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Orm family proteins mediate sphingolipid homeostasis

Orm family proteins mediate sphingolipid homeostasis. David K. Breslow et al. Nature , 2010. Presentation by: Gillian Dekkers and Soledad Ordoñez Supervisor: Joost Holthuis. Sphingolipids. Are needed for: Membrane impermeability Molecular signalling Sorting

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Orm family proteins mediate sphingolipid homeostasis

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  1. Orm family proteins mediate sphingolipid homeostasis David K. Breslow et al. Nature, 2010 Presentation by: Gillian Dekkers and Soledad Ordoñez Supervisor: JoostHolthuis

  2. Sphingolipids • Are needed for: • Membrane impermeability • Molecular signalling • Sorting • Cell-cell recognition • Block in synthesis compromises cell growth and survival • However, intermediates of synthesis are mediators of cell stress pathways • Accumulation results in growth arrest and cell death

  3. How do cells solve the dilemma of sphingolipid regulation? Previously known:

  4. 1. Sphingolipid synthesis Toxicintermediates ER Vitalspingolipids Golgi Figure 1d

  5. 2. Saccharomycescerevisiae • Budding yeast with short generation time • Easily cultured • Entire genome sequenced in 1996 • Complete set of deletion mutants is available

  6. 3. Orm proteins • Connection whithchilhood asthma. • Humans: ORMDL1/2/3, S. cerevisiae: ORM1/2 • Trans membrane protein located in ER membrane. • No known functional domains • Strongly conserved between organisms

  7. E-MAPs • Epistatic mini-array profiles • Finding functional relationships between genes by: • Mapping genetic interactions

  8. E-maps method • Take a knock out of one gene of interest • Make double knock outs with a variety of genes • Check the growth rate of this double knock outs Gene1Δ x Gene2Δ

  9. E-Maps: outcome

  10. Problem: Essential genes • Essential genes cannot be screened • Solution: the use of hypomorphic alleles • mRNA of gene is destabilized • Lower expression

  11. E-Map of the ER • Orm1/2Δ versus 1400 genes of the ER

  12. A strong anti correlation with LCB1/2 • Lcb’s: subunits of LCB synthase • Suggesting that ORM2 and LCB1/2 have opposing cellular roles * * Figure 1a * hypormorphic alleles

  13. Orm1/2 overexpression • Orm expression phenocopies reduced Lcb1/2 expression Figure 1a

  14. Orm’s: negative regulator of the sphingolipid pathway * • LCB Synthase Figure 1d *Serinepalmoyltransferase, SPT

  15. Orm proteins control LCB levels • ORM1/2 overexpression causes reduced levels of LCB’s and ceramides • ORM1/2 deletion causes increased flux throughout the pathway Figure 1b

  16. Growth defect in Orm1/2 Δcells is relievedbymyriosin • Myriosin: LCB synthase inhibitor Figure 1c

  17. Ormproteinsform a complexwith LCB synthase SPOTS

  18. SPOTS isconservedfromyeasttohumans

  19. Whatisknownuntilhere? • Ormproteins are part of a conservedcomplexcalled SPOTS. • Orm1/2 negativelyregulateLCBssynthesisbyactingdirectlyon Lcb1/2. • Ormfunctionisconserved in humancells. Whywouldcellsincludenegativeregulators of LCB Synthase, Orm1/2?

  20. Whatisknownuntilhere? • Ormproteins are part of a conservedcomplexcalled SPOTS. • Orm1/2 negativelyregulateLCBssynthesisbyactingdirectlyon Lcb1/2. • Ormfunctionisconserved in humancells. Whywouldcellsincludenegativeregulators of LCB Synthase, Orm1/2? Homeostaticregulation !

  21. Orm 1/2 are regulated in response todistribution of sphingolipidsynthesis Myriocin = LCB synthaseinhibitor

  22. Orm 1/2 are regulated in response todistribution of sphingolipidsynthesis Ormeffectcanceled

  23. Orm 1/2 undergo phosphorylation and respondtomyriocintreatment • Ormregulation : • Alteration in Orm1/2 expresionlevels. X • Block of theinteractionwith Lcb1/2 and Sac1

  24. Orm 1/2 undergo phosphorylation and respondtomyriocintreatment • Ormregulation : • Alteration in Orm1/2 expresionlevels. X • Block of theinteractionwith Lcb1/2 and Sac1

  25. Phosphorylation of Ormproteinsis a homeostatic response todisruption of earlyprecursors. IncreaseOrmphosphorilation. No phosphorilation response.

  26. Mapping phosphorylation sites

  27. Blocking phosphorylation doesnotdisruptthecomplex Lcb1 interactionwithOrm 1/2 ismantainedevenwithoutOrmphosphorilation. Blocking of phosphorilationpreventsthechange in higherorderassemblyof the SPOTS complex.

  28. Orm phosphorylation removesthenegative control of LCB synthase.

  29. Ormproteins control sphingolipid homeostasis in theEndoplasmicReticulum • Ormproteins are homeostaticregulators of sphingolipidmetabolism. • WithoutOrmmediatedbrakeon SPT thereistoxicaccumulation of sphingolipids. • Normal intermidiate phosphorylation willgive chance to a rapidresponce.

  30. Ormproteins control sphingolipid homeostasis in theEndoplasmicReticulum • SPOTS couldact as a coordinating center thatcoupleschanges in sphingolipids and nutrientstotheactivity and localization of keyenzymes of lipidmetabolism and trafficiking.

  31. Articlerelevance • Provides a startingpointforstudyinghowprotein and lipidsynthesisiscoordinatedduringmembranebiogenesis. • Disregulation of sphingolipidmetabolismmaycontributetothedevelopment of childhoodasthma. • New Approach, whichallowstudies in proteinswithknowfunction.

  32. Questionsstilltobesolved • How do Ormproteinsregulate SPT? • Whichkinases and phosphatases control Ormphosphorilation? • Whatistheidentity of thesphingolipidintermediatewhoselevels are sensedtoregulateOrmactivity.

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