PaperPlayer biorxiv biochemistry

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

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

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

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

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

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

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

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

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

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

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.18.388496v1?rss=1 Authors: Hallin, E. I., Markusson, S., Bottger, L., Torda, A. E., Bramham, C. R., Kursula, P. Abstract: Synaptic plasticity is vital for brain function and memory formation. One of the key proteins in long-term synaptic plasticity and memory is the activity-regulated cytoskeleton-associated protein (Arc). Mammalian Arc forms virus-like capsid-like structures in a process requiring the N-terminal domain and contains two C-terminal lobes that are structural homologues to retroviral capsids. Drosophila has two isoforms of Arc, dArc1 and dArc2, with low sequence similarity to mammalian Arc, but lacking the mammalian Arc N-terminal domain. Both dArc isoforms have a capsid homology domain consisting of N- and C-terminal lobes. We carried out structural characterization of the four individual dArc lobe domains. As opposed to the corresponding mammalian Arc lobe domains, which are monomeric, the dArc lobes were all oligomeric in solution, indicating a strong propensity for homophilic interactions. The N-lobe from dArc2 formed a domain-swapped dimer in the crystal structure, resulting in a novel dimer interaction that could be relevant for capsid assembly or other dArc functions. This domain-swapped structure resembles the dimeric protein C of flavivirus capsids, as well as the structure of histones dimers, domain-swapped transcription factors, and membrane-interacting BAK domains. The strong oligomerization properties of the isolated dArc lobe domains explain the ability of dArc to form capsids in the absence of any large N-terminal domain, in contrast to the mammalian protein. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.18.389155v1?rss=1 Authors: Guo, S., Vance, T., Zahiri, H., Eves, R., Stevens, C., Hehemann, J.-H., Vidal-Melgosa, S., Davies, P. Abstract: Carbohydrate recognition by lectins governs critical host-microbe interactions. MpPA14 lectin is a domain of a 1.5-MDa adhesin responsible for a symbiotic bacterium-diatom interaction in Antarctica. Here we show MpPA14 binds various monosaccharides, with L-fucose and N-acetyl glucosamine being the strongest ligands (Kd ~ 150 uM). High-resolution structures of MpPA14 with 15 different sugars bound elucidated the molecular basis for the lectin's apparent binding promiscuity but underlying selectivity. MpPA14 mediates strong Ca2+-dependent interactions with the 3, 4 diols of L-fucopyranose and glucopyranoses, and binds other sugars via their specific minor isomers. Thus, MpPA14 only binds polysaccharides like branched glucans and fucoidans with these free end-groups. Consistent with our findings, adhesion of MpPA14 to diatom cells was selectively blocked by L-fucose, but not by N-acetyl galactosamine. With MpPA14 lectin homologs present in adhesins of several pathogens, our work gives insight into an anti-adhesion strategy to block infection via ligand-based antagonists. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.17.387910v1?rss=1 Authors: Okumura, M., Kanemura, S., Matsusaki, M., Kinoshita, M., Saio, T., Ito, D., Hirayama, C., Kumeta, H., Watabe, M., Amagai, Y., Lee, Y.-H., Akiyama, S., Inaba, K. Abstract: P5, also known as PDIA6, is a PDI-family member that plays an important role in the ER quality control. Herein, we revealed that P5 dimerizes via a unique adhesive motif contained in the N-terminal thioredoxin-like domain. This motif is apparently similar to, but radically different from conventional leucine-zipper motifs, in that the former includes a periodic repeat of leucine or valine residues at the third or fourth position spanning five helical turns on 15-residue anti-parallel -helices, unlike the latter of which the leucine residues appear every two helical turns on ~30-residue parallel -helices at dimer interfaces. A monomeric P5 mutant with the impaired adhesive motif showed structural instability and local unfolding, and behaved as an aberrant protein that induces the ER stress response. Disassembly of P5 to monomers compromised its ability to inactivate IRE1 via reduction of intermolecular disulfide bonds and its Ca2+-dependent regulation of chaperone function in vitro. Thus, the leucine-valine adhesive motif supports structure and physiological function of P5. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.17.387829v1?rss=1 Authors: Huang, X., Kim, D. S., Huang, P., Vater, A. W., Siegel, J. B. Abstract: Computational protein design is growing in popularity as a means to engineer enzymes. Currently, protein design algorithms can predict the stability and function of the enzymes to only a limited degree. Thus, further experimental data is required for training software to more accurately characterize the structure-function relationship of enzymes. To date, the Design2Data (D2D) database holds 129 single point mutations of {beta}-glucosidase B (BglB) characterized by kinetic and thermal stability biophysical parameters. In this study, we introduced six mutants into the BglB database and examined their catalytic activity and thermal stability: L171M, H178M, M221L, E406W, N160E, and F415M. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.18.388215v1?rss=1 Authors: Vandebroek, L., Noguchi, H., Kamata, K., Tame, J. R. H., Van Meervelt, L., Parac-Vogt, T. N., Voet, A. R. D. Abstract: The controlled formation of protein supramolecular assemblies is challenging but it could provide an important route for the development of hybrid biomaterials. In this work, we demonstrate formation of well-defined complexes formed between the 8-fold symmetrical designer protein Tako8 and soluble metal-oxo clusters from the family of Anderson-Evans, Keggin and ZrIV-substituted Wells-Dawson polyoxometalates. A combination of x-ray crystallography and solution studies showed that metal-oxo clusters are able to serve as linker nodes for the bottom-up creation of protein based supramolecular assemblies. Our findings indicate that clusters with larger size and negative charge are capable of modulating the crystal packing of the protein, highlighting the need for a size and shape complementarity with the protein node for optimal alteration of the crystalline self-assembly. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.16.385856v1?rss=1 Authors: Oliveira Bortot, L., Lopes Rangel, V., A. Pavlovici, F., El Omari, K., Wagner, A., Brandao-Neto, J., Talon, R., von Delft, F., G Reidenbach, A., M Vallabh, S., Vallabh Minikel, E., Schreiber, S., Cristina Nonato, M. Abstract: Prion disease is caused by the misfolding of the cellular prion protein, PrPC, into a self-templating conformer, PrPSc. Nuclear magnetic resonance (NMR) and X-ray crystallography revealed the 3D structure of the globular domain of PrPC and the possibility of its dimerization via an interchain disulfide bridge that forms due to domain swap or by non-covalent association of two monomers. On the contrary, PrPSc is composed by a complex and heterogeneous ensemble of poorly defined conformations and quaternary arrangements that are related to different patterns of neurotoxicity. Targeting PrPC with molecules that stabilize the native conformation of its globular domain emerged as a promising approach to develop anti-prion therapies. One of the advantages of this approach is employing structure-based drug discovery methods to PrPC. Thus, it is essential to expand our structural knowledge about PrPC as much as possible to aid such drug discovery efforts. In this work, we report a crystallographic structure of the globular domain of human PrPC that shows a novel dimeric form and a novel oligomeric arrangement. We use molecular dynamics simulations to explore its structural dynamics and stability and discuss potential implications of these new quaternary structures to the conversion process. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.17.386979v1?rss=1 Authors: Bantis, L. E., Parente, D. J., Fenton, A. W., Swint-Kruse, L. Abstract: Amino acid variation at "rheostat" positions provides opportunity to modulate various aspects of protein function - such as binding affinity or allosteric coupling - across a wide range. Previously a subclass of "multiplex" rheostat positions was identified at which substitutions simultaneously modulated more than one functional parameter. Using the Miller laboratory's dataset of ~4000 variants of lactose repressor protein (LacI), we compared the structural properties of multiplex rheostat positions with (i) "single" rheostat positions that modulate only one functional parameter, (ii) "toggle" positions that follow textbook substitution rules, and (iii) "neutral" positions that tolerate any substitution without changing function. The combined rheostat classes comprised >40% of LacI positions, more than either toggle or neutral positions. Single rheostat positions were broadly distributed over the structure. Multiplex rheostat positions structurally overlapped with positions involved in allosteric regulation. When their phenotypic outcomes were interpreted within a thermodynamic framework, functional changes at multiplex positions were uncorrelated. This suggests that substitutions lead to complex changes in the underlying molecular biophysics. Bivariable and multivariable analyses of evolutionary signals within multiple sequence alignments could not differentiate single and multiplex rheostat positions. Phylogenetic analyses - such as ConSurf - could distinguish rheostats from toggle and neutral positions. Multivariable analyses could also identify a subset of neutral positions with high probability. Taken together, these results suggest that detailed understanding of the underlying molecular biophysics, likely including protein dynamics, will be required to discriminate single and multiplex rheostat positions from each other and to predict substitution outcomes at these sites. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.17.386177v1?rss=1 Authors: Cuevas-Navarro, A., Van, R., Cheng, A., Urisman, A., Castel, P., McCormick, F. Abstract: The spindle assembly checkpoint (SAC) is an evolutionarily conserved safety mechanism that maintains genomic stability. However, despite the understanding of the fundamental mechanisms that control the SAC, it remains unknown how signaling pathways directly interact with and regulate the mitotic checkpoint activity. In response to extracellular stimuli, a diverse network of signaling pathways involved in cell growth, survival, and differentiation are activated and this process is prominently regulated by the Ras family of GTPases. Here we show that RIT1, a Ras-related GTPase, is essential for timely progression through mitosis and proper chromosome segregation. Furthermore, pathogenic levels of RIT1 silence the SAC, accelerate transit through mitosis, and promote chromosome segregation errors through direct association with SAC proteins MAD2 and p31comet. Our results highlight a unique function of RIT1 compared to other Ras GTPases and elucidate a direct link between a signaling pathway and the SAC through a novel regulatory mechanism. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.17.387241v1?rss=1 Authors: Alexander, L. E., Gilbertson, J. S., Xie, B., Song, Z., Nikolau, B. J. Abstract: The extensive collection of glossy (gl) and eceriferum (cer) mutants of maize and Arabidopsis have proven invaluable in dissecting the branched metabolic pathways that support cuticular lipid deposition. This branched pathway integrates the fatty acid elongation-decarbonylative branch and the fatty acid elongation-reductive branch that has the capacity to generate hundreds of cuticular lipid metabolites. In this study a combined transgenic and biochemical strategy was implemented to explore and compare the physiological function of three homologous genes, Gl2, Gl2-like and CER2 in the context of this branched pathway. These biochemical characterizations integrated new extraction-chromatographic procedures with high-spatial resolution mass spectrometric imaging methods to profile the cuticular lipids on developing floral tissues transgenically expressing these transgenes in wild-type or cer2 mutant lines of Arabidopsis. Collectively, these datasets establish that both the maize Gl2 and Gl2-like genes are functional homologs of the Arabidopsis CER2 gene. In addition, the dynamic distribution of cuticular lipid deposition follows distinct floral organ localization patterns indicating that the fatty acid elongation-decarbonylative branch of the pathway is differentially localized from the fatty acid elongation-reductive branch of the pathway. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.17.387142v1?rss=1 Authors: Zhang, W., Kim, C., Tam, C. P., Lelyveld, V., Bala, S., Chaput, J., Szostak, J. W. Abstract: The prebiotic synthesis of ribonucleotides is likely to have been accompanied by the synthesis of noncanonical nucleotides including the threo-nucleotide building blocks of TNA. Here we examine the ability of activated threo-nucleotides to participate in nonenzymatic template-directed polymerization. We find that primer extension by multiple sequential threo-nucleotide monomers can occur but is strongly disfavored relative to ribo-nucleotides. Kinetic, NMR and crystallographic studies suggest that this is due in part to the slow formation of the imidazolium-bridged TNA intermediate in primer extension, and in part because of the greater distance between the attacking RNA primer 3'-hydroxyl and the phosphate of the incoming threo-nucleotide intermediate. Even a single activated threo-nucleotide in the presence of an activated downstream RNA oligonucleotide is added to the primer ten-fold more slowly than an activated ribonucleotide. In contrast, a single activated threo-nucleotide at the end of an RNA primer or in an RNA template results in only a modest decrease in the rate of primer extension, consistent with the minor and local structural distortions revealed by crystal structures. Our results are consistent with a model in which heterogeneous primordial oligonucleotides would, through cycles of replication, have given rise to increasingly homogeneous RNA strands. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.16.379206v1?rss=1 Authors: Takahashi, M., Seino, R., Ezoe, T., Ishiyama, M., Ueno, Y. Abstract: The plasma membrane (PM) plays a critical role in many cellular processes, and PM dysfunction is a key biomarker related to the cell status and several diseases. Imaging techniques using small fluorescent probes have become increasingly important tools for visualizing living cells, and particularly, their PMs. Among the commercially available PM-specific probes, PKH dyes are widely used; however, the utility of these dyes is limited by their short membrane retention times and high cytotoxicity. Herein, PlasMem Bright Green and Red are implemented as new PM-specific fluorescent probes, which employ a polycyclic aromatic fluorophore to improve their retention ability and a strong acid moiety to reduce their transmembrane diffusion and cytotoxicity. We demonstrate that the long retention and low cytotoxicity of the PlasMem Bright dyes enable them to be applied for observing neuronal PMs and monitoring PM dynamics involving the endocytic pathway. Furthermore, we successfully detected mitochondria in nerve axons over long periods of time using PlasMem Bright dyes. Finally, the combined use of exosome staining probes and PlasMem Bright dyes allowed clear visualization of the endocytic pathway. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.16.385187v1?rss=1 Authors: Oganesyan, I., Lento, C., Tandon, A., Wilson, D. J. Abstract: Both normal and pathological functions of -synuclein (SN), an abundant protein in the central and peripheral nervous system, have been linked to its interaction with membrane lipid bilayers. The ability to characterize structural transitions of SN upon membrane complexation will clarify molecular mechanisms associated with SN-linked pathologies, including Parkinsons disease (PD), Multiple Systems Atrophy and other synucleinopathies. In this work, Time-Resolved ElectroSpray Ionization Hydrogen/ Deuterium Exchange Mass Spectrometry (TRESI-HDX-MS) was employed to acquire a detailed picture of SNs conformational transitions as it undergoes complexation with nanodisc membrane mimics. Using this approach, SN interactions with DMPC nanodiscs were shown to be rapid exchanging and to have a little impact on the SN conformational ensemble. Interactions with nanodiscs containing lipids known to promote amyloidogenesis (e.g., POPG), on the other hand, were observed to induce substantial and specific changes in the SN conformational ensemble. Ultimately, we identify a region corresponding residues 19-28 and 45-57 of the SN sequence that is uniquely impacted by interactions with amyloidogenic lipid membranes and may therefore play a critical role in pathogenic aggregation. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.16.384339v1?rss=1 Authors: Ngah, Y. E., Cho, F. N., Etagha, B. S., Fusi, N. G., Francisca, N., Ikomey, M. G., Ibrahim, N. Abstract: Introduction The incidence of hepatotoxicity is life-threatening and can result to an end-stage liver disease in long-term patients on combined antiretroviral therapy (cART). Our study sought to evaluate the incidence and predictors of cART-induced hepatotoxicity (CIH) among long term users on cART in a rural District hospital. Methods This was a hospital-based cross-sectional study in the Bali District Hospital. Spectrophotometric method was use for the quantitative measurement of alanine-aminotransferase (ALT) and aspartate-aminotransferase (AST) levels. Patients with elevations of both ALT and AST were considered CIH. The Chi ({chi} 2 ) square test, ANOVA and Kaplan Meier log-ranked/ survival analyses were used to analyse the data. Results Of the 350 participants enrolled [156 (44.6%) males and 194 (55.4%) females], aged 43.87 {+/-} 0.79 years (range 20 - 84 years) included in this analysis, 26 (4.4%) experienced moderate CIH. We observed 57 (16.3%), 62 (17.7%) and 238 (68%) elevated levels ALT + AST, ALT and AST respectively. Two independent predictive factors of CIH were, the male sex and alcoholism during the study period. Conclusion The prevalence of CIH in HIV-infected patients in Bali was lower than that observed in previous studies. The duration of therapy had no influence on the frequency of CIH. Alcoholism and smoking showed significant differences in the development of CIH. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.16.384594v1?rss=1 Authors: Brun, J., Vasiljevic, S., Gangadharan, B., Hensen, M., Chandran, A. V., Hill, M. L., Kiappes, J. L., Dwek, R. A., Alonzi, D. S., Struwe, W. B., Zitzmann, N. Abstract: Severe acute respiratory syndrome coronavirus 2 is the causative pathogen of the COVID-19 pandemic which as of Nov 15, 2020 has claimed 1,319,946 lives worldwide. Vaccine development focuses on the viral trimeric spike glycoprotein as the main target of the humoral immune response. Viral spikes carry glycans that facilitate immune evasion by shielding specific protein epitopes from antibody neutralisation. Immunogen integrity is therefore important for glycoprotein-based vaccine candidates. Here we show how site-specific glycosylation differs between virus-derived spikes and spike proteins derived from a viral vectored SARS-CoV-2 vaccine candidate. We show that their cellular secretion pathways are unique, resulting in different protein glycosylation and secretion, which may have implications for the resulting immune response and future vaccine design. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.15.383679v1?rss=1 Authors: Knight, K. M., Ghosh, S., Campbell, S. L., Lefevre, T. J., Olsen, R. H. J., Smrcka, A. V., Valentin, N. H., Yin, G., Vaidehi, N., Dohlman, H. G. Abstract: G proteins play a central role in signal transduction and pharmacology. Signaling is initiated by cell-surface receptors, which promote GTP binding and the dissociation of G from the G{beta}{gamma} subunits. Structural studies have revealed the molecular basis for subunit association with receptors, RGS proteins and downstream effectors. In contrast, the mechanism of subunit dissociation is poorly understood. We used cell signaling assays, MD simulations, biochemistry and structural analysis to identify a conserved network of amino acids that dictates subunit release. In the presence of the terminal phosphate of GTP, a glycine forms a polar network with an arginine and glutamate, putting torsional strain on the subunit binding interface. This "G-R-E motif" secures GTP and, through an allosteric link, discharges the G{beta}{gamma} dimer. Replacement of network residues prevents subunit dissociation, regardless of agonist or GTP binding. These findings reveal the molecular basis for the final committed step of G protein activation. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.14.363911v1?rss=1 Authors: Li, J., Huang, Q. Abstract: Oat protein isolate (OPI) is among the plant proteins with valuable functionalities (e.g. emulsification) during daily supplement intake. Understanding their structures helps to manipulate oat proteins at small scale, which enables the appropriate deployment of their functions. Based upon such understanding, the molecular structure of oat protein isolate (OPI) in aqueous medium was investigated by synchrotron small angle X ray scattering (SAXS), and this study allows a structural reconstitution of OPI in aqueous medium. Besides, this SAXS study is complimentary to the previous study (Liu et al. J. Agric. Food Chem. 2009, 57, 4552 to 4558). From form factor fitting, we confirmed that OPI aqueous solutions at low concentrations (0.3~2 mg/mL) obtained a disk conformation (41.4*41.4*10.2 A3). Once protein concentration increased to 5 mg/mL and 10 mg/mL, the individual disk proteins formed large dimensional rod like aggregates, which was evidenced by the analyses of effective structure factor and pair distribution function (PDF). Based on the PDF results, the ab initio models of OPI particles at low concentrations (0.3 mg/mL to 2.0 mg/mL) were restored by using GASBOR algorithm. Finally, we found that weak attraction between OPI particles occurred, which was verified by second virial coefficient and pair potential. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.14.381277v1?rss=1 Authors: Johansson, E., Diffley, J. F. X. Abstract: Single-stranded DNA breaks, including simple nicks, are amongst the most common forms of DNA damage in cells. They can be readily repaired by ligation; however, if a nick occurs just ahead of an approaching replisome, the outcome is a 'collapsed' replication fork in which the nick is converted into a single-ended double-strand DNA break. Attention has largely focused on the processes by which this broken end is used to prime replication restart. We realized that in eukaryotic cells, where replication initiates from multiple replication origins, a second fork converging on the collapsed fork offers additional opportunities for repair, but also generates a substrate that can promote localized re-replication. We have modelled this with purified proteins in vitro and have found that there is, indeed, an additional hazard that eukaryotic replisomes face. We discuss how this problem might be mitigated. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.13.381293v1?rss=1 Authors: Nguyen, G. T. T., Sutinen, A., Raasakka, A., Muruganandam, G., Loris, R., Kursula, P. Abstract: Charcot-Marie-Tooth disease (CMT) is one of the most common inherited neurological disorders. Despite the common involvement of ganglioside-induced differentiation-associated protein 1 (GDAP1) in CMT, the protein structure and function, as well as the pathogenic mechanisms, remain unclear. We determined the crystal structure of the complete human GDAP1 core domain, which shows a novel mode of dimerization within the glutathione S-transferase (GST) family. The long GDAP1-specific insertion forms an extended helix and a flexible loop. GDAP1 is catalytically inactive towards classical GST substrates. Through metabolite screening, we identified a ligand for GDAP1, the fatty acid hexadecanedioic acid, which is relevant for mitochondrial membrane permeability and Ca2+ homeostasis. The fatty acid binds to a pocket next to a CMT-linked residue cluster, increases protein stability, and induces changes in protein conformation and oligomerization. The closest homologue of GDAP1, GDAP1L1, is monomeric in its full-length form. Our results highlight the uniqueness of GDAP1 within the GST family and point towards allosteric mechanisms in regulating GDAP1 oligomeric state and function. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.13.381962v1?rss=1 Authors: Cheng, C. L., Wong, M. K., Li, Y., Hochstrasser, M. Abstract: The proteasome is a large protease complex that degrades both misfolded and regulatory proteins. In eukaryotes, the 26S proteasome contains six different AAA+ ATPase subunits, Rpt1-Rpt6, which form a hexameric ring as part of the base subcomplex that drives unfolding and translocation of substrates into the proteasome core. Archaeal proteasomes contain only a single type of ATPase subunit, the proteasome-activating nucleotidase (PAN), which forms a trimer-of-dimers and is homologous to the eukaryotic Rpt subunits. A key PAN proline residue (P91) forms cis and trans peptide bonds in successive subunits around the ring, allowing efficient dimerization through upstream coiled coils. The importance of the equivalent Rpt prolines in eukaryotic proteasome assembly was unknown. We show an equivalent proline is strictly conserved in Rpt3 (in S. cerevisiae, P93) and Rpt5 (P76), well conserved in Rpt2 (P103), and loosely conserved in Rpt1 (P96) in deeply divergent eukaryotes, but in no case is its mutation strongly deleterious to yeast growth. However, the rpt2-P103A, rpt3-P93A, and rpt5-P76A mutations all cause synthetic defects with specific base assembly chaperone deletions. The Rpt5-P76A mutation decreases the levels of the protein and induces a mild proteasome assembly defect. The yeast rpt2-P103A rpt5-P76A double mutant has strong growth defects attributable to defects in proteasome base formation. Several Rpt subunits in this mutant form aggregates that are cleared, at least in part, by the Hsp42-mediated protein quality control (PQC) machinery. We propose that the conserved Rpt linker prolines promote efficient 26S proteasome base assembly by facilitating specific ATPase heterodimerization. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.13.381988v1?rss=1 Authors: Saumya, K. U., Gadhave, K., Kumar, A., Giri, R. Abstract: Capsid-anchor (CA) of Zika virus (ZIKV) is a small, single-pass transmembrane sequence that separates the capsid (C) protein from downstream pre-membrane (PrM) protein. During ZIKV polyprotein processing, CA is cleaved-off from C and PrM and left as a membrane-embedded peptide. CA plays an essential role in the assembly and maturation of the virus. However, its independent folding behavior is still unknown. Since misfolding and aggregation propensity of transmembrane proteins are now increasingly recognized and has been linked to several proteopathic disorders. Therefore, in this study, we investigated the amyloid-forming propensity of CA at physiological conditions. We observed aggregation behavior of CA peptide using dye-binding assays and ThT kinetics. The morphological analysis of CA aggregates explored by high-resolution microscopy (TEM and AFM) revealed characteristic amyloid-like fibrils. Further, the effect on mammalian cells exhibited the cytotoxic nature of the CA amyloid-fibrils. Our findings collectively shed light on the amyloidogenic phenomenon of flaviviral protein, which may contribute to their infection. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.12.379131v1?rss=1 Authors: Sun, W., Liu, X., Shi, Q., Sun, H., Li, J., Tang, X., Guo, Z., Liu, Y., Ma, J., Ren, N., Jin, F., He, C., Song, W. Abstract: Previous studies reported that gender and age could influence urine metabolomics, which should be considered in biomarker discovery. As a consequence, for the baseline of urine metabolomics characteristics, it becomes critical to avoid confounding effects in clinical cohort studies. In this study, we provided a comprehensive lifespan characterization of urine metabolomics in a cohort of 348 healthy children and 315 adults aged 1 to 70 years using liquid chromatography coupled with high resolution mass spectrometry. Our results suggested that gender-dependent urine metabolites are much greater in adults than in children. The pantothenate and CoA biosynthesis and alanine metabolism pathways were enriched in early life. Androgen and estrogen metabolism showed high activity during adolescence and youth stages. Pyrimidine metabolism was enriched in the old stage. Based on the above analysis, metabolomic characteristics of each age stage were provided. This work could help us understand the baseline of urine metabolism characteristics and contribute to further studies of clinical disease biomarker discovery. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.11.377309v1?rss=1 Authors: Ahn, S. B., Kamath, K. S., Mohamedali, A., Noor, Z., Wu, J. X., Pascovici, D., Adhikari, S., Cheruku, H. R., Guillemin, G. J., McKay, M. J., Nice, E. C., Baker, M. S. Abstract: Credible detection and quantification of low abundance proteins from human blood plasma is a major challenge in precision medicine biomarker discovery when using mass spectrometry (MS). Here, we employed a mixture of recombinant proteins in DDA libraries to subsequently detect cancer-associated low abundance plasma proteins using SWATH/DIA. The exemplar DDA recombinant protein spectral library (rPSL) was derived from tryptic digestion of 36 human recombinant proteins that had been previously implicated as possible cancer biomarkers in both our own and other studies. The rPSL was then used to identify proteins from non-depleted colorectal cancer (CRC) plasmas by SWATH-MS. Most (32/36) of the proteins in the rPSL were reliably identified in plasma samples, including 8 proteins (BTC, CXCL10, IL1B, IL6, ITGB6, TGF, TNF, TP53) not previously detected using high-stringency MS in human plasmas according to PeptideAtlas. The rPSL SWATH-MS protocol was compared to DDA-MS using MARS-depleted and post-digestion peptide fractionated plasmas (here referred to as a human plasma DDA library). Of the 32 proteins identified using rPSL SWATH, only 12 were identified using DDA-MS. The 20 additional proteins exclusively identified by using the rPSL approach with SWATH were mostly lower abundance (i.e.,

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.12.379768v1?rss=1 Authors: Daly, L. A., Brownridge, P. J., See, V., Eyers, C. E. Abstract: Adaption of cells to low oxygen environments is an essential process mediated in part by the Hypoxia Inducible Factors (HIFs). Like other transcription factors, the stability and transcriptional activity of HIFs, and consequently the hypoxic response, are regulated by post-translational modification (PTM) and changes in biomolecular interactions. However, our current understanding of PTM-mediated regulation of HIFs is primarily based on in vitro protein fragment-based studies, with validation typically having been conducted by in cellulo fragment expression and hypoxia mimicking drugs. Consequently, we still lack an understanding of true oxygen deprivation signaling via HIF. Using an immunoprecipitation-based, mass spectrometry approach, we characterize the regulation of in cellulo expressed full-length HIF-1 and HIF-2, in terms of both PTM and binding partners, in response to normoxia (21% oxygen) and hypoxia (1% oxygen). These studies revealed that a change in oxygen tension significantly alters the complexity and composition of HIF- protein interaction networks, with HIF-2 in particular having an extended hypoxia-induced interactome, most notably with mitochondrial-associated proteins. Both HIF isoforms are heavily covalently modified: we define ~40 different sites of PTM on each of HIF-1 and HIF-2, comprising 13 different PTM types, including multiple cysteine modifications and a highly unusual phosphocysteine. Over 80% of the PTMs identified are novel, and approximately half exhibit oxygen-dependency under these conditions. Combined with domain and evolutionary analysis of >225 vertebrate species, we validate Ser31 phosphorylation on HIF-1 as a regulator of transcription, and propose functional roles for Thr406, Thr528 and Ser581 on HIF-2. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.12.380584v1?rss=1 Authors: Hatstat, A. K., Pupi, M. D., McCafferty, D. G. Abstract: The Nedd4 family contains several structurally related but functionally distinct HECT-type ubiquitin ligases. The members of the Nedd4 family are known to recognize substrates through their multiple WW domains, which recognize PY motifs (PPxY, LPxY) or phospho-threonine or phospho-serine residues. To better understand substrate specificity across the Nedd4 family, we report the development and implementation of a python-based tool, PxYFinder, to identify PY motifs in the primary sequences of previously identified interactors of Nedd4 and related ligases. Using PxYFinder, we find that, on average, half of Nedd4 family interactions are PY-motif mediated. Further, we find that PPxY motifs are more prevalent than LPxY motifs and are more likely to occur in proline-rich regions. Further, PPxY regions are more disordered on average relative to LPxY-containing regions. Informed by consensus sequences for PY motifs across the Nedd4 interactome, we rationally designed a peptide library and employed a computational screen, revealing sequence- and biomolecular interaction-dependent determinants of WW-domain/PY-motif interactions. Cumulatively, our efforts provide a new bioinformatic tool and expand our understanding of sequence and structural factors that contribute to PY-motif mediated substrate recognition across the Nedd4 family. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.11.378018v1?rss=1 Authors: Eberle, R. J., Olivier, D. S., Amaral, M. S., Willbold, D., Arni, R. K., Coronado, M. A. Abstract: Since the first report of a new pneumonia disease in December 2019 (Wuhan, China) up to now WHO reported more than 50 million confirmed cases and more than one million losses, globally. The causative agent of COVID-19 (SARS-CoV-2) has spread worldwide resulting in a pandemic of unprecedented magnitude. To date, no clinically safe drug or vaccine is available and the development of molecules to combat SARS-CoV-2 infections is imminent. A well-known strategy to identify molecules with inhibitory potential against SARS-CoV-2 proteins is the repurposing of clinically developed drugs, e.g., anti-parasitic drugs. The results described in this study demonstrate the inhibitory potential of quinacrine and suramin against SARS-CoV-2 main protease (3CLpro). Quinacrine and suramin molecules present a competitive and non-competitive mode of inhibition, respectively, with IC50 and KD values in low M range. Using docking and molecular dynamics simulations we identified a possible binding mode and the amino acids involved in these interactions. Our results suggested that suramin in combination with quinacrine showed promising synergistic efficacy to inhibit SARS-CoV-2 3CLpro. The identification of effective, synergistic drug combinations could lead to the design of better treatments for the COVID-19 disease. Drug repositioning offers hope to the SARS-CoV-2 control. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.11.378463v1?rss=1 Authors: Leandro, J., Bender, A., Dodatko, T., Argmann, C., Yu, C., Houten, S. M. Abstract: The glutaric acidurias are a group of inborn errors of metabolism with different etiologies. Glutaric aciduria type 3 (GA3) is a biochemical phenotype with uncertain clinical relevance caused by a deficiency of succinyl-CoA:glutarate-CoA transferase (SUGCT). SUGCT catalyzes the succinyl-CoA-dependent conversion of glutaric acid into glutaryl-CoA preventing urinary loss of the organic acid. Here, we describe the presence of a GA3 trait in mice of 129 substrains due to SUGCT deficiency, which was identified by screening of urine organic acid profiles obtained from different inbred mouse strains including 129S2/SvPasCrl. Molecular and biochemical analyses in an F2 population of the parental C57BL/6J and 129S2/SvPasCrl strains (B6129F2) confirmed that the GA3 trait occurred in Sugct129/129 animals. We evaluated the impact of SUGCT deficiency on metabolite accumulation in the glutaric aciduria type 1 (GA1) mouse model. We found that GA1 mice with SUGCT deficiency have decreased excretion of urine 3-hydroxyglutaric acid and decreased levels glutarylcarnitine in urine, plasma and kidney. Our work demonstrates that SUGCT contributes to the production of glutaryl-CoA under conditions of low and pathologically high glutaric acid levels. Our work also highlights the notion that unexpected biochemical phenotypes can occur in widely used inbred animal lines. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.11.378711v1?rss=1 Authors: Liaci, A. M., Steigenberger, B., Tamara, S., Telles de Souza, P. C., Grollers-Mulderij, M., Ogrissek, P., Marrink, S.-J., Scheltema, R. A., Foerster, F. Abstract: The signal peptidase complex (SPC) is an essential membrane complex in the endoplasmic reticulum (ER), where it removes signal peptides (SPs) from a large variety of secretory pre-proteins with exquisite specificity. Although the determinants of this process have been established empirically, the molecular details of SP recognition and removal remain elusive. Here, we show that the human SPC exists in two functional paralogs with distinct proteolytic subunits. We determined the atomic structures of both paralogs using electron cryo-microscopy and structural proteomics. The active site is formed by a catalytic triad and abuts the ER membrane, where a transmembrane window collectively formed by all subunits locally thins the bilayer. This unique architecture generates specificity for thousands of SPs based on the length of their hydrophobic segments. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.11.378570v1?rss=1 Authors: Nie, L., Pike, A. C., Pascoa, T. C., Bushell, S. R., Quigley, A., Ruda, G. F., Chu, A., Cole, V., Speedman, D., Moreira, T., Shrestha, L., Mukhopadhyay, S. M., Burgess-Brown, N. A., Love, J. D., Brennan, P. E., Carpenter, E. P. Abstract: Very long chain fatty acids (VLCFAs) are essential building blocks for synthesis of the ceramides and sphingolipids required for nerve, skin and retina function and 3-keto acyl-CoA synthases (ELOVL elongases) perform the first step in the FA elongation cycle. Although ELOVLs are implicated in common diseases including insulin resistance, hepatic steatosis and Parkinson's, their underlying molecular mechanisms are unknown. Here we report the structure of the human ELOVL7 elongase, which includes an inverted transmembrane barrel structure surrounding a 35 [A] long tunnel containing a covalently-attached product analogue. The structure reveals the substrate binding sites in the tunnel and an active site deep in the membrane including the canonical ELOVL HxxHH sequence. This indicates a ping-pong mechanism for catalysis, involving unexpected covalent histidine adducts. The unusual substrate-binding arrangement and chemistry suggest mechanisms for selective ELOVL inhibition, relevant for diseases where VLCFAs accumulate such as X-linked adrenoleukodystrophy. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.11.377994v1?rss=1 Authors: Makarov, M., Meng, J., Tretyachenko, V., Srb, P., Brezinova, A., Giacobelli, V. G., Bednarova, L., Vondrasek, J., Dunker, A. K., Hlouchova, K. Abstract: It is well-known that the large diversity of protein functions and structures is derived from the broad spectrum of physicochemical properties of the 20 canonical amino acids. According to the generally accepted hypothesis, protein evolution was continuously associated with enrichment of this alphabet, increasing stability, specificity and spectrum of catalytic functions. Aromatic amino acids are considered the latest addition to genetic code. The main objective of this study was to test whether enzymatic catalysis can spare the aromatic amino acids (aromatics) by determining the effect of amino acid alphabet reduction on structure and function of dephospho-CoA kinase (DPCK). We designed two mutant variants of a putative DPCK from Aquifex aeolicus by substituting (i) Tyr, Phe and Trp or (ii) all aromatics (including His), i.e. ~10% of the total sequence. Their structural characterization indicates that removal of aromatic amino acids may support rich secondary structure content although inevitably impairs a firm globular arrangement. Both variants still possess ATPase activity, although with 150-300 times lower efficiency in comparison with the wild-type phosphotransferase activity. The transfer of the phosphate group to the dephospho-CoA substrate is however heavily uncoupled and only one of the variants is still able to perform the reaction. Here we provide support to the hypothesis that proteins in the early stages of life could support at least some enzymatic activities, despite lower efficiencies resulting from the lack of a firm hydrophobic core. Based on the presented data we hypothesize that further protein scaffolding role may be provided by ligands upon binding. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.11.378067v1?rss=1 Authors: Sato, K., Martin-Pintado, N., Post, H., Altelaar, M., Knipscheer, P. Abstract: G-quadruplex (or G4) structures are non-canonical DNA structures that form in guanine-rich sequences and threaten genome stability when not properly resolved. G4 unwinding occurs during S phase via an unknown mechanism. Using Xenopus egg extracts, we define a three-step G4 unwinding mechanism that is coupled to DNA replication. First, the replicative helicase (CMG) stalls at a leading strand G4 structure. Second, the DHX36 helicase mediates the bypass of the CMG past the intact G4 structure, which allows approach of the leading strand to the G4. Third, G4 structure unwinding by the FANCJ helicase enables the DNA polymerase to synthesize past the G4 motif. A G4 on the lagging strand template does not stall CMG, but still requires DNA replication for unwinding. DHX36 and FANCJ have partially redundant roles, conferring robustness to this pathway. Our data reveal a novel genome maintenance pathway that promotes faithful G4 replication thereby avoiding genome instability. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.09.375329v1?rss=1 Authors: Tomko, E. J., Luyties, O., Rimel, J. K., Tsai, C.-L., Fuss, J. O., Fishburn, J., Hahn, S., Tsutakawa, S. E., Taatjes, D. J., Galburt, E. A. Abstract: The general transcription factor TFIIH contains three ATP-dependent catalytic activities. TFIIH functions in nucleotide excision repair primarily as a DNA helicase and in Pol II transcription initiation as a dsDNA translocase and protein kinase. During initiation, the XPB/Ssl2 subunit of TFIIH couples ATP hydrolysis to dsDNA translocation facilitating promoter opening and the kinase module phosphorylates the C-terminal domain to facilitate the transition to elongation. These functions are conserved between metazoans and yeast; however, yeast TFIIH also drives transcription start-site scanning in which Pol II scans downstream DNA to locate productive start-sites. The ten-subunit holo-TFIIH from S. cerevisiae has a processive dsDNA translocase activity required for scanning and a structural role in scanning has been ascribed to the three-subunit TFIIH kinase module. Here, we assess the dsDNA translocase activity of ten-subunit holo- and core-TFIIH complexes (i.e. seven subunits, lacking the kinase module) from both S. cerevisiae and H. sapiens. We find that neither holo nor core human TFIIH exhibit processive translocation, consistent with the lack of start-site scanning in humans. Furthermore, in contrast to holo-TFIIH, the S. cerevisiae core-TFIIH also lacks processive translocation and its dsDNA-stimulated ATPase activity was reduced ~5-fold to a level comparable to the human complexes, potentially explaining the reported upstream shift in start-site observed in the absence of the S. cerevisiae kinase module. These results suggest that neither human nor S. cerevisiae core-TFIIH can translocate efficiently, and that the S. cerevisiae kinase module functions as a processivity factor to allow for robust transcription start-site scanning. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.10.376327v1?rss=1 Authors: Frank, F., Liu, X., Ortlund, E. A. Abstract: The glucocorticoid receptor (GR) is a ligand-regulated transcription factor (TF) that controls the tissue- and gene-specific transactivation and transrepression of thousands of target genes. Distinct GR DNA binding sequences with activating or repressive activities have been identified, but how they modulate transcription in opposite ways is not known. We show that GR forms phase-separated condensates that specifically concentrate known co-regulators via their intrinsically disordered regions (IDRs) in vitro. A combination of dynamic, multivalent (between IDRs) and specific, stable interactions (between LxxLL motifs and the GR ligand binding domain) control the degree of recruitment. Importantly, GR DNA-binding directs the selective partitioning of co-regulators within GR condensates such that activating DNAs cause enhanced recruitment of co-activators. Our work shows that condensation controls GR function by modulating co-regulator recruitment and provides a mechanism for the up- and down-regulation of GR target genes controlled by distinct DNA recognition elements. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.10.376251v1?rss=1 Authors: Martinez-Alarcon, D., Pieters, R. J., VARROT, A. Abstract: Scedosporium apiospermum is an emerging opportunistic fungal pathogen responsible for life-threatening infections in immunocompromised patients. This fungus exhibits limited susceptibility to all current antifungals and, due its emerging character, its pathogenicity and virulence factors remain largely unknown. Carbohydrate binding proteins such as lectins are involved in host-pathogen interactions and may constitute valuable therapeutic targets to inhibit microbial adhesion to the host cells by using carbohydrate mimics. However, such lectins are still unidentified in S. apiospermum. Here, we present the first report of the identification and characterization of a lectin from S. apiospermum named SapL1. SapL1 is homologous to the conidial surface lectin FleA from Aspergillus fumigatus, known to be involved in the adhesion to host glycoconjugates present in human lung epithelium. The present report includes a detailed strategy to achieve SapL1 soluble expression in bacteria, its biochemical characterization, an analysis of its specificity and affinity by glycan arrays and isothermal titration calorimetry (ITC), as well as the structural characterization of its binding mode by X-ray crystallography. The information gathered here contributes to the understanding of glycosylated surface recognition by Scedosporium species and is essential for the design and development of antiadhesive glycodrugs targeting SapL1. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.10.376822v1?rss=1 Authors: Walter, J. D., Hutter, C. A. J., Garaeva, A. A., Scherer, M., Zimmermann, I., Wyss, M., Rheinberger, J., Ruedin, Y., Earp, J. C., Egloff, P., Sorgenfrei, M., Hürlimann, L., Gonda, I., Meier, G., Remm, S., Thavarasah, S., Zimmer, G., Slotboom, D. J., Paulino, C., Plattet, P., Seeger, M. A. Abstract: The COVID-19 pandemic has resulted in a global crisis. Here, we report the generation of synthetic nanobodies, known as sybodies, against the receptor-binding domain (RBD) of SARS-CoV-2 spike protein. We identified a sybody pair (Sb#15 and Sb#68) that can bind simultaneously to the RBD, and block ACE2 binding, thereby neutralizing pseudotyped and live SARS-CoV-2 viruses. Cryo-EM analyses of the spike protein in complex with both sybodies revealed symmetrical and asymmetrical conformational states. In the symmetric complex each of the three RBDs were bound by both sybodies, and adopted the up conformation. The asymmetric conformation, with three Sb#15 and two Sb#68 bound, contained one down RBD, one up-out RBD and one up RBD. Bispecific fusions of the sybodies increased the neutralization potency 100-fold, as compared to the single binders. Our work demonstrates that linking two binders that recognize spatially-discrete binding sites result in highly potent SARS-CoV-2 inhibitors for potential therapeutic applications. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.10.363747v1?rss=1 Authors: Johe, P., Jaenicke, E., Neuweiler, H., Schirmeister, T., Kersten, C., Hellmich, U. A. Abstract: Rhodesain is the lysosomal cathepsin L-like cysteine protease of T. brucei rhodesiense, the causative agent of Human African Trypanosomiasis. The enzyme is essential for the proliferation and pathogenicity of the parasite as well as its ability to overcome the blood-brain barrier of the host. Lysosomal cathepsins are expressed as zymogens with an inactivating pro-domain that is cleaved under acidic conditions. A structure of the uncleaved maturation intermediate from a trypanosomal cathepsin L-like protease is currently not available. We thus established the heterologous expression of T. brucei rhodesiense pro-rhodesain in E. coli and determined its crystal structure. The trypanosomal pro-domain differs from non-parasitic pro-cathepsins by a unique, extended -helix that blocks the active site and whose interactions resemble that of the antiprotozoal inhibitor K11777. Interdomain dynamics between pro- and core protease domain as observed by photoinduced electron transfer fluorescence correlation spectroscopy increase at low pH, where pro-rhodesain also undergoes autocleavage. Using the crystal structure, molecular dynamics simulations and mutagenesis, we identify a conserved interdomain salt bridge that prevents premature intramolecular cleavage at higher pH values and may thus present a control switch for the observed pH-sensitivity of pro-enzyme cleavage in (trypanosomal) CathL-like proteases. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.09.374082v1?rss=1 Authors: Musico, A., Frigerio, R., Mussida, A., Barzon, L., Sinigaglia, A., Riccetti, S., Gobbi, F., Piubelli, C., Bergamaschi, G., Chiari, M., Gori, A., Cretich, M. Abstract: A workflow for SARS-CoV-2 epitope discovery on peptide microarrays is herein reported. The process started with a proteome-wide screening of immunoreactivity based on the use of a high-density microarray followed by a refinement and validation phase on a restricted panel of probes using microarrays with tailored peptide immobilization through a click-based strategy. Progressively larger, independent cohorts of Covid-19 positive sera were tested in the refinement processes, leading to the identification of immunodominant regions on SARS-CoV-2 Spike (S), Nucleocapsid (N) protein and Orf1ab polyprotein. A summary study testing 50 serum samples highlighted an epitope of the N protein (region 155-171) providing 92% sensitivity and 100% specificity of IgG detection in Covid-19 samples thus being a promising candidate for rapid implementation in serological tests. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.08.373522v1?rss=1 Authors: Tanner, N. K., Yeter-Alat, H., Belgareh-Touze, N., Huvelle, E., Mokdadi, M., Banroques, J. Abstract: The DEAD-box RNA helicase Ded1 is an essential yeast protein involved in translation initiation that belongs to the DDX3 subfamily. The purified Ded1 protein is an ATP-dependent RNA binding protein and an RNA-dependent ATPase, but it lacks substrate specificity and enzymatic regulation. Here we demonstrate by yeast genetics, in situ localization and in vitro biochemical approaches that Ded1 is associated with and regulated by the signal recognition particle (SRP), which is a universally conserved ribonucleoprotein complex required for the co-translational translocation of polypeptides into the endoplasmic reticulum lumen and membrane. Ded1 is physically associated with SRP components in vivo and in vitro. Ded1 is genetically linked with SRP proteins. Finally, the enzymatic activity of Ded1 is inhibited by SRP21 with SCR1 RNA. We propose a model where Ded1 actively participates in the translocation of proteins during translation. Our results open a new comprehension of the role of Ded1 during translation. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.09.373951v1?rss=1 Authors: Petroff, A., Weir, R. L., Yates, C. R., Ng, J. D., Baudry, J. Abstract: Stearoyl-CoA desaturase-1 (SCD1 or delta-9 desaturase, D9D) is a key metabolic protein that modulates cellular inflammation and stress, but overactivity of SCD1 is associated with diseases including cancer and metabolic syndrome. This transmembrane endoplasmic reticulum protein converts saturated fatty acids into monounsaturated fatty acids, primarily stearoyl-CoA into oleoyl-CoA, which are critical products for energy metabolism and membrane composition. The present computational molecular dynamics study characterizes the molecular dynamics of SCD1 with substrate, product, and as apoprotein. The modeling of SCD1:fatty acid interactions suggests that 1) SCD1:CoA moiety interactions open the substrate binding tunnel, 2) SCD1 stabilizes a substrate conformation favorable for desaturation, and 3) SCD1:product interactions result in an opening of the tunnel, possibly allowing product exit into the surrounding membrane. Together, these results describe a highly dynamic series of SCD1 conformations resulting from the enzyme:cofactor:substrate interplay that inform drug-discovery efforts. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.09.375618v1?rss=1 Authors: Kim, T. Y., Cha, J. S., Kim, H., Choi, Y., Cho, H.-S., Kim, H.-S. Abstract: A protein binder with a desired epitope and binding affinity is critical to the development of therapeutic agents. Here we present computationally-guided design and affinity improvement of a protein binder recognizing a specific site on domain IV of human epidermal growth factor receptor 2 (HER2). As a model, a protein scaffold composed of Leucine-rich repeat (LRR) modules was used. We designed protein binders which appear to bind a target site on domain IV using a computational method. Top 10 designs were expressed and tested with binding assays, and a lead with a low micro-molar binding affinity was selected. Binding affinity of the selected lead was further increased by two-orders of magnitude through mutual feedback between computational and experimental methods. The utility and potential of our approach was demonstrated by determining the binding interface of the developed protein binder through its crystal structure in complex with the HER2 domain IV. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.09.375048v1?rss=1 Authors: Galvagnion, C., Cerri, S., Schapira, A. H. V., Blandini, F., Di Monte, D. A. Abstract: Intraneuronal accumulation of aggregated -synuclein is a pathological hallmark of Parkinson's disease. Therefore, mechanisms capable of promoting -synuclein deposition bear important pathogenetic implications. Mutations of the glucocerebrosidase 1 (GBA) gene represent a prevalent Parkinson's disease risk factor. They are associated with loss of activity of a key enzyme involved in lipid metabolism, glucocerebrosidase, supporting a mechanistic relationship between abnormal -synuclein-lipid interactions and the development of Parkinson pathology. In this study, the lipid membrane composition of fibroblasts isolated from control subjects, patients with idiopathic Parkinson's disease (iPD) and Parkinson patients carrying the L444P GBA mutation (PD-GBA) was assayed using shotgun lipidomics. The lipid profile of PD-GBA fibroblasts differed significantly from that of control and iPD cells. It was characterized by an overall increase in sphingolipid levels. It also featured a significant change in the proportion of ceramide, sphingomyelin and hexosylceramide molecules with shorter and longer hydrocarbon chain length; levels of shorter-chain molecules were increased while the percent of longer-chain sphingolipids was decreased in PD-GBA lipid extracts. The extent of this shift was correlated to the degree of reduction of fibroblast glucocerebrosidase activity. In a second set of experiments, lipid extracts from control and PD-GBA fibroblasts were added to incubations of recombinant -synuclein. The kinetics of -synuclein aggregation, as assessed by the binding of thioflavin T to amyloid structures, was significantly accelerated after addition of PD-GBA extracts as compared to control samples. Amyloid fibrils collected at the end of these incubations contained lipids, indicating -synuclein-lipid co-assembly. Lipids extracted from -synuclein fibrils were also analysed by shotgun lipidomics. Data revealed that the lipid content of these fibrils was significantly enriched of shorter-chain sphingolipids. Taken together, findings of this study indicate that the L444P GBA mutation and consequent enzymatic loss are associated with a distinctly altered membrane lipid profile that provides a biological fingerprint of this mutation in Parkinson fibroblasts. This altered lipid profile, which includes an increased content of shorter-chain sphingolipids, could also be an indicator of increased risk for -synuclein aggregate pathology. Shorter-chain molecules may act as preferred reactants during lipid-induced -synuclein fibrillation. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.09.374355v1?rss=1 Authors: Pearl, L. H., Pal, M., Munoz-Hernandez, H., Bjorklund, D., Zhou, L., Degliesposti, G., Skehel, J. M., Hesketh, E. L., Thompson, R. F., Llorca, O., Prodromou, C. Abstract: The R2TP (RUVBL1-RUVBL2-RPAP3-PIH1D1) complex, in collaboration with HSP90, functions as a chaperone for the assembly and stability of protein complexes, including RNA polymerases, snRNPs and PI3 kinase-like kinases (PIKK) such as TOR and SMG1. PIKK stabilisation depends on an additional complex of TELO2, TTI1 and TTI2 (TTT), whose structure and function are poorly understood. We have now determined the cryo-EM structure of the human R2TP-TTT complex that together with biochemical experiments reveals the mechanism of TOR recruitment to the R2TP-TTT chaperone. The HEAT-repeat TTT complex binds the kinase domain of TOR, without blocking its activity, and delivers TOR to the R2TP chaperone. In addition, TTT regulates the R2TP chaperone by inhibiting RUVBL1-RUVBL2 ATPase activity and by modulating the conformation and interactions of the PIH1D1 and RPAP3 components of R2TP. Together, our results show how TTT couples the recruitment of TOR to R2TP with the regulation of this chaperone system. Copy rights belong to original authors. Visit the link for more info