A common T17A polymorphism in the signal peptide of the cytotoxic T-lymphocyte antigen 4 (CTLA-4), a T-cell receptor that negatively regulates immune responses, is associated with risk for autoimmune disease. Because the polymorphism is absent from the mature protein, we hypothesized that its biological effect must involve early stages of protein processing, prior to signal peptide cleavage. Constructs representing the two alleles were compared by in vitro translation, in the presence of endoplasmic reticulum membranes. We studied glycosylation by endoglycosidase H digestion and glycosylation mutant constructs, cleavage of peptide with inhibitors, and membrane integration by ultracentrifugation and proteinase K sensitivity. A major cleaved and glycosylated product was seen for both alleles of the protein but a band representing incomplete glycosylation was markedly more abundant in the predisposing Ala allele (32.7 ؎ 1.0 versus 10.6% ؎ 1.2 for Thr, p < 10 ؊9 ). In addition, differential intracellular/surface partitioning was studied with co-transfection of the alleles fused to distinct fluorescent proteins in COS-1 cells. By quantitative confocal microscopy we found a higher ratio of cell surface/total CTLAThr 17 versus CTLAAla 17 (p ‫؍‬ 0.01). Our findings corroborate observations, in other proteins, that the signal peptide can determine the efficiency of post-translational modifications other than cleavage and suggest inefficient processing of the autoimmunity predisposing Ala allele as the explanation for the genetic effect.

No abstract

A recent study mapped the known association of type 1 diabetes with the cytotoxic T lymphocyte-associated antigen 4 gene to a polymorphism at the 3'end (+6230G>A), but could not rule out additional contribution from the 5' end of the gene. To examine this possibility, we analyzed four polymorphisms at the 5'-flanking region for effects independent of +6230G>A. We confirm, by the transmission disequilibrium test, in 496 family trios overtransmission of the susceptibility allele (G) at +6230 (217/168; P = 0.013). Of the four promoter polymorphisms, one (-319C>T) showed overtransmission of the C allele (97/58; P = 0.0017). Because the undertransmitted T at the promoter is in linkage disequilibrium with the overtransmitted G at +6230G>A, the effect observed at the promoter cannot be accounted for by linkage disequilibrium with the +6230G>A. We confirm this by showing that parents heterozygous at the promoter but homozygous at +6230 overtransmit the C promoter allele even more significantly (53/24; P = 9 x 10(-4)). In vitro, the T promoter allele directs higher luciferase expression in Jurkat cells by 42% (P = 0.006), a difference also found in lymphocyte mRNA from eight individuals heterozygous at the promoter, but homozygous at +6230 (P = 1.3 x 10(-4)). Thus, the +6230G>A cannot be the sole functional variant. Either the two polymorphisms define a haplotype carrying the (yet unexamined) functional variant or the -319C>T contributes to the genetic association independently, a possibility suggested by the functional evidence we present.

No abstract

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause CF. The most common mutation, F508 deletion, causes CFTR misfolding and endoplasmic reticulum retention, preventing it from trafficking to the cell surface. One approach to CF treatment is to identify compounds that correct the trafficking defect. We screened a marine extract collection and, after extract, deconvolution identified the latonduines as F508del-CFTR trafficking correctors that give functional correction in vivo. Using a biotinylated azido derivative of latonduine, we identified the poly(ADP-ribose) polymerase (PARP) family as latonduine target proteins. We show that latonduine binds to PARPs 1, 2, 3, 4, 5a, and 5b and inhibits PARP activity, especially PARP-3. Thus, latonduine corrects F508del-CFTR trafficking by modulating PARP activity. Latonduines represent pharmacologic agents for F508del-CFTR correction, and PARP-3 is a pathway for the development of CF treatments.