Extended exome sequencing identifies BACH2 as a novel major risk locus for Addison's disease.

BACKGROUND: Autoimmune disease is one of the leading causes of morbidity and mortality worldwide. In Addison's disease, the adrenal glands are targeted by destructive autoimmunity. Despite being the most common cause of primary adrenal failure, little is known about its aetiology. METHODS: To understand the genetic background of Addison's disease, we utilized the extensively characterized patients of the Swedish Addison Registry. We developed an extended exome capture array comprising a selected set of 1853 genes and their potential regulatory elements, for the purpose of sequencing 479 patients with Addison's disease and 1394 controls. RESULTS: We identified BACH2 (rs62408233-A, OR = 2.01 (1.71-2.37), P = 1.66 × 10-15 , MAF 0.46/0.29 in cases/controls) as a novel gene associated with Addison's disease development. We also confirmed the previously known associations with the HLA complex. CONCLUSION: Whilst BACH2 has been previously reported to associate with organ-specific autoimmune diseases co-inherited with Addison's disease, we have identified BACH2 as a major risk locus in Addison's disease, independent of concomitant autoimmune diseases. Our results may enable future research towards preventive disease treatment.

Identification of yeast deletion strains that are hypersensitive to brefeldin A or monensin, two drugs that affect intracellular transport.

We have screened the Eurofan deletion strain collection for mutants that are either sensitive or resistant to three drugs known to affect intracellular transport: brefeldin A, monensin and C(2)-ceramide. Drug-sensitive mutants were analysed by complementation with cognate clones and tetrad analysis to confirm that the phenotypes are linked to the deletions. Out of 620 deletion strains, we found 18 mutants that were sensitive to either brefeldin A, monensin or both. Several of these mutants are deleted for genes that are known to be involved in intracellular transport, membrane biogenesis and/or cell wall biosynthesis. Among such previously known genes were VAM6, VAC7, SYS1, TLG2, RCY1, ERG4, ALG9 and ALG12. Some other genes recovered in our screen were not previously implicated in intracellular transport, but are related to other yeast genes with such a function. Still other genes encode proteins with no obvious link to intracellular transport. Several of these are putative transcription factors or RNA-binding proteins, which suggests that they may affect drug sensitivity by modulating the expression of other genes or proteins.

Mutational alterations in the homotetrameric chaperone SecB that implicate the structure as dimer of dimers.

Variant forms of SecB with substitutions of aminoacyl residues in the region from 74 to 80 were analyzed with respect to their ability to bind a physiological ligand, precursor galactose-binding protein, and to their oligomeric states. SecBL75Q and SecBE77K are tetramers with affinity for ligand indistinguishable from that of the wild-type SecB, and thus the export defect exhibited by strains producing these variants must result from an effect on interactions between SecB and other components. SecBF74I is tetrameric but binds ligand with a lower affinity. Substitutions at positions 76, 78, and 80 cause a shift in the equilibrium so that the SecB tetramer dissociates into dimers. We conclude that the tetramer is a dimer of dimers and that the residues Cys76, Val78, and Gln80 must be involved either directly or indirectly in forming the interface between dimers. These variant species are defective in binding ligand; however, because their oligomeric state is altered no conclusion can be drawn concerning the direct role of these residues in ligand binding.

Structural comparison of the precursor and the mature form of napin, the 2S storage protein in Brassica napus.

The 2S storage protein napin from Brassica napus var. L. is synthesised as a precursor protein at the endoplasmic reticulum and transported along a gradient of decreasing pH to the vacuole, where two propeptides are removed to produce mature napin. The structures of pronapin expressed in insect cells and mature napin from rape seed were characterised. Limited proteolysis with several endoproteases cleaved primarily in the propeptides, suggesting that the propeptides are exposed to the exterior of the protein. Immunological comparison in parallel with circular dichroic spectrometry, both at neutral and acid pH, indicated that the propeptides had only a minor influence on the conformation of the regions of the molecule that correspond to mature napin.

Processing in vitro of pronapin, the 2S storage-protein precursor of Brassica napus produced in a baculovirus expression system.

The maturation of the 2S albumin, napin, in Brassica napus L. involves removal of an amino-terminal and an internal propeptide. Pulse-chase experiments with B. napus embryos showed that intermediates are detectable during the pronapin processing. Intact pronapin was expressed by baculovirus in Spodoptera frugiperda insect cells in order to obtain substrate for studying the processing event. Processing of pronapin with a crude B. napus embryo protein extract resulted in several fragments of similar sizes to those of napin heavy and light chains. The character of the major processing activity in the B. napus extract suggested that it was due to an aspartic proteinase. A secondary activity indicated an additional endoproteinase involved in the pronapin processing. Limited proteolysis of pronapin with a purified aspartic proteinase from Hordeum vulgare showed that cleavage occurred exclusively in the prosequences. The cleavage products formed in-vitro requires additional trimming of the propeptides in order to obtain the subunits of mature napin.

Processing of the 2S storage protein pronapin in Brassica napus and in transformed tobacco.

The 2S protein napin in Brassica napus is synthesized as a proprotein from which an N-terminal an an internal propeptide are removed. In order to investigate the mechanism of 2S storage-protein processing, N-terminal sequences were determined for the light and heavy chains of all major napin isoforms isolated. Mutants of a napin gene, with deletions of different portions of the propeptides, were transformed into tobacco and napin protein was isolated. Napin light and heavy chains were separated and their N-terminal amino acid sequences determined. Further, the C-terminal residues of one napin isoform isolated from B. napus and one mutant napin isolated from tobacco were deduced from molecular-mass determinations of the constituents chains. Analyses suggested that the two propeptides are exposed at the surface of the proprotein. The light chain is processed to the correct length independent of the amino acid sequence in the N-terminal propeptide and the processing site. The internal propeptide is attacked by endoproteases. Aminopeptidases and carboxypeptidases then digest portions of the propeptide to the extent allowed by the primary and the three-dimensional structures, often resulting in 2S protein chains with partly frayed ends.

Analysis of the promoter region of napin genes from Brassica napus demonstrates binding of nuclear protein in vitro to a conserved sequence motif.

Napin is a seed storage protein from Brassica napus (rape) that is encoded by a gene family. We have isolated and characterized a novel napin gene, napB. Comparisons of the 5'-upstream region of napB to the promoter regions of previously published napin genes reveal that certain sequence motives are evolutionary conserved and may be implicated in gene regulation. These consensus motives, that overlap with purine/pyrimidine stretches, are TACACAT and CATGCA both of which frequently occur as overlapping, direct repeats. Related or identical sequences are also found in the upstream regions of the homologous genes of Arabidopsis thaliana. One copy of the CATGCA motif occurs in close proximity to the TATA box in all the above genes. In this case it overlaps with an octamer sequence (ATGCAAAT) which is a sequence element common in many eukaryotic promoters and enhancers. The TACACAT sequence, as part of a longer purine/pyrimidine stretch, was found to interact with a protein present in crude nuclear extracts from developing B. napus seeds. Napin genes appear to be methylated to almost equal extents whether present in expressing or non-expressing tissue.

Export of alpha-amylase by Bacillus amyloliquefaciens requires proton motive force.

The secretion of protein directly into the extracellular medium by Bacillus amyloliquefaciens, a gram-positive bacterium, was shown to be dependent on proton motive force. When the electrochemical membrane potential gradient of protons was dissipated either by uncouplers or by valinomycin in combination with K+, a precursor form of alpha-amylase accumulated on the cellular membrane. The proton motive force could be dissipated without altering the intracellular level of ATP, indicating that the observed inhibition of export was not the result of decreased ATP concentration.

Effects of growth conditions and mutations in RNA polymerase on translational activity in vitro in Escherichia coli.

The translational capacity in vitro in Escherichia coli, using RNA from phage R17 or Q beta as messenger, is several times higher if the extracts are prepared from cells harvested in early exponential phase or grown under conditions of good aeration compared to if extracts are prepared from cells harvested in a later growth phase or grown under semi-aerobic conditions. In low activity extracts the production of phage replicase protein is preferentially affected. Growth of a wild type strain under semi-aerobic conditions has a less pronounced effect on translational capacity in vitro using crude mRNA from normal or T4 infected cells or with poly(U). Mutants were fortuitously found which did not show a lowered translational activity in vitro as a result of entering late phase of growth. Two of these were changed in RNA polymerase. Two different translational inhibitors can be demonstrated in the ribosomal wash fraction obtained from semi-aerobically grown wild type cells, whereas only one was found in the case of aerobically grown cells. The low translational activity of semi-aerobically grown cells in vitro is implied to be dependent on the induction or activation of a translational inhibitor. It behaves like a protein but is not likely to be a protease or RNAse.