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A CDK-regulated chromatin segregase promoting chromosome replication

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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.20.390914v1?rss=1 Authors: Chacin, E., Bansal, P., Reusswig, K.-U., Diaz-Santin, L., Ortega, P., Vizjak, P., Gomez-Gonzalez, B., Mueller-Planitz, F., Aguilera, A., Pfander, B., Cheung, A., Kurat, C. Abstract: The replication of chromosomes during S phase is critical for cellular and organismal function. Replicative stress can result in genome instability, which is a major driver of cancer. Yet how chromatin is made accessible during eukaryotic DNA synthesis is poorly understood. Here, we report the identification of a novel class of chromatin remodeling enzyme, entirely distinct from classical SNF2-ATPase family remodelers. Yta7 is a AAA+-ATPase that assembles into ~ 1 MDa hexameric complexes capable of segregating histones from DNA. Yta7 chromatin segregase promotes chromosome replication both in vivo and in vitro. Biochemical reconstitution experiments using purified proteins revealed that enzymatic activity of Yta7 is regulated by S phase-forms of Cyclin-Dependent Kinase (S-CDK). S-CDK phosphorylation stimulates ATP hydrolysis by Yta7, promoting nucleosome disassembly and chromatin replication. Our results present a novel mechanism of how cells orchestrate chromatin dynamics in co-ordination with the cell cycle machinery to promote genome duplication during S phase. Copy rights belong to original authors. Visit the link for more info

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    Public LC-Orbitrap-MSMS Spectral Library for Metabolite Identification

    Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.21.392266v1?rss=1 Authors: Phapale, P., Palmer, A., Gathungu, R. M., Kale, D., Brugger, B., Alexandrov, T. Abstract: Liquid chromatography-mass spectrometry(LC-MS)-based untargeted metabolomics studies require high-quality spectral libraries for reliable metabolite identification. We have constructed EMBL-MCF, an open LC-MS/MS spectral library that currently contains over 1600 fragmentation spectra from 435 authentic standards of endogenous metabolites and lipids. The unique features of the library are presence of chromatographic profiles acquired with different LC-MS methods and coverage of different adduct ions. The library covers many biologically important metabolites with some unique metabolites and lipids as compared to other public libraries. The EMBL-MCF spectral library is created and shared using an in-house developed web-application at https://curatr.mcf.embl.de/. The library is freely available online and also integrated with other mass spectral repositories. Copy rights belong to original authors. Visit the link for more info
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    Crosstalk between repair pathways elicits Double-Strand Breaks in alkylated DNA: implications for the action of temozolomide

    Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.21.391326v1?rss=1 Authors: FUCHS, R. P., Isogawa, A., Paulo, J. A., Onizuka, K., Takahashi, T., Amunugama, R., Duxin, J. P., Fujii, S. Abstract: Temozolomide, a DNA methylating agent, is the primary chemotherapeutic drug used in glioblastoma treatment. TMZ induces mostly N-alkylation adducts (N7-methylguanine and N3-methyladenine) and some O6-methylguanine (O6mG). Current models propose that during DNA replication, thymine is incorporated across from O6mG, promoting a futile cycle of mismatch repair (MMR) that leads to DNA double strand breaks (DSBs). To revisit the mechanism of O6mG processing, we reacted plasmid DNA with N-Methyl-N-nitrosourea (MNU), a temozolomide mimic, and incubated it in Xenopus egg extracts. We show that in this system, mismatch repair (MMR) proteins are enriched on MNU-treated DNA and we observe robust, MMR-dependent, repair synthesis. Our evidence also suggests that MMR, initiated at O6mG:C sites, is strongly stimulated in cis by repair processing of other lesions, such as N-alkylation adducts. Importantly, MNU-treated plasmids display DSBs in extracts, the frequency of which increased linearly with the square of alkylation dose. We suggest that DSBs result from two independent repair processes, one involving MMR at O6mG:C sites and the other involving BER acting at a nearby N-alkylation adducts. We propose a new, replication-independent mechanism of action of TMZ, that operates in addition to the well-established cell cycle dependent mode of action. Copy rights belong to original authors. Visit the link for more info
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    CM1-driven assembly and activation of Yeast γ-Tubulin Small Complex underlies microtubule nucleation

    Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.21.392803v1?rss=1 Authors: Brilot, A. F., Lyon, A., Zelter, A., Viswanath, S., Maxwell, A., MacCoss, M. J., Muller, E. G., Sali, A., Davis, T. N., Agard, D. A. Abstract: Microtubule (MT) nucleation is regulated by the {gamma}-tubulin ring complex ({gamma}TuRC), conserved from yeast to humans. In Saccharomyces cerevisiae, {gamma}TuRC is composed of seven identical {gamma}-tubulin small complex ({gamma}TuSC) sub-assemblies which associate helically to template microtubule growth. {gamma}TuRC assembly provides a key point of regulation for the MT cytoskeleton. Here we combine cross-linking mass spectrometry (XL-MS), X-ray crystallography and cryo-EM structures of monomeric and dimeric {gamma}TuSC and open and closed helical {gamma}TuRC assemblies in complex with Spc110p to elucidate the mechanisms of {gamma}TuRC assembly. {gamma}TuRC assembly is substantially aided by the evolutionarily conserved CM1 motif in Spc110p spanning a pair of adjacent {gamma}TuSCs. By providing the highest resolution and most complete views of any {gamma}TuSC assembly, our structures allow phosphorylation sites to be mapped, suggesting their role in regulating spindle pole body attachment and ring assembly. We further identify a structurally analogous CM1 binding site in the human {gamma}TuRC structure at the interface between GCP2 and GCP6, which allows for the interpretation of significant structural changes arising from CM1 helix binding to metazoan {gamma}TuRC. Copy rights belong to original authors. Visit the link for more info
  • PaperPlayer biorxiv biochemistry podcast

    A CDK-regulated chromatin segregase promoting chromosome replication

    Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.20.390914v1?rss=1 Authors: Chacin, E., Bansal, P., Reusswig, K.-U., Diaz-Santin, L., Ortega, P., Vizjak, P., Gomez-Gonzalez, B., Mueller-Planitz, F., Aguilera, A., Pfander, B., Cheung, A., Kurat, C. Abstract: The replication of chromosomes during S phase is critical for cellular and organismal function. Replicative stress can result in genome instability, which is a major driver of cancer. Yet how chromatin is made accessible during eukaryotic DNA synthesis is poorly understood. Here, we report the identification of a novel class of chromatin remodeling enzyme, entirely distinct from classical SNF2-ATPase family remodelers. Yta7 is a AAA+-ATPase that assembles into ~ 1 MDa hexameric complexes capable of segregating histones from DNA. Yta7 chromatin segregase promotes chromosome replication both in vivo and in vitro. Biochemical reconstitution experiments using purified proteins revealed that enzymatic activity of Yta7 is regulated by S phase-forms of Cyclin-Dependent Kinase (S-CDK). S-CDK phosphorylation stimulates ATP hydrolysis by Yta7, promoting nucleosome disassembly and chromatin replication. Our results present a novel mechanism of how cells orchestrate chromatin dynamics in co-ordination with the cell cycle machinery to promote genome duplication during S phase. Copy rights belong to original authors. Visit the link for more info
  • PaperPlayer biorxiv biochemistry podcast

    Dietary zinc restriction induces nociceptive pain with reduced inflammation in mice

    Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.21.392548v1?rss=1 Authors: de Lima, C. K. F., Sisnande, T., da Silva, R. V., da Silva, V. D., do Amaral, J. J., Ochs, S. M., Roedel dos Santos, B. L., Miranda, A. L. P., Lima, L. M. T. d. R. Abstract: Zinc (Zn) is an essential micronutrient involved in a large diversity of cellular metabolism, included in the physiology of nervous system and pain modulation. There is little evidence for the role of Zn nutritional alternations to the onset and progression of neuropathic and inflammatory pain. We investigate the effects of a zinc restricted diet on the development of pain. Weaned mice were submitted to different diets: AIN-93 (38mg/kg of Zn) and Zn-deficient (AIN-93 with 11mg/kg of Zn), during four weeks. Mechanical allodynia was measured weekly using Von Frey hairs. Plantar assays for cold and heat allodynia, formalin-induced nociception and carrageenan-induced mechanical allodynia were performed at the 4th week. Plasma, DRG and livers samples were obtained for biochemical and metabolomics analysis. Zn deficient diet completely changed mice sensitivity pattern, inducing an intense allodynia evoked by mechanical, cold and heat stimulus since weaning and during four weeks. Showed also an increased sensitivity of neurogenic phase of formalin test but the inflammatory pain behavior was drastically reduced. Zn restriction increased the ATF-3 and SOD-1 levels at DRG and reduced that of GFAP, leading an increase of neuronal activation and oxidative stress, and reduced neuroimmune activity. Plasma TNF was also reduced and metabolomics analyses suggest a downregulation of lipid metabolism of arachidonic acid, reinforcing the impact of Zn restriction to the inflammatory response. Reduction of Zn intake interferes in pain circuits, reducing inflammatory pain, however enhancing nociceptive pain. Accordingly, Zn imbalance could be predisposing factor for NP development. Therefore, dietary zinc supplementation and its monitoring present clinical relevance. Copy rights belong to original authors. Visit the link for more info
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    Comparative biochemistry of four polyester (PET) hydrolases

    Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.20.392019v1?rss=1 Authors: Arnling Baath, J., Borch, K., Jensen, K., Brask, J., Westh, P. Abstract: The potential of bioprocessing in a circular plastic economy has strongly stimulated research in enzymatic degradation of different synthetic resins. Particular interest has been devoted to the commonly used polyester, poly(ethylene terephthalate) (PET), and a number of PET hydrolases have been described. However, a kinetic framework for comparisons of PET hydrolases (or other plastic degrading enzymes) acting on the insoluble substrate, has not been established. Here, we propose such a framework and test it against kinetic measurements on four PET hydrolases. The analysis provided values of kcat and KM, as well as an apparent specificity constant in the conventional units of M-1s-1. These parameters, together with experimental values for the number of enzyme attack sites on the PET surface, enabled comparative analyses. We found that the PET hydrolase from Ideonella sakaiensis was the most efficient enzyme at ambient conditions, and that this relied on a high kcat rather than a low KM. Moreover, both soluble and insoluble PET fragments were consistently hydrolyzed much faster than intact PET. This suggests that interactions between polymer strands slow down PET degradation, while the chemical steps of catalysis and the low accessibility associated with solid substrate were less important for the overall rate. Finally, the investigated enzymes showed a remarkable substrate affinity, and reached half the saturation rate on PET, when the concentration of attack sites in the suspension was only about 50 nM. We propose that this is linked to nonspecific adsorption, which promotes the nearness of enzyme and attack sites. Copy rights belong to original authors. Visit the link for more info
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    Extracellular vesicles from human adipose stem cells are neuroprotective after stroke in rats

    Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.18.388355v1?rss=1 Authors: Rohden, F., Teixeira, L. V., Bernardi, L. P., Marques, N. P. F., Colombo, M., Teixeira, G. R., de Oliveira, F. d. S., Cirne Lima, E. O., Guma, F. C. R., Souza, D. O. Abstract: Ischemic stroke is a prominent cause of death and disability, demanding innovative therapeutic strategies. Accordingly, extracellular vesicles (EVs) released from mesenchymal stem cells are promising tools for stroke treatment. In this study, we evaluated the potential neuroprotective properties of EVs released from human adipose tissue stem cells (hAT-MSC), which were obtained from a healthy individual submitted to liposuction. A single intranasal EVs administration was performed 24 h after the ischemic stroke in rats. The EVs brain penetration and the tropism to brain zone of ischemia was observed 18 h after administration. Thus, we measured EVs neuroprotection against the ischemic stroke-induced impairment on long-term motor and behavioral performance. Indeed, one single intranasal EVs administration reversed the stroke damages on: i) front paws symmetry; ii) working memory, short- and long-term memory; iii) anxiety-like behavior. These findings highlight hAT-MSC-derived EVs as a promising therapeutic strategy in stroke. Copy rights belong to original authors. Visit the link for more info
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    Common allotypes of ER aminopeptidase 1 have substrate- dependent and highly variable enzymatic properties

    Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.19.389403v1?rss=1 Authors: Jonathan, H. P., Temponeras, I., Kuiper, J., Cortes, A., Korczynska, J., Kitchen, S., Stratikos, E. Abstract: Objective: Polymorphic variation of immune system proteins can drive variability of individual immune responses. ER aminopeptidase 1 (ERAP1) generates antigenic peptides for presentation by MHC class I molecules. Coding single nucleotide polymorphisms (SNPs) in ERAP1 have been associated with predisposition to inflammatory rheumatic disease and shown to affect functional properties of the enzyme, but the interplay between combinations of these SNPs as they exist in allotypes, has not been thoroughly explored. Methods: We used phased genotype data to estimate ERAP1 allotype frequency in 2,504 individuals across five major human populations, generated highly pure recombinant enzymes corresponding to the 10 most common ERAP1 allotypes and systematically characterized their in vitro enzymatic properties. Results: We find that ERAP1 allotypes possess a wide range of enzymatic activities, whose ranking is substrate-dependent. Strikingly, allotype 10, previously associated with Behcet's disease, is consistently a low-activity outlier, suggesting that a significant percentage of individuals carry a sub-active ERAP1 gene. Enzymatic analysis revealed that ERAP1 allotypes can differ in both catalytic efficiency and substrate affinity, differences that can change intermediate accumulation in multi-step trimming reactions. Alterations in efficacy of an allosteric inhibitor that targets the regulatory site of the enzyme suggest that allotypic variation influences the communication between the regulatory and the active site. Conclusion: Our work defines the wide landscape of ERAP1 activity in human populations and demonstrates how common allotypes can induce substrate-dependent variability in antigen processing, thus contributing, in synergy with MHC haplotypes, to immune response variability and to predisposition to chronic inflammatory conditions Copy rights belong to original authors. Visit the link for more info
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    CHD7 interacts with the nucleosome acidic patch for its efficient activity via its N-terminal region

    Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.19.389429v1?rss=1 Authors: Lee, E., Kang, C., Purhonen, P., Hebert, H., Bouazoune, K., Hohng, S., Song, J.-J. Abstract: Chromodomain-Helicase DNA binding protein 7 (CHD7) is an ATP dependent chromatin remodeler involved in maintaining open chromatin structure. Mutations of CHD7 gene causes multiple developmental disorders, notably CHARGE syndrome. However, there is not much known about the molecular mechanism by which CHD7 remodels nucleosomes. Here, we performed integrative biophysical analysis on CHD7 chromatin remodeler using crosslinking-mass spectrometry (XL-MS), cryo-Electron Microscopy (cryo-EM) and single-molecule Forster Resonance Energy Transfer (smFRET). We uncover that N-terminal to the Chromodomain (N-CRD) interacts with nucleosome. Importantly, this region is required for efficient ATPase stimulation and nucleosome remodeling activity of CHD7. The cryo-EM analysis on the N-CRD_Chromodomain bound to nucleosome reveals that the N-CRD interacts with the acidic patch of nucleosome. Furthermore, smFRET analysis shows the mutations in the N-CRD result in slow or highly-fluctuating remodeling activity. Collectively, our results uncover the functional importance of a previously unidentified N-terminal region in CHD7 and implicate that the multiple domains in chromatin remodelers are involved in regulating their activities. Copy rights belong to original authors. Visit the link for more info
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    Improved production of SARS-CoV-2 spike receptor-binding domain (RBD) for serology assays

    Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.18.388868v1?rss=1 Authors: Mehalko, J., Drew, M., Snead, K., Denson, J.-P., Wall, V., Taylor, T., Sadtler, K., Messing, S., Gillette, W., Esposito, D. Abstract: The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a commonly used antigen for serology assays critical to determining the extent of SARS-CoV-2 exposure in the population. Different versions of the RBD protein have been developed and utilized in assays, with higher sensitivity attributed to particular forms of the protein. To improve the yield of these high-sensitivity forms of RBD and support the increased demand for this antigen in serology assays, we investigated several protein expression variables including DNA elements such as promoters and signal peptides, cell culture expression parameters, and purification processes. Through this investigation, we developed a simplified and robust purification strategy that consistently resulted in high levels of the high-sensitivity form of RBD and demonstrated that a carboxyterminal tag is responsible for the increased sensitivity in the ELISA. These improved reagents and processes produce high-quality proteins which are functional in serology assays and can be used to investigate seropositivity to SARS-CoV-2 infection. Copy rights belong to original authors. Visit the link for more info

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