Avsnitt
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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.
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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.
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Saknas det avsnitt?
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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.
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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.
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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.
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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.
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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.
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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
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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