We analyzed the mechanism by which a Graves' disease-associated C/T polymorphism in the Kozak sequence of CD40 affects CD40 expression. CD40 expression levels on B cells in individuals with CT and TT genotypes were decreased by 13.3 and 39.4%, respectively, compared with the levels in CC genotypes (P = 0.012). Similarly, Rat-2 fibroblasts transfected with T-allele cDNA expressed 32.2% less CD40 compared with their C-allele-transfected counterparts (P = 0.004). Additionally, an in vitro transcription/translation system showed that the T-allele makes 15.5% less CD40 than the C-allele (P < 0.001), demonstrating that the effect of the single-nucleotide polymorphism (SNP) on CD40 expression is at the level of translation. However, the SNP did not affect transcription, because the mRNA levels of CD40, as measured by quantitative RT-PCR, were independent of genotype. Therefore, our results may suggest that the C allele of the CD40 Kozak SNP, which is associated with Graves' disease, could predispose to disease by increasing the efficiency of translation of CD40 mRNA.

Graves' disease (GD) is associated with HLA-DR3 (DRB1*03) in Caucasians, but the exact amino-acid sequence in the DR b1 chain conferring susceptibility to GD is unknown. Therefore, the aim of our study was to identify the critical sequence among the HLA-DRB1 amino-acid residues occupying the peptide-binding pocket, which conferred susceptibility to GD. We sequenced the HLA-DRB1 locus in 208 Caucasian GD patients and 149 Caucasian controls. Sequence analysis showed an increased frequency of DR b-Arg-74 in GD patients compared to controls (41.8 and 13.4%, respectively; P ¼ 2.3 Â 10 À8 , OR ¼ 4.6). Moreover, subset analyses showed that DR b-Arg-74 was also significantly more frequent in the HLA-DR3 negative GD patients than in controls (7.6 vs 0.8%, P ¼ 0.02, OR ¼ 10.5), suggesting that the association with DR b-Arg-74 is independent of the association with HLA-DR3. Structural modeling studies demonstrated that the change at position 74 from the neutral amino acids Ala or Gln to the positively charged amino-acid Arg significantly modifies the three-dimensional structure of the DR peptide-binding pocket. Our results suggested that structural heterogeneity of the DR b-chain peptidebinding pocket P4 at residue 74 predispose some at risk individuals to GD.

Hashimoto's thyroiditis (HT) is associated with HLA, but the associated allele is still controversial. We hypothesized that specific HLA-DR pocket-sequence variants are associated with HT and that similar variants in the murine I-E locus (homologous to HLA-DR) predispose to experimental autoimmune thyroiditis (EAT), a classical mouse model of HT. Therefore, we sequenced the polymorphic exon 2 of the HLA-DR gene in 94 HT patients and 149 controls. In addition, we sequenced exon 2 of the I-E gene in 22 strains of mice, 12 susceptible to EAT and 10 resistant. Using logistic regression analysis, we identified a pocket amino acid signature, Tyr-26, Tyr-30, Gln-70, Lys-71, strongly associated with HT (P ‫؍‬ 6.18 ؋ 10 ؊5 , OR ‫؍‬ 3.73). Lys-71 showed the strongest association (P ‫؍‬ 1.7 ؋ 10 ؊8 , OR ‫؍‬ 2.98). This association was seen across HLA-DR types. The 5-aa haplotype Tyr-26, Tyr-30, Gln-70, Lys-71, Arg-74 also was associated with HT (P ‫؍‬ 3.66 ؋ 10 ؊4 ). In mice, the I-E pocket amino acids Val-28, Phe-86, and Asn-88 were strongly associated with EAT. Structural modeling studies demonstrated that pocket P4 was critical for the development of HT, and pockets P1 and P4 influenced susceptibility to EAT. Surprisingly, the structures of the HTand EAT-susceptible pockets were different. We conclude that specific MHC II pocket amino acid signatures determine susceptibility to HT and EAT by causing structural changes in peptidebinding pockets that may influence peptide binding, selectivity, and presentation. Because the HT-and EAT-associated pockets are structurally different, it is likely that distinct antigenic peptides are associated with HT and EAT.gene ͉ Hashimoto's thyroiditis ͉ HLA ͉ major histocompatibility complex H ashimoto's thyroiditis (HT) is among the most common human autoimmune diseases with a population prevalence in the United States of 1-4.6% (1, 2). HT is characterized by infiltration of the thyroid by autoreactive T and B cells causing thyroid cell death and production of anti-thyroid peroxidase (TPO) and antithyroglobulin (Tg) antibodies (reviewed in ref.3). Clinically, the disease manifests by hypothyroidism requiring thyroid hormone supplementation, and most patients develop goiter. The pathogenesis of HT is believed to involve a complex interaction between inborn genetic susceptibility (reviewed in ref. 4) and an external trigger such as infection (5) or iodine (6). As a result, thyroidspecific T cells become activated and infiltrate the thyroid. The thyroid-infiltrating T cells induce thyroid cell death, causing gradual destruction of the thyroid gland, hypothyroidism, and goiter (reviewed in ref.3).The MHC gene locus encoding the HLA glycoproteins in humans consists of a complex of genes located on chromosome 6p21 (reviewed in ref. 4). Because the HLA region is highly polymorphic and contains many immune response genes, it was the first candidate genetic region to be studied for association with HT. However, in contrast to the clear association of Graves' disease (GD) with HLA-DR3, data on HL...

SCF (Skp1⅐CUL1⅐F-box protein⅐ROC1) E3 ubiquitin ligase and Cdc34 E2-conjugating enzyme catalyze polyubiquitination in a precisely regulated fashion. Here, we describe biochemical evidence suggesting an autoinhibitory role played by the human CUL1 ECTD (extreme C-terminal domain; spanning the C-terminal 50 amino acids), a region that is predicted to contact the ROC1 RING finger protein by structural studies. We showed that ECTD did not contribute to CUL1's stable association with ROC1. Remarkably, deletion of ECTD, or missense mutations designed to disrupt the predicted ECTD⅐ROC1 interaction, markedly increased the ability of SCF ␤TrCP2 to promote IB␣ polyubiquitination and polyubiquitin chain assembly by Cdc34 in vitro. Thus, disruption of ECTD yields in vitro effects that parallel SCF activation by Nedd8 conjugation to CUL1. We propose that SCF may be subject to autoinhibitory regulation, in which Nedd8 conjugation acts as a molecular switch to drive the E3 into an active state by diminishing the inhibitory ECTD⅐ROC1 interaction.autoinhibition ͉ E3 ubiquitin ligase ͉ neddylation

We have previously reported on the functional interaction of Lipid II with human alpha-defensins, a class of antimicrobial peptides. Lipid II is an essential precursor for bacterial cell wall biosynthesis and an ideal and validated target for natural antibiotic compounds. Using a combination of structural, functional and in silico analyses, we present here the molecular basis for defensin-Lipid II binding. Based on the complex of Lipid II with Human Neutrophil peptide-1, we could identify and characterize chemically diverse low-molecular weight compounds that mimic the interactions between HNP-1 and Lipid II. Lead compound BAS00127538 was further characterized structurally and functionally; it specifically interacts with the N-acetyl muramic acid moiety and isoprenyl tail of Lipid II, targets cell wall synthesis and was protective in an in vivo model for sepsis. For the first time, we have identified and characterized low molecular weight synthetic compounds that target Lipid II with high specificity and affinity. Optimization of these compounds may allow for their development as novel, next generation therapeutic agents for the treatment of Gram-positive pathogenic infections.