TTBK2 kinase substrate specificity and the impact of spinocerebellar-ataxia-causing mutations on expression, activity, localization and development

Mutations that truncate the C-terminal non-catalytic moiety of TTBK2 (tau tubulin kinase 2) cause the inherited, autosomal dominant, SCA11 (spinocerebellar ataxia type?11) movement disorder. In the present study we first assess the substrate specificity of TTBK2 and demonstrate that it has an unusual preference for a phosphotyrosine residue at the +2 position relative to the phosphorylation site. We elaborate a peptide substrate (TTBKtide, RRKDLHDDEEDEAMSIYpA) that can be employed to quantify TTBK2 kinase activity. Through modelling and mutagenesis we identify a putative phosphate-priming groove within the TTBK2 kinase domain. We demonstrate that SCA11 truncating mutations promote TTBK2 protein expression, suppress kinase activity and lead to enhanced nuclear localization. We generate an SCA11-mutation-carrying knockin mouse and show that this leads to inhibition of endogenous TTBK2 protein kinase activity. Finally, we find that, in homozygosity, the SCA11 mutation causes embryonic lethality at embryonic day 10. These findings provide the first insights into some of the intrinsic properties of TTBK2 and reveal how SCA11-causing mutations affect protein expression, catalytic activity, localization and development. We hope that these findings will be helpful for future investigation of the regulation and function of TTBK2 and its role in SCA11.     

The driver of malignancy in KG-1a leukemic cells, FGFR1OP2-FGFR1, encodes an HSP90 addicted oncoprotein

The KG-1a cell line is developed from a human stem cell myeloproliferative neoplasm as the result of intragenic disruption and a chromosomal translocation of the FGFR1 gene and the FGFR1OP2 gene encoding a protein of unknown function called FOP2 (FGFR1 Oncogene Partner 2). The resulting fusion protein FOP2-FGFR1 is soluble and has constitutive tyrosine kinase activity. Since the heat shock protein HSP90 and its co-chaperone CDC37 have been shown to stabilize many oncogenic proteins, we investigated the requirement for HSP90 or HSP90-CDC37 assistance to maintain the stability or activity of FOP2-FGFR1 expressed in KG-1a cells. We found that HSP90-CDC37 forms a permanent complex with FOP2-FGFR1. This results in protection against degradation of FOP2-FGFR1 and holds the oncoprotein in a permanently active conformation. Inhibition of HSP90 or depletion of CDC37 or heat shock factor 1 (HSF1) reduced the expression level of FOP2-FGFR1 and was sufficient to block the oncoprotein induced proliferation of KG-1a cells. We conclude that the driver of malignancy in KG-1a leukemic cells, FOP2-FGFR1, is an HSP90 addicted oncoprotein. This provides a rationale for the therapeutic use of HSP90 inhibitors in myeloid leukemias that contain FGFR fusion proteins.     

Canard-induced complex oscillations in an excitatory network

Journal of Mathematical Biology - In Neuroscience, mathematical modelling involving multiple spatial and temporal scales can unveil complex oscillatory activity such as excitable responses to an...     

Development of a Novel Heart Failure Risk Tool: The Barcelona Bio-Heart Failure Risk Calculator (BCN Bio-HF Calculator)

Background

A combination of clinical and routine laboratory data with biomarkers reflecting different pathophysiological pathways may help to refine risk stratification in heart failure (HF). A novel calculator (BCN Bio-HF calculator) incorporating N-terminal pro B-type natriuretic peptide (NT-proBNP, a marker of myocardial stretch), high-sensitivity cardiac troponin T (hs-cTnT, a marker of myocyte injury), and high-sensitivity soluble ST2 (ST2), (reflective of myocardial fibrosis and remodeling) was developed.

Methods

Model performance was evaluated using discrimination, calibration, and reclassification tools for 1-, 2-, and 3-year mortality. Ten-fold cross-validation with 1000 bootstrapping was used.

Results

The BCN Bio-HF calculator was derived from 864 consecutive outpatients (72% men) with mean age 68.2±12 years (73%/27% New York Heart Association (NYHA) class I-II/III-IV, LVEF 36%, ischemic etiology 52.2%) and followed for a median of 3.4 years (305 deaths). After an initial evaluation of 23 variables, eight independent models were developed. The variables included in these models were age, sex, NYHA functional class, left ventricular ejection fraction, serum sodium, estimated glomerular filtration rate, hemoglobin, loop diuretic dose, β-blocker, Angiotensin converting enzyme inhibitor/Angiotensin-2 receptor blocker and statin treatments, and hs-cTnT, ST2, and NT-proBNP levels. The calculator may run with the availability of none, one, two, or the three biomarkers. The calculated risk of death was significantly changed by additive biomarker data. The average C-statistic in cross-validation analysis was 0.79.

Conclusions

A new HF risk-calculator that incorporates available biomarkers reflecting different pathophysiological pathways better allowed individual prediction of death at 1, 2, and 3 years.     

Identification of Key Residues That Confer Rhodobacter sphaeroides LPS Activity at Horse TLR4/MD-2

The molecular determinants underpinning how hexaacylated lipid A and tetraacylated precursor lipid IVa activate Toll-like receptor 4 (TLR4) are well understood, but how activation is induced by other lipid A species is less clear. Species specificity studies have clarified how TLR4/MD-2 recognises different lipid A structures, for example tetraacylated lipid IVa requires direct electrostatic interactions for agonism. In this study, we examine how pentaacylated lipopolysaccharide from Rhodobacter sphaeroides (RSLPS) antagonises human TLR4/MD-2 and activates the horse receptor complex using a computational approach and cross-species mutagenesis. At a functional level, we show that RSLPS is a partial agonist at horse TLR4/MD-2 with greater efficacy than lipid IVa. These data suggest the importance of the additional acyl chain in RSLPS signalling. Based on docking analysis, we propose a model for positioning of the RSLPS lipid A moiety (RSLA) within the MD-2 cavity at the TLR4 dimer interface, which allows activity at the horse receptor complex. As for lipid IVa, RSLPS agonism requires species-specific contacts with MD-2 and TLR4, but the R2 chain of RSLA protrudes from the MD-2 pocket to contact the TLR4 dimer in the vicinity of proline 442. Our model explains why RSLPS is only partially dependent on horse TLR4 residue R385, unlike lipid IVa. Mutagenesis of proline 442 into a serine residue, as found in human TLR4, uncovers the importance of this site in RSLPS signalling; horse TLR4 R385G/P442S double mutation completely abolishes RSLPS activity without its counterpart, human TLR4 G384R/S441P, being able to restore it. Our data highlight the importance of subtle changes in ligand positioning, and suggest that TLR4 and MD-2 residues that may not participate directly in ligand binding can determine the signalling outcome of a given ligand. This indicates a cooperative binding mechanism within the receptor complex, which is becoming increasingly important in TLR signalling.     

Personal Trust Increases Cooperation beyond General Trust

In this paper we present a new methodology which, while allowing for anonymous interaction, it also makes possible to compare decisions of cooperating or defecting when playing games within a group, according to whether or not players personally trust each other. The design thus goes beyond standard approaches to the role of trust in fostering cooperation, which is restricted to general trust. It also allows considering the role of the topology of the social network involved may play in the level of cooperation found. The results of this work support the idea that personal trust promotes cooperation beyond the level of general trust. We also found that this effect carries over to the whole group, making it more cohesive, but that higher levels of cohesion rely on a particular topology. As a conclusion, we hypothesize that personal trust is a psychological mechanism evolved to make human social life possible in the small groups our ancestors lived in, and that this mechanism persists and plays a role in sustaining cooperation and social cohesion.     

Effect of exercise intensity and training on antioxidants and cholesterol profile in cyclists

The aim of this work was to evaluate the effect of different intensity of exercise and different training status on antioxidants and cholesterol profile in cyclists. 33 male cyclists (17 amateur and 16 professional cyclists) participated in this study. The amateurs all trained 14 +/- 1 h each week, and their VO(2) max was 62.5 +/- 1.8 ml/Kg x min; the professionals all trained 24 +/- 1 h each week, and their VO(2) max was 80.2 +/- 1.6 ml/Kg x min. Amateurs were submitted to the maximal and submaximal prolonged exercise tests. Professionals were submitted to a mountain stage (170 km) of cycling competition. Serum lipid and cholesterol profile (triglycerides, total cholesterol, HDL-cholesterol, LDL-cholesterol and VLDL-cholesterol) and plasma antioxidant capacity (ascorbic acid, alpha-tocopherol, retinol, beta-carotene and others) were measured before and after exercise tests. Hematological determinations (number of erythrocytes, hematocrit and hemoglobin concentration) and dietary intake were also measured. No significant differences were observed in basal values (before exercise tests) of amateur and professional cyclists. Negligible differences were found between dietary intake of amateur and professional cyclists, and also the results of hematological values showed there was no effect of degree of hydration or dietary intake on blood levels of studied antioxidant and lipid parameters. An increase in plasma levels of vitamin C, vitamin E, triglycerides and VLDL-cholesterol levels, and also a decrease of beta-carotene and LDL-cholesterol. were observed in well-trained professional cyclists after the cycling stage - an endurance exercise--but not in amateur cyclists. Amateur cyclists showed only mild increases in total cholesterol after maximal and submaximal exercise, while a rise in HDL-cholesterol was only observed after maximal exercise; none of these changes were observed in professional cyclists. Plasma levels of antioxidant vitamins and carotenes, and also serum lipids, total cholesterol and lipoprotein-cholesterol showed an overall response to exercise, and their increase and/or decrease must be explained as a consequence of the different training status of sportsmen and intensity and duration of exercise tests.     

Applications of single nucleotide polymorphisms in crop genetics

The discovery of single nucleotide polymorphisms (SNPs) and insertions/deletions, which are the basis of most differences between alleles, has been simplified by recent developments in sequencing technology. SNP discovery in many crop species, such as corn and soybean, is relatively straightforward because of their high level of intraspecific nucleotide diversity, and the availability of many gene and expressed sequence tag (EST) sequences. For these species, direct readout of SNP haplotypes is possible. Haplotype-based analysis is more informative than analysis based on individual SNPs, and has more power in analyzing association with phenotypes. The elite germplasm of some crops may have been subjected to bottlenecks relatively recently, increasing the amount of linkage disequilibrium (LD) present and facilitating the association of SNP haplotypes at candidate gene loci with phenotypes. Whole-genome scans may help identify genome regions that are associated with interesting phenotypes if sufficient LD is present. Technological improvements make the use of SNP and indel markers attractive for high-throughput use in marker-assisted breeding, EST mapping and the integration of genetic and physical maps.     

X-ray crystallographic studies on butyryl-ACP reveal flexibility of the structure around a putative acyl chain binding site

Acyl carrier protein (ACP) is an essential cofactor in biosynthesis of fatty acids and many other reactions that require acyl transfer steps. We have determined the first crystal structures of an acylated form of ACP from E. coli, that of butyryl-ACP. Our analysis of the molecular surface of ACP reveals a plastic hydrophobic cavity in the vicinity of the phosphopantethylated Ser36 residue that is expanded and occupied by the butyryl and beta-mercaptoethylamine moieties of the acylated 4'-phosphopantetheine group in one of our crystal forms. In the other form, the cavity is contracted, and we propose that the protein has adopted the conformation after delivery of substrate into the active site of a partner enzyme.