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Results for “Biochemistry”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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At least 217 records · Page 12Linked to original sources

bioRxiv2016

Metabolome Identification by Systematic Stable Isotope Labeling Experiments and False Discovery Analysis with a Target-Decoy Strategy

We introduce a formula-based strategy and algorithm (JUMPm) for global metabolite identification and false discovery analysis in untargeted mass spectrometry-based metabolomics. JUMPm determines the chemical formulas of metabolites from unlabeled and stable-isotope labeled metabolome data, and derives the most likely metabolite identity by searching structure databases. JUMPm also estimates the false discovery rate (FDR) with a target-decoy strategy based on the octet rule of chemistry. With systematic stable isotope labeling of yeast, we identified 2,085 chemical formulas (10% FDR), 892 of which were assigned with metabolite structures. We evaluated JUMPm with a library of synthetic standards, and found that 96% of the formulas were correctly identified. We extended the method to mammalian cells with direct isotope labeling and by heavy yeast spike-in. This strategy and algorithm provide a powerful a practical solution for global identification of metabolites with a critical measure of confidence.

biochemistry↗

bioRxiv2016

Evaluation of quantum dot conjugated antibodies for immunofluorescent labelling of cellular targets

Semiconductor quantum dots (Qdots) have been utilised as probes in fluorescent microscopy and provide an alternative to fluorescent dyes and fluorescent proteins, due to their brightness, photostability, and the possibility to excite different Qdots with a single wavelength. In spite of these attractive properties, their take up by biologists has been somewhat limited and only a few Qdot conjugates are commercially available for the labelling of cellular targets. Although, many protocols have been reported for the specific labelling of proteins with Qdots, the majority of these relied on Qdot-conjugated antibodies synthesised specifically by the authors and therefore not broadly available, which limits the scope of applications and complicates replication. Here, the specificity of a commercially available Qdot conjugated secondary antibody (Qdot-Ab), for different antigens, was tested. Antigens were labelled simultaneously with a fluorescent dye coupled to a secondary antibody (Dye-Ab) and the Qdot-Ab. Although, the Dye-Ab labelled all of the intended target proteins, the Qdot-Ab only bound to some of the protein targets in the cytosol and could not reach the nucleus even after extensive cell permeabilisation.

biochemistry↗

bioRxiv2016

Preliminary investigation of Incorporation and Viability of Crystal Violet in Aspergillus flavus

The abusive use of antimicrobials have caused resistance in the microorganisms. Therefore, inovations in research of modern microbiology developed the photodynamic inactivation that is non-toxic and do not promote microbian resistance since it has multiples action sites. When a photoactive molecule is activated by light, it becomes more toxic for the microorganism than for the human cells, increasing the phototoxic effect against such cells. We tested the incorporation of the photosensitizer crystal violet by Aspergillus flavus and its viability in different periods of time of incubation (6 and 18 hours), concentrations of crystal violet (4M, 10M e 15 M) and different conditions (resting and agitation). We also tested the photoinactivation of Aspergillus flavus containing crystal violet. The incorporation rate does not depend on the concentration of CV neither on the condition of incubation. It depends exclusively on the medium, since it presented higher values in lack ingredients medium. The assays of viability of fungi after exposure to crystal violet show that there is fungic growth when samples are maintained in the dark. However, when the culture were submitted to excitation via laser, there was a decrease in the biomass growth, which indicates that there was photodynamic inactivation of the fungi.

biochemistry↗

bioRxiv2016

Cushing’s Syndrome mutant PKAL205R exhibits altered substrate specificity

The PKAL205R hotspot mutation has been implicated in Cushings Syndrome through hyperactive gain-of-function PKA signaling, however its influence on substrate specificity has not been investigated. Here, we employ the Proteomic Peptide Library (ProPeL) approach to create high-resolution models for PKAWT and PKAL205R substrate specificity. We reveal that the L205R mutation reduces canonical hydrophobic preference at the substrate P+1 position, and increases acidic preference in downstream positions. Using these models, we designed peptide substrates that exhibit altered selectivity for specific PKA variants, and demonstrate the feasibility of selective PKAL205R loss-of-function signaling. Through these results, we suggest that substrate rewiring may contribute to Cushings Syndrome disease etiology, and introduce a powerful new paradigm for investigating mutation-induced kinase substrate rewiring in human disease.

biochemistry↗

bioRxiv2016

Mechanism of ribosome rescue by ArfA and RF2

ArfA rescues ribosomes stalled on truncated mRNAs by recruiting the release factor RF2, which normally binds stop codons to catalyze peptide release. We report two 3.2-[A] resolution cryo-EM structures - determined from a single sample - of the 70S ribosome with ArfA*RF2 in the A site. In both states, the ArfA C-terminus occupies the mRNA tunnel downstream of the A site. One state contains a compact inactive RF2 conformation, hitherto unobserved in 70S termination complexes. Ordering of the ArfA N-terminus in the second state rearranges RF2 into an extended conformation that docks the catalytic GGQ motif into the peptidyl-transferase center. Our work thus reveals the structural dynamics of ribosome rescue. The structures demonstrate how ArfA \"senses\" the vacant mRNA tunnel and activates RF2 to mediate peptide release without a stop codon, allowing stalled ribosomes to be recycled.

biochemistry↗

bioRxiv2016

Reprogramming protein kinase substrate specificity through synthetic mutations

Protein kinase specificity is largely imparted through substrate binding pocket motifs. Missense mutations in these regions are frequently associated with human disease, and in some cases can alter substrate specificity. However, current efforts at decoding the influence of mutations on substrate specificity have been focused on disease-associated mutations. Here, we adapted the Proteomic Peptide Library (ProPeL) approach for determining kinase specificity to the task of exploring structure-function relationships in kinase specificity by interrogating the effects of synthetic mutation. We established a specificity model for the wild-type DYRK1A kinase with unprecedented resolution. Using existing crystallographic and sequence homology data, we rationally designed mutations that precisely reprogrammed the DYRK1A kinase at the P+1 position to mimic the substrate preferences of a related kinase, CK II. This study illustrates a new synthetic biological approach to reprogram kinase specificity by design, and a powerful new paradigm to investigate structure-function relationships underpinning kinase substrate specificity.

biochemistry↗

bioRxiv2016

Lysine methyltransferase SETD6 modifies histones and non-histone proteins

Although central to regulating the access to genetic information, most lysine methyltransferases remain poorly characterised relative to other family of enzymes. Herein, we report new substrates for the lysine methyltransferase SETD6. Based on the SETD6- catalysed site on the histone variant H2AZ, we identified similar sequences in the canonical histones H2A, H3, and H4 that are modified by SETD6 in vitro, and putative non-histone substrates. We herein expend the repertoire of substrates for methylation by SETD6.

biochemistry↗

bioRxiv2016

VIP36 preferentially binds to core-fucosylated N-glycans: a molecular docking study

Alpha 1-6 fucosyltransferase (Fut8) is known for its properties as an enhancer of nonsmall cell lung cancer metastasis and as a suppressor in hepatocellular carcinoma cells (Hep3B). Promising candidates of affected molecules include E-cadherin. In its absence, during epithelial-mesenchymal transition, the pathway triggers signaling to the nucleus via {beta}-catenin-TCF/LEF. Contrarily, in less metastatic tumors, Fut8 stimulates cell-cell adhesion. Regulated classes of molecules could also include the sorting machinery of polarized epithelial cells, sorted ligands or both, that may be altered in cellular transformation. I have analyzed here the cargo receptor VIP36 (Vesicular-integral membrane protein of 36 kD) for carbohydrate interaction. It has been described as a lectin in the ERGIC (endoplasmic reticulum-Golgi intermediate compartment), Golgi apparatus and plasma membrane. The docking reveals top-interacting carbohydrates of the N-glycan and O-glycan class that encompass N-linked glycans of high mannose and equally complex type which likely function as sorted ligands in epithelial cells. O-glycans score lower and include core 2 residue binding. I show that fucose core modifications by Fut8 stimulate binding of N-linked glycans to VIP36, which is known to be different from binding of galectins 3 and 9. This suggests that Fut8-upregulation may directly alter the affinity of sorted cargo and may enhance the sorting to the apical pathway as exemplified in hepatocytes and traffic to bile. High affinity binding of the ganglioside GM1 carbohydrate headgroup to VIP36 suggests a linkage with protein and glycosphingolipid apical transfer in epithelial cells. Thus, this fundamental approach with large scale docking of 165 carbohydrates including 19 N-glycan high mannose, 17 Nglycan hybrid, 9 N-glycan complex, 17 O-glycan core, 27 Sialoside, 25 Fucoside and 51 other glycan residues suggests, that linked cargo-receptor apical transport may provide a path to epithelial polarization that may be modulated by core fucosylation.

biochemistry↗

bioRxiv2016

Genetic variation and RNA structure regulate microRNA biogenesis

MiRNA biogenesis is highly regulated at the post-transcriptional level; however, the role of sequence and secondary RNA structure in this process has not been extensively studied. A single G to A substitution present in the terminal loop of pri-mir-30c-1 in breast cancer patients had been previously described to result in increased levels of mature miRNA. Here, we report that this genetic variant directly affects Drosha-mediated processing of pri-mir-30c-1 in vitro and in cultured cells. Structural analysis of this variant revealed an altered RNA structure that facilitates the interaction with SRSF3, an SR protein family member that promotes pri-miRNA processing. Our results are compatible with a model whereby a genetic variant in pri-mir-30c-1 leads to a secondary RNA structure rearrangement that facilitates binding of SRSF3 resulting in increased levels of miR-30c. These data highlights that primary sequence determinants and RNA structure are key regulators of miRNA biogenesis.

biochemistry↗

bioRxiv2016

Vitrification after multiple rounds of sample application and blotting improves particle density on cryo-electron microscopy grids.

Single particle cryo-electron microscopy (cryoEM) is becoming widely adopted as a tool for structural characterization of biomolecules at near-atomic resolution. Vitrification of the sample to obtain a dense distribution of particles within a single field of view remains a major bottleneck for the success of such experiments. Here, we describe a simple and cost-effective method to increase the density of frozen-hydrated particles on grids with holey carbon support films. It relies on performing multiple rounds of sample application and blotting prior to plunge freezing in liquid ethane. We show that this approach is generally applicable and significantly increases particle density for a range of samples, such as small protein complexes, viruses and filamentous assemblies. The method is versatile, easy to implement, minimizes sample requirements and can enable characterization of samples that would otherwise resist structural studies using single particle cryoEM.

biochemistry↗

bioRxiv2016

Cryo-EM structure of the ATP-sensitive potassium channel illuminates mechanisms of assembly and gating

ATP-sensitive potassium (KATP) channels are metabolic sensors that couple cell energetics to membrane excitability. In pancreatic {beta}-cells, channels formed by SUR1 and Kir6.2 regulate insulin secretion and are the targets of antidiabetic sulfonylureas. Here, we used cryo-EM to elucidate structural basis of channel assembly and gating. The structure, determined in the presence of ATP and the sulfonylurea glibenclamide, at ~6[A] resolution reveals a closed Kir6.2 tetrameric core with four peripheral SUR1s each anchored to a Kir6.2 by its N-terminal transmembrane domain (TMD0). Intricate interactions between TMD0, the loop following TMD0, and Kir6.2 near the proposed PIP2 binding site, and where ATP density is observed, suggest SUR1 may contribute to ATP and PIP2 binding to enhance Kir6.2 sensitivity to both. The SUR1-ABC core is found in an unusual inward-facing conformation whereby the two nucleotide binding domains are misaligned along a two-fold symmetry axis, revealing a possible mechanism by which glibenclamide inhibits channel activity.

biochemistry↗

bioRxiv2016

Familial Mutations and Post-translational Modifications of UCH-L1 in Parkinson’s Disease and Neurodegenerative Disorders

Parkinsons disease (PD) is one of the most common progressive neurodegenerative disorders in modern society. The disease involves many genetic risk factors as well as a sporadic pathogenesis that is age- and environment-dependent. Of particular interest is the formation of intra-neural fibrillar aggregates, namely Lewy bodies (LBs), the histological hallmark of PD, which results from aberrant protein homeostasis or misfolding that results in neurotoxicity. A better understanding of the molecular mechanism and composition of these cellular inclusions will help shed light on the progression of misfolding-associated neurodegenerative disorders. Ubiquitin carbonyl-terminal hydrolase L1 (UCH-L1) is found to co-aggregate with -synuclein (S), the major component of LBs. Several familial mutations of UCH-L1, namely p.Ile93Met (p.I93M), p.Glu7Ala (p.E7A), and p.Ser18Tyr (p.S18Y), are associated with PD and other neurodegenerative disorders. Here, we review recent progress and recapitulate the impact of PD-associated mutations of UCH-L1 in the context of their biological functions gleaned from biochemical and biophysical studies. Finally, we summarize the effect of these genetic mutations and post-translational modifications on the association of UCH-L1 and PD in terms of loss of cellular functions or gain of cellular toxicity.

biochemistry↗

bioRxiv2016

In culture cross-linking of bacterial cells reveals proteome scale dynamic protein-protein interactions at peptide level

Identification of dynamic protein-protein interactions at the peptide level on a proteomic scale is a challenging approach that is still in its infancy. We have developed a system to cross-link cells directly in culture with the special lysine cross-linker bis(succinimidyl)-3-azidomethyl-glutarate (BAMG). We used the Gram positive model bacterium Bacillus subtilis as an exemplar system. Within 5 min extensive intracellular cross-linking was detected, while intracellular cross-linking in a Gram-negative species, Escherichia coli, was still undetectable after 30 min, in agreement with the low permeability in this organism for lipophilic compounds like BAMG. We were able to identify 82 unique inter-protein cross-linked peptides with less than a 1% false discovery rate by mass spectrometry and genome-wide data base searching. Nearly 60% of the inter-protein cross-links occur in assemblies involved in transcription and translation. Several of these interactions are new, and we identified a binding site between the {delta} and {beta}' subunit of RNA polymerase close to the downstream DNA channel, providing a clue into how {delta} might regulate promoter selectivity and promote RNA polymerase recycling. Our methodology opens new avenues to investigate the functional dynamic organization of complex protein assemblies involved in bacterial growth.

biochemistry↗

bioRxiv2016

HILAQ: A Novel Strategy for Newly Synthesized Protein Quantification

Here we describe a new strategy, HILAQ (Heavy Isotope Labeled Azidohomoalanine Quantification), to rapidly quantify the molecular vulnerability profile to oxytosis. HILAQ quantified more than 1,900 Newly Synthesized Proteins (NSP) after 1h pulse labeling in HEK293T cell line. Compared with the previous published QuaNCAT protocol, the HILAQ strategy simplifies newly synthesized proteome analysis and achieves higher sensitivity.

biochemistry↗

bioRxiv2016

Fenton-type chemistry by a copper enzyme: molecular mechanism of polysaccharide oxidative cleavage

The discovery of Lytic Polysaccharide Monooxygenases (LPMOs) has been instrumental for the development of economically sustainable lignocellulose biorefineries. Despite the obvious importance of these exceptionally powerful redox enzymes, their mode of action remains enigmatic and their activity and stability under process conditions are hard to control. By using enzyme assays, mass spectrometry and experiments with labeled oxygen atoms, we show that H2O2, and not O2 as previously thought, is the co-substrate of LPMOs. By controlling H2O2 supply, stable reaction kinetics and high enzymatic rates are achieved, the LPMOs work under anaerobic conditions, and the need for adding stoichiometric amounts of reductants is alleviated. These results offer completely new perspectives regarding the mode of action of these unique mono-copper enzymes, the enzymatic conversion of biomass in Nature, and industrial biorefining.

biochemistry↗

bioRxiv2016

Environment driven conformational changes modulate H-NS DNA bridging activity

Bacteria frequently need to adapt to altered environmental conditions. Adaptation requires changes in gene expression, often mediated by global regulators of transcription. The nucleoid-associated protein H-NS is a key global regulator in Gram-negative bacteria, and is believed to be a crucial player in bacterial chromatin organization via its DNA bridging activity. H-NS activity in vivo is modulated by physico-chemical factors (osmolarity, pH, temperature) and interaction partners. Mechanistically it is unclear how functional modulation of H-NS by such factors is achieved. Here, we show that a diverse spectrum of H-NS modulators alter the ability of H-NS to bridge DNA. Changes in monovalent and divalent ion concentrations drive an abrupt switch between a bridging and non-bridging DNA binding mode. Similarly, synergistic and antagonistic co-regulators modulate the DNA bridging efficiency. Structural studies suggest a conserved mechanism: H-NS switches between a \"closed\" and an \"open\", bridging competent, conformation driven by environmental cues and interaction partners.

biochemistry↗

bioRxiv2017

In vitro assembly of the Rous Sarcoma Virus capsid protein into hexamer tubes under physiological conditions.

During a proteolytically-driven maturation process, the ortho-retroviral capsid protein (CA) assembles to form the convex shell that surrounds the viral genome. In some orthoretroviruses, including Rous Sarcoma Virus (RSV), CA carries a short and hydrophobic spacer peptide (SP) at its C-terminus early in the maturation process, which is progressively removed as maturation proceeds. In this work, we show that RSV CA assembles in vitro at physiological temperatures, forming hexamer tubes that effectively model the mature capsid surface. Tube assembly is strongly influenced by electrostatic effects, and is a nucleated process that remains thermodynamically favored at lower temperatures, but is effectively arrested by the large Gibbs energy barrier associated with nucleation. RSV CA tubes are multi-layered, being formed by nested and concentric tubes of capsid hexamers. However the spacer peptide acts as a layering determinant during tube assembly. If only a minor fraction of CA-SP is present, multi-layered tube formation is blocked, and single-layered tubes predominate. This likely prevents formation of biologically aberrant multi-layered capsids in the virion. The generation of single-layered hexamer tubes facilitated 3D helical image reconstruction from cryo-electron microscopy data, revealing the basic tube architecture.

biochemistry↗

bioRxiv2017

Polymer dynamics of Alp7A reveals two ‘critical’ concentrations that govern dynamically unstable actin-like proteins

ABSTRACTDynamically unstable polymers capture and move cellular cargos in both bacteria and eukaryotes, but the regulation of their assembly remains poorly understood. Here we describe polymerization of Alp7A, a bacterial Actin-Like Protein (ALP) that segregates the low copy-number plasmid pLS20 in Bacillus subtilis. Purified Alp7A forms dynamically unstable polymers with two critical points: an intrinsic critical concentration (0.6 M), observed when ATP hydrolysis is blocked, and a dynamic critical concentration (10.3 M), observed when ATP hydrolysis occurs. From biochemical and kinetic analysis, the intrinsic critical concentration reflects a balance between filament elongation and shortening, while the dynamic critical concentration reflects a balance between filament nucleation and catastrophic disassembly. Although Alp7A does not form stable polymers at physiological concentrations, rapid nucleation by an accessory factor, Alp7R, decreases the dynamic critical concentration into the physiological range. Intrinsic and dynamic critical concentrations are fundamental parameters that can be used to describe the behavior of all dynamically unstable polymers.

biochemistry↗