Synthesis of a Fluorogenic Analogue of Sphingosine‐1‐Phosphate and Its Use to Determine Sphingosine‐1‐Phosphate Lyase Activity

Bedia, Carmen; Camacho, Luz; Casas, Josefina; Abad, José Luı́s; Delgado, Antonio; Veldhoven, Paul P. Van; Fabriás, Gemma

doi:10.1002/cbic.200800809

Cited by 30 publications

(21 citation statements)

References 30 publications

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“…2 B and C), strongly suggesting similar enzymatic activity for LpSpl. We therefore analyzed whether LpSpl exhibited S1P lyase activity by using the coumarinic sphinganine 1-phosphate analog, a fluorogenic substrate that is cleaved by SPL in a highly specific manner, producing umbelliferone whose fluorescence was quantified (30). Expression of LpSpl in HEK-293T cells induced significant lyase activity as did mouse SPL (mSPL) used as positive control (Fig.…”

Section: Resultsmentioning

confidence: 99%

Legionella pneumophila S1P-lyase targets host sphingolipid metabolism and restrains autophagy

Rolando

Escoll

Nora

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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116

142

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Autophagy is an essential component of innate immunity, enabling the detection and elimination of intracellular pathogens. Legionella pneumophila, an intracellular pathogen that can cause a severe pneumonia in humans, is able to modulate autophagy through the action of effector proteins that are translocated into the host cell by the pathogen's Dot/Icm type IV secretion system. Many of these effectors share structural and sequence similarity with eukaryotic proteins. Indeed, phylogenetic analyses have indicated their acquisition by horizontal gene transfer from a eukaryotic host. Here we report that L. pneumophila translocates the effector protein sphingosine-1 phosphate lyase (LpSpl) to target the host sphingosine biosynthesis and to curtail autophagy. Our structural characterization of LpSpl and its comparison with human SPL reveals high structural conservation, thus supporting prior phylogenetic analysis. We show that LpSpl possesses S1P lyase activity that was abrogated by mutation of the catalytic site residues. L. pneumophila triggers the reduction of several sphingolipids critical for macrophage function in an LpSpl-dependent and -independent manner. LpSpl activity alone was sufficient to prevent an increase in sphingosine levels in infected host cells and to inhibit autophagy during macrophage infection. LpSpl was required for efficient infection of A/J mice, highlighting an important virulence role for this effector. Thus, we have uncovered a previously unidentified mechanism used by intracellular pathogens to inhibit autophagy, namely the disruption of host sphingolipid biosynthesis.Legionella pneumophila | sphingosine-1-phosphate lyase | autophagy | sphingolipids | virulence T he Gram-negative intracellular bacterium Legionella pneumophila is an opportunistic human pathogen responsible for Legionnaires' disease. The bacteria are naturally found in freshwater systems where they replicate within protozoan hosts (1). It is thought that the adaptation to replication within amoebas has equipped L. pneumophila with the factors required to replicate successfully within human macrophages following opportunistic infection (2). Through genome sequencing, we have discovered that L. pneumophila encodes a high number and variety of proteins similar in sequence to eukaryotic proteins that are never or rarely found in other prokaryotic genomes (3). Subsequent phylogenetic analyses have suggested that many of these proteins were acquired by horizontal gene transfer (3, 4). One of these proteins exhibits a high degree of similarity to eukaryotic sphingosine-1 phosphate lyase (SPL). The L. pneumophila SPL homolog (LpSpl encoded by gene lpp2128, lpg2176, or legS2) is conserved in all L. pneumophila strains sequenced to date, but absent from Legionella longbeachae (SI Appendix, Table S1). Phylogenetic analysis of SPL sequences showed that the L. pneumophila spl gene was most likely acquired early during evolution by horizontal gene transfer from a protozoan organism (4, 5). With the increase in genome sequences available...

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Section: Resultsmentioning

confidence: 99%

Legionella pneumophila S1P-lyase targets host sphingolipid metabolism and restrains autophagy

Rolando

Escoll

Nora

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

116

142

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“…SPL activity determination was conducted as described (23), with little modifications. The assay was carried out in 96-well plates.…”

Section: Spl Activitymentioning

confidence: 99%

First Evidence of Sphingosine 1-Phosphate Lyase Protein Expression and Activity Downregulation in Human Neoplasm: Implication for Resistance to Therapeutics in Prostate Cancer

Brizuela

Ader

Mazerolles

et al. 2012

Molecular Cancer Therapeutics

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This is the first report of sphingosine 1-phosphate lyase (SPL) protein expression and enzymatic activity in human neoplasm. This enzyme drives irreversible degradation of sphingosine 1-phosphate (S1P), a bioactive lipid associated with resistance to therapeutics in various cancers, including prostate adenocarcinoma. In fresh human prostatectomy specimens, a remarkable decrease in SPL enzymatic activity was found in tumor samples, as compared with normal adjacent tissues. A significant relationship between loss of SPL expression and higher Gleason score was confirmed in tissue microarray (TMA) analysis. Moreover, SPL protein expression and activity were inversely correlated with those of sphingosine kinase-1 (SphK1), the enzyme producing S1P. SPL and SphK1 expressions were independently predictive of aggressive cancer on TMA, supporting the relevance of S1P in prostate cancer. In human C4-2B and PC-3 cell lines, silencing SPL enhanced survival after irradiation or chemotherapy by decreasing expression of proteins involved in sensing and repairing DNA damage or apoptosis, respectively. In contrast, enforced expression of SPL sensitized cancer cells to irradiation or docetaxel by tilting the ceramide/S1P balance toward cell death. Interestingly, the S1P degradation products failed to sensitize to chemo-and radiotherapy, supporting the crucial role of ceramide/ S1P balance in cancer. Of note, the combination of SPL enforced expression with a SphK1 silencing strategy by further decreasing S1P content made prostate cancer cells even more sensitive to anticancer therapies, suggesting that a dual strategy aimed at stimulating SPL, and inhibiting SphK1 could represent a future approach to sensitize cancer cells to cancer treatments.

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“…Lysates from HEK293T cells heterologously expressing mSGPL1 showed robust activity with a fluorogenic S1P substrate analog 36 compared to control cell lysates (Fig. 4g, left) and this activity was blocked in a concentration-dependent manner by miconazole (IC 50 value = 3.2 μM; 95% CI, 2.4 – 4.1 μM) (Fig.…”

Section: Resultsmentioning

confidence: 98%

Mapping Protein Targets of Bioactive Small Molecules Using Lipid-Based Chemical Proteomics

et al. 2017

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Lipids play critical roles in cell biology, often through direct interactions with proteins. We recently described the use of photoreactive lipid probes combined with quantitative mass spectrometry to globally map lipid-protein interactions, and the effects of drugs on these interactions, in cells. Here, we investigate the broader potential of lipid-based chemical proteomic probes for determining the cellular targets of biologically active small molecules, including natural product derivatives and repurposed drugs of ill-defined mechanisms. We identify the prostaglandin-regulatory enzyme PTGR2 as a target of the anti-diabetic hops derivative KDT501 and show that miconazole—an anti-fungal drug that attenuates disease severity in preclinical models of multiple sclerosis—inhibits SGPL1, an enzyme that degrades the signaling lipid sphingosine-1-phosphate, drug analogues of which are used to treat multiple sclerosis in humans. Our findings highlight the versatility of lipid-based chemical proteomics probes for mapping small molecule-protein interactions in human cells to gain mechanistic understanding of bioactive compounds.

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Synthesis of a Fluorogenic Analogue of Sphingosine‐1‐Phosphate and Its Use to Determine Sphingosine‐1‐Phosphate Lyase Activity

Abstract: Illuminating an ER enzyme: We report on the design and synthesis of a fluorogenic chemical sensor (1) to measure sphingosine‐1‐phosphate lyase activity in high‐throughput screening formats, as well as its validation using lyase knockout (Sgpl1−/−) cells.magnified image

Cited by 30 publications

References 30 publications

Legionella pneumophila S1P-lyase targets host sphingolipid metabolism and restrains autophagy

Legionella pneumophila S1P-lyase targets host sphingolipid metabolism and restrains autophagy

First Evidence of Sphingosine 1-Phosphate Lyase Protein Expression and Activity Downregulation in Human Neoplasm: Implication for Resistance to Therapeutics in Prostate Cancer

Mapping Protein Targets of Bioactive Small Molecules Using Lipid-Based Chemical Proteomics

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