Transcript Dia 1

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:
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–
–
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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
Toxic
intermediates
ER
Vital
spingolipids
Golgi
Figure 1d
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2. Saccharomyces cerevisiae
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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
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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
* 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
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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
Blocking phosphorylation does not
disrupt the complex
Blocking of phosphorilation
prevents the change in
higher order assembly of
the SPOTS complex.
Lcb1 interaction with Orm 1/2 is mantained
even without Orm phosphorilation.
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.