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

The unfolded protein response signals through high-order assembly of Ire1. Alexei V. Korennykh 1 , 3 , Pascal F. Egea 1 , Andrei A. Korostelev 4 , Janet Finer-Moore 1 , Chao Zhang 2 , 3 , Kevan M. Shokat 2 , 3 , Robert M. Stroud 1 & Peter Walter 1 , 3 Alysia Birkholz 1.15.2010.

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

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  1. 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

  2. ~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

  3. 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

  4. 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

  5. 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)

  6. Aim • Authors didn’t 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

  7. RNase activity assay: Construct cleaved is labeled with P32 and is derived from Xbp1 mRNA

  8. Linker controls the oligomerization Oligomerization is inhibited by increasing salt concentrations- repressing RNase activity

  9. Ire1KR32 shows fold increase in speed of cleavage

  10. Structure of the oligomer • Difficult to crystalize • ADP association wasn’t working • Tried using kinase inhibitors – insert into ADP binding site Kinase inhibitors actually activated the RNase activity

  11. Resolution of crystals

  12. 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

  13. Electron density map for APY29 bound to IRE1KR32Δ28 3 possible hydrogen bonds formed Although APY29 fits in the ADP site reasonably well, it doesn’t have the divalent Mg 2+ RNase activity of APY29 doesn’t change with addition of EDTA

  14. 14 Ire molecules formed a crystal lattice with back to back dimers

  15. Double Helix

  16. Why is this model attractive? • Kinasedimers 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

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

  18. 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

  19. The mechanism Ire1 has no preference for single or double loops

  20. 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

  21. Model

  22. 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

  23. 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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