Solution of the structure of tetrameric human glucose 6-phosphate dehydrogenase by molecular replacement

Recombinant human glucose 6-phosphate dehydrogenase (G6PD) has been crystallized and its structure solved by molecular replacement. Crystals of the natural mutant R459L grow under similar conditions in space groups P212121 and C2221 with eight or four 515-residue molecules in the asymmetric unit, respectively. A non-crystallographic 222 tetramer was found in the C2221 crystal form using a 4 A resolution data set and a dimer of the large beta + alpha domains of the Leuconostoc mesenteroides enzyme as a search model. This tetramer was the only successful search model for the P212121 crystal form using data to 3 A. Crystals of the deletion mutant DeltaG6PD grow in space group F222 with a monomer in the asymmetric unit; 2.5 A resolution data have been collected. Comparison of the packing of tetramers in the three space groups suggests that the N-terminal tail of the enzyme prevents crystallization with exact 222 molecular symmetry.published_or_final_versio

Molecular crowding defines a common origin for the Warburg effect in proliferating cells and the lactate threshold in muscle physiology

Aerobic glycolysis is a seemingly wasteful mode of ATP production that is seen both in rapidly proliferating mammalian cells and highly active contracting muscles, but whether there is a common origin for its presence in these widely different systems is unknown. To study this issue, here we develop a model of human central metabolism that incorporates a solvent capacity constraint of metabolic enzymes and mitochondria, accounting for their occupied volume densities, while assuming glucose and/or fatty acid utilization. The model demonstrates that activation of aerobic glycolysis is favored above a threshold metabolic rate in both rapidly proliferating cells and heavily contracting muscles, because it provides higher ATP yield per volume density than mitochondrial oxidative phosphorylation. In the case of muscle physiology, the model also predicts that before the lactate switch, fatty acid oxidation increases, reaches a maximum, and then decreases to zero with concomitant increase in glucose utilization, in agreement with the empirical evidence. These results are further corroborated by a larger scale model, including biosynthesis of major cell biomass components. The larger scale model also predicts that in proliferating cells the lactate switch is accompanied by activation of glutaminolysis, another distinctive feature of the Warburg effect. In conclusion, intracellular molecular crowding is a fundamental constraint for cell metabolism in both rapidly proliferating- and non-proliferating cells with high metabolic demand. Addition of this constraint to metabolic flux balance models can explain several observations of mammalian cell metabolism under steady state conditions

On the pulmonary toxicity of oxygen : III. The induction of oxygen dependency by oxygen use

Author Posting. © The Author(s), 2010. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Experimental and Molecular Pathology 89 (2010): 36-45, doi:10.1016/j.yexmp.2010.05.004.Oxygen is central to the development of neonatal lung injury. The increase in oxygen exposure of the neonatal lung during the onset of extrauterine air breathing is an order of magnitude, from a range of 10-12 to 110-120 Torr. The contributions of oxygen and the volume and pressure relationships of ventilatory support to lung injury are not easily distinguished in the clinical setting. Sequential changes in inspired air or 100% oxygen were studied in 536 newborn rabbits without ventilatory support. Bilateral cervical vagotomies (BCV) were performed at 24 hours post natal to induce ventilatory distress which eventuates in hyaline membrane disease. The sequences applied yielded evidence for an induced state of oxygen dependency from oxygen use which was reflected in differences in survival and the extent of pulmonary injury. The median survival for animals kept in air throughout was 3 hours. Oxygen before vagotomy or during the first 3 hours afterwards extended the survival significantly but produced more extensive, more severe, and more rapid lung lesions. Returning animals to air after prior oxygen exposure reduced the number of survivors past 10 hours and shortened the maximum survival in those groups. These features indicate the development of a dependency of the defense mechanisms on the availability of oxygen at the higher level for metabolic and possibly other aspects of the pulmonary and systemic response to injury, beyond the usual physiological need. Subset analysis revealed additive and latent effects of oxygen and demonstrated a remarkable rapidity in onset of severe lesions under some circumstances, illustrating the toxicity of oxygen per se.John A. Hartford Foundation, New York, N.Y. 10022-171

Identification of an intronic regulatory element in the human protein C (PROC) gene

Regulatory DNA elements responsible for human protein C (PROC) gene expression have previously been identified in the upstream promoter region and first (untranslated) exon of the gene. Here we show that an additional sequence element located more than 500 bp downstream of the core promoter within intron 1 further enhances PROC promoter-driven reporter gene expression in human hepatoma cells. In common with core promoter constructs used in previous studies, the activity of this 3'-extended regulatory region is diminished by a naturally occurring promoter mutation. However, in contrast to constructs lacking intronic sequence, the promoter/intron regulatory region is repressed rather than activated by the transcription factor HNF-1. Using both conventional alignment procedures and complexity analysis to study the human and canine PROC sequences, we identified two conserved intronic regions, which were tested for their involvement in gene regulation. High-level gene expression from the intron-coupled promoter was dependent upon the integrity of a 142 bp sequence element, a duplicate copy of which is located in an upstream region of the PROC gene that possesses enhancer activity. These findings emphasise the potential importance of intragenic sequences for gene regulation and serve to illustrate that the results of PROC promoter/reporter gene experiments are critically dependent upon the sequence context. The identification of such intragenic elements is relevant to the analysis of human genetic disease since it will facilitate the detection and functional evaluation of regulatory mutations and polymorphisms