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Major histocompatibility complex (MHC) class I molecules are found at the cell surface of all nucleated cells. Crystallographic studies of MHC class I molecules from mouse9-12, human13, duck14 and chicken15 reveal that the ternary complex comprises of two polypeptide chains: a polymorphic heavy chain with a transmembrane domain, a non-covalently linked invariant light chain Î²2-microglobulin (Î²2m) and an immunogenic oligopeptide when present at the cell surface. The MHC class I heavy chain has three domains; one structural and two that contain the peptide binding cleft that comprises of two Î±-helices and a Î²-sheet and is localized outermost from the plasma membrane.
The main function of MHC class I molecules is to present single, short peptide fragments from intracellular proteins to cytotoxic T lymphocytes (CTL) as a part of antigen recognition process in the cell mediated immune response. Upon recognition of an antigenic peptide, the CTLs become activated and release cytotoxic effectors locally and thereby kill altered self-cells e.g. cells infected with viruses or bacteria as well as cancer cells. Other lymphocytes release cytokines which enhance the cytotoxic response and activate cellular agents such as macrophages and natural killer cells and complement cascade as well as stimulate B cells to produce antibodies.
The "highly dynamic and remarkably inefficient" 16 process of generating MHC class I molecules associated with antigenic peptides is conducted in a series of steps. It is considered that 10 000 proteins need to be degraded to form a stable MHC class I - peptide ligand complex 19. Intracellular endogenous antigenic proteins destined for degradation by proteasome arise from standard reading frames but contain transcriptional, splicing, translational and post-translational errors 16. Within 10 minutes of synthesis, a third of newly synthesized proteins are degraded16 to oligopeptides in either the ubiquitin-proteasome pathway in cytoplasm or less commonly by tripeptidyl peptidase (TTP II) which is capable of performing some functions of that of proteasome6. Complex foreign antigens are also degraded to peptides but the efficiency of their immunogeneicity is capped by the phenomenon of immunodominance with only 0.5% being potentially immunogenic when bound to any given MHC class I allele 18. A limited number of peptides are transported to the endoplasmic reticulum (ER) with the majority being cleaved to amino acids by thimet oligopeptidase (TOP) 7. Compatible length peptides are transported to endoplasmic reticulum in ATP-dependent manner by transporter for antigen processing (TAP) 1 which is a member of macromolecular peptide-loading complex (PLC) 20. Since the MHC class I molecule is capable to accommodate peptides of 8-10 amino acids in length some extended peptides that are to be presented need to be trimmed to the preferred length by cytosolic aminopeptidases IFNÎ³-inducible leucyl aminopeptidase (LAP) or bleomycin hydrolase (BH) and puromycin-sensitive aminopeptidase (PSA) 5 and/or by concerted action of ER aminopeptidases ERAP1 and ERAP2 (ER aminopeptidase 1 and 2) in humans 6 and ERAAP (ER aminopeptidase associated with antigen processing) in mice8. These larger degradation products are more commonly trimmed at the N-terminal end since 26S proteasomes and immunoproteasomes were shown to produce mainly N-extended versions of an antigenic peptide 17. The assembly of a stable MHC class I-peptide complex is accompanied and stabilized by other members of the PLC: molecular chaperones tapasin, calreticulin and ERp6020. Once loaded with a peptide, the MHC class I molecule is transported via Golgi to the cell surface.
The chaperones play an important role in the selection of the peptides that are to be presented on the cell surface but the exact mechanism of the selection is yet to be understood. Tapasin influences the stability of the MHC class I-peptide complex by assisting the selection of peptides with "lower-off rate" or by creating complexes that are more "compact and stable" temporally which facilitates their transport to the cell surface10. The MHC class I molecules associated with peptides referred to as sub-optimal or general indiscriminate peptides are found on the cell surface if the chaperones are absent 10, 20. Peptide-free MHC class I molecules are too unstable to be exported out of the ER. There is compelling evidence20 that the peptide binding/loading induces a conformational change in the MHC class I complex, stabilizing it to enable export through Golgi apparatus to the cell surface. The five C-terminal residues of the antigenic peptide have been determined to be sufficient to induce such a conformational change which suggests that an "assembly intermediate" may be involved 10. The penultimate residue carbonyl oxygen forms a hydrogen bond which acts as an anchor with one of six specificity-determining pockets in the peptide-binding groove10, 20. The N terminal is not thought to stabilize the complex to a comparable extent10.
Increasing our knowledge about how the peptide selection occurs and elucidating the exact molecular mechanism and the dynamics/kinetics of the peptide association and dissociation from the MHC class I molecule is of paramount importance. This is because knowing this could enable the efficient targeting of virally transfected or aberrant cells and the possibility of engineering novel therapies to fight cancer. The aim of this project is to determine the importance and function of the penultimate base carbonyl oxygen in both the formation of the hydrogen bond and in peptide selection by substituting it with sulphur or by completely removing it before analysing its effect on conformation of both murine H-2Db and H-2Kb in the crystal structure.
Generation of soluble H-2Db/Kb -b2m-peptide MHC complexes
Preparation of the samples
Cell lysis - detergent wash and resolubilization
MHC ternary complex refolding
MHC ternary complex concentrating
MHC ternary complex purification
Crystallization of H-2Db/Kb -b2m-peptide MHC complexes
Data collection and structure refinement
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