Dual client binding sites in the ATP-independent chaperone SurA.

The ATP-independent chaperone SurA protects unfolded outer membrane proteins (OMPs) from aggregation in the periplasm of Gram-negative bacteria, and delivers them to the ß-barrel assembly machinery (BAM) for folding into the outer membrane (OM). Precisely how SurA recognises and binds its different OMP clients remains unclear. Escherichia coli SurA comprises three domains: a core and two PPIase domains (P1 and P2). Here, by combining methyl-TROSY NMR, single-molecule Förster resonance energy transfer (smFRET), and bioinformatics analyses we show that SurA client binding is mediated by two binding hotspots in the core and P1 domains. These interactions are driven by aromatic-rich motifs in the client proteins, leading to SurA core/P1 domain rearrangements and expansion of clients from collapsed, non-native states. We demonstrate that the core domain is key to OMP expansion by SurA, and uncover a role for SurA PPIase domains in limiting the extent of expansion. The results reveal insights into SurA-OMP recognition and the mechanism of activation for an ATP-independent chaperone, and suggest a route to targeting the functions of a chaperone key to bacterial virulence and OM integrity.

CEP192 localises mitotic Aurora-A activity by priming its interaction with TPX2.

Aurora-A is an essential cell-cycle kinase with critical roles in mitotic entry and spindle dynamics. These functions require binding partners such as CEP192 and TPX2, which modulate both kinase activity and localisation of Aurora-A. Here we investigate the structure and role of the centrosomal Aurora-A:CEP192 complex in the wider molecular network. We find that CEP192 wraps around Aurora-A, occupies the binding sites for mitotic spindle-associated partners, and thus competes with them. Comparison of two different Aurora-A conformations reveals how CEP192 modifies kinase activity through the site used for TPX2-mediated activation. Deleting the Aurora-A-binding interface in CEP192 prevents centrosomal accumulation of Aurora-A, curtails its activation-loop phosphorylation, and reduces spindle-bound TPX2:Aurora-A complexes, resulting in error-prone mitosis. Thus, by supplying the pool of phosphorylated Aurora-A necessary for TPX2 binding, CEP192:Aurora-A complexes regulate spindle function. We propose an evolutionarily conserved spatial hierarchy, which protects genome integrity through fine-tuning and correctly localising Aurora-A activity.

Profiling sorghum-microbe interactions with a specialized photoaffinity probe identifies key sorgoleone binders in Acinetobacter pittii.

Interactions between plants and soil microbial communities that benefit plant growth and enhance nutrient acquisition are driven by the selective release of metabolites from plant roots, or root exudation. To investigate these plant-microbe interactions, we developed a photoaffinity probe based on sorgoleone (sorgoleone diazirine alkyne for photoaffinity labeling, SoDA-PAL), a hydrophobic secondary metabolite and allelochemical produced in Sorghum bicolor root exudates. We applied SoDA-PAL to the identification of sorgoleone-binding proteins in Acinetobacter pittii SO1, a potential plant growth-promoting microbe isolated from sorghum rhizosphere soil. Competitive photoaffinity labeling of A. pittii whole cell lysates with SoDA-PAL identified 137 statistically enriched proteins, including putative transporters, transcriptional regulators, and a subset of proteins with predicted enzymatic functions. We performed computational protein modeling and docking with sorgoleone to prioritize candidates for experimental validation and then confirmed binding of sorgoleone to four of these proteins in vitro: the α/ß fold hydrolase SrgB (OH685_09420), a fumarylacetoacetase (OH685_02300), a lysophospholipase (OH685_14215), and an unannotated hypothetical protein (OH685_18625). Our application of this specialized sorgoleone-based probe coupled with structural bioinformatics streamlines the identification of microbial proteins involved in metabolite recognition, metabolism, and toxicity, widening our understanding of the range of cellular pathways that can be affected by a plant secondary metabolite.IMPORTANCEHere, we demonstrate that a photoaffinity-based chemical probe modeled after sorgoleone, an important secondary metabolite released by sorghum roots, can be used to identify microbial proteins that directly interact with sorgoleone. We applied this probe to the sorghum-associated bacterium Acinetobacter pittii and showed that probe labeling is dose-dependent and sensitive to competition with purified sorgoleone. Coupling the probe with proteomics and computational analysis facilitated the identification of putative sorgoleone binders, including a protein implicated in a conserved pathway essential for sorgoleone catabolism. We anticipate that discoveries seeded by this workflow will expand our understanding of the molecular mechanisms by which specific metabolites in root exudates shape the sorghum rhizosphere microbiome.

Invasive Trichoderma longibrachiatum infection in a neutropaenic patient.

Invasive fungal infection is a life-threatening complication of chemotherapy and neutropaenia in the haematology population. Trichoderma species rarely cause human disease but have been reported to cause invasive infection in the immunosuppressed. We present a case of invasive Trichoderma longibrachiatum pulmonary infection with fatal outcome in a neutropaenic patient with acute myeloid leukaemia. 2012 Elsevier Ltd. All rights reserved.

Understanding ß-strand mediated protein-protein interactions: tuning binding behaviour of intrinsically disordered sequences by backbone modification.

A significant challenge in chemical biology is to understand and modulate protein-protein interactions (PPIs). Given that many PPIs involve a folded protein domain and a peptide sequence that is intrinsically disordered in isolation, peptides represent powerful tools to understand PPIs. Using the interaction between small ubiquitin-like modifier (SUMO) and SUMO-interacting motifs (SIMs), here we show that N-methylation of the peptide backbone can effectively restrict accessible peptide conformations, predisposing them for protein recognition. Backbone N-methylation in appropriate locations results in faster target binding, and thus higher affinity, as shown by relaxation-based NMR experiments and computational analysis. We show that such higher affinities occur as a consequence of an increase in the energy of the unbound state, and a reduction in the entropic contribution to the binding and activation energies. Thus, backbone N-methylation may represent a useful modification within the peptidomimetic toolbox to probe ß-strand mediated interactions.

Clinical utility of investigations in triple-negative thrombocytosis: A real-world, multicentre evaluation of UK practice.

Diagnosis of essential thrombocythaemia (ET) is challenging in patients lacking JAK2/CALR/MPL mutations. In a retrospective evaluation of 320 patients with 'triple-negative thrombocytosis', we assessed utility of bone marrow histology (90.9% of patients) and myeloid gene panel (MGP, 55.6%). Supportive histology ('myeloproliferative neoplasm-definite/probable', 36.8%) was associated with higher platelet counts and varied between centres. 14.6% MGP revealed significant variants: 3.4% JAK2/CALR/MPL and 11.2% other myeloid genes. Final clinical diagnosis was strongly predicted by histology, not MGP. 23.7% received cytoreduction (17.6% under 60 years). Real-world 'triple-negative' ET diagnosis currently depends heavily on histology; we advocate caution in MGP-negative cases and that specific guidelines are needed.

Suppressing Competing Solvent Reduction in CO2 Electroreduction with a Magnetic Field.

The presence of an external magnetic field is found to affect the competition between the H2O and CO2 reduction reactions by increasing mass transport via the Lorentz force. Increasing the magnetic field strength at the electrode surface from 0 to 325 mT increases the selectivity of CO over H2 by 3×, while an increase in current density from 0.5 to 5 mA/cm2 increases the selectivity of CO production by 5×. Cyclic voltammetry and finite-element simulations reveal that the origin of the enhanced CO selectivity is attributable to a magnetic field lowering the electrode-electrolyte interfacial pH. A drop in interfacial pH enables increased production of CO from CO2 reduction due to a decrease in the activity of H2O reduction and increase in CO2 solubility near the electrode surface. The insight provided in this study offers new opportunities to control reaction selectivity in electrocatalysis with magnetic field vectors.

Peptide macrocyclisation via intramolecular interception of visible-light-mediated desulfurisation.

Synthetic methods that enable the macrocyclisation of peptides facilitate the development of effective therapeutic and diagnostic tools. Herein we report a peptide cyclisation strategy based on intramolecular interception of visible-light-mediated cysteine desulfurisation. This method allows cyclisation of unprotected peptides in an aqueous solution via the installation of a hydrocarbon linkage. We explore the limits of this chemistry using a range of model peptides of increasing length and complexity, including peptides of biological/therapeutic relevance. The method is applied to replace the native disulfide of the peptide hormone, oxytocin, with a proteolytically/redox-stable hydrocarbon, and internal macrocyclisation of an MCL-1-binding peptide.

Biophysical and structural analyses of the interaction between the SHANK1 PDZ domain and an internal SLiM.

The PDZ (Postsynaptic density protein-95[PSD-95]/Discs-large) domain, prevalent as a recognition module, has attracted significant attention given its ability to specifically recognize ligands with consensus motifs (also termed PDZ binding motifs [PBMs]). PBMs typically bear a C-terminal carboxylate as a recognition handle and have been extensively characterized, whilst internal ligands are less well known. Here we characterize a short linear motif (SLiM) - EESTSFQGP - as an internal PBM based on its strong binding affinity towards the SHANK1 PDZ domain (SHANK1656-762 hereafter referred to as SHANK1). Using the acetylated analogue Ac-EESTSFQGP-CONH2 as a competitor for the interaction of SHANK1 with FAM-Ahx-EESTSFQGP-CONH2 or a typical fluorophore-labelled C-terminal PBM - GKAP - FITC-Ahx-EAQTRL-COOH - the internal SLiM was demonstrated to show comparable low-micromolar IC50 by competition fluorescent anisotropy. To gain further insight into the internal ligand interaction at the molecular level, we obtained the X-ray co-crystal structure of the Ac-EESTSFQGP-CONH2/SHANK1 complex and compared this to the Ac-EAQTRL-COOH/SHANK1 complex. The crystallographic studies reveal that the SHANK1 backbones for the two interactions overlap significantly. The main structural differences were shown to result from the flexible loops which reorganize to accommodate the two PBMs with distinct lengths and terminal groups. In addition, the two C-terminal residues Gly and Pro in Ac-EESTSFQGP-CONH2 were shown not to participate in interaction with the target protein, implying further truncation and structural modification using peptidomimetic approaches on this sequence may be feasible. Taken together, the SLiM Ac-EESTSFQGP-CONH2 holds potential as an internal ligand for targeting SHANK1.

Quantification and description of photothermal heating effects in plasmon-assisted electrochemistry.

A growing number of reports have demonstrated plasmon-assisted electrochemical reactions, though debate exists around the mechanisms underlying the enhanced activity. Here we address the impact of plasmonic photothermal heating with cyclic voltammetry measurements and finite-element simulations. We find that plasmonic photothermal heating causes a reduction in the hysteresis of the anodic and cathodic waves of the voltammograms along with an increase in mass-transport limiting current density due to convection induced by a temperature gradient. At slow scan rates, a temperature difference as low as 1 K between the electrode surface and bulk electrolytic solution enhances the current density greater than 100%. Direct interband excitation of Au exclusively enhances current density by photothermal heating, while plasmon excitation leads to photothermal and nonthermal enhancements. Our study reveals the role of temperature gradients in plasmon-assisted electrochemistry and details a simple control experiment to account for photothermal heating.

Visible-Light Driven Control Over Triply and Quadruply Hydrogen-Bonded Supramolecular Assemblies.

Supramolecular polymers offer tremendous potential to produce new "smart" materials, however, there remains a need to develop systems that are responsive to external stimuli. In this work, visible-light responsive hydrogen-bonded supramolecular polymers comprising photoresponsive supramolecular synthons (I-III) consisting of two hydrogen bonding motifs (HBMs) connected by a central ortho-tetrafluorinated azobenzene have been characterized by DOSY NMR and viscometry. Comparison of different hydrogen-bonding motifs reveals that assembly in the low and high concentration regimes is strongly influenced by the strength of association between the HBMs. I, Incorporating a triply hydrogen-bonded heterodimer, was found to exhibit concentration dependent switching between a monomeric pseudo-cycle and supramolecular oligomer through intermolecular hydrogen bonding interactions between the HBMs. II, Based on the same photoresponsive scaffold, and incorporating a quadruply hydrogen-bonded homodimer was found to form a supramolecular polymer which was dependent upon the ring-chain equilibrium and thus dependent upon both concentration and photochemical stimulus. Finally, III, incorporating a quadruply hydrogen-bonded heterodimer represents the first photoswitchable AB type hydrogen-bonded supramolecular polymer. Depending on the concentration and photostationary state, four different assemblies dominate for both monomers II and III, demonstrating the ability to control supramolecular assembly and physical properties triggered by light.

Tumor therapy by targeting extracellular hydroxyapatite using novel drugs: A paradigm shift.

BACKGROUND: It has been shown that tumor microenvironment (TME) hydroxyapatite (HAP) is typically associated with many malignancies and plays a role in tumor progression and growth. Additionally, acidosis in the TME has been reported to play a key role in selecting for a more aggressive tumor phenotype, drug resistance and desensitization to immunotherapy for many types of cancers. TME-HAP is an attractive target for tumor detection and treatment development since HAP is generally absent from normal soft tissue. We provide strong evidence that dissolution of hydroxyapatite (HAP) within the tumor microenvironment (TME-HAP) using a novel therapeutic can be used to kill cancer cells both in vitro and in vivo with minimal adverse effects. METHODS: We developed an injectable cation exchange nano particulate sulfonated polystyrene solution (NSPS) that we engineered to dissolve TME-HAP, inducing localized acute alkalosis and inhibition of tumor growth and glucose metabolism. This was evaluated in cell culture using 4T1, MDA-MB-231 triple negative breast cancer cells, MCF10 normal breast cells, and H292 lung cancer cells, and in vivo using orthotopic mouse models of cancer that contained detectable microenvironment HAP including breast (MMTV-Neu, 4T1, and MDA-MB-231), prostate (PC3) and colon (HCA7) cancer using 18 F-NaF for HAP and 18 F-FDG for glucose metabolism with PET imaging. On the other hand, H292 lung tumor cells that lacked detectable microenvironment HAP and MCF10a normal breast cells that do not produce HAP served as negative controls. Tumor microenvironment pH levels following injection of NSPS were evaluated via Chemical Exchange Saturation (CEST) MRI and via ex vivo methods. RESULTS: Within 24 h of adding the small concentration of 1X of NSPS (~7 µM), we observed significant tumor cell death (~ 10%, p < 0.05) in 4T1 and MDA-MB-231 cell cultures that contain HAP but ⟨2% in H292 and MCF10a cells that lack detectable HAP and in controls. Using CEST MRI, we found extracellular pH (pHe) in the 4T1 breast tumors, located in the mammary fat pad, to increase by nearly 10% from baseline before gradually receding back to baseline during the first hour post NSPS administration. in the tumors that contained TME-HAP in mouse models, MMTV-Neu, 4T1, and MDA-MB-231, PC3, and HCA7, there was a significant reduction (p<0.05) in 18 F-Na Fuptake post NSPS treatment as expected; 18 F- uptake in the tumor = 3.8 ± 0.5 %ID/g (percent of the injected dose per gram) at baseline compared to 1.8 ±0.5 %ID/g following one-time treatment with 100 mg/kg NSPS. Of similar importance, is that 18 F-FDG uptake in the tumors was reduced by more than 75% compared to baseline within 24 h of treatment with one-time NSPS which persisted for at least one week. Additionally, tumor growth was significantly slower (p < 0.05) in the mice treated with one-time NSPS. Toxicity showed no evidence of any adverse effects, a finding attributed to the absence of HAP in normal soft tissue and to our therapeutic NSPS having limited penetration to access HAP within skeletal bone. CONCLUSION: Dissolution of TME-HAP using our novel NSPS has the potential to provide a new treatment paradigm to enhance the management of cancer patients with poor prognosis.

Cardiovascular risk in a contemporary cohort of patients with myeloproliferative neoplasms'.

BACKGROUND: Myeloproliferative neoplasms (MPNs) are a group of disorders of clonal haemopoiesis associated with an inherent risk of arterial and venous thrombotic complications. The prevalence of thrombotic complications and the impact of cardiovascular risk factors (CVRFs) in contemporary patient cohorts within the current era of MPN treatments have not been completely defined. OBJECTIVES: We aim to characterise the cardiovascular risk of patients with MPN by identifying the prevalence of CVRFs and describing the pattern of thrombotic events. We also aim to utilise the QRISK3 algorithm, which is a validated model used to estimate an individual's risk of developing cardiovascular disease, to further phenotype this cohort of patients. METHODS: We perform a retrospective analysis on a single-centre cohort of 438 patients with MPN. RESULTS: MPN patients continue to carry a high burden of vascular morbidity with a prevalence of arterial thrombotic events in 15.8 % (69/438) and venous thrombotic events in 13.2 % (58/438) of the cohort. The novel use of the QRISK3 algorithm, which showed a mean score of 13.7 % across the MPN population, provides further evidence to suggest an increased cardiovascular risk in MPN patients. CONCLUSION: With an increased risk of cardiovascular disease in patients with MPN, we propose an integrated approach between primary and specialised healthcare services using risk stratification tools such as QRISK3, which will allow aggressive optimisation of CVRFs to prevent thrombosis and reduce the overall morbidity and mortality in patients with MPN.

Inhibition of Aurora-A/N-Myc Protein-Protein Interaction Using Peptidomimetics: Understanding the Role of Peptide Cyclization.

Using N-Myc61-89 as a starting template we showcase the systematic use of truncation and maleimide constraining to develop peptidomimetic inhibitors of the N-Myc/Aurora-A protein-protein interaction (PPI); a potential anticancer drug discovery target. The most promising of these - N-Myc73-94-N85C/G89C-mal - is shown to favour a more Aurora-A compliant binding ensemble in comparison to the linear wild-type sequence as observed through fluorescence anisotropy competition assays, circular dichroism (CD) and nuclear magnetic resonance (NMR) experiments. Further in silico investigation of this peptide in its Aurora-A bound state, by molecular dynamics (MD) simulations, imply (i) the bound conformation is more stable as a consequence of the constraint, which likely suppresses dissociation and (ii) the constraint may make further stabilizing interactions with the Aurora-A surface. Taken together this work unveils the first orthosteric N-Myc/Aurora-A inhibitor and provides useful insights on the biophysical properties and thus design of constrained peptides, an attractive therapeutic modality.

Directing the size and dispersity of silver nanoparticles with kudzu leaf extracts.

Kudzu is an abundant and invasive species in the Southeastern United States. The prospective use of kudzu as a non-toxic, green and biocompatible reducing and stabilizing agent for one-pot Ag nanoparticle synthesis was investigated. Ag nanoparticles were synthesized using aqueous and ethanolic kudzu leaf and stem extracts. The size and dispersity of the synthesized nanoparticles were found to depend on the extract used. Ultraviolet-visible and Fourier transform infrared spectroscopies were used to characterize the extracts. Surface-enhanced fluorescence and Raman scattering were used to characterize the surface species on synthesized Ag nanoparticles. The primary reducing and stabilizing agents in aqueous kudzu leaf extracts were determined to be reducing sugars and saponins which result in Ag nanoparticles with average diameters of 21.2 ± 4.8 nm. Ethanolic kudzu leaf extract was determined to be composed of chlorophyll, reducing sugars and saponins, producing Ag nanoparticles with average diameters of 9.0 ± 1.6 nm. Control experiments using a chlorophyllin standard as the reducing and stabilizing agent reveal that chlorophyll has a key role in the formation of small and monodisperse Ag nanoparticles. Experiments carried out in the absence of light demonstrate that reducing sugars and saponins also contribute to the formation of Ag nanoparticles in ethanolic kudzu leaf extracts. We propose a mechanism by which reducing sugars donate electrons to reduce Ag+ leading to the formation of Ag nanoparticles, forming carboxylic acid sugars which stabilize and partially stabilize Ag nanoparticles synthesized with aqueous and ethanolic kudzu leaf extracts, respectively. In the ethanolic extract, photoexcited chlorophyll serves as a co-reducing and co-stabilizing agent, leading to small and monodisperse Ag nanoparticles.

Potential-Dependent Temporal Dynamics of CO Surface Concentration in Electrocatalytic CO2 Reduction.

Beyond the identity and structure of an intermediate, changes in its concentration on and near the electrode surface with time are a critical component to understand and improve selectivity and reactivity in electrochemical transformations. We apply pulsed-potential electrochemical Raman scattering microscopy to measure the potential-dependent temporal evolution of CO formed during electrocatalytic CO2 reduction in acetonitrile on Ag electrodes. At driving potentials positive of the onset potential as determined by cyclic voltammetry, CO accumulates on the electrode surface at time scales longer than 1 s. Near the ensemble onset potential, CO resides on the electrode surface for approximately 100 ms. At potentials known to evolve CO from the electrode surface, CO remains adsorbed on the electrode for less than 10 ms. The time scales accessible in our strategy are nearly 3 orders of magnitude faster than transient Raman or infrared measurements, allowing direct measurement of the temporal evolution of intermediates.