MHC class I molecules in the endoplasmic reticulum (ER) bind antigenic peptides translocated from the cytosol by the Transporters associated with Antigen Processing (TAP). The assembly of a class I molecule involves two chaperones, calnexin and calreticulin, and the thiol oxido-reductase, ERp57. Calreticulin- and ERp57-associated class I molecules physically associate with TAP molecules, with another protein, tapasin, serving as a bridge. Peptide binding releases the class I molecules from the “peptide loading complex”, and a disulfide-linked dimer of tapasin and ERp57 within the complex catalyzes peptide loading. The peptides can come from extracellular proteins in the case of dendritic cells, a process called cross-presentation. Calnexin, calreticulin and ERp57 are also involved in the assembly of CD1d molecules, which bind lipids.
MHC class II molecules form a complex in the ER with the invariant chain. This complex is targeted to lysosomes where invariant chain is degraded and a residual fragment eliminated by HLA-DM, allowing peptide binding. A gamma interferon-inducible lysosomal thiol reductase (GILT) plays a role in peptide generation, shown using a GILT “knock-out” mouse. Reduction of disulfide bonds by GILT helps unfold protein antigens to MHC class II molecules. Recent work has shown that cross-presentation of disulfide-containing antigens can be facilitated by GILT.
Other work centers on antiviral mechanisms of proteins inducible by interferons. The interferon-inducible viperin protein plays a role in resistance to influenza virus.
Extensive Research Description
The main interests of this laboratory are in two areas; antigen processing and the mechanisms of action of antiviral proteins stimulated by interferons. Studies of MHC class I molecules center onto problems. The first is the mechanism(s) governing the association of antigen-derived peptides with class I molecules in the endoplasmic reticulum (ER). Peptides are transported into the ER by the Transporter associated with Antigen Processing (TAP) after their generation from cytosolic proteins by proteasomal degradation. Peptide binding occurs in the context of the peptide loading complex, which consists of TAP, an associated transmembrane glycoprotein called tapasin, and an associated 'empty' class I molecule which is a heterodimer of the class I heavy chain and ß2microglobulin. Also in the complex are two housekeeping molecules, the chaperone calreticulin which associates with the glycan on the class I heavy chain, and ERp57, a thiol oxidoreductase that is disulfide linked to tapasin. How the peptide loading complex promotes peptide loading of class I molecules at the molecular level is under intensive investigation, but it is clear that the tapasin-ERp57 heterodimer is critically involved in peptide binding and the process of peptide editing, which ensures that MHC class I molecules associate with peptides of the highest affinity before leaving the ER for the cell surface.
The second area of interest in MHC class I lies in the phenomenon of cross-presentation, which is essential for priming the response of naïve CD8-positive T cells to viral antigens in vivo. Cross-presentation is a particular property of dendritic cells that allows them to generate MHC class I complexes with peptides derived from protein antigens that are internalized by phagocytosis or pinocytosis. These two processes ultimately allow entry of external proteins into the cytosol, either by crossing the phagosomal membrane or the ER membrane after they gain access to the ER. The mechanisms allowing protein access to the ER and the translocation mechanism responsible for entry into the cytosol are two problems being studied. Recent work shows that unfolding of the antigenic protein in the endocytic pathway facilitates its translocation into the cytosol. If the protein contains disulfide bonds reduction by Gamma interferon-inducible lysosomal thiol reductase (GILT) may be required.
Work on MHC class II currently focuses on the role of GILT in MHC class II-restricted antigen processing. The latter is important for the recognition by CD4-positive T cells of protein antigens containing disulfide bonds, demonstrated in a GILT knockout mouse.
A third area of interest within the antigen processing field centers on CD1d molecules, which are structurally similar to MHC class I molecules but bind lipids rather than peptides. We are trying to understand the role of lipid binding and of ER chaperones in the assembly of CD1 molecules, as well as the precise mechanisms governing lipid binding in endosomes and lysosomes. We have shown that saposins, small molecules essential for the degradation of sphingolipids, are required for lipid binding to CD1d molecules in the endocytic pathway. Saposins are capable of mobilizing monomeric lipid molecules from the endosomal or lysosomal bilayer which then allows them to bind to theCD1d molecules. The importance of CD1d in antiviral responses is highlighted by the efforts made by a number of viruses to reduce CD1dexpression upon infection. We found that Herpes simplexvirus-1 (HSV-1) does this by inhibiting CD1d re-expression on the surface of cells during recycling through the endosomal/lysosomal system. Upon HSV-1 infection, CD1d molecules accumulate in lysosomes and disappear from the cell surface.
Finally, we are interested in understanding the functions of interferon-inducible proteins, particularly a protein we identified and called viperin which inhibits the growth of a number of viruses, including influenza virus. We originally showed that viperin has an inhibitory effect on the growth of human cytomegalovirus (CMV), even though it is induced by CMV upon infection. We have recently shown that viperin is relocalized by a CMV-encoded protein to mitochondria, where it has an effect on cellular metabolism that facilitates CMV replication.
- Dong, G., Wearsch, P.A., Peaper, D.R., Cresswell, P., Reinisch, K.M. Insights into MHC class I peptide loading from the structure of the tapasin/ERp57 heterodimer. Immunity 30(1):21-32, 2009. PMCID:2650231
- Singh, R., Cresswell, P. Defective cross-presentation of viral antigens in GILT-free mice. Science 328:1394-1398, 2010. PMCID:2925227