Folding and assembly of peptide-filled class I major histocompatibility (MHC) molecules occurs in the endoplasmic reticulum (ER) and is a multi-step process mediated by association of the heavy chain (HC) and beta2microglobulin (beta2m) subunits with the chaperones calnexin and calreticulin, the transporter for antigen processing (TAP), and the protein tapasin, prior to binding antigenic peptides. Although the key proteins involved along the folding pathway have been identified, it is unclear just how they interact with each other and which particular functions they fulfill. This proposal seeks to understand many critical aspects related to the formation of a specific intermediary along this pathway; the HC/beta2m/calreticulin complex. We will use a combination of biochemical and crystallographic approaches to: demonstrate the direct binding between the soluble HC/beta2m heterodimer and the soluble chaperone calreticulin; determine the parameters governing this association; assess the stability and protease sensitivity of the chaperone-associated HC/beta2m heterodimer; probe the sites of interaction; clarify the role and mode of action of calreticulin as it pertains to its association with class I MHC molecules; solve the crystal structure of the HC/beta2m/calreticulin complex; provide a detailed structural description of the peptide binding site prior to being occupied with a peptide ligand; and assess the implications of the results (1) in the context of the overall mechanism governing the assembly of peptide-filled class I MHC molecules, and (2) in relation to other ER proteins known to depend on calreticulin to properly fold. The long term objective of this study is to provide a molecular understanding of a cellular process critical for maintaining effective immune responses against viruses and pathogens. The principles which evolve have implications to understand development of T cells in the thymus, tumor immunology, and autoimmunity.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI045070-01A1
Application #
6051293
Study Section
Allergy and Immunology Study Section (ALY)
Program Officer
Prasad, Shiv A
Project Start
2000-03-01
Project End
2005-02-28
Budget Start
2000-03-01
Budget End
2001-02-28
Support Year
1
Fiscal Year
2000
Total Cost
$221,768
Indirect Cost
Name
University of Connecticut
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
City
Storrs-Mansfield
State
CT
Country
United States
Zip Code
06269