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 SnijderVera 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

MHCII-ß is oligo-ubiquitinated

Ubiquitination takes place in the MVB after peptide loading

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

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

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

Fig 4B

• Wild-type Ak-β was ubiquitinated – Not mutant Ak- βK225A

Fig 4A• Distribution of Ab-α in Ab-β-KO

+ Ak-β-(wt) BMDC equal to WT BMDC

• In Ab- β KO no MHCII expression

• Ak-b-K225 accumulates at PM– Does not get ubiquitinated

Fig 4C

• Exogenous mixes haplotypes are properly assembled in transduced WT– ubiquitination-deficient Ak-

βK225A located at plasma membrane

Fig 5

• Endogenous MHC II in MVB was associated 74% with LV (74%)

• In Ak-bK225A, 16% was found located in LV– Sorting is drastically changed

Sorting steps

• Where is ubiquitination involved or required?– Is it endocytosis or

recycling?

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

• Less Ak-bK225A was detected intracellularly (vs Ab-b) (ratio 5.5 ± 1.4)

• Due to reduced uptake or accelerated recyling?

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