The conversion of isopentenyl pyrophosphate to phytoene in Neurospora crassa requires both a soluble and a particulate fraction. Soluble and particulate enzyme factions obtained from light-treated and dark-grown wid type, albino-i, alZbi-2, albino-3, and white collar-I strains were mixed in various combinations, and the activity for conversion of 11-'4Clisopen-tenyl pyrophosphate to phytoene was assayed. From such experiments it can be concluded that (a) albino-3 is defective in the soluble fraction; (b) albino-2 is defective in the particulate fraction; (c) the in vivo lght treatment increases the enzyme actvity in the particulate fraction; (d) this light effect occurs in wild type, albno-I, and albin-3 straiw and (e) enzyme activity is present in the particulate faction obtained from the white collar-i mutant, but the in vivo light treatment does not cause an increase in this activity. To measure directly the level of particulate enzyme activity,
Identification of the targets of autoreactive T cells is important for understanding the pathogenesis of many autoimmune diseases. In multiple sclerosis, myelin proteins are thought to be the targets of autoreactive T-cell responses. To date only major histocompatibility complex class 11-restricted CD4+ T-cell responses to myelin proteins have been investigated. In the present study, the ability of self peptides derived from human myelin proteins to induce autoreactive CD8+ T-cell responses has been assessed. Peptide sequences from human myelin basic protein (MBP), proteolipid protein (PLP), myelin-associated glycoprotein (MAG), and myelin oligodendrocyte glycoprotein have been identified that bind to and form stable complexes with HLA-A2. MBP 110-118, PLP 80-88, MAG 287-295, MAG 509-517, and MAG 556-564 were all able to induce peptide-specific HLA-A2-restricted CD8+ cytotoxic T-lymphocyte (CTL) responses in vitro in HLA-A2+ individuals. CTLs specific for MBP 110-118 and MAG 556-564 could recognize endogenously processed antigens presented by HLA-A2. CTL clones reactive to MBP 110-118 and MAG 556-564 produced tumor necrosis factor a and a subset ofthese clones also produced interferon y. These results demonstrate that (i) self peptides derived from human myelin proteins can induce autoreactive CD8+ CTLs and (ii) these CD8+ T cells produce cytokines thought to be important in mediating demyelinating disease. These studies provide an experimental approach for the assessment of CD8+ T-cell responses in such autoimmune diseases.sively by MHC class I molecules (17, 18), and adult oligodendrocytes constitutively express MHC class I molecules but do not express class II MHC molecules, even when stimulated by interferon y (IFN-y) (19). Thus, if adult oligodendrocytes are targets for T-cell reactivity, they will most likely be recognized by MHC class I-restricted CD8+ T cells but not by class II-restricted CD41 T cells. (iii) In an animal model of demyelinating disease, experimental allergic encephalomyelitis (EAE), CD8+ T cells have been shown to have an immunoregulatory effect on the course of the disease (20, 21). Despite these implications that autoreactive CD8+ T-cell responses may play an important role in MS, a successful experimental approach to this question has not yet been reported.To investigate the possible role(s) of CD8+ T-cell responses to myelin proteins in the pathogenesis of demyelinating disorders, we have taken advantage of recent advances in the ability to predict peptide epitopes presented by the HLA class I molecule, . A computer-based algorithm has been devised that predicts the stability of HLA-A2-peptide complexes by quantitating positive and negative effects on binding by each amino acid within a nonamer peptide (22). This algorithm has been used to predict HLA-A2 binding nonamer peptides from the myelin proteins MBP, PLP, MAG, and MOG. In this report we have identified HLA-A2-restricted epitopes derived from these proteins that are capable of inducing CD8+ cytotoxic T-lymphocyte (CTL)...
Structural studies have shown that class I major histocompatibility complex (MHC)-restricted peptide-specific T cell receptor (TCR)-α/βs make multiple contacts with the α1 and α2 helices of the MHC, but it is unclear which or how many of these interactions contribute to functional binding. We have addressed this question by performing single amino acid mutagenesis of the 15 TCR contact sites on the human histocompatibility leukocyte antigen (HLA)-A2 molecule recognized by the A6 TCR specific for the Tax peptide presented by HLA-A2. The results demonstrate that mutagenesis of only three amino acids (R65, K66, and A69) that are clustered on the α1 helix affected T cell recognition of the Tax/HLA-A2 complex. At least one of these three mutants affected T cell recognition by every member of a large panel of Tax/HLA-A2–specific T cell lines. Biacore measurements showed that these three HLA-A2 mutations also altered A6 TCR binding kinetics, reducing binding affinity. These results show that for Tax/HLA-A2–specific TCRs, there is a location on the central portion of the α1 helix that provides interactions crucial to their function with the MHC molecule.
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