MHC MOLECULES 1) Describe three important differences between class I and class II MHC proteins? Class I is a single chain bound to an invariant partner, 2-microglobulin, whereas class II is a heterodimer Class II is expressed only on APCs, whereas class II is ubiquitously expressed Class I present 8-10 amino acid peptides, whereas class II presents 14-18 amino acid Peptides 2) The following set of peptides are eluted from a purified MHC molecule. Which class of MHC molecule was it and what are its "anchor residues?" VFTSKAINK SFTIRTALK QFTYFIVAK RFTQKAIAK RFTQFIVNK EFTGKYFKK F in position 2, T in position 3, and K in position 9. 3). Why would it make sense that Interferons would activate the expression of LMP2 and LMP7?? LMP2 and LMP 7 are involved in Class I antigen processing which is important in activating CD8+ T cells which are most effective against viruses. Interferons are produced in response to viral infections 4) Class II MHC molecules will traffic through the same ER that Class I molecules are in yet they do not normally bind endogenous peptides. a) How are Class II Molecules prevented from binding endogenous peptides in the ER? Through Invariant chain binding to peptide cleft. Ii is cleaved to leave the CLIP in the Class II MHC groove. b) How is this "blocking protein" removed from the Class II MHC groove to allow an exogenous peptide to bind? Class II MHC is loaded in the endosomes and lysosomes of APCs. The low pH in these endosomes along with HLA-DM enables foreign peptide exchange with CLIP. 5) What is retrograde translocation and why do cells need this ability for Class I antigen processing and presentation? Retrograde translocation is the ability of proteins to transfer into cytoplasm from the ER. This is necessary in the antigen processing of membrane proteins in the cytoplasm so that they may become Class I antigens. 6) Tapasin deficient mice have Class I MHC that exit the ER but they do not have normal peptide loading. How do you think this deficiency causes this result? Tapasin plays a critical role in retaining Class I MHC molecules in the ER and allows for proper loading of Class I MHC with peptide. 7) Why would CD1 (Class I like molecules) be found in the same endosomes as Class II MHC molecules?CD1 primarily present bacterial glycolipids which would be found in endosomes (with exogenous antigens T-CELL RECEPTOR 8) Compare the T cell receptor and the B cell receptor for antigen with regard to: a) protein structure usual picture of IgM vs. TCR b) genetic of diversity generated by V(D)J recombination. T cells do not undergo somatic mutation or class switching. c) associated polypeptides TCR associates with CD3 complex and BCR associates with Ig- a and Ig- b d) co-receptors TCR uses CD4 and CD8, BCR uses CD19 recruited by CD21 (CR2, the complement receptor) e) recognition of antigen BCR recognizes native antigen, usually a 3-dimensional surface TCR recognizes a peptide-MHC complex where the peptide is a stretch of contiguous amino acids. 9) What roles do CD4 and CD8 play in T cell antigen recognition? They play essential roles by binding non-polymorphic regions of either MHC class II or MHC class I respectively. 10) What is cDNA? What is the principle underlying subtractive cDNA cloning? cDNA is a DNA copy of an RNA transcript. It can be either single or double stranded. Subtractive cDNA cloning uses DNA hybridization to remove cDNA sequences in common between two populations of cells allowing one to clone gene copies which are uniquely expressed in a given cell type and not its "neighbor 11) How many different peptides can a class I MHC molecule bind if it requires the structure XXXX(F or Y)XX(I, L, or M) ? 20 x 20 x 20 x 20 x 2 x 20 x 20 x 3 = 3.84 x 108T-CELL SIGNALLING 3) Describe the general structure of a Src-family tyrosine kinase, indicating its various functional domains. Know about SH1, SH2 and SH3 domains and what they do. Also, regulatory phosphorylation sites and myristylation site a) Which Src family kinases are involved in coupling TCR engagement to changes in gene activity? Lck, fyn b) Which Src family kinases are involved in coupling BCR engagement to changes in gene activity? Blk, lyn, fyn c)What PTK in B cells plays a similar role to that of Zap-70 in T cells? syk 4) How is NF-AT transcription factor activated? Phospholipase Cg (PLC)is activated by phosphorylation. Active PLC hydrolyzes a phospholipids component of the plasma membrane to generate the second messenger, DAG and IP3. Ip3 mediates the release of Ca2+ from the endoplasmic reticulum. Ca2+ binds the protein calmodulin, which then associates with and activates the Ca2+/calmodulin dependent phosphatase calcineurin. Active calcineurin removes a phosphate group from NFAT, which allow this transcription factor to tranbslocate into the nucleus. 5) What is an "adapter" molecule and how are adapters involved in signal transduction? Adapters lack PTK activity, but contain both SH2 domains and phosphorylatable tyrosines. They serve as scaffolds for the building of signaling complexes. 6) What T cell signaling phenotype would you predict for a tyrosine to phenylalanine mutation in an ITAM of the CD3γ chain? The ITAMs on the CD3 chains get phosphorylated upon T cell activation by Lck and serve as docking sites for the PTK named Zap-70. Zap-70 is the critical PTK for T cell activation since in its absence, there is little phosphorylation and activation of PLCgamma and the ras pathway doesn't turn on. The question is how will mutating the CD3 gamma chain to an unphosphorylatable form effect these steps. I don't really know the answer since I have never seen the experiment done, but I expect it will decreasequantitatively TCR signaling, but won't abolish it since several of the other CD3 chains have ITAMs which could still be phosphorylated 7) What is an SH2 domain and how does it work? Src-homology 2 domain. Binds to specific phosphotyrosine-containing motifs in signaling molecues (such as ITAMs) and helps PTKs find targets and scaffolds assemble signaling complexes. 8) Why is cyclosporin used in the setting of organ transplantation? Would you expect that a transplant patient would have to take cyclosporin forever, or just around the time of the transplant? Blocks TCR signal transduction by preventing NFAT activation (know the pathway). Thus, host T cells would not activate against allograft material.. You must take the drug forever to prevent