Structural and functional characterization of human ERp44: a closer look at a member of PDI family regulating protein quality control in the early secretory pathway

Abstract

The Endoplasmic Reticulum (ER) is the site of folding and assembly of secretory proteins. Fidelity of protein-based intracellular communication is guaranteed by protein quality control mechanisms located at the Early Secretory Compartment (ESC), which restricts forward transport to native proteins. ERp44 plays a key role in the Thiol-Mediated Retention (TMR) of variety of client proteins (Ero1, SUMF1, Adiponectin, IgM) thus regulating their transport and localization. Little is known about the molecular mechanisms of ERp44-TMR and how it is regulated in living cells. Hence, the overall aim of this work is to investigate the structure-function relationship of ERp44. In collaboration with Wang's group the crystal structure of ERp44 was determined. The structure of ERp44 most likely represents a non-reactive conformation of the protein. Indeed, Cys29 is shielded from the bulk solvent by C-terminal tail and almost inaccessible for the formation of intermolecular disulfide bonds with client proteins. Based on the obtained structural data and functional studies, a panel of mutants of ERp44 has been characterized in order to understand how C-terminal tail rearrangements expose substrate binding site, thus modulating substrates binding/ release in view of its role in TMR. Moreover, the pH gradient between ESC organelles was investigated as major determinant of C-terminal tail rearrangements. Given the known pH differences in the ESC, the data support the hypothesis of a role of the pH variation in governing ERp44-TMR activity in vivo. A deeper knowledge of the structure/function relationship of ERp44 will shed light on the protein quality control mechanisms thus providing essential knowledge of ESC processing diseases and in biotechnology, improving the production of man-made therapeutic proteins