Structure and activity of the NAD(P)+‐dependent succinate semialdehyde dehydrogenase YneI from Salmonella typhimurium

Aldehyde dehydrogenases are found in all organisms and play an important role in the metabolic conversion and detoxification of endogenous and exogenous aldehydes. Genomes of many organisms including Escherichia coli and Salmonella typhimurium encode two succinate semialdehyde dehydrogenases with low sequence similarity and different cofactor preference (YneI and GabD). Here, we present the crystal structure and biochemical characterization of the NAD(P)⁺‐dependent succinate semialdehyde dehydrogenase YneI from S. typhimurium. This enzyme shows high activity and affinity toward succinate semialdehyde and exhibits substrate inhibition at concentrations of SSA higher than 0.1 mM. YneI can use both NAD⁺ and NADP⁺ as cofactors, although affinity to NAD⁺ is 10 times higher. High resolution crystal structures of YneI were solved in a free state (1.85 Å) and in complex with NAD⁺ (1.90 Å) revealing a two domain protein with the active site located in the interdomain interface. The NAD⁺ molecule is bound in the long channel with its nicotinamide ring positioned close to the side chain of the catalytic Cys268. Site‐directed mutagenesis demonstrated that this residue, as well as the conserved Trp136, Glu365, and Asp426 are important for activity of YneI, and that the conserved Lys160 contributes to the enzyme preference to NAD⁺. Our work has provided further insight into the molecular mechanisms of substrate selectivity and activity of succinate semialdehyde dehydrogenases. © 2012 Wiley Periodicals, Inc.     

Selenium Supplementation Alters Gene Expression Profiles Associated with Innate Immunity in Whole-Blood Neutrophils of Sheep

Footrot (FR) is a common, contagious bacterial disease of sheep that results in lameness and significant economic losses for producers. We previously reported that sheep affected with FR have lower whole-blood (WB) selenium (Se) concentrations and that Se supplementation in conjunction with routine control practices accelerates recovery from FR. To determine whether oral Se-yeast administered at supranutritional levels (>4.9 mg Se/week) alters the ability of sheep to resist or recover from FR infection, 60 ewes with and 60 ewes without FR were drenched once weekly for 62.5 weeks with 0, 4.9, 14.7, or 24.5 mg organic Se-yeast (30 ewes per treatment group). Footrot prevalence and severity were measured at 0, 20, 28, 40, and 60 weeks of Se supplementation. Genomic expression of eight WB-neutrophil genes for selenoproteins and seven WB-neutrophil genes for proteins involved in innate immunity was determined at the end of the treatment period using SYBR Green and quantitative polymerase chain reaction methodology. Supranutritional Se-yeast supplementation successfully increased Se status in sheep but did not prevent FR. Supranutritional Se-yeast supplementation increased WB-neutrophil expression of genes involved in innate immunity: l-selectin, interleukin-8 receptor, and toll-like receptor 4, which were or tended to be lower in ewes affected with FR. Furthermore, supranutritional Se-yeast supplementation altered the expression of selenoprotein genes involved in innate immunity, increasing selenoprotein S and glutathione peroxidase 4 and decreasing iodothyronine deiodinases 2 and 3. In conclusion, supranutritional Se-yeast supplementation does not prevent FR, but does alter WB-neutrophil gene expression profiles associated with innate immunity, including reversing those impacted by FR.     

77Se Enrichment of Proteins Expands the Biological NMR Toolbox

Sulfur, a key contributor to biological reactivity, is not amendable to investigations by biological NMR spectroscopy. To utilize selenium as a surrogate, we have developed a generally applicable ⁷⁷Se isotopic enrichment method for heterologous proteins expressed in Escherichia coli. We demonstrate ⁷⁷Se NMR spectroscopy of multiple selenocysteine and selenomethionine residues in the sulfhydryl oxidase augmenter of liver regeneration (ALR). The resonances of the active-site residues were assigned by comparing the NMR spectra of ALR bound to oxidized and reduced flavin adenine dinucleotide. An additional resonance appears only in the presence of the reducing agent and disappears readily upon exposure to air and subsequent reoxidation of the flavin. Hence, ⁷⁷Se NMR spectroscopy can be used to report the local electronic environment of reactive and structural sulfur sites, as well as changes taking place in those locations during catalysis.     

LY2228820 Dimesylate,a Selective Inhibitor of p38 Mitogen-activated Protein Kinase,Reduces Angiogenic Endothelial Cord Formation in Vitro and in Vivo

LY2228820 dimesylate is a highly selective small molecule inhibitor of p38α and p38β mitogen-activated protein kinases (MAPKs) that is currently under clinical investigation for human malignancies. p38 MAPK is implicated in a wide range of biological processes, in particular those that support tumorigenesis. One such process, angiogenesis, is required for tumor growth and metastasis, and many new cancer therapies are therefore directed against the tumor vasculature. Using an in vitro co-culture endothelial cord formation assay, a surrogate of angiogenesis, we investigated the role of p38 MAPK in growth factor- and tumor-driven angiogenesis using LY2228820 dimesylate treatment and by shRNA gene knockdown. p38 MAPK was activated in endothelial cells upon growth factor stimulation, with inhibition by LY2228820 dimesylate treatment causing a significant decrease in VEGF-, bFGF-, EGF-, and IL-6-induced endothelial cord formation and an even more dramatic decrease in tumor-driven cord formation. In addition to involvement in downstream cytokine signaling, p38 MAPK was important for VEGF, bFGF, EGF, IL-6, and other proangiogenic cytokine secretion in stromal and tumor cells. LY2228820 dimesylate results were substantiated using p38α MAPK-specific shRNA and shRNA against the downstream p38 MAPK effectors MAPKAPK-2 and HSP27. Using in vivo models of functional neoangiogenesis, LY2228820 dimesylate treatment reduced hemoglobin content in a plug assay and decreased VEGF-A-stimulated vascularization in a mouse ear model. Thus, p38α MAPK is implicated in tumor angiogenesis through direct tumoral effects and through reduction of proangiogenic cytokine secretion via the microenvironment.     

GPCR-induced dissociation of G-protein subunits in early stage signal transduction

G-protein coupled receptors (GPCRs) form a ternary complex of agonist, receptor and G-proteins during primary signal transduction at the cell membrane. Downstream signalling is thought to be preceded by the process of dissociation of Gα and Gβγ subunits, thus exposing new surfaces to interact with downstream effectors. We demonstrate here for the first time, the dissociation of heterotrimeric G-protein subunits (i.e., Gα and Gβγ) following agonist-induced GPCR (α_2A-adrenergic receptor; α_2A-AR) activation in a cell-free assay system. α_2A-AR membranes were reconstituted with the G-proteins (±hexahistidine-tagged) Gα_i1 and Gβ₁γ₂ and functional signalling was determined following activation of the reconstituted receptor:G-protein complex with the potent agonist UK-14304, and [³⁵S]GTPγS. In the presence of Ni²⁺-coated agarose beads, the activated his-tagged Gα_i1his-[³⁵S]GTPγS complex was captured on the Ni²⁺-presenting surface. When his-tagged Gβ₁γ₂ (Gβ₁γ₂his) was used with Gα_i1, the [³⁵S]GTPγS-bound Gα_i1 was not present on the Ni²⁺-coated beads, but rather, it was separated from the β₁γ₂(his)-beads, demonstrating receptor-induced dissociation of Gα and Gβγ subunits. Treatment of the reconstituted α_2A-AR membranes containing Gβ₁γ₂his:Gα_i1 with imidazole confirmed the specificity for the Ni²⁺:G-protein surface dissociation of Gα_i1 from Gβ₁γ₂his. These data demonstrate for the first time, the complete dissociation of the G-protein subunits and extend observations on the role of G-proteins in the assembly and disassembly of the ternary complex in the primary events of GPCR signalling.     

Lignified and nonlignified fiber cables in the lacunae of <Emphasis Type="Italic">Typha angustifolia</Emphasis>

The leaves of Typha are noteworthy in terms of their mechanical properties. We determined the mechanical properties of the fiber cables within the leaf. We found that in vegetative plants, the lignified fiber cables isolated from the leaf sheath and nonlignified fiber cables isolated from the leaf blade of Typha angustifolia differ in their diameter, swelling capacity, Young’s modulus, tensile strength, and break load. These differing properties are related to their contributions to stability in the two regions of the leaf.     

Whither life? Conjectures on the future evolution of biochemistry

Life has existed on the Earth for approximately four billion years. The sheer depth of evolutionary time, and the diversity of extant species, makes it tempting to assume that all the key biochemical innovations underpinning life have already happened. But we are only a little over halfway through the trajectory of life on our planet. In this Opinion piece, we argue: (i) that sufficient time remains for the evolution of new processes at the heart of metabolic biochemistry and (ii) that synthetic biology is providing predictive insights into the nature of these innovations. By way of example, we focus on engineered solutions to existing inefficiencies in energy generation, and on the complex, synthetic regulatory circuits that are currently being implemented.