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bioRxiv2018

Clinical assay for direct assessment of IgG galactosylation in serum using endoglycosidase S

Over the past 30 years, it has been firmly established that a wide spectrum of (autoimmune) diseases such as rheumatoid arthritis, Crohns and lupus, but also other pathologies like alcoholic and non-alcoholic steatohepatitis (ASH and NASH) are driven by chronic inflammation and are hallmarked by a reduced level of serum IgG galactosylation. IgG (under)galactosylation is a promising biomarker to assess disease severity, and monitor and adjust therapy. However, this biomarker has not been implemented in routine clinical chemistry due to a complex analytical procedure that necessitates IgG purification, which is difficult to perform and validate at high throughput.\n\nWe addressed this issue by using endo-{beta}-N-acetylglucosaminidase from Streptococcus pyogenes (endoS) to specifically release Fc N-glycans in whole serum. The entire assay can be completed in a few hours and only entails adding endoS and labeling the glycans with APTS. Glycans are then readily analyzed through capillary electrophoresis. We demonstrate in two independent patient cohorts that IgG undergalactosylation levels obtained with this assay correlate very well with scores calculated from PNGaseF-released glycans of purified antibodies. Our new assay allows to directly and specifically measure the degree of IgG galactosylation in serum through a fast and completely liquid phase protocol, without the requirement for antibody purification. This should help advancing this biomarker towards clinical implementation.

biochemistry↗

bioRxiv2018

Quantitative solid-phase assay to measure deoxynucleoside triphosphate pools

Deoxyribonucleoside triphosphates (dNTPs) are the reduced nucleotides used as the building blocks and energy source for DNA replication and maintenance in all living systems. They are present in highly regulated amounts and ratios in the cell, and their balance has been implicated in the most important cell processes, from determining the fidelity of DNA replication to affecting cell fate. Furthermore, many cancer drugs target biosynthetic enzymes in dNTP metabolism, and mutations in genes directly or indirectly affecting these pathways are the cause of devastating diseases. The accurate and systematic measurement of these pools is key to understand the mechanisms behind these diseases and their treatment. We present a new method for measuring dNTP pools from biological samples, utilising the current state-of-the-art polymerase method, modified to a solid-phase setting and optimised for larger scale measurements.

biochemistry↗

bioRxiv2018

Structural basis of human 5,10-methylenetetrahydrofolate reductase (MTHFR) regulation by phosphorylation and S-adenosylmethionine inhibition

The folate and methionine cycles are crucial to the biosynthesis of lipids, nucleotides and proteins, and production of the global methyl donor S-adenosylmethionine (SAM). 5,10-methylenetetrahydrofolate reductase (MTHFR) represents a key regulatory connection between these cycles, generating 5-methyltetrahydrofolate for initiation of the methionine cycle, and undergoing allosteric inhibition by its end product SAM. Our 2.5 [A] resolution crystal structure of human MTHFR reveals a unique architecture, appending the well-conserved catalytic TIM-barrel to a eukaryote-only SAM-binding domain. The latter domain of novel fold provides the predominant interface for MTHFR homo-dimerization, positioning the N-terminal serine-rich phosphorylation region into proximity with the C-terminal SAM-binding domain. This explains how MTHFR phosphorylation, identified on 11 N-terminal residues (16-total), increases sensitivity to SAM binding and inhibition. Finally, we demonstrate the 25-amino-acid inter-domain linker enables conformational plasticity and propose it to be a key mediator of SAM regulation.

biochemistry↗

bioRxiv2018

Femtomolar and selective dopamine detection by a gold nanoparticle enhanced surface plasmon resonance aptasensor

Ultrasensitive and selective detection and quantification of dopamine (DA) plays a key role in monitoring neurodegenerative diseases. However, the detection limit reported for DA detection is typically in the lower nM range. Pushing the detection limit to pM or lower for this particular target to cover the physiological levels (< 130 pM) is significant. Herein, DA DNA aptamer (DAAPT) gold nanoparticle (AuNP) conjugate is utilized to enhance the surface plasmon resonance (SPR) signal, which enables to detect and quantify DA in the femtomolar (200 fM) to picomolar range. To the best of our knowledge, this is the lowest detection limit achieved for SPR sensing of dopamine. The as-prepared 10 nm DAAPT-AuNP conjugate demonstrates strong binding affinity (Kd = 3.1 {+/-} 1.4 nM) to the complementary DNA (cDNA) probe on gold chip. The cDNA probe is immobilized to the chip via polydopamine surface chemistry, which allows the Michael addition of any primary amine-terminated biomolecules. By adjusting the concentration of the DAAPT-AuNP conjugate, two calibration curves are generated with dynamic ranges from 100 {micro}M to 2 mM, and from 200 fM to 20 nM, respectively. Both calibration curves have negative slopes, showing good agreement to a dose-response curve in an enzyme inhibition assay. In addition, the sensing strategy is evaluated to be specific for DA detection using a series of DA analogs and other metabolites as potential interferences.

biochemistry↗

bioRxiv2018

Deep mutational analysis reveals functional trade-offs in the sequences of EGFR autophosphorylation sites

Upon activation, the epidermal growth factor receptor (EGFR) phosphorylates tyrosine residues in its cytoplasmic tail, which triggers the binding of Src Homology 2 (SH2) and Phosphotyrosine Binding (PTB) domains and initiates downstream signaling. The sequences flanking the tyrosine residues (referred to as phosphosites) must be compatible with phosphorylation by the EGFR kinase domain and the recruitment of adapter proteins, while minimizing phosphorylation that would reduce the fidelity of signal transmission. In order to understand how phosphosite sequences encode these functions within a small set of residues, we carried out high-throughput mutational analysis of three phosphosite sequences in the EGFR tail. We used bacterial surface-display of peptides, coupled with deep sequencing, to monitor phosphorylation efficiency and the binding of the SH2 and PTB domains of the adapter proteins Grb2 and Shc1, respectively. We found that the sequences of phosphosites in the EGFR tail are restricted to a subset of the range of sequences that can be phosphorylated efficiently by EGFR. Although efficient phosphorylation by EGFR can occur with either acidic or large hydrophobic residues at the -1 position with respect to the tyrosine, hydrophobic residues are generally excluded from this position in tail sequences. The mutational data suggest that this restriction results in weaker binding to adapter proteins, but also disfavors phosphorylation by the cytoplasmic tyrosine kinases c-Src and c-Abl. Our results show how EGFR-family phosphosites achieve a trade-off between minimizing off-pathway phosphorylation while maintaining the ability to recruit the diverse complement of effectors required for downstream pathway activation.

biochemistry↗

bioRxiv2018

Structural and functional studies of the RBPJ-SHARP complex reveal conserved corepressor binding site

The Notch pathway is a conserved signaling mechanism that is essential for cell fate decisions during pre and postnatal development. Dysregulated signaling underlies the pathophysiology of numerous human diseases, most notably T-cell acute lymphoblastic leukemia. Receptor-ligand interactions result in changes in gene expression, which are regulated by the transcription factor CSL. CSL forms a complex with the intracellular domain of the Notch receptor and the transcriptional coactivator Mastermind, which is required to activate transcription of all Notch target genes. CSL can also function as repressor by interacting with corepressor proteins, e.g. SHARP in mammals and Hairless in Drosophila melanogaster; however, its role as a transcriptional repressor is not well understood. Here we determine the high-resolution structure of RBPJ, the mouse CSL ortholog, bound to the corepressor SHARP and DNA, which reveals a new mode of corepressor binding to CSL and an interesting example for how ligand binding sites evolve in proteins. Based on the structure, we designed and tested a number of mutants in biophysical, biochemical, and cellular assays to characterize the role of RBPJ as a repressor of Notch target genes. Our cellular studies clearly demonstrate that RBPJ mutants that are deficient for binding SHARP are incapable of repressing transcription from genes responsive to Notch signaling. Altogether, our structure-function studies of the RBPJ-SHARP corepressor complex bound to DNA provide significant insights into the repressor function of RBPJ and identify a new binding pocket on RBPJ that could be targeted for therapeutic benefit.

biochemistry↗

bioRxiv2018

The Shape of the Ribosome Exit Tunnel Affects Cotranslational Protein Folding

The E.coli ribosome exit tunnel can accommodate small folded proteins, while larger ones fold outside. It remains unclear, however, to what extent the geometry of the tunnel influences protein folding. Here, using E. coli ribosomes with deletions in loops in proteins uL23 and uL24 that protrude into the tunnel, we investigate how tunnel geometry determines where proteins of different sizes fold. We find that a 29-residue zinc-finger domain normally folding close to the uL23 loop folds deeper in the tunnel in uL23 {Delta}loop ribosomes, while two ~100-residue protein normally folding close to the uL24 loop near the tunnel exit port fold at deeper locations in uL24 {Delta}loop ribosomes, in good agreement with results obtained by coarse-grained molecular dynamics simulations. This supports the idea that cotranslational folding commences once a protein domain reaches a location in the exit tunnel where there is sufficient space to house the folded structure.

biochemistry↗

bioRxiv2018

MethylSight: Taking a wider view of lysine methylation through computer-aided discovery to provide insight into the human methyl-lysine proteome

Protein Lys methylation plays a critical role in numerous cellular processes, yet it has been challenging to identify Lys methylation in a systematic manner. We present here an approach combining in silico prediction with targeted mass spectrometry (MS) to identify Lys methylation (Kme) sites at the proteome level. We have developed MethylSight, a program that predicts Kme events solely on physicochemical and biochemical properties of putative methylation sites, which can then be validated by targeted MS. Using this approach, we have identified 70 new histone Kme marks with a 90% validation rate. H2BK43me2, which undergoes dynamic changes during stem cell differentiation, is found to be a substrate of KDM5b. Furthermore, MethylSight predicts ~50,000 Kme sites in non-histone proteins with high confidence, suggesting that Lys methylation is a prevalent post-translational modification. Our work provides a useful resource for systematic exploration of the role of Lys methylation in human health and disease.

biochemistry↗

bioRxiv2018

Critical comparison of ultracentrifugation and a commercial kit for isolation of exosomes derived from glioblastoma and breast cancer cell lines

Exosomes are small extracellular vesicles around 30-100 nm in diameter that are secreted from cells and can be found in most body fluids. Exosomes can be a vital source of biomarkers as they contain various substances (e.g. lipids, RNAs, metabolites and proteins) that can reflect the cell of origin (e.g. cancer cells). For isolation of exosomes present in biological matrices, ultracentrifugation (UC)-based procedures are most common. Other approaches exist, including commercial kits developed for easy and low sample volume isolation. In this study, differential UC and an isolation kit from a major vendor (Total Exosome Isolation Reagent from Thermo Fisher Scientific) were compared. Exosomes were isolated from cell culture media of two different cell sources (patient derived cells from glioblastoma multiforme and the breast cancer cell line MDA-MB-231). For both isolation methods, transmission electron microscopy, dynamic light scattering and western blotting indicated the presence of exosomes. The kit- and UC isolates contained similar amounts of protein measured by the bicinchoninic acid (BCA) assay with absorbance at 562 nm. Using western blot, positive exosome markers were identified in all isolates, and additional exosome markers were identified using MS-based proteomics. For the glioblastoma exosome isolates, the number of proteins identified with liquid chromatography tandem MS (LC-MS/MS) was higher for the UC isolates than the kit isolates when injecting equal protein amounts, contrary to that for the breast cancer exosome isolates. However, negative exosome markers were also found in glioblastoma isolates using LC-MS/MS. Thus, we would not use the term \"exosome isolation\" as impurities may be present with both isolation methods. Notably, potential biomarkers for both diseases were identified in the isolates using LS-MS/MS. In our opinion, the two isolation methods had rather similar performance, although with some minor differences based on cell of origin.

biochemistry↗

bioRxiv2018

Near-Infrared Spectroscopy for metabolite quantification and species identification

The aim of the study was to investigate the accuracy of near-infrared spectroscopy (NIRS) in determining triglyceride level and species of wild caught Drosophila. NIRS is a remote sensing method that uses the near-infrared region of the electromagnetic spectrum. It detects the absorption of light by molecular bonds and can be used with live insects. We employ the chemometric approach to combine spectra and reference data from a known sample to produce a multivariate calibration model. Once the calibration model was developed, we used an independent set to validate the accuracy of the calibration model. The optimized calibration model for triglyceride quantification yielded an accuracy of 73%. Simultaneously, we used NIRS to discriminate two species of Drosophila. Flies from independent sets were correctly classified into D. melanogaster and D. simulans with accuracy higher than 80%. Finally, we show that the biological interpretations derived from reference data and the NIRS predictions do not differ. These results suggest that NIRS has the potential to be used as a high throughput screening method to assess a live individual insects triglyceride level and taxonomic status.

biochemistry↗

bioRxiv2018

Comprehensive peptide quantification for data independent acquisition mass spectrometry using chromatogram libraries

Data independent acquisition (DIA) mass spectrometry is a powerful technique that is improving the reproducibility and throughput of proteomics studies. We introduce a new experimental workflow that uses this technique to construct chromatogram libraries that capture fragment ion chromatographic peak shape and retention time for every detectable peptide in an experiment. These coordinates calibrate information in spectrum libraries or protein databases to a specific mass spectrometer and chromatography setup, and enable sensitive peptide detection in quantitative experiments. We also present EncyclopeDIA, a software tool for generating and searching chromatogram libraries, and demonstrate the performance of our workflow by quantifying proteins in human and yeast cells. We find that by exploiting calibrated retention time and fragmentation specificity in chromatogram libraries, EncyclopeDIA can detect and quantify >50% more peptides from DIA experiments than with DDA-based spectrum libraries alone.

biochemistry↗

bioRxiv2018

Inhibition of acetyl-CoA carboxylase by spirotetramat causes lipid depletion and surface coat deficiency in nematodes

Plant-parasitic nematodes pose a significant threat to agriculture causing annual yield losses worth more than 100 billion US$. Nematode control often involves the use of nematicides, but many of them including non-selective fumigants have been phased out, particularly due to ecotoxicological concerns. Thus new control strategies are urgently needed. Spirotetramat (SPT) is used as phloem-moble systemic insecticide targeting acetyl-CoA carboxylase (ACC) of pest insects and mites upon foliar application. Our studies revealed that SPT known to be activated in planta to SPT-enol acts as a developmental inhibitor of the free-living nematode Caenorhabditis elegans and the plant-parasitic nematode Heterodera schachtii. Exposure to SPT-enol leads to larval arrest and disruption of the life cycle. Furthermore, SPT-enol inhibits nematode ACC activity, affects storage lipids, fatty acid composition and disrupts surface coat synthesis. Silencing of H. schachtii ACC by RNAi induced similar phenotypes and thus mimics the effects of SPT-enol, supporting the conclusion that SPT-enol acts on nematodes by inhibiting ACC. Our studies demonstrated that the inhibition of de novo lipid biosynthesis by interfering with nematode ACC is a new nematicidal mode of action addressed by spirotetramat, a well-known systemic insecticide for sucking pest control.

biochemistry↗

bioRxiv2018

Spontaneous emergence of self-replication in chemical reaction systems

Explaining origins of life requires us to explain how self-replication arises. Specifically, how can self-replicating entities develop spontaneously from chemical reaction systems in which no reaction is self-replicating? Previous models either supply a framework for minimal living systems or only consider catalyzed reactions, and thus fail to provide a comprehensive theory. We establish a general model for chemical reaction systems that properly accounts for energetics, kinetics and conservation laws. We find that (1) some systems are collectively-catalytic (e.g., the citric acid cycle), while others self-replicate as a whole (e.g., the formose reaction); (2) side reactions do not always inhibit such systems; (3) many alternative chemical universes often contain one or more such systems; (4) in some self-replicating systems, the entropy of certain parts spontaneously decreases; (5) complex self-replicating molecules emerge spontaneously from simple reaction systems through a sequence of transitions. Together these results start to explain origins of prebiotic evolution.

biochemistry↗

bioRxiv2018

Extraction of a recombinant full-length NADPH-cytochrome P450 oxidoreductase from bacterial membranes: effect of detergents and additives

NADPH-cytochrome P450 oxidoreductase (POR) is a membrane protein in the endoplasmic reticulum of eukaryotic cells. POR is as a key reducing partner for a number of cytochrome P450 proteins involved in different metabolic degradation and signaling pathways. Preparation of the full-length recombinant POR expressed in bacteria has been reported and, typically, involved the use of Triton X-100 detergent for extraction of the overexpressed POR from bacterial membranes. However, extraction efficiency is always relatively low hindering structural studies, particularly--the NMR spectroscopy requiring isotopic enrichment. In this paper, we assessed the effect of a variety of detergents and additives on the efficiency of the membrane-extraction step in POR preparation protocol. We evaluated non-ionic detergents with the variable hydrophobicity (Triton X-100, X-114, and X-405) and structure (Triton X-100, TWEEN-20, Brij-35), a zwitterionic/non-ionic detergent combination (Triton X-100 and CHAPS), as well as a range of alkylamines and polyamines as additives to the conventional extraction buffer containing Triton X-100. None of the detergents or detergent-additive combinations yielded better extraction efficiency than the conventional protocol with the Triton X-100. Lack of variation of the extraction yield allows to hypothesize that the conventional protocol extracts all of the available natively-folded monomeric POR while the remaining fraction is possibly an unfolded aggregated POR, which did not insert in the membranes during expression. We propose that the yield of soluble POR may be increased by a careful optimization of expression conditions while monitoring the distribution of POR between soluble and insoluble fractions in the detergent extraction step.

biochemistry↗

bioRxiv2018

A proteome-based design of bitter peptide digestion regime to attenuate cod-bone soup bitterness: comparison with a rainbow trout extract-mediated bitter taste masking approach

BACKGROUNDThe fresh bones (with some meat on them; frequently discarded as a large quantity of industry garbage) of marine fish such as cod and salmon are good materials for manufacture of food additives (taste adjusters). However, such fish-bone originated additives often have apparent bitter taste and need additional debittering regime.\n\nRESULTSIn this study, 46 known bitter peptides in the cod proteome were targeted for specific protease digestion to eliminate bitter taste from the cod bone soup. Though the debittering effect was apparent, the bitter taste was not completely removed. However, the bitter taste can be removed by addition of trout extract to a complete extent. The strong debittering power of rainbow trout extract was further confirmed by the debittering experiments on salmon bone soup and bitter melon, both with perfect results.\n\nCONCLUSIONThese results indicated that the cod bone soup bitterness comes not only from bitter peptide but also from other substances that can be masked by trout extract. Considering the fact that trout proteome has more potential bitter peptides than cod, trout extract shall have a strong bitter masking substance to be determined in the future.

biochemistry↗

bioRxiv2018

Molecular Dynamics Simulation of the E. coli FtsZ

Previous molecular dynamics studies of the FtsZ protein revealed that the protein has high intrinsic flexibility which the crystal structures were unable to reveal. The initial configuration in these studies was based on the available crystal structure data and therefore, the effect of the C-terminal Intrinsically Disordered Region (IDR) of FtsZ could not be observed in these previous studies. Recent investigations have revealed that the C-terminal IDR is crucial for FtsZ assembly in vitro and Z ring formation in vivo. Therefore, in this study, we simulate FtsZ with the IDR.\n\nSimulations of the FtsZ monomer in different nucleotide bound forms (without nucleotide, GTP, GDP) were performed. In the conformations of FtsZ monomer with GTP, GTP binds variably with the protein. Such variable interaction with the monomer has not been observed in any previous simulation studies of FtsZ and not observed in crystal structures. The central helix bends towards the C-terminal domain in the GTP bound form, thus making way for polymerization. Nucleotide dependent small shift/rotation of the C-terminal domain was observed in average structures.

biochemistry↗

bioRxiv2018

Molecular Dynamics simulation of the E. coli FtsZ dimer

FtsZ dimer was studied to gain insights into FtsZ protofilament formation. In a previous simulation study of the M.janaschii dimer it was found that the monomer-monomer contacts in the GDP bound dimer is lower which results in the high curvature of the GDP bound protofilaments. In this study, we have simulated the E.coli FtsZ dimer. The initial structure was obtained from our previous study in which we had simulated the E.coli FtsZ monomer with its C-terminal IDR (Intrinsically Disordered region) built by homology modelling. The M.janaschii FtsZ dimer subunit contacts were used in the initial configuration. Simulations of the dimer were performed with GTP, GDP and ATP. We observed that the C-terminal domain rotates considerably during dimerization. We also observed the different dynamics of the GTP, GDP and ATP bound dimers due to which assembly into straight protofilaments is favoured only in the presence of GTP.

biochemistry↗

bioRxiv2018

Molecular Dynamics Simulations of the FtsZ mutant G105S

In our previous studies we simulated FtsZ monomer and dimer in different nucleotide binding states. In our simulations, we had used the E.coli FtsZ homology model including the FtsZ Intrinsically Disordered Region (IDR). Our simulations revealed that FtsZ dynamics involves a key stage in which GTP binds to monomeric FtsZ and opens its nucleotide binding site which in turn favours polymerization. During dimerization, the C-terminal of the top monomer rotates considerably towards the bottom monomer. Such a rotation of the C-terminal domain leads to capture of the nucleotide by its N-terminal domain. In this study we simulate the FtsZ G105S mutant to see if it may have ATPase activity which was reported in a previous study.

biochemistry↗