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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2017

Skin biomarkers for cystic fibrosis: a potential noninvasive approach for patient screening.

BackgroundCystic fibrosis is a disabling genetic disease with an increased prevalence in populations with European heritage. Currently, the most used technique for collection of cystic fibrosis samples and diagnosis is provided through uncomfortable tests, with uncertain results, mostly based on chloride concentration in sweat. Since cystic fibrosis mutation induces many metabolic changes in patients, exploring these alterations might be an alternative to visualize potential biomarkers that could be used as interesting tools for further diagnostic upgrade, prioritizing simplicity, low cost and quickness.\n\nMethodsThis contribution describes an accurate strategy to provide potential biomarkers related to cystic fibrosis, which may be understood as a potential tool for new diagnostic approaches and/or for monitoring disease evolution. Therefore, the present proposal consists of using skin imprints on silica plates as a way of sample collection, followed by direct-infusion high-resolution mass spectrometry and multivariate data analysis, intending to identify metabolic changes in skin composition of cystic fibrosis patients.\n\nResultsMetabolomics analysis allowed identifying chemical markers that can be traced back to cystic fibrosis in patients skin imprints, differently from control subjects. Seven chemical markers from several molecular classes were elected, represented by bile acids, a glutaric acid derivative, thyrotropin releasing hormone, an inflammatory mediator, a phosphatidic acid, and diacylglycerol isomers, all reflecting metabolic disturbances that occur due to of cystic fibrosis.\n\nConclusionThe comfortable method of sample collection combined with the identified set of biomarkers represent potential tools that open the range of possibilities to manage cystic fibrosis and follow the disease evolution. This exploratory approach points to new perspectives about cystic fibrosis management and maybe to further development of a new diagnostic assay based on them.

biochemistry↗

bioRxiv2017

Hsf1 and Hsp70 constitute a two-component feedback loop that regulates the yeast heat shock response

Models for regulation of the eukaryotic heat shock response typically invoke a negative feedback loop consisting of the transcriptional activator Hsf1 and a molecular chaperone encoded by an Hsf1 target gene. Previously, we identified Hsp70 as the chaperone responsible for Hsf1 repression in Saccharomyces cerevisiae and constructed a mathematical model based on Hsp70-mediated negative feedback that recapitulated the dynamic activity of Hsf1 during heat shock. The model was based on two assumptions: dissociation of Hsp70 activates Hsf1, and transcriptional induction of Hsp70 deactivates Hsf1. Here we validated these assumptions. First, we severed the feedback loop by uncoupling Hsp70 expression from Hsf1 regulation. As predicted by the model, Hsf1 was unable to efficiently deactivate in the absence of Hsp70 transcriptional induction. Next we mapped a discrete Hsp70 binding site on Hsf1 to a motif in the C-terminal activation domain known as conserved element 2 (CE2). Removal of CE2 resulted in increased Hsf1 activity under non-heat shock conditions and delayed deactivation kinetics. In addition, we uncovered a role for the N-terminal domain of Hsf1 in negatively regulating DNA binding. These results reveal the quantitative control mechanisms underlying the feedback loop charged with maintaining cytosolic proteostasis.

biochemistry↗

bioRxiv2017

Polysaccharide-mediated synthesis of melanins from serotonin and other 5-hydroxy indoles

As a continuation of our research on the melanin formation from catecholamines, we studied the polysaccharide-mediated oxidation of serotonin and other 5-hydroxy indoles into melanin-like materials. As for the catecholamines, we observed that many polysaccharides promote the oxidation of such compounds, particularly in the presence of Cu2+. The reactions were monitored using RP-HPLC and SEC techniques. Melanin-like materials were purified through dialysis and evaluated using UV_Vis and FT_IR spectroscopic techniques. One such material, synthesized from chondroitin sulfate type A and serotonin in the presence of Cu2+ was found to affect the release of IL-l{beta} and IL-6 cytokines from immune cells.

biochemistry↗

bioRxiv2017

Synthesis of a eukaryotic chromosome reveals a role for N6-methyladenine in nucleosome organization

Biochemical studies of chromatin have typically used either artificial DNA templates with unnaturally high affinity for histones, or small genomic DNA fragments deprived of their cognate physical environment. It has thus been difficult to dissect chromatin structure and function within fully native DNA substrates. Here, we circumvent these limitations by exploiting the minimalist genome of the eukaryote Oxytricha trifallax, whose notably small ~3kb chromosomes mainly encode single genes. Guided by high-resolution epigenomic maps of nucleosome organization, transcription, and DNA N6-methyladenine (m6dA) locations, we reconstruct full-length Oxytricha chromosomes in vitro and use these synthetic facsimiles to dissect the influence of m6dA and histone post-translational modifications on nucleosome organization. We show that m6dA directly disfavors nucleosomes in a quantitative manner, leading to local decreases in nucleosome occupancy that are synergistic with histone acetylation. The effect of m6dA can be partially reversed by the action of an ATP-dependent chromatin remodeler. Furthermore, erasing m6dA marks from Oxytricha chromosomes leads to proportional increases in nucleosome occupancy across the genome. This work showcases Oxytricha chromosomes as powerful yet practical models for studying eukaryotic chromatin and transcription in the context of biologically relevant DNA substrates.\n\nHighlightsO_LIDe novo synthesis of complete, epigenetically defined Oxytricha chromosomes\nC_LIO_LIEpigenomic profiles of chromatin organization in Oxytrichas miniature chromosomes\nC_LIO_LIm6dA directly disfavors nucleosome occupancy in natural and synthetic chromosomes\nC_LIO_LIHistone acetylation and chromatin remodelers temper the impact of m6dA on chromatin\nC_LI

biochemistry↗

bioRxiv2017

Interleukin-1β and -6 release from immune cells by DOPA-based melanin as free pigment or complexed to carboxymethylcellulose

We have observed that many polysaccharides can promote the oxidation of 3,4-dihydroxyphenylalanine (DOPA) into melanin-like pigments leading to the formation of water-soluble polysaccharide/melanin complexes. These pigments were characterized by size exclusion chromatography and FT-IR spectroscopy. The effect on the secretion of interleukin (IL)-l{beta} and IL-6 from immune cells by DOPA-based melanin synthesized in the presence or absence of carboxymethylcellulose (CMC) was evaluated. We observed that the melanin/CMC complex had a more potent effect on both IL secretions compared to the melanin prepared from DOPA in the absence of any polysaccharide. The study of the effect of melanins on the IL secretion by immune or other cells will help illuminate the potential contributions of this broad class of pigments to pathological conditions like Parkinsons disease or ochronosis.

biochemistry↗

bioRxiv2017

Crystal structure of BrlR reveals a potential pyocyanin binding site

The transcriptional regulator BrlR from Pseudomonas aeruginosa is a member of the MerR family of multidrug transport activators. Studies have shown BrlR plays an important role in high level drug tolerance of P. aeruginosa in biofilm. Its drug tolerance ability can be enhanced by 3',5'-cyclic diguanylic acid (c-di-GMP). Here, we show the apo structure of BrlR and the direct binding between GyrI-like domain of BrlR and P. aeruginosa toxin pyocyanin. Furthermore, pyocyanin can enhance the binding between BrlR and DNA in vitro. These findings suggest BrlR can serve as the binding partner for both c-di-GMP and pyocyanin.

biochemistry↗

bioRxiv2017

Nucleolin internalizes Bothrops asper Lys49 phospholipase A2 forming cell surface amyloid-like assemblies

Phospholipases A2 (PLA2s) are a major component of snake venoms. Some of them cause severe muscle necrosis through a still unknown mechanism. Phospholipid hydrolysis is a possible explanation of their toxic action, but catalytic and toxic properties of PLA2s are not directly connected. In addition, viperid venoms contain PLA2-like proteins, which are very toxic even if they lack catalytic activity due to a critical mutation in position 49. Nucleolin, a main component of the nucleolus, is a disordered protein involved in many protein assembly and phase separation phenomena. In some circumstances nucleolin is exposed on the cell surface from where it is involved in the internalization of many ligands.\n\nIn this work we demonstrate that Bothrops asper myotoxin II (Mt-II), a Lys49 PLA2-like toxin, interacts with, and is internalized in cells by nucleolin. The internalization process is functional to the toxicity of the protein, as both an antibody and an aptamer specific for nucleolin protect cells from intoxication. We identified central RRM and the C-terminal R/F-GG domain of nucleolin as the regions involved in the interaction with Mt-II. Finally we observed that Mt-II forms, on the cell surface, amyloid-like assemblies that colocalize with nucleolin and that can be involved in the activation of the internalization process. The presence, in the three dimensional structure of Mt-II and related PLA2 homologues, of four exposed loops enriched in prion-like amino acid sequences reinforces this hypothesis.\n\nPhospholipases A2 | Lys49 myotoxins | nucleolin | amyloid-like | molecular assemblies\n\nSIGNIFICANCEThe main finding of this work, the role of nucleolin as Bothrops asper Mt-II receptor, is a remarkable step forward in understanding the mechanism of action of cytotoxic PLA2s. It may suggest new strategies for anti-venom therapies and explain the anti-tumoral and anti-viral pharmacological action of snake PLA2s, since nucleolin is a receptor for many growth factors and virus.\n\nThe proposed internalization mechanism, via formation of molecular assemblies among Mt-II amyloid-like structures and other proteins, including nucleolin, can be of general validity. Cell surface molecular assemblies couldbepointsofselectionandconcentrationnotonlyofsnake,butalsoofmammaliansecretedPLA2s, proteins involved in different pathologies, and trigger the internalization pathway only when their molarity exceeds a threshold dose.

biochemistry↗

bioRxiv2017

A Cdk1 phosphomimic mutant of MCAK impairs microtubule end recognition.

The microtubule depolymerising kinesin-13, MCAK, is phosphorylated at residue T537 by Cdk1. This is the only known phosphorylation site within MCAKs motor domain. To understand the impact of phosphorylation by Cdk1 microtubule depolymerisation activity, we have investigated the molecular mechanism of the phosphomimic mutant T537E. This mutant significantly impairs microtubule depolymerisation activity and when transfected into cells causes metaphase arrest and misaligned chromosomes. We show that the molecular mechanism underlying the reduced depolymerisation activity of this phosphomimic mutant is an inability to recognise the microtubule end. The microtubule-end residence time is reduced relative to wild-type MCAK, whereas the lattice residence time is unchanged by the phosphomimic mutation. Further, the microtubule-end specific stimulation of ADP dissociation, characteristic of MCAK, is abolished by this mutation. Our data shows that T537E is unable to distinguish between the microtubule end and the microtubule lattice.

biochemistry↗

bioRxiv2017

Biomarker evaluation of plasma microRNA-122, high-mobility group box-1 and keratin-18 in acute gallstone disease

BackgroundA biomarker that stratifies patients with complications of gallstone disease from the denominator pool of people with acute biliary-type symptoms is needed. Circulating microRNA-122 (miRNA-122), high-mobility group box1 (HMGB1), full-length keratin-18 (flk-18) and caspase-cleaved keratin-18 (cck-18) are established hepatocyte injury biomarkers. The aim of this study was to evaluate the discriminatory power of these biomarkers in plasma to identify potential biliary complications that may require acute intervention.\n\nMethodAn observational biomarker cohort study was carried out in a University teaching hospital for 12 months beginning 3rd September 2014. Blood samples were collected from adults referred with acute biliary-type symptoms. miRNA-122 was measured by quantitative real-time polymerase chain reaction, and HMGB1, cck-18 and flk-18 by ELISA.\n\nResults300 patients were screened and 289 patients were included. Plasma miRNA-122, cck-18 and flk-18 concentrations were increased in patients with gallstones compared to those without (miRNA-122: median: 2.89 x 104 copies/ml vs. 0.95 x 104 copies/ml [P<0.001]; cck-18: 121.9 U/L vs. 104.6 U/L [P = 0.041]; flk-18: 252.4 U/L vs. 151.8 U/L [P<0.001]). Uncomplicated gallstone disease was associated with higher miRNA-122 and cck-18 concentrations than complicated disease (miRNA-122: 5.72 x 104 copies/ml vs. 2.26x104 copies/ml [P=0.022]; cck-18: 139.7 U/L vs. 111.4 U/L [P=0.049]). There was no significant difference in HMGB1 concentration between patients with and without gallstones [P=0.480]. Separation between groups for all biomarkers was modest.\n\nConclusionmicroRNA-122 and keratin-18 plasma concentrations are elevated in patients with gallstones. However, these biomarkers were not sufficiently discriminatory to be progressed as clinically useful biomarkers in this context.

biochemistry↗

bioRxiv2017

Unique ATP-cone-driven allosteric regulation of ribonucleotide reductase via the radical-generating subunit

Ribonucleotide reductases (RNRs) are key enzymes in DNA synthesis and repair, with sophisticated allosteric mechanisms controlling both substrate specificity and overall activity. In RNRs, the activity master-switch, the ATP-cone, has been found exclusively in the catalytic subunit. In two class I RNR subclasses whose catalytic subunit lacks the ATP-cone, we discovered ATP-cones in the radical-generating subunit. The ATP-cone in the Leewenhoekiella blandensis radical-generating subunit regulates activity via modifications of quaternary structure induced by binding of nucleotides. ATP induces enzymatically competent dimers, whereas dATP induces non-productive tetramers, resulting in different holoenzyme complexes. The tetramer forms solely by interactions between ATP-cones, as evidenced by a 2.45 [A] crystal structure. We also present evidence for an MnIIIMnIV metal center. In summary, lack of an ATP-cone domain in the catalytic subunit was compensated by evolutionary capture of the domain by the radical-generating subunit. Our findings present a novel opportunity for dATP-regulation of engineered proteins.

biochemistry↗

bioRxiv2017

RecBCD possesses strong coupling between DNA and nucleotides binding that may propel stepping mechanism during translocation

Double-strand DNA breaks are the severest type of genomic damage, requiring rapid response to ensure survival. RecBCD helicase in prokaryotes initiates processive and rapid DNA unzipping essential for break repair. Yet, the energetics of RecBCD during translocation along the DNA track needs to be quantitatively clarified. Specifically, its essential to understand how RecBCD switches between its binding states to enable its translocation. Here we determine, by systematic affinity measurements, the degree of coupling between DNA and nucleotide binding to RecBCD. We show that, in the presence of ADP, RecBCD binds weakly to DNA that harbors a double overhang mimicking an unwinding intermediate. Consistently, RecBCD binds weakly to ADP in the presence of the same DNA. We did not observe coupling between DNA and nucleotide binding for DNA molecules having only a single overhang, suggesting that RecBCD subunits must both bind DNA to "sense" the nucleotide state. Excitingly, we observed weak coupling for AMPpNp as RecBCD remains strongly bound to DNA in its presence. Detailed thermodynamic analysis of RecBCD reaction mechanism suggests an energetic compensation between RecB and RecD, which may be essential for rapid unwinding. Our findings provide the basis for a stepping mechanism during the processive translocation of RecBCD.

biochemistry↗

bioRxiv2017

Biogenesis of a mitochondrial DNA inheritance machinery in the mitochondrial outer membrane

Mitochondria cannot form de novo but require mechanisms that mediate their inheritance to daughter cells. The parasitic protozoan Trypanosoma brucei has a single mitochondrion with a single-unit genome that is physically connected across the mitochondrial membranes to the basal body of the flagellum. This connection, termed tripartite attachment complex (TAC), is essential for the segregation of the replicated mitochondrial genomes prior to cytokinesis. Here we identify a protein complex consisting of three integral mitochondrial outer membrane proteins - TAC60, TAC42 and TAC40 - which are essential subunits of the TAC. TAC60 contains separable mitochondrial import and TAC-sorting signals and its biogenesis depends on the main outer membrane protein translocase. TAC40 is a member of the mitochondrial porin family, whereas TAC42 represents a novel class of mitochondrial outer membrane {beta}-barrel proteins. Consequently TAC40 and TAC42 contain C-terminal {beta}-signals. Thus in trypanosomes the highly conserved {beta}-barrel protein assembly machinery plays a major role in the biogenesis of its unique mitochondrial genome segregation system.

biochemistry↗

bioRxiv2017

Out-of-Register Aβ42 Assemblies as Models for Neurotoxic Oligomers and Fibrils

We propose a variant of the recently found S-shaped A{beta}1-42-motif that is characterized by out-of-register C-terminal {beta}-strands. We show that chains with this structure can not only form fibrils that are compatible with the NMR signals, but also barrel-shaped oligomers that resemble the ones formed by the much smaller cylindrin peptides. Running at physiological temperatures long all-atom molecular dynamics simulations with an explicit solvent, we study the stability of these constructs and show that they are plausible models for neurotoxic oligomers. Analyzing the transitions between different assemblies we suggest a mechanism for amyloid formation in Alzheimers disease.

biochemistry↗

bioRxiv2017

Lipid-Uptake Pathways and Lipid-Protein interactions in P-glycoprotein Revealed by Coarse-Grained Molecular Dynamics Simulations

P-glycoprotein (P-gp) exports a broad range of dissimilar compounds, including drugs, lipids and lipid-like molecules. Due to its substrate promiscuity, P-gp is a key player in the development of cancer multidrug resistance (MDR). Although P-gp is one of the most studied members of ABC-transporters, the mechanism of how its substrates access the cavity remains unclear. In this work, we performed coarse-grained (CG) molecular dynamics (MD) simulations to explore possible pathways of lipid-uptake in the inward-facing conformation of P-gp embedded in bilayers with different PC:PE lipid ratios. Our results show that in the inward facing orientation only lipids from the lower leaflet are taken up by the transporter. We identify positively charged residues at the portals of P-gp that favor lipid entrance to the cavity, as well as lipid binding sites, in good agreement with previous experimental studies. Our results show no selectivity for PC vs. PE lipids. We offer several examples of lipid uptake-pathways for PC and PE lipids that help to elucidate the molecular mechanism of substrate-uptake in P-gp.

biochemistry↗

bioRxiv2017

PyFolding: An open-source software package for graphing, analysis and simulation of thermodynamic and kinetic models of protein folding

Abstract]For many years, curve fitting software has been heavily utilized to fit simple models to various types of biophysical data. Although such software packages are easy to use for simple functions, they are often expensive and present substantial impediments to applying more complex models or for the analysis of large datasets. One field that is relient on such data analysis is the thermodynamics and kinetics of protein folding. Over the past decade, increasingly sophisticated analytical models have been generated, but without simple tools to enable routine analysis. Consequently, users have needed to generate their own tools or otherwise find willing collaborators. Here we present PyFolding, a free, open source, and extensible Python framework for graphing, analysis and simulation of the biophysical properties of proteins. To demonstrate the utility of PyFolding, we have used it to analyze and model experimental protein folding and thermodynamic data. Examples include: (i) multi-phase kinetic folding fitted to linked equations, (ii) global fitting of multiple datasets and (iii) analysis of repeat protein thermodynamics with Ising model variants. Moreover, we demonstrate how Pyfolding is easily extensible to novel functionality beyond applications in protein folding via the addition of new models. Example scripts to perform these and other operations are supplied with the software, and we encourage users to contribute notebooks and models to create a community resource. Finally, we show that PyFolding can be used in conjunction with Jupyter notebooks as an easy way to share methods and analysis for publication and amongst research teams.

biochemistry↗

bioRxiv2017

Dephosphorylation is the Mechanism of Fibroblast Growth Factor Inhibition of Guanylyl Cyclase-B

Activating mutations in fibroblast growth factor receptor 3 (FGFR3) and inactivating mutations of guanylyl cyclase-B (GC-B, also called NPRB or NPR2) cause dwarfism. FGF exposure inhibits GC-B activity in a chondrocyte cell line, but the mechanism of the inactivation is not known. Here, we report that FGF exposure causes dephosphorylation of GC-B in rat chondrosarcoma cells, which correlates with a rapid, potent and reversible inhibition of C-type natriuretic peptide-dependent activation of GC-B. Cells expressing a phosphomimetic mutant of GC-B that cannot be inactivated by dephosphorylation because it contains glutamate substitutions for all known phosphorylation sites showed no decrease in GC-B activity in response to FGF. We conclude that FGF rapidly inactivates GC-B by a reversible dephosphorylation mechanism, which may contribute to the signaling network by which activated FGFR3 causes dwarfism.\n\nHighlightsO_LIGuanylyl Cyclase-B is expressed in rat chondrosarcoma cells\nC_LIO_LIFGF2 induces a rapid, potent, and reversible inhibition of GC-B\nC_LIO_LIFGF2 treatment causes GC-B dephosphorylation\nC_LIO_LIFGF2 does not inhibit a dephosphorylation-resistant form of GC-B\nC_LIO_LIDephosphorylation is the mechanism of FGF2-dependent inhibition of GC-B\nC_LI\n\nAbbreviations

biochemistry↗

bioRxiv2017

Molecular basis for the maintenance of lipid asymmetry in the outer membrane of Escherichia coli

A distinctive feature of the Gram-negative bacterial cell envelope is the asymmetric outer membrane (OM), where lipopolysaccharides (LPS) and phospholipids (PLs) reside in the outer and inner leaflets, respectively. This unique lipid asymmetry renders the OM impermeable to external insults. In Escherichia coli, the OmpC-MlaA complex is believed to maintain lipid asymmetry by removing mislocalized PLs from the outer leaflet of the OM. How it performs this function is unknown. Here, we define the molecular architecture of the OmpC-MlaA complex to gain insights into its role in PL transport. We establish that MlaA sits entirely within the bilayer in complex with OmpC and provides a hydrophilic channel possibly for PL translocation across the OM. Furthermore, we show that flexibility in a hairpin loop adjacent to the channel modulates MlaA activity. Finally, we demonstrate that OmpC plays an active role in maintaining OM lipid asymmetry together with MlaA. Our work offers glimpses into how the OmpC-MlaA complex transports PLs across the OM and has important implications for future antimicrobial drug development.

biochemistry↗

bioRxiv2017

Fibril branching dominates self-assembly of mutant huntingtin exon-1 aggregates in vitro

Huntingtin (HTT) fragments with extended polyglutamine (polyQ) tracts self-assemble into amyloid-like fibrillar aggregates. Elucidating the fibril formation mechanism is critical for understanding Huntingtons disease pathology and for developing novel therapeutic strategies. Here, we performed systematic experimental and theoretical studies to examine the self-assembly of an aggregation-prone N-terminal HTT exon-1 fragment with 49 glutamines (Ex1Q49). Using high resolution imaging techniques such as electron microscopy and atomic force microscopy, we show that Ex1Q49 fragments in cell-free assays spontaneously convert into large, highly complex bundles of amyloid fibrils with multiple ends and fibril branching points. Furthermore, we present experimental evidence that two nucleation mechanisms control spontaneous Ex1Q49 fibrillogenesis: (1) a relatively slow primary fibril-independent nucleation process, which involves the spontaneous formation of aggregation-competent fibrillary structures, and (2) a fast secondary fibril-dependent nucleation process, which involves nucleated branching and promotes the rapid assembly of highly complex fibril bundles with multiple ends. The proposed aggregation mechanism is supported by studies with the small molecule O4, which perturbs early events in the aggregation cascade and delays Ex1Q49 fibril assembly, comprehensive mathematical and computational modelling studies, and seeding experiments with small, preformed fibrillar Ex1Q49 aggregates that promote the assembly of amyloid fibrils. Together, our results suggest that nucleated branching in vitro plays a critical role in the formation of complex fibrillar HTT exon-1 aggregates with multiple ends.

biochemistry↗