SOBP is mutated in syndromic and nonsyndromic intellectual disability and is highly expressed in the brain limbic system

Intellectual disability (ID) affects 1%-3% of the general population. We recently reported on a family with autosomal-recessive mental retardation with anterior maxillary protrusion and strabismus (MRAMS) syndrome. One of the reported patients with ID did not have dysmorphic features but did have temporal lobe epilepsy and psychosis. We report on the identification of a truncating mutation in the SOBP that is responsible for causing both syndromic and nonsyndromic ID in the same family. The protein encoded by the SOBP, sine oculis binding protein ortholog, is a nuclear zinc finger protein. In mice, Sobp (also known as Jxc1) is critical for patterning of the organ of Corti; one of our patients has a subclinical cochlear hearing loss but no gross cochlear abnormalities. In situ RNA expression studies in postnatal mouse brain showed strong expression in the limbic system at the time interval of active synaptogenesis. The limbic system regulates learning, memory, and affective behavior, but limbic circuitry expression of other genes mutated in ID is unusual. By comparing the protein content of the +/jc to jc/jc mice brains with the use of proteomics, we detected 24 proteins with greater than 1.5-fold differences in expression, including two interacting proteins, dynamin and pacsin1. This study shows mutated SOBP involvement in syndromic and nonsyndromic ID with psychosis in humans.     

Staphylococcus aureus NrdH Redoxin Is a Reductant of the Class Ib Ribonucleotide Reductase

Staphylococci contain a class Ib NrdEF ribonucleotide reductase (RNR) that is responsible, under aerobic conditions, for the synthesis of deoxyribonucleotide precursors for DNA synthesis and repair. The genes encoding that RNR are contained in an operon consisting of three genes, nrdIEF, whereas many other class Ib RNR operons contain a fourth gene, nrdH, that determines a thiol redoxin protein, NrdH. We identified a 77-amino-acid open reading frame in Staphylococcus aureus that resembles NrdH proteins. However, S. aureus NrdH differs significantly from the canonical NrdH both in its redox-active site, C-P-P-C instead of C-M/V-Q-C, and in the absence of the C-terminal [WF]SGFRP[DE] structural motif. We show that S. aureus NrdH is a thiol redox protein. It is not essential for aerobic or anaerobic growth and appears to have a marginal role in protection against oxidative stress. In vitro, S. aureus NrdH was found to be an efficient reductant of disulfide bonds in low-molecular-weight substrates and proteins using dithiothreitol as the source of reducing power and an effective reductant for the homologous class Ib RNR employing thioredoxin reductase and NADPH as the source of the reducing power. Its ability to reduce NrdEF is comparable to that of thioredoxin-thioredoxin reductase. Hence, S. aureus contains two alternative thiol redox proteins, NrdH and thioredoxin, with both proteins being able to function in vitro with thioredoxin reductase as the immediate hydrogen donors for the class Ib RNR. It remains to be clarified under which in vivo physiological conditions the two systems are used.Ribonucleotide reductases (RNRs) are essential enzymes in all living cells, providing the only known de novo pathway for the biosynthesis of deoxyribonucleotides, the immediate precursors of DNA synthesis and repair. RNRs catalyze the controlled reduction of all four ribonucleotides to maintain a balanced pool of deoxyribonucleotides during the cell cycle (29). Three main classes of RNRs are known. Class I RNRs are oxygen-dependent enzymes, class II RNRs are oxygen-independent enzymes, and class III RNRs are oxygen-sensitive enzymes. Class I RNRs are divided into two subclasses, subclasses Ia and Ib.Staphylococcus aureus is a Gram-positive facultative aerobe and a major human pathogen (24). S. aureus contains class Ib and class III RNRs, which are essential for aerobic and anaerobic growth, respectively (26). The class Ib NrdEF RNR is encoded by the nrdE and nrdF genes: NrdE contains the substrate binding and allosteric binding sites, and NrdF contains the catalytic site for ribonucleotide reduction. The S. aureus nrdEF genes form an operon containing a third gene, nrdI, the product of which, NrdI, is a flavodoxin (5, 33). Many other bacteria such as Escherichia coli (16), Lactobacillus lactis (17), and Mycobacterium and Corynebacterium spp. possess class Ib RNR operons that contain a fourth gene, nrdH (30, 44, 50), whose product, NrdH, is a thiol-disulfide redoxin (16, 17, 40, 43, 49). More-complex situations are found for some bacteria, where the class Ib RNR operon may be duplicated and one or more of the nrdI and nrdH genes may be missing or located in another part of the chromosome (29).NrdH proteins are glutaredoxin-like protein disulfide oxidoreductases that alter the redox state of target proteins via the reversible oxidation of their active-site dithiol proteins. NrdH proteins function with high specificity as electron donors for class I RNRs (9, 16-18). They are widespread in bacteria, particularly in those bacteria that lack glutathione (GSH), where they function as a hydrogen donor for the class Ib RNR (12, 16, 17). In E. coli, which possesses class Ia and class Ib RNRs, NrdH functions in vivo as the primary electron donor for the class Ib RNR, whereas thioredoxin or glutaredoxin is used by the class Ia NrdAB RNR (12, 17). NrdH redoxins contain a conserved CXXC motif, have a low redox potential, and can reduce insulin disulfides. NrdH proteins possess an amino acid sequence similar to that of glutaredoxins but behave functionally more like thioredoxins. NrdH proteins are reduced by thioredoxin reductase but not by GSH and lack those residues in glutaredoxin that bind GSH and the GSH binding cleft (39, 40). The structures of the E. coli and Corynebacterium ammoniagenes NrdH redoxins reveal the presence of a wide hydrophobic pocket at the surface, like that in thioredoxin, that is presumed to be involved in binding to thioredoxin reductase (39, 40). NrdI proteins are flavodoxin proteins that function as electron donors for class Ib RNRs and are involved in the maintenance of the NrdF diferric tyrosyl radical (5, 33). In Streptococcus pyogenes, NrdI is essential for the activity of the NrdHEF system in a heterologous E. coli in vivo complementation assay (33). Class Ib RNRs are proposed to depend on two specific electron donors, NrdH, which provides reducing power to the NrdE subunit, and NrdI, which supplies electrons to the NrdF subunit (33).In this report we identify an open reading frame (ORF) in S. aureus encoding an NrdH-like protein with partial sequence relatedness to the E. coli, Salmonella enterica serovar Typhimurium, L. lactis, and C. ammoniagenes NrdH proteins. In contrast to these bacteria, the S. aureus nrdH gene does not form part of the class Ib RNR operon. The S. aureus NrdH protein differs in its structure from the canonical NrdH in its redox-active site, C-P-P-C instead of C-M/V-Q-C, and in the absence of the C-terminal [WF]SGFRP[DE] structural motif. We show that in vitro, S. aureus NrdH reduces protein disulfides and is an electron donor for the homologous class Ib NrdEF ribonucleotide reductase.     

Gene-Gene Interactions Lead to Higher Risk for Development of Type 2 Diabetes in an Ashkenazi Jewish Population

Background

Evidence has accumulated that multiple genetic and environmental factors play important roles in determining susceptibility to type 2 diabetes (T2D). Although variants from candidate genes have become prime targets for genetic analysis, few studies have considered their interplay. Our goal was to evaluate interactions among SNPs within genes frequently identified as associated with T2D.

Methods/Principal Findings

Logistic regression was used to study interactions among 4 SNPs, one each from HNF4A[rs1884613], TCF7L2[rs12255372], WFS1[rs10010131], and KCNJ11[rs5219] in a case-control Ashkenazi sample of 974 diabetic subjects and 896 controls. Nonparametric multifactor dimensionality reduction (MDR) and generalized MDR (GMDR) were used to confirm findings from the logistic regression analysis. HNF4A and WFS1 SNPs were associated with T2D in logistic regression analyses [P<0.0001, P<0.0002, respectively]. Interaction between these SNPs were also strong using parametric or nonparametric methods: the unadjusted odds of being affected with T2D was 3 times greater in subjects with the HNF4A and WFS1 risk alleles than those without either (95% CI = [1.7–5.3]; P≤0.0001). Although the univariate association between the TCF7L2 SNP and T2D was relatively modest [P = 0.02], when paired with the HNF4A SNP, the OR for subjects with risk alleles in both SNPs was 2.4 [95% CI = 1.7–3.4; P≤0.0001]. The KCNJ11 variant reached significance only when paired with either the HNF4A or WFSI SNPs: unadjusted ORs were 2.0 [95% CI = 1.4–2.8; P≤0.0001] and 2.3 [95% CI = 1.2-4.4; P≤0.0001], respectively. MDR and GMDR results were consistent with the parametric findings.

Conclusions

These results provide evidence of strong independent associations between T2D and SNPs in HNF4A and WFS1 and their interaction in our Ashkenazi sample. We also observed an interaction in the nonparametric analysis between the HNF4A and KCNJ11 SNPs (P≤0.001), demonstrating that an independently non-significant variant may interact with another variant resulting in an increased disease risk.     

Identification of the N-terminal peptide binding site of glucose-regulated protein 94

Because the stress protein GRP94 can augment presentation of peptides to T cells, it is important to define how it, as well as all other HSP90 family members, binds peptides. Having previously shown that the N-terminal half of GRP94 can account for the peptide binding activity of the full-length protein, we now locate this binding site by testing predictions of a molecular docking model. The best predicted site was on the opposite face of the beta sheet from the pan-HSP90 radicicol-binding pocket, in close proximity to a deep hydrophobic pocket. The peptide and radicicol-binding sites are distinct, as shown by the ability of a radicicol-refractive mutant to bind peptide. When the fluorophore acrylodan is attached to Cys117 within the hydrophobic pocket, its fluorescence is reduced upon peptide binding, consistent with proximity of the two ligands. Substitution of His125, which contacts the bound peptide, compromises peptide-binding activity. We conclude that peptide binds to the concave face of the beta sheet of the N-terminal domain, where binding is regulated during the action cycle of the chaperone.     

Meaning-making in the immune system

Meaning-making is the process by which a system responds to an indeterminate signal. This article focuses on meaning-making in living systems. It proposes several guidelines for studying the process of meaning-making in living systems in general, and in the immune system in particular. Drawing on a general framework for studying meaning-making in living systems, I suggest three basic organizing concepts for studying meaning-making-variability of the signal, context markers, and transgradience. Those concepts present a radical alternative to the information-processing approach that governs biological research and may shed new light on biological processes.     

A flux model of glycolysis and the oxidative pentosephosphate pathway in developing Brassica napus embryos

Developing oilseeds synthesize large quantities of triacylglycerol from sucrose and hexose. To understand the fluxes involved in this conversion, a quantitative metabolic flux model was developed and tested for the reaction network of glycolysis and the oxidative pentose phosphate pathway (OPPP). Developing Brassica napus embryos were cultured with [U-13C6]glucose, [1-13C]glucose, [6-13C]glucose, [U-13C12]sucrose, and/or [1,2-13C2]glucose and the labeling patterns in amino acids, lipids, sucrose, and starch were measured by gas chromatography/mass spectrometry and NMR. Data were used to verify a reaction network of central carbon metabolism distributed between the cytosol and plastid. Computer simulation of the steady state distribution of isotopomers in intermediates of the glycolysis/OPPP network was used to fit metabolic flux parameters to the experimental data. The observed distribution of label in cytosolic and plastidic metabolites indicated that key intermediates of glycolysis and OPPP have similar labeling in these two compartments, suggesting rapid exchange of metabolites between these compartments compared with net fluxes into end products. Cycling between hexose phosphate and triose phosphate and reversible transketolase velocity were similar to net glycolytic flux, whereas reversible transaldolase velocity was minimal. Flux parameters were overdetermined by analyzing labeling in different metabolites and by using data from different labeling experiments, which increased the reliability of the findings. Net flux of glucose through the OPPP accounts for close to 10% of the total hexose influx into the embryo. Therefore, the reductant produced by the OPPP accounts for at most 44% of the NADPH and 22% of total reductant needed for fatty acid synthesis.     

The folate precursor p-aminobenzoate is reversibly converted to its glucose ester in the plant cytosol

Plants synthesize p-aminobenzoate (pABA) in chloroplasts and use it for folate synthesis in mitochondria. It has generally been supposed that pABA exists as the free acid in plant cells and that it moves between organelles in this form. Here we show that fruits and leaves of tomato and leaves of a diverse range of other plants have a high capacity to convert exogenously supplied pABA to its beta-D-glucopyranosyl ester (pABA-Glc), whereas yeast and Escherichia coli do not. High performance liquid chromatography analysis indicated that much of the endogenous pABA in fruit and leaf tissues is esterified and that the total pool of pABA (free plus esterified) varies greatly between tissues (from 0.2 to 11 nmol g-1 of fresh weight). UDP-glucose:pABA glucosyltransferase activity was readily detected in fruit and leaf extracts, and the reaction was found to be freely reversible. p-Aminobenzoic acid beta-D-glucopyranosyl ester esterase activity was also detected in extracts. Subcellular fractionation indicated that the glucosyltransferase and esterase activities are predominantly if not solely cytosolic. Taken together, these results show that reversible formation of pABA-Glc in the cytosol is interposed between pABA production in chloroplasts and pABA consumption in mitochondria. As pABA is a hydrophobic weak acid, its uncharged form is membrane-permeant, and its anion is consequently prone to distribute itself spontaneously among subcellular compartments according to their pH. Esterification of pABA may eliminate such errant behavior and provide a readily reclaimable storage form of pABA as well as a substrate for membrane transporters.