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Results for “Biochemistry”

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At least 1,207 records · Page 67Linked to original sources

bioRxiv2018

Deciphering copper coordination in the animal prion protein amyloidogenic domain

Prions are pathological isoforms of the cellular prion protein (PrPC) responsible for transmissible spongiform encephalopathies (TSE). PrPC interacts with copper through unique octarepeat and non-octarepeat (non-OR) binding sites. Previous works on human PrPC suggest that copper binding to the non-OR region may have a role during prion conversion. The molecular details of copper coordination within the non-OR region are not well characterized. By means of small angle X-ray scattering (SAXS) and extended X-ray absorption fine structure (EXAFS) spectroscopy, we have investigated the Cu(II) structural effects on the protein folding and its coordination geometries when bound to the non-OR region of recombinant PrPC (recPrP) from animal species considered high or less resistant to TSE. As TSE-resistant model, we used ovine PrPC carrying the protective polymorphism at residues A136, R154 and R171 (OvPrP ARR); while as highly TSE-susceptible PrPC models we employed OvPrP with polymorphism V136, R154 and Q171 (OvPrP VRQ) and Bank vole recPrP (BvPrP). Our results reveal that Cu(II) affects the structural plasticity of the non-OR region leading to a more compacted conformation of recPrP. We also identified two Cu(II) coordinations in the non-OR region of these animal species. In type-1 coordination present in OvPrP ARR, Cu(II) is coordinated by four residues (S95, Q98, M109 and H111). Conversely, the type-2 coordination is present in OvPrP VRQ and BvPrP, where Cu(II) is coordinated by three residues (Q98, M109 and H111) and by one water molecule, making the non-OR region more flexible and open to the solvent. These changes in copper coordination in prion resistant and susceptible species provide new insights into the molecular mechanisms governing the resistance or susceptibility of certain species to TSE.

biochemistry↗

bioRxiv2018

A tRNA modification balances carbon and nitrogen metabolism by regulating phosphate homeostasis, to couple metabolism to cell cycle progression.

Cells must appropriately sense and integrate multiple metabolic resources to commit to proliferation. Here, we report that cells regulate carbon and nitrogen metabolic homeostasis through tRNA U34-thiolation. Despite amino acid sufficiency, tRNA-thiolation deficient cells appear amino acid starved. In these cells, carbon flux towards nucleotide synthesis decreases, and trehalose synthesis increases, resulting in a starvation-like metabolic signature. Thiolation mutants have only minor translation defects. However, these cells exhibit strongly decreased expression of phosphate homeostasis genes, resulting in an effectively phosphate-limited state. Reduced phosphate enforces a metabolic switch, where glucose-6-phosphate is routed towards storage carbohydrates. Notably, trehalose synthesis, which releases phosphate and thereby restores phosphate availability, is central to this metabolic rewiring. Thus, cells use thiolated tRNAs to perceive amino acid sufficiency, and balance carbon and amino acid metabolic flux to maintain metabolic homeostasis, by controlling phosphate availability. These results further biochemically explain how phosphate availability determines a switch to a starvation-state.

biochemistry↗

bioRxiv2018

Cation binding to SERCA

Sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) is a transmembrane pump that plays an important role in transporting calcium into the sarcoplasmic reticulum (SR). While calcium (Ca2+) binds SERCA with micro-molar affinity, magnesium (Mg2+) and potassium (K+) also compete with calcium (Ca2+) binding. However, the molecular bases for these competing ions influence on SERCA function and the selectivity of the pump for Ca2+ are not well-established. We therefore used in silico methods to resolve molecular determinants of cation binding in the canonical site I and II Ca2+ binding sites: 1) triplicate molecular dynamics (MD) simulations of Mg2+, Ca2+and K+-bound SERCA. 2) mean spherical approximation (MSA) theory to determine the affinity and selectivity of cation binding to the MD-resolved structures and 3) state models of SERCA turnover informed from MSA-derived affinity data. Our key findings are that a) coordination at sites I and II are optimized for Ca2+and to a lesser extent for Mg2+ and K+, as determined by MD-derived cation-amino acid oxygen and bound water configurations, b) the impaired coordination and high desolvation cost for Mg2+ precludes favorable Mg2+ binding relative to Ca2+, while K+ has limited capacity to bind site I, c) Mg2+ most likely acts as inhibitor and K+ as intermediate in SERCAs reaction cycle, based on a best-fit state model of SERCA turnover. These findings provide a quantitative basis for SERCA function that leverages molecular-scale thermodynamic data and rationalize enzyme activity across broad ranges of K+, Ca2+ and Mg2+ concentrations.

biochemistry↗

bioRxiv2018

The third-generation tetracycline, KBP-7072, exploits and reveals a new potential of the primary tetracycline binding pocket

Antibiotic resistance is a growing threat to human health requiring the discovery or development of new anti-infectives. As such, KBP-7072 is a novel tetracycline derivative that exhibits broad-spectrum activity against Gram-positive and -negative bacterial strains. To determine the mechanism of action of KBP-7072 and understand how its unique C9 extension can be used to combat the growing problem of antibiotic resistance we determined the structure of KBP-7072 bound to the bacterial 30S ribosomal subunit, the inhibitory target of typical tetracyclines. We show that KBP-7072 binds to the primary tetracycline binding site on the 30S ribosomal subunit consistent with it acting as a protein synthesis inhibitor that blocks A-site occupation. Moreover, the unique chemical nature of KBP-7072{acute}s C9 extension leads to a distinctive interaction pattern with the 30S subunit that distinguishes KBP-7072 from the third-generation tetracycline, Tigecycline, and thus expands the interaction potential of the primary tetracycline binding pocket.

biochemistry↗

bioRxiv2018

Structural basis of sterol recognition by human hedgehog receptor PTCH1

Hedgehog signaling is central in embryonic development and tissue regeneration. Disruption of the pathway is linked to genetic diseases and cancer. Binding of the secreted ligand, Sonic hedgehog (ShhN) to its receptor Patched (PTCH1) activates the signaling pathway. Here, we describe a 3.4 [A] cryo-EM structure of the human PTCH1 bound to ShhNC24II, a modified hedgehog ligand mimicking its palmitoylated form. The membrane-embedded part of PTCH1 is surrounded by ten sterol molecules at the inner and outer lipid bilayer portion of the protein. The annular sterols interact at multiple sites with both the sterol sensing domain, SSD, and the SSD-like domain, SSDL, which are located on opposite sides of PTCH1. The structure reveals a possible route for sterol translocation across the lipid bilayer by PTCH1 and homologous transporters.

biochemistry↗

bioRxiv2019

Cysteine oxidation triggers amyloid fibril formation of the tumor suppressor p16INK4A

Accumulation of the CDK4/6 inhibitor p16INK4A in response to oncogenic transformation leads to cell cycle arrest and senescence and is therefore frequently lost in cancer. p16INK4A is also known to accumulate under conditions of oxidative stress and thus could potentially be regulated by the reversible oxidation of cysteines (redox signaling). Indeed, oxidation of the single cysteine in p16INK4A in human cells occurs under relatively mild oxidizing conditions and leads to disulfide-dependent dimerization. p16INK4A is an all alpha-helical protein, but here we report that upon cysteine-dependent dimerization, p16INK4A undergoes a dramatic structural rearrangement and forms aggregates that have the typical features of amyloid fibrils, including binding of diagnostic dyes, presence of cross-{beta} sheet structure, and typical dimensions found in electron microscopy. p16INK4A amyloid formation abolishes its function as a CDK4/6 inhibitor. Collectively, these observations mechanistically link the cellular redox state to the inactivation of p16INK4A through the formation of amyloid fibrils.

biochemistry↗

bioRxiv2019

Simultaneous proteomics and three PTMomics characterization of pro-inflammatory cytokines stimulated INS-1E cells using TiO2 enrichment strategy

Diverse protein post-translational modifications (PTMs) in proteins form complex combinatorial patterns to regulate the protein function and biological processes in a fine-tuning manner. Reversible phosphorylation, cysteines (Cys) modification, and N-linked glycosylation are essentially involved in cellular signaling pathways of pro-inflammatory cytokines, which can induce beta cell death and diabetes. Here we developed a novel mass spectrometry-based proteomic strategy (termed TiCPG) for the simultaneous comprehensive characterization of the proteome and three post-translational modifications (PTMomes) by applying TiO2 enrichment of peptides with reversibly modified Cysteine (rmCys), Phosphorylation, and sialylated N-linked (SAN-) Glycosylation from low amount of sample material with largely minimized sample loss. We applied this TiCPG strategy to quantitatively study the change of the three PTMs in {beta}-cell-like INS-1E cells subject to pro-inflammatory cytokines stimulation. It enabled efficient enrichment and quantitative analysis of 8346 rmCys sites, 10321 phosphosites and 1906 SAN-glycosylation sites from 5853 proteins. Significant regulation was found on 100 proteins at the total protein level, while much higher degree of regulation was identified on 3025 peptides with PTMs from 1490 proteins. The three PTMs were co-regulated in proteins, but demonstrated differential spatial and temporal patterns related to protein cellular localization and function in the time course of cytokines stimulation, and they were extensively involved in essential signaling pathways related to pro-inflammatory cytokine mediated {beta}-cell apoptosis, such as the inducible NO synthase (NOS2) signaling pathway, Overall, the TiCPG strategy is proved as a straight forward and powerful tool for multiple PTMomics studies.

biochemistry↗

bioRxiv2018

A complex containing lysine-acetylated actin inhibits the formin INF2

INF2 is a member of the formin family of actin assembly factors. Dominant mis-sense mutations in INF2 link to two diseases: focal segmental glomerulosclerosis (FSGS), a kidney disease; and Charcot-Marie-Tooth disease (CMTD), a neuropathy. All disease mutations map to the autoinhibitory Diaphanous Inhibitory Domain (DID). Curiously, purified INF2 is not autoinhibited, suggesting the existence of additional cellular inhibitors. We purified an INF2 inhibitor from mouse brain, and identified it as a complex between lysine-acetylated actin (KAc-actin) and cyclase-associated protein (CAP). Inhibition of INF2 by CAP/KAc-actin requires INF2 DID. Treatment of CAP/KAc-actin with histone deacetylase 6 (HDAC6) releases INF2 inhibition, while HDAC6 inhibitors block cellular INF2 activation. INF2 disease mutants are poorly inhibited by CAP/KAc-actin, suggesting that FSGS and CMTD result from reduced CAP/KAc-actin binding. This is the first demonstrated role for lysine-acetylated actin: regulation of an actin assembly factor by a novel mechanism, which we call facilitated auto-inhibition.

biochemistry↗

bioRxiv2019

Generating quantitative binding landscapes through fractional binding selections, deep sequencing and data normalization

Quantifying the effects of various mutations on binding free energy is crucial for understanding the evolution of protein-protein interactions and would greatly facilitate protein engineering studies. Yet, measuring changes in binding free energy ({Delta}{Delta}Gbind) remains a tedious task that requires expression of each mutant, its purification, and affinity measurements. We developed a new approach that allows us to quantify {Delta}{Delta}Gbind for thousands of protein mutants in one experiment. Our protocol combines protein randomization, Yeast Surface Display technology, Next Generation Sequencing, and a few experimental {Delta}{Delta}Gbind data points on purified proteins to generate {Delta}{Delta}Gbind values for the remaining numerous mutants of the same protein complex. Using this methodology, we comprehensively map the single-mutant binding landscape of one of the highest-affinity interaction between BPTI and Bovine Trypsin. We show that {Delta}{Delta}Gbind for this interaction could be quantified with high accuracy over the range of 12 kcal/mol displayed by various BPTI single mutants.

biochemistry↗

bioRxiv2019

Kinetics of enzymatic mercury methylation at nanomolar concentrations catalyzed by HgcAB

Methylmercury (MeHg) is a potent neurotoxin that bioaccumulates in fish. MeHg is generated by anaerobic bacteria and archaea possessing the gene pair hgcAB. Although bacterial mercury (Hg) methylation has been characterized in vivo, the specific role of HgcAB in catalyzing Hg methylation is not well understood. Here we report the kinetics of HgcAB-mediated Hg methylation in cell lysates of Desulfovibrio desulfuricans ND132 at nanomolar Hg concentrations. The enzymatic Hg methylation mediated by HgcAB is highly oxygen-sensitive, irreversible, and follows Michaelis-Menten kinetics with an apparent KM of 3.2 nM and Vmax of 19.7 fmol{middle dot}min-1{middle dot}mg-1 total protein for the substrate Hg(II). Although the abundance of HgcAB in the cell lysates is extremely low, Hg(II) was quantitatively converted to MeHg at subnanomolar substrate concentrations. Supplementation with ATP, methyltetrahydrofolate, or pyruvate did not enhance MeHg production under the experimental conditions. Insight into the kinetics of Hg methylation catalyzed by HgcAB advances our understanding of the complex global Hg cycle.

biochemistry↗

bioRxiv2019

In vitro analysis of RNA polymerase II elongation complex dynamics

RNA polymerase II elongation complexes (ECs) were assembled from nuclear extract on immobilized DNA templates and analyzed by quantitative mass spectrometry. Time course experiments showed that initiation factor TFIIF can remain bound to early ECs, while levels of core elongation factors Spt4-Spt5, Paf1C, Spt6-Spn1, and Elf1 levels remain steady. Importantly, the dynamic phosphorylation patterns of the Rpb1 C-terminal domain (CTD), and the factors that recognize them, change as a function of post-initiation time, rather than distance elongated. Chemical inhibition of Kin28/Cdk7 blocks both Serine 5 and Serine 2 phosphorylation, affects initiation site choice, and inhibits elongation efficiency. EC components dependent on CTD phosphorylation include capping enzyme, Cap Binding Complex, Set2, and the PAF1 complex. By recapitulating many known features of in vivo elongation, this system reveals new details that clarify how EC-associated core elongation factors, chromatin regulators, and RNA processing factors change at each step of transcription.

biochemistry↗

bioRxiv2019

The UTX Tumor Suppressor Directly Senses Oxygen to Control Chromatin and Cell Fate

Mammalian cells express multiple 2-oxoglutarate (OG)-dependent dioxygenases, including many chromatin regulators. The oxygen affinities, and hence oxygen sensing capabilities, of the 2-oxoglutarate (OG)-dependent dioxygenases reported to date vary widely. Hypoxia can affect chromatin, but whether this reflects a direct effect on chromatin-modifying dioxygenases, or indirect effects caused by the hypoxic-induction of the HIF transcription factor or the endogenous 2-OG competitor 2-hydroxyglutarate (2-HG), is unclear. Here we report that hypoxia induces a HIF- and 2-HG-independent histone modification signature consistent with KDM inactivation. We also show that the H3K27 histone demethylase KDM6A (also called UTX), but not its paralog KDM6B, is oxygen-sensitive. KDM6A loss, like hypoxia, prevented H3K27me3 erasure and blocked differentiation. Conversely, restoring H3K27me3 homeostasis in hypoxic cells reversed these effects. Therefore, oxygen directly affects chromatin regulators to control cell fate.\n\nOne Sentence SummaryKDM6A demethylase activity is diminished under hypoxic conditions and causes changes in gene expression programs that govern cell fate.

biochemistry↗

bioRxiv2019

A staphylococcal cyclophilin carries a single domain and unfolds via the formation of an intermediate that preserves cyclosporin A binding activity

Cyclophilin (Cyp), a peptidyl-prolyl cis-trans isomerase (PPIase), acts as a virulence factor in many bacteria including Staphylococcus aureus. The enzymatic activity of Cyp is inhibited by cyclosporin A (CsA), an immunosuppressive drug. To precisely determine the unfolding mechanism and the domain structure of Cyp, we have investigated a chimeric S. aureus Cyp (rCyp) using various probes. Our limited proteolysis and the consequent analysis of the proteolytic fragments indicate that rCyp is composed of one domain with a short flexible tail at the C-terminal end. We also show that the urea-induced unfolding of both rCyp and rCyp-CsA is completely reversible and proceeds via the synthesis of at least one stable intermediate. The secondary structure, tertiary structure, and the hydrophobic surface area of no intermediate are fully identical to those of other intermediate or the related native protein. Further analyses reveal no loss of CsA binding activity in rCyp intermediate. The thermodynamic stability of rCyp was also significantly increased in the presence of CsA, recommending that this protein could be employed to screen new CsA derivatives in future.

biochemistry↗

bioRxiv2019

Structure and function of a 9.6 megadalton bacterial iron storage compartment

Iron storage proteins are essential for maintaining intracellular iron homeostasis and redox balance. Iron is generally stored in a soluble and bioavailable form inside ferritin protein compartments. However, some organisms do not encode ferritins and thus rely on alternative storage strategies. Encapsulins, a class of protein-based organelles, have recently been implicated in microbial iron and redox metabolism. Here, we report the structural and mechanistic characterization of a 42 nm two-component encapsulin-based iron storage compartment from Quasibacillus thermotolerans. Using cryo-electron microscopy and x-ray crystallography, we reveal the assembly principles of a thermostable T = 4 shell topology and its catalytic ferroxidase cargo. We show that the cargo-loaded compartment has an exceptionally large iron storage capacity storing over 23,000 iron atoms. These results form the basis for understanding alternate microbial strategies for dealing with the essential element iron.

biochemistry↗

bioRxiv2019

WDR76 promotes MLL-rearranged leukemia via selective recognition of 5-hydroxymethylcytosine in DNA

Although rare, the distribution of the 5-hydroxymethylcytosine (hmC) modification in mammalian DNA is tissue- and gene-specific, yet distinct from its transcriptionally-repressive methylcytosine (mC) precursor, suggesting unique signaling potential. To examine this possibility, we fractionated mammalian brain extracts to discover binding partners specific for oxidized states of mC. We demonstrate that one such factor, WDR76, is a highly hmC-specific binding protein that modulates gene expression within chromosomal regions enriched in hmC where it binds. We demonstrate direct transcriptional activation of several target genes in mouse embryonic stem cells as a function of hmC levels and contingent upon WDR76. In human cell lines and mouse models, WDR76 recruitment by hmC is critical for the initiation and maintenance of MLL-rearranged leukemias. Beyond its canonical role as an intermediate in mC remediation, we show that hmC can be an epigenetic mark whose recognition drives leukemogenesis, portending analogous signaling pathways for other rare DNA modifications.

biochemistry↗

bioRxiv2019

Identification of Factors Complicating Bioluminescence Imaging

In vivo bioluminescence imaging (BLI) has become a standard, non-invasive imaging modality for following gene expression or the fate of proteins and cells in living animals. Currently, bioluminescent reporters used in laboratories are mostly derivatives of two major luciferase families: ATP-dependent insect luciferases and ATP-independent marine luciferases. Inconsistent results have been reported for experiments using different bio-luminescent reporters and users are often confused when trying to choose an optimal bioluminescent reporter for a given research purpose. Herein, we re-examined inconsistency in several experimental settings and identified factors, such as ATP dependency, serum stability, and molecular size, which significantly affected BLI results. We expect this study will make the research community aware of these factors and facilitate more accurate interpretation of BLI data by considering the nature of each bioluminescent reporter.

biochemistry↗

bioRxiv2019

Interaction of HelQ helicase with RPA modulates RPA-DNA binding and stimulates HelQ to unwind DNA through a protein roadblock.

Cells reactivate compromised DNA replication forks using enzymes that include DNA helicases for separating DNA strands and remodelling protein-DNA complexes. HelQ helicase promotes replication-coupled DNA repair in mammals in a network of interactions with other proteins. We report newly identified HelQ helicase activities, when acting alone and when interacting with RPA. HelQ helicase was strongly inhibited by a DNA-protein barrier (BamHIE111A), and by an abasic site in the translocating DNA strand. Interaction of HelQ with RPA activated DNA unwinding through the protein barrier, but not through the abasic site. Activation was lost when RPA was replaced with bacterial SSB or DNA binding-defective RPA, RPAARO1. We observed stable HelQ-RPA-DNA ternary complex formation, and present evidence that an intrinsically disordered N-terminal region of HelQ (N-HelQ) interacts with RPA, destabilising RPA-DNA binding. Additionally, SEC-MALS showed that HelQ multimers are converted into catalytically active dimers when ATP-Mg2+ is bound. HelQ and RPA are proposed to jointly promote replication fork recovery by helicase-catalysed displacement of DNA-bound proteins, after HelQ gains access to ssDNA through its N-terminal domain interaction with RPA.

biochemistry↗

bioRxiv2019

Systematic Development Of Sandwich Immunoassays For The Plasma Secretome

The plasma proteome offers a clinically useful window into human health and disease. With recent progress made on the development of highly multiplexed immunoassays with high sample throughput, a remaining need is to establish a pipeline for validating the individual proteins that build such bio-signatures by using targeted assays. In order to streamline such efforts, we developed a workflow to build dual binder sandwich immunoassays (SIA) and chose to evaluate this on proteins predicted to be secreted form cells and tissues. Utilizing the multiplexing capacities of the bead array technology, we first screened ~ 1,800 unique antibody pairs against 209 protein targets and collected data from dilution series of recombinant proteins as well as EDTA plasma. Employing 624 unique antibodies from the Human Protein Atlas, we obtained dilution-dependent curves in plasma and concentration-dependent curves of full-length proteins for 102 (49%) of the targets. For 22 protein assays, the longitudinal, inter-individual and technical performance was determined in a set of plasma samples collected from 18 healthy subjects every third month over one year. Lastly, we compared 14 of these assays with SIAs composed of other binders, proximity extension assays and affinity-free targeted mass spectrometry. Our workflow provides a multiplexed approach to screen for SIA pairs that suggests using at least three antibodies per target. This design is applicable for a wider range of targets of the plasma proteome, while the assays can be applied for discovery but also to validate emerging candidates derived from other platforms.

biochemistry↗