Oligosaccharide recognition and binding to the carbohydrate binding module of AMP-activated protein kinase

AbstractThe AMP-activated protein kinase (AMPK) contains a carbohydrate-binding module (β1-CBM) that is conserved from yeast to mammals. β1-CBM has been shown to localize AMPK to glycogen in intact cells and in vitro. Here we use Nuclear Magnetic Resonance spectroscopy to investigate oligosaccharide binding to 15N labelled β1-CBM. We find that β1-CBM shows greatest affinity to carbohydrates of greater than five glucose units joined via α,1→4 glycosidic linkages with a single, but not multiple, glucose units in an α,1→6 branch. The near identical chemical shift profile for all oligosaccharides whether cyclic or linear suggest a similar binding conformation and confirms the presence of a single carbohydrate-binding site

Patients with aortic stenosis exhibit early improved endothelial function following transcatheter aortic valve replacement: The eFAST study

BACKGROUND: Patients with severe aortic stenosis (AS) exhibit systemic endothelial dysfunction, which can be associated with myocardial ischaemia in absence of obstructive coronary disease. Transcatheter aortic valve replacement (TAVR) is used to treat severe AS in patients with high or prohibitive surgical risk. However, it remains unknown whether endothelial function recovers post-TAVR. We therefore sought to assess the early and late changes in flow-mediated dilation (FMD), a measure of endothelial function, following TAVR. METHODS: Patients undergoing TAVR for severe AS had ultrasound assessment of brachial endothelial-independent and -dependent FMD. Measurements were performed pre-TAVR, at early follow-up (<48 h post-TAVR) and late follow-up (4-6 weeks post-TAVR). RESULTS: 27 patients (mean age 82.0 ± 7.0; 33.3% female) were recruited; 37.0% had diabetes mellitus and 59.3% had hypertension. Brachial artery FMD increased from 4.2 ± 1.6% (pre-TAVR) to 9.7 ± 3.5% at early follow-up (p < 0.0001). At late follow-up, improvement compared with early follow-up was sustained (8.7 ± 1.9%, p = 0.27). Resting brachial arterial flow velocities decreased significantly at late follow-up (11.24 ± 5.16 vs. 7.73 ± 2.79 cm/s, p = 0.003). Concordantly, at late follow-up, there was decrease in resting wall shear stress (WSS; 14.8 ± 7.8 vs. 10.6 ± 4.8dyne/cm2, p = 0.01), peak WSS (73.1 ± 34.1 vs. 58.8 ± 27.8dyne/cm2, p = 0.03) and cumulative WSS (3543 ± 1852 vs. 2504 ± 1089dyne·s/cm2, p = 0.002). Additionally, a favourable inverse correlation between cumulative WSS and FMD was restored at late follow-up (r = -0.21 vs. r = 0.49). CONCLUSION: Endothelial function in patients with AS improves early post-TAVR and this improvement is sustained. This likely occurs as a result of improved arterial haemodynamics, leading to lower localised WSS and release of vasoactive mediators that may also alleviate myocardial ischaemia

Crystallization and preliminary X-ray diffraction studies of FHA domains of Dun1 and Rad53 protein kinases

Forkhead-associated (FHA) domains are modular protein–protein interaction domains of ~130 amino acids present in numerous signalling proteins. FHA-domain-dependent protein interactions are regulated by phosphorylation of target proteins and FHA domains may be multifunctional phosphopeptide-recognition modules. FHA domains of the budding yeast cell-cycle checkpoint protein kinases Dun1p and Rad53p have been crystallized. Crystals of the Dun1-FHA domain exhibit the symmetry of the space group P6122 or P6522, with unit-cell parameters a = b = 127.3, c = 386.3 Å; diffraction data have been collected to 3.1 Å resolution on a synchrotron source. Crystals of the N-terminal FHA domain (FHA1) of Rad53p diffract to 4.0 Å resolution on a laboratory X-ray source and have Laue-group symmetry 4/mmm, with unit-cell parameters a = b = 61.7, c = 104.3 Å

Cell-free protein synthesis as a tool to study RXFP3- Relaxin-3 protein interactions

With the discovery of the relaxin family peptide receptors there is interest in obtaining a clearer understanding of the structure of these proteins and the molecular mechanism of receptor-ligand interaction. As G-protein coupled receptors, obtaining milligram quantities for structural investigations is hampered by the inherent instability of these integral membrane proteins. In the current context, understanding of GPCR structural biology has increased dramatically with crystal structures of several inactive and now active forms solved. In addition, the first nuclear magnetic resonance structure of a GPCR was obtained which is of crucial importance to studying these receptors in a more “biologically relevant” setting. However despite this expansion in the field, most structures have been solved on modified systems so as to increase stability and are not necessarily representative of the native receptors. In relation to the relaxin family peptide receptors, we chose to investigate relaxin-family peptide receptor-3 expressed by cellfree protein synthesis. In contrast to in-vivo expression, cell-free was capable of producing large amounts of native receptor which makes it amenable to demanding structural studies

Patient-specific Computer Simulation: An Emerging Technology for Guiding the Transcatheter Treatment of Patients with Bicuspid Aortic Valve.

Transcatheter aortic valve implantation (TAVI) is increasingly being used to treat younger, lower-risk patients, many of whom have bicuspid aortic valve (BAV). As TAVI begins to enter these younger patient cohorts, it is critical that clinical outcomes from TAVI in BAV are matched to those achieved by surgery. Therefore, the identification of patients who, on an anatomical basis, may not be suitable for TAVI, would be desirable. Furthermore, clinical outcomes of TAVI in BAV might be improved through improved transcatheter heart valve sizing and positioning. One potential solution to these challenges is patient-specific computer simulation. This review presents the methodology and clinical evidence surrounding patient-specific computer simulation of TAVI in BAV

Development of a scaffold displaying exoloops of RXFP1

Relaxin family peptide receptor 1 (RXFP1), the cognate receptor for relaxin, is a G-protein coupled receptor (GPCR) possessing a unique extracellular region consisting of a domain of 10 leucine rich repeats (LRRs) linked to an N-terminal low density lipoprotein Class A module. Relaxin binds to its receptor primarily by a high affinity interaction with the LRRs. An additional low-affinity interaction has been proposed to occur between relaxin and the the exoloops (ELs) of the transmembrane domain, however the molecular detail of this interaction remains undefined. While site directed mutagenesis and subsequent functional characterisation of these mutants traditionally allows identification of residues contributing to receptor function, in this case results are complicated by the presence of the high affinity binding site in the LRRs. To create a tool to investigate the low-affinity interaction, a protein scaffold system displaying exoloops 1 and 2 from RXFP1 was designed. This was achieved by inserting RXFP1 exoloops 1 and 2 into the native loops of a thermostabilised 6 kDa GB1 protein creating EL1/EL2-GB1. This protein has been expressed and purified in milligram quantities and used in conjunction with biophysical techniques such as NMR to explore relaxin binding to the exoloops of RXFP1

Feasibility and Validity of Computed Tomography-Derived Fractional Flow Reserve in Patients With Severe Aortic Stenosis: The CAST-FFR Study

BACKGROUND: Coronary artery disease is common in patients with severe aortic stenosis. Computed tomography-derived fractional flow reserve (CT-FFR) is a clinically used modality for assessing coronary artery disease, however, its use has not been validated in patients with severe aortic stenosis. This study assesses the safety, feasibility, and validity of CT-FFR in patients with severe aortic stenosis. METHODS: Prospectively recruited patients underwent standard-protocol invasive FFR and coronary CT angiography (CTA). CTA images were analyzed by central core laboratory (HeartFlow, Inc) for independent evaluation of CT-FFR. CT-FFR data were compared with FFR (ischemia defined as FFR ≤0.80). RESULTS: Forty-two patients (68 vessels) underwent FFR and CTA; 39 patients (92.3%) and 60 vessels (88.2%) had interpretable CTA enabling CT-FFR computation. Mean age was 76.2±6.7 years (71.8% male). No patients incurred complications relating to premedication, CTA, or FFR protocol. Mean FFR and CT-FFR were 0.83±0.10 and 0.77±0.14, respectively. CT calcium score was 1373.3±1392.9 Agatston units. On per vessel analysis, there was positive correlation between FFR and CT-FFR (Pearson correlation coefficient, R=0.64, P<0.0001). Sensitivity, specificity, positive predictive value, and negative predictive values were 73.9%, 78.4%, 68.0%, and 82.9%, respectively, with 76.7% diagnostic accuracy. The area under the receiver-operating characteristic curve for CT-FFR was 0.83 (0.72-0.93, P<0.0001), which was higher than that of CTA and quantitative coronary angiography (P=0.01 and P<0.001, respectively). Bland-Altman plot showed mean bias between FFR and CT-FFR as 0.059±0.110. On per patient analysis, the sensitivity, specificity, positive predictive, and negative predictive values were 76.5%, 77.3%, 72.2%, and 81.0% with 76.9% diagnostic accuracy. The per patient area under the receiver-operating characteristic curve analysis was 0.81 (0.67-0.95, P<0.0001). CONCLUSIONS: CT-FFR is safe and feasible in patients with severe aortic stenosis. Our data suggests that the diagnostic accuracy of CT-FFR in this cohort potentially enables its use in clinical practice and provides the foundation for future research into the use of CT-FFR for coronary evaluation pre-aortic valve replacement

Thienopyridone Drugs Are Selective Activators of AMP-Activated Protein Kinase β1-Containing Complexes

SummaryThe AMP-activated protein kinase (AMPK) is an αβγ heterotrimer that plays a pivotal role in regulating cellular and whole-body metabolism. Activation of AMPK reverses many of the metabolic defects associated with obesity and type 2 diabetes, and therefore AMPK is considered a promising target for drugs to treat these diseases. Recently, the thienopyridone A769662 has been reported to directly activate AMPK by an unexpected mechanism. Here we show that A769662 activates AMPK by a mechanism involving the β subunit carbohydrate-binding module and residues from the γ subunit but not the AMP-binding sites. Furthermore, A769662 exclusively activates AMPK heterotrimers containing the β1 subunit. Our findings highlight the regulatory role played by the β subunit in modulating AMPK activity and the possibility of developing isoform specific therapeutic activators of this important metabolic regulator

A Phase I/II Study of Chemotherapy Followed by Donor Lymphocyte Infusion plus Interleukin-2 for Relapsed Acute Leukemia after Allogeneic Hematopoietic Cell Transplantation

The efficacy of donor lymphocyte infusion (DLI) for treatment of relapsed acute leukemia after allogeneic hematopoietic cell transplantation is limited. We hypothesized that interleukin-2 (IL-2) combined with DLI after chemotherapy might augment graft-versus-leukemia effects. To identify a safe and effective IL-2 regimen, a phase I/II study of DLI plus IL-2 therapy was performed for such patients. After chemotherapy, 17 patients received DLI (1 × 108 CD3/kg for patients with related donors, and 0.1 × 108 CD3/kg for those with unrelated donors) and an escalating dose of induction IL-2 (1.0, 2.0, or 3.0 × 106 IU/m2/day representing levels I [n = 7], Ia [n = 9], and II [n = 1]) for 5 days followed by maintenance (1.0 × 106 IU/m2/day) for 10 days as a continuous intravenous infusion. Unacceptable IL-2–related toxicities developed in 1 patient at level I, 2 at level Ia, and 1 at level II. Grades III-IV acute graft-versus-host disease (aGVHD) developed in 5 patients, and extensive chronic GVHD (cGVHD) developed in 8. Eight patients had a complete remission after chemotherapy prior to DLI, and 2 additional patients had a complete remission after DLI plus IL-2 therapy. In conclusion, the maximal tolerated induction dose of IL-2 combined with DLI appears to be 1.0 × 106 IU/m2/day. IL-2 administration after DLI might increase the incidence of cGVHD