Substrate binding to Src: A new perspective on tyrosine kinase substrate recognition from NMR and molecular dynamics

Most signal transduction pathways in humans are regulated by protein kinases through phosphorylation of their protein substrates. Typical eukaryotic protein kinases are of two major types: those that phosphorylate‐specific sequences containing tyrosine (~90 kinases) and those that phosphorylate either serine or threonine (~395 kinases). The highly conserved catalytic domain of protein kinases comprises a smaller N lobe and a larger C lobe separated by a cleft region lined by the activation loop. Prior studies find that protein tyrosine kinases recognize peptide substrates by binding the polypeptide chain along the C‐lobe on one side of the activation loop, while serine/threonine kinases bind their substrates in the cleft and on the side of the activation loop opposite to that of the tyrosine kinases. Substrate binding structural studies have been limited to four families of the tyrosine kinase group, and did not include Src tyrosine kinases. We examined peptide‐substrate binding to Src using paramagnetic‐relaxation‐enhancement NMR combined with molecular dynamics simulations. The results suggest Src tyrosine kinase can bind substrate positioning residues C‐terminal to the phosphoacceptor residue in an orientation similar to serine/threonine kinases, and unlike other tyrosine kinases. Mutagenesis corroborates this new perspective on tyrosine kinase substrate recognition. Rather than an evolutionary split between tyrosine and serine/threonine kinases, a change in substrate recognition may have occurred within the TK group of the human kinome. Protein tyrosine kinases have long been therapeutic targets, but many marketed drugs have deleterious off‐target effects. More accurate knowledge of substrate interactions of tyrosine kinases has the potential for improving drug selectivity.     

E.E.G. and Personality Factors in Baby Batterers

Out of 35 parents who battered their children eight had an abnormal E.E.G. All of these were found to be psychopathic, of low intelligence, and to be persistent batterers. The presence of an abnormal E.E.G. strongly suggests that some baby batterers are more closely related to those who commit acts of violence and that taken as a whole they are not a homogenous group about whom it is safe to generalize. The possibility of a separate subgroup among baby batterers, therefore, needs close attention.     

Microbial Communities Associated with Zones of Elevated Magnetic Susceptibility in Hydrocarbon-Contaminated Sediments

Recent studies suggest that magnetic susceptibility (MS) measurements can play an important role in identifying zones where microbial-mediated iron mineral transformations are occurring. Here we investigated the microbial community variations within zones of elevated MS in a petroleum hydrocarbon-contaminated aquifer near Bemidji, Minnesota, USA. Our main objective was to 1) identify the key microbial populations that may play a role in hydrocarbon degradation, 2) analyze which microbial populations could be connected to the elevated MS and 3) explore the use of non-destructive geophysical techniques as a tool to guide microbial sampling. Clone libraries based on the 16S rRNA gene revealed the presence of iron-reducing β-Proteobacteria in the vadose zone, whereas the free petroleum phase on the water table was characterized by a methanogenic consortium, in which the syntrophic δ-proteobacterium Smithella and the hydrogenotrophic Methanoregula predominated. Nonmetric multidimensional scaling (NMDS) found a close relationship between elevated MS values and the methanogenic hydrocarbon-degrading consortium. Our results suggest that magnetic susceptibility measurements can guide microbiologists to zones of active microbial biodegradation in aged petroleum spills.     

Genetic Variants in the Bone Morphogenic Protein Gene Family Modify the Association between Residential Exposure to Traffic and Peripheral Arterial Disease

There is a growing literature indicating that genetic variants modify many of the associations between environmental exposures and clinical outcomes, potentially by increasing susceptibility to these exposures. However, genome-scale investigations of these interactions have been rarely performed particularly in the case of air pollution exposures. We performed race-stratified genome-wide gene-environment interaction association studies on European-American (EA, N = 1623) and African-American (AA, N = 554) cohorts to investigate the joint influence of common single nucleotide polymorphisms (SNPs) and residential exposure to traffic (“traffic exposure”)—a recognized vascular disease risk factor—on peripheral arterial disease (PAD). Traffic exposure was estimated via the distance from the primary residence to the nearest major roadway, defined as the nearest limited access highways or major arterial. The rs755249-traffic exposure interaction was associated with PAD at a genome-wide significant level (P = 2.29x10^-8) in European-Americans. Rs755249 is located in the 3’ untranslated region of BMP8A, a member of the bone morphogenic protein (BMP) gene family. Further investigation revealed several variants in BMP genes associated with PAD via an interaction with traffic exposure in both the EA and AA cohorts; this included interactions with non-synonymous variants in BMP2, which is regulated by air pollution exposure. The BMP family of genes is linked to vascular growth and calcification and is a novel gene family for the study of PAD pathophysiology. Further investigation of BMP8A using the Genotype Tissue Expression Database revealed multiple variants with nominally significant (P < 0.05) interaction P-values in our EA cohort were significant BMP8A eQTLs in tissue types highlight relevant for PAD such as rs755249 (tibial nerve, eQTL P = 3.6x10^-6) and rs1180341 (tibial artery, eQTL P = 5.3x10^-6). Together these results reveal a novel gene, and possibly gene family, associated with PAD via an interaction with traffic air pollution exposure. These results also highlight the potential for interactions studies, particularly at the genome scale, to reveal novel biology linking environmental exposures to clinical outcomes.     

Effect of nitrogen and phosphorus addition on phenanthrene biodegradation in four soils

Phenanthrene mineralization rates were found to vary widely among four soils; differences in soil nutrient levels was one hypothesis to explain this variation. To test this hypothesis, phenanthrene mineralization rates were measured in these soils with, and without, added nitrogen and phosphorus. Mineralization rates either remained unchanged or were depressed by the addition of nitrogen and phosphorus. Phenanthrene degradation rates remained unchanged in the soil which had the highest indigenous levels of nitrogen and phosphorus and which showed the largest increase in phosphorus levels after nutrients were added. The soils in which degradation rates were depressed had lower initial phosphorus concentrations and showed much smaller or no measurable increase in phosphorus levels after nutrients were added to the soils. To understand the response of phenanthrene degradation rates to added nitrogen and phosphorus, it may be necessary to consider the bioavailability of added nutrients and nutrient induced changes in microbial metabolism and ecology.     

Transmembrane segment 6 of the Glut1 glucose transporter is an outer helix and contains amino acid side chains essential for transport activity

Experimental data and homology modeling suggest a structure for the exofacial configuration of the Glut1 glucose transporter in which 8 transmembrane helices form an aqueous cavity in the bilayer that is stabilized by four outer helices. The role of transmembrane segment 6, predicted to be an outer helix in this model, was examined by cysteine-scanning mutagenesis and the substituted cysteine accessibility method using the membrane-impermeant, sulfhydryl-specific reagent, p-chloromercuribenzene-sulfonate (pCMBS). A fully functional Glut1 molecule lacking all 6 native cysteine residues was used as a template to produce a series of 21 Glut1 point mutants in which each residue along helix 6 was individually changed to cysteine. These mutants were expressed in Xenopus oocytes, and their expression levels, functional activities, and sensitivities to inhibition by pCMBS were determined. Cysteine substitutions at Leu(204) and Pro(205) abolished transport activity, whereas substitutions at Ile(192), Pro(196), Gln(200), and Gly(201) resulted in inhibition of activity that ranged from approximately 35 to approximately 80%. Cysteine substitutions at Leu(188), Ser(191), and Leu(199) moderately augmented specific transport activity relative to the control. These results were dramatically different from those previously reported for helix 12, the structural cognate of helix 6 in the pseudo-symmetrical structural model, for which none of the 21 single-cysteine mutants exhibited reduced activity. Only the substitution at Leu(188) conferred inhibition by pCMBS, suggesting that most of helix 6 is not exposed to the external solvent, consistent with its proposed role as an outer helix. These data suggest that helix 6 contains amino acid side chains that are critical for transport activity and that structurally analogous outer helices may play distinct roles in the function of membrane transporters.     

Seed Germination Biology of the Narrowly Endemic Species Lesquerella stonensis (Brassicaceae)

Abstract Lesquerella stonensis (Brassicaceae) is an obligate winter annual endemic to a small portion of Rutherford County in the Central Basin of Tennessee, where it grows in disturbed habitats. This species forms a persistent seed bank, and seeds remain viable in the soil for at least 6 years. Seeds are dormant at maturity in May and are dispersed as soon as they ripen. Some of the seeds produced in the current year, as well as some of those in the persistent seed bank, afterripen during late spring and summer; others do not afterripen and thus remain dormant. Seeds require actual or simulated spring/summer temperatures to come out of dormancy. Germination occurs in September and October. Fully afterripened seeds germinate over a wide range of thermoperiods (15/6–35/20°C) and to a much higher percentage in light (14 h photoperiod) than in darkness. The optimum daily thermoperiod for germination was 30/15°C. Nondormant seeds that do not germinate in autumn are induced back into dormancy (secondary dormancy) by low temperatures (e.g., 5°C) during winter, and those that are dormant do not afterripen; thus seeds cannot germinate in spring. These seed dormancy/ germination characteristics of L. stonensis do not differ from those reported for some geographically widespread, weedy species of winter annuals and thus do not help account for the narrow endemism of this species.