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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

Activity of ATX in the presence of selected steroids.(a) A visual guide to the annotations used for indicating specific functional groups in the bile acids chemical formulas (b–h) Inhibition of lysoPLD ATX activity measured as released choline by LPC(18:1) hydrolysis in the presence of specific bile salts with different steroid moiety hydroxylation patterns and acidic tail conjugations. The 12-OH substitution is not tolerated while the conjugation does not affect activity. The grey, blue and red circles highlight the hydroxylation of the steroid backbone; the orange and blue boxes highlight the conjugation partner. The error bars represent s.e.m. from triplicate experiments. Conc, concentration.
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f3: Activity of ATX in the presence of selected steroids.(a) A visual guide to the annotations used for indicating specific functional groups in the bile acids chemical formulas (b–h) Inhibition of lysoPLD ATX activity measured as released choline by LPC(18:1) hydrolysis in the presence of specific bile salts with different steroid moiety hydroxylation patterns and acidic tail conjugations. The 12-OH substitution is not tolerated while the conjugation does not affect activity. The grey, blue and red circles highlight the hydroxylation of the steroid backbone; the orange and blue boxes highlight the conjugation partner. The error bars represent s.e.m. from triplicate experiments. Conc, concentration.

Mentions: As sterols were not present during ATX purification, while ATX was selective for a 7α-substituted steroid presumably acquired from the cells and/or cell culture medium, we went on to investigate how distinct steroids may modulate ATX activity (Fig. 3). Hydroxysterols like 7HCS or 7α,25-dihydroxycholesterol, as well as various other steroids (testosterone, dexamethasone, prednisolone), had no effect on ATX-mediated LPC hydrolysis (Fig. 3b and Supplementary Fig. 3). Strikingly, however, the conjugated bile salt tauroursodeoxycholate (TUDCA) inhibited ATX activity with an apparent IC50 of 10 μM (Fig. 3c). Structure–activity relationship studies using bile salts with different substitutions of the steroid rings and different conjugates of the acidic tail, revealed that: (i) bile salts with the C7 hydroxyl in the α-configuration (taurochenodeoxycholate) or in the β-configuration (TUDCA) inhibit ATX activity with similar potency (Fig. 3 c,d); (ii) the presence of a hydroxyl in C12 of the C ring, either in the presence of the C7 hydroxyl (that is, taurocholate) or in the absence of the C7 hydroxyl (that is, taurodeoxycholate) abrogate the inhibitory activity of TUDCA (Fig. 3e,f); and (iii) the conjugation of the acidic tail did not significantly change potency, as shown by using glycochenodeoxycholate (which is conjugated to glycine instead of taurine) or ursodeoxycholate (lacking conjugation; Fig. 3g,h).


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)

Activity of ATX in the presence of selected steroids.(a) A visual guide to the annotations used for indicating specific functional groups in the bile acids chemical formulas (b–h) Inhibition of lysoPLD ATX activity measured as released choline by LPC(18:1) hydrolysis in the presence of specific bile salts with different steroid moiety hydroxylation patterns and acidic tail conjugations. The 12-OH substitution is not tolerated while the conjugation does not affect activity. The grey, blue and red circles highlight the hydroxylation of the steroid backbone; the orange and blue boxes highlight the conjugation partner. The error bars represent s.e.m. from triplicate experiments. Conc, concentration.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4834639&req=5

f3: Activity of ATX in the presence of selected steroids.(a) A visual guide to the annotations used for indicating specific functional groups in the bile acids chemical formulas (b–h) Inhibition of lysoPLD ATX activity measured as released choline by LPC(18:1) hydrolysis in the presence of specific bile salts with different steroid moiety hydroxylation patterns and acidic tail conjugations. The 12-OH substitution is not tolerated while the conjugation does not affect activity. The grey, blue and red circles highlight the hydroxylation of the steroid backbone; the orange and blue boxes highlight the conjugation partner. The error bars represent s.e.m. from triplicate experiments. Conc, concentration.
Mentions: As sterols were not present during ATX purification, while ATX was selective for a 7α-substituted steroid presumably acquired from the cells and/or cell culture medium, we went on to investigate how distinct steroids may modulate ATX activity (Fig. 3). Hydroxysterols like 7HCS or 7α,25-dihydroxycholesterol, as well as various other steroids (testosterone, dexamethasone, prednisolone), had no effect on ATX-mediated LPC hydrolysis (Fig. 3b and Supplementary Fig. 3). Strikingly, however, the conjugated bile salt tauroursodeoxycholate (TUDCA) inhibited ATX activity with an apparent IC50 of 10 μM (Fig. 3c). Structure–activity relationship studies using bile salts with different substitutions of the steroid rings and different conjugates of the acidic tail, revealed that: (i) bile salts with the C7 hydroxyl in the α-configuration (taurochenodeoxycholate) or in the β-configuration (TUDCA) inhibit ATX activity with similar potency (Fig. 3 c,d); (ii) the presence of a hydroxyl in C12 of the C ring, either in the presence of the C7 hydroxyl (that is, taurocholate) or in the absence of the C7 hydroxyl (that is, taurodeoxycholate) abrogate the inhibitory activity of TUDCA (Fig. 3e,f); and (iii) the conjugation of the acidic tail did not significantly change potency, as shown by using glycochenodeoxycholate (which is conjugated to glycine instead of taurine) or ursodeoxycholate (lacking conjugation; Fig. 3g,h).

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