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We report the sequence of several MAL-activator genes, including inducible, constitutive, and noninducible alleles of MAL23, MAL43, MAL63, and mal64. Constitutive alleles of MAL23 and MAL43 vary considerably from inducible alleles in their C-terminal domain, with many of the alterations clustered and common to both alleles. The 27 alterations from residues 238-461 of Mal43-C protein are sufficient for constitutivity, but the minimal number of alterations needed for the constitutive phenotype could not be determined. The sequence of mal64, a nonfunctional homologue of MAL63, revealed that Mal64p is 85% identical to Mal63p. Two mutations that activate mal64 and cause constitutivity are nonsense mutations resulting in truncated proteins of 306 and 282 residues. We conclude that the C-terminal region of the MAL-activator, from residues 283-470, contains a maltose-responsive negative regulatory domain, and that extensive mutation or deletion of the entire region causes loss of the negative regulatory function. Additionally, certain sequence elements in the region appear to be necessary for efficient induction of the full-length Mal63 activator protein. These studies highlight the role of ectopic recombination as an important mechanism of mutagenesis of the telomere-associated family of MAL loci.

A practical and scaleable synthesis of the [gamma]-secretase inhibitor, which consists of a central trisubtituted cyclohexane core with appended propionic acid, 2-5-difluorophenyl, and 4-chlorophenylsulfonyl moieties, is presented.

FasL expression on human immune cells and cancer cells plays important roles in immune homeostasis and in cancer development. Our previous study suggests that polymorphisms in the FasL promoter can significantly affect the gene expression in human cells. In addition to the functional FasL SNP -844C>T (rs763110), three other SNPs (SNP -756A>G or rs2021837, SNP -478A>T or rs41309790, and SNP -205 C>G or rs74124371) exist in the proximal FasL promoter. In the current study, we established three major FasL hyplotypes in humans. Interestingly, a transcription motif search revealed that the FasL promoter possessed two consensus T-cell factor (TCF/LEF1) binding elements (TBEs), which is either polymorphic (SNP -205C>G) or close to the functional SNP -844C>T. Subsequently, we demonstrate that both FasL TBEs formed complexes with the TCF-4 and β-catenin transcription factors in vitro and in vivo. Co-transfection of LEF-1 and β-catenin transcription factors significantly increased FasL promoter activities, suggesting that FasL is a target gene of the β-catenin/T-cell factor pathway. More importantly, we found that the rare allele (-205G) of the polymorphic FasL TBE (SNP -205C>G) failed to bind the TCF-4 transcription factor and that SNP -205 C>G significantly affected the promoter activity. Furthermore, promoter reporter assays revealed that FasL SNP haplotypes influenced promoter activities in human colon cancer cells and in human T cells. Finally, β-catenin knockdown significantly decreased the FasL expression in human SW480 colon cancer cells. Collectively, our data suggest that β-catenin may be involved in FasL gene regulation and that FasL expression is influenced by FasL SNP haplotypes, which may have significant implications in immune response and tumorigenesis. [ABSTRACT FROM AUTHOR]

Ligand specificity characterizes receptors for Abs and many other immune receptors, but the common use of the FcR γ-chain as their signaling subunit challenges the concept that these receptors are functionally distinct. We hypothesized that elements for specificity might be determined by the unique cytoplasmic domain (CY) sequences of the ligand-binding α-chains of γ-chain-associated receptors. Among Fey receptors, a protein kinase C (PKC) phosphorylation consensus motif [RSSTR], identified within the FcYRIIIa (CD16A) CY by in silico analysis, is specifically phosphorylated by PKCs, unlike other FcRs. Phosphorylated CD16A mediates a more robust calcium flux, tyrosine phosphorylation of Syk, and proinflammatory cytokine production, whereas non-phosphorylatable CD16A is more effective at activation of the Gab2/PI3K pathway, leading to enhanced degranulation. S100A4, a specific protein-binding partner for CD16A-CY newly identified by yeast two-hybrid analysis, inhibits phosphorylation of CD16A-CY by PKC in vitro, and reduction of S100A4 levels in vivo enhances receptor phosphorylation upon cross-linking. Taken together, PKC-mediated phosphorylation of CD16A modulates distinct signaling pathways engaged by the receptor. Calcium-activated binding of S100A4 to CD16A, promoted by the initial calcium flux, attenuates the phosphorylation of CY, and, acting as a molecular switch, may both serve as a negative feedback on cytokine production pathways during sustained receptor engage-ment and favor a shift to degranulation, consistent with the importance of granule release following conjugate formation between CD16A+ effector cells and target cells. This switch mechanism points to new therapeutic targets and provides a framework for understanding novel receptor polymorphisms. [ABSTRACT FROM AUTHOR]

The cytoplasmic domain (CY) of the ligand-binding alpha-chain of the gamma-chain-associated FcRs can modulate receptor function such as phagocytosis, endocytosis, and intracellular trafficking of receptor-Ag complexes. To assess the potential role of the CY domain of human FcgammaRIa (CD64) alpha-chain in the transcriptional regulation of receptor-induced gene expression, we developed stably transfected murine macrophage cell lines expressing a full-length or a CY deletion mutant (tail-less) of human FcgammaRIa to analyze gene expression in response to receptor-specific cross-linking. Using the Affymetrix murine genome U74Av2 GeneChip array, we observed >100 candidate genes having > or =2-fold difference expression at 1.5 and 3 h after stimulation. Focusing on several immunologically related genes, we confirmed differential expression of M-CSF, macrophage inhibitory cytokine-1, leukocyte-specific protein 1, MIP-2, and IL-1R antagonist by RT-PCR and RNase protection assays. Analysis of mRNA stability indicated that the differential regulation of gene expression by the CY of the CD64 alpha-chain is at the level of gene transcription. Our results indicate that the CY of the CD64 alpha-chain modulates transcriptional activity induced by receptor-specific engagement in macrophages and provides a framework for understanding distinct expression profiles elicited by different Fc gamma-chain-associated receptors.

A single-nucleotide polymorphism (SNP), identified at nucleotide position -844 in the 5' promoter of the FasL gene, lies within a putative binding motif for CAAT/enhancer-binding protein beta (C/EBPbeta). Electrophoretic mobility shift and supershift assays confirmed that this element binds specifically to C/EBPbeta and demonstrated that the two alleles of this element have different affinities for C/EBPbeta. In luciferase reporter assays, the -844C genotype had twice the basal activity of the -844T construct, and basal expression of Fas ligand (FasL) on peripheral blood fibrocytes was also significantly higher in -844C than in -844T homozygous donors. FasL is located on human chromosome 1q23, a region that shows linkage to the systemic lupus autoimmune phenotype. Analysis of 211 African American systemic lupus erythematosus patients revealed enrichment of the -844C homozygous genotype in these systemic lupus erythematosus patients compared with 150 ethnically matched normal controls (p = 0.024). The -844C homozygous genotype may lead to the increased expression of FasL, to altered FasL-mediated signaling in lymphocytes, and to enhanced risk for autoimmunity. This functionally significant SNP demonstrates the potential importance of SNPs in regulatory regions and suggests that differences in the regulation of FasL expression may contribute to the development of the autoimmune phenotype.