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bioRxiv2017

Understanding inhibitor resistance in Mps1 kinase through novel biophysical assays and structures

Monopolar spindle 1 (Mps1/TTK) is a protein kinase essential in mitotic checkpoint signalling, preventing anaphase until all chromosomes are properly attached to spindle microtubules. Mps1 has emerged as a potential target for cancer therapy, and a variety of compounds have been developed to inhibit its kinase activity. Mutations in the catalytic domain of Mps1 that give rise to inhibitor resistance, but retain catalytic activity and do not display cross-resistance to other Mps1 inhibitors, have been described. Here we characterize the interactions of two such mutants, Mps1 C604Y and C604W, which raise resistance to two closely related compounds, NMS-P715 and its derivative Cpd-5, but not to the well-characterised Mps1 inhibitor, reversine. We show that estimates of the IC50 (employing a novel specific and efficient assay that utilizes a fluorescently labelled substrate) and of the binding affinity (KD) indicate that in both mutants, Cpd-5 should be better tolerated than the closely related NMS-P715. To gain further insight, we determined the crystal structure of the Mps1 kinase mutants bound to Cpd-5 and NMS-P715, and compare the binding modes of Cpd-5, NMS-P715 and reversine. The difference in steric hindrance between Tyr/Trp604 and the trifluoromethoxy moiety of NMS-P715, the methoxy moiety of Cpd-5, and complete absence of such a group in reversine, account for differences we observe in vitro. Our analysis enforces the notion that inhibitors targeting Mps1 drug-resistant mutations can emerge as a feasible intervention strategy based on existing scaffolds, if the clinical need arises.\n\nSummary statementThe inhibition of specific Mps1 kinase inhibitors towards the wild-type protein and inhibitor-resistant mutants is explained by a novel specific activity assay, biophysical characterisation, and X-ray structures.\n\nAbbreviations

biochemistry↗

bioRxiv2017

The stoichiometric signature of Rhodopsin-family G protein coupled receptors

Whether Rhodopsin-family G protein-coupled receptors (GPCRs) form dimers is highly controversial, with much data both for and against emerging from studies of mostly individual receptors. The types of large-scale comparative studies from which a consensus could eventually emerge have not previously been attempted. Here, we sought to determine the stoichiometric \"signatures\" of 60 GPCRs expressed by a single human cell-line using orthogonal bioluminescence resonance energy transfer-based and single-molecule microscopy assays. We observed that a relatively small fraction of Rhodopsin-family GPCRs behaved as dimers and that these receptors otherwise appeared to be monomeric. Mapped onto the entire family the analysis predicted that fewer than 20% of the ~700 Rhodopsin- family receptors form dimers. The clustered distribution of Rhodopsin-family dimers, and a striking correlation between receptor stoichiometry and GPCR family-size that we also identified, suggested that evolution has tended to favor the lineage expansion of monomers rather than dimers.\n\nOne Sentence SummaryAnalysis of 71 GPCRs from a single cell reveals the strong tendency of Rhodopsin-family receptors to exist as monomers rather than form dimers.

biochemistry↗

bioRxiv2017

Structural characterization of human Vaccinia-Related Kinases (VRK) bound to small-molecule inhibitors identifies different P-loop conformations

The human genome encodes two active Vaccinia-related protein kinases (VRK), VRK1 and VRK2. These proteins have been implicated in a number of cellular processes and linked to a variety of tumors. However, understanding the cellular role of VRKs and establishing their potential use as targets for therapeutic intervention has been limited by the lack of tool compounds that can specifically modulate the activity of these kinases in cells. Here we identified BI-D1870, a dihydropteridine inhibitor of RSK kinases, as a promising starting point for the development of chemical probes targeting the active VRKs. We solved co-crystal structures of both VRK1 and VRK2 bound to BI-D1870 and of VRK1 bound to two broad-spectrum inhibitors. These structures revealed that both VRKs can adopt a P-loop folded conformation, which is stabilized by different mechanisms on each protein. Based on these structures, we suggest modifications to the dihydropteridine scaffold that can be explored to produce potent and specific inhibitors towards VRK1 and VRK2.

biochemistry↗

bioRxiv2017

Recombinant expression in E. coli of human FGFR2 with its transmembrane and extracellular domains

Fibroblast growth factor receptors (FGFRs) are a family of receptor tyrosine kinases containing three domains: an extracellular receptor domain, a single transmembrane helix, and an intracellular tyrosine kinase domain. FGFRs are activated by fibroblast growth factors (FGFs) as part of complex signal transduction cascades regulating angiogenesis, skeletal formation, cell differentiation, proliferation, cell survival, and cancer. We have developed the first recombinant expression system in E. coli to produce a construct of human FGFR2 containing its transmembrane and extracellular receptor domains. We demonstrate that the expressed construct is functional in binding heparin and dimerizing. Size exclusion chromatography demonstrates that the purified FGFR2 does not form a complex with FGF1 or adopts an inactive dimer conformation. Progress towards the successful recombinant production of intact FGFRs will facilitate further biochemical experiments and structure determination that will provide insight into how extracellular FGF binding activates intracellular kinase activity.

biochemistry↗

bioRxiv2017

Early biomarker discovery in urine of Walker 256 subcutaneous rat model

Despite advances in cancer treatments, early detection of cancer is still the most promising way to improve outcomes. Without homeostatic control, urine reflects early changes in the body and can potentially be used for early cancer diagnosis. In this study, a Walker 256 tumor rat model was established by subcutaneous injection of Walker 256 tumor cells. To identify urinary proteome changes during cancer development, urine samples from Walker 256 tumor-bearing rats were collected at five time points corresponding to before cancer cell implantation, before tumor mass palpability, at tumor mass appearance, during rapid tumor growth, and at cachexia. The urinary protein patterns changed significantly as the tumors progressed, as measured using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The urinary proteome of tumor-bearing rats was identified using Fusion Lumos mass spectrometry with label-free quantification. Then, 30 dynamically changed urinary proteins during cancer progression were selected as more reliable cancer biomarkers, and they were validated by targeted proteomics. Combined with the results of label-free and targeted proteome quantification, a total of 10 urinary proteins (HPT, APOA4, CO4, B2MG, A1AG, CATC, VCAM1, CALB1, CSPG4, and VTDB) changed significantly even before a tumor mass was palpable, and these early changes in urine could also be identified with differential abundance at late stages of cancer. Our study indicated that urine is a sensitive biomarker source for early detection of cancer.

biochemistry↗

bioRxiv2017

Prophage genomics reveals patterns in phage genome organization and replication

Temperate phage genomes are highly variable mosaic collections of genes that infect a bacterial host, integrate into the hosts genome or replicate as low copy number plasmids, and are regulated to switch from the lysogenic to lytic cycles to generate new virions and escape their host. Genomes from most Bacterial phyla contain at least one or more prophages. We updated our PhiSpy algorithm to improve detection of prophages and to provide a web-based framework for PhiSpy. We have used this algorithm to identify 36,488 prophage regions from 11,941 bacterial genomes, including almost 600 prophages with no known homology to any proteins. Transfer RNA genes were abundant in the prophages, many of which alleviate the limits of translation efficiency due to host codon bias and presumably enable phages to surpass the normal capacity of the hosts translation machinery. We identified integrase genes in 15,765 prophages (43% of the prophages). The integrase was routinely located at either end of the integrated phage genome, and was used to orient and align prophage genomes to reveal their underlying organization. The conserved genome alignments of phages recapitulate early, middle, and late gene order in transcriptional control of phage genes, and demonstrate that gene order, presumably selected by transcription timing and/or coordination among functional modules has been stably conserved throughout phage evolution.

biochemistry↗

bioRxiv2017

Structural basis for EarP-mediated arginine glycosylation of translation elongation factor EF-P

Glycosylation is a universal strategy to post-translationally modify proteins. The recently discovered arginine rhamnosylation activates the polyproline specific bacterial translation elongation factor EF-P. EF-P is rhamnosylated on arginine 32 by the glycosyltransferase EarP. However, the enzymatic mechanism remains elusive. In the present study, we solved the crystal structure of EarP from Pseudomonas putida. The enzyme is composed of two opposing domains with Rossmann-folds, thus constituting a GT-B glycosyltransferase. While TDP-rhamnose is located within a highly conserved pocket of the C-domain, EarP recognizes the EF-P via its KOW-like N-domain. Based on our structural data combined with an in vitro /in vivo enzyme characterization, we propose a mechanism of inverting arginine glycosylation. As EarP is essential for pathogenicity in P. aeruginosa our study provides the basis for targeted inhibitor design.

biochemistry↗

bioRxiv2017

Long-term dysbiosis promotes insulin resistance during obesity despite rapid diet-induced changes in the gut microbiome of mice

The intestinal microbiota and insulin sensitivity are rapidly altered in response to a high fat diet (HFD). It is unclear if gut dysbiosis precedes insulin resistance or vice versa. The initial triggers of diet-induced insulin resistance can differ from mechanisms underlying chronic dysglycemia during prolonged obesity. It is not clear if intestinal dysbiosis contributes to insulin resistance during short-term or long-term HFD-feeding. We found that diet-induced changes in the composition of the fecal microbiome preceded changes in glucose and insulin tolerance at both the onset and removal of a HFD in mice. Dysbiosis occurred after 1-3 days of HFD-feeding, whereas insulin and glucose intolerance manifested by 3-4 days. Antibiotic treatment did not alter glucose tolerance during this short-term HFD period. Conversely, antibiotics improved glucose tolerance in mice with protracted obesity caused by long-term HFD feeding for over 2 months. We also found that microbiota transmissible glucose intolerance only occurred after prolonged diet-induced dysbiosis. Germ-free mice had impaired glucose tolerance when reconstituted with the microbiota from long-term, but not short-term HFD-fed animals. Our results are consistent with intestinal microbiota contributing to chronic insulin resistance and dysglycemia during prolonged obesity, despite rapid diet-induced changes in the taxonomic composition of the fecal microbiota.

biochemistry↗

bioRxiv2017

Pyroxasulfone Resistance in Lolium rigidum Conferred by Enhanced Metabolic Capacity

Author ContributionsRB and AP designed and performed the experiments. RB, AP, TG, SP wrote the manuscript.\n\nOne sentence summaryThis study provides novel insight into herbicide resistance conferred by GST-based detoxification to allow proactive intervention to minimize weed resistance evolution.\n\nAbstractThe evolution of herbicide-resistant weed populations in response to synthetic herbicide selective pressure is threatening safe weed control practices achieved by these molecules. In Australia multiple-resistant populations of annual ryegrass (Lolium rigidum) are effectively controlled by soil-applied herbicides which provide adequate weed control.\n\nIn this study we define the mechanistic basis of the experimentally-evolved resistance to the soil-applied herbicide pyroxasulfone in a L. rigidum population. TLC and HPLC-MS provide biochemical confirmation that pyroxasulfone resistance is metabolism-based with identification and quantification of pyroxasulfone metabolites formed via a glutathione conjugation pathway in pyroxasulfone-resistant L. rigidum plants. The observed over-expression of two putative resistance-endowing GST genes is consistent with pyroxasulfone-resistance in parental plants (P6) and positively correlated to pyroxasulfone resistance in F1 pair-cross progenies. Thus, a major detoxification mechanism involves glutathione conjugation to pyroxasulfone and GST over-expression in pyroxasulfone-resistant L. rigidum plants. The definition of the genetic basis of metabolic resistance in weeds can be a first crucial step towards chemical means to reverse resistance and improve long-term weed resistance management.

biochemistry↗

bioRxiv2017

Solution Structure Of Human STARD1 Protein And Its Interaction With Fluorescently-Labeled Cholesterol Analogues With Different Position Of The NBD-Group

Intracellular cholesterol transfer to mitochondria, a bottleneck of adrenal and gonadal steroidogenesis, relies on the functioning of the steroidogenic acute regulatory protein (StAR, STARD1), for which many disease-associated mutations have been described. Despite significant progress in the field, the exact mechanism of cholesterol binding and transfer by STARD1 remains debatable, and the solution conformation of STARD1 is insufficiently characterized, partially due to its poor solubility. Although cholesterol binding to STARD1 was widely studied by commercially available fluorescent NBD-analogues, the effect of the NBD group position on binding remained unexplored. Here, we analyzed in detail the hydrodynamic properties and solution conformation of STARD1 and its interaction with cholesterol-like steroids bearing 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group in different position, namely 22-NBD-cholesterol (22NC), 25-NBD-cholesterol (25NC), 20-((NBDamino)-pregn-5-en-3-ol (20NP) and 3-(NBDamino)-cholestane (3NC). The small-angle X-ray scattering (SAXS)-based modeling and docking simulations show that, apart from movements of the flexible {Omega}1-loop, STARD1 unlikely undergoes significant structural rearrangements proposed earlier as a gating mechanism for cholesterol binding. While being able to stoichiometrically bind 22NC and 20NP with high fluorescence yield and quantitative exhaustion of fluorescence of some protein tryptophans, STARD1 binds 25NC and 3NC with much lower affinity and poor fluorescence yield. In contrast to 3NC, binding of 20NP leads to STARD1 stabilization and increases the NBD fluorescence lifetime. Remarkably, in terms of fluorescence response, 20NP outperforms commonly used 22NC and is recommended for future studies. Our study benefits from state-of-the-art techniques and revisits the results of the STARD1 research over the last 20 years, revealing important novel information.

biochemistry↗

bioRxiv2017

A toolbox of immunoprecipitation-grade monoclonal antibodies against human transcription factors.

A key component to overcoming the reproducibility crisis in biomedical research is the development of readily available, rigorously validated and renewable protein affinity reagents. As part of the NIH Protein Capture Reagents Program (PCRP), we have generated a collection of 1406 highly validated, immunoprecipitation (IP) and/or immunoblotting (IB) grade, mouse monoclonal antibodies (mAbs) to 736 human transcription factors. We used HuProt human protein microarrays to identify mAbs that recognize their cognate targets with exceptional specificity. Using an integrated production and validation pipeline, we validated these mAbs in multiple experimental applications, and have distributed them to the Developmental Studies Hybridoma Bank (DSHB) and several commercial suppliers. This study allowed us to perform a meta-analysis that identified critical variables that contribute to the generation of high quality mAbs. We find that using full-length antigens for immunization, in combination with HuProt analysis, provides the highest overall success rates. The efficiencies built into this pipeline ensure substantial cost savings compared to current standard practices.

biochemistry↗

bioRxiv2017

Gene-centric constraint of metabolic models

MotivationA number of approaches have been introduced in recent years allowing gene expression data to be integrated into the standard flux Balance Analysis (FBA) technique. This additional information permits greater accuracy in the prediction of intracellular fluxes, even when knowledge of the growth medium and biomass composition is incomplete, and allows exploration of organisms metabolism under wide-ranging conditions. However, existing techniques still focus on the reaction as the fundamental unit of their modelling. This carries the advantages of incorporating expression measurements, but discounts the fact that genes (and their associated proteins) may be involved in the catalysis of multiple reactions through the formation of alternative protein complexes.\n\nResultsWe demonstrate an approach focusing not on reactions or genes as the fundamental unit, but on the Gene Complex (GC), a set of genes that is sufficient to catalyse a given reaction. We define expression-based limits in such a way that proteins cannot do double duty: no single molecule is permitted to contribute to the catalysis of more than one reaction at a time. Using experimentally determined RNA expression and intracellular fluxes, we validate this novel and more conceptually sound approach.\n\nAvailability and ImplementationAn implementation of the GC-FO_SCPLOWLUXC_SCPLOW algorithm is available as part of the Pyabolism python module. https://github.com/nickfyson/pyabolism\n\nContactnickfyson@gmail.com

biochemistry↗

bioRxiv2017

Catalytic properties of RNA polymerases IV and V: accuracy, nucleotide incorporation and rNTP/dNTP discrimination

Catalytic subunits of DNA-dependent RNA polymerases of bacteria, archaea and eukaryotes share hundreds of ultra-conserved amino acids. Remarkably, the plant-specific RNA silencing enzymes, Pol IV and Pol V differ from Pols I, II and III at ~140 of these positions, yet remain capable of RNA synthesis. Whether these amino acid changes in Pols IV and V alter their catalytic properties in comparison to Pol II, from which they evolved, is unknown. Here, we show that Pols IV and V differ from one another, and Pol II, in nucleotide incorporation rate, transcriptional accuracy and the ability to discriminate between ribonucleotides and deoxyribonucleotides. Pol IV transcription is notably error-prone, which may be tolerable, or even beneficial, for biosynthesis of siRNAs targeting transposon families in trans. By contrast, Pol V exhibits high fidelity transcription, suggesting a need for Pol V transcripts to faithfully reflect the DNA sequence of target loci in order to recruit siRNA-Argonaute protein silencing complexes.

biochemistry↗

bioRxiv2017

Early Candidate Biomarkers Found From Urine Of Astrocytoma Rat Before Changes In MRI

Astrocytoma is the most common aggressive glioma and its early diagnosis remains difficult. Biomarkers are changes associated with the disease. Urine, which is not regulated by homeostatic mechanisms, accumulates changes and therefore is a better source for biomarker discovery. In this study, C6 cells were injected into Wistar rats brain as astrocytoma model. Urine samples were collected at day 2, day 6, day 10 and day 13 after injection, and the urinary proteomes were analyzed. On the 10th day, lesions appeared in magnetic resonance imaging. On the 13th day, clinical symptoms started. But differential urinary proteins were changed with the development of the astrocytoma, and can provide clues even on the 2nd and 6th day. Twenty-seven differential proteins with human orthologs had been reported to associate with astrocytoma. Thirty-nine proteins were verified in four more rats as candidate biomarkers of astrocytoma using multiple-reaction monitoring. A panel of differential urinary proteins may provide early biomarkers for diagnose of astrocytoma.

biochemistry↗

bioRxiv2017

Synthesis And Modification Of A Mesoporous Material Type MCM-41 By An Amine For The Adsorption Of Organic Pollutants: Anionic And Cationic Dyes.

The main objective of this work was to synthesize the MCM-41 material from optimized protocols. In the second step, the pore size and the specific surface area of the parent material was increased by the incorporation of swelling agents (long chain carbon amines N-N, Dimethyl-dodecylamine << DMDDA >>) in post-synthesis. Then the selective extraction of the amine and the calcination allowed us to obtain materials with pore sizes and even larger surfaces than those of the starting materials (parent materials). The adsorbents, identified as MCM-41 (P), DMDDA-41 (A), DMDDA-41 (B), DMDDA-41 (C) and MCM-41 (P/C), were characterized regarding their texture, mesoscopic ordering and chemical surface and finally their adsorption capacity was evaluated using two diffrent dyes (anionic: Orange II sodium and cationic: Janus Green B). The results obtained during the adsorption study show the efficiency of these materials, in particular the \"amine and calcined\" materials for decolorizing aqueous media contaminated with organic dyes. The kinetic studies and the adsorption isotherms were carried out to clarify the method of fixing each of the two dyes on the two materials tested. The experiments showed that the amine material had a maximum capacity for fixing Orange II (221.06 mg / g) (anionic dye), whereas for a maximum adsorption capacity (455.23 mg / g) Of the Janus GB (cationic dye), the calcined material is more efficient.

biochemistry↗

bioRxiv2017

A library of structurally homogeneous human N-glycans synthesized from microbial oligosaccharide precursors

Synthesis of homogenous glycans in quantitative yields represents a major bottleneck to the production of molecular tools for glycoscience, such as glycan microarrays, affinity resins, and reference standards. Here, we describe a combined biological/enzymatic method termed bioenzymatic synthesis that is capable of efficiently converting microbially-derived precursor oligosaccharides into structurally uniform human-type N-glycans. Unlike starting material obtained by chemical synthesis or direct isolation from natural sources, which can be time consuming and costly to generate, bioenzymatic synthesis involves precursors derived from renewable sources including wild-type Saccharomyces cerevisiae glycoproteins and lipid-linked oligosaccharides from glycoengineered Escherichia coli. Following deglycosylation of these biosynthetic precursors, the resulting microbial oligosaccharides are subjected to a greatly simplified purification scheme followed by structural remodeling using commercially available and recombinantly produced glycosyltransferases including key N- acetylglucosaminyltransferases (e.g., GnTI, GnTII, and GnTIV) involved in early remodeling of glycans in the mammalian glycosylation pathway. Using this approach, preparative quantities of hybrid and complex-type N-glycans including asymmetric multi-antennary structures were generated all without the need of a specialized skillset. Collectively, our results reveal bioenzymatic synthesis to be a user-friendly methodology for rapidly supplying homogeneous oligosaccharide structures that can be used to understand the human glycome and probe the biological roles of glycans in health and disease.

biochemistry↗

bioRxiv2017

Resveratrol Induces Mitochondrial Dysfunction And Decreases Chronological Life Span Of Saccharomyces cerevisiae In A Glucose-Dependent Manner

A broad range of health benefits have been attributed to resveratrol (RSV) supplementation in mammalian systems, including the increases in longevity. Nonetheless, despite the growing number of studies performed with RSV, the molecular mechanism by which it acts still remains unknown. Recently, it has been proposed that inhibition of the oxidative phosphorylation activity is the principal mechanism of RSV action. This mechanism suggests that RSV might induce mitochondrial dysfunction resulting in oxidative damage to cells with a concomitant decrease of cell viability and cellular life span. To prove this hypothesis, the chronological life span (CLS) of Saccharomyces cerevisiae was studied as it is accepted as an important model of oxidative damage and aging. In addition, oxygen consumption, mitochondrial membrane potential, and hydrogen peroxide (H2O2) release were measured in order to determine the extent of mitochondrial dysfunction. The results demonstrated that the supplementation of S. cerevisiae cultures with 100 M RSV decreased CLS in a glucose-dependent manner. At high-level glucose, RSV supplementation increased oxygen consumption during the exponential phase yeast cultures, but inhibited it in chronologically aged yeast cultures. However, at low-level glucose, oxygen consumption was inhibited in yeast cultures in the exponential phase as well as in chronologically aged cultures. Furthermore, RSV supplementation promoted the polarization of the mitochondrial membrane in both cultures. Finally, RSV decreased the release of H2O2 with high-level glucose and increased it at low-level glucose. Altogether, this data supports the hypothesis that RSV supplementation decreases CLS as a result of mitochondrial dysfunction and this phenotype occurs in a glucose-dependent manner.

biochemistry↗

bioRxiv2017

Structural insights into the light-driven auto-assembly process of the water-oxidizing Mn4CaO5-cluster in photosystem II

In plants, algae and cyanobacteria, Photosystem II (PSII) catalyzes the light-driven splitting of water at a protein-bound Mn4CaO5-cluster, the water-oxidizing complex (WOC). In the photosynthetic organisms, the light-driven formation of the WOC from dissolved metal ions is a key process because it is essential in both initial activation and continuous repair of PSII. Structural information is required for understanding of this chaperone-free metal-cluster assembly. For the first time, we obtained a structure of PSII from Thermosynechococcus elongatus without the Mn4CaO5-cluster. Surprisingly, cluster-removal leaves the positions of all coordinating amino acid residues and most nearby water molecules largely unaffected, resulting in a pre-organized ligand shell for kinetically competent and error-free photo-assembly of the Mn4CaO5-cluster. First experiments initiating (i) partial disassembly and (ii) partial re-assembly after complete depletion of the Mn4CaO5-cluster agree with a specific bi-manganese cluster, likely a di--oxo bridged pair of Mn(III) ions, as an assembly intermediate.

biochemistry↗