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Rxivist combines preprints from bioRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 67,588 bioRxiv papers from 298,004 authors.

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in category biochemistry

1,955 results found. For more information, click each entry to expand.

1801: Zinc-α2-Glycoprotein Is An Inhibitor Of Amine Oxidase Copper-Containing 3
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Posted to bioRxiv 07 Aug 2019

Zinc-α2-Glycoprotein Is An Inhibitor Of Amine Oxidase Copper-Containing 3
104 downloads biochemistry

Matthias Romauch

Zinc-alpha2-glycoprotein (ZAG) is a major plasma protein whose levels increase in chronic energy-demanding diseases and thus serves as an important clinical biomarker in the diagnosis and prognosis of the development of cachexia. Current knowledge suggests that ZAG mediates progressive weight loss through beta-adrenergic signaling in adipocytes, resulting in the activation of lipolysis and fat mobilization. Here, through crosslinking experiments, amine oxidase copper-containing 3 (AOC3) is identified as a novel ZAG binding partner. AOC3, also known as vascular adhesion protein 1 (VAP-1) and semicarbazide sensitive amine oxidase (SSAO), deaminates primary amines, thereby generating the corresponding aldehyde, H2O2 and HN3. It is an ectoenzyme largely expressed by adipocytes and induced in endothelial cells during inflammation. Extravasation of immune cells depends on amine oxidase activity and AOC3-derived H2O2 has an insulinogenic effect. The observations described here suggest that ZAG acts as an allosteric inhibitor of AOC3 and interferes with the associated pro-inflammatory and anti-lipolytic functions. Thus, inhibition of the deamination of lipolytic hormone octopamine by AOC3 represents a novel mechanism by which ZAG might stimulate lipolysis. Furthermore, experiments involving overexpression of recombinant ZAG reveal that its glycosylation is co-regulated by oxygen availability and that the pattern of glycosylation affects its inhibitory potential. The newly identified protein interaction between AOC3 and ZAG highlights a previously unknown functional relationship, which may be relevant to inflammation, energy metabolism and the development of cachexia.

1802: The nuclear transport receptor TNPO1 binds macrophage immunometabolism regulator MACIR via a PY-NLS motif
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Posted to bioRxiv 14 Nov 2019

The nuclear transport receptor TNPO1 binds macrophage immunometabolism regulator MACIR via a PY-NLS motif
103 downloads biochemistry

Gavin McGauran, Emma Dorris, Razvan Borza, Niamh Morgan, Denis C. Shields, David Matallanas, Anthony G Wilson, David J. O’Connell

Expression of the macrophage immunometabolism regulator gene (MACIR) is associated with severity of autoimmune disease pathology and the regulation of macrophage biology through unknown mechanisms. The 206 amino acid protein lacks homology to any characterized protein sequence and is a disordered protein according to structure prediction algorithms. Here we identify specific interactions of MACIR using a fragment complementation-based affinity pull down of cellular proteins prepared with a membrane solubilization buffer. Quantitative mass spectrometry showed enrichment of nuclear and mitochondrial proteins and of 63 significant interacting proteins, binding to the nuclear transport receptor TNPO1 and trafficking proteins UNC119 homolog A and B were validated by immunoprecipitation. Analysis of mutations in two candidate recognition motifs in the MACIR amino acid sequence confirmed TNPO1 binds via a PY-NLS motif (aa98-117). Characterizing nuclear MACIR activity in macrophage and fibroblasts is a priority with respect to developing strategies for treatment of autoimmune disease.

1803: Laccase-mediated catalyzed fluorescent reporter deposition for live cell imaging
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Posted to bioRxiv 10 Aug 2019

Laccase-mediated catalyzed fluorescent reporter deposition for live cell imaging
103 downloads biochemistry

Brandon Cisneros, Neal K. Devaraj

Catalyzed reporter deposition (CARD) is a widely established method for labeling biological samples analyzed using microscopy. Horseradish peroxidase, commonly used in CARD to amplify reporter signals, requires the addition of hydrogen peroxide which may perturb samples used in live-cell microscopy. Herein we describe an alternative method of performing CARD using a laccase enzyme, which does not require exogenous hydrogen peroxide. Laccase is an oxidative enzyme which can carry out single-electron oxidations of phenols and related compounds by reducing molecular oxygen. We demonstrate proof-of-concept for this technique through the non-targeted covalent labeling of bovine serum albumin using a fluorescently-labeled ferulic acid derivative as the laccase reporter substrate. We further demonstrate the viability of this approach by performing live-cell CARD with an antibody-conjugated laccase against a surface bound target. CARD using laccase produces an amplified fluorescence signal by labeling cells without the need for exogenous hydrogen peroxide.

1804: Unprocessed serum glycosylphosphatidylinositol-anchored proteins are correlated to metabolic states
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Posted to bioRxiv 28 Nov 2018

Unprocessed serum glycosylphosphatidylinositol-anchored proteins are correlated to metabolic states
103 downloads biochemistry

Günter A. Müller, Andreas W. Herling, Kerstin Stemmer, Andreas Lechner, Matthias H Tschöp

To study the possibility that components of eukaryotic plasma membranes are released in spontaneous or controlled fashion, a chip-based sensor was developed for complete glycosylphosphatidylinositol-anchored proteins (GPI-AP), which may form together with (phospho)lipids so far unknown (non-vesicular) extracellular complexes (GLEC). The sensor relies on changes in phase shift and amplitude of surface acoustic waves propagating over the chip surface upon specific capturing of the GPI-AP and detection of associated phospholipids and renders isolation of the labile GLEC unnecessary. GLEC were found to be released from isolated rat adipocyte plasma membranes immobilized on the chip, dependent on the flow rate and composition of the buffer stream. Moreover, incubation medium of isolated adipocytes and serum of rats are sources for GLEC which enables their differentiation according to cell size and genotype or body weight, respectively, as well as human serum.

1805: A selective and rapid cell-permeable inhibitor of human caspase-3
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Posted to bioRxiv 18 Jul 2019

A selective and rapid cell-permeable inhibitor of human caspase-3
102 downloads biochemistry

Angelo Solania, Gonzalo E. Gonzalez-Paez, Dennis W. Wolan

The individual roles and overlapping functionalities the twelve human caspases have during apoptosis and other cellular processes remain poorly resolved primarily due to a lack of chemical tools. Here we present a new selective caspase-3 inhibitor, termed Ac-ATS010-KE, with rapid and irreversible binding kinetics. Relative to previously designed caspase-3-selective molecules that have tremendously abated inhibitory rates and thus limited use in biological settings, the improved kinetics of Ac-ATS010-KE permit its use in a cell-based capacity. We demonstrate that Ac-ATS010-KE prevents apoptosis with comparable efficacy to the general caspase inhibitor Ac-DEVD-KE and surprisingly does so without side-chain methylation. This observation is in contrast to the well-established peptide modification strategy typically employed for improving cellular permeability. Ac-ATS010-KE protects against extrinsic apoptosis, which demonstrates the utility of a thiophene carboxylate leaving group in biological settings, challenges the requisite neutralization of free carboxylic acids to improve cell permeability, and provides a tool-like compound to interrogate the role of caspase-3 in a variety of cellular processes.

1806: Hat1-Dependent Lysine Acetylation Targets Diverse Cellular Functions
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Posted to bioRxiv 31 Oct 2019

Hat1-Dependent Lysine Acetylation Targets Diverse Cellular Functions
102 downloads biochemistry

Paula A Agudelo Garcia, Prabakaran Nagarajan, Mark R. Parthun

Lysine acetylation has emerged as one of the most important post-translational modifications, regulating different biological processes. However, its regulation by lysine acetyltransferases is still unclear in most cases. Hat1 is a lysine acetyltransferase originally identified based on its ability to acetylate histones. Using an unbiased proteomics approach, we have determined how loss of Hat1 affects the mammalian acetylome. Hat1+/+ and Hat1−/− mouse embryonic fibroblast (MEF) cells lines were grown in both glucose- and galactose-containing media, as Hat1 is required for growth on galactose and Hat1−/− cells exhibit defects in mitochondrial function. Following trypsin digestion of whole cell extracts, acetylated peptides were enriched by acetyllysine affinity purification and acetylated peptides were identified and analyzed by label-free quantitation. Comparison of the acetylome from Hat1+/+ cells grown on galactose and glucose demonstrated that there are large carbon source-dependent changes in the mammalian acetylome where the acetylation of enzymes involved in glycolysis was the most affected. Comparisons of the acetylomes from Hat1+/+ and Hat1−/− cells identified 65 proteins whose acetylation decreased by at least 2.5-fold in cells lacking Hat1. In Hat1−/− cells, acetylation of the auto regulatory loop of CBP was the most highly affected, decreasing by up to 20-fold. In addition to proteins involved in chromatin structure, Hat1-dependent acetylation was also found in a number of transcriptional regulators, including p53, and mitochondrial proteins. Hat1 mitochondrial localization suggests that it may be directly involved in the acetylation of mitochondrial proteins.

1807: Characterization of Sorbitol Dehydrogenase SmoS from Sinorhizobium meliloti 1021
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Posted to bioRxiv 01 Jul 2019

Characterization of Sorbitol Dehydrogenase SmoS from Sinorhizobium meliloti 1021
102 downloads biochemistry

MacLean G. Kohlmeier, Ben A. Bailey-Elkin, Brian L. Mark, Ivan J. Oresnik

Sinorhizobium meliloti 1021 is a Gram-negative alphaproteobacterium with a robust capacity for carbohydrate metabolism. The enzymes that facilitate these reactions assist in the survival of the bacterium across a range of environmental niches, and they may also be suitable for use in industrial processes. SmoS is a dehydrogenase that catalyzes the oxidation of the commonly occurring sugar alcohols sorbitol and galactitol into fructose and tagatose respectively using NAD+ as a cofactor. The main objective of this study is to evaluate SmoS using biochemical techniques. The nucleotide sequence was codon optimized for heterologous expression in E. coli BL21 (DE3) GOLD cells, the protein was subsequently overexpressed and purified. Size exclusion chromatography and X-ray diffraction experiments suggest that SmoS is a tetrameric peptide. SmoS was crystallized to 2.1 Å in the absence of substrate and 2.0 Å in complex with sorbitol. SmoS was characterized kinetically and shown to have a preference for sorbitol despite a higher affinity for galactitol. Computational ligand docking experiments suggest that galactitol oxidation proceeds slowly because tagatose binds the protein in a more energetically favorable complex than fructose, and is retained in the active site for a longer time frame following oxidation which reduces the rate of the reaction. These results supplement the inventory of biomolecules with the potential for industrial applications and enhance our understanding of metabolism in the model organism S. meliloti.

1808: MCH4 is a multicopy suppressor of glycine toxicity in Saccharomyces cerevisiae
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Posted to bioRxiv 29 May 2019

MCH4 is a multicopy suppressor of glycine toxicity in Saccharomyces cerevisiae
101 downloads biochemistry

Artem V Melnykov, Elliot L Elson

Saccharomyces cerevisiae can either import amino acids from the surrounding or synthesize inside the cell, and both processes are tightly regulated. Disruption of such regulation can result in amino acid toxicity to the cell through mechanisms that are poorly understood. In this study we make use of a mutant strain with deregulated general amino acid permease gene whose growth is inhibited by low concentrations of several amino acids. We carry out multicopy suppression screen with several toxic amino acids and identify MCH4 as a gene that suppresses inhibitory effects of glycine. We find that expression of MCH4 is regulated by osmotic shock but not other kinds of stress. These findings are discussed in the context of possible mechanisms of amino acid toxicity.

1809: Salt-free fractionation of complex isomeric mixtures of glycosaminoglycan oligosaccharides compatible with ESI-MS and microarray analysis
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Posted to bioRxiv 09 Sep 2019

Salt-free fractionation of complex isomeric mixtures of glycosaminoglycan oligosaccharides compatible with ESI-MS and microarray analysis
101 downloads biochemistry

Hao Liu, Apoorva Joshi, Pradeep Chopra, Lin Liu, Geert-Jan Boons, Joshua S. Sharp

Heparin and heparan sulfate (Hp/HS) are linear complex glycosaminoglycans which are involved in diverse biological processes. The structural complexity brings difficulties in separation, making the study of structure-function relationships challenging. Here we present a separation method for Hp/HS oligosaccharide fractionation with cross-compatible solvent and conditions, combining size exclusion chromatography (SEC), ion-pair reversed phase chromatography (IPRP), and hydrophilic interaction chromatography (HILIC) as three orthogonal separation methods that do not require desalting or extensive sample handling. With this method, the final eluent is suitable for structure-function relationship studies, including tandem mass spectrometry and microarray printing. Our data indicate that high resolution is achieved on both IPRP and HILIC for Hp/HS isomers. In addition, the fractions co-eluted in IPRP could be further separated by HILIC, with both separation dimensions capable of resolving some isomeric oligosaccharides. We demonstrate this method using both unpurified reaction products from isomeric synthetic hexasaccharides and an octasaccharide fraction from enoxaparin, identifying isomers resolved by this multi-dimensional separation method. We demonstrate both structural analysis by MS, as well as functional analysis by microarray printing and screening using a prototypical Hp/HS binding protein: basic-fibroblast growth factor (FGF2). Collectively, this method provides a strategy for efficient Hp/HS structure-function characterization.

1810: Defining how oncogenic and developmental mutations of PIK3R1 alter the regulation of class IA phosphoinositide 3-kinases
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Posted to bioRxiv 16 Jul 2019

Defining how oncogenic and developmental mutations of PIK3R1 alter the regulation of class IA phosphoinositide 3-kinases
101 downloads biochemistry

Gillian L Dornan, Jordan TB Stariha, Manoj K. Rathinaswamy, Cameron J Powell, Martin J Boulanger, John E Burke

The class IA PI3Ks are key signalling enzymes composed of a heterodimer of a p110 catalytic subunit and a p85 regulatory subunit, with PI3K mutations being causative of multiple human diseases including cancer, primary immunodeficiencies, and developmental disorders. Mutations in the p85α regulatory subunit encoded by PIK3R1 can both activate PI3K through oncogenic truncations in the iSH2 domain, or inhibit PI3K through developmental disorder mutations in the cSH2 domain. Using a combined biochemical and hydrogen deuterium exchange mass spectrometry approach we have defined the molecular basis for how these mutations activate both the p110α/p110δ catalytic subunits. We find that the oncogenic Q572* truncation of PIK3R1 disrupts all inhibitory inputs, with p110α being hyper-activated compared to p110δ. In addition, we find that the R649W mutation in the cSH2 of PIK3R1 decreases sensitivity to activation by receptor tyrosine kinases. This work reveals novel insight into isoform specific regulation of p110s by p85α.

1811: The distinct biochemical property enables thymidylate kinase as a drug target and participates in pyrimidine drug sensitivity in Candida albicans
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Posted to bioRxiv 09 Nov 2018

The distinct biochemical property enables thymidylate kinase as a drug target and participates in pyrimidine drug sensitivity in Candida albicans
101 downloads biochemistry

Chang Yu Huang, Yee-Chun Chen, Betty A. Wu-Hsieh, Jim-Min Fang, Zee-Fen Chang

The ability to overcome drug resistance in outbreaks of Candida albicans infection is an unmet need in health management. Here, we investigated CDC8, which encodes thymidylate kinase (TMPK), as a potential drug target for the treatment of C. albicans infection. In this study, we found that the specific region spanning amino acids 106-123, namely, the Ca-loop of C. albicans TMPK (CaTMPK) contributes to the hyperactivity of this enzyme compared to the human enzyme (hTMPK) and to the utilization of deoxy-uridine monophosphate (dUMP)/ deoxy-5-Fluorouridine monophosphate (5-FdUMP) as a substrate. Notably, CaTMPK but not hTMPK enables dUTP/5-FdUTP-mediated DNA toxicity in yeast. CRISPR-mediated deletion of this Ca-loop in C. albicans demonstrated the critical role of this Ca-loop in fungal growth and susceptibility to 5-Fluorouridine (5-FUrd). Moreover, pathogenic and drug-resistant C. albicans clones were similarly sensitive to 5-FUrd. Thus, this study not only identified a target site for the development of CaTMPK-selective drugs but also revealed 5-FUrd to be a potential drug for the treatment of C. albicans infection.

1812: ILEX PARAGUARIENSIS, EXERCISE AND CARDIOPROTECTION: A retrospective analysis
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Posted to bioRxiv 25 Oct 2018

ILEX PARAGUARIENSIS, EXERCISE AND CARDIOPROTECTION: A retrospective analysis
101 downloads biochemistry

Fábio Cahuê, José Hamilton Matheus Nascimento, Luciane Barcellos, Veronica P. Salerno

Studies on strategies to generate cardioprotective effects have been on the rise. Previous work by our group with an ex vivo model of ischemia/reperfusion has shown that both the short-term consumption of yerba mate and exercise can each induce protection of cardiac function independently. Surprising, the two strategies together do not, with an apparent loss of their respective cardioprotection activity. To improve our understanding of the mechanisms involved without reperforming the experiments, we have conducted a retrospective data science-analysis that have produced new insights. The analysis shows that yerba mate generated reductive stress. Alone, this stress increased redox damage in the heart that appears to have led to a protective conditioning. In combination with exercise, the effects of mate inhibited the intermittent ROS generation promoted by exercise alone, which diminished the adaptive response in the heart. These results suggest that an understanding of the molecular mechanisms involved with the yerba mate-promoted reductive stress in cardiac tissue could lead to improved strategies to induce cardioprotection.

1813: Musculoskeletal fat imaging and quantification by high-resolution metabolite cycling magnetic resonance spectroscopic imaging at 3 T: A fast method to generate separate distribution maps of lipid components
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Posted to bioRxiv 12 Aug 2019

Musculoskeletal fat imaging and quantification by high-resolution metabolite cycling magnetic resonance spectroscopic imaging at 3 T: A fast method to generate separate distribution maps of lipid components
101 downloads biochemistry

Ahmad A Alhulail, Debra A Patterson, Pingyu Xia, Xiaopeng Zhou, Chen Lin, M. Albert Thomas, Ulrike Dydak, Uzay Emir

Purpose: To provide a rapid, non-invasive fat quantification technique capable of producing separate lipid component maps. Methods: The calf muscles in 5 healthy adolescents (age 12-16 years; BMI = 20 ± 3 Kg/m2) were scanned by two different fat fraction (FF) quantification methods. A high-resolution, density-weighted concentric ring trajectory (DW-CRT) metabolite cycling (MC) magnetic resonance spectroscopic imaging (MRSI) technique was implemented to collect data with 0.25 mL resolution within 3 minutes and 16 seconds. For comparative purposes, the standard Dixon technique was performed. The two techniques were compared using structural similarity (SSIM) analysis. Additionally, the difference in the distribution of each lipid over the adolescent calf muscles was assessed based on the MRSI data. Results: The proposed MRSI technique provided individual FF maps for eight musculoskeletal lipids identified by LCModel analysis (L09, L11, L13, L15, L21, L23, L53, and L55) with mean SSIM indices of 0.19, 0.04, 0.03, 0.50, 0.45, 0.04, 0.07, and 0.12, respectively compared to that of Dixon FF map. Further analysis of voxels with zero SSIM demonstrated an increased sensitivity of FF lipid maps from data acquired using this MRSI technique over the standard Dixon technique. The trend of lipid spatial distribution over calf muscles was consistent with previously published findings in adults. Conclusion: The advantages of this MRSI technique make it a useful tool when individual lipid FF maps are desired within a short scanning time.

1814: Histone chaperone Nucleophosmin regulates transcription of key genes involved in oral tumorigenesis.
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Posted to bioRxiv 22 Nov 2019

Histone chaperone Nucleophosmin regulates transcription of key genes involved in oral tumorigenesis.
101 downloads biochemistry

Parijat Senapati, Suchismita Dey, Deepthi Sudarshan, Aditya Bhattacharya, Shyla G., Sadhan Das, Surabhi Sudevan, Tessy Thomas Maliekal, Tapas Kumar Kundu

Nucleophosmin (NPM1) is a multifunctional histone chaperone that can activate RNA Polymerase II-driven chromatin transcription. Acetylation of NPM1 by acetyltransferase p300 has been shown to further enhance its transcription activation potential. Moreover, its total and acetylated pools are increased in oral squamous cell carcinoma. However, the role of NPM1 or its acetylated form (AcNPM1) in transcriptional regulation in cells is not fully elucidated. Using ChIP-seq analyses, we show that AcNPM1 co-occupies marks of active transcription at promoters and DNase I hypersensitive sites at enhancers. Moreover, NPM1 interacts with a multitude of proteins involved in transcription, including RNA Pol II, general transcription factors, mediator subunits, histone acetyltransferase complexes, and chromatin remodelers. Moreover, its histone chaperone also contributes to transcriptional activation. We further show that AcNPM1 regulates key genes required for proliferation, migration and invasion potential of oral cancer cells and knockdown of NPM1 mitigates these processes in cells as well as orthotopic tumors in mice. Collectively, these results establish that AcNPM1 functions as a coactivator and regulates the expression of key genes involved in oral tumorigenesis.

1815: Protein Acetylation Induced by Dichloroacetate (DCA) Treatment is Associated with Decreased Respiration in Cultured Hepatocytes: Preliminary Results
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Posted to bioRxiv 06 Jun 2019

Protein Acetylation Induced by Dichloroacetate (DCA) Treatment is Associated with Decreased Respiration in Cultured Hepatocytes: Preliminary Results
101 downloads biochemistry

Jesse G. Meyer

Protein post-translational modification (PTM) by acetylation at the ε-amine on lysine residues in proteins regulates various cellular processes including transcription and metabolism. Several metabolic and genetic perturbations are known to increase acetylation of various proteins. Hyper-acetylation can also be induced using deacetylase inhibitors. While there is much interest in discovering drugs that can reverse protein acetylation, pharmacological tools that increase non-enzymatic protein acetylation are needed in order to understand the physiological role of excess protein acetylation. In this study, I assessed whether inhibition of pyruvate dehydrogenase kinase (PDHK) could cause protein hyper-acetylation due to excess production of acetyl-CoA by pyruvate dehydrogenase (PDH). Western blot of total protein from dichloroacetate (DCA) treated hepatocytes with anti-acetyl-lysine antibody showed increased protein acetylation, and seahorse respirometry of DCA pretreated hepatocytes indicated a subtle decrease in basal and maximal respiratory capacity.

1816: Activation of phospholipase C β by Gβγ and Gαq involves C-terminal rearrangement to release auto-inhibition
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Posted to bioRxiv 18 Oct 2019

Activation of phospholipase C β by Gβγ and Gαq involves C-terminal rearrangement to release auto-inhibition
101 downloads biochemistry

Isaac Fisher, Meredith Jenkins, Greg Tall, John E Burke, Alan V. Smrcka

Phospholipase C (PLC) enzymes hydrolyse phosphoinositide lipids to inositol phosphates and diacylglycerol. Direct activation of PLCβ by Gαq and/or Gβγ subunits mediates signalling by Gq and some Gi coupled G protein-coupled receptors (GPCRs), respectively. PLCβ isoforms contain a unique C-terminal extension, consisting of proximal and distal C-terminal domains (CTD) separated by a flexible linker. The structure of PLCβ3 bound to Gαq is known, however, for both Gαq and Gβγ, the mechanism for PLCβ activation on membranes is unknown. We examined PLCβ2 dynamics on membranes using hydrogen deuterium exchange mass spectrometry (HDX-MS). Gβγ caused a robust increase in dynamics of the distal C-terminal domain (CTD). Gαq showed decreased deuterium incorporation at the Gαq binding site on PLCβ. In vitro Gβγ-dependent activation of PLC is inhibited by the distal CTD. The results suggest that disruption of auto-inhibitory interactions with the CTD, respectively, leads to increased PLCβ hydrolase activity.

1817: Selectivity and engineering of the sialoglycan-binding spectrum in Siglec-like adhesins
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Posted to bioRxiv 07 Oct 2019

Selectivity and engineering of the sialoglycan-binding spectrum in Siglec-like adhesins
100 downloads biochemistry

Barbara Bensing, Lioudmila V Loukachevitch, Rupesh Agarwal, Izumi Yamakawa, Kelvin Luong, Azadeh Hadadianpour, Hai Yu, Kevin Fialkowski, Manuel Castro, Zdzislaw Wawrzak, Xi Chen, Jerome Baudry, Jeremy Smith, Paul Sullam, T M Iverson

The Siglec-like Serine-Rich Repeat (SRR) adhesins mediate bacterial attachment to mammalian hosts via sialoglycan receptors. Here, we combine structural, computational, biochemical, and phylogenetic approaches to elucidate the determinants of the sialoglycan-binding spectrum across the family of Siglec-like SRR adhesins. We further identified mutable positions that disproportionately affect sialoglycan selectivity, as demonstrated by increases in binding to alternative ligands of 2- to 3- orders of magnitude. Biologically, these studies highlight how bacteria nimbly modulate the receptor interaction during coevolution of host and pathogen. These studies additionally created binding proteins specific for sialyl-T antigen or 6S-sialyl LewisX that can recognize glycosylation of human plasma proteins. The engineered binding proteins can facilitate the characterization of normal cellular glycan modifications or may be used as diagnostic tools in disease states with altered glycosylation.

1818: Structural elements that modulate the substrate specificity of plant purple acid phosphatases: avenues for improved phosphorus acquisition in crops
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Posted to bioRxiv 28 Aug 2019

Structural elements that modulate the substrate specificity of plant purple acid phosphatases: avenues for improved phosphorus acquisition in crops
100 downloads biochemistry

Daniel Feder, Ross P McGeary, Natasa Mitić, Thierry Lonhienne, Agnelo Furtado, Benjamin L. Schulz, Robert J Henry, Susanne Schmidt, Luke W Guddat, Gerhard Schenk

Phosphate acquisition by plants is an essential process that is directly implicated in the optimization of crop yields. Purple acid phosphatases (PAPs) are ubiquitous metalloenzymes, which catalyze the hydrolysis of a wide range of phosphate esters and anhydrides. While some plant PAPs display a preference for ATP as the substrate, others are efficient in hydrolyzing phytate or 2-phosphoenolpyruvate (PEP). PAP from red kidney bean (rkbPAP) is an efficient ATP- and ADPase, but has no activity towards phytate. The crystal structure of this enzyme in complex with an ATP analogue (to 2.20 A resolution) provides insight into the amino acid residues that play an essential role in binding this substrate. Homology modelling was used to generate three-dimensional structures for the active sites of PAPs from tobacco (NtPAP) and Arabidopsis thaliana (AtPAP12 and AtPAP26) that are efficient in hydrolyzing phytate and PEP as substrates, respectively. In combination with substrate docking simulations and a phylogenetic analysis of 49 plant PAP sequences (including the first PAP sequences reported from Eucalyptus), several active site residues were identified that are important in defining the substrate specificities of plant PAPs. These results may inform bioengineering studies aimed at identifying and incorporating suitable plant PAP genes into crops to improve phosphorus use efficiency. Organic phosphorus sources increasingly supplement or replace inorganic fertilizer, and efficient phosphorus use of crops will lower the environmental footprint of agriculture while enhancing food production.

1819: Integrative model to coordinate the oligomerization and aggregation mechanisms of CCL5
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Posted to bioRxiv 03 Sep 2019

Integrative model to coordinate the oligomerization and aggregation mechanisms of CCL5
100 downloads biochemistry

Yi-Chen Chen, Siou-Pei Chen, Jin-Ye Li, Pei-Chun Chen, Yi-Zong Lee, Kun-Mou Li, Raz Zarivach, Yuh-Ju Sun, Shih-Che Sue

The CC-type chemokine ligand 5 (CCL5) is involved in the pathogenesis of many inflammatory conditions. The oligomerization and aggregation of CCL5 are considered to be responsible for its inflammatory properties. The CC-type dimer acts as the basic unit to constitute the oligomer. However, the structural basis of CCL5 oligomerization remains controversial. In this study, NMR and biophysical analyses proposed evidence that no single dimer-dimer interaction dominates in the oligomerization process of CCL5. CCL5 could oligomerize alternatively through two different interactions, E66-K25 and E66-R44/K45. In addition, a newly determined trimer structure reported an interfacial interaction through the N-terminal 12FAY14 sequence. The interaction contributes to aggregation and precipitation. In accordance with the observations, an integrative model explains the CCL5 oligomerization and aggregation process. CCL5 assembly consists of two types of dimer-dimer interactions and one aggregation mechanism. For full-length CCL5, the molecular accumulation triggers oligomerization through the E66-K25 interaction, and the 12FAY14 interaction acts as a secondary effect to derive aggregation. The E66-R44/K45 interaction dominates in CCL5 N-terminal truncations. The interaction would lead to filament-like formation in solution.

1820: Dissecting the structural and functional roles of a vicinal iron-binding site in encapsulated ferritins
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Posted to bioRxiv 27 Sep 2019

Dissecting the structural and functional roles of a vicinal iron-binding site in encapsulated ferritins
99 downloads biochemistry

Cecilia Piergentili, Didi He, Jennifer Ross, Will A Stanley, Laurène Adam, C Logan Mackay, Kevin J Waldron, Dave J Clarke, Jon Marles-Wright

The Encapsulated ferritin-like proteins belong to the universally distributed ferritin superfamily, which function as iron detoxification and storage systems. The encapsulated ferritins have a distinct annular structure and must associate with an encapsulin nanocage to form a competent iron store capable of holding significantly more iron than classical ferritins. The catalytic mechanism of iron oxidation in the ferritin family is still an open question due to differences in organisation of the ferroxidase catalytic site and secondary metal binding sites vicinal to this. We have previously identified a putative metal binding site on the inner surface of Rhodospirillum rubrum encapsulated ferritin at the interface between the two-helix subunits and proximal to the ferroxidase centre. Here we present a comprehensive structural and functional study to investigate the functional relevance of this putative iron entry site by means of enzymatic assays, mass-spectrometry, and X-ray crystallography. We show that catalysis occurs in the ferroxidase centre and suggest a dual role for the secondary site as an electrostatic trap guiding ferrous ions toward the ferroxidase centre and, at the same time, acting as a barrier protecting the ferroxidase site against non-cognate inhibiting species. Moreover, confinement of encapsulated ferritins within the encapsulin nanocage, while enhancing the ferritin ability to undergo catalysis, does not influence the specific function of the secondary site.

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