Regulation of the action of phospholipases on lipid membranes by HePC

ZULUETA DÍAZ, YENISLEIDY DE LAS MERCEDES; AMBROGGIO, ERNESTO; FANANI, MARÍA LAURA

San Luis

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Biofísica; 2019

Sociedad Argentina de Biofísica

Miltefosine (HePC) is an amphiphile that is currently used as a cytotoxic for anti-tumor treatments or against infection diseases such as Leishmaniasis 1-5. It has been reported that the action of enzymes related to lipid metabolism, such as phospholipases, are very susceptible to changes in the physical environment of the membrane. In turn the action of phospholipases indirectly modulate other metabolic pathways through structural, electrostatic, and phase states changes of the biomembranes where they act 6. Our group previously demonstrated that the incorporation of HePC into model lipid membranes resulted in changes in the physical properties of the host membrane making them more easily compressible7. Based on these reports, we hypothesize that HePC could exert its pharmacological function by affecting the action of phospholipases (Sphingomyelinase: SMase and Phospholipase C: PLC) indirectly by modulating these membrane properties. Therefore, we study the effect of HePC on changes either at the level of enzymatic activity or on the changes that the formation of products has on the physical properties of those membranes.In the present study, we used large unilamellar vesicles (LUVs) composed of DOPC (PLC substrate): DOPE or SM16 (SMase substrate): DOPE in the absence and presence of HePC. In these systems, the activity of both enzymes was determined by thin plate chromatography (TLC). Simultaneously, we studied the change in turbidity of the LUV suspensions by spectrophotometry and the changes induced in the particle size distribution by Dynamic Light Dispersion (DLS).Our results indicated that the presence of HePC did not affect the initial hydrolysis rate of DOPC or SM16 (or the production of DAG or Cer) at short times. However, in the case of PLC, the addition of 20 mol % of HePC inhibited the restructuring of membranes, characteristic of the DAG enzymatic formation process. A drastic decrease in vesicle aggregation, DAG formation and generation of smaller of LUVs populations compared to the control was observed at long times (40 min). These results show for the first time that HePC is able to inhibit the enzymatic action and therefore the aggregation induced by the PLC in a remarkable way. In the case of SMase, a modest effect on vesicle restructuring was observed.In general, results obtained appears not to be related to an enzyme inhibition or interaction of HePC with the substrates or products of the reaction but with a non-specific geometric compensation. HePC has an effective cone-shaped geometry, which when inserted in bilayer can induce a positive curvature counteracting the effect caused by DAG. Therefore, these results gain relevance in the understanding of the mechanism of action by which the HePC regulates the lipid metabolism and signal transduction pathways in which these enzymes are involve.Abbreviations: DOPC: dioleoylphosphatidylcholine; DOPE: dioleoylphosphatidylethanolamine; DAG: diacyl glycerol; Cer: ceramide; Hexadecylphosphocholine: HePC.Bibliographic references(1) Pachioni, J. D. A.; Magalhães, J. G.; Juliane, E.; Lima, C.; Bueno, L. D. M. Alkylphospholipids ? A Promising Class of Chemotherapeutic Agents with a Broad Pharmacological Spectrum. 2013, 16 (5), 742?759.(2) Rakotomanga, M.; Blanc, S.; Gaudin, K.; Chaminade, P.; Loiseau, P. M. Miltefosine Affects Lipid Metabolism in Leishmania Donovani Promastigotes ᰔ. 2007, 51 (4), 1425?1430.(3) Dorlo, T. P. C.; Balasegaram, M.; Beijnen, J. H.; Vries, P. J. De. Miltefosine: A Review of Its Pharmacology and Therapeutic Efficacy in the Treatment of Leishmaniasis. 2012, No. July, 2576?2597.(4) Berardi, M.; Paula, A.; Elisabete, M.; Zaniquelli, D.; Ulises, A.; Siuiti, A. Biophysical Chemistry Miltefosine and BODIPY-Labeled Alkylphosphocholine with Leishmanicidal Activity : Aggregation Properties and Interaction with Model Membranes. Biophys. Chem. 2015, 196, 92?99.(5) Goñi, F. M.; Montes, L.; Alonso, A. Progress in Lipid Research Phospholipases C and Sphingomyelinases : Lipids as Substrates and Modulators of Enzyme Activity. 2012, 51, 238?266.(6) Tullio, L. De; Maggio, B.; Fanani, M. L. Sphingomyelinase Acts by an Area-Activated Mechanism on the Liquid-Expanded Phase of Sphingomyelin Monolayers. 2008, 49.(7) Zulueta Díaz, Y. de las M.; Fanani, M. L. Crossregulation between the Insertion of Hexadecylphosphocholine (Miltefosine) into Lipid Membranes and Their Rheology and Lateral Structure. Biochim. Biophys. Acta - Biomembr. 2017, 1859 (10), 1891?1899.