40 These results are consistent with our own, as CatG is known to have a chymotrysin-like activity,

although digestion patterns of other substrates by these BAY 57-1293 in vivo two proteases are not always identical.38 The finding that cleavage of MHC II occurs after L is consistent with published data on CatG specificity, the preferred P1 amino acids for CatG cleavage being Y, F, R, L, and K.41,42 Both in vitro and ex vivo data initially suggested, but did not prove, that CatG might be involved in physiological MHC II turnover. The DR loop that harbours the cleavage site is physically close to the DM interaction site of DR, and a subset of adjacent mutations that impair DM interaction also confer resistance to CatG-mediated proteolysis. DM is known to stabilize empty Pictilisib nmr MHC II molecules against degradation during endosomal peptide exchange, and this protective effect might be attributable to protection of DM-associated empty DR molecules from CatG cleavage. We were unable to reproduce this effect with DM/DR complexes formed in vitro (data not shown), but this negative result might reflect the fact that these are reversible, non-covalent

complexes. Furthermore, the inverse relationships between changes in CatG activity and MHC II levels during immune cell activation were consistent with a role for CatG in MHC II turnover. Previous work has shown that CatG accumulates in endocytic compartments of primary APCs and contributes to endosomal processing of autoantigens,38,43 so its subcellular location would be compatible with participation of CatG in endosomal MHC II turnover. However, three independent experiments failed

to provide positive evidence that would implicate CatG in MHC II turnover in APCs. First, pharmacological inhibition of CatG for extended periods of time in primary human APCs failed to cause accumulation of HLA-DR molecules or of large degradation intermediates. In some preliminary Non-specific serine/threonine protein kinase experiments, we noticed that endogenous CatG activity appeared to cause DR degradation following detergent lysis of cells (data not shown); however, inclusion of the CatG inhibitor in the lysis buffer prevented this artifact, and this precaution was adopted in the experiments shown here. Similarly, genetic ablation of CatG in mice had no effect on steady-state levels of murine MHC II molecules. Collectively, our data suggest that CatG acts enzymatically upon detergent-solubilized, but not upon membrane-embedded native MHC II molecules. We considered two possible explanations for the lack of CatG cleavage in live APCs. One possibility is that the resistance of MHC II molecules to endosomal CatG cleavage reflected the neutral, rather than endosomal, pH optimum of CatG cleavage of MHC II.