Sebastián Montealegre

• The purpose of this work is to establish a correlation between the biochemistry (i.e., the protein structure) and the cell biology (i.e., the intracellular transport and modification) of MHC class I endocytosis. I present some experimental evidence about the role of beta 2 microglobulin (β2m) in the endocytic destruction of the MHC class I allotype H 2Kb. The most important conclusion of my work is that the dissociation of β2m from the heavy chain of H 2Kb is the key requirement for the endocytic destruction of the complex. My experiments suggest that the dissociation of β2m from the heavy chain occurs mostly inside the cell, that is, in an endosomal compartment. I show here that in a fibroblast cell line, there are very few MHC class I free heavy chains (FHC) at steady state at the surface. This is somewhat unexpected, given the wealth of literature describing the presence of FHC at the cell surface of different cell lines, but the apparent discrepancy could be resolved. I have also corroborated that an effect known in the MHC class I field as “empty molecules coming out in the cold” is not correctly named. Rather, the statement should be that “reduced endocytosis causes the low-temperature cell surface accumulation of suboptimally loaded MHC class I molecules”. This accumulation is due to the increased affinity of β2m for the heavy chain of Kb at low temperature. Finally, I present a different but related on the structural features of β2m by fluorescence spectroscopy. In this part of the work, I provide experimental evidence, using the small molecule reducing agent TCEP, that β2m is a flexible protein. The work can be a starting point for a more detailed investigation on protein folding in general and especially the potential to correlate in vitro experiments with in silico molecular dynamics simulations.