A pH-Dependent Kinetic Model of Dihydrolipoamide Dehydrogenase from Multiple Organisms

AbstractDihydrolipoamide dehydrogenase is a flavoenzyme that reversibly catalyzes the oxidation of reduced lipoyl substrates with the reduction of NAD+ to NADH. In vivo, the dihydrolipoamide dehydrogenase component (E3) is associated with the pyruvate, α-ketoglutarate, and glycine dehydrogenase complexes. The pyruvate dehydrogenase (PDH) complex connects the glycolytic flux to the tricarboxylic acid cycle and is central to the regulation of primary metabolism. Regulation of PDH via regulation of the E3 component by the NAD+/NADH ratio represents one of the important physiological control mechanisms of PDH activity. Furthermore, previous experiments with the isolated E3 component have demonstrated the importance of pH in dictating NAD+/NADH ratio effects on enzymatic activity. Here, we show that a three-state mechanism that represents the major redox states of the enzyme and includes a detailed representation of the active-site chemistry constrained by both equilibrium and thermodynamic loop constraints can be used to model regulatory NAD+/NADH ratio and pH effects demonstrated in progress-curve and initial-velocity data sets from rat, human, Escherichia coli, and spinach enzymes. Global fitting of the model provides stable predictions to the steady-state distributions of enzyme redox states as a function of lipoamide/dihydrolipoamide, NAD+/NADH, and pH. These distributions were calculated using physiological NAD+/NADH ratios representative of the diverse organismal sources of E3 analyzed in this study. This mechanistically detailed, thermodynamically constrained, pH-dependent model of E3 provides a stable platform on which to accurately model multicomponent enzyme complexes that implement E3 from a variety of organisms

Evidence for Hysteretic Substrate Channeling in the Proline Dehydrogenaseand ∆\u3csup\u3e1\u3c/sup\u3e-Pyrroline-5-carboxylate Dehydrogenase Coupled Reaction of Proline UtilizationA(PutA)

Background: PutA from Escherichia coli is a bifunctional enzyme and transcriptional repressor in proline catabolism. Results: Steady-state and transient kinetic data revealed a mechanism in which the two enzymatic reactions are coupled by an activation step. Conclusion: Substrate channeling in PutA exhibits hysteretic behavior. Significance: This is the first kinetic model of bi-enzyme activity in PutA and reveals a novel mechanism of channeling activation

DJ-1 is not a deglycase and makes a modest contribution to cellular defense against methylglyoxal damage in neurons

Human DJ-1 is a cytoprotective protein whose absence causes Parkinson\u27s disease and is also associated with other diseases. DJ-1 has an established role as a redox-regulated protein that defends against oxidative stress and mitochondrial dysfunction. Multiple studies have suggested that DJ-1 is also a protein/nucleic acid deglycase that plays a key role in the repair of glycation damage caused by methylglyoxal (MG), a reactive α-keto aldehyde formed by central metabolism. Contradictory reports suggest that DJ-1 is a glyoxalase but not a deglycase and does not play a major role in glycation defense. Resolving this issue is important for understanding how DJ-1 protects cells against insults that can cause disease. We find that DJ-1 reduces levels of reversible adducts of MG with guanine and cysteine in vitro. The steady-state kinetics of DJ-1 acting on reversible hemithioacetal substrates are fitted adequately with a computational kinetic model that requires only a DJ-1 glyoxalase activity, supporting the conclusion that deglycation is an apparent rather than a true activity of DJ-1. Sensitive and quantitative isotope-dilution mass spectrometry shows that DJ-1 modestly reduces the levels of some irreversible guanine and lysine glycation products in primary and cultured neuronal cell lines and whole mouse brain, consistent with a small but measurable effect on total neuronal glycation burden. However, DJ-1 does not improve cultured cell viability in exogenous MG. In total, our results suggest that DJ-1 is not a deglycase and has only a minor role in protecting neurons against methylglyoxal toxicity

Comparison of the Contributions of Heat-Labile Enterotoxin and Heat-Stable Enterotoxin b to the Virulence of Enterotoxigenic \u3ci\u3eEscherichia coli \u3c/i\u3ein F4ac Receptor-Positive Young Pigs

In swine, the most common and severe enterotoxigenic Escherichia coli (ETEC) infections are caused by strains that express K88 (F4)+ fimbriae, heat-labile enterotoxin (LT), heat-stable enterotoxin b (STb), and enteroaggregative E. coli heat-stable toxin 1. Previous studies based on a design that involved enterotoxin genes cloned into a nontoxigenic fimbriated strain have suggested that LT but not STb plays an important role in dehydrating diarrheal disease in piglets study, we compared these two toxins in terms of importance for piglets \u3e1 week old with a study design that involved construction of isogenic single- and double-deletion mutants and inoculation of 9-day-old F4ac receptor-positive gnotobiotic piglets. Based on the postinoculation percent weight change per h and serum bicarbonate concentrations, the virulence of the STb- mutant (ΔestB) did not significantly differ from that of the parent. However, deletion of the LT genes (ΔeltAB) in the STb- mutant resulted in a complete abrogation of weight loss, dehydration, and metabolic acidosis in inoculated pigs, and LT complementation restored the virulence of this strain. These results support the hypothesis that LT is a more significant contributor than STb to the virulence of F4+ ETEC infections in young F4ac receptor-positive pigs less than 2 weeks old. However, in contrast to previous studies with gnotobiotic piglets, there was no evidence that the expression of LT enhanced the ability of the F4+ ETEC strain to colonize the small intestine

Evidence for Hysteretic Substrate Channeling in the Proline Dehydrogenaseand ∆\u3csup\u3e1\u3c/sup\u3e-Pyrroline-5-carboxylate Dehydrogenase Coupled Reaction of Proline UtilizationA(PutA)

Background: PutA from Escherichia coli is a bifunctional enzyme and transcriptional repressor in proline catabolism. Results: Steady-state and transient kinetic data revealed a mechanism in which the two enzymatic reactions are coupled by an activation step. Conclusion: Substrate channeling in PutA exhibits hysteretic behavior. Significance: This is the first kinetic model of bi-enzyme activity in PutA and reveals a novel mechanism of channeling activation

Google haul out : Earth observation imagery and digital aerial surveys in coastal wildlife management and abundance estimation

© The Author(s), 2017. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Bioscience 67 (2017): 760–768, doi:10.1093/biosci/bix059.As the sampling frequency and resolution of Earth observation imagery increase, there are growing opportunities for novel applications in population monitoring. New methods are required to apply established analytical approaches to data collected from new observation platforms (e.g., satellites and unmanned aerial vehicles). Here, we present a method that estimates regional seasonal abundances for an understudied and growing population of gray seals (Halichoerus grypus) in southeastern Massachusetts, using opportunistic observations in Google Earth imagery. Abundance estimates are derived from digital aerial survey counts by adapting established correction-based analyses with telemetry behavioral observation to quantify survey biases. The result is a first regional understanding of gray seal abundance in the northeast US through opportunistic Earth observation imagery and repurposed animal telemetry data. As species observation data from Earth observation imagery become more ubiquitous, such methods provide a robust, adaptable, and cost-effective solution to monitoring animal colonies and understanding species abundances.We would like to thank generous support from International Fund for Animal Welfare, the Bureau of Ocean Energy, and the Oak Foundation for funding support for the telemetry devices

Evidence review of the potential wider impacts of climate change mitigation options: agriculture, forestry, land use and waste sectors

A report prepared for Scottish Government. Greenhouse gas (GHG) mitigation is a central policy objective in Scotland. The Climate Change (Scotland) Act 2009 sets an interim 42% reduction target for 2020 and an 80% target for 2050 across all sectors of society (1990 baseline). As a priority policy area, it has become vital to better understand the co-benefits and adverse impacts arising from mitigation actions on our environment, economy and society. Integrated assessment is key in prioritising environmental actions, reducing adverse impacts and enhancing positive co-effects. This report aims to summarise evidence on the wider impacts (WI) of GHG mitigation options (MO) in the Agriculture, land use, land use change and forestry sectors (ALULUCF) and those related waste management. The key findings of the review, are a summary of the wider impacts and an overview of the challenges in quantifying and monetising these impacts

Pan-Cancer Analysis of lncRNA Regulation Supports Their Targeting of Cancer Genes in Each Tumor Context

Long noncoding RNAs (lncRNAs) are commonly dys-regulated in tumors, but only a handful are known toplay pathophysiological roles in cancer. We inferredlncRNAs that dysregulate cancer pathways, onco-genes, and tumor suppressors (cancer genes) bymodeling their effects on the activity of transcriptionfactors, RNA-binding proteins, and microRNAs in5,185 TCGA tumors and 1,019 ENCODE assays.Our predictions included hundreds of candidateonco- and tumor-suppressor lncRNAs (cancerlncRNAs) whose somatic alterations account for thedysregulation of dozens of cancer genes and path-ways in each of 14 tumor contexts. To demonstrateproof of concept, we showed that perturbations tar-geting OIP5-AS1 (an inferred tumor suppressor) andTUG1 and WT1-AS (inferred onco-lncRNAs) dysre-gulated cancer genes and altered proliferation ofbreast and gynecologic cancer cells. Our analysis in-dicates that, although most lncRNAs are dysregu-lated in a tumor-specific manner, some, includingOIP5-AS1, TUG1, NEAT1, MEG3, and TSIX, synergis-tically dysregulate cancer pathways in multiple tumorcontexts

Pan-cancer Alterations of the MYC Oncogene and Its Proximal Network across the Cancer Genome Atlas

Although theMYConcogene has been implicated incancer, a systematic assessment of alterations ofMYC, related transcription factors, and co-regulatoryproteins, forming the proximal MYC network (PMN),across human cancers is lacking. Using computa-tional approaches, we define genomic and proteo-mic features associated with MYC and the PMNacross the 33 cancers of The Cancer Genome Atlas.Pan-cancer, 28% of all samples had at least one ofthe MYC paralogs amplified. In contrast, the MYCantagonists MGA and MNT were the most frequentlymutated or deleted members, proposing a roleas tumor suppressors.MYCalterations were mutu-ally exclusive withPIK3CA,PTEN,APC,orBRAFalterations, suggesting that MYC is a distinct onco-genic driver. Expression analysis revealed MYC-associated pathways in tumor subtypes, such asimmune response and growth factor signaling; chro-matin, translation, and DNA replication/repair wereconserved pan-cancer. This analysis reveals insightsinto MYC biology and is a reference for biomarkersand therapeutics for cancers with alterations ofMYC or the PMN