7C, D) and did not vary in BAT for acetyl-CoA (supplementary Fig. IIIB). For the nuclear thoroughly fractions from Them1+/+ mice, apparent Km values in BAT and liver were higher than their respective homogenates (Fig. 7A, B). However, no activity was detected in nuclei of BAT or liver harvested from Them1?/? mice (Fig. 7 and supplementary Fig. III). This suggested that Them1 was the major source of fatty acyl-CoA thioesterase activity in nuclei, even though it was expressed there at relatively low levels. Although the isolated nuclear fractions were determined to be free of other organelle markers (4), we used immunofluorescence (supplementary Fig. IV) and immunohistochemistry (supplementary Fig. V) to confirm nuclear localization. By confocal immunofluorescence microscopy (supplementary Fig.

IV), Them1 in BAT was observed to colocalize with a fluorescent nuclear stain in Them1+/+ but not Them1?/? mice. Because high levels of autofluorescence from lipid droplets prevented adequate visualization of cytoplasmic Them1 and because immunofluorescence microscopy failed to detect Them1 in liver, we also used immunohistochemistry to image the protein. This revealed nuclear staining, as well as abundant cytoplasmic Them1 in BAT (supplementary Fig. VA) and liver (supplementary Fig. VB) of Them1+/+ but not Them1?/? mice. DISCUSSION Genetic ablation of Them1 in the mouse produced dramatic increases in energy expenditure and improvements in glucose and lipid metabolism (4). To gain insights into regulatory mechanisms, the current study was designed to elucidate biochemical characteristics of Them1.

3 The main findings were that Them1 dimerized to form an Acot with relative preference for long-chain fatty acyl-CoAs and that the START domain optimized enzyme activity. In mouse BAT and liver, loss of Them1 expression had the greatest impact on the acyl-CoA thioesterase activity of the nuclear and microsomal fractions, suggesting cellular functions. Dimerization of recombinant Them1 is in keeping with other native and recombinant type II Acots for which hot dog-fold domains oligomerize to create active sites that reside at the interfaces between the domains (1, 2). Structural analyses have revealed that the hot dog domains of Acot7 (20), Acot12 (1), Acot13/Them2 (21), Them4 (22), and Acot15/Them5 (23) form dimers or oligomers of dimers.

However, these structures are not uniformly representative of the enzymes in solution. For example, Acot12 functions as a dimer and a tetramer in solution (24) even though the crystal structure of its thioesterase domains reveals a trimer of hot dog-fold dimers (1). Batimastat This apparent discrepancy could reflect the influence of the START domain on oligomerization of Acot12. The balance between Acot12 dimers and tetramers depends upon enzyme concentration and temperature and on the presence of acetyl-CoA, ATP, and ADP (24).