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Steroid binding to Autotaxin links bile salts and lysophosphatidic acid signalling.

Keune WJ, Hausmann J, Bolier R, Tolenaars D, Kremer A, Heidebrecht T, Joosten RP, Sunkara M, Morris AJ, Matas-Rico E, Moolenaar WH, Oude Elferink RP, Perrakis A - Nat Commun (2016)

Bottom Line: Autotaxin (ATX) generates the lipid mediator lysophosphatidic acid (LPA).ATX has a tripartite active site, combining a hydrophilic groove, a hydrophobic lipid-binding pocket and a tunnel of unclear function.Furthermore, our findings suggest potential clinical implications in the use of steroid drugs.

View Article: PubMed Central - PubMed

Affiliation: Division of Biochemistry, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.

ABSTRACT
Autotaxin (ATX) generates the lipid mediator lysophosphatidic acid (LPA). ATX-LPA signalling is involved in multiple biological and pathophysiological processes, including vasculogenesis, fibrosis, cholestatic pruritus and tumour progression. ATX has a tripartite active site, combining a hydrophilic groove, a hydrophobic lipid-binding pocket and a tunnel of unclear function. We present crystal structures of rat ATX bound to 7α-hydroxycholesterol and the bile salt tauroursodeoxycholate (TUDCA), showing how the tunnel selectively binds steroids. A structure of ATX simultaneously harbouring TUDCA in the tunnel and LPA in the pocket, together with kinetic analysis, reveals that bile salts act as partial non-competitive inhibitors of ATX, thereby attenuating LPA receptor activation. This unexpected interplay between ATX-LPA signalling and select steroids, notably natural bile salts, provides a molecular basis for the emerging association of ATX with disorders associated with increased circulating levels of bile salts. Furthermore, our findings suggest potential clinical implications in the use of steroid drugs.

No MeSH data available.


Related in: MedlinePlus

Physiological effects of bile salts on human serum and LPA receptor activation.(a) Inhibition of lysoPLD activity is human serum supplemented by TUDCA, measured as released choline by LPC(18:1) hydrolysis. The error bars represent s.e.m. from three different samples. (b) Representative confocal images of untreated and TUDCA-treated HeLa cells transfected with GFP-LPA1; the number of GFP-LPA1-containing endosomes is lower in TUDCA-treated cells. Scale bar, 10 μm. (c) Quantification of LPA1 internalization shows that TUDCA reduces the number of endosomes compared with untreated cells; error bars correspond to s.e.m. from 27 images in two independent experiments; ***P<0.001 according to Student's t-test.
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f6: Physiological effects of bile salts on human serum and LPA receptor activation.(a) Inhibition of lysoPLD activity is human serum supplemented by TUDCA, measured as released choline by LPC(18:1) hydrolysis. The error bars represent s.e.m. from three different samples. (b) Representative confocal images of untreated and TUDCA-treated HeLa cells transfected with GFP-LPA1; the number of GFP-LPA1-containing endosomes is lower in TUDCA-treated cells. Scale bar, 10 μm. (c) Quantification of LPA1 internalization shows that TUDCA reduces the number of endosomes compared with untreated cells; error bars correspond to s.e.m. from 27 images in two independent experiments; ***P<0.001 according to Student's t-test.

Mentions: To validate that bile salts act as allosteric inhibitors in a physiological setting, we examined their effect on modulating LPA production in human serum. Consistent with our in vitro findings, TUDCA inhibits endogenous ATX activity in human serum ex vivo (Fig. 6a) with an apparent IC50 of ∼30 μM. Also in serum, TUDCA behaves as a partial inhibitor, leaving ∼40% residual activity. Next, we examined the ability of bile salts to modulate ATX-LPA signalling, by examining their effect on ATX-dependent LPA receptor activation in cultured cells (in situ), using agonist-induced LPA1 receptor internalization as readout1718. As shown in Fig. 6b,c, at low LPC substrate levels, TUDCA reduces ATX-mediated LPAR1 endocytosis in HeLa cells, consistent with reduced and/or delayed LPA production and delivery.


Steroid binding to Autotaxin links bile salts and lysophosphatidic acid signalling.

Keune WJ, Hausmann J, Bolier R, Tolenaars D, Kremer A, Heidebrecht T, Joosten RP, Sunkara M, Morris AJ, Matas-Rico E, Moolenaar WH, Oude Elferink RP, Perrakis A - Nat Commun (2016)

Physiological effects of bile salts on human serum and LPA receptor activation.(a) Inhibition of lysoPLD activity is human serum supplemented by TUDCA, measured as released choline by LPC(18:1) hydrolysis. The error bars represent s.e.m. from three different samples. (b) Representative confocal images of untreated and TUDCA-treated HeLa cells transfected with GFP-LPA1; the number of GFP-LPA1-containing endosomes is lower in TUDCA-treated cells. Scale bar, 10 μm. (c) Quantification of LPA1 internalization shows that TUDCA reduces the number of endosomes compared with untreated cells; error bars correspond to s.e.m. from 27 images in two independent experiments; ***P<0.001 according to Student's t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4834639&req=5

f6: Physiological effects of bile salts on human serum and LPA receptor activation.(a) Inhibition of lysoPLD activity is human serum supplemented by TUDCA, measured as released choline by LPC(18:1) hydrolysis. The error bars represent s.e.m. from three different samples. (b) Representative confocal images of untreated and TUDCA-treated HeLa cells transfected with GFP-LPA1; the number of GFP-LPA1-containing endosomes is lower in TUDCA-treated cells. Scale bar, 10 μm. (c) Quantification of LPA1 internalization shows that TUDCA reduces the number of endosomes compared with untreated cells; error bars correspond to s.e.m. from 27 images in two independent experiments; ***P<0.001 according to Student's t-test.
Mentions: To validate that bile salts act as allosteric inhibitors in a physiological setting, we examined their effect on modulating LPA production in human serum. Consistent with our in vitro findings, TUDCA inhibits endogenous ATX activity in human serum ex vivo (Fig. 6a) with an apparent IC50 of ∼30 μM. Also in serum, TUDCA behaves as a partial inhibitor, leaving ∼40% residual activity. Next, we examined the ability of bile salts to modulate ATX-LPA signalling, by examining their effect on ATX-dependent LPA receptor activation in cultured cells (in situ), using agonist-induced LPA1 receptor internalization as readout1718. As shown in Fig. 6b,c, at low LPC substrate levels, TUDCA reduces ATX-mediated LPAR1 endocytosis in HeLa cells, consistent with reduced and/or delayed LPA production and delivery.

Bottom Line: Autotaxin (ATX) generates the lipid mediator lysophosphatidic acid (LPA).ATX has a tripartite active site, combining a hydrophilic groove, a hydrophobic lipid-binding pocket and a tunnel of unclear function.Furthermore, our findings suggest potential clinical implications in the use of steroid drugs.

View Article: PubMed Central - PubMed

Affiliation: Division of Biochemistry, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.

ABSTRACT
Autotaxin (ATX) generates the lipid mediator lysophosphatidic acid (LPA). ATX-LPA signalling is involved in multiple biological and pathophysiological processes, including vasculogenesis, fibrosis, cholestatic pruritus and tumour progression. ATX has a tripartite active site, combining a hydrophilic groove, a hydrophobic lipid-binding pocket and a tunnel of unclear function. We present crystal structures of rat ATX bound to 7α-hydroxycholesterol and the bile salt tauroursodeoxycholate (TUDCA), showing how the tunnel selectively binds steroids. A structure of ATX simultaneously harbouring TUDCA in the tunnel and LPA in the pocket, together with kinetic analysis, reveals that bile salts act as partial non-competitive inhibitors of ATX, thereby attenuating LPA receptor activation. This unexpected interplay between ATX-LPA signalling and select steroids, notably natural bile salts, provides a molecular basis for the emerging association of ATX with disorders associated with increased circulating levels of bile salts. Furthermore, our findings suggest potential clinical implications in the use of steroid drugs.

No MeSH data available.


Related in: MedlinePlus