Biomedical subjects

Marie-Eve Paquet

Publications and source records attributed to Marie-Eve Paquet.

3 recordsLinked to original sources

PubMed2005

In vitro and in vivo assays to assess the functions of calnexin and calreticulin in ER protein folding and quality control.

Newly synthesized polypeptides entering the endoplasmic reticulum (ER) encounter a large array of molecular chaperones and folding factors that facilitate proper folding as well as assess folding status, retaining non-native proteins within the ER. Calnexin (CNX), an ER membrane protein, and its soluble homologue, calreticulin (CRT), are two important molecular chaperones that contribute to both processes. They are highly unusual chaperones in that they act as lectins, binding the Asn-linked oligosaccharides of newly synthesized glycoproteins, as well as recognizing the polypeptide segments of glycoproteins. Furthermore, they associate with ERp57, a thiol oxidoreductase, that is thought to enhance the oxidative folding of glycoproteins bound to CNX/CRT. These characteristics of CNX and CRT as well as their mode of action have been elucidated though the use of multiple in vitro and in vivo approaches. This chapter will focus on the description of a number of in vitro assays that have been used to characterize the lectin and ERp57-binding functions of CNX/CRT and also their abilities to act as molecular chaperones to suppress protein aggregation. In addition, we will describe insect and mammalian expression systems in which major histocompatibility complex class I molecules are used as model glycoprotein substrates for CNX and CRT. These systems have been valuable in assessing folding and quality control events in vivo that are influenced by CNX or CRT as well as in characterizing the spectrum of substrates that are recognized by these chaperones.

Animals↗

PubMed2004

Bap29/31 influences the intracellular traffic of MHC class I molecules.

In this study, we examine the role of the putative cargo receptor B cell-associated protein (Bap)29/31 in the export of MHC class I molecules out of the endoplasmic reticulum (ER). We show that Bap31 binds to two allotypes of mouse class I molecules, with the interaction initiated at the time of H chain association with beta(2)-microglobulin and maintained until the class I molecule has left the ER. We also show that Bap31 is part of the peptide-loading complex, although is not required for its formation. Bap31 binds not only to class I molecules, but can bind to tapasin in the absence of class I. Consistent with an important role in recruiting class I molecules to transport vesicles, we show that in the absence of Bap29/31, there is a loss of class I colocalization with mSec31 (p137), a component of mammalian coat protein complex II coats. This observation is also associated with a delay in class I traffic from ER to Golgi. Our results are consistent with the view that class I molecules are largely recruited to ER exit sites by Bap29/31, and that Bap29/31 is a cargo receptor for MHC class I molecules.

Animals↗

PubMed2002

Mutant MHC class I molecules define interactions between components of the peptide-loading complex.

Class I histocompatibility molecules, consisting of a heavy chain, beta2-microglobulin and peptide, are assembled in the endoplasmic reticulum (ER) with the assistance of several molecular chaperones and accessory proteins. Peptide binding occurs when assembling class I molecules associate with a loading complex consisting of the transporter associated with antigen processing (TAP) peptide transporter, tapasin, ERp57 and calreticulin (CRT)/calnexin. To assess the physical organization of this complex, we generated a series of mutants in the murine H-2Dd heavy chain and assessed their association with components of the complex. Seven mutations, clustered between amino acids 122 and 136 in the heavy chain alpha2 domain plus one mutation at position 222 in the alpha3 domain, resulted in loss of interaction with tapasin. Association with TAP was always lost simultaneously, supporting the view that tapasin acts as an obligatory bridge between class I molecules and TAP. Compared with previous studies on the HLA-A2 molecule, some differences in points of tapasin interaction were observed. Failure of the H-2Dd mutants to bind tapasin resulted in low cell-surface expression and altered intracellular transport. Most mutants retained a substantial degree of peptide loading, consistent with the view that although tapasin may promote peptide binding to class I, it is not required. A surprising observation was that all mutants lacking tapasin interaction retained normal association with CRT. This contrasts with previous observations on other class I molecules and, combined with differences in tapasin interaction, suggests that the organization of the ER peptide-loading complex can vary depending on the specific class I molecule examined.

ATP Binding Cassette Transporter, Subfamily B, Mem↗