Novel mutation in hexokinase 2 confers resistance to 2-deoxyglucose by altering protein dynamics

Glucose is central to many biological processes, serving as an energy source and a building block for biosynthesis. After glucose enters the cell, hexokinases convert it to glucose-6-phosphate (Glc-6P) for use in anaerobic fermentation, aerobic oxidative phosphorylation, and the pentose-phosphate pathway. We here describe a genetic screen in Saccharomyces cerevisiae that generated a novel spontaneous mutation in hexokinase-2, hxk2^G238V, that confers resistance to the toxic glucose analog 2-deoxyglucose (2DG). Wild-type hexokinases convert 2DG to 2-deoxyglucose-6-phosphate (2DG-6P), but 2DG-6P cannot support downstream glycolysis, resulting in a cellular starvation-like response. Curiously, though the hxk2^G238V mutation encodes a loss-of-function allele, the affected amino acid does not interact directly with bound glucose, 2DG, or ATP. Molecular dynamics simulations suggest that Hxk2^G238V impedes sugar binding by altering the protein dynamics of the glucose-binding cleft, as well as the large-scale domain-closure motions required for catalysis. These findings shed new light on Hxk2 dynamics and highlight how allosteric changes can influence catalysis, providing new structural insights into this critical regulator of carbohydrate metabolism. Given that hexokinases are upregulated in some cancers and that 2DG and its derivatives have been studied in anti-cancer trials, the present work also provides insights that may apply to cancer biology and drug resistance.     

Spatial separation of protein domains is not necessary for catalytic activity or substrate binding in a xylanase.

Xylanase A (XYLA) from Pseudomonas fluorescens subspecies cellulosa shows sequence conservation with two endoglucanases from the same organism. The conserved sequence in XYLA, consisting of the N-terminal 234 residues, is not essential for catalytic activity. Full-length XYLA and a fusion enzyme, consisting of the N-terminal 100 residues of XYLA linked to mature alkaline phosphatase, bound tightly to crystalline cellulose (Avicel), but not to xylan. The capacity of truncated derivatives of the xylanase to bind polysaccharides was investigated. XYLA lacking the first 13 N-terminal amino acids did not bind to cellulose. However, a catalytically active XYLA derivative (XYLA'), in which residues 100-234 were deleted, bound tightly to Avicel. Substrate specificity, cellulose-binding capacity, specific activity and Km for xylan hydrolysis were evaluated for each of the xylanases. No differences in any of these parameters were detected for the two enzymes. It is concluded that XYLA contains a cellulose-binding domain consisting of the N-terminal 100 residues which is distinct from the active site. Spatial separation of the catalytic and cellulose-binding domains is not essential for the enzyme to function normally.     

Exploration of the structure-activity relationship of the diaryl anilide class of ligands for translocator protein-potential novel positron emitting tomography imaging agents

A series of novel ligands based on the diaryl anilide (DAA) class of translocator protein (TSPO) ligands was synthesised and evaluated as potential positron emitting tomography (PET) ligands for imaging TPSO in vivo. Fluorine-18 labelling of the molecules was achieved using direct radiolabelling or synthon based labelling approaches. Several of the ligands prepared have promising profiles as potential TSPO PET imaging ligands and will be evaluated further as potential clinical imaging agents.     

An algorithm for analyzing linkages affected by heterozygous translocations: QuadMap

Heterozygous chromosome rearrangements such as reciprocal translocations are most accurately displayed as two-dimensional linkage maps. Standard linkage mapping software packages, such as MapMaker, generate only one-dimensional maps and so reciprocal translocations appear as clusters of markers, even though they originate from two nonhomologous chromosomes. To more accurately map these regions, researchers have developed statistical methods that use the variance in map distance to distinguish among the four segments (two translocation, two interstitial) of the translocation. In this study, we describe modifications to one of these protocols, that proposed by Livingstone et al. (2000). We also introduce QuadMap, a new software application for dissecting heterozygous translocation-affected linkage maps.     

Microaerophilic–aerobic sequential decolourization/biodegradation of textile azo dyes by a facultative Klebsiella sp. strain VN-31

Four different azo dyes were decolourized and biodegraded in a sequential microaerophilic–aerobic treatment by a facultative Klebsiella sp. strain VN-31, a bacterium isolated from activated sludge process of the textile industry. Dye decolourization was performed under microaerophilic conditions until no colour was observed (decolourization percentage >94%). The medium was then aerated to promote the biodegradation of the amines produced. The presence of aromatic amine in the microaerophilic stage and its absence in the aerobic stage demonstrate azo bond reduction and an oxidative biodegradation process, respectively. Total Organic Carbon (TOC) reduction for the growth medium plus dyes was ～50% in the microaerophilic stage and ～80% in the aerobic stage. The degradation products were also characterized by FT-IR and UV–vis techniques and their toxicity measured using Daphnia magna. The results provide evidence that the successive microaerophilic/aerobic stages, using a single Klebsiella sp. strain VN-31 in the same bioreactor, were able to form aromatic amines by the reductive break down of the azo bond and to oxidize them into non-toxic metabolites.     

New methods to identify conserved microsatellite loci and develop primer sets of high cross-species utility - as demonstrated for birds

We have developed a new approach to create microsatellite primer sets that have high utility across a wide range of species. The success of this method was demonstrated using birds. We selected 35 avian EST microsatellite loci that had a high degree of sequence homology between the zebra finch Taeniopygia guttata and the chicken Gallus gallus and designed primer sets in which the primer bind sites were identical in both species. For 33 conserved primer sets, on average, 100% of loci amplified in each of 17 passerine species and 99% of loci in five non-passerine species. The genotyping of four individuals per species revealed that 24-76% (mean 48%) of loci were polymorphic in the passerines and 18-26% (mean 21%) in the non-passerines. When at least 17 individuals were genotyped per species for four Fringillidae finch species, 71-85% of loci were polymorphic, observed heterozygosity was above 0.50 for most loci and no locus deviated significantly from Hardy-Weinberg proportions. This new set of microsatellite markers is of higher cross-species utility than any set previously designed. The loci described are suitable for a range of applications that require polymorphic avian markers, including paternity and population studies. They will facilitate comparisons of bird genome organization, including genome mapping and studies of recombination, and allow comparisons of genetic variability between species whilst avoiding ascertainment bias. The costs and time to develop new loci can now be avoided for many applications in numerous species. Furthermore, our method can be readily used to develop microsatellite markers of high utility across other taxa.     

Genomic aberrations in lung adenocarcinoma in never smokers

Background

Lung cancer in never smokers would rank as the seventh most common cause of cancer death worldwide.

Methods and Findings

We performed high-resolution array comparative genomic hybridization analysis of lung adenocarcinoma in sixty never smokers and identified fourteen new minimal common regions (MCR) of gain or loss, of which five contained a single gene (MOCS2, NSUN3, KHDRBS2, SNTG1 and ST18). One larger MCR of gain contained NSD1. One focal amplification and nine gains contained FUS. NSD1 and FUS are oncogenes hitherto not known to be associated with lung cancer. FISH showed that the amplicon containing FUS was joined to the next telomeric amplicon at 16p11.2. FUS was over-expressed in 10 tumors with gain of 16p11.2 compared to 30 tumors without that gain. Other cancer genes present in aberrations included ARNT, BCL9, CDK4, CDKN2B, EGFR, ERBB2, MDM2, MDM4, MET, MYC and KRAS. Unsupervised hierarchical clustering with adjustment for false-discovery rate revealed clusters differing by the level and pattern of aberrations and displaying particular tumor characteristics. One cluster was strongly associated with gain of MYC. Another cluster was characterized by extensive losses containing tumor suppressor genes of which RB1 and WRN. Tumors in that cluster frequently harbored a central scar-like fibrosis. A third cluster was associated with gains on 7p and 7q, containing ETV1 and BRAF, and displayed the highest rate of EGFR mutations. SNP array analysis validated copy-number aberrations and revealed that RB1 and WRN were altered by recurrent copy-neutral loss of heterozygosity.

Conclusions

The present study has uncovered new aberrations containing cancer genes. The oncogene FUS is a candidate gene in the 16p region that is frequently gained in never smokers. Multiple genetic pathways defined by gains of MYC, deletions of RB1 and WRN or gains on 7p and 7q are involved in lung adenocarcinoma in never smokers.     

Long-term response of Sitka spruce (Picea sitchensis (Bong.) Carr.) to CO2 enrichment and nitrogen supply. I. Growth,biomass allocation and physiology

ABSTRACT

After a 3-year exposure to elevated CO₂, young trees of Sitka spruce (Picea sitchensis (Bong.) Carr.) were planted in native, nutrient-deficient forest soil and grown for two more years with three CO₂ treatments in open-top chambers, and with two nutrient treatments (with and without supplied N). Elevated CO₂ resulted in larger fresh mass, dry mass, leaf area and leaf thickness in two-year old needles, but had no effect on one-year old and current needles. Tree height, basal diameter and biomass production also increased, regardless of N supply. In trees without added N, elevated CO₂ resulted in higher root-to-shoot and absorbing roots-to-stump ratios. Regardless of N supply, trees grown in elevated CO₂ had lower photosynthetic rates on a leaf area basis. Photosynthesis reduction was accompanied by a decline in Rubisco activity and leaf N concentration. Under elevated CO₂, added N elevated photosynthesis and Rubisco activity, suggesting a dependence on N availability of the photosynthetic response to elevated CO₂. Stomatal conductance of trees grown with added N decreased in response to elevated CO₂. This may account for the larger reduction in intercellular CO₂ concentration, and hence photosynthesis, in the trees supplied with N than in those without N supply.     

Potato virus Y NIa protease activity is not sufficient for elicitation of Ry-mediated disease resistance in potato

Ry confers extreme resistance (ER) to all strains of potato virus Y (PVY). In previous work, we have shown that the protease domain of the nuclear inclusion a protease (NIaPro) from PVY is the elicitor of the Ry-mediated resistance and that integrity of the protease active site is required for the elicitation of the resistance response. Two possibilities arise from these results: first, the structure of the active protease has elicitor activity; second, NIa-mediated proteolysis is required to elicit the resistance response. To resolve these possibilities, the NIaPro from PVY was randomly mutagenised and the clones obtained were screened for elicitation of cell death as an indicator of resistance and proteolytic activity. We did not find any mutants that had retained the ability to elicit cell death but had lost protease activity, as measured by processing of the NIa cleavage site in the viral genome. This was consistent with the idea that protease activity is necessary for elicitor activity. However, protease activity was not sufficient because we found three elicitor-defective mutants in which there was a high level of protease activity in this assay.