyl‐CoA Thioesterase 2

Acyl‐CoA thioesterases (Acots) hydrolyze fatty acyl‐CoA esters into free fatty acid and CoA. This group of understudied enzymes is found in several cellular compartments including mitochondria where Acots have been hypothesized, but never shown, to prevent a CoA limitation and mitigate beta‐oxidation overload. Before embarking on the study of the functional role of mitochondrial Acots we sought to determine some basic characteristics of mitochondrial Acots (Acot2, 7, 9 13) that were unknown but would be important for the interpretation of physiology in models with Acot depletion. First we determined that Acot7, 9 and 13 localize to the mitochondrial matrix, joining Acot2 that was already shown to localize there. Then, we determined that Acot13 activity is powerfully inhibited by CoA, as was previously shown for Acot7 and Acot9, whereas Acot2 activity is unaffected by CoA, and was not substantially altered by any of the other potential allosteric regulators we tested. Thus Acot2 may be the only matrix Acot that is constitutively active and, importantly, remains fully active when CoA is plentiful. Finally, using measurements of protein abundance and activity in isolated mitochondria from multiple mouse tissues, we found that the highest thioesterase activity is towards short chain acyl‐CoA in brown adipose tissue, kidney and skeletal muscle, mediated by Acot9; however CoA inhibition would largely suppress this activity when CoA is plentiful. The next most significant thioesterase activity was towards C14:0‐ and C16:0‐CoA, in heart and skeletal muscle, and we found that this activity was shared equally between Acot2 and Acot13. However, as for Acot9, Acot13 activity would be suppressed when CoA is plentiful, leaving Acot2 as the sole source of long‐chain acyl‐CoA thioesterase activity in heart and skeletal muscle when CoA is replete, suggesting that it should have functional relevance in these tissues. Indeed, using Acot2 whole‐body and muscle‐specific knockout mouse models, we find that cardiac ejection fraction is decreased when Acot2 is depleted, but only in female mice. This was accompanied by higher cardiac acyl‐CoA levels, across almost all chain lengths, in female mice with Acot2 depletion whereas Acot2 depleted male hearts had acyl‐CoA levels that were similar to control mice. Substrate oxidation studies in isolated heart mitochondria reveal differences with fatty acid substrates but not with pyruvate, suggesting that beta‐oxidation is the major substrate pathway affected by Acot2 deletion. Ongoing studies aim to further understand the functional relevance of Acot2 in the heart. Also, because quantitative analyses suggest that Acot2 activity could theoretically outstrip acyl‐CoA dehydrogenase activity which has a similar Kd for long‐chain acyl‐CoAs, we are interested to understand where, within the matrix, Acot2 localizes and, in this regard, are investigating the binding partners of Acot2.