PROTEUS · The role of the intracellular peptidases in the immune system and in the cellular metabolism
FP6 — Marie Curie Actions (Human Resources and Mobility)
- Duration
- 2006-12-01 → 2008-11-30
- EU contribution
- €149,275
- Participants
- 1
- Scheme
- EIF
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Results in brief
Final Activity Report Summary - PROTEUS (The role of the intracellular peptidases in the immune system and in the cellular metabolism)
The novel membrane associated Ring-CH (MARCH) family proteins are transmembrane E3 ubiquitin ligases involved in a variety of cellular processes. Nine MARCH members have been cloned and characterised in humans so far. Apart from MARCH 2 and 3, which seem to be involved in the regulation of endosomal trafficking, the remaining members of the family target transmembrane proteins for ubiquitination leading to rapid endocytosis and degradation. We made green fluorescent protein (GFP)-fusion constructs of several MARCH and over-expressed them in the melanoma cell line MelJuSo, which endogenously expressed the antigen presenting machinery of a professional antigen presenting cell. Using flowcytometry, we measured the surface expression level of MHC class I and found that MARCH 1, 4, 8, and 9 were able to decrease the membrane expression of MHC class I. To further investigate the fine specificity of these MARCH proteins for MHC class I, we measured their capacity to downregulate the MHC class I heavy chain devoid of ß2-microglobulin which was also expressed in the surface. We observed that all MARCH proteins were also able to internalise the heavy chain of MHC class I. In parallel, we analysed the effect of MARCH proteins on MHC class II presentation. MARCH 1 and 8 downregulated MHC class II both loaded with canonical peptide and CLIP peptide. Thrugh cotransfecting MARCH with dominant negative form of dynamin we found that the endocytosis of MHC class I, but not MHC class II, by MARCH was a dynamin-dependent process. Surprisingly, MARCH 1 and 8 recognised an unrelated receptor, i.e. transferring receptor, apart from recognising MHC class II. This was a dynamin-independent process in both cases. Following the observations that the same MARCHs were able to recognise two structurally different proteins, we analysed if the MARCH proteins which downregulated MHC class I recognised, and thus downregulated, the structurally very related protein CD1d. Surprisingly, only MARCH 2 was able to recognise CD1d, while MARCH 1 could recognise only MHC class I. In order to elucidate this phenomenon we generated two chimeras by switching the intracellular domains of MARCH 1 and 2. By analysing the effect on the membrane expression of CD1d, some mutants of CD1d and MHC class I we anticipated to achieve a better knowledge on the specificity of MARCH proteins.
Data: CORDIS, © European Union
Project objective
MHC class I molecules present peptides from intracellular antigens to the immune system. The peptides are produced by the intracellular proteasome and further trimmed and destroyed by various unrelated peptidases. Only a fraction (0.01%) of these peptides escape peptidase destruction through import into the ER by the peptide transporter TAP before they can bind to resident MHC class I molecules for presentation at the plasma membrane. Since the peptidase activity plays an important role in the selection of peptides for presentation, we propose to study these using microinjection of internally quenched peptides and inhibitors, to measure peptidase activity in living cells. I aim to: 1. Test with a large set of internally quenched peptides the subspecializati on of intracellular peptidases for substrate (peptide) length and sequence. 2. Identify the respective peptidases responsible for the detected subspecializations. Chemical inhibitors and RNAi for TOP, BH and TPPII have been defined and their effects on pep tide destruction will be measured. 3. Test the effect of the peptidases on the petpidome as presented by MHC class I molecules. Peptidases will be inhibited and the resulting peptidome will be assayed by comparative mass spectrometry using differently labelled amino acids. 4. Test whether different cells employ different peptidase activities. The Neefjes lab has indications that dendritic cells change their peptidase activity and I will study this and the effect of various activation stages of these cells in more detail. 5. Combine these findings with the results on manipulation for peptidase activity to determine the effect of peptidase activity on the outcome of MHC class I response in dendritic cells. The experiments should reveal the role of cytosolic peptidase activities in antigen presentation by MHC class I molecules and add information to better predict the MHC class I peptidome on the basis of the primary sequence of the antigen.
Original text from CORDIS.
Participants
- Nederlands Kanker Instituut · AmsterdamCoordinatorNetherlands
Links
Data: CORDIS, © European Union