Rxivist logo

Rxivist combines preprints from bioRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 89,516 bioRxiv papers from 383,511 authors.

Most downloaded bioRxiv papers, all time

in category biochemistry

2,907 results found. For more information, click each entry to expand.

1841: A myopic perspective on the future of protein diagnostics
more details view paper

Posted to bioRxiv 04 Dec 2017

A myopic perspective on the future of protein diagnostics
258 downloads biochemistry

Ulf Landegren, Rasel A. Al-Amin, Johan Björkesten

Plasma proteome analyses of the future promise invaluable insights into states of health, not only by measuring proteins whose role it is to ensure blood homeostasis, but increasingly also as a window into the health of practically any tissue in the body via so-called leakage protein biomarkers. Realizing more of this vast potential will require progress along many lines. Here we discuss the main ones, such as optimal selection of target proteins, affinity reagents, immunoassay formats, samples, and applications, with a view from ongoing work in our laboratory.

1842: Dissecting the structural and functional roles of a vicinal iron-binding site in encapsulated ferritins
more details view paper

Posted to bioRxiv 27 Sep 2019

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

Cecilia Piergentili, Jennifer Ross, Didi He, Kelly J Gallagher, Will A Stanley, Laurène Adam, C Logan Mackay, Kevin J. Waldron, David James 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. ### Competing Interest Statement The authors have declared no competing interest.

1843: The biochemical activities of the Saccharomyces cerevisiae Pif1 helicase are regulated by its N-terminal domain
more details view paper

Posted to bioRxiv 02 Apr 2019

The biochemical activities of the Saccharomyces cerevisiae Pif1 helicase are regulated by its N-terminal domain
257 downloads biochemistry

David G. Nickens, Christopher W. Sausen, Matthew L. Bochman

PIF1 family helicases represent a highly conserved class of enzymes involved in multiple aspects of genome maintenance. Many PIF1 helicase are multi-domain proteins, but the functions of their non-helicase domains are poorly understood. Here, we characterized how the N-terminal domain (NTD) of the Saccharomyces cerevisiae Pif1 helicase affects its functions both in vivo and in vitro . Removal of the Pif1 NTD alleviated the toxicity associated with Pif1 over-expression in yeast. Biochemically, the N-terminally truncated Pif1 (Pif1ΔN) retained in vitro DNA binding, DNA unwinding, and telomerase regulation activities, but these activities differed markedly from those displayed by full-length recombinant Pif1. However, Pif1ΔN was still able to synergize with the Hrq1 helicase to inhibit telomerase activity in vitro , similar to full-length Pif1. These data impact our understanding of PIF1 helicase evolution and the roles of these enzymes in the maintenance of genome integrity.

1844: PyXlinkViewer: a flexible tool for visualisation of protein chemical crosslinking data within the PyMOL molecular graphics system
more details view paper

Posted to bioRxiv 17 Jun 2020

PyXlinkViewer: a flexible tool for visualisation of protein chemical crosslinking data within the PyMOL molecular graphics system
257 downloads biochemistry

Bob Schiffrin, Sheena E. Radford, David. J. Brockwell, Antonio N. Calabrese

Chemical crosslinking-mass spectrometry (XL-MS) is a valuable technique for gaining insights into protein structure and the organization of macromolecular complexes. XL-MS data yields inter-residue restraints that can be compared with high-resolution structural data. Distances greater than the crosslinker spacer-arm can reveal lowly-populated 'excited' states of proteins/protein assemblies, or crosslinks can be used as restraints to generate structural models in the absence of structural data. Despite increasing uptake of XL-MS, there are few tools to enable rapid and facile mapping of XL-MS data onto high-resolution structures or structural models. PyXlinkViewer is a user-friendly plugin for PyMOL v2 that maps intra-protein, inter-protein and dead-end crosslinks onto protein structures/models and automates the calculation of inter-residue distances for the detected crosslinks. This enables rapid visualisation of XL-MS data, assessment of whether a set of detected crosslinks is congruent with structural data, and easy production of high-quality images for publication. ### Competing Interest Statement The authors have declared no competing interest.

1845: High accuracy protein structures from minimal sparse paramagnetic solid-state NMR restraints
more details view paper

Posted to bioRxiv 05 Nov 2018

High accuracy protein structures from minimal sparse paramagnetic solid-state NMR restraints
256 downloads biochemistry

Alberto Perez, Kari Gaalswyk, Christopher P. Jaroniec, Justin L. MacCallum

There is a pressing need for new computational tools to integrate data from diverse experimental approaches in structural biology. We present a strategy that combines sparse paramagnetic solid-state NMR restraints with physics-based atomistic simulations. Our approach explicitly accounts for uncertainty in the interpretation of experimental data through the use of a semi-quantitative mapping between the data and the restraint energy that is calibrated by extensive simulations. We apply our approach to solid-state NMR data for the model protein GB1 labeled with Cu2+-EDTA at six different sites. We are able to determine the structure to ca. 1 Å accuracy within a single day of computation on a modest GPU cluster. We further show that in 4 of 6 cases, the data from only a single paramagnetic tag are sufficient to fold the protein to high accuracy.

1846: The N-terminus of Sec61p plays key roles in ER protein import and ERAD
more details view paper

Posted to bioRxiv 05 Feb 2018

The N-terminus of Sec61p plays key roles in ER protein import and ERAD
256 downloads biochemistry

Francesco Elia, Thomas Tretter, Karin Römisch

Sec61p is the channel-forming subunit of the heterotrimeric Sec61 complex that mediates co-translational protein import into the endoplasmic reticulum (ER). In yeast, proteins can also be post-translationally translocated by the hetero-heptameric Sec complex, composed of the Sec61 and the Sec63 complexes. The Sec61 channel is also a candidate for the dislocation channel for misfolded proteins from the ER to the cytosol during ER-associated degradation (ERAD). The structure of the Sec61 complex is highly conserved, but the roles of its N-terminal acetylation and its amphipathic N-terminal helix are unknown so far. To gain insight into the function of the Sec61p N-terminus, we mutated its N-acetylation site, deleted its amphipathic helix, or both the helix and the N-acetylation site. Mutation of the N-acetylation site on its own had no effect on protein import into the ER in intact cells, but resulted in an ERAD defect. Yeast expressing sec61 without the N-terminal amphipathic helix displayed severe growth defects and had profound defects in post-translational protein import into the ER. Nevertheless the formation of the hetero-heptameric Sec complex was not affected. Instead, the lack of the N-terminal amphipathic helix compromised the integrity of the heterotrimeric Sec61 complex. We conclude that the N-terminal helix of Sec61p is required for post-translational protein import into the ER and Sec61 complex stability, whereas N-terminal acetylation of Sec61p plays a role in ERAD.

1847: Normal development and fertility of Fut1, Fut2, and Sec1 triple knockout mice
more details view paper

Posted to bioRxiv 21 Apr 2019

Normal development and fertility of Fut1, Fut2, and Sec1 triple knockout mice
256 downloads biochemistry

Jiaxi Chen, Zhipeng Su, Chunlei Zhang, Fenge Li, Patrick Hwu, Zhen Wang, Yanping Wang, Yunsen Li, Jiao Tong, Chunchao Chen, Dapeng Zhou

The fucose alpha(1,2) galactose structure (H antigen) is synthesized by alpha 1,2 fucosyltransferases Fut1, Fut2 and Sec1. H antigen has been reported to be involved in cancer progression, neurite migration, synaptic plasticity, host-microbe interaction, blastocyst implantation, and the maintenance of gut microbiome. Genetic depletion of Fut1 or Fut2 only cause defects of alpha1,2 fucosylation in limited tissues because of enzyme redundancy. In this study, we generated mice with deficiencies in Fut1, Fut2, and Sec1 genes to deplete H antigen through BAC Engineering for the generation of ES Cell-Targeting construct. The homogenous triple knockout mice showed no significant decrease of viability or development. Mass spectrometry and Western blot analysis confirmed the absence of H blood group antigen in multiple organs. These results indicate normal development and fertility of mice devoid of blood group H. The fine pathophysiological alterations in these mice remain to be carefully studied, and they may serve as valuable tools to study gut microbiome and host-microbe interactions.

1848: LanD-like Flavoprotein-Catalyzed Aminovinyl-Cysteine Formation through Oxidative Decarboxylation and Cyclization of a Peptide at the C-Terminus
more details view paper

Posted to bioRxiv 14 Feb 2020

LanD-like Flavoprotein-Catalyzed Aminovinyl-Cysteine Formation through Oxidative Decarboxylation and Cyclization of a Peptide at the C-Terminus
256 downloads biochemistry

Jingyu Liu, Yanping Qiu, Tao Fu, Miao Li, Yuqing Li, Qian Yang, Zhijun Tang, Haoyu Tang, Guangyu Li, Lifeng Pan, Wen Liu

Aminovinyl-cysteine residues arise from processing the C-terminal L-Cys and an internal L-Ser/L-Thr or L-Cys of a peptide. Formation of these nonproteinogenic amino acids, which occur in a macrocyclic ring of diverse ribosomally synthesized lanthipeptides and non-lanthipeptides, remains poorly understood. Here, we report that LanD-like flavoproteins in the biosynthesis of distinct non-lanthipeptides share an unexpected dual activity for aminovinyl-cysteine formation. Each flavoprotein catalyzes oxidative decarboxylation of the C-terminal L-Cys and couples the resulting enethiol nucleophile with the internal residue to afford a thioether linkage for peptide cyclization. The cyclization step, which largely depends on proximity effect by positioning the enethiol intermediate with a bent conformation at the active site, can be substrate-dependent, proceeding inefficiently through nucleophilic substitution for an unmodified peptide or efficiently through Michael addition for a dehydrated/dethiolated peptide. Uncovering this unusual flavin-dependent paradigm for thioether residue formation advances the understanding in the biosynthesis of aminovinyl-cysteine-containing RiPPs and renews interest in flavoproteins, particularly those involved in non-redox transformations. LanD-like flavoproteins activity, which is flexible in peptide substrate and amenable for evolution by engineering, can be combined with different post-translational modifications for structural diversity, thereby holding promise for peptide macrocyclization/functionalization in drug development by chemoenzymatic or synthetic biology approaches.

1849: A canstatin-derived peptide provides insight into the role of Capillary Morphogenesis Gene 2 in angiogenic regulation and matrix uptake
more details view paper

Posted to bioRxiv 17 Jul 2019

A canstatin-derived peptide provides insight into the role of Capillary Morphogenesis Gene 2 in angiogenic regulation and matrix uptake
256 downloads biochemistry

Jordan G. Finnell, Tsz-Ming Tsang, Lorna Cryan, Samuel Garrard, Sai-Lun Lee, P. Christine Ackroyd, Michael S. Rogers, Kenneth A. Christensen

Capillary Morphogenesis Gene 2 protein (CMG2) is a transmembrane, integrin-like receptor and the primary receptor for the anthrax toxin. In addition to its role as an anthrax toxin receptor, CMG2 has been repeatedly shown to play a role in angiogenic processes. However, the molecular mechanism mediating observed CMG2-related angiogenic effects has not been fully elucidated. Previous studies have found that CMG2 binds type IV collagen (Col-IV), a key component of the vascular basement membrane, as well as other ECM proteins. Currently, no link has been made between these CMG2-ECM interactions and angiogenesis; however, ECM fragments are known to play a role in regulating angiogenesis. Here, we further characterize the CMG2-Col-IV interaction and explore the effect of this interaction on angiogenesis. Using a peptide array, we observed that CMG2 preferentially binds peptide fragments of the NC1 (non-collagenous domain 1) domains of Col-IV. These domains are also known as the fragments arresten (from the α1 chain) and canstatin (from the α2 chain) and have documented antiangiogenic properties. A second peptide array was probed to map a putative binding epitope. A top hit from the initial array, a canstatin-derived peptide, binds to the CMG2 ligand-binding von Willebrand factor A (vWA) domain with sub-micromolar affinity (peptide S16, Kd = 400 ± 200 nM). This peptide competes with anthrax protective antigen (PA) for CMG2 binding, and does not bind CMG2 in the presence of EDTA. Together these data suggest that, like PA, S16 interacts with CMG2 at the metal-ion dependent adhesion site (MIDAS) of its vWA domain. We demonstrate that CMG2 specifically mediates endocytic uptake of S16, since CMG2-/- endothelial cells show markedly reduced S16 uptake, without reducing total endocytosis. Furthermore, we show that S16 reduces endothelial migration but not cell proliferation. Taken together, our data demonstrate that a Col IV-derived anti-angiogenic peptide acts via CMG2, suggesting a possible link between CMG2-Col IV interactions and angiogenesis.

1850: Phosphatidylinositol 3,5-Bisphosphate Regulates Yeast Vacuole Fusion at the Transition between trans-SNARE Complex Formation and Hemifusion
more details view paper

Posted to bioRxiv 10 Aug 2018

Phosphatidylinositol 3,5-Bisphosphate Regulates Yeast Vacuole Fusion at the Transition between trans-SNARE Complex Formation and Hemifusion
255 downloads biochemistry

Gregory E Miner, Katherine D Sullivan, Annie Guo, Brandon C Jones, Matthew L Starr, Rutilio A. Fratti

Phosphoinositides (PIs) regulate myriad cellular functions including membrane fusion, as exemplified by the yeast vacuole, which uses various PIs at different stages of fusion. In light of this, the effect of phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] on vacuole fusion remains unknown. PI(3,5)P2 is made by the PI3P 5-kinase Fab1/PIKfyve and has been characterized as a regulator of vacuole fission during hyperosmotic shock where it interacts with the TRP family Ca2+ channel Yvc1. Here we demonstrate that exogenously added dioctanoyl (C8) PI(3,5)P2 abolishes homotypic vacuole fusion. This effect was not linked to interactions with Yvc1, as fusion was equally affected using yvc1Δ vacuoles. Thus, the effects of C8-PI(3,5)P2 on fusion versus fission operate through distinct mechanisms. Further testing showed that C8-PI(3,5)P2 inhibited vacuole fusion after the formation of trans-SNARE pairs. Although SNARE complex formation was unaffected we found that C8-PI(3,5)P2 strongly inhibited hemifusion. Overproduction of endogenous PI(3,5)P2 by the fab1T2250A hyperactive kinase mutant also inhibited at the hemifusion stage, bolstering the model in which PI(3,5)P2 inhibits fusion when present elevated levels. Taken together, this work identifies a novel function for PI(3,5)P2 as a negative regulator of vacuolar fusion. Moreover, it suggests that this lipid acts as a molecular switch between fission and fusion.

1851: Iron is a ligand of SecA-like metal-binding domains in vivo
more details view paper

Posted to bioRxiv 19 Apr 2019

Iron is a ligand of SecA-like metal-binding domains in vivo
255 downloads biochemistry

Tamar Cranford-Smith, Mohammed Jamshad, Mark Jeeves, Rachael A Chandler, Jack Yule, Ashley Robinson, Farhana Alam, Karl A. Dunne, Edwin H. Aponte Angarita, Mashael Alanazi, Cailean Carter, Ian R. Henderson, Janet E Lovett, Peter Winn, Timothy Knowles, Damon Huber

The ATPase SecA is an essential component of the bacterial Sec machinery, which transports proteins across the cytoplasmic membrane. Most SecA proteins contain a long C-terminal tail (CTT). In Escherichia coli , the CTT contains a structurally flexible linker domain and a small metal-binding domain (MBD). The MBD coordinates zinc via a conserved cysteine-containing motif and binds to SecB and ribosomes. In this study, we screened a high-density transposon library for mutants that affect the susceptibility of E. coli to sodium azide, which inhibits SecA-mediated translocation. Results from sequencing this library suggested that mutations removing the CTT make E. coli less susceptible to sodium azide at subinhibitory concentrations. Copurification experiments suggested that the MBD binds to iron and that azide disrupts iron binding. Azide also disrupted binding of SecA to membranes. Two other E. coli proteins that contain SecA-like MBDs, YecA and YchJ, also copurified with iron, and NMR spectroscopy experiments indicated that YecA binds iron via its MBD. Competition experiments and equilibrium binding measurements indicated that the SecA MBD binds preferentially to iron and that a conserved serine is required for this specificity. Finally, structural modelling suggested a plausible model for the octahedral coordination of iron. Taken together, our results suggest that SecA-like MBDs likely bind to iron in vivo . * BEST : band-selective short transient excitation DIPSI : decoupling in the presence of scalar interactions DTT : dithiothreitol EDTA : ethylene diamine tetra-acetic acid EPR : electron paramagnetic resonance HSQC : heteronuclear single quantum coherence ICP : inductively coupled plasma IPTG : isopropyl-β-thiogalactoside ITC : isothermal titration calorimetry MS : mass spectrometry NMR : nuclear magnetic resonance NTA : nitrilotriacetic acid OES : optical emission spectrometry SUMO : small ubiquitin-like modifier TCEP : tris(2-carboxyethyl)phosphine TOCSY : total correlated spectroscopy TROSY : transverse relaxation optimised spectroscopy UPF : unidentified protein function UV : ultraviolet

1852: Molecules inhibit the enzyme activity of 3-chymotrypsin-like cysteine protease of SARS-CoV-2 virus: the experimental and theory studies
more details view paper

Posted to bioRxiv 29 May 2020

Molecules inhibit the enzyme activity of 3-chymotrypsin-like cysteine protease of SARS-CoV-2 virus: the experimental and theory studies
255 downloads biochemistry

Zhesheng He, Wencong Zhao, Wenchao Niu, Xuejiao Gao, Xingfa Gao, Yong Gong, Xueyun Gao

SARS-CoV-2 has emerged as a world public health threat. Herein, we report that the clinical approved auranofin could perfectly inhibit the activity of 3-chymotrypsin-like cysteine protease (Mpro or 3CLpro) of SARS-CoV-2. Gold cluster could significantly inhibit 3CLpro of SARS-COV-2. Phenyl isothiocyanate and Vitamin K3 could well suppress the activity of 3CLpro. For Mpro inhibition, IC50 of auranofin, Vitamin K3, phenyl isothiocyanate, gold cluster are about 0.51 micromolar/L, 7.96 micromolar/L, 10.13 micromolar/L, 1.61 micromolar/L, respectively. These compounds may be with potentials for treatment SARS-CoV-2 virus replication. Especially for FDA approved auranofin, it is an anti-inflammation drug in clinic, thus it may with strong potential to inhibit virus replication and suppress the inflammation damage in COVID-19 patients. Gold cluster is with better safety index and well anti-inflammation in vitro/vivo, therefore it is with potential to inhibit virus replication and suppress the inflammation damage caused by COVID-19 virus. As Au(I) ion is active metabolism specie derived from gold compounds or gold clusters in vivo, further computational studies revealed Au ion could tightly bind thiol group of Cys145 residue of 3CLpro thus inhibit enzyme activity. Also, phenyl isothiocyanate and Vitamin K3 may interact with thiol group of Cys145 via Michael addition reaction, molecular dynamic (MD) theory studied are applied to confirmed these small molecules are stable in the pocket and inhibit Mpro activity. ### Competing Interest Statement The authors have declared no competing interest.

1853: Standard Flow Multiplexed Proteomics (SFloMPro). An Accessible and Cost-Effective Alternative to NanoLC Workflows
more details view paper

Posted to bioRxiv 25 Feb 2020

Standard Flow Multiplexed Proteomics (SFloMPro). An Accessible and Cost-Effective Alternative to NanoLC Workflows
255 downloads biochemistry

Conor Jenkins, Benjamin C. Orsburn

Multiplexed proteomics using isobaric tagging allows for simultaneously comparing the proteomes of multiple samples. In this technique, digested peptides from each sample are labeled with a chemical tag prior to pooling sample for LC-MS/MS with nanoflow chromatography (NanoLC). The isobaric nature of the tag prevents deconvolution of samples until fragmentation liberates the isotopically labeled reporter ions. To ensure efficient peptide labeling, large concentrations of labeling reagents are included in the reagent kits to allow scientists to use high ratios of chemical label per peptide. The increasing speed and sensitivity of mass spectrometers has reduced the peptide concentration required for analysis, leading to most of the label or labeled sample to be discarded. In conjunction, improvements in the speed of sample loading, reliable pump pressure, and stable gradient construction of analytical flow HPLCs has continued to improve the sample delivery process to the mass spectrometer. In this study we describe a method for performing multiplexed proteomics without the use of NanoLC by using offline fractionation of labeled peptides followed by rapid standard flow HPLC gradient LC-MS/MS. Standard Flow Multiplexed Proteomics (SFloMPro) enables high coverage quantitative proteomics of up to 16 mammalian samples in about 24 hours. In this study, we compare NanoLC and SFloMPro analysis of fractionated samples. Our results demonstrate that comparable data is obtained by injecting 20 micrograms of labeled peptides per fraction with SFloMPro, compared to 1 microgram per fraction with NanoLC. We conclude that, for experiments where protein concentration is not strictly limited, SFloMPro is a competitive approach to traditional NanoLC workflows with improved up-time, reliability and at a lower relative cost per sample.

1854: Co-Occurrence of Enzyme Domains Guides the Discovery of an Oxazolone Synthetase
more details view paper

Posted to bioRxiv 12 Jun 2020

Co-Occurrence of Enzyme Domains Guides the Discovery of an Oxazolone Synthetase
254 downloads biochemistry

Tristan de Rond, Julia E. Asay, Bradley S. Moore

Multi-domain enzymes are cellular machines that orchestrate two or more catalytic activities to carry out metabolic transformations with increased control and speed. Here, we report the development of a new genome mining approach for the targeted discovery of new metabolic pathways based on the co-occurrence of enzyme domains (CO-ED) in a single protein. CO-ED was designed to identify unannotated multifunction proteins for accelerated functional characterization and discovery based on the premise that linked enzyme domains have evolved to function collaboratively. Guided by CO-ED, we targeted an unannotated predicted ThiF-nitroreductase di-domain enzyme found in more than 50 proteobacteria. Through heterologous expression and biochemical reconstitution of this enzyme, we discovered a series of new natural products containing the rare oxazolone heterocycle, and identified the first reported oxazolone synthetase in biology. This proof-of-principle experiment validates CO-ED-guided genome mining as a new method with potential broad utility for both the discovery of novel enzymatic transformations and the functional gene annotation of multi-domain enzymes. ### Competing Interest Statement The authors have declared no competing interest.

1855: Buffering Agent Induced Lactose Content Increases via Growth Hormone-Mediated Activation of Gluconeogenesis in Lactating Goats
more details view paper

Posted to bioRxiv 25 Jun 2017

Buffering Agent Induced Lactose Content Increases via Growth Hormone-Mediated Activation of Gluconeogenesis in Lactating Goats
254 downloads biochemistry

Lin Li, MeiLin He, Ying Liu, Yuanshu Zhang

Dairy goats are often fed a high-concentrate (HC) diet to meet lactation demands; however, long-term concentrate feeding is unhealthy and decreases milk yield and lactose content. Therefore, we tested whether a buffering agent increases the output of glucose in the liver and influences of lactose synthesis. In this study, sixteen lactating goats were randomly assigned to two groups: one group received a HC diets (Concentrate : Forage = 6:4, HG), and the other group received the same diet with a buffering agent added (0.2% NaHCO3, 0.1% MgO, BG) as a treatment for 19-weeks experimental period. The results showed that the total volatile fatty acids and lipopolysaccharide (LPS) declined in the rumen leading to the rumen pH was stabilized in the BG group. Milk yield and lactose content increased. The alanine aminotransferase, aspartate transaminase, alkaline phosphatase, pro-inflammatory cytokines, LPS and lactate content in the plasma was significantly decreased, whereas prolactin and growth hormone levels were increased. The hepatic vein content of glucose was increased. In addition, the expression of pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6PC) in the liver was significantly up-regulated. In mammary gland, the glucose transporter type-1, 8, 12 and sodium-glucose cotransporter-1 levels were increased. Cumulatively, the buffering agent treatment increased blood concentrations of glucose via the gluconeogenes and promoting their synthesis in the liver.

1856: CLICK-enabled analogues reveal pregnenolone interactomes in cancer and immune cells
more details view paper

Posted to bioRxiv 16 Oct 2019

CLICK-enabled analogues reveal pregnenolone interactomes in cancer and immune cells
254 downloads biochemistry

Sougata Roy, James Sipthorp, Bidesh Mahata, Jhuma Pramanik, Marco L Hennrich, Anne-Claude Gavin, Steven V. Ley, Sarah A. Teichmann

Pregnenolone (P5) promotes prostate cancer cell growth, and de novo synthesis of intratumoural P5 is a potential cause of development of castration-resistance. Immune cells can also synthesize P5 de novo. Despite its biological importance, little is known about P5s mode of actions, which appears to be context-dependent and pleiotropic. A comprehensive proteome-wide spectrum of P5-binding proteins that are involved in its trafficking and functionality remains unknown. Here, we describe an approach that integrates chemical biology for probe synthesis with chemoproteomics to map P5-protein interactions in live prostate cancer cells and murine CD8+ T cells. We subsequently identified P5-binding proteins potentially involved in P5-trafficking, and in P5s non-genomic action that may drive the promotion of castrate-resistance prostate cancer and regulate CD8+ T cell function. We envisage that this methodology could be employed for other steroids to map their interactomes directly in a broad range of living cells, tissues and organisms.

1857: Autonomous bioluminescence imaging of single mammalian cells with the bacterial bioluminescence system
more details view paper

Posted to bioRxiv 08 Oct 2019

Autonomous bioluminescence imaging of single mammalian cells with the bacterial bioluminescence system
254 downloads biochemistry

Carola Gregor, Jasmin K. Pape, Klaus C. Gwosch, Tanja Gilat, Steffen J. Sahl, Stefan W. Hell

Bioluminescence based imaging of living cells has become an important tool in biological and medical research. However, many bioluminescence imaging applications are limited by the requirement of an externally provided luciferin substrate and the low bioluminescence signal which restricts the sensitivity and spatiotemporal resolution. The bacterial bioluminescence system is fully genetically encodable and hence produces autonomous bioluminescence without an external luciferin, but its brightness in cell types other than bacteria has so far not been sufficient for imaging single cells. We coexpressed codon-optimized forms of the bacterial luxCDABE and frp genes from multiple plasmids in different mammalian cell lines. Our approach produces high luminescence levels that are comparable to firefly luciferase, thus enabling autonomous bioluminescence microscopy of mammalian cells.

1858: Presence of WH2 like domain in VgrG-1 toxin of Vibrio cholerae reveals the molecular mechanism of actin cross-linking
more details view paper

Posted to bioRxiv 29 Nov 2017

Presence of WH2 like domain in VgrG-1 toxin of Vibrio cholerae reveals the molecular mechanism of actin cross-linking
253 downloads biochemistry

Priyanka Dutta, A.S. Jijumon, Mohit Mazumder, Drisya Dileep, Asish K Mukhopadhyay, Samudrala Gourinath, Sankar Maiti

Type VI secretion systems (T6SS) plays a crucial role in Vibrio cholerae mediated pathogenicity and predation. Tip of T6SS is homologous to gp27/gp5 complex or tail spike of T4 bacteriophage. VgrG-1 of V. cholerae T6SS is unusual among other VgrG because its effector domain is trans-located into the cytosol of eukaryotic cells with an additional actin cross-linking domain (ACD) at its C terminal end. ACD of VgrG-1 (VgrG-1-ACD) causes T6SS dependent host cell cytotoxicity through actin cytoskeleton disruption to prevent bacterial engulfment by macrophages. ACD mediated actin cross-linking promotes survival of the bacteria in the small intestine of humans, along with other virulence factors; establishes successful infection with the onset of diarrhoea in humans. Our studies demonstrated VgrG-1-ACD can bind to actin besides actin cross-linking activity. Computational analysis of ACD revealed the presence of WH2 domain through which it binds actin. Mutations in WH2 domain lead to loss of actin binding in vitro. VgrG-1-ACD having the mutated WH2 domain cannot cross-link actin efficiently in vitro and manifests less actin cytoskeleton disruption when transfected in HeLa cells.

1859: Mechanochemical Coupling and Bi-Phasic Force-Velocity Dependence in the Ultra-Fast Ring ATPase SpoIIIE
more details view paper

Posted to bioRxiv 22 Nov 2017

Mechanochemical Coupling and Bi-Phasic Force-Velocity Dependence in the Ultra-Fast Ring ATPase SpoIIIE
253 downloads biochemistry

Ninning Liu, Gheorghe Chistol, Yuanbo Cui, Carlos Bustamante

Multi-subunit ring-shaped ATPases are molecular motors that harness chemical free energy to perform vital mechanical tasks such as polypeptide translocation, DNA unwinding, and chromosome segregation. Previously we reported the intersubunit coordination and stepping behavior of the hexameric ring-shaped ATPase SpoIIIE (Liu et al., 2015). Here we use optical tweezers to characterize the motor’s mechanochemistry. Analysis of the motor response to external force at various nucleotide concentrations identifies phosphate release as the likely force-generating step. Analysis of SpoIIIE pausing indicates that pauses are off-pathway events. Characterization of SpoIIIE slipping behavior reveals that individual motor subunits engage DNA upon ATP binding. Furthermore, we find that SpoIIIE’s velocity exhibits an intriguing bi-phasic dependence on force. We hypothesize that this behavior is an adaptation of ultra-fast motors tasked with translocating DNA from which they must also remove DNA-bound protein roadblocks. Based on these results, we formulate a comprehensive mechanochemical model for SpoIIIE.

1860: The WD40-repeat protein WDR-48 promotes the stability of the deubiquitinating enzyme USP-46 by inhibiting its ubiquitination and degradation
more details view paper

Posted to bioRxiv 21 Jun 2019

The WD40-repeat protein WDR-48 promotes the stability of the deubiquitinating enzyme USP-46 by inhibiting its ubiquitination and degradation
253 downloads biochemistry

Molly Hodul, Rakesh Ganji, Caroline L Dahlberg, Malavika Raman, Peter Juo

Ubiquitination is a reversible post-translational modification that has emerged as a critical regulator of synapse development and function. However, mechanisms that regulate the deubiquitinating enzymes (DUBs) that are responsible for the removal of ubiquitin from target proteins are poorly understood. We previously showed that the DUB USP-46 removes ubiquitin from the glutamate receptor GLR-1 and regulates it trafficking and degradation in C. elegans . We found that WD40-repeat proteins WDR-20 and WDR-48 bind and stimulate the catalytic activity of USP-46. Here, we identify another mechanism by which WDR-48 regulates USP-46. We found that increased expression of WDR-48, but not WDR-20, promotes USP-46 abundance in mammalian cells in culture and in C. elegans neurons in vivo. Inhibition of the proteasome promotes the abundance of USP-46, and this effect is non-additive with increased expression of WDR-48. We found that USP-46 is ubiquitinated, and expression of WDR-48 reduces the levels of ubiquitin-USP-46 conjugates and increases the half-life of USP-46. A point mutant version of WDR-48 that disrupts binding to USP-46 is unable to promote USP-46 abundance in vivo. Together, these data support a model in which WDR-48 binds and stabilizes USP-46 protein levels by preventing the ubiquitination and degradation of USP-46 in the proteasome. Given that a large number of USPs interact with WDR proteins, we propose that stabilization of DUBs by their interacting WDR proteins may be a conserved and widely used mechanism to control DUB availability and function.

Previous page 1 . . . 91 92 93 94 95 96 97 . . . 146 Next page

PanLingua

Sign up for the Rxivist weekly newsletter! (Click here for more details.)


News