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Dendritic Cells Regulate Exposure of MHC Class II at Their Plasma Membrane by Oligoubiquitination

Dendritic Cells Regulate Exposure of MHC Class II at Their Plasma Membrane by Oligoubiquitination. Joost Snijder Vera Verhage Supervisor: Prof. W. Stoorvogel. Proteolytic processing of MHC-II. Research Aim. To establish what role ubiquitination has in sorting of MHC-II complexes in DC’s.

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Dendritic Cells Regulate Exposure of MHC Class II at Their Plasma Membrane by Oligoubiquitination

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  1. Dendritic Cells Regulate Exposure of MHC Class II at Their Plasma Membrane by Oligoubiquitination JoostSnijder Vera Verhage Supervisor: Prof. W. Stoorvogel

  2. Proteolytic processing of MHC-II

  3. Research Aim To establish what role ubiquitination has in sorting of MHC-II complexes in DC’s

  4. MHCII-ß is oligo-ubiquitinated

  5. Ubiquitination takes place in the MVB after peptide loading

  6. Proteolytic processing of Ii is required for Ubiquitination, DC maturation interferes

  7. Summary • MHCII-ß is oligo-ubiquitinated • Ubiquitination takes place in the MVB after peptide loading • Proteolytic processing of Ii is required for ubiquitination • LPS-induced maturation interferes with ubiquitination

  8. Target residue 225 • Conserved Lysine residue 225 • Ak-βK225A= mutant Lysine  Alanine • 2 MHCII Haplotypes • Ab-α/β = Wild type • Ak-α/β = Transduced haplotype

  9. Fig 4B • Wild-type Ak-β was ubiquitinated • Not mutant Ak- βK225A

  10. Fig 4A • Distribution of Ab-α in Ab-β-KO + Ak-β-(wt) BMDC equal to WT BMDC • In Ab- βKO no MHCII expression • Ak-b-K225 accumulates at PM • Does not get ubiquitinated

  11. Exogenous mixes haplotypes are properly assembled in transduced WT ubiquitination-deficient Ak-βK225A located at plasma membrane Fig 4C

  12. Endogenous MHC II in MVB was associated 74% with LV (74%) In Ak-bK225A, 16% was found located in LV Sorting is drastically changed Fig 5

  13. Where is ubiquitination involved or required? Is it endocytosis or recycling? Sorting steps

  14. Fig 6A • Transfer from EEA1 to DM positive structure • EEA1, early endosomal marker • DM class II compartment • Delay in Mutant Ak-bK225A • 20°C, endocytosis occurs, but no recycling and transport to lysosomes

  15. Less Ak-bK225A was detected intracellularly (vs Ab-b) (ratio 5.5 ± 1.4) Due to reduced uptake or accelerated recyling?

  16. Conclusion • Ubiquitination of MHC II-b • occurs at lysine 225 and is required for intracellular retention of MHC II in immature DCs. • is required for sorting at MVB, failure result in transfer by default to the plasma membrane • Is required for efficient endocytosis

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