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After the Great East Japan Earthquake in March 2011, providing support for older adults who continued to live in the affected areas has been a critical issue. We examined the effects of programs to...

High-affinity MHC I-peptide interactions are considered essential for immunogenicity. However, some neo-epitopes with low affinity for MHC I have been reported to elicit CD8 T cell dependent tumor rejection in immunization-challenge studies. Here we show in a mouse model that a neo-epitope that poorly binds to MHC I is able to enhance the immunogenicity of a tumor in the absence of immunization. Fibrosarcoma cells with a naturally occurring mutation are edited to their wild type counterpart; the mutation is then re-introduced in order to obtain a cell line that is genetically identical to the wild type except for the neo-epitope-encoding mutation. Upon transplantation into syngeneic mice, all three cell lines form tumors that are infiltrated with activated T cells. However, lymphocytes from the two tumors that harbor the mutation show significantly stronger transcriptional signatures of cytotoxicity and TCR engagement, and induce greater breadth of TCR reactivity than those of the wild type tumors. Structural modeling of the neo-epitope peptide/MHC I pairs suggests increased hydrophobicity of the neo-epitope surface, consistent with higher TCR reactivity. These results confirm the in vivo immunogenicity of low affinity or ‘non-binding’ epitopes that do not follow the canonical concept of MHC I-peptide recognition., The immunogenicity of peptides is believed to be determined by their high-affinity binding to MHC I. Here authors show that low-affinity MHC I-peptide interactions are also able to trigger robust T cell response and anti-tumour immunity in vivo.

A high- affinity MHC I-peptide interaction is considered essential for immunogenicity. However, some neoepitopes with low affinities for MHC have been reported to elicit CD8-dependent tumor rejection in immunization-challenge studies. Here, we ask if a non-binder, tumor-rejection- mediating neoepitope influences the natural immunogenicity of a tumor in vivo, in the absence of artificial immunization. A mutation in tumor MUT1 was edited to its WT counterpart; the mutation was then re-introduced into the WT tumor, recapitulating the mutation in a tumor MUT2. TILs from all three tumors show T cell activation. However, TILs of MUT1 and MUT2 show significantly stronger transcriptional signatures of cytotoxicity and TCR engagement as well as the greater breadth of TCR reactivity than those of WT. Structural modeling of the Kd-neoepitope complex suggests increased hydrophobicity of the neoepitope surface consistent with higher TCR reactivity. These results reveal the immunogenicity in vivo of low affinity or “non-binding” epitopes that do not follow the canonical view of MHC I-peptide recognition.

Neoantigens have attracted attention as biomarkers or therapeutic targets. However, accurate prediction of neoantigens is still challenging, especially in terms of its accuracy and cost. Variant detection using RNA sequencing (RNA‐seq) data has been reported to be a low‐accuracy but cost‐effective tool, but the feasibility of RNA‐seq data for neoantigen prediction has not been fully examined. In the present study, we used whole‐exome sequencing (WES) and RNA‐seq data of tumor and matched normal samples from six breast cancer patients to evaluate the utility of RNA‐seq data instead of WES data in variant calling to detect neoantigen candidates. Somatic variants were called in three protocols using: (i) tumor and normal WES data (DNA method, Dm); (ii) tumor and normal RNA‐seq data (RNA method, Rm); and (iii) combination of tumor RNA‐seq and normal WES data (Combination method, Cm). We found that the Rm had both high false‐positive and high false‐negative rates because this method depended greatly on the expression status of normal transcripts. When we compared the results of Dm with those of Cm, only 14% of the neoantigen candidates detected in Dm were identified in Cm, but the majority of the missed candidates lacked coverage or variant allele reads in the tumor RNA. In contrast, about 70% of the neoepitope candidates with higher expression and rich mutant transcripts could be detected in Cm. Our results showed that Cm could be an efficient and a cost‐effective approach to predict highly expressed neoantigens in tumor samples., We evaluated the utility of RNA‐seq data instead of WES data in variant calling to detect neoantigen candidates. We found that the method combining tumor RNA‐seq data and normal WES data (Combination method) could detect neoantigen candidates that have higher expression and rich variant transcripts, and this method may be an efficient and cost‐effective strategy alternative to the conventional method (DNA method).

Introduction: We developed MHC-peptide interaction assay that measures cell-surface expression of covalently linked peptide-MHC II complex. The utility of the assay has been validated for known CD4+ T-cell epitopes of Mycobacterium tuberculosis antigens (Miyadera et al. submitted). Using this assay (named “delta MHC (ΔMHC) assay”), we screened HLA II-binding regions in SARS-CoV-2 Spike protein to identify potential T-cell epitopes. Method: ΔMHC assay uses engineered fibroblast cell line that stably expresses DPA1. The cell line is transduced with pMXs-IG (Kitamura et al. 2003 Exp Hematol) that carries DPB1*05:01-peptide fusion. The assay measures cell-surface HLA expression through flow cytometry and calculates the expression levels normalized to the internal control GFP and control peptide. The peptides (15-mer) that covered the entire Spike protein were analyzed in this study. Results: We performed the assay for HLA-DP5 (DPA1*02:02-DPB1*05:01), which is present at high frequency in East Asian populations. Among the 211 Spike peptides analyzed, we found > 8 peptides that showed strong binding to DP5, indicating that these regions might act as T-cell epitopes. Additional >10 regions showed intermediate binding. Part of binding regions were overlapped with DR-restricted T-cell epitopes reported for SARS-CoV (Yang et al. 2009, Int Imm). Some of the DP5-binding regions were also overlapped with viral mutation sites of contagious variants (VOC 2020 12/01 (UK), 501Y.V2 (South Africa), B1.1.24B (Brazil)). The binding analyses of the variants for DP5 and DP0401 are underway.

Human leukocyte antigens (HLA)-DQA1*01:07 was identified as an HLA-DQ blank specificity that segregated with the serological HLA-A2, -B7, -DR14, -DR52 haplotype, which carried DQB1*05:03. The blank specificity of DQA1*01:07-DQB1*05:03 may be because of lack of reactivity of available typing sera, or disruption of proper assembly of DQ heterodimer. The cDNA sequence of DQA1*01:07 is nearly identical to DQA1*01:04 except for a variant at position 304, which results in the replacement of an arginine with a cysteine at 79α. To determine whether the DQA1*01:07 product can be expressed on cell-surface, we co-expressed DQA1*01:07 with various DQB1*05 or *06 alleles in fibroblast cells. Cell-surface expression of DQ was detectable when DQA1*01:07 was co-expressed with DQB1*06:04 but undetectable with other DQB1*05 and DQB1*06 alleles, including DQB1*05:03, to which DQA1*01:07 was encoded in cis. These data suggest that DQA1*01:07 may act as a phenotypically null allele in the DQA1*01:07-DQB1*05:03 haplotype, while it can be expressed at a low level in the presences of certain DQB1*06 alleles, such as DQB1*06:04, in trans. Based on the null or low expression of DQA1*01:07 as shown in the previous and present studies, DQA1*01:07 has recently been renamed to DQA1*01:07Q, indicating its questionable expression.

The mechanism of genetic associations between human leukocyte antigen (HLA) and susceptibility to autoimmune disorders has remained elusive for most of the diseases, including rheumatoid arthritis (RA) and type 1 diabetes (T1D), for which both the genetic associations and pathogenic mechanisms have been extensively analyzed. In this review, we summarize what are currently known about the mechanisms of HLA associations with RA and T1D, and elucidate the potential mechanistic basis of the HLA-autoimmunity associations. In RA, the established association between the shared epitope (SE) and RA risk has been explained, at least in part, by the involvement of SE in the presentation of citrullinated peptides, as confirmed by the structural analysis of DR4-citrullinated peptide complex. Self-peptide(s) that might explain the predispositions of variants at 11β and 13β in DRB1 to RA risk have not currently been identified. Regarding the mechanism of T1D, pancreatic self-peptides that are presented weakly on the susceptible HLA allele products are recognized by self-reactive T cells. Other studies have revealed that DQ proteins encoded by the T1D susceptible DQ haplotypes are intrinsically unstable. These findings indicate that the T1D susceptible DQ haplotypes might confer risk for T1D by facilitating the formation of unstable HLA-self-peptide complex. The studies of RA and T1D reveal the two distinct mechanistic basis that might operate in the HLA-autoimmunity associations. Combination of these mechanisms, together with other functional variations among the DR and DQ alleles, may generate the complex patterns of DR-DQ haplotype associations with autoimmunity.

Genes encoding the human leukocyte antigens (HLA) are associated with diverse immunological disorders, including autoimmune diseases and infections. Recently, significant progresses have been made in the HLA typing technologies through the use of next generation sequencers. The reliable platforms for the SNP-based imputation of HLA genotypes have also been established. These technical advancements should enable further identification of HLA associations with diseases. One of the remaining questions is the mechanism through which HLA confer disease susceptibility. As a first step toward comprehensive understanding of functional variations among HLA allele products, we established a protocol to analyze the HLA-binding peptides through quantification of cell-surface HLA expression in an engineered cell line. In this article, we summarize the overview of the cell-surface HLA expression assay, which we plan to use for screening and collection of HLA-peptide interaction profiles for large sets of HLA alleles and peptides.

Epstein-Barr virus (EBV) infects B cells and ~95% of adults are infected. EBV glycoprotein gp42 is essential for entry of virus into B cells. EBV gp42 binds to the β1 chain of HLA-DQ, -DR, and -DP on B cells, and uses these molecules for infection. To investigate if certain HLA-DQ alleles are associated with EBV seronegativity, we recruited ~3,300 healthy adult blood donors, identified 106 EBV-seronegative individuals, and randomly selected a control group of EBV-seropositive donors from the donor pool. A larger than expected proportion of EBV-seronegative subjects were HLA-DQ β1 *04/*05 and *06/*06, and to a lesser extent, *02/*03, compared with the control group, while a larger than expected portion of EBV-seropositive persons were HLA-DQ β1 *02/*02. We examined the ability of EBV gp42 to bind to different HLA-DQ molecules using human and mouse cells stably expressing these alleles. EBV gp42 bound less effectively to cells expressing HLA-DQ β1 *04/*05, *06/*06, or *03/*03 than to cells expressing HLA-DQ β1 *02/*02. These data are consistent with our observations of increased EBV seronegativity with DQ β1 *04/*05 or *06/*06 alleles. These findings emphasize the importance of a single genetic locus (HLA-DQ β1) to influence infectivity with EBV.

UTokyo Research掲載「免疫タンパク質の不安定さが、自己免疫疾患のかかりやすさに関係」 URI: http://www.u-tokyo.ac.jp/ja/utokyo-research/research-news/autoimmunity-associated-genes-encode-exceptionally-unstable-proteins/, UTokyo Research "Autoimmunity associated genes encode exceptionally unstable proteins" URI: http://www.u-tokyo.ac.jp/en/utokyo-research/research-news/autoimmunity-associated-genes-encode-exceptionally-unstable-proteins/

Introduction Accurate prediction of T-cell epitopes have remained a bottleneck for developing peptide vaccines for infections and cancer immunotherapy, especially for major histocompatibility complex class II (MHC II). We previously found that cell-surface expression level of the covalently linked peptide-MHC II complex is a good indicator of MHC-peptide interaction (Miyadera et al. 2015 J Clin Invest). We named this assay as “delta MHC (dMHC) assay” and validated its utility in the prediction of CD4+ T-cell epitopes. Method dMHC assay was performed using engineered fibroblast cells that stably express DRA- or DQA1. The cell line was transduced with pMXs-IG (Kitamura et al. 2003 Exp Hematol) that carries DRB1/3/4/5- or DQB1-peptide fusion construct. Cell surface HLA expression was measured by flow cytometry and normalized to the internal control GFP (Miyadera et al. 2015 J Clin Invest). Results and Discussion We conducted systematic binding analysis for HLA class II (27 alleles: HLA-DRB1,3,4,5, and HLA-DQ) and nine peptides derived from Mycobacterium tuberculosis antigens CFP-10 and ESAT-6 that contain potential CD4+ T-cell epitopes (Nagai et al. 2014 J Immunol Res). The cell surface HLA expression level in the presence of the test peptide were normalized to the expression level of the negative control peptide (g9). The normalized expression level (dMHC-g9) showed overall good correlation with the predicted binding affinity (IC50). Moreover, dMHC-g9 outperforms IC50 in predicting potential T-cell epitopes. These data indicate that dMHC assay may be suitable as a platform for large scale analysis of MHC-peptide interaction and data sharing, which should facilitate improved prediction of CD4+ T-cell epitopes.

In Japanese, Koreans and Caucasians, narcolepsy/hypocretin deficiency is tightly associated with the DRB1*15:01-DQA1*01:02-DQB1*06:02 haplotype. Studies in African-Americans suggest a primary effect of DQB1*06:02, but this observation has been difficult to confirm in other populations because of high linkage disequilibrium between DRB1*15:01/3 and DQB1*06:02 in most populations. In this study, we studied human leucocyte antigen (HLA) class II in 202 Chinese narcolepsy patients (11% from South China) and found all patients to be DQB1*06:02 positive. Comparing cases with 103 unselected controls, and 110 and 79 controls selected for the presence of DQB1*06:02 and DRB1*15:01, we found that the presence of DQB1*06:02 and not DRB1*15:01 was associated with narcolepsy. In particular, Southern Chinese haplotypes such as the DRB1*15:01-DQA1*01:02-DQB1*06:01 and DRB1*15:01-DQA1*01:02-DQB1*05 were not associated with narcolepsy. As reported in Japanese, Koreans, African-Americans and Caucasians, additional protective effects of DQA1*01 (non-DQA1*01:02) and susceptibility effects of DQB1*03:01 were observed. These results illustrate the extraordinary conservation of HLA class II effects in narcolepsy across populations and show that DRB1*15:01 has no effect on narcolepsy susceptibility in the absence of DQB1*06:02. The results are also in line with a previously proposed 'HLA-DQ allelic competition model' that involves competition between non-DQA1*01:02, non-DQB1*06:02 'competent' (able to dimerize together) DQ1 alleles and the major DQα*01:02/ DQβ*06:02 narcolepsy heterodimer to reduce susceptibility.

BACKGROUND Narcolepsy is a sleep disorder characterized by excessive daytime sleepiness, cataplexy, and REM sleep abnormalities. A genome-wide association study identified a novel narcolepsy-related single nucleotide polymorphism (SNP), rs5770917, which is located adjacent to CPT1B (carnitine palmitoyltransferase 1B). In this study, we analyzed the CPT1B expression level and measured the carnitine fractions in blood samples obtained from narcolepsy patients and control subjects to test the hypothesis that fatty acid β-oxidation is altered in narcolepsy. METHODOLOGY AND RESULTS We measured CPT1B mRNA expression in white blood cells of 38 narcolepsy patients and 56 healthy control subjects. The serum carnitine fractions (total carnitine, free carnitine, and acylcarnitine) were measured in the 38 narcolepsy patients and in 30 of 56 control subjects. Stepwise multiple regression analysis revealed that the risk allele (C) for SNP rs5770917 was significantly associated with decreased CPT1B mRNA expression (P = 1.0 × 10(-9)), and the CPT1B expression was higher in the narcolepsy patients than in the controls (P = 0.005). The acylcarnitine levels were abnormally low in 21% of the narcolepsy patients while those of all the controls were within the normal range. Stepwise multiple regression analysis using the dichotomous variable for acylcarnitine (normal or abnormal) as an objective variable revealed that the diagnosis of narcolepsy but not CPT1B expression level and BMI was associated with abnormally low acylcarnitine levels (P = 0.006). CONCLUSIONS Our results indicate that multiple factors are involved in the regulation of serum acylcarnitine levels. Abnormally low levels of acylcarnitine observed in narcolepsy suggest dysfunctional fatty acid β-oxidation pathway.

Nafuredin, a δ-lactone antibiotic, is a fungal metabolite showing selective helminth NADH-fumarate reductase inhibition, and whose target had been revealed as complex I. We found that nafuredin is easily converted to nafuredin-γ by weak alkaline treatment. The structure of nafuredin-γ was elucidated as a γ-lactone form of nafuredin with keto-enol tautomerism. Nafuredin-γ shows similar complex I inhibitory activity as nafuredin, and it also possesses anthelmintic activity in vivo.

We recently reported that Ascaris suum mitochondria express stage-specific isoforms of complex II: the flavoprotein subunit and the small subunit of cytochrome b (CybS) of the larval complex II differ from those of adult enzyme, while two complex IIs share a common iron-sulfur cluster subunit (Ip). In the present study, A. suum larval complex II was highly purified to characterize the larval cytochrome b subunits in more detail. Peptide mass fingerprinting and N-terminal amino acid sequencing showed that the larval and adult cytochrome b (CybL) proteins are identical. In contrast, cDNA sequences revealed that the small subunit of larval cytochrome b (CybS(L)) is distinct from the adult CybS (CybS(A)). Furthermore, Northern analysis and immunoblotting showed stage-specific expression of CybS(L) and CybS(A) in larval and adult mitochondria, respectively. Enzymatic assays revealed that the ratio of rhodoquinol-fumarate reductase (RQFR) to succinate-ubiquinone reductase (SQR) activities and the K(m) values for quinones are almost identical for the adult and larval complex IIs, but that the fumarate reductase (FRD) activity is higher for the adult form than for the larval form. These results indicate that the adult and larval A. suum complex IIs have different properties than the complex II of the mammalian host and that the larval complex II is able to function as a RQFR. Such RQFR activity of the larval complex II would be essential for rapid adaptation to the dramatic change of oxygen availability during infection of the host.

Stevens-Johnson syndrome (SJS) and its severe variant, toxic epidermal necrolysis (TEN), are acute inflammatory vesiculobullous reactions of the skin and mucous membranes. Cold medicines including non-steroidal anti-inflammatory drugs (NSAIDs) and multi-ingredient cold medications are reported to be important inciting drugs. We used two sample sets of Japanese patients to investigate the association between HLA genotypes and cold medicine-related SJS/TEN (CM-SJS/TEN), including acetaminophen-related SJS/TEN (AR-SJS/TEN) with severe mucosal involvement such as severe ocular surface complications (SOC). HLA-A*02:06 was strongly associated with CM-SJS/TEN with SOC and AR-SJS/TEN with SOC. HLA-B*44:03 was also detected as an independent risk allele for CM-, including AR-SJS/TEN with SOC. Analyses using data obtained from CM-SJS/TEN patients without SOC and patients with CM-unrelated SJS/TEN with SOC suggested that these two susceptibility alleles are involved in the development of only CM-SJS/TEN with SOC patients.

Ubiquinone (UQ) is a lipid found in most biological membranes and is a co-factor in many redox processes including the mitochondrial respiratory chain. UQ has been implicated in protection from oxidative stress and in the aging process. Consequently, it is used as a dietary supplement and to treat mitochondrial diseases. Mutants of the clk-1 gene of the nematode Caenorhabditis elegans are fertile and have an increased life span, although they do not produce UQ but instead accumulate a biosynthetic intermediate, demethoxyubiquinone (DMQ). DMQ appears capable to partially replace UQ for respiration in vivo and in vitro. We have produced a vertebrate model of cells and tissues devoid of UQ by generating a knockout mutation of the murine orthologue of clk-1(mclk1). We find that mclk1−/− embryonic stem cells and embryos accumulate DMQ instead of UQ. As in the nematode mutant, the activity of the mitochondrial respiratory chain of −/− embryonic stem cells is only mildly affected (65% of wild-type oxygen consumption). However, mclk1−/− embryos arrest development at midgestation, although earlier developmental stages appear normal. These findings indicate that UQ is necessary for vertebrate embryonic development but suggest that mitochondrial respiration is not the function for which UQ is essential when DMQ is present.

Sparganum proliferum is a larval cestode for which the adult stage is unknown. It is characterized by the continuous branching and budding when parasitized to humans, and causes fatal human sparganosis. However, the biological features of S. proliferum, including its taxonomic status, still remain obscure. Our previous investigation suggested that S. proliferum might be phylogenetically distinct from Spirometra erinaceieuropaei, by the analysis on mitochondrial NADH dehydrogenase subunit 3 (ND3) gene. However, mitochondrial DNA sequence in Platyhelminth is known to have heteroplasmy within a species. Therefore, in the present study, we have investigated the complete nucleotide sequences of mitochondrial cytochrome c oxidase subunit I (COI) gene and the partial nucleotide sequences of nuclear coded succinate dehydrogenase iron-sulfur protein subunit gene (sdhB). The results clearly demonstrated that S. proliferum is a distinct species from S. erinaceieuropaei, and that S. proliferum belongs to the order Pseudophyllidea.

The survival of parasites is dependent on that of the host. It is considered that parasites originated from nonparasitic ancestors and adapted to the environment in the host during the evolutional process, and developed host-and organ-specificities. Regarding energy metabolism, which is an essential factor for the survival, parasites adapt to the environment under a low oxygen tension in the host using metabolic systems which are very different from that of the host mammals. In such systems, parasite mitochondria play diverse roles. Especially, marked changes in the morphology and components of the mitochondria in the life cycle are very interesting in biological aspects such as developmental control and environmental adaptation. Such unique properties of parasite mitochondria could be promising targets for chemotherapy.

Sparganum proliferum is characterized by continuous branching and budding, the resulting progeny invading all tissues of the human body, causing fatal sparganosis. Its life cycle, definitive hosts and the route of infection to humans have not yet been disclosed. Because its morphology is similar to Spirometra erinacei, the phylogeny of S. proliferum has been thought to be identical to or closely related to S. erinacei. However, the taxonomy of S. proliferum has not been established up to present due to the lack of definitive observations. In order to clarify the phylogenetic relationship between S. proliferum and S. erinacei, nucleotide sequences of mitochondrial NADH dehydrogenase subunit 3 gene (ND3) and four mitochondrial tRNA coding genes of S. proliferum and other pseudophyllidean cestodes were analyzed. The sequences of S. proliferum showed high similarity to those of S. erinacei, although they were clearly different from each other, indicating that the phylogeny of S. proliferum and S. erinacei is distinct. This is the first report showing the phylogenetic relationship among S. proliferum and other pseudophyllidean cestodes at the DNA sequence level.

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are severe drug hypersensitivities with high mortality. Typical over-the-counter drugs of cold medicines are suggested to be causative. As multiple ingredients are generally contained in cold medicines, it is of particular interest to investigate which ingredients are responsible for SJS/TEN. However, experimental examination of causal relationships between SJS/TEN and a particular drug molecule is not straightforward. Significant association between HLA-A*02:06 and SJS/TEN with severe ocular surface complications has been observed in the Japanese. In the present study, we have undertakenin silicodocking simulations between various ingredients contained in cold medicines available in Japan and the HLA-A*02:06 molecule. We use the composite risk index (CRI) that is the absolute value of the binding affinity multiplied by the daily dose to assess the potential risk of the adverse reactions. The drugs which have been recognized as causative drugs of SJS/TEN in Japan have revealed relatively high CRI, and the association between SJS/TEN and HLA-A*02:06 has been qualitatively verified. The results have also shown that some drugs whose links to SJS/TEN have not been clinically recognized in Japan show the high CRI and suggested that attention should be paid to their adverse drug reactions.

Infections with parasitic helminths are important causes of morbidity and mortality worldwide. New drugs that are parasite specific and minimally toxic to the host are needed to counter these infections effectively. Here we report the finding of a previously unidentified compound, nafuredin, from Aspergillus niger . Nafuredin inhibits NADH-fumarate reductase (complexes I + II) activity, a unique anaerobic electron transport system in helminth mitochondria, at nM order. It competes for the quinone-binding site in complex I and shows high selective toxicity to the helminth enzyme. Moreover, nafuredin exerts anthelmintic activity against Haemonchus contortus in in vivo trials with sheep. Thus, our study indicates that mitochondrial complex I is a promising target for chemotherapy, and nafuredin is a potential lead compound as an anthelmintic isolated from microorganisms.

The interaction of HLA proteins with peptides is usually analyzed by the in vitro binding assay or the T-cell activation assay. Although widely used, there are some limitations to these assays, such as the difficulty to handle hydrophobic peptides and the potential inter-assay variations of the binding parameters. In this study, we have devised a cell-surface expression assay, which estimates the peptide-HLA interaction by measuring the cell-surface expression levels of HLA. This assay utilizes the general property of MHC to be expressed at high levels when stabilized by peptides. Methods HLA-DQ α -stable cells were established using retroviral vector pMXs-puro (Kitamura et al. (2003) Exp Hematol 31:1007) and packaging cells PLAT-E (Morita et al. (2000) Gene Ther 7:1063). The HLA-DQ α -stable cells were transduced with pMXs-IG/HLA-DQ β , in which the model peptide was inserted between the signal sequence and the mature protein sequence of HLA-DQB1 (modified from Kozono, et al. (1994) Nature 369:151). The cell-surface HLA-DQ expression was measured by flow cytometry using GFP as an internal control. Results Using this assay, we validated the interactions of HLA-DQ with the known high- and low-affinity peptides. The cell-surface HLA-DQ increased greatly in the presences of the high-affinity peptides such as insulin B (1-15) for DQ0602 (Ettinger et al. (1998) J Immunol 160: 2365) compared to the low-affinity peptides or to the potential non-binders. The interaction of HLA-DQ with a series of self- and non-self-peptides such as CLIP and insulin B were also analyzed. The surface-expression assay will be useful to estimate the HLA-peptide interactions for a large numbers of alleles and peptides, and to identify the self- and non-self-peptides that bind to the disease-associated HLA allele products.

Parasites have exploited unique energy metabolic pathways as adaptations to the natural host habitat. In fact, the respiratory systems of parasites typically show greater diversity in electron transfer pathways than do those of host animals. These unique aspects of parasite mitochondria and related enzymes may represent promising targets for chemotherapy. Natural products have been recognized as a source of the candidates of the specific inhibitors for such parasite respiratory chains. Chalcones was recently evaluated for its antimalarial activity in vitro and in vivo. However, its target is still unclear in malaria parasites. In this study, we investigated that licochalcone A inhibited the bc1 complex (ubiquinol-cytochrome c reductase) as well as complex II (succinate ubiquinone reductase, SQR) of Plasmodium falciparum mitochondria. In particular, licochalcone A inhibits bc1 complex activity at very low concentrations. Because the property of the P. falciparum bc1 complex is different from that of the mammalian host, chalcones would be a promising candidate for a new antimalarial drug.

Nafuredin, a delta-lactone antibiotic, is a fungal metabolite showing selective helminth NADH-fumarate reductase inhibition, and whose target had been revealed as complex I. We found that nafuredin is easily converted to nafuredin-gamma by weak alkaline treatment. The structure of nafuredin-gamma was elucidated as a gamma-lactone form of nafuredin with keto-enol tautomerism. Nafuredin-gamma shows similar complex I inhibitory activity as nafuredin, and it also possesses anthelmintic activity in vivo.

It has long been accepted that bacterial quinol-fumarate reductase (QFR) generally uses a low-redox-potential naphthoquinone, menaquinone (MK), as the electron donor, whereas mitochondrial QFR from facultative and anaerobic eukaryotes uses a low-redox-potential benzoquinone, rhodoquinone (RQ), as the substrate. In the present study, we purified novel complex II from the RQ-containing phototrophic purple bacterium, Rhodoferax fermentans that exhibited high rhodoquinol-fumarate reductase activity in addition to succinate-ubiquinone reductase activity. SDS/PAGE indicated that the purified R. fermentans complex II comprises four subunits of 64.0, 28.6, 18.7 and 17.5 kDa and contains 1.3 nmol heme per mg protein. Phylogenetic analysis and comparison of the deduced amino acid sequences of R. fermentans complex II with pro/eukaryotic complex II indicate that the structure and the evolutional origins of R. fermentans complex II are closer to bacterial SQR than to mitochondrial rhodoquinol-fumarate reductase. The results strongly indicate that R. fermentans complex II and mitochondrial QFR might have evolved independently, although they both utilize RQ for fumarate reduction.

Enzymes in the mitochondrial respiratory chain are involved in various physiological events in addition to their essential role in the production of ATP by oxidative phosphorylation. The use of specific and potent inhibitors of complex I (NADH-ubiquinone reductase) and complex III (ubiquinol-cytochrome c reductase), such as rotenone and antimycin, respectively, has allowed determination of the role of these enzymes in physiological processes. However, unlike complexes I, III, and IV (cytochrome c oxidase), there are few potent and specific inhibitors of complex II (succinate-ubiquinone reductase) that have been described. In this article, we report that atpenins potently and specifically inhibit the succinate-ubiquinone reductase activity of mitochondrial complex II. Therefore, atpenins may be useful tools for clarifying the biochemical and structural properties of complex II, as well as for determining its physiological roles in mammalian tissues.

Ubiquinone (UQ) (coenzyme Q) is a lipophilic redox-active molecule that functions as an electron carrier in the mitochondrial electron transport chain. Electron transfer via UQ involves the formation of semiubiquinone radicals, which causes the generation of superoxide radicals upon reaction with oxygen. In the reduced form, UQ functions as a lipid-soluble antioxidant, and protects cells from lipid peroxidation. Thus, UQ is also important as a lipophilic regulator of oxidative stress. Recently, a study on long-lived clk-1 mutants of Caenorhabditis elegans demonstrated that biosynthesis of UQ is dramatically altered in mutant mitochondria. Demethoxy ubiquinone (DMQ), that accumulates in clk-1 mutants in place of UQ, may contribute to the extension of life span. Here we elucidate the possible mechanisms of life span extension in clk-1 mutants, with particular emphasis on the electrochemical property of DMQ. Recent findings on the biochemical function of CLK-1 are also discussed.

Parasites have developed a variety of physiological functions necessary for existence within the specialized environment of the host. Regarding energy metabolism, which is an essential factor for survival, parasites adapt to low oxygen tension in host mammals using metabolic systems that are very different from that of the host. The majority of parasites do not use the oxygen available within the host, but employ systems other than oxidative phosphorylation for ATP synthesis. In addition, all parasites have a life cycle. In many cases, the parasite employs aerobic metabolism during their free-living stage outside the host. In such systems, parasite mitochondria play diverse roles. In particular, marked changes in the morphology and components of the mitochondria during the life cycle are very interesting elements of biological processes such as developmental control and environmental adaptation. Recent research has shown that the mitochondrial complex II plays an important role in the anaerobic energy metabolism of parasites inhabiting hosts, by acting as quinol–fumarate reductase.

Mutations in the clk-1 gene of Caenorhabditis elegans result in an extended life span and an average slowing down of developmental and behavioral rates. However, it has not been possible to identify biochemical changes that might underlie the extension of life span observed in clk-1 mutants, and therefore the function of CLK-1 in C. elegans remains unknown. In this report, we analyzed the effect of clk-1 mutation on ubiquinone (UQ(9)) biosynthesis and show that clk-1 mutants mitochondria do not contain detectable levels of UQ(9). Instead, the UQ(9) biosynthesis intermediate, demethoxyubiquinone (DMQ(9)), is present at high levels. This result demonstrates that CLK-1 is absolutely required for the biosynthesis of UQ(9) in C. elegans. Interestingly, the activity levels of NADH-cytochrome c reductase and succinate-cytochrome c reductase in mutant mitochondria are very similar to those in the wild-type, suggesting that DMQ(9) can function as an electron carrier in the respiratory chain. To test this possibility, the short side chain derivative DMQ(2) was chemically synthesized. We find that DMQ(2) can act as an electron acceptor for both complex I and complex II in clk-1 mutant mitochondria, while another ubiquinone biosynthesis precursor, 3-hydroxy-UQ(2), cannot. The accumulation of DMQ(9) and its use in mutant mitochondria indicate, for the first time in any organism, a link between the alteration in the quinone species used in respiration and life span.

Allelic diversity in human leukocyte antigens (HLA) have been explained by balancing selection acting on polymorphic residues in the peptide binding groove of HLA that diversify binding spectrum for pathogenic epitopes. Here we show that HLA class II (HLA-DQ) allele products are extremely diverse in protein stability levels. We have profiled the relative stability of DQ proteins by a cell-based assay that measures cell surface MHC protein density (surface density index; SDI). The SDI varies over 100-fold among DQ allele products, indicating large stability differences. Through mutagenesis, we identified polymorphic residues that regulate the intrinsic stability of DQ protein, which includes variations on the exterior of the peptide-binding groove that mediate interdomain hydrogen bonds. One of the major stability regulator in DQA1 achieves high ratio of synonymous to non-sysnonymous substitutions per site (ds/dn), suggesting that variations at this site have been maintained through balancing selection. DQ genes of non-human primates and mammals also maintain these variatns. These data suggest that HLA and MHC have been evolving through diversifications in protein stability levels. Our study provides new functional insights into the mechanism of HLA evolution.

Type 1 diabetes (T1D) is an autoimmune disorder that mediates destruction of insulin-producing pancreatic cells. Previous genetic association studies reported HLA II to be the major factor for increased susceptibility to T1D. Yet, knowledge remained limited on disease mechanism in Japanese. The purpose of current study is to elucidate immunological mechanism of risk and protective alleles and to determine the peptide binding repertoire of T1D susceptible DR alleles in the Japanese for Zinc Transporter 8 (ZnT8), one of the established auto-antigenic peptides. We expressed DR9 (DRA*01:01-DRB1*09:01), DR4(DRA*01:01-DRB1*04:05), DR8 (DRA*01:01-DRB1*08:02) contributing to susceptibility to T1D in the Japanese and DR15 (DRA1*01:01-DRB1*15:01) that are associated with protection in mammalian cells. A plate based peptide binding assay system was established using nickel coated microtiter plates to measure the binding of synthetic peptides to recombinant DR proteins. Established DR9 binding motif was searched in ZnT8 sequences. Binding characteristics of risk and protective DR allele products including DR9 are examined in utilization of a 20mer ZnT8 peptide library.

HLA-DQ provide the strongest genetic contributions to susceptibility and resistance to type 1 diabetes (T1D). In Europeans, haplotypes carrying non-Asp57β polymorphism in DQB1 (DQA1*03-DQB1*03:02, DQA1*05-DQB1*02) associate strongly to the susceptibility to T1D, while T1D-risk haplotypes in East Asians encode Asp57β (DQA1*03-DQB1*03:03/*04:01). The distinct haplotype associations are ascribed to differences in HLA-DQ-DR haplotype composition and distribution across populations. To understand the mechanistic basis of T1D pathogenesis in East Asian population, we evaluated the disease association of DQA1-DQB1 combinations in trans in T1D case/control samples in Japanese population, which was collected and genotyped by the Committee on Type 1 Diabetes, Japan Diabetes Society. The DQA1 alleles were estimated from DRB1-DQB1 haplotype distributions in general populations, and trans HLA-DQ dimer formation was estimated from the dimerization profile for the entire HLA-DQ alleles (data not shown). Homozygous individuals were also assumed to form trans dimers (dimers identical to cis haplotype) and counted as "trans", in evaluating the associations to T1D. The preliminary result do not suggest the contributions of particular trans DQA1-DQB1 combinations to T1D. The association analyses using the larger sample size will be presented and discussed with the potential mechanistic basis of T1D in Asians.

HLA (Human Leukocyte Antigen) is the most important genetic loci in relation to disease predisposition with autoimmune, infectious, and numerous other disorders, as revealed by genome wide association studies. Although allele specific antigen presentation has been believed as the major functional determinant implicated in diseases, the highly promiscuous antigen binding to disease associated and non-associated alleles in many autoimmune disorders suggest the contributions of multiple other factors in HLA-predisposition to diseases. The precise mechanisms by which particular HLA alleles exert disease condition thus remain uncovered for most of these disorders. In this study we have established a cell-based, highly quantitative assay to measure the cell surface protein expression levels of HLA class II, that might reflect their protein stability levels. The comprehensive picture on the HLA-DQ protein stability profile will be presented and discussed with regard to the allele specific polymorphisms and disease associations, as well as the haplotype evolution of HLA-DQ loci.

Allelic polymorphisms of HLA class II are associated to the susceptibility/resistance to various diseases. In particular, the susceptibility to sleep disorder narcolepsy is strongly linked to DQB1*0602. In addition, some other DQB1 alleles also affect the susceptibility to the disease when present in addition to DQB1*0602. In order to understand how these alleles contribute to the susceptibility/resistance to narcolepsy, we have analyzed the abilities of these and other DQB1 alleles to form dimer with various DQA1 alleles. The residues which may contribute to the association of DQA1 and DQB1 were also identified and will be presented.