Orm family proteins mediate sphingolipid homeostasis

Presentation by: Gillian Dekkers and Soledad Ordoñez

Supervisor: Joost Holthuis

David K. Breslow et al. Nature, 2010

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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

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How do cells solve the dilemma of sphingolipid regulation?

Previously known:

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1. Sphingolipid synthesis

ER

Golgi

Toxic intermediates

Vital spingolipids

Figure 1d

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2. Saccharomyces cerevisiae

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

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3. Orm proteins

• Connection whith chilhood asthma. • Humans: ORMDL1/2/3, S. cerevisiae: ORM1/2 • Trans membrane protein located in ER

membrane.• No known functional domains• Strongly conserved between organisms

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E-MAPs

• Epistatic mini-array profiles• Finding functional relationships between

genes by:– Mapping genetic interactions

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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

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E-Maps: outcome

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Problem: Essential genes

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

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E-Map of the ER

• Orm1/2Δ versus 1400 genes of the ER

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A strong anti correlation with LCB1/2

• Lcb’s: subunits of LCB synthase

• Suggesting that ORM2 and LCB1/2 have opposing cellular roles

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* hypormorphic alleles Figure 1a

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Orm1/2 overexpression

• Orm expression phenocopies reduced Lcb1/2 expression

Figure 1a

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Orm’s: negative regulator of the sphingolipid pathway

• LCB Synthase *

* Serine palmoyltransferase, SPT Figure 1d

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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

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Growth defect in Orm1/2 Δ cells is relieved by myriosin

• Myriosin: LCB synthase inhibitor

Figure 1c

Orm proteins form a complex with LCB synthase

SPOTS

SPOTS is conserved from yeast to humans

What is known until here?– Orm proteins are part of a conserved complex

called SPOTS. – Orm1/2 negatively regulate LCBs synthesis by

acting directly on Lcb1/2. – Orm function is conserved in human cells.

Why would cells include negative regulators of LCB Synthase, Orm1/2?

What is known until here?– Orm proteins are part of a conserved complex

called SPOTS. – Orm1/2 negatively regulate LCBs synthesis by

acting directly on Lcb1/2. – Orm function is conserved in human cells.

Why would cells include negative regulators of LCB Synthase, Orm1/2?

Homeostatic regulation !

Orm 1/2 are regulated in response to distribution of sphingolipid synthesis

Myriocin = LCB synthase inhibitor

Orm 1/2 are regulated in response to distribution of sphingolipid synthesis

Orm effect canceled

Orm 1/2 undergo phosphorylation and respond to myriocin treatment

• Orm regulation : • Alteration in Orm1/2 expresion levels. X• Block of the interaction with Lcb1/2 and Sac1

Orm 1/2 undergo phosphorylation and respond to myriocin treatment

• Orm regulation : • Alteration in Orm1/2 expresion levels. X• Block of the interaction with Lcb1/2 and Sac1

Phosphorylation of Orm proteins is a homeostatic response to disruption of

early precursors.

Increase Orm phosphorilation.

No phosphorilation response.

Mapping phosphorylation sites

Lcb1 interaction with Orm 1/2 is mantained even without Orm phosphorilation.

Blocking phosphorylation does not disrupt the complex

Blocking of phosphorilation prevents the change in higher order assembly of the SPOTS complex.

Orm phosphorylation removes the negative control of LCB synthase.

Orm proteins control sphingolipid homeostasis in the Endoplasmic Reticulum

• Orm proteins are homeostatic regulators of sphingolipid metabolism. •Without Orm mediated brake on SPT there is toxic accumulation of sphingolipids.•Normal intermidiate phosphorylation will give chance to a rapid responce.

Orm proteins control sphingolipid homeostasis in the Endoplasmic Reticulum

• SPOTS could act as a coordinating center that couples changes in sphingolipids and nutrients to the activity and localization of key enzymes of lipid metabolism and trafficiking.

Article relevance

• Provides a starting point for studying how protein and lipid synthesis is coordinated during membrane biogenesis.

• Disregulation of sphingolipid metabolism may contribute to the development of childhood asthma.

• New Approach, which allow studies in proteins with know function.

Questions still to be solved

–How do Orm proteins regulate SPT?

–Which kinases and phosphatases control Orm phosphorilation?

–What is the identity of the sphingolipid intermediate whose levels are sensed to regulate Orm activity.