The loading of peptides into class I molecules in the ER has turned out to be a fascinating story of molecular interactions. Prior to peptide binding, class I H chains assemble with beta2m and are physically associated with TAP. Furthermore, this association has been reported to promote efficient presentation by class I of intracellular peptides. In the same complex with class I and TAP are the proteins tapasin (gp48) and calreticulin. There is suggestive evidence that each member of this complex (TAP/tapasin/calreticulin) plays a role in the assembly of class I H chains with beta2m and peptide. Thus, we propose that each member of the TAP complex should be considered a molecular chaperone. However, the individual contribution of TAP, tapasin, and calreticulin in class I subunit assembly and their precise chaperone function remains undefined. The experiments in this application will test an extensive series of site-specific mutants to determine whether class I heavy chains and beta2m bind directly and independently to TAP, tapasin and calreticulin. Mutations found to ablate specific interactions will provide key insights into the geometry of the interactions within the complex, as well as the separate functions of TAP, tapasin and calreticulin in peptide loading of class I. Additional experiments will specifically define the separate roles of calreticulin, TAP and tapasin in i) the stabilization of the beta2m/H chain heterodimer prior to peptide binding, and ii) the release from ER retention of the beta2m/H chain heterodimer after peptide binding. Our knowledge of the precise mechanism of peptide loading in the ER is key to understanding how viral pathogens abrogate immune recognition and how we might design future protocols for constructive intervention.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI042793-05
Application #
6627865
Study Section
Allergy and Immunology Study Section (ALY)
Program Officer
Kirkham, Perry M
Project Start
1999-08-01
Project End
2005-01-31
Budget Start
2003-02-01
Budget End
2005-01-31
Support Year
5
Fiscal Year
2003
Total Cost
$301,765
Indirect Cost
Name
Washington University
Department
Genetics
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130