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bioRxiv2017

Computational Investigation of O2 Diffusion Through an Intra-molecular Tunnel in AlkB; Influence of Polarization on O2 Transport

E. Coli AlkB catalyzes the direct dealkylation of various alkylated bases in damaged DNA. The diffusion of molecular Oxygen to the active site in AlkB is an essential step for the oxidative dealkylation activity. Despite detailed studies on the stepwise oxidation mechanism of AlkB, there is no conclusive picture of how O2 molecules reach the active site of the protein. Yu et al. (Nature, 439, 879) proposed the existence of an intra-molecular tunnel based on their initial crystal structures of AlkB. We have employed computational simulations to investigate possible migration pathways inside AlkB for O2 molecules. Extensive molecular dynamics (MD) simulations, including explicit ligand sampling and potential of mean force (PMF) calculations, have been performed to provide a microscopic description of the O2 delivery pathway in AlkB. Analysis of intra-molecular tunnels using the CAVER software indicates two possible pathways for O2 to diffuse into the AlkB active site. Explicit ligand sampling simulations suggests that only one of these tunnels provides a viable route. The free energy path for an oxygen molecule to travel along each of these tunnels has been determined with AMBER and AMOEBA. Both PMFs indicate passive transport of O2 from the surface of the protein. However, the inclusion of explicit polarization shows a very large barrier for diffusion of the co-substrate out of the active site, compared with the non-polarizable potential. In addition, our results suggest that the mutation of a conserved residue along the tunnel, Y178, has dramatic effects on the dynamics of AlkB and on the transport of O2 along the tunnel.

biochemistry↗

bioRxiv2017

Tolloid cleavage activates latent GDF8 by priming the pro-complex for dissociation

Growth differentiation factor 8 (GDF8)/Myostatin is a latent TGF{-}{beta} family member that potently inhibits skeletal muscle growth. Here, we compared the conformation and dynamics of precursor, latent, and Tolloid{-}cleaved GDF8 pro{-}complexes to understand structural mechanisms underlying latency and activation of GDF8. Negative stain electron microscopy (EM) of precursor and latent pro{-}complexes reveals a V{-}shaped conformation that is unaltered by furin cleavage and sharply contrasts with the ring{-}like, cross{-}armed conformation of latent TGF{-}{beta}1. Surprisingly, Tolloid{-}cleaved GDF8 does not immediately dissociate, but in EM exhibits structural heterogeneity consistent with partial dissociation. Hydrogen-deuterium exchange was not affected by furin cleavage. In contrast, Tolloid cleavage, in the absence of prodomain-growth factor dissociation, increased exchange in regions that correspond in pro-TGF-{beta}1 to the 1-helix, latency lasso, and {beta}1 strand in the prodomain and to the {beta}6-7 strands in the growth factor. Thus, these regions are important in maintaining GDF8 latency. Our results show that Tolloid cleavage activates latent GDF8 by destabilizing specific prodomain-growth factor interfaces and primes the growth factor for release from the prodomain.

biochemistry↗

bioRxiv2017

Buffering Agent Induced Lactose Content Increases via Growth Hormone-Mediated Activation of Gluconeogenesis in Lactating Goats

Dairy goats are often fed a high-concentrate (HC) diet to meet lactation demands; however, long-term concentrate feeding is unhealthy and decreases milk yield and lactose content. Therefore, we tested whether a buffering agent increases the output of glucose in the liver and influences of lactose synthesis. In this study, sixteen lactating goats were randomly assigned to two groups: one group received a HC diets (Concentrate: Forage = 6:4, HG), and the other group received the same diet with a buffering agent added (0.2% NaHCO3, 0.1% MgO, BG) as a treatment for 19-weeks experimental period. The results showed that the total volatile fatty acids and lipopolysaccharide (LPS) declined in the rumen leading to the rumen pH was stabilized in the BG group. Milk yield and lactose content increased. The alanine aminotransferase, aspartate transaminase, alkaline phosphatase, pro-inflammatory cytokines, LPS and lactate content in the plasma was significantly decreased, whereas prolactin and growth hormone levels were increased. The hepatic vein content of glucose was increased. In addition, the expression of pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6PC) in the liver was significantly up-regulated. In mammary gland, the glucose transporter type-1, 8, 12 and sodium-glucose cotransporter-1 levels were increased. Cumulatively, the buffering agent treatment increased blood concentrations of glucose via the gluconeogenes and promoting their synthesis in the liver. It may contribute to the increase in milk yield and lactose synthesis of lactating goats.

biochemistry↗

bioRxiv2017

Molecular characterization of latent GDF8 reveals mechanisms of activation

Growth/differentiation factor 8 (GDF8) or myostatin negatively regulates muscle mass. GDF8 is held in a latent state through interactions with its N-terminal prodomain, much like TGF-{beta} Using a combination of small angle X-ray scattering and mutagenesis, we characterized the interactions of GDF8 with its prodomain. Our results show that the prodomain:GDF8 complex can exist in a fully latent state and an activated or triggered state where the prodomain remains in complex with the mature domain. However, these states are not reversible, indicating the latent GDF8 is spring-loaded. Structural analysis shows that the prodomain:GDF8 complex adopts an open configuration, distinct from the latency state of TGF- and more similar to the open state of Activin A and BMP9 (non-latent complexes). We determined that GDF{beta}8 maintains similar features for latency, including the alpha-1 helix and fastener elements, and identified a series of mutations in the prodomain of GDF8 that alleviate latency, including I56E, which does not require activation by the protease Tolloid. In vivo, active GDF8 variants were potent negative regulators of muscle mass, compared to wild-type GDF8. Collectively, these results help characterize the latency and activation mechanisms of GDF8.

biochemistry↗

bioRxiv2017

Systems-Level Annotation of Metabolomics Data Reduces 25,000 Features to Fewer than 1,000 Unique Metabolites

When using liquid chromatography/mass spectrometry (LC/MS) to perform untargeted metabolomics, it is now routine to detect tens of thousands of features from biological samples. Poor understanding of the data, however, has complicated interpretation and masked the number of unique metabolites actually being measured in an experiment. Here we place an upper bound on the number of unique metabolites detected in Escherichia coli samples analyzed with one untargeted metabolomic method. We first group multiple features arising from the same analyte, which we call \"degenerate features\", using a context-driven annotation approach. Surprisingly, this analysis revealed thousands of previously unreported degeneracies that reduced the number of unique analytes to ~2,961. We then applied an orthogonal approach to remove non-biological features from the data by using the 13C-based credentialing technology. This further reduced the number of unique analytes to less than 1,000.

biochemistry↗

bioRxiv2017

MS Western, a method of multiplexed absolute protein quantification is a practical alternative to western blotting

Absolute quantification of proteins elucidates the molecular composition, regulation and dynamics of multiprotein assemblies and networks. Here we report on a method termed MS Western that accurately determines the molar abundance of dozens of user-selected proteins at the sub-femtomole level in whole cell or tissue lysates without metabolic or chemical labelling and without using specific antibodies. MS Western relies upon GeLC-MS/MS and quantifies proteins by in-gel co-digestion with an isotopically labelled QconCAT protein chimera composed of concatenated proteotypic peptides. It requires no purification of the chimera and relates the molar abundance of all proteotypic peptides to a single reference protein. In comparative experiments, MS Western outperformed immunofluorescence Western blotting by the protein detection specificity, linear dynamic range and sensitivity of protein quantification. To validate MS Western in an in vivo experiment, we quantified the molar content of zebrafish core histones H2A, H2B, H3 and H4 during ten stages of early embryogenesis. Accurate quantification (CV<10%) corroborated the anticipated histones equimolar stoichiometry and revealed an unexpected trend in their total abundance.

biochemistry↗

bioRxiv2017

A mannitol/sorbitol receptor stimulates dietary intake in Tribolium castaneum

Perception of chemical stimuli by insects aids in accepting or rejecting food. Gustatory receptors (Grs) regulating external signals in chemosensory organs have been found in many insects. Tribolium castaneum, a major pest of stored products, possesses over 200 Gr genes. An expanded repertoire of Gr genes appears to be required for diet recognition in generalist feeders; however, it remains unclear whether T. castaneum recognizes a suite of chemicals common to many products or whether it is attracted to specific chemicals, and whether its Grs are involved in its feeding behavior. It is difficult to determine the food preference of T. castaneum based on its dietary intake due to a lack of appropriate methodology. This study established a novel dietary intake estimation method using gypsum, designated the TribUTE (Tribolium Urges To Eat) assay. T. castaneum adults were fed gypsum block without added organic compounds. Sugar preference was determined by adding sweeteners and measuring the amount of gypsum in the excreta. Mannitol was the strongest attractant of T. castaneum dietary intake; in addition, TcGr20 was responsible for mannitol and sorbitol responses in Xenopus oocyte expression, but did not respond to any other non-volatile compounds tested. The EC50 values of TcGr20 for mannitol and sorbitol were 72.6 mM and 90.6 mM, respectively, suggesting that TcGr20 is a feasible receptor for the recognition of mannitol in lower concentrations. TcGr20 was expressed in the antennae, where the perception of mannitol would occur. We examined whether TcGr20 was involved in mannitol recognition using RNAi and the TribUTE assay. The amounts of excreta in TcGr20 dsRNA-injected adults decreased significantly despite the presence of mannitol, compared to that of the control adults. Taken together, our results suggest that T. castaneum adults recognized mannitol/sorbitol using TcGr20 receptors, thereby facilitating their dietary intake.\n\nAbbreviation

biochemistry↗

bioRxiv2017

Allosteric Logic of the V. vulnificus Adenine Riboswitch Resolved by Four-dimensional Chemical Mapping

The structural logic that define the functions of gene regulatory RNA molecules may be radically different from classic models of allostery, but the relevant structural correlations have remained elusive in even intensively studied RNA model systems. Here, we present a four-dimensional expansion of chemical mapping called lock-mutate-map-rescue (LM2R), which integrates multiple layers of mutation with nucleotide-resolution chemical mapping. This technique resolves the core mechanism of the adenine-responsive V. vulnificus add riboswitch including its gene expression platform, a paradigmatic system for which both Monod-Wyman-Changeux (MWC) conformational selection models and non-MWC alternatives have been proposed. To discriminate amongst these models, we locked each functionally important helix through designed mutations and assessed formation or depletion of other helices via high-throughput compensatory rescue experiments. These LM2R measurements give strong support to the pre-existing correlations predicted by MWC models, disfavor alternative models, and reveal new structural heterogeneities that may be general across ligand-free riboswitches.

biochemistry↗

bioRxiv2017

Mass spectrometry based qualification ofantibodies for plasma proteomics.

There is a strong need for procedures that enable context and application dependent validation of antibodies. Here we describe the foundation for a resource aiding more detailed assessment antibody selectivity for capturing endogenous proteins from human plasma. In 414 immunoprecipitation (IP) experiments with EDTA plasma, data was generated by mass spectrometry (LC-MS) with 157 antibodies (targeting 120 unique proteins). Out of a total of 1,313 unique proteins, 426 proteins (33%) were detected in > 20% of the assays and indicate a background comprised of mainly proteins from the complement system. For all proteins identified either in heat-treated or untreated EDTA plasma, frequencies of occurrence were derived. We determined z-scores for each IP as a measure of enrichment to annotate the antibodies into four categories (ON-target, CO-target, OFF-target and NO-target). For 45% (70/157) of the tested antibodies, the expected target proteins were enriched (z-score [≥]3) above background. There were 84% (59/70) of binders that co-enriched other proteins beside the intended target, either due to OFF-target binding or predicted interactions. Comparing several antibodies raised against IGFBP2, the established library allowed us to describe protein complexes in plasma, and we employed multiplexed sandwich immunoassays to confirm these. In summary, the generated resource of plasma enrichment profiles and background proteins adds a very useful and yet lacking starting point for the assessment of antibody selectivity in this clinically important body fluid. The provided insights will contribute to a more informed use of validated affinity reagents and may lead to further advancements of plasma proteomics assays.

biochemistry↗

bioRxiv2017

Methods for the Refolding of Disulfide-Rich Proteins.

Eschericia coli remains the workhorse producing recombinant proteins given its ease of handling, access and genetic manipulation using standard laboratory techniques. However, disulfide-rich proteins can be difficult to produce in E. coli, in large part due to the reducing environment of the bacterial cytoplasm. Refolding from insoluble inclusion bodies can be a viable strategy for generating substantial quantities of disulfide-rich protein. For the best chance of successfully refolding a protein, it is vital to carry out a variety of small-scale test refolds under a swathe of conditions including altering the concentration of urea, salts, reduced and oxidized glutathione, temperature, length of refold time and protein dilution factor. Once a protein has undergone refolding it is vital to determine that the final product is natively folded given the chance of soluble misfolded protein. For determination of correct folding, a variety of techniques can be employed, and ideally, numerous should be used together. For proteins that possess enzymatic function the gold standard to assess correct folding is an activity assay. Non-enzymatic proteins can be assessed using a combination of circular dichroism and nuclear magnetic resonance spectroscopy. These techniques should be utilized alongside mass spectrometry, Western blotting and SDS-PAGE.

biochemistry↗

bioRxiv2017

Biochemical characterization and essentiality of Plasmodium fumarate hydratase

Plasmodium falciparum (Pf), the causative agent of malaria has an iron-sulfur cluster-containing class I fumarate hydratase (FH) that catalyzes the interconversion of fumarate to malate, a well-known reaction in the tricarboxylic acid cycle. In humans, the same reaction is catalyzed by class II FH that has no sequence or structural homology with the class I enzyme. Fumarate, generated in large quantities in the parasite as a byproduct of AMP synthesis is converted to malate by the action of FH, and subsequently used in the generation of the key metabolites oxaloacetate, aspartate and pyruvate. Here we report on the kinetic characterization of purified recombinant PfFH, functional complementation of fh deficiency in Escherichia coli and mitochondrial localization in the parasite. The substrate analog, mercaptosuccinic acid was found to be a potent inhibitor of PfFH with a Ki value in the nanomolar range. Knockout of the fh gene was not possible in P. berghei when drug-selection of the transfectants was performed in BALB/c mice while the gene was amenable to knockout when C57BL/6 mice were used as host, thereby indicating mouse-strain dependent essentiality of the fh gene to the parasite.

biochemistry↗

bioRxiv2017

Genome-wide identification of intrasplicing events in the human transcriptome and hints to their regulatory potential

Alternative splicing is one of the major regulators of both, transcriptome diversity and individual isoform abundance. Therefore regulation of alternative splicing is crucial and yet, due to the complexity of the human genome (23.000 genes, most of which can be alternatively spliced) diverse and multileveled. Identifying and understanding the scope and variety of splicing events is still an ongoing process. We established a novel pipeline to extract splicing events from specific RNA-sequencing datasets and identified numerous splicing events that did not span the entirety of the respective annotated intron but used intronic splice sites. These splicing events could be generally categorized into three groups: 5recursive (using the exonic splice donor and intronic acceptor), 3recursive (using an intronic splice donor and an exonic acceptor) and nested (using two intronic splice sites). Surprisingly, the splicing events we found occurred in introns of all lengths, but generally followed the abundance scheme of all introns, i.e. most were found in introns between 500 and 5000 bps. After confirmation of these splice events by conventional methodologies, we further analyzed the impact of intrasplicing on full intron removal. For this we established a luciferase-based reporter which showed that these intronic splicing steps can be beneficial, deleterious or neutral for full intron removal. Thus intrasplicing events can be crucial for determining the transcriptional output. This in part confirms recent findings on recursive splicing events in humans and other vertebrates and further uncovers an additional level of transcriptome regulation based on a yet undiscovered level of flexibility and regulation of splice site selection and its impact on gene expression.

biochemistry↗

bioRxiv2017

RXFP1 expression is regulated by miR-144-3p in Fibroblasts from Patients with Idiopathic Pulmonary Fibrosis

Relaxin has been considered as a potential therapy for patients with pulmonary fibrosis. We have previously shown, however, that a potential limitation of relaxin-based therapy for Idiopathic Pulmonary Fibrosis (IPF) is the loss of expression of the relaxin receptor Relaxin/Insulin Like Receptor 1 (RXFP1) expression in fibroblasts. The molecular mechanism for RXFP1 down-regulation in IPF patients remains unclear. To determine whether microRNAs play a role in RXFP1 gene expression, we employed a bioinformatics approach to identify microRNAs (miRs) that are predicted to target RXFP1. By in silico analysis, we identified a putative target site in the RXFP1 mRNA for the miR-144 family. We found that miR-144-3p was upregulated in IPF fibroblasts compared to donor lung fibroblast controls. Forced miR-144-3p mimic expression reduced RXFP1 mRNA and protein levels and increased expression of the myofibroblast marker alpha-smooth muscle actin (-SMA) in donor lung fibroblasts. IPF lung fibroblasts transfected with a miR-144-3p inhibitor increased RXFP1 expression and reduced -SMA expression. A lentiviral luciferase reporter vector carrying the WT 3UTR of RXFP1 was repressed more in lung fibroblasts whereas vector carrying a mutated miR-144-3p binding site exhibited less sensitivity to endogenous miR-144-3p expression, suggesting that RXFP1 is a direct target of miR-144-3p. Thus, miR-144-3p is highly expressed in IPF fibroblasts and acts as a negative regulator of RXFP1 protein expression.

biochemistry↗

bioRxiv2017

CRISPR/Cas9-APEX-mediated proximity labeling enables discovery of proteins associated with a predefined genomic locus in living cells

The activation or repression of a genes expression is primarily controlled by changes in the proteins that occupy its regulatory elements. The most common method to identify proteins associated with genomic loci is chromatin immunoprecipitation (ChIP). While having greatly advanced our understanding of gene expression regulation, ChIP requires specific, high quality, IP-competent antibodies against nominated proteins, which can limit its utility and scope for discovery. Thus, a method able to discover and identify proteins associated with a particular genomic locus within the native cellular context would be extremely valuable. Here, we present a novel technology combining recent advances in chemical biology, genome targeting, and quantitative mass spectrometry to develop genomic locus proteomics, a method able to identify proteins which occupy a specific genomic locus.

biochemistry↗

bioRxiv2017

Enhanced proofreading governs CRISPR-Cas9 targeting accuracy

The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing1-4. High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity were unknown5-9. Using single-molecule Forster resonance energy transfer (smFRET) experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state10 when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target mismatches and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we identified residues within REC3 involved in mismatch sensing and designed a new hyper-accurate Cas9 variant (HypaCas9) that retains robust on-target activity in human cells. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing.

biochemistry↗

bioRxiv2017

Bending of the BST-2 coiled-coil during viral budding

BST-2/tetherin is a human extracellular transmembrane protein that serves as a host defense factor against HIV-1 and other viruses by inhibiting viral spreading. Structurally, BST-2 is a homodimeric coiled-coil that is connected to the host cell membrane by N and C terminal transmembrane anchors. The C-terminal membrane anchor of BST-2 is inserted into the budding virus while the N-terminal membrane anchor remains in the host cell membrane creating a viral tether. The structural mechanism of viral budding and tethering as mediated by BST-2 is not clear. To more fully describe the mechanism of viral tethering, we created a model of BST-2 embedded in a membrane and used steered molecular dynamics to simulate the transition from the host cell membrane associated BST-2 and the cell-virus membrane bridging form. We observed that BST-2 did not transition as a rigid structure, but instead bent at sites with a reduced interface between the helices of the coiled-coil. The simulations for the human BST-2 were then compared with simulations on the mouse homolog, which has a more stable coiled-coil. We observed that the mouse homolog spread the bending across the ectodomain, rather than breaking at discrete points as observed with the human homolog. These simulations support previous biochemical and cellular work suggesting some flexibility in the coiled-coil is necessary for viral tethering, while also highlighting how subtle changes in protein sequence can influence the dynamics and stability of proteins with overall similar structure.

biochemistry↗

bioRxiv2017

Association of Vancomycin with Lipid Vesicles

Antibiotics plays a pivotal role in modern medicine for the treatment of bacterial infection in patients. Membrane defines the boundary between single cell and its environment and is a main target for antibacterial agents. To better understand the mechanism of antibiotics action on microbes, we utilized liposome as membrane mimic model to study antibiotics interaction with bacterial membrane by variety of biophysical methods. Isothermal calorimetry and fluorescence photometry experiments were performed to examine interaction between antibiotics and liposome. We found that vancomycin, one of the most important antibiotics for the treatment of serious infections by gram-positive bacteria, binds to the liposome. The association between the drug and the liposome does not involve the tail part of the lipids. Moreover, the binding affinity increases along with the increment of liposome size. Of three major lipid components, phosphatidylglycerol is the preferential target for vancomycin binding. We also showed that vancomycin associates with vesicle derived from Staphylococcus aureus membrane in a similar manner as the binding to liposome. Our data suggested that vancomycin associates with bacterial membrane through direct interaction with lipid head groups with the extent of the association depending very much on specific type of lipids and curvature of local membrane structure.

biochemistry↗

bioRxiv2017

Overcoming insecticide resistance through computational inhibitor design

Insecticides allow control of agricultural pests and disease vectors and are vital for global food security and health. The evolution of resistance to insecticides, such as organophosphates (OPs), is a serious and growing concern. OP resistance often involves sequestration or hydrolysis of OPs by carboxylesterases. Inhibiting carboxylesterases could therefore restore the effectiveness of OPs for which resistance has evolved. Here, we use covalent computational design to produce nano/pico-molar boronic acid inhibitors of the carboxylesterase E7 from the agricultural pest Lucilia cuprina, as well as a common Gly137Asp E7 mutant that confers OP resistance. These inhibitors, with high selectivity against human acetylcholinesterase, and low to no toxicity in human cells and mice, act synergistically with the OPs diazinon and malathion to reduce the amount of OP required to kill L. cuprina by up to 16-fold, and abolish resistance. The compounds exhibit broad utility in significantly potentiating another OP, chlorpyrifos against the common pest, the peach-potato aphid (Myzus persicae). These compounds represent a solution to OP resistance as well as to environmental concerns regarding overuse of OPs, allowing significant reduction of use without compromising efficacy.

biochemistry↗