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Recent evidence indicates that genes required for antigen processing are present in the class II region of the MHC. Two new classes of MHC genes that may encode much of the machinery required in the class I (endogenous) antigen-processing pathway have been discovered. There is evidence that genes required in the class II pathway also reside in this region.
The cbb3-type cytochrome c oxidases as members of the heme-copper oxidase superfamily are involved in microaerobic respiration in both pathogenic and non-pathogenic proteobacteria. The biogenesis of these multisubunit enzymes, encoded by the ccoNOQP operon, depends on the ccoGHIS gene products, which are proposed to be specifically required for co-factor insertion and maturation of cbb3-type cytochrome c oxidases. Here, the assembly of the cbb3-type cytochrome c oxidase from the facultative photosynthetic model organism Rhodobacter capsulatus was investigated using blue-native polyacrylamide gel electrophoresis. This process involves the formation of a stable but inactive 210 kDa sub-complex consisting of the subunits CcoNOQ and the assembly proteins CcoH and CcoS. By recruiting monomeric CcoP, this sub-complex is converted into an active 230 kDa CcoNOQP complex. Formation of these complexes and the stability of the monomeric CcoP are impaired drastically upon deletion of ccoGHIS. In a ccoI deletion strain, the 230 kDa complex was absent, although monomeric CcoP was still detectable. In contrast, neither of the complexes nor the monomeric CcoP was found in a ccoH deletion strain. In the absence of CcoS, the 230 kDa complex was assembled. However, it exhibited no enzymatic activity, suggesting that CcoS might be involved in a late step of biogenesis. Based on these data, we propose that CcoN, CcoO and CcoQ assemble first into an inactive 210 kDa sub-complex, which is stabilized via its interactions with CcoH and CcoS. Binding of CcoP, and probably subsequent dissociation of CcoH and CcoS, then generates the active 230 kDa complex. The insertion of the heme cofactors into the c-type cytochromes CcoP and CcoO precedes sub-complex formation, while the cofactor insertion into CcoN could occur either before or after the 210 kDa sub-complex formation during the assembly of the cbb3-type cytochrome c oxidase.
Cytochrome b of R. sphaeroides R-26 has been purified from the isolated cytochrome b-c1 complex to homogeneity. The purification procedure involves Triton X-100 and urea solubilization, calcium phosphate column chromatography at different pH values, and ammonium sulfate fractionation. The purified protein contains 23 nmol heme per mg protein and has an apparent molecular weight of 43,000, as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis. The spectral characteristics of purified cytochrome b are similar to those of cytochrome b in the active cytochrome b-c1 complex but with a lower absorbance. The amino acid composition has been determined and compared with cytochrome b purified from other sources.
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Low molecular weight polypeptide (LMP) genes are located within the major histocompatibility complex and have been associated with autoimmune diseases such as ankylosing spondylitis. In order to define the distribution of LMP genes in Mexican populations, the LMP2 and LMP7 polymorphism was analyzed in 312 Mexican individuals (95 Mexican Mestizos, 48 Nahuas, 56 Mazatecans, 50 Teenek, and 63 Mayos) belonging to different ethnic groups. In Mexican populations both Mestizos and Amerindians presented similar distribution of LMP2 and LMP7 polymorphisms, except Nahuas and Mayos who presented the higher frequencies of LMP2-H/H and the lowest frequencies of LMP2-H/R genotypes (P < 0.05 when compared with Mexican Mestizos). The LMP7-K/K genotype was absent in Nahuas, Teenek and Mayos and only one Mazatecan individual presented this genotype. Differences with other populations were found in Mexicans. An increased frequency of LMP2-H and a decreased frequency of LMP2-R alleles were observed in Mexican Amerindians (Nahuas and Mayos) when compared with Brazilian Amerindians (Kaingang and Guarani) and Caucasians (Spaniards) (P < 0.05). All Mexican populations (Mestizos and Amerindians) presented an increased frequency of LMP7-Q allele and a decreased frequency of LMP7-K allele when compared to Brazilian Amerindians (Kaingang), Caucasians (United States) and Asian (Japan) populations (P < 0.05). Genetic distances showed that Mexican Mestizos have an important relation with Spaniards and with all Mexican Amerindians. The present data corroborate the influence of Spaniard and Amerindian genes in the Mexican Mestizo population and could help to define the true significance of LMP polymorphism as genetic and evolutive marker in the Amerindian populations.
Processing of antigens for presentation by major histocompatibility complex (MHC) class I molecules requires the activity of the proteasome. The 20S proteasome complex is composed of 14 different subunits, 2 of which can be substituted by the interferon gamma (IFN-gamma)-inducible and MHC-encoded subunits LMP2 and LMP7 (low molecular mass poylpeptides 2 and 7). A third subunit, MECL-1, is inducible by IFN-gamma but is encoded outside the MHC. Here we show by cotransfection experiments that the incorporation of MECL-1 into the 20S proteasome is directly dependent on the expression of LMP2 but independent of LMP7. Conversely, the uptake of LMP2 is strongly enhanced by MECL-1 expression. The expression of MECL-1 caused a replacement of the homologous subunit Z in the 20S proteasome complex. LMP2 is required for MECL-1 incorporation at the level of proteasome precursor formation that guarantees the concerted incorporation of two IFN-gamma-inducible proteasome subunits encoded inside and outside the MHC. The obligatory coincorporation of MECL-1 and LMP2 is an important parameter for the interpretation of results obtained with LMP2-deficient cell lines and mice as well as for the design of experiments addressing the function of MECL-1 in antigen presentation.
Recently, expression of the immunoproteasome subunits low molecular protein (LMP) 2 or LMP7 was shown to reduce the presentation of certain major histocompatibility complex (MHC) class I-restricted tumour peptide epitopes in renal cell carcinoma and melanoma cells. This may provide the tumour cells with an immune escape mechanism. To test the relevance of this hypothesis, we have taken advantage of the fact that spontaneous regression of human primary melanoma is thought to be the result of a successful peptide-specific cellular immune response in vivo. Immunohistochemical staining with anti-LMP2 and anti-LMP7 xenoantibodies showed a significantly higher expression of these immunoproteasome subunits in primary melanoma lesions exhibiting histological signs of tumour regression than in primary melanoma lesions without regression phenomena. In spontaneously regressing melanoma lesions, LMP2 and LMP7 expression was significantly associated with the presence of tumour-infiltrating lymphocytes. Our results are compatible with the possibility that the expression of the immunoproteasome subunits LMP2 and LMP7 rather than their downregulation in melanoma cells is associated with the presence of a successful anti-melanoma immune response.
A cytochrome b/c1 complex which catalyses the reduction of cytochrome c by ubiquinol has been isolated from Rhodopseudomonas sphaeroides GA. It contains two hemes b and substoichiometric amounts of ubiquinone-10 and of the Rieske Fe-S center per cytochrome c1, and is essentially free of reaction center and bacteriochlorophyll. The complex consists of three major polypeptides with apparent molecular masses of 40, 34 and 25 kDa. The 34-kDa polypeptide carries heme. Cytochrome c1 has a midpoint potential of 285 mV. For cytochrome b two midpoint potentials, at 50 and -60 mV, at pH 7.4, can be derived if one assumes two components of equal amount. Ubiquinol--cytochrome c oxidoreductase activity is specific for ubiquinol and bacterial cytochromes c, and is inhibited by antimycin A and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole. The complex shows oxidant-induced reduction of cytochrome b.
An early biochemical change in the Parkinsonian substantia nigra (SN) is reduction in total glutathione (GSH + GSSG) levels in affected dopaminergic neurons prior to depletion in mitochondrial complex I activity, dopamine loss, and cell death. We have demonstrated using dopaminergic PC12 cell lines genetically engineered to inducibly down-regulate glutathione synthesis that total glutathione depletion in these cells results in selective complex I inhibition via a reversible thiol oxidation event. Here, we demonstrate that inhibition of complex I may occur either by direct nitric oxide (NO) but not peroxinitrite-mediated inhibition of complex I or through H2O2-mediated inhibition of the tricarboxylic acid (TCA) cycle enzyme alpha-ketoglutarate dehydrogenase (KGDH) which supplies NADH as substrate to the complex; activity of both enzymes are reduced in PD. While glutathione depletion causes a reduction in spare KGDH enzymatic capacity, it produces a complete collapse of complex I reserves and significant effects on mitochondrial function. Our data suggest that NO is likely the primary agent involved in preferential complex I inhibition following acute glutathione depletion in dopaminergic cells. This may have major implications in terms of understanding mechanisms of dopamine cell death associated with PD especially as they relate to complex I inhibition.
Listeria monocytogenes (L. monocytogenes) secretes proteins associated with its virulence into the cytosol of infected cells. These secreted proteins are degraded by host cell proteasomes and processed into peptides that are bound by MHC class I molecules in the endoplasmic reticulum. We have found that the MHC class I antigen-processing pathway is very efficient at generating the epitopes that are presented to cytolytic T lymphocytes (CTL). Depending on which antigen is investigated, from 3 to 30% of degraded antigens are processed into nonamer peptides that are bound by MHC class I molecules. Surprisingly, neither the efficiency of epitope generation nor the absolute number of epitopes per infected cell determines the magnitude of the in vivo CTL response. One of the least prevalent epitopes, derived from an antigen that is virtually undetectable in infected cells, primes the immunodominant CTL response in L. monocytogenes-infected mice. Our studies suggest that immunodominant and subdominant T-cell responses cannot be predicted by the prevalence of antigens or epitopes alone, and that additional factors, yet to be determined, are involved.
Experiments with crude extracts of Pseudomonas carboxydohydrogena revealed that a quinone is necessary for CO oxidation, and that cytochromes of the a, b, and c types and functional terminal oxidase(s) are found in cells grown on CO as the sole source of carbon and energy. CO dehydrogenase delivers electrons to the electron transport system at the level of quinone, and nicotinamide adenine dinucleotide (phosphate) is not involved in CO oxidation.
The immune system can recognize antigenic peptides derived from tumors by their presentation on MHC class I complexes to CTLs. Immunoproteasomes (i20S) can substantially enhance the MHC class I peptide repertoire, making down-regulation of i20S an important strategy of tumor cells in manipulating immune surveillance. Here, we report that human cancer cells express the nonfunctional immunosubunit-variant LMP7E1, in addition to, or instead of LMP7E2, in response to IFN-gamma. This preferential expression of LMP7E1 and the consequent down-regulation of LMP7E2 results in i20S deficiency. The molecular explanation for this phenomenon is the incapacity of LMP7E1 to interact efficiently with the proteasome maturation protein, which regularly recruits LMP7E2 into nascent i20S precursor complexes. In contrast to previous reports, i20S formation in these cancer cells cannot be restored by IFN-gamma treatment. However, expression of LMP7E2 in these cells restores the i20S-deficient phenotype. Thus, our data describe a novel mechanism that contributes to the process of oncogenesis.
The mouse pancreatic beta TC3 and beta TC6-F7 cell lines were used to characterize the effects of interferon-gamma (IFN-y) on beta-cell phenotype and function. Initially, intracellular and secreted insulin were compared in glucose-stimulated cells over time. A significant reduction in insulin content and secretion was observed on a per-cell basis in glucose-stimulated beta TC3 and beta TC6-F7 cells after 12 h of exposure to IFN-gamma. The steadystate level of pre-proinsulin mRNA expression was not affected by IFN-gamma. Thus, we postulate that IFN-gamma's inhibitory actions occur after transcription of pre-proinsulin genes. Time-course analysis of IFN-gamma-regulated mRNA expression of the two intra-MHC-encoded subunits of the proteasome (low-molecular-mass polypeptide [Lmp]-2 and Lmp-7) revealed a correlation between their induction and the inhibitory effects of IFN-gamma on glucose-stimulated insulin production. Increased expression of Lmp-2 and Lmp-7 mRNA was accompanied by a corresponding induction of LMP2 and LMP7 protein expression. Subsequently, major histocompatibility complex (MHC) class I cell-surface expression was significantly increased in IFN-gamma-treated beta TC3 and beta TC6-F7 cells. Exposure of IFN-gamma-treated beta-cells to a peptide aldehyde inhibitor of the proteasome (MG132) significantly attenuated MHC class I cell-surface expression but did not prevent the negative effects of IFN-gamma on glucose responsiveness. Enhanced expression of the MHC class I antigen processing and presentation pathway and diminished insulin production appear to be distinct pathological alterations in beta-cells exposed to the insulitic cytokine IFN-gamma.
To identify prostate cancer-associated Ags, tumor-reactive T lymphocytes were generated using iterative stimulations of PBMC from a prostate cancer patient with an autologous IFN-gamma-treated carcinoma cell line in the presence of IL-2. A CD8+ T cell line and TCR alphabeta+ T cell clone were isolated that secreted IFN-gamma and TNF-alpha in response to autologous prostate cancer cells but not to autologous fibroblasts or lymphoblastoid cells. However, these T cells recognized several normal and malignant prostate epithelial cell lines without evidence of shared classical HLA molecules. The T cell line and clone also recognized colon cancers, but not melanomas, sarcomas, or lymphomas, suggesting recognition of a shared epithelium-associated Ag presented by nonclassical MHC or MHC-like molecules. Although Ag recognition by T cells was inhibited by mAb against CD8 and the TCR complex (anti-TCR alphabeta, CD3, Vbeta12), it was not inhibited by mAb directed against MHC class Ia or MHC class II molecules. Neither target expression of CD1 molecules nor HLA-G correlated with T cell recognition, but beta2-microglobulin expression was essential. Ag expression was diminished by brefeldin A, lactacystin, and cycloheximide, but not by chloroquine, consistent with an endogenous/cytosolic Ag processed through the classical class I pathway. These results suggest that prostate cancer and colon cancer cells can process and present a shared peptidic Ag to TCR alphabeta+ T cells via a nonclassical MHC I-like molecule yet to be defined.
The function of major histocompatibility complex (MHC) class I molecule is to sample peptides derived from intracellular proteins and to present these peptides to CD8+ T cells. We analyzed twenty-nine human cancer cell lines for expression of the TAP-1/2 and LMP-2/7 genes and identified whether defects in these genes were associated with the level of expression of MHC molecules in cancer cells. Expression of TAP-1 was positive in 86% (25/29) of the cell lines, TAP-2 expression was positive in 82% (24/29), LPM2 was positive in 86% (25/29), and LMP-7 was positive in 52% (15/29) at the cell lines. In fifteen cell lines that expressed LMP-7, the mean fluorescence intensity (MFI) was 825.1 +/- 123.2; however, in the cell lines that lacked LMP-7, the MFI was 314.7 +/- 77.2. These data suggest that the expression level of MHC class I molecules is associated, in most cases, with the loss of the LMP-7 gene but without the loss of the TAP-1, TAP-2 or LMP-2 genes.
OBJECTIVE: To determine if a polymorphism in the immunoproteasome subunit LMP7 was associated with juvenile rheumatoid arthritis (JRA) and had functional significance. METHODS: The frequency of LMP7QQ+ vs QQ- (QK and KK genotypes) among 207 patients with JRA and 50 controls was determined. JRA subtypes were pauciarticular (53%), polyarticular (33%), and systemic (14%). Onset was before age 6 (early onset) in 60% of patients. The functional significance of the LMP7 polymorphism was determined by comparing incorporation of LMP7Q vs LMP7K into proteasomes. RESULTS: There was an increased frequency of LMP7QQ in patients vs controls (73 vs 56%; p = 0.016), mainly due to the pauciarticular and systemic JRA subtypes (p = 0.037), and more pronounced in early onset disease (77 vs 56%; p = 0.006). The association persisted with stratification for HLA-DR5(11) and -DPB 1 *0201 (p = 0.002 and 0.013). We found no difference in the relative incorporation of LMP7Q and LMP7K into proteasomes. CONCLUSIONS: These results support an association between LMP7QQ homozygosity and JRA, particularly early onset disease. The difference persists with stratification, at least for DR5(11) and DPB1*0201, suggesting that this effect is unlikely to be due to linkage disequilibrium with HLA alleles known to be associated with early onset pauciarticular JRA. Importantly, as there does not appear to be functional significance associated with the LMP7 polymorphism, this may be a marker for another as yet unidentified susceptibility locus.
It is proved by using the Dixon plot and the Lineweaver-Burk plot that thenoyltrifluoroacetone (TTFA) has two inhibitive sites affecting the reduction of ubiquinone catalyzed by succinate-ubiquinone reductase. The high affinity site (inhibited at the concentration of thenoyltrifluoroacetone less than 20 mumol/L) shows noncompetitive with substrate Q2, while the low affinity site (inhibited at the concentration of TTFA over 20 mumol/L) shows competitive. It is suggested that both the reducing steps of Q----QH-. and QH-.----QH2 are inhibited by thenoyltrifluoroacetone.
The amino acid sequence of the ubiquinone binding protein (QP-C) in the cytochrome bc1 region of the mitochondrial electron transfer chain was determined by analysis of peptides obtained by cyanogen bromide cleavage and staphylococcal protease digestion of succinylated derivatives. It was found to consist of 110 amino acid residues and its amino terminus to be blocked by an acetyl group, as determined by mass spectrometry of the amino-terminal peptide and a comparison with peptides chemically synthesized on high-performance liquid chromatography. The molecular weight of this ubiquinone binding protein including the acetyl group was calculated to be 13,389. The predicted secondary structure of QP-C has alpha-helical content of about 50% and QP-C was classified as an "all-alpha" or "alpha + beta" protein. This is the first report describing the amino acid sequence of the ubiquinone binding protein. A comparison of this sequence with that of the 14-kDa subunit of the yeast ubiquinol-cytochrome c reductase complex from the nucleotide sequence showed these two sequences to be quite similar.