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

Vavassori, Stefano (2010). 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. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000ed8e

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.