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Results for “Biochemistry”

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At least 145 records · Page 8Linked to original sources

bioRxiv2016

REVIEW AND ANALYSIS OF MERCURY LEVELS IN BLUE MARLIN (Makaira nigricans, Lacepede 1802) AND SWORDFISH (Xiphias gladius, Linnaeus 1758)

Mercury in the environment comes from natural processes and industrial activities. The inorganic form associates with the organic matter resulting on methylmercury (MeHg), the most toxic form to man. Mercury occurs in fishes mostly in organic form, the methylmercury. The billfishes appear to have high mercury levels because they are large predators that occupy top levels in the trophic chain. The mercury biomagnificates through the trophic chain and reaches high concentrations in top predators. The present study summarized the information about mercury levels in billfishes available in the literature, with emphasis on swordfish and blue marlin and made an analysis of data from several studies using the multivariate technique Agglomerative Hierarchical Cluster (AHC). The total mercury (THg) ranged from 0.7g/g to 12.2g/g in blue marlin and from 0.04g/g to 5.1g/g in swordfish, with data from 121 blue marlin specimens and 354 of swordfish. While a large part of the mercury content of the blue marlin was above 0.5g/g allowed by WHO, the specimens of swordfish summarized on the present study showed generally lower values within or slightly above this limit, but the swordfish is still consumed nowadays, and large amounts consumed oftentimes may represent a risk to human health.

Biochemistry↗

bioRxiv2016

Hormetic Shifting of Redox Environment by Pro-Oxidative Resveratrol Protects Cells Against Stress

Resveratrol has gained tremendous interest owing to multiple reported health-beneficial effects. However, the underlying key mechanism of action of resveratrol remained largely controversial. Here, we demonstrate that under physiologically relevant conditions major biological effects of resveratrol can be attributed to the generation of oxidation products such as reactive oxygen species (ROS). At low hormetic concentrations (< 50 M), treatment with resveratrol increased cell viability in a set of representative cell models, whereas application of quenchers of ROS completely truncated these beneficial effects. Notably, application of resveratrol led to mild, Nrf2-specific cellular gene expression reprogramming. For example, in primary human epidermal keratinocytes this resulted in a 1.3-fold increase of endogenous metabolites such as gluthathione (GSH) and subsequently in a quantitative reduction of the cellular redox environment by 2.61 mV mmol GSH (g protein)-1. In particular in resveratrol pre-treated cells, after external application of oxidative stress by using 0.8 % ethanol, endogenous ROS generation was consequently reduced by 24 %. In contrast to the common perception that resveratrol acts mainly as a chemical antioxidant or as a target protein-specific ligand, we propose that effects from resveratrol treatment are essentially based on oxidative triggering of cells. In relevant physiological microenvironments this effect can lead to hormetic shifting of cellular defence towards a more reductive state to improve resilience to oxidative stress in a manner that can be exactly defined by the redox-environment of the cell.

Biochemistry↗

bioRxiv2016

Multi-site internal modification of long DNA substrates for single-molecule studies

Single-molecule studies of protein-nucleic acid interactions frequently require site-specific modification of long DNA substrates. DNA isolated from bacteriophage {lambda} ({lambda}-DNA) is a convenient source of high quality long (48.5 kb) DNA. However, introducing specific DNA sequences, tertiary structures, and chemical modifications into {lambda}-DNA remains technically challenging. Most current approaches rely on multi-step ligations with low yields and incomplete products. Here, we describe a molecular toolkit for rapid preparation of modified {lambda}-DNA. A set of PCR cassettes facilitates the introduction of recombinant DNA sequences into {lambda}-DNA with 90-100% yield. Furthermore, various DNA structures and chemical modifications can be inserted at user-defined sites via an improved nicking enzyme-based strategy. As a proof-of-principle, we explore the interactions of Proliferating Cell Nuclear Antigen (PCNA) with modified DNA sequences and structures incorporated within {lambda}-DNA. Our results demonstrate that PCNA can load on both 5-ssDNA flaps and a 13xCAG triplet repeat. However, PCNA remains trapped on the 13xCAG structure, confirming a proposed mechanism for triplet repeat expansion. Although we focus on {lambda}-DNA, this method is applicable to all long DNA substrates. We anticipate that this molecular toolbox will be broadly useful for both ensemble- and single-molecule studies that require site-specific modification of long DNA substrates.

Biochemistry↗

bioRxiv2016

Structural and kinetic analysis of the COP9-Signalosome activation and the cullin-RING ubiquitin ligase deneddylation cycle

The COP9-Signalosome (CSN) regulates cullin-RING ubiquitin ligase (CRL) activity and assembly by cleaving Nedd8 from cullins. Free CSN is autoinhibited, and it remains unclear how it becomes activated. We combine structural and kinetic analyses to identify mechanisms that contribute to CSN activation and Nedd8 deconjugation. Both CSN and neddylated substrate undergo large conformational changes upon binding, with important roles played by the N-terminal domains of Csn2 and Csn4 and the RING domain of Rbx1 in enabling formation of a high affinity, fully active complex. The RING domain is crucial for deneddylation, and works in part through conformational changes involving insert-2 of Csn6. Nedd8 deconjugation and re-engagement of the active site zinc by the autoinhibitory Csn5 glutamate-104 diminish affinity for Cul1/Rbx1 by ~100-fold, resulting in its rapid ejection from the active site. Together, these mechanisms enable a dynamic deneddylation-disassembly cycle that promotes rapid remodeling of the cellular CRL network.

Biochemistry↗

bioRxiv2016

Allosteric inhibition of Aurora-A kinase by a synthetic VNAR nanobody

The vast majority of clinically-approved protein kinase inhibitors target the ATP binding pocket directly. Consequently, many inhibitors have broad selectivity profiles and most have significant off-target effects. Allosteric inhibitors are generally more selective, but are difficult to identify because allosteric binding sites are often unknown or poorly characterized, and there is no clearly preferred approach to generating hit matter. Aurora-A is activated through binding of TPX2 to an allosteric site on the kinase catalytic domain, and this knowledge could be exploited to generate an inhibitor. However, it is currently unclear how to design such a compound because a small molecule or peptide mimetic of TPX2 would be expected to activate, not inhibit the kinase. Here, we generated an allosteric inhibitor of Aurora-A kinase based on a synthetic, VNAR single domain nanobody scaffold, IgNARV-D01. Biochemical studies and a crystal structure of the Aurora-A/IgNARV-D01 complex show that the nanobody overlaps with the TPX2 binding site. In contrast with the binding of TPX2, which stabilizes an active conformation of the kinase, binding of the nanobody stabilizes an inactive conformation, in which the C-helix is distorted, the canonical Lys-Glu salt bridge is broken, and the regulatory (R-) spine is disrupted by an additional hydrophobic side chain from the activation loop. These studies illustrate how nanobodies can be used to characterize the regulatory mechanisms of kinases and provide a rational basis for structure-guided design of allosteric Aurora-A kinase inhibitors.\n\nSignificanceProtein kinases are commonly dysregulated in cancer and inhibitors of protein kinases are key therapeutic drugs. However, this strategy is often undermined by a lack of selectivity since the ATP binding pocket that kinase inhibitors usually target is highly conserved. Inhibitors that target allosteric sites are more selective but more difficult to generate. Here we identify a single domain antibody (nanobody) to target an allosteric pocket on the catalytic domain of Aurora-A kinase and demonstrate that the mechanism is antagonistic to a physiologically-relevant allosteric activator, TPX2. This work will enable the development of allosteric Aurora-A inhibitors as potential therapeutics, and provide a model for the development of tools to investigate allosteric modes of kinase inhibition.

Biochemistry↗

bioRxiv2016

Magnetite biomineralization in Magnetospirillum magneticum is regulated by a switch-like behavior in the HtrA protease MamE

Magnetotactic bacteria are aquatic organisms that produce subcellular magnetic particles in order to orient in the earths geomagnetic field. MamE, a predicted HtrA protease required to produce magnetite crystals in the magnetotactic bacterium Magnetospirillum magneticum AMB-1, was recently shown to promote the proteolytic processing of itself and two other biomineralization factors in vivo. Here, we have analyzed the in vivo processing patterns of three proteolytic targets and used this information to reconstitute proteolysis with a purified form of MamE in vitro. MamE cleaves a custom peptide substrate with positive cooperativity, and its auto-proteolysis can be stimulated with exogenous substrates or peptides that bind to either of its PDZ domains. A misregulated form of the protease that circumvents specific genetic requirements for proteolysis causes biomineralization defects, showing that proper regulation of its activity is required during magnetite biosynthesis in vivo. Our results represent the first reconstitution of MamEs proteolytic activity and show that its behavior is consistent with the previously proposed checkpoint model for biomineralization.

Biochemistry↗

bioRxiv2016

Assembly and Activation of Dynein-Dynactin by the Cargo Adaptor Protein Hook3

Metazoan cytoplasmic dynein moves processively along microtubules with the aid of dynactin and an adaptor protein that joins dynein and dynactin into a stable ternary complex. Here, we have examined how Hook3, a cargo adaptor involved in Golgi and endosome transport, forms a motile dynein-dynactin complex. We show that the conserved Hook domain interacts directly with the dynein light intermediate chain 1 (LIC1). By solving the crystal structure of the Hook domain and using structure-based mutagenesis, we identify two conserved surface residues that are each critical for LIC1 binding. Hook proteins with mutations in these residues fail to form a stable dynein-dynactin complex, revealing a crucial role for LIC1 in this interaction. We also identify a region of Hook3 specifically required for an allosteric activation of processive motility. Our work reveals the structural details of Hook3s interaction with dynein and offers insight into how cargo adaptors form processive dynein-dynactin motor complexes.

Biochemistry↗

bioRxiv2016

The Higher Plant Plastid Complex I (NDH) is a Reversible Proton Pump that increases ATP production by Cyclic Electron Flow Around Photosystem I

Cyclic electron flow around photosystem I (CEF) is critical for balancing the photosynthetic energy budget of the chloroplast, by generating ATP without net production of NADPH. We demonstrate that the chloroplast NADPH dehydrogenase complex (NDH), a homolog to respiratory Complex I, pumps approximately two protons from the chloroplast stroma to the lumen per electron transferred from ferredoxin to plastoquinone, effectively increasing the efficiency of ATP production via CEF by two-fold compared to CEF pathways involving non-proton-pumping plastoquinone reductases. Under certain physiological conditions, the coupling of proton and electron transfer reactions within NDH should enable a non-canonical mode of photosynthetic electron transfer, allowing electron transfer from plastoquinol to NADPH to be driven by the thylakoid proton motive force possibly helping to sense or remediate mismatches in the photosynthetic budget.

Biochemistry↗

bioRxiv2016

Htm1p-Pdi1p is a folding sensitive mannosidase that marks N-glycoproteins for ER-associated protein degradation

Our understanding of how the endoplasmic reticulum-associated protein degradation (ERAD) machinery efficiently targets terminally misfolded proteins while avoiding the misidentification of nascent polypeptides and correctly folded proteins is limited. For luminal N-glycoproteins, demannosylation of their N-glycan to expose a terminal 1,6-linked mannose is necessary for their degradation via ERAD, but whether this modification is specific to misfolded proteins is unknown. Here we report that the Htm1p-Pdi1p complex acts as a folding-sensitive mannosidase for catalyzing this first committed step. We reconstitute this step in vitro with Htm1p-Pdi1p and model glycoprotein substrates whose structural states we can manipulate. We find that Htm1p-Pdi1p is a glycoprotein-specific mannosidase, which preferentially targets nonnative glycoproteins trapped in partially structured states. As such, Htm1p-Pdi1p is suited to act as a licensing factor that monitors folding in the ER lumen and preferentially commits glycoproteins trapped in partially structured states for degradation.

Biochemistry↗

bioRxiv2016

Automated structure refinement of macromolecular assemblies from cryo-EM maps using Rosetta

Cryo-EM has revealed many challenging yet exciting macromolecular assemblies at near-atomic resolution (3-4.5[A]), providing biological phenomena with molecular descriptions. However, at these resolutions accurately positioning individual atoms remains challenging and may be error-prone. Manually refining thousands of amino acids - typical in a macromolecular assembly - is tedious and time-consuming. We present an automated method that can improve the atomic details in models manually built in near-atomic-resolution cryo-EM maps. Applying the method to three systems recently solved by cryo-EM, we are able to improve model geometry while maintaining or improving the fit-to-density. Backbone placement errors are automatically detected and corrected, and the refinement shows a large radius of convergence. The results demonstrate the method is amenable to structures with symmetry, of very large size, and containing RNA as well as covalently bound ligands. The method should streamline the cryo-EM structure determination process, providing accurate and unbiased atomic structure interpretation of such maps.

Biochemistry↗

bioRxiv2016

On the validity and errors of the pseudo-first-order kinetics in ligand-receptor binding

The simple bimolecular ligand-receptor binding interaction is often linearized by assuming pseudo-first-order kinetics when one species is present in excess. Here, a phase-plane analysis allows the derivation of a new condition for the validity of pseudo-first-order kinetics that is independent of the initial receptor concentration. The validity of the derived condition is analyzed from two viewpoints. In the first, time courses of the exact and approximate solutions to the ligand-receptor rate equations are compared when all rate constants are known. The second viewpoint assess the validity through the error induced when the approximate equation is used to estimate kinetic constants from data. Although these two interpretations of validity are often assumed to coincide, we show that they are distinct, and that large errors are possible in estimated kinetic constants, even when the linearized and exact rate equations provide nearly identical solutions.

Biochemistry↗

bioRxiv2016

Sub-atomic resolution X-ray diffraction of the SH3 domain from the post-synaptic density protein Shank3

The post-synaptic density multidomain scaffolding proteins of the Shank family are structurally poorly characterised. The Shank family consists of three members, and domain-specific interactions of Shank are involved in forming a network of proteins at the post-synaptic region for intracellular signalling and cellular scaffolding. While X-ray crystallography has provided some information on individual Shank domains, the structural basis of Shank interactions is still largely unknown. In this study, the production and crystallisation of the previously uncharacterised Shank3 SH3 domain is presented. The highly twinned crystals diffracted synchrotron X-rays to a resolution higher than 0.9 [A], and these crystals will eventually have the potential to provide an ultrahigh-resolution view into the Shank family SH3 domains and their interactions.

Biochemistry↗

bioRxiv2016

Acidic pH is a Metabolic Switch for 2‐Hydroxyglutarate Generation and Signaling

2-hydroxyglutarate (2-HG) is an important epigenetic regulator, with potential roles in cancer and stem cell biology. The D (R) enantiomer (D-2-HG) is an oncometabolite generated from ketoglutarate (-KG) by mutant isocitrate dehydrogenase (ICDH), while L (S) 2-HG is generated by lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) in response to hypoxia. Since acidic pH is a common feature of hypoxia, as well as tumor and stem cell microenvironments, we hypothesized that pH may regulate cellular 2-HG levels. Herein we report that cytosolic acidification under normoxia moderately elevated 2-HG in cells, and boosting endogenous substrate -KG levels further stimulated this elevation. Studies with isolated LDH-1 and MDH-2 revealed that generation of 2-HG by both enzymes was stimulated several-fold at acidic pH, relative to normal physiologic pH. In addition, acidic pH was found to inhibit the activity of the mitochondrial L-2-HG removal enzyme L-2-HG dehydrogenase, and to stimulate the reverse reaction of ICDH (carboxylation of KG to isocitrate). Furthermore, since acidic pH is known to stabilize hypoxia-inducible factor (HIF), and 2-HG is a known inhibitor of HIF prolyl hydroxylases, we hypothesized that 2-HG may be required for acid-induced HIF stabilization. Accordingly, cells stably over-expressing L-2HGDH exhibited a blunted HIF response to acid. Together these results suggest that acidosis is an important and previously overlooked regulator of 2-HG accumulation and other oncometabolic events, with implications for HIF signaling.

Biochemistry↗

bioRxiv2016

The relative values of the turnover number and the dissociation rate constant determine the definition of the Michaelis-constant

Abstract.The Km value is widely used and defined as a measure of the stability of the ES-complex, however, this is true if the first reaction step exists in rapid equilibrium and the reaction is activation controlled. This paper will discuss the theoretical limitation of each case of the quasi-steady-state approximation using the statistical thermodynamic and the diffusion theory to define the Km value.

Biochemistry↗

bioRxiv2016

Blind testing cross-linking/mass spectrometry under the auspices of the 11th critical assessment of methods of protein structure prediction (CASP11)

Determining the structure of a protein by any method requires varies contributions from experimental and computational sides. In a recent study, high-density cross-linking/mass spectrometry data in combination with ab initio structure prediction by conformational space search determined the structure of human serum albumin (HSA) domains, with an RMSD to X-ray structure of up to 2.53 [A], or 3.38 [A] in the context of blood serum. This paper reports the blind test on the readiness of this technology through the help of Critical Assessment of protein Structure Prediction (CASP). We identified between 201-381 unique residue pairs at an estimated 5% FDR (at link level albeit with missing site assignment precision evaluation), for the four proteins that we provided data for. This equates to between 0.63-1.20 proximal residues per residue, which is comparable to that obtained in the HSA study (0.85 links per residue at 5% FDR). Nevertheless, initial results of CASP11 have suggested that improvements in structure prediction using cross-link data are slight. Most significantly, however, CASP11 revealed to us some of the current limitations of cross-linking, spelling out areas in which the method must develop in future: links spread unevenly over sequence and beta sheets both lacked links and suffered from weak definition of observed links over structure. With CASP12 taking place this year and biannually in the future, blind testing low-resolution structure analysis tools is a worthwhile and feasible undertaking. Data are available via ProteomeXchange with identifier PXD003643.\n\nThe abbreviations used are

Biochemistry↗

bioRxiv2016

Glu-370 in the Large Subunit Influences the Substrate Binding, Allosteric, and Heat Stability Properties of Potato ADP-glucose Pyrophosphorylase

ADP-glucose pyrophosphorylase (AGPase) is a key allosteric enzyme in plant starch biosynthesis. Plant AGPase is a heterotetrameric enzyme that consists of large (LS) and small subunits (SS), which are encoded by two different genes. In this study, we showed that the conversion of Glu to Gly at position 370 in the LS of AGPase alters the heterotetrameric stability along with the binding properties of substrate and effectors of the enzyme. Kinetic analyses revealed that the affinity of the LSE370GSSWT AGPase for glucose-1-phosphate is 3-fold less than for wild type (WT) AGPase. Additionally, the LSE370GSSWT AGPase requires 3-fold more 3-phosphogyceric acid to be activated. Finally, the LSE370GSSWT AGPase is less heat stable compared with the WT AGPase. Computational analysis of the mutant Gly-370 in the 3D modeled LS AGPase showed that this residue changes charge distribution of the surface and thus affect stability of the LS AGPase and overall heat stability of the heterotetrameric AGPase. In summary, our results show that LSE370 intricately modulate the heat stability and enzymatic activity of the AGPase.

Biochemistry↗

bioRxiv2016

Quantitative cross-linking/mass spectrometry reveals subtle protein conformational changes

We have developed quantitative cross-linking/mass spectrometry (QCLMS) to interrogate conformational rearrangements of proteins in solution. Our workflow was tested using a structurally well-described reference system, the human complement protein C3 and its activated cleavage product C3b. We found that small local conformational changes affect the yields of cross-linking residues that are near in space while larger conformational changes affect the detectability of cross-links. Distinguishing between minor and major changes required robust analysis based on replica analysis and a label-swapping procedure. By providing workflow, code of practice and a framework for semi-automated data processing, we lay the foundation for QCLMS as a tool to monitor the domain choreography that drives binary switching in many protein-protein interaction networks.\n\nAbbreviations

Biochemistry↗

bioRxiv2016

Specificity Principles in RNA-Guided Targeting

RNA-guided nucleases (RGNs) provide sequence-specific gene regulation through base-pairing interactions between a small RNA guide and target RNA or DNA. RGN systems, which include CRISPR-Cas9 and RNA interference (RNAi), hold tremendous promise as programmable tools for engineering and therapeutic purposes. However, pervasive targeting of sequences that closely resemble the intended target has remained a major challenge, limiting the reliability and interpretation of RGN activity and the range of possible applications. Efforts to reduce off-target activity and enhance RGN specificity have led to a collection of empirically derived rules, which often paradoxically include decreased binding affinity of the RNA-guided nuclease to its target. Here we demonstrate that simple kinetic considerations of the targeting reaction can explain these and other literature observations. The kinetic models described provide a foundation for understanding RGN systems and a necessary physical and functional framework for their rational engineering.

Biochemistry↗