N- and C-Terminal Truncations to Enhance Protein Solubility and Crystallization: Predicting Protein Domain Boundaries with Bioinformatics Tools

Soluble protein expression is a key requirement for biochemical and structural biology approaches to study biological systems in vitro. Production of sufficient quantities may not always be achievable if proteins are poorly soluble which is frequently determined by physico-chemical parameters such as intrinsic disorder. It is well known that discrete protein domains often have a greater likelihood of high-level soluble expression and crystallizability. Determination of such protein domain boundaries can be challenging for novel proteins. Here, we outline the application of bioinformatics tools to facilitate the prediction of potential protein domain boundaries, which can then be used in designing expression construct boundaries for parallelized screening in a range of heterologous expression systems

Characterization of designed, synthetically accessible bryostatin analog HIV latency reversing agents.

HIV latency in resting CD4+ T cell represents a key barrier preventing cure of the infection with antiretroviral drugs alone. Latency reversing agents (LRAs) can activate HIV expression in latently infected cells, potentially leading to their elimination through virus-mediated cytopathic effects, host immune responses, and/or therapeutic strategies targeting cells actively expressing virus. We have recently described several structurally simplified analogs of the PKC modulator LRA bryostatin (termed bryologs) designed to improve synthetic accessibility, tolerability in vivo, and efficacy in inducing HIV latency reversal. Here we report the comparative performance of lead bryologs, including their effects in reducing cell surface expression of HIV entry receptors, inducing proinflammatory cytokines, inhibiting short-term HIV replication, and synergizing with histone deacetylase inhibitors to reverse HIV latency. These data provide unique insights into structure-function relationships between A- and B-ring bryolog modifications and activities in primary cells, and suggest that bryologs represent promising leads for preclinical advancement

Fragment Hotspot Mapping to Identify Selectivity-Determining Regions between Related Proteins.

Funder: ExscientiaFunder: Diamond Light SourceFunder: Kungliga Tekniska HoegskolanFunder: Chinese Center for Disease Control and PreventionFunder: European Federation of Pharmaceutical Industries and AssociationsFunder: European CommissionFunder: Kennedy Trust for Rheumatology ResearchFunder: Ontario Institute for Cancer ResearchFunder: Royal Institution for the Advancement of Learning McGill UniversityFunder: UCBSelectivity is a crucial property in small molecule development. Binding site comparisons within a protein family are a key piece of information when aiming to modulate the selectivity profile of a compound. Binding site differences can be exploited to confer selectivity for a specific target, while shared areas can provide insights into polypharmacology. As the quantity of structural data grows, automated methods are needed to process, summarize, and present these data to users. We present a computational method that provides quantitative and data-driven summaries of the available binding site information from an ensemble of structures of the same protein. The resulting ensemble maps identify the key interactions important for ligand binding in the ensemble. The comparison of ensemble maps of related proteins enables the identification of selectivity-determining regions within a protein family. We applied the method to three examples from the well-researched human bromodomain and kinase families, demonstrating that the method is able to identify selectivity-determining regions that have been used to introduce selectivity in past drug discovery campaigns. We then illustrate how the resulting maps can be used to automate comparisons across a target protein family

BLAST: the Redshift Survey

The Balloon-borne Large Aperture Submillimeter Telescope (BLAST) has recently surveyed ~8.7 deg^2 centered on GOODS-South at 250, 350, and 500 microns. In Dye et al. (2009) we presented the catalogue of sources detected at 5-sigma in at least one band in this field and the probable counterparts to these sources in other wavebands. In this paper, we present the results of a redshift survey in which we succeeded in measuring redshifts for 82 of these counterparts. The spectra show that the BLAST counterparts are mostly star-forming galaxies but not extreme ones when compared to those found in the Sloan Digital Sky Survey. Roughly one quarter of the BLAST counterparts contain an active nucleus. We have used the spectroscopic redshifts to carry out a test of the ability of photometric redshift methods to estimate the redshifts of dusty galaxies, showing that the standard methods work well even when a galaxy contains a large amount of dust. We have also investigated the cases where there are two possible counterparts to the BLAST source, finding that in at least half of these there is evidence that the two galaxies are physically associated, either because they are interacting or because they are in the same large-scale structure. Finally, we have made the first direct measurements of the luminosity function in the three BLAST bands. We find strong evolution out to z=1, in the sense that there is a large increase in the space-density of the most luminous galaxies. We have also investigated the evolution of the dust-mass function, finding similar strong evolution in the space-density of the galaxies with the largest dust masses, showing that the luminosity evolution seen in many wavebands is associated with an increase in the reservoir of interstellar matter in galaxies.Comment: Accepted for publication in the Astrophysical Journal. Maps and associated results are available at http://blastexperiment.info

Gas phase characterization of the noncovalent quaternary structure of Cholera toxin and the Cholera toxin B subunit pentamer

Cholera toxin (CTx) is an AB5 cytotonic protein that has medical relevance in cholera and as a novel mucosal adjuvant. Here, we report an analysis of the noncovalent homopentameric complex of CTx B chain (CTx B5) using electrospray ionization triple quadrupole mass spectrometry and tandem mass spectrometry and the analysis of the noncovalent hexameric holotoxin usingelectrospray ionization time-of-flight mass spectrometry over a range of pH values that correlate with those encountered by this toxin after cellular uptake. We show that noncovalent interactions within the toxin assemblies were maintained under both acidic and neutral conditions in the gas phase. However, unlike the related Escherichia coli Shiga-like toxin B5 pentamer (SLTx B), the CTx B5 pentamer was stable at low pH, indicating that additional interactions must be present within the latter. Structural comparison of the CTx B monomer interface reveals an additional α-helix that is absent in the SLTx B monomer. In silico energy calculations support interactions between this helix and the adjacent monomer. These data provide insight into the apparent stabilization of CTx B relative to SLTx B

Solar Wind at 6.8 Solar Radii from UVCS Observation of Comet C/1996Y1

The comet C/1996Y1, a member of the Kreutz family of Sun-grazing comets, was observed with the Ultraviolet Coronagraph Spectrometer (UVCS) aboard the Solar and Heliospheric Observatory (SOHO) satellite. The Lyα line profile and spatial distribution are interpreted in terms of the theory of bow shocks driven by mass-loading. At the time of the observation, the comet was 6.8 R☉ from the Sun in a region of high-speed wind, a region difficult to observe directly with the SOHO instruments but an important region for testing models of solar wind acceleration and heating. We find a solar wind speed below 640 km s-1 and a constraint on the combination of solar wind speed and proton temperature. The total energy per proton at 6.8 R☉ is 50%-75% of the energy at 1 AU, indicating that significant heating occurs at larger radii. The centroid and width of the Lyα line generally confirm the predictions of models of the cometary bow shock driven by mass-loading as cometary molecules are ionized and swept up in the solar wind. We estimate an outgassing rate of 20 kg s-1, which implies an active area of the nucleus only about 6.7 m in diameter at 6.8 R☉. This is likely to be the size of the nucleus, because any inert mantle would have probably been blown off during the approach to the Sun

A poised fragment library enables rapid synthetic expansion yielding the first reported inhibitors of PHIP(2), an atypical bromodomain

Research into the chemical biology of bromodomains has been driven by the development of acetyl-lysine mimetics. The ligands are typically anchored by binding to a highly conserved asparagine residue. Atypical bromodomains, for which the asparagine is mutated, have thus far proven elusive targets, including PHIP(2) whose parent protein, PHIP, has been linked to disease progression in diabetes and cancers. The PHIP(2) binding site contains a threonine in place of asparagine, and solution screening have yielded no convincing hits. We have overcome this hurdle by combining the sensitivity of X-ray crystallography, used as the primary fragment screen, with a strategy for rapid follow-up synthesis using a chemically-poised fragment library, which allows hits to be readily modified by parallel chemistry both peripherally and in the core. Our approach yielded the first reported hit compounds of PHIP(2) with measurable IC50 values by an AlphaScreen competition assay. The follow-up libraries of four poised fragment hits improved potency into the sub-mM range while showing good ligand efficiency and detailed structural data

The scientific impact of the Structural Genomics Consortium: a protein family and ligand-centered approach to medically-relevant human proteins

As many of the structural genomics centers have ended their first phase of operation, it is a good point to evaluate the scientific impact of this endeavour. The Structural Genomics Consortium (SGC), operating from three centers across the Atlantic, investigates human proteins involved in disease processes and proteins from Plasmodium falciparum and related organisms. We present here some of the scientific output of the Oxford node of the SGC, where the target areas include protein kinases, phosphatases, oxidoreductases and other metabolic enzymes, as well as signal transduction proteins. The SGC has aimed to achieve extensive coverage of human gene families with a focus on protein–ligand interactions. The methods employed for effective protein expression, crystallization and structure determination by X-ray crystallography are summarized. In addition to the cumulative impact of accelerated delivery of protein structures, we demonstrate how family coverage, generic screening methodology, and the availability of abundant purified protein samples, allow a level of discovery that is difficult to achieve otherwise. The contribution of NMR to structure determination and protein characterization is discussed. To make this information available to a wide scientific audience, a new tool for disseminating annotated structural information was created that also represents an interactive platform allowing for a continuous update of the annotation by the scientific community

Integrative GWAS and co-localisation analysis suggests novel genes associated with age-related multimorbidity

Abstract Advancing age is the greatest risk factor for developing multiple age-related diseases. Therapeutic approaches targeting the underlying pathways of ageing, rather than individual diseases, may be an effective way to treat and prevent age-related morbidity while reducing the burden of polypharmacy. We harness the Open Targets Genetics Portal to perform a systematic analysis of nearly 1,400 genome-wide association studies (GWAS) mapped to 34 age-related diseases and traits, identifying genetic signals that are shared between two or more of these traits. Using locus-to-gene (L2G) mapping, we identify 995 targets with shared genetic links to age-related diseases and traits, which are enriched in mechanisms of ageing and include known ageing and longevity-related genes. Of these 995 genes, 128 are the target of an approved or investigational drug, 526 have experimental evidence of binding pockets or are predicted to be tractable, and 341 have no existing tractability evidence, representing underexplored genes which may reveal novel biological insights and therapeutic opportunities. We present these candidate targets for exploration and prioritisation in a web application

Advanced quantitative evaluation of PET systems using the ACR phantom and NiftyPET software

Purpose: A novel phantom-imaging platform, a set of software tools, for automated and high-precision imaging of the American College of Radiology (ACR) positron emission tomography (PET) phantom for PET/magnetic resonance (PET/MR) and PET/computed tomography (PET/CT) systems is proposed. Methods: The key feature of this platform is the vector graphics design that facilitates the automated measurement of the knife-edge response function and hence image resolution, using composite volume of interest templates in a 0.5 mm resolution grid applied to all inserts of the phantom. Furthermore, the proposed platform enables the generation of an accurate μ -map for PET/MR systems with a robust alignment based on two-stage image registration using specifically designed PET templates. The proposed platform is based on the open-source NiftyPET software package used to generate multiple list-mode data bootstrap realizations and image reconstructions to determine the precision of the two-stage registration and any image-derived statistics. For all the analyses, iterative image reconstruction was employed with and without modeled shift-invariant point spread function and with varying iterations of the ordered subsets expectation maximization (OSEM) algorithm. The impact of the activity outside the field of view (FOV) was assessed using two acquisitions of 30 min each, with and without the activity outside the FOV. Results: The utility of the platform has been demonstrated by providing a standard and an advanced phantom analysis including the estimation of spatial resolution using all cylindrical inserts. In the imaging planes close to the edge of the axial FOV, we observed deterioration in the quantitative accuracy, reduced resolution (FWHM increased by 1–2 mm), reduced contrast, and background uniformity due to the activity outside the FOV. Although it slows convergence, the PSF reconstruction had a positive impact on resolution and contrast recovery, but the degree of improvement depended on the regions. The uncertainty analysis based on bootstrap resampling of raw PET data indicated high precision of the two-stage registration. Conclusions: We demonstrated that phantom imaging using the proposed methodology with the metric of spatial resolution and multiple bootstrap realizations may be helpful in more accurate evaluation of PET systems as well as in facilitating fine tuning for optimal imaging parameters in PET/MR and PET/CT clinical research studies