Expression, purification and preliminary crystallographic analysis of human thyroid hormone responsive protein

Tianjin Key Laboratory of Protein Science, College of Life Sciences, Nankai University, Tianjin, People's Republic of China.
Acta Crystallographica Section F Structural Biology and Crystallization Communications (Impact Factor: 0.57). 08/2011; 67(Pt 8):941-6. DOI: 10.1107/S1744309111021099
Source: PubMed

ABSTRACT Thyroid hormone responsive protein (Thrsp, also known as Spot 14 and S14) is a carbohydrate-inducible and thyroid-hormone-inducible nuclear protein specific to liver, adipose and lactating mammary tissues. Thrsp functions to activate genes encoding fatty-acid synthesis enzymes. Recent studies have shown that in some cancers human Thrsp (hS14) localizes to the nucleus and is amplified, suggesting that it plays a role in the regulation of lipogenic enzymes during tumourigenesis. Thrsp, a member of the Spot 14 superfamily, is an acidic homodimeric protein with no sequence similarity to other mammalian gene products and its biochemical function is elusive. To shed light on the structure-function relationship of this protein, human Thrsp was crystallized. Recombinant human Thrsp (hThrsp), the N-terminally truncated human Thrsp(10-146) (hThrsp9) and their selenomethionyl (SeMet) derivatives were expressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. Diffraction-quality crystals were grown at 293 K using Li(2)SO(4) as a precipitant. Using synchrotron radiation, data for the hThrsp SeMet derivative, hThrsp9 and its SeMet derivative were collected to 4.0, 3.0 and 3.6 Å resolution, respectively, at 100 K. The crystals of full-length hThrsp and its SeMet derivative belonged to space group P4(1)2(1)2, with approximate unit-cell parameters a = b = 123.9, c = 242.1 Å, α = β = γ = 90.0°. In contrast, the crystals of the truncated hThrsp9 and its SeMet derivative belonged to space group P2(1)2(1)2(1), with approximate unit-cell parameters a = 91.6, b = 100.8, c = 193.7 Å, α = β = γ = 90.0°. A molecular-replacement solution calculated using a murine Spot 14 structure as a search model indicated the presence of six molecules per asymmetric unit, comprising three hThrsp homodimers.

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    ABSTRACT: Spot 14 (S14) is a protein that is abundantly expressed in lipogenic tissues and is regulated in a manner similar to other enzymes involved in fatty acid synthesis. Deletion of S14 in mice decreased lipid synthesis in lactating mammary tissue, but the mechanism of S14's action is unknown. Here we present the crystal structure of S14 to 2.65 Å and biochemical data showing that S14 can form heterodimers with MIG12. MIG12 modulates fatty acid synthesis by inducing the polymerization and activity of acetyl-CoA carboxylase, the first committed enzymatic reaction in the fatty acid synthesis pathway. Coexpression of S14 and MIG12 leads to heterodimers and reduced acetyl-CoA carboxylase polymerization and activity. The structure of S14 suggests a mechanism whereby heterodimer formation with MIG12 attenuates the ability of MIG12 to activate ACC.
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    ABSTRACT: We generated a Spot 14 null mouse to assess the role of Spot 14 in de novo lipid synthesis and report the Spot 14 null mouse exhibits a phenotype in the lactating mammary gland. Spot 14 null pups nursed by Spot 14 null dams gain significantly less weight than wild-type pups nursed by wild-type dams. In contrast, Spot 14 null pups nursed by heterozygous dams show similar weight gain to wild-type littermates. We found the triglyceride content in Spot 14 null milk is significantly reduced. We demonstrate this reduction is the direct result of decreased de novo lipid synthesis in lactating mammary glands, corroborated by a marked reduction of medium-chain fatty acids in the triglyceride pool. Importantly, the reduced lipogenic rate is not associated with significant changes in the activities or mRNA of key lipogenic enzymes. Finally, we report the expression of a Spot 14-related gene in liver and adipose tissue, which is absent in the lactating mammary gland. We suggest that expression of both the Spot 14 and Spot 14-related proteins is required for maximum efficiency of de novo lipid synthesis in vivo and that these proteins impart a novel mechanism regulating de novo lipogenesis.
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