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.
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.