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Rxivist combines preprints from bioRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 93,254 bioRxiv papers from 397,991 authors.

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in category biochemistry

3,091 results found. For more information, click each entry to expand.

1941: Global analysis of protein degradation in prion infected cells
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Posted to bioRxiv 23 Jul 2019

Global analysis of protein degradation in prion infected cells
255 downloads biochemistry

Charles Hutti, Kevin A Welle, Jennifer R Hryhorenko, Sina Ghaemmaghami

Prion diseases are rare neurological disorders caused by the misfolding of the cellular prion protein (PrPC) into cytotoxic fibrils (PrPSc). Intracellular PrPSc aggregates primarily accumulate within late endosomes and lysosomes, organelles that participate in the degradation and turnover of a large subset of the proteome. Thus, intracellular accumulation of PrPSc aggregates have the potential to globally influence protein degradation kinetics within an infected cell. We analyzed the proteome-wide effect of prion infection on protein degradation rates in N2a neuroblastoma cells by dynamic stable isotopic labeling with amino acids in cell culture (dSILAC) and bottom-up proteomics. The analysis quantified the degradation rates of more than 4,700 proteins in prion-infected and uninfected cells. As expected, the degradation rate of the prion protein is significantly decreased upon aggregation in infected cells. In contrast, the degradation kinetics of the remainder of the N2a proteome generally increases upon prion infection. This effect occurs concurrently with increases in the cellular activities of autophagy and lysosomal hydrolases. The resulting enhancement in proteome flux may play a role in the survival of N2a cells upon prion infection.

1942: Dimerization of the Pragmin pseudo-kinase regulates protein tyrosine phosphorylation
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Posted to bioRxiv 27 Nov 2017

Dimerization of the Pragmin pseudo-kinase regulates protein tyrosine phosphorylation
255 downloads biochemistry

Céline Lecointre, Valérie simon, Clément kerneur, Frédéric Allemand, Aurélie Fournet, Ingrid Montarras, Jean-Luc Pons, Muriel Gelin, Constance Brignatz, Serge Urbach, Gilles Labesse, Serge Roche

The pseudo-kinase and signaling protein Pragmin has been linked to cancer by regulating protein tyrosine phosphorylation via unknown mechanisms. Here we present the crystal structure of the Pragmin 906-1368 amino acids C-terminus, which encompasses its kinase domain. We show that Pragmin contains a classical protein kinase fold devoid of catalytic activity. A particular inhibitory triad, conserved in a Pragmin/SgK269/PEAK1/C19orf35 superfamily, tightly holds the catalytic lysine (K997) to prevent ATP binding. By proteomics, we discovered that this pseudo-kinase uses the tyrosine kinase CSK to induce protein tyrosine phosphorylation in human cells. Interestingly, the protein kinase domain is bordered by N- and C-terminal extensions forming an original dimerization domain that regulates Pragmin self-association and stimulates CSK activity. A1329E mutation in the C-terminal extension destabilizes Pragmin dimerization and reduces CSK activation. Thus, our results reveal a new dimerization mechanism by which a pseudo-kinase can induce protein tyrosine phosphorylation.

1943: Multiplexable fluorescence lifetime imaging (FLIM) probes for Abl and Src-family kinases
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Posted to bioRxiv 30 May 2019

Multiplexable fluorescence lifetime imaging (FLIM) probes for Abl and Src-family kinases
255 downloads biochemistry

Nur P. Damayanti, Sampreeti Jena, Jackie Tan, Joseph M. K. Irudayaraj, L. L. Parker

Most commonly employed strategies to map kinase activities in live cells require expression of genetically-encoded proteins (e.g. FRET sensors). In this work, we describe development and preliminary application of a set of cell-penetrating, fluorophore labelled peptide substrates for fluorescence lifetime imaging (FLIM) of Abl and Src-family kinase activities. These probes do not rely on FRET pairs or genetically-encoded protein expression. We also demonstrate image-by-image and pixel-by-pixel quantification of probe phosphorylation ratio, suggesting that this strategy will be useful for detailed mapping of single cell and subcellular kinase activity in live cells.

1944: Sterols as dietary markers for Drosophila melanogaster
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Posted to bioRxiv 29 Nov 2019

Sterols as dietary markers for Drosophila melanogaster
255 downloads biochemistry

Oskar Knittelfelder, Elodie Prince, Susanne Sales, Eric Fritzsche, Thomas Wöhner, Marko Brankatschk, Andrej Shevchenko

During cold acclimation fruit flies switch their feeding from yeast to plant food, however there are no robust markers to monitor it in the wild. Drosophila melanogaster is a sterol auxotroph and relies on dietary sterols to produce lipid membranes, lipoproteins and molting hormones. We employed shotgun lipidomics to quantify eight major food sterols in total extracts of heads, female and male genital tracts of adult flies. We found that their sterol composition is dynamic and reflective of flies diet in an organ-specific manner. Season-dependent changes observed in the organs of wild-living flies suggested that the molar ratio between yeast (ergosterol, zymosterol) and plant (sitosterol, stigmasterol) sterols is a quantifiable, generic and unequivocal marker of their feeding behavior, including cold acclimation. It provides technically simpler and more contrast readout compared to the full lipidome analysis and is suitable for ecological and environmental population-based studies.

1945: Towards the application of Tc toxins as a universal protein translocation system
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Posted to bioRxiv 18 Jul 2019

Towards the application of Tc toxins as a universal protein translocation system
255 downloads biochemistry

Daniel Roderer, Evelyn Schubert, Oleg Sitsel, Stefan Raunser

Tc toxins are large bacterial protein complexes that inject cytotoxic enzymes into target cells using a sophisticated syringe-like mechanism. Tc toxins are composed of a membrane translocator and a cocoon that encapsulates a toxic enzyme. The toxic enzyme varies between Tc toxins from different species and is not conserved. Here, we investigated whether the toxic enzyme can be replaced by other small proteins of different origin and properties, namely human Cdc42, herpes simplex virus ICP47, Arabidopsis thaliana iLOV, Escherichia coli DHFR, human Ras-binding domain of CRAF kinase, and tobacco etch virus protease. Using a combination of electron microscopy, X-ray crystallography and in vitro translocation assays, we demonstrate that it is possible to turn Tc toxins into customizable molecular syringes for delivering proteins of interest across membranes. We also infer the guidelines that protein cargos must obey in terms of size, charge, and fold in order to successfully take advantage of this new universal protein translocation system.

1946: The inter-dimeric interface controls function and stability of Ureaplasma urealiticum methionine S-adenosyltransferase
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Posted to bioRxiv 12 Jun 2019

The inter-dimeric interface controls function and stability of Ureaplasma urealiticum methionine S-adenosyltransferase
254 downloads biochemistry

Daniel Kleiner, Fannia Shmulevich, Raz Zarivach, Anat Shahar, Michal Sharon, Gili Ben-Nissan, Shimon Bershtein

Methionine S-adenosyltransferases (MATs) are predominantly homotetramers, comprised of dimers of dimers. The highly conserved dimeric interface harbors two active sites, making the dimer the obligatory functional unit. Yet, functionality of the recently evolved inter-dimeric interface remains unknown. Here, we show that the inter-dimeric interface of U. urealiticum MAT has evolved to control the catalytic activity and structural integrity of the homotetramer in response to product accumulation. When all four active sites are occupied with the product, S-adenosylmethionine (SAM), binding of four additional SAM molecules to the inter-dimeric interface prompts a ~45° shift in the dimer orientation and a concomitant ~60% increase in the interface area. This rearrangement inhibits the enzymatic activity by locking the flexible active site loops in a closed state and renders the tetramer resistant to proteolytic degradation. Our findings suggest that the inter-dimeric interfaces of MATs are recruited by evolution to tune the molecular properties of the entire homotetramer.

1947: Two Novel α-L-Arabinofuranosidases from Bifidobacterium longum subsp. longum belonging to Glycoside Hydrolase Family 43 Cooperatively Degrade Arabinan
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Posted to bioRxiv 29 Jul 2018

Two Novel α-L-Arabinofuranosidases from Bifidobacterium longum subsp. longum belonging to Glycoside Hydrolase Family 43 Cooperatively Degrade Arabinan
254 downloads biochemistry

Masahiro Komeno, Honoka Hayamizu, Kiyotaka Fujita, Hisashi Ashida

Arabinose-containing poly- or oligosaccharides are suitable carbohydrate sources for Bifidobacterium longum subsp. longum, though their degradation pathways are poorly understood. In this study, we found that the gene expression levels of bllj_1852 and bllj_1853 from B. longum subsp. longum JCM 1217 were enhanced in the presence of arabinan. Both genes encode previously uncharacterized glycoside hydrolase (GH) family 43 enzymes. Subsequently, we cloned those genes and characterized the recombinant enzymes expressed in Escherichia coli. Both enzymes exhibited α-L-arabinofuranosidase activity toward synthetic p-nitrophenyl glycoside, but the specificities for L-arabinofuranosyl linkages were different. BLLJ_1852 catalyzed the hydrolysis of α1,2- and α1,3-L-arabinofuranosyl linkages found in the side chains of arabinan and arabinoxylan. BLLJ_1852 released L-arabinose 100 times faster from arabinan than from arabinoxylan but did not act on arabinogalactan. BLLJ_1853 catalyzed the hydrolysis of α1,5-L-arabinofuranosyl linkages found on the arabinan backbone. BLLJ_1853 released L-arabinose from arabinan but not from arabinoxylan or arabinogalactan. Both enzyme activities were largely suppressed with EDTA treatment, suggesting that they require divalent metal ions. BLLJ_1852 was moderately activated in the presence of all divalent cations tested, whereas BLLJ_1853 activity was inhibited by Cu2+. The GH43 domains of BLLJ_1852 and BLLJ_1853 are classified into GH43 subfamilies 27 and 22, respectively, but hardly share similarity with other biochemically characterized members in the corresponding subfamilies.

1948: Untargeted metabolite analysis of Ocimum leaves shows species specific variations
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Posted to bioRxiv 17 Jun 2019

Untargeted metabolite analysis of Ocimum leaves shows species specific variations
254 downloads biochemistry

Manu Shree, Ranjan K Nanda, Shyam K. Masakapalli

Tulsi (Ocimum species), the queen of herbs is a common ingredient in beverages with perceived health benefits. Recently published Ocimum genome highlighted the presence of several genes that contributes to important phytochemicals but a comprehensive metabolite profiling to study the water soluble metabolites of Ocimum is lacking. In this study, untargeted metabolic profiling of hot water extract of fresh and air dried leaves of O. basilicum, O. sanctum and O. kilimandscharicum species employing gas chromatography and mass spectrometry (GC-MS) was attempted. Analysis of hot water extracts of Ocimum leaves will provide details of molecules consumed and species specific differences, if any. Several metabolic features including amino acids (glycine, serine glutamate), organic and other acids (succinic acid, fumaric acid, 4-amino butanoic acid), sugars and their derivatives (glucose, sucrose, mannitol, fructose) and secondary metabolites (shikimic acid, quinic acid, catechol, gamma amino butyric acid, eugenol) were identified. Multivariate statistical analysis of GC-MS data indicated several species specific metabolic similarities and differences. Based on variable importance parameter score of >1, it was observed that in case of air dried extracts, glucose, fumaric acid, and D-mannitol displayed as important variables for species specific variation. Whereas in case of fresh leaves extracts, the variation was prominent due to xylose, D-allose and an unknown metabolic feature detected at 24 min (metabolite@24 with highest m/z 75). Phytochemical phenotype of Ocimum leaves not only shows species specific variations but these may partly explain their difference in taste and health benefits from their use as hot beverages.

1949: Genetic ablation of the mitochondrial ribosome in Plasmodium falciparum sensitizes the human malaria parasite to antimalarial drugs targeting mitochondrial functions
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Posted to bioRxiv 14 Jan 2020

Genetic ablation of the mitochondrial ribosome in Plasmodium falciparum sensitizes the human malaria parasite to antimalarial drugs targeting mitochondrial functions
254 downloads biochemistry

Liqin Ling, Maruthi Mulaka, Justin Munro, Swati Dass, Michael W. Mather, Michael K. Riscoe, Manuel Llinás, Jing Zhou, Hangjun Ke

The mitochondrion of malaria parasites contains clinically validated drug targets. Within Plasmodium spp., the mitochondrial DNA (mtDNA) is only 6 kb long, being the smallest mitochondrial genome among all eukaryotes. The mtDNA encodes only three proteins of the mitochondrial electron transport chain and ~ 27 small, fragmented rRNA genes in length of 22-195 nucleotides. The rRNA fragments are thought to form a mitochondrial ribosome (mitoribosome), together with ribosomal proteins imported from the cytosol. The mitoribosome of Plasmodium falciparum has been shown to be essential for maintenance of the mitochondrial membrane potential and parasite viability. However, the role of mitoribosomes in sustaining the metabolic status of the parasite mitochondrion remains unknown. Here, among the 14 annotated mitoribosomal proteins of the small subunit of P. falciparum, we verified the localization and tested the essentiality of three candidates (PfmtRPS12, PfmtRPS17, PfmtRPS18), employing a CRISPR/Cas9 mediated conditional knockdown tool. Using immuno-electron microscopy, we provided evidence that the mitoribosome is closely associated with the mitochondrial inner membrane in the parasite. Upon knockdown of the mitoribosome, the parasites became hypersensitive to inhibitors targeting the bc1 complex, dihydroorotate dehydrogenase and F1Fo ATP synthase complex. Furthermore, knockdown of the mitoribosome blocked the pyrimidine biosynthesis pathway and reduced the pool of pyrimidine nucleotides. Together, our data suggest that disruption of the P. falciparum mitoribosome compromises the metabolic capability of the mitochondrion, rendering the parasite hypersensitive to a panel of inhibitors targeting mitochondrial functions.

1950: The WD40-repeat protein WDR-48 promotes the stability of the deubiquitinating enzyme USP-46 by inhibiting its ubiquitination and degradation
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Posted to bioRxiv 21 Jun 2019

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

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

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

1951: The proteomic inventory reveals the chloroplast ribosome as nexus within a diverse protein network
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Posted to bioRxiv 16 Dec 2019

The proteomic inventory reveals the chloroplast ribosome as nexus within a diverse protein network
254 downloads biochemistry

Lisa Désirée Westrich, Vincent Leon Gotsmann, Claudia Herkt, Fabian Ries, Tanja Kazek, Raphael Trösch, Silvia Ramundo, Jörg Nickelsen, Laura Armbruster, Markus Wirtz, Zuzana Storchová, Markus Raeschle, Felix Willmund

Chloroplast gene expression is tightly regulated and majorly controlled on the level of protein synthesis. Fine-tuning of translation is vital for plant development, acclimation to environmental challenges and for the assembly of major protein complexes such as the photosynthesis machinery. However, many regulatory mediators and the interaction network of chloroplast ribosomes are not known to date. We report here on a deep proteomic analysis of the plastidic ribosome interaction network in Chlamydomonas reinhardtii cells. Affinity-purification of ribosomes was achieved via endogenous affinity tagging of the chloroplast-encoded protein Rpl5, yielding a specific enrichment of >650 chloroplast-localized proteins. The ribosome interaction network was validated for several proteins and provides a new source of mainly conserved factors directly linking translation with central processes such as protein folding, photosystem biogenesis, redox control, RNA maturation, energy and metabolite homeostasis. Our approach provided the first evidence for the existence of a plastidic co-translational acting N-acetyltransferase (cpNAT1). Expression of tagged cpNAT1 confirmed its ribosome-association, and we demonstrated the ability of cpNAT1 to acetylate substrate proteins at their N-terminus. Our dataset establishes that the chloroplast protein synthesis machinery acts as nexus in a highly choreographed, spatially interconnected protein network and underscores its wide-ranging regulatory potential during gene expression.

1952: Tetramerisation of the CRISPR ring nuclease Csx3 facilitates cyclic oligoadenylate cleavage
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Posted to bioRxiv 29 Apr 2020

Tetramerisation of the CRISPR ring nuclease Csx3 facilitates cyclic oligoadenylate cleavage
254 downloads biochemistry

Januka S. Athukoralage, Stuart McQuarrie, Sabine Grüschow, Shirley Graham, Tracey M Gloster, Malcolm F White

Type III CRISPR systems detect foreign RNA and activate the cyclase domain of the Cas10 subunit, generating cyclic oligoadenylate (cOA) molecules that act as a second messenger to signal infection, activating nucleases that degrade the nucleic acid of both invader and host. This can lead to dormancy or cell death; to avoid this, cells need a way to remove cOA from the cell once a viral infection has been defeated. Enzymes specialised for this task are known as ring nucleases, but are limited in their distribution. Here, we demonstrate that the widespread CRISPR associated protein Csx3, previously described as an RNA deadenylase, is a ring nuclease that rapidly degrades cyclic tetra-adenylate (cA4). The enzyme has an unusual cooperative reaction mechanism involving an active site that spans the interface between two dimers, sandwiching the cA4 substrate. We propose the name Crn3 (CRISPR associated ring nuclease 3) for the Csx3 family.

1953: Identification of activators of human fumarate hydratase by quantitative high-throughput screening
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Posted to bioRxiv 02 Jul 2019

Identification of activators of human fumarate hydratase by quantitative high-throughput screening
254 downloads biochemistry

Hu Zhu, Olivia W Lee, Pranav Shah, Ajit Jadhav, Xin Xu, Samarjit Patnaik, Min Shen, Matthew D. Hall

Fumarate hydratase (FH) is a metabolic enzyme that is part of the Krebs-cycle, and reversibly catalyzes the hydration of fumarate to malate. Mutations of the FH gene have been associated with fumarate hydratase deficiency (FHD), hereditary leiomyomatosis, renal cell cancer (HLRCC), and other diseases. Currently there are no high-quality small molecule probes for studying human fumarate hydratase. To address this, we developed a quantitative high throughput screening (qHTS) FH assay and screened a total of 57,037 compounds from in-house libraries in dose-response. While no inhibitors of FH were confirmed, a series of phenyl-pyrrolo-pyrimidine-diones were identified as activators of human fumarate hydratase. These compounds were not substrates of fumarate hydratase, were inactive in a malate dehydrogenase counter screen, and showed no detectable reduction–oxidation activity. The binding of two compounds from the series to human fumarate hydratase was confirmed by microscale thermophoresis. The low hit rate in this screening campaign confirmed that FH is a ‘tough target’ to modulate, and the small molecule activators of human fumarate hydratase reported here may serve as a starting point for further optimization and development into cellular probes of human FH and potential drug candidates.

1954: Molecular control of gene expression by Brucella BaaR, an IclR-family repressor
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Posted to bioRxiv 22 Jan 2018

Molecular control of gene expression by Brucella BaaR, an IclR-family repressor
253 downloads biochemistry

Julien Herrou, Daniel M. Czyż, Aretha Fiebig, Jonathan W Willett, Youngchang Kim, Ruiying Wu, Gyorgy Babnigg, Sean Crosson

The Brucella abortus general stress response sigma factor, σE1, directly and indirectly regulates the transcription of dozens of genes that influence stress survival and host infection. Characterizing the functions of σE1-regulated genes therefore contributes to understanding of B. abortus physiology and infection biology. Transcription of the IclR family regulator, Bab2_0215, is indirectly activated by σE1 but its function remains undefined. We present a structural and functional characterization of Bab2_0215, which we have named Brucella adipic acid activated regulator (BaaR). BaaR adopts a classic IclR-family fold and directly regulates the transcription of two operons with predicted roles in carboxylic acid oxidation. BaaR binds two sites on chromosome II between baaR and a divergently transcribed hydratase/dehydrogenase (acaD2), and represses transcription. We identified three carboxylic acids (adipic acid tetradecanedioic acid, ε-aminocaproic acid) and a lactone (ε-caprolactone) that enhance transcription from the baaR and acaD2 promoters. However, neither the activating acids nor caprolactone enhance transcription by binding directly to BaaR. Induction of baaR transcription by adipic acid requires the gene bab2_0213, which encodes a major facilitator superfamily transporter, suggesting that Bab2_0213 transports adipic acid across the inner membrane. We conclude that a set of structurally related organic molecules activate transcription of genes repressed by BaaR. Our study provides molecular-level understanding of a gene expression program regulated downstream of σE1.

1955: Unraveling the interaction between doxorubicin and DNA origami nanostructures for customizable chemotherapeutic drug release
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Posted to bioRxiv 15 May 2020

Unraveling the interaction between doxorubicin and DNA origami nanostructures for customizable chemotherapeutic drug release
253 downloads biochemistry

Heini Ijäs, Boxuan Shen, Amelie Heuer-Jungemann, Adrian Keller, Mauri A. Kostiainen, Tim Liedl, Janne A. Ihalainen, Veikko Linko

Doxorubicin (DOX) is a commonly employed drug in cancer chemotherapy, and its high DNA-binding affinity can be harnessed in preparing DOX-loaded DNA nanostructures for targeted delivery and therapeutics. Although DOX has been widely studied, the existing literature of promising DOX-loaded DNA nanocarriers remains limited and incoherent. Here, based on an in-depth spectroscopic analysis, we characterize and optimize the DOX loading into different 2D and 3D scaffolded DNA origami nanostructures. In our experimental conditions, all of our DNA origami designs show similar DOX binding capacities, which are, however, remarkably lower than previously reported. To simulate the possible physiological degradation pathways, we examine the stability and DOX release properties of the complexes upon DNase I digestion, revealing customizable drug release profiles related to the DNA origami superstructure and the loaded DOX content. In addition, we identify major DOX aggregation mechanisms and spectral changes linked to pH, magnesium, and DOX concentration that have been largely ignored in experimenting with DNA nanostructures. Therefore, we believe this work can act as a guide to tailoring the release profiles and developing better drug delivery systems based on DNA carriers. ### Competing Interest Statement The authors have declared no competing interest.

1956: Substrate Channeling via a Transient Protein-Protein Complex: The case of D-Glyceraldehyde-3-Phosphate Dehydrogenase and L-Lactate Dehydrogenase
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Posted to bioRxiv 23 Jan 2020

Substrate Channeling via a Transient Protein-Protein Complex: The case of D-Glyceraldehyde-3-Phosphate Dehydrogenase and L-Lactate Dehydrogenase
253 downloads biochemistry

Željko M. Svedružić, Ivica Odorčić, Christopher H. Chang, Draženka Svedružić

Background: D-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and L-lactate dehydrogenase (LDH) can form a complex that can regulate the major metabolic pathways, however, the exact mechanism remains unknown. We analyzed a possibility of NADH-channeling from GAPDH-NADH complex to LDH isozymes using enzymes from different cells. Results: Enzyme-kinetics and NADH-binding studies showed that LDH can use GAPDH-NADH complex as a substrate. LDH activity with GAPDH-NADH complex was challenged with anti-LDH antibodies to show that the channeled and the diffusive reactions always take place in parallel. The channeling path is dominant only in assays with limiting free-NADH concertation that mimic cytosolic conditions. Analytical ultracentrifugation showed that the channeling does not require a high affinity complex. Molecular dynamics calculations and protein-protein interaction studies showed that LDH and GAPDH can form a leaky channeling complex only at subsaturating NADH concentrations. The interaction sites are conserved between LDH isozymes from heart and muscle, and between GAPDH molecules from rabbit and yeast cells. Positive electric fields between the NAD(H) binding sites on LDH and GAPDH tetramers, showed that NAD(H)-channeling within the LDH-GAPDH complex, can be an extension of NAD(H)-channeling between the adjacent subunits in each tetramer. Conclusions: In the case of a transient (GAPDH-NADH)-LDH complex, the relative contribution from the channeled and the diffusive paths depends on the overlap between off-rates for the transient (GAPDH-NADH)-LDH complex and off-rates for the GAPDH-NADH complex. Molecular evolution or metabolic engineering protocols can exploit substrate channeling for metabolic flux control by fine-tuning substrate-binding affinity for the key enzymes in the competing reaction paths.

1957: AR phosphorylation and CHK2 kinase activity regulates IR stabilized AR - CHK2 interaction and prostate cancer survival
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Posted to bioRxiv 05 Sep 2019

AR phosphorylation and CHK2 kinase activity regulates IR stabilized AR - CHK2 interaction and prostate cancer survival
253 downloads biochemistry

Huy Q Ta, Natalia Dworak, Melissa L Ivey, Devin G Roller, Daniel Gioeli

We have previously demonstrated that checkpoint kinase 2 (CHK2) is a critical negative regulator of androgen receptor (AR) transcriptional activity, prostate cancer (PCa) cell growth, and androgen sensitivity. We have now uncovered that the AR directly interacts with CHK2 and ionizing radiation (IR) increases this interaction. This IR-induced increase in AR-CHK2 interactions requires AR phosphorylation and CHK2 kinase activity. PCa associated CHK2 mutants with impaired kinase activity reduced IR-induced AR-CHK2 interactions. The destabilization of AR-CHK2 interactions induced by CHK2 variants impairs CHK2 negative regulation of cell growth. CHK2 depletion increases transcription of DNAPK and RAD54, increases clonogenic survival, and increases resolution of DNA double strand breaks. The data support a model where CHK2 sequesters the AR through direct binding decreasing AR transcription and suppressing PCa cell growth. CHK2 mutation or loss of expression thereby leads to increased AR transcriptional activity and survival in response to DNA damage. ### Competing Interest Statement The authors have declared no competing interest.

1958: Indirect Sexual Selection Drives Rapid Sperm Protein Evolution
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Posted to bioRxiv 29 Jun 2019

Indirect Sexual Selection Drives Rapid Sperm Protein Evolution
253 downloads biochemistry

Damien B. Wilburn, Lisa M. Tuttle, Rachel E. Klevit, Willie J. Swanson

Sexual selection can explain rapid evolution of fertilization proteins, yet sperm proteins evolve rapidly even if they are not directly involved in fertilization. Here we demonstrate that FITZAP, an intrinsically disordered sperm protein in the marine mollusk abalone, exploits differences in the intracellular and oceanic ionic environments to package the fertilization protein lysin at extraordinary concentrations inside sperm by forming Fuzzy Interacting Transient Zwitterion (FITZ) complexes. FITZAP binds lysin at the same protein interface as its egg receptor VERL, and as sexual selection rapidly alters the lysin-VERL interface, FITZAP coevolves rapidly to maintain lysin binding. Consequently, FITZAP-lysin interactions exhibit a similar species-specificity as lysin-VERL interactions. Thus, tethered molecular arms races driven by sexual selection can generally explain rapid sperm protein evolution. One Sentence Summary Structural study of sperm proteins reveals a novel protein packaging/dispersion system embedded in a coevolutionary arms race.

1959: Estrogen represses Tgfbr1 and Bmpr1a expression via estrogen receptor beta in MC3T3-E1 cells
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Posted to bioRxiv 29 Jul 2017

Estrogen represses Tgfbr1 and Bmpr1a expression via estrogen receptor beta in MC3T3-E1 cells
253 downloads biochemistry

Hanliang He, Chunqing Wang, Qifeng Tang, Fan Yang, Youjia Xu

MC3T3-E1 is a clonal pre-osteoblastic cell line derived from newborn mouse calvaria, which is commonly used in osteoblast studies. To investigate the effects of estrogen on osteoblasts, we treated MC3T3-E1 cells with various concentrations of estrogen and assessed their proliferation. Next, we performed RNA deep sequencing to investigate the effects on estrogen target genes. Bmpr1a and Tgfbr1, important participants in the TGF-beta signaling pathway, were down-regulated in our deep sequencing results. Bioinformatics analysis revealed that estrogen receptor response elements (EREs) were present in the Bmpr1a and Tgfbr1 promoters. Culturing the cells with the estrogen receptor (ER) alpha or beta antagonists 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride (MPP) or 4-[2-phenyl-5,7-bis(trifluoromethyl) pyrazolo[1,5-alpha]pyrimidin-3-yl] phenol (PTHPP), respectively, demonstrated that ER beta is involved in the estrogen-mediated repression of Tgfbr1 and Bmpr1a.The chromatin immunoprecipitation (ChIP) results were consistent with the conclusion that E2 increased the binding of ER beta at the EREs located in the Tgfbr1 and Bmpr1a promoters. Our research provides new insight into the role of estrogen in bone metabolisms.

1960: Genome-wide effects of the antimicrobial peptide apidaecin on translation termination
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Posted to bioRxiv 20 May 2020

Genome-wide effects of the antimicrobial peptide apidaecin on translation termination
253 downloads biochemistry

Kyle Mangano, Tanja Florin, Xinhao Shao, Dorota Klepacki, Irina Chelysheva, Zoya Ignatova, Yu Gao, Alexander Mankin, Nora Vázquez-Laslop

Biochemical studies had shown that the antimicrobial peptide apidaecin (Api) inhibits protein synthesis by binding in the nascent peptide exit tunnel and trapping the release factor associated with a terminating ribosome. The mode of Api action in bacterial cells had remained unknown. Genome-wide analysis revealed that Api leads to pronounced ribosome arrest at stop codons and ribosome queuing. In addition, Api causes a dramatic increase of stop codon bypass by ribosomes paused in a pre-release state, resulting in accumulation of proteins with C-terminal extensions. Stop codon bypass occurs in 0-frame, by misincorporating near-cognate aminoacyl-tRNAs, or via frameshifting. Api-mediated pervasive stalling of pre-release ribosomes futilely activates the ribosome rescue systems. Understanding the unique mechanism of Api action in living cells may contribute to development of new treatments for infections and genetic diseases, and research tools for genome exploration.

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