Development of an Activity‐Based Probe for Autotaxin

Cavalli, Silvia; Houben, Anna J.S.; Albers, Harald M. H. G.; Tilburg, Erica W. van; Ru, Arnoud de; Aoki, Junken; Veelen, Peter A. van; Moolenaar, Wouter H.; Ovaa, Huib

doi:10.1002/cbic.201000349

Cited by 11 publications

(12 citation statements)

References 31 publications

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“…As observed in the choline release assay, activation by 18:1 LPA resulted in an AC 50 value of 1.1 ± 0.3 µM (Fig.4D). Consistently, pre-incubating ATX with LPA for 30 minutes before supplying the LPC substrate reduced the length of the lag phase (Fig.4E-F).Taken together, these assays point out to two separate LPA sites: an orthosteric high affinity (~50 nM) site, virtually identical to the primary LPC binding site that leads to catalysis or competition with minimal inhibitors and has been well characterized (22,23,33); and an allosteric low affinity (~1.5 µM) site that leads to an activation of LPC hydrolysis. These observations are of key importance to start understanding the lag phase we described above.…”

supporting

confidence: 68%

“…Taken together, these assays point out to two separate LPA sites: an orthosteric high affinity (~50 nM) site, virtually identical to the primary LPC binding site that leads to catalysis or competition with minimal inhibitors and has been well characterized (22,23,33); and an allosteric low affinity (~1.5 µM) site that leads to an activation of LPC hydrolysis. These observations are of key importance to start understanding the lag phase we described above.…”

Section: Lpa Modulates Atx Catalysismentioning

confidence: 99%

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Lysophosphatidic acid produced by autotaxin acts as an allosteric modulator of its catalytic efficiency

Salgado-Polo

Fish

Matsoukas

et al. 2018

Journal of Biological Chemistry

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Autotaxin is a secreted glycoprotein and the only member of the ectonucleotide pyrophosphatase/phosphodiesterase family that converts lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA). LPA controls key responses such as cell migration, proliferation, and survival, implicating ATX-LPA signalling in various (patho)physiological processes and establishing it as a drug target. ATX structural and functional studies have revealed an orthosteric and an allosteric site, called the "pocket" and the "tunnel," respectively. However, the mechanisms in allosteric modulation of ATX's activity as a lysophospholipase D are unclear. Here, using the physiological LPC substrate, a new fluorescent substrate, and diverse ATX inhibitors, we revisited the kinetics and allosteric regulation of the ATX catalytic cycle, dissecting the different steps and pathways leading to LPC hydrolysis. We found that ATX activity is stimulated by LPA and that LPA activates ATX lysophospholipase D activity by binding to the ATX tunnel. A consolidation of all experimental kinetics data yielded a comprehensive catalytic model supported by molecular modelling simulations, and suggested a positive feedback mechanism that is regulated by the abundance of the LPA products activating hydrolysis of different LPC species. Our results complement and extend the current understanding of ATX hydrolysis in light of the allosteric regulation by ATX-produced LPA species, and have implications for the design and application of both orthosteric and allosteric ATX inhibitors.Autotaxin (ATX or ENPP2) is a secreted glycoprotein and a unique member of the ectonucleotide pyrophosphatase / phosphordiesterase (ENPP) family (1). It is the only ENPP family member with lysophospholipase D (lysoPLD) activity (EC 3.1.4.39), and it is the main enzyme responsible for the hydrolysis of lysophosphatidylcholine (2-acyl-sn-glycero-3-phosphocholine or LPC) to produce the bioactive lipid lysophosphatidic acid (monoacyl-snglycerol-3-phosphate or LPA) (2-4). LPA acts as a ligand for several LPA receptors (LPARs) showing overlapping activities. The ATX-LPA signalling axis is vital for embryonic development and has been implicated in many (patho)physiological processes, which include vascular development (5), cancer metastasis (6), and other human diseases, such as fibrosis (7) and cholestatic pruritus (8). ATX is translated as a preproenzyme that is secreted to plasma upon its proteolytic processing, resulting in its native structural domains (9, 10). Close to the Nterminus, ATX presents two somatomedin B (SMB)-like domains, which are followed by the central catalytic phosphodiesterase (PDE) domain, and an inactive nuclease-like domain. Catalysis occurs in a bimetallic active site presenting two Zn 2+ atoms, and resembles that of other members of the alkaline phosphatase family (11). The catalytic site of ATX is organized in a tripartite binding site (Fig.1), where the active site is followed by a shallow hydrophilic groove that (11)(12)(13), and a tunnel, also called ...

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supporting

confidence: 68%

Section: Lpa Modulates Atx Catalysismentioning

confidence: 99%

Lysophosphatidic acid produced by autotaxin acts as an allosteric modulator of its catalytic efficiency

Salgado-Polo

Fish

Matsoukas

et al. 2018

Journal of Biological Chemistry

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“…Therefore, the exploring of ATX inhibitors is highly focused in cancer therapy, and several ATX specific inhibitors have been developed in recent years [43-45]. In the present paper, we have demonstrated that inhibition of ATX lysoPLD activity with ATX inhibitor (such as BrP-LPA and S32826) could enhance the TSA-induced apoptosis in cancer cells.…”

Section: Discussionmentioning

confidence: 57%

Autotaxin is induced by TSA through HDAC3 and HDAC7 inhibition and antagonizes the TSA-induced cell apoptosis

Wang

et al. 2011

Mol Cancer

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Background: Autotaxin (ATX) is a secreted glycoprotein with the lysophospholipase D (lysoPLD) activity to convert lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA), a bioactive lysophospholipid involved in diverse biological actions. ATX is highly expressed in some cancer cells and contributes to their tumorigenesis, invasion, and metastases, while in other cancer cells ATX is silenced or expressed at low level. The mechanism of ATX expression regulation in cancer cells remains largely unknown. Results: In the present study, we demonstrated that trichostatin A (TSA), a well-known HDAC inhibitor (HDACi), significantly induced ATX expression in SW480 and several other cancer cells with low or undetectable endogenous ATX expression. ATX induction could be observed when HDAC3 and HDAC7 were down-regulated by their siRNAs. It was found that HDAC7 expression levels were low in the cancer cells with high endogenous ATX expression. Exogenous over-expression of HDAC7 inhibited ATX expression in these cells in a HDAC3-dependent manner. These data indicate that HDAC3 and HDAC7 collaboratively suppress ATX expression in cancer cells, and suggest that TSA induce ATX expression by inhibiting HDAC3 and HDAC7. The biological significance of this regulation mechanism was revealed by demonstrating that TSA-induced ATX protected cancer cells against TSAinduced apoptosis by producing LPA through its lysoPLD activity, which could be reversed by BrP-LPA and S32826, the inhibitors of the ATX-LPA axis.

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“…http://dx.doi.org/10.1101/346288 doi: bioRxiv preprint first posted online Jun. 13, 2018; Taken together, these assays point out to two separate LPA sites: an orthosteric high affinity (~50nM) site, virtually identical to the primary LPC binding site that leads to catalysis or competition with minimal inhibitors and has been well characterized 25,26,34 ; and an allosteric low affinity (~1.5 µM) site that leads to an activation of LPC hydrolysis. These observations are of key importance to start understanding the lag phase we described above.…”

Section: Resultsmentioning

confidence: 99%

Lysophosphatidic acid produced by Autotaxin acts as an allosteric modulator of its catalytic efficiency

Salgado-Polo

Fish

Matsoukas

et al. 2018

Preprint

View full text Add to dashboard Cite

Autotaxin is a secreted phosphodiesterase that converts lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA). LPA controls key cellular responses such as migration, proliferation and survival, implicating ATX-LPA signalling in various (patho)physiological processes and establishing it as a drug target. ATX structural and functional studies have revealed an orthosteric and an allosteric site, the "pocket" and the "tunnel". Here, we revisit the kinetics of the ATX catalytic cycle in light of allosteric regulation, dissecting the different steps and pathways that lead to LPC hydrolysis. Consolidating all experimental kinetics data to a comprehensive catalytic model supported by molecular modelling simulations, suggests a positive feedback mechanism, regulated by the abundance of the LPA products activating hydrolysis of different LPC species. Our results complement and extend current understanding of ATX hydrolysis in light of the allosteric regulation by produced LPA species, and have implications for the design and application of orthosteric and allosteric ATX inhibitors.

show abstract

Development of an Activity‐Based Probe for Autotaxin

Cited by 11 publications

References 31 publications

Lysophosphatidic acid produced by autotaxin acts as an allosteric modulator of its catalytic efficiency

Lysophosphatidic acid produced by autotaxin acts as an allosteric modulator of its catalytic efficiency

Autotaxin is induced by TSA through HDAC3 and HDAC7 inhibition and antagonizes the TSA-induced cell apoptosis

Lysophosphatidic acid produced by Autotaxin acts as an allosteric modulator of its catalytic efficiency

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