The unfolded protein response signals through high-order assembly of Ire1

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The unfolded protein response signals through
high-order assembly of Ire1

• Alexei V. Korennykh1,3, Pascal F. Egea1, Andrei
  A. Korostelev4, Janet Finer-Moore1, Chao
  Zhang2,3, Kevan M. Shokat2,3, Robert M. Stroud1
  Peter Walter1,3
• Alysia Birkholz
• 1.15.2010

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• 33 of proteins fold in the ER
• Need a mechanism to detect misfolded proteins
• UPR
• Ire1 is the crucial factor
• Has been shown to dimerize
• Organized into UPR induced foci
• RNase
• Trans-autophosphorylation
• Binding of ADP

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Ire1
This pathway is really a sort of duckbilled
platypus. There have been so many surprises. -
Feroz Papa UCSF

• Inositol-requiring protein-1
• ER-membrane-resident kinase/RNase
• a non-spliceosomal mRNA splicing reaction
• In yeast HAC1 mRNA
• HAC1p proteins are then translated
• In metazoans, Xbp1 mRNA
• Xbp1 proteins are then translated
• These proteins adjust protein folding

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In stressed cells, IRE-1 binds to unfolded
proteins, dimerizes and trans-autophosphorylates
in kinase domain This activates the RNase domain
which splices XBP1 or HAC1 The spliced form of
these genes leads to activation of the UPR
BIOC/MCB 568 -- University of Arizona
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Activation of Ire1 by self-association.
Oligomerization has been suggested to promote the
activation the kinase and RNase domains
Binding sites of Ire1 in metazoans (right) and
yeast (left)
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Aim

• Authors didnt like the proposed model that had
  the activation loops too far away for
  trans-autophosphorylation
• Previous model had no linker component
• A different Ire1 dimer is needed for the
  trans-autophosphorylation

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RNase activity assay Construct cleaved is
labeled with P32 and is derived from Xbp1 mRNA
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Linker controls the oligomerization
Oligomerization is inhibited by increasing salt
concentrations- repressing RNase activity
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Ire1KR32 shows fold increase in speed of cleavage
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Structure of the oligomer

• Difficult to crystalize
• ADP association wasnt working
• Tried using kinase inhibitors insert into ADP
  binding site

Kinase inhibitors actually activated the RNase
activity
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Resolution of crystals
Resolution (Å) Structural features observable in a good map
5.5 Overall shape of the molecule. Helices as rods of strong intensity.
3.5 The main chain (usually with some ambiguities).
3.0 The side chains partly resolved.
2.5 Side chains well resolved. The plane of the peptide bond resolved. Atoms located to about 0.4 Å.
1.5 Atoms located to about 0.1 Å (the limit of protein crystallography as of 1985--it's a little bit lower now)
0.77 Bond lengths in small crystals measured to 0.005 Å.
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• Initially got a Ire1- APY29 complex of 3.9 A
• When they deleted 28 amino acids forming a loop,
  got a resolution of 3.2 A

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Electron density map for APY29 bound to
IRE1KR32?28 3 possible hydrogen bonds formed
Although APY29 fits in the ADP site reasonably
well, it doesnt have the divalent Mg 2
RNase activity of APY29 doesnt change with
addition of EDTA
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14 Ire molecules formed a crystal lattice with
back to back dimers
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(No Transcript)
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Double Helix
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Why is this model attractive?

• Kinase dimers are side by side, making
  trans-autophosphorylation more logical
• Kinase inactivating mutant caused no
  phosphorylation to occur, indicating
  phosphorylation activity is regulated through
  self
• Phosphorylation sites are on activation loops
  which face IF3c
• IF3c proposed to transfer phosphates

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Do all three interfaces control RNase activity?
Four monomers combined
Contacts within the different interfaces
Single monomer
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The mechanism

• Initially showed that the IreKR32 seemed to show
  preference for double stem loop mRNA
• To look at this, generated a 354 ntd RNA with a
  single stem loop, and a 58 ntd RNA with a double
  stem loop

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The mechanism
Ire1 has no preference for single or double loops
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Localization of Helix loop helix near IF2
interface (dimerization interface) may control
RNase activity
Cavity is created that is characteristic for
substrate binding pockets of enzymes
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Model
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Conclusions

• Old model is incomplete
• Ire1 kinase, Rnase, and partial linker forms a
  supermolecular structure
• Co-factor binding and phosphorylation enhance
  self-association of Ire1 but neither is required
• Linkers are necessary for oligomerization

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Future work

• Although the authors used an increased structure
  for crystallization, we are still missing the
  full structure
• Need to have evidence that these supermolecular
  structures are actually present in in vitro/ in
  vivo models

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