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Targeting protein-ligand neosurfaces using a generalizable deep learning approach bioRxiv. Biochem. Pub Date : 2024-03-28 Anthony Marchand, Stephen Buckley, Arne Schneuing, Martin Pacesa, Pablo Gainza, Evgenia Elizarova, Rebecca Manuela Neeser, Pao-Wan Lee, Luc Reymond, Maddalena Elia, Leo Scheller, Sandrine Georgeon, Joseph Schmidt, Philippe Schwaller, Sebastian Josef Maerkl, Michael Bronstein, Bruno Emmanuel Correia
Molecular recognition events between proteins drive biological processes in living systems. However, higher levels of mechanistic regulation have emerged, where protein-protein interactions are conditioned to small molecules. Here, we present a computational strategy for the design of proteins that target neosurfaces, i.e. surfaces arising from protein-ligand complexes. To do so, we leveraged a deep
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Intrinsically disordered RNA-binding motifs cooperate to catalyze RNA folding and drive phase separation bioRxiv. Biochem. Pub Date : 2024-03-27 Annika Niedner-Boblenz, Thomas Monecke, Janosch Hennig, Melina Klostermann, Mario Hofweber, Elena Davydova, Andre P Gerber, Irina Anosova, Wieland Mayer, Marisa Mueller, Roland Heym, Robert Janowski, Jean-Christophe Paillart, Dorothee Dormann, Kathi Zarnack, Michael Sattler, Dierk Niessing
RNA-binding proteins are essential for gene regulation and the spatial organization of cells. Here, we report that the yeast ribosome biogenesis factor Loc1p is an intrinsically disordered RNA-binding protein with eight repeating positively charged, unstructured nucleic acid binding (PUN) motifs. While a single of these previously undefined motifs stabilizes folded RNAs, multiple copies strongly cooperate
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Structural basis for recognition of the FLAG-1 tag by anti-FLAG M2 bioRxiv. Biochem. Pub Date : 2024-03-27 J. Wouter Beugelink, Els Sweep, Bert J.C. Janssen, Joost Snijder, Matti F. Pronker
The FLAG-tag/anti-FLAG system is a widely used biochemical tool for protein detection and purification. Anti-FLAG M2 is the most popular antibody against the FLAG-tag, due to its ease of use, versatility, and availability in pure form or as bead conjugate. M2 binds N-terminal, C-terminal and internal FLAG-tags and binding is calcium-independent, but the molecular basis for the FLAG-tag specificity
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Carbohydrate-active enzymes from Akkermansia muciniphila breakdown mucin O-glycans to completion bioRxiv. Biochem. Pub Date : 2024-03-27 Lucy I Crouch, Cassie R Bakshani, Taiwo O Ojuri, Bo Pilgaard, Jesper Holck, Ross McInnes, Maria Zakhour, Manon Kerouedan, Emily Newton, David N Bolam
Akkermansia muciniphila is a human microbial symbiont residing in the mucosal layer of the large intestine. Its main carbon source is the highly heterogeneous mucin glycoprotein that constitutes the majority of the mucus dry weight. A. muciniphila uses an array of Carbohydrate-Active enZymes (CAZymes) and sulfatases to access this complex energy source. Here we describe the biochemical characterisation
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Crosslinking by ZapD drives the assembly of short FtsZ filaments into toroidal structures in solution bioRxiv. Biochem. Pub Date : 2024-03-27 Adrián Merino-Salomón, Jonathan Schneider, Leon Babl, Jan-Hagen Krohn, Marta Sobrinos-Sanguino, Tillman Schäfer, Juan R Luque-Ortega, Carlos Alfonso, Mercedes Jiménez, Marion Jasnin, Petra Schwille, Germán Rivas
In most bacteria, division depends on a cytoskeletal structure, the Z ring, which serves as a scaffold for recruiting additional proteins, with which it forms the machinery responsible for division, the divisome. The detailed architecture of the ring, in particular the mechanisms of assembly, stabilization, and disassembly, are still largely unknown. Here, we highlight the role of FtsZ-associated proteins
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Application of chimeric antigens to paper-based diagnostics for detection of West Nile virus infections of Crocodylus porosus: a novel animal test case bioRxiv. Biochem. Pub Date : 2024-03-27 Ryan A Johnston, Gervais Habarugira, Jessica J Harrison, Sally R Isberg, Jasmin Moran, Mahali Morgan, Steven S Davis, Lorna F Melville, Christopher B Howard, Charles S Henry, Joanne Macdonald, Helle A Bielefeldt-Ohmann, Roy A Hall, Jody Hobson-Peters
Laboratory based diagnostics like plaque reduction neutralization tests (PRNT) and ELISA are commonly used to detect seroconversion to flavivirus infections. However, faster, qualitative screening methods are needed for quicker diagnosis and better patient outcomes. Lateral flow assays (LFAs) can provide rapid results (5-15 mins) at the point of care, yet few commercial flavivirus antibody detection
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Multi-omics characterization of highly enriched human plasma extracellular vesicles. bioRxiv. Biochem. Pub Date : 2024-03-27 Huaqi Su, Christopher Fowler, Colin L Masters, Kevin J Barnham, Gavin E. Reid, Laura J Vella
Extracellular vesicles (EVs) in blood plasma offer a valuable reservoir of intracellular cellular cargo, making them a promising source of liquid based biomarkers. The molecular cargo of small EVs (sEVs) is of particular interest because some EV subtypes encapsulate cargo from organelles including mitochondria, endosomes, and the autophagy pathways, which are implicated in multiple diseases. However
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A domain-swapped CaMKII conformation facilitates linker-mediated allosteric regulation bioRxiv. Biochem. Pub Date : 2024-03-27 Viet Chi Bao Nguyen, Can Özden, Kairong Dong, Ana Pamela Torres-Ocampo, Noelle Dziedzic, Daniel Flaherty, Jian Huang, Saketh Sankura, Nikki Lyn Abromson, Diana R. Tomchick, Jianhan Chen, Scott C Garman, Margaret Stratton
Ca2+ signaling plays a key role in physiological processes such as memory formation and cardiac function. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is the primary kinase that responds to Ca2+ inputs in these cells. There are four CaMKII paralogs in mammals which are alternatively spliced in the variable linker region to create upwards of 70 different variants. In this study, we systematically
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Comparison of alternative solvents for in situ extraction of hydrocarbons from the colonial green alga Botryococcus braunii race B (Showa) bioRxiv. Biochem. Pub Date : 2024-03-27 Takehiro A. Ozawa-Uyeda, Sebastian J. Overmans, Barbara Bastos de Freitas, Edmundo Lozoya-Gloria, Kyle J. Lauersen
The colony–forming, green microalga Botryococcus braunii secretes petroleum–like hydrocarbons, which enables the non–destructive continuous in situ extraction, ′milking′, of these extracellular products during culture growth without cell lysis. This work compares the suitability of 15 different solvents, including alkanes, halogenated solvents, and green solvents, for in situ extraction of B. braunii
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Redox Regulation of Brain Selective Kinases BRSK1/2: Implications for Dynamic Control of the Eukaryotic AMPK family through Cys-based mechanisms bioRxiv. Biochem. Pub Date : 2024-03-26 George Bendzunas, Dominic P. Byrne, Safal Shrestha, Leonard A. Daly, Sally O Oswald, Samiksha Katiyar, Aarya Venkat, Wayland Yeung, Claire E. Eyers, Patrick A Eyers, Natarajan Kannan
In eukaryotes, protein kinase signaling is regulated by a diverse array of post-translational modifications (PTMs), including phosphorylation of Ser/Thr residues and oxidation of cysteine (Cys) residues. While regulation by activation segment phosphorylation of Ser/Thr residues is well understood, relatively little is known about how oxidation of cysteine residues modulate catalysis. In this study
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Structural Basis for Oxidized Glutathione Recognition by the Yeast Cadmium Factor 1 bioRxiv. Biochem. Pub Date : 2024-03-26 Tik Hang Soong, Clare F Hotze, Nitesh Kumar Khandelwal, Thomas M Tomasiak
Transporters from the ABCC family have an essential role in detoxifying electrophilic compounds including metals, drugs, and lipids, often through conjugation with glutathione complexes. The Yeast Cadmium Factor 1 (Ycf1) transports glutathione alone as well as glutathione conjugated to toxic heavy metals including Cd2+, Hg2+, and As3+. To understand the complicated selectivity and promiscuity of heavy
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Bioorthogonal labeling and enrichment of histone monoaminylation reveal its accumulation and regulatory function in cancer cell chromatin bioRxiv. Biochem. Pub Date : 2024-03-26 Nan Zhang, Jinghua Wu, Farzana Hossain, Haidong Peng, Huapeng Li, Connor Gibson, Min Chen, Huan Zhang, Shuaixin Gao, Xinru Zheng, Yongdong Wang, Jiangjiang Zhu, Jing J. Wang, Ian Maze, Qingfei Zheng
Histone monoaminylation (i.e., serotonylation and dopaminylation) is an emerging category of epigenetic mark occurring on the fifth glutamine (Q5) residue of H3 N-terminal tail, which plays significant roles in gene transcription. Current analysis of histone monoaminylation is mainly based on site-specific antibodies and mass spectrometry, which either lacks high resolution or is time-consuming. In
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Unmasking Hidden Systemic Effects of Neurodegenerative Diseases: A Two-Pronged Approach to Biomarker Discovery bioRxiv. Biochem. Pub Date : 2024-03-26 Sandra I. Anjo, Miguel Rosado, Inês Baldeiras, Andreia Gomes, Diana Pires, Cátia Santa, Joana Pinto, Cristina Januário, Isabel Santana, Ana Verdelho, Alexandre de Mendonça, Miguel Castelo-Branco, Bruno Manadas
Background: The identification of reliable blood biomarkers for neurodegenerative Diseases (NDs) has been of pivotal importance in translational/clinical research. However, conventional omics struggle with the complexity of blood samples, which makes it difficult to achieve the desired goal. To address this, in this work the potential of High Molecular Weight (HMW) fractionation under non-denaturing
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Functional metagenomics reveals an alternative, broad-specificity pathway for metabolism of carbohydrates in human gut commensal bacteria bioRxiv. Biochem. Pub Date : 2024-03-25 Seyed Amirhossein Nasseri, Aleksander C. Lazarski, Imke L. Lemmer, Chloe Y. Zhang, Eva Brencher, Hong-Ming Chen, Lyann Sim, Leo Betschart, Liam J. Worral, Natalie C. J. Strynadka, Stephen G. Withers
The vast majority of the glycosidases characterised so far follow one of the variations of the 'Koshland' mechanisms to hydrolyse glycosidic bonds. Herein we describe a large-scale screen of a human gut microbiome metagenomic library using an assay that selectively identifies non-Koshland glycosidase activities. This screen led to identification of a commonly occurring cluster of enzymes with unprecedentedly
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Diverse Database and Machine Learning Model to narrow the generalization gap in RNA structure prediction bioRxiv. Biochem. Pub Date : 2024-03-25 Silvi Rouskn, Alberic de Lajarte, Yves Martin Des Taillades, Colin Kalicki, Federico Fuchs, Justin Aruda, Dragui Salazar, Matthew Allan, Casper LEsperance-Kerckhoff, Alex Kashi, Fabrice Jossinet
Understanding macromolecular structures of proteins and nucleic acids is critical for discerning their functions and biological roles. Advanced techniques as crystallography, NMR, and CryoEM have facilitated the determination of over 180,000 protein structures, all cataloged in the Protein Data Bank (PDB). This comprehensive repository has been pivotal in developing deep learning algorithms for predicting
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Structural characterization of ligand binding and pH-specific enzymatic activity of mouse Acidic Mammalian Chitinase bioRxiv. Biochem. Pub Date : 2024-03-25 Roberto Efraín Díaz, Andrew K. Ecker, Galen J Correy, Pooja Asthana, Iris D Young, Bryan Faust, Michael C Thompson, Ian B Seiple, Steven J Van Dyken, Richard M Locksley, James S Fraser
Chitin is an abundant biopolymer and pathogen-associated molecular pattern that stimulates a host innate immune response. Mammals express chitin-binding and chitin-degrading proteins to remove chitin from the body. One of these enzymes, Acidic Mammalian Chitinase (AMCase), is known for its ability to function under acidic conditions in the stomach but is also active in tissues with more neutral pHs
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Exploring Liquid-Liquid Phase Separation in the Organization of Golgi Matrix Proteins bioRxiv. Biochem. Pub Date : 2024-03-25 Luis Felipe Santos Mendes, Carolina Gimenes Oliveira, Antonio Jose Costa Filho, Emanuel Kava
The Golgi apparatus is a critical organelle in protein sorting and lipid metabolism. Characterized by its stacked, flattened cisternal structure, the Golgi exhibits distinct polarity with its cis- and trans-faces orchestrating various protein maturation and transport processes. At the heart of its structural integrity and organization are the Golgi Matrix Proteins (GMPs), predominantly comprising Golgins
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Cryo-EM structure of the tetra-phosphorylated R-domain in Ycf1 reveals key interactions for transport regulation bioRxiv. Biochem. Pub Date : 2024-03-24 Rodolpho Souza Amado de Carvalho, Md Shamiul Islam Rasel, Nitesh K Khandelwal, Thomas M Tomasiak
Many ATP-binding cassette (ABC) transporters are regulated by phosphorylation on long and disordered loops. which makes their interactions a challenge to visualize. We have trapped an activated state of the regulatory domain (R-domain) of Yeast Cadmium Factor 1 (Ycf1) by enzymatically enriching the phosphorylated state. A 3.2 angstrom cryo-EM structure reveals an R-domain structure with four phosphorylated
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Sulfoquinovosyl diacylglycerol is required for dimerization of the Rhodobacter sphaeroides RC-LH1 core complex bioRxiv. Biochem. Pub Date : 2024-03-24 Elizabeth C Martin, Adam GM Bowie, Taylor Wellfare Reid, C Neil Hunter, Andrew Hitchcock, David J K Swainsbury
The reaction centre-light harvesting 1 (RC-LH1) core complex is indispensable for anoxygenic photosynthesis. In the purple bacterium Rhodobacter (Rba.) sphaeroides RC-LH1 is produced both as a monomer in which 14 LH1 subunits form a crescent-shaped antenna around one RC, and as a dimer, where 28 LH1 subunits form an S-shaped antenna surrounding two RCs. The PufX polypeptide augments the five RC and
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In tissue spatial single-cell metabolomics by coupling mass spectrometry imaging and immunofluorescences bioRxiv. Biochem. Pub Date : 2024-03-23 Landry Blanc, Florent Grelard, Michael Tuck, Veronique Dartois, Antonio Peixoto, Nicolas Desbenoit
In this work, we introduce a multimodal imaging workflow that integrates Matrix-assisted Laser Desorption Ionization Mass Spectrometry Imaging (MALDI-MSI) combined with Immunofluorescence (IF) microscopy to enhance in tissue spatial single-cell metabolomics. The workflow allows to correlate cell populations with associated small molecule distributions by conducting on the same tissue section MSI before
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A candidate reference method for the quantification of α-synuclein in cerebrospinal fluid using an SI traceable primary calibrator and multiple reaction monitoring bioRxiv. Biochem. Pub Date : 2024-03-23 Leran Zhang, Eva Iles-Toth, Adam Cryar, Giles Drinkwater, Lucia Di Vagno, Marie-Laure Pons, Julia Mateyka, Bryan McCullough, Eli Achtar, Cailean Clarkson, Laura Goeschel, Peter Koertvelyessy, Chris Mussell, Christopher Hopley, Agnes Floeel, Christophe Hirtz, Sylvain Lehmann, Milena Quaglia
Objectives α-synuclein aggregation is an indicator of neurodegenerative diseases such as Parkinson's disease (PD) and recent advances have suggested that this protein could serve as a potential biomarker. It has been indicated that soluble and oligomeric α-synuclein in biological fluids could have diagnostic applications for PD. Clinical laboratories currently rely on antibody-based assays to detect
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The priming phosphorylation of KaiC is activated by the release of its autokinase autoinhibition bioRxiv. Biochem. Pub Date : 2024-03-23 Yoshihiko Furuike, Yasuhiro Onoue, Shinji Saito, Toshifumi Mori, Shuji Akiyama
KaiC, a cyanobacterial circadian clock protein with autokinase activity, catalyzes the dual phosphorylation of its own S431 and T432 residues in a circadian manner in the presence of KaiA and KaiB. Priming phosphorylation at T432 is a key step that promotes secondary phosphorylation at S431. Although KaiA binding is considered essential for KaiC phosphorylation, the mechanisms underlying the activation
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Trace Amines are Essential Metabolites for the Autocrine Regulation of β-Cell Signaling and Insulin Secretion bioRxiv. Biochem. Pub Date : 2024-03-23 Kaya Keutler, Sebastian Hauke, Aurelien Laguerre, Mireia Andreu-Carbo, Jona Rada, David Grandy, Dmytro A. Yushchenko, Carsten Schultz
Secretion of insulin in response to extracellular stimuli, such as elevated glucose levels and small molecules that act on G-protein coupled receptors (GPCRs), is the hallmark of β-cell physiology. Trace amines (TAs) are small aromatic metabolites that were identified as low-abundant ligands of the trace amine-associated receptor 1 (TAAR1) in the central nervous system (CNS), a GPCR that is also expressed
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Snapshots of Pseudomonas aeruginosa SOS response activation complex reveal structural prerequisites for LexA engagement and cleavage bioRxiv. Biochem. Pub Date : 2024-03-23 Filippo Vascon, Sofia De Felice, Matteo Gasparotto, Stefan Huber, Claudio Catalano, Monica Chinellato, Alessandro Grinzato, Francesco Filippini, Lorenzo Maso, Arjen J. Jakobi, Laura Cendron
Antimicrobial resistance represents a major threat to human health and Pseudomonas aeruginosa stands out among the pathogens responsible for this emergency. The SOS response to DNA damage plays a pivotal role in bacterial evolution, driving the development of resistance mechanisms and influencing the adaptability of bacterial populations to challenging environments, particularly in the context of antibiotic
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Physics-informed machine learning for automatic model reduction in chemical reaction networks bioRxiv. Biochem. Pub Date : 2024-03-23 Joseph Pateras, Preetam Ghosh, Colin Zhang, Shriya Majumdar, Ayush Pal
Physics-informed machine learning emerges as a transformative approach, bridging the gap between the high fidelity of mechanistic models and the adaptive, data-driven insights afforded by artificial intelligence and machine learning. In the realm of chemical reaction network modeling, this synergy is particularly valuable. It offers a solution to the prohibitive computational costs associated with
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Identification of Novel Allosteric Sites of SARS-CoV-2 Papain-Like Protease (PLpro) for the Development of COVID-19 Antivirals bioRxiv. Biochem. Pub Date : 2024-03-23 Juliana C. Ferreira, Kenana Al Adem, Samar Fadl, Adrian J. Villanueva, Lara Alzyoud, Mohammad A. Ghattas, Wael M. Rabeh
Coronaviruses such as SARS-CoV-2 encode a conserved papain-like protease (PLpro) that is crucial for viral replication and immune evasion, making it a prime target for antiviral drug development. In this study, three surface pockets on SARS-CoV-2 PLpro that may function as sites for allosteric inhibition were computationally identified. To evaluate the effects of these pockets on proteolytic activity
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Structure, substrate selectivity determinants and membrane interactions of a Glutamate-specific TAXI TRAP binding protein from Vibrio cholerae. bioRxiv. Biochem. Pub Date : 2024-03-23 Joseph FS Davies, Andrew Daab, Nicholas Massouh, Corey Kirkland, Bernadette Strongitharm, Andrew Leech, Marta Farré, Gavin Hugh Thomas, Christopher Mulligan
Tripartite ATP independent periplasmic (TRAP) transporters are widespread in prokaryotes and are responsible for the transport of a variety of different ligands, primarily organic acids. TRAP transporters are secondary active transporters that employ a substrate binding protein to bind and present the substrate to membrane embedded translocation component. TRAP transporters can be divided into two
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VASP phase separation with priming proteins of fast endophilin mediated endocytosis modulates actin polymerization bioRxiv. Biochem. Pub Date : 2024-03-22 Karthik B Narayan, Honey Priya James, Jonathan Cope, Samsuzzoha Mondal, Laura Baeyens, Francesco Milano, Jason Zheng, Matthias Krause, Tobias Baumgart
Actin polymerization is essential in several clathrin-independent endocytic pathways including fast endophilin mediated endocytosis (FEME), however the actin machinery involved in FEME has been elusive. Here, we show that the actin polymerase VASP, colocalizes and interacts directly with the FEME priming complex. We identify endophilin (EDP) as a VASP binding partner and establish novel non-canonical
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The cytoplasmic tail of myelin protein zero induces morphological changes in lipid membranes bioRxiv. Biochem. Pub Date : 2024-03-22 Oda C. Krokengen, Christine Touma, Anna Mularski, Aleksi Sutinen, Ryan Dunkel, Marie Ytterdal, Arne Raasakka, Haydyn D.T. Mertens, Adam Cohen Simonsen, Petri Kursula
The major myelin protein expressed by the peripheral nervous system Schwann cells is protein zero (P0), representing 50% of the total protein content in myelin. This 30-kDa integral membrane protein consists of an immunoglobulin (Ig)-like domain, a transmembrane helix, and a 69-residue C-terminal cytoplasmic tail (P0ct). The basic residues in P0ct contribute to the tight packing of the myelin lipid
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Discovery, Characterization and Synthetic Potential of Two Novel Bacterial Alcohol Oxidases bioRxiv. Biochem. Pub Date : 2024-03-22 Paula Cinca-Fernando, Christian Ascaso-Alegre, Emma Sevilla, Marta Martinez-Julvez, Juan Mangas-Sanchez, Patricia Ferreira
The search for novel synthetic tools to prepare industrial chemicals in a safer and greener manner is a continuing challenge in synthetic chemistry. In this manuscript, we report the discovery, characterization, and synthetic potential of two novel aryl-alcohol oxidases from bacteria which are able to oxidize a variety of aliphatic and aromatic alcohols in high efficiencies (up to 4970 min-1mM-1).
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Discovery of a cell-active chikungunya virus nsP2 protease inhibitor using a covalent fragment-based screening approach bioRxiv. Biochem. Pub Date : 2024-03-22 Eric M. Merten, John D. Sears, Tina M. Leisner, Paul B. Hardy, Anirban Ghoshal, Mohammad Anwar Hossain, Kesatebrhan Haile Asressu, Peter J. Brown, Michael A. Stashko, Laura E. Herring, Angie L. Mordant, Thomas S. Webb, Christine A. Mills, Natalie K. Barker, Jamie J. Arnold, Craig E. Cameron, Daniel N. Streblow, Nathaniel J. Moorman, Mark Heise, Timothy M. Willson, Konstantin I. Popov, Kenneth H. Pearce
Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that has been responsible for numerous large-scale outbreaks in the last twenty years. Currently, there are no FDA-approved therapeutics for any alphavirus infection. CHIKV non-structural protein 2 (nsP2), which contains a cysteine protease domain, is essential for viral replication, making it an attractive target for a drug discovery campaign
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Delving into human α1,4-galactosyltransferase acceptor specificity: the role of enzyme dimerization bioRxiv. Biochem. Pub Date : 2024-03-22 Krzysztof Mikolajczyk, Karol Wroblewski, Sebastian Kmiecik
Glycosyltransferases (GTs) exhibit precise donor and acceptor specificities, governed by intricate mechanisms, including protein assembly. Human α1,4-galactosyltransferase (A4galt), a Golgi apparatus-resident GT, catalyzes the synthesis of Gb3 glycosphingolipid (GSL) and P1 glycotope on glycoproteins (GPs), receptors for Shiga toxin type 1 (Stx1) and 2 (Stx2). These toxins are produced by enterohemorrhagic
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Cohesin supercoils DNA during loop extrusion bioRxiv. Biochem. Pub Date : 2024-03-22 Iain F Davidson, Roman Barth, Sabrina Horn, Richard Janissen, Kota Nagasaka, Gordana Wutz, Roman R Stocsits, Benedikt Bauer, Cees Dekker, Jan-Michael Peters
Cohesin extrudes genomic DNA into loops that promote chromatin assembly, gene regulation and recombination. Here we show that cohesin introduces negative supercoils into extruded DNA. Supercoiling requires engagement of cohesin's ATPase heads, DNA clamping by these heads, and a DNA binding site on cohesin's hinge, indicating that cohesin supercoils DNA when constraining it between the hinge and the
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A PTER-dependent pathway of taurine metabolism linked to energy balance bioRxiv. Biochem. Pub Date : 2024-03-22 Wei Wei, Xuchao Lyu, Andrew L Markhard, Sipei Fu, Rachel E Mardjuki, Peter E Cavanagh, Xianfeng Zeng, Jakub Rajniak, Nannan Lu, Shuke Xiao, Meng Zhao, Maria Dolores Moya-Garzon, Steven D Truong, ChiuChun J Chou, Lianna W Wat, Saranya Chidambaranathan Reghupaty, Laetitia Voilquin, Duo Xu, Fangfang Shen, Wentao Huang, Cuauhtemoc B Ramirez, Cholsoon Jang, Katrin J Svensson, Michael A Fischbach, Jonathan
Taurine is a conditionally essential micronutrient and one of the most abundant amino acids in humans1-3. In endogenous taurine metabolism, dedicated enzymes are involved in biosynthesis of taurine from cysteine as well as the downstream derivatization of taurine into secondary taurine metabolites4,5. One such taurine metabolite is N-acetyltaurine6. Levels of N-acetyltaurine are dynamically regulated
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Paradigm shift in biomarker translation: a pipeline to generate clinical grade biomarker candidates from DIA-MS discovery bioRxiv. Biochem. Pub Date : 2024-03-22 Qin Fu, Manasa Vegesna, Niveda Sundararaman, Eugen Damoc, Tabiwang N. Arrey, Anna Pashkova, Emebet Mengesha, Philip Debbas, Sandy Joung, Dalin Li, Susan Cheng, Jonathan Braun, Dermot P.B. McGovern, Christopher Murray, Yue Xuan, Jennifer E. Van Eyk
Clinical biomarker development has been stymied by inaccurate protein quantification from mass spectrometry (MS) discovery data and a prolonged validation process. To mitigate these issues, we created the Targeted Extraction Assessment of Quantification (TEAQ) software package. This innovative tool uses the discovery cohort analysis to select precursors, peptides, and proteins that adhere to established
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Activity of zebrafish THAP9 transposase and zebrafish P element-like transposons bioRxiv. Biochem. Pub Date : 2024-03-22 Nitzan Kutnowski, George E Ghanim, Yeon Lee, Donald Rio
Transposable elements are mobile DNA segments that are found ubiquitously across the three domains of life. One family of transposons, called P elements, were discovered in the fruit fly Drosophila melanogaster. Since their discovery, P element transposase-homologous genes (called THAP-domain containing 9 or THAP9) have been discovered in other animal genomes. Here, we show that the zebrafish (Danio
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Translational impacts of enzymes that modify ribosomal RNA around the peptidyl transferase centre bioRxiv. Biochem. Pub Date : 2024-03-22 Letian Bao, Josefine Liljeruhm, Ruben Crespo Blanco, Gerrit Brandis, Jaanus Remme, Anthony C. Forster
Large ribosomal RNAs (rRNAs) are modified heavily post-transcriptionally in functionally-important regions but, paradoxically, individual knockouts (KOs) of the modification enzymes have minimal impact on Escherichia coli growth. Furthermore, we recently constructed a strain with combined KOs of five modification enzymes (RluC, RlmKL, RlmN, RlmM and RluE) of the "critical region" of the peptidyl transferase
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Estimating Nutrient Concentration in Food Using Untargeted Metabolomics bioRxiv. Biochem. Pub Date : 2024-03-21 Michael L Sebek, Giulia Menichetti, Albert-László Barabási
Untargeted metabolomics can detect hundreds of biochemicals in food, yet without standards, it cannot quantify them. Here we show that we can take advantage of the universal scaling of nutrient concentrations to estimate the concentration of all biochemicals detected by untargeted metabolomics. We validate our method on 20 raw foods, finding an excellent agreement between the predicted and the experimentally
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Neurogranin Modulates the Rate of Association between Calmodulin and Target Peptides bioRxiv. Biochem. Pub Date : 2024-03-21 John A Putkey, Laurel Hoffman, Vladimir Berka, Xu Wang
The best-known mode of action of calmodulin (CaM) is binding of Ca2+ to its N- and C-domains, followed by binding to target proteins. An underappreciated facet of this process is that CaM is typically bound to proteins at basal levels of free Ca2+, including the small, intrinsically disordered, neuronal IQ-motif proteins called PEP-19 and neurogranin (Ng). PEP-19 and Ng would not be effective competitive
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Structural insight into bacterial co-transcriptional translation initiation bioRxiv. Biochem. Pub Date : 2024-03-21 Takeshi Yokoyama, Yuko Murayama, Tomomi Uchikubo-Kamo, Yuri Tomabechi, Asuteka Nagao, Tsutomu Suzuki, Mikako Shirouzu, Shun-ichi Sekine
In bacteria, transcription and translation are tightly coupled, forming a transcription-translation complex (TTC) between RNA polymerase (RNAP) and the ribosome. As nascent mRNA emerging from RNAP is susceptible to ribonuclease digestion, undesired RNA folding, and R-loop formation, immediate TTC formation is important. Here, we report the cryo-electron microscopy structures that capture the translation
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RNA Chemical Probing Reagents and Protein Amino Acids: a Double-Edged Sword bioRxiv. Biochem. Pub Date : 2024-03-21 Lucy Fallon, David Klingler, Daniel Cohn, Liza Marcus, Alisha N Jones
RNA chemical probing experiments are a broadly used method for revealing the structure of RNA, as well as for identifying protein binding sites. This is beneficial for expanding our understanding of biological processes governed by protein-RNA complex interactions, as well as facilitating the identification of complex inhibiting molecules. The reagents commonly used in chemical probing experiments
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Redirecting the pioneering function of FOXA1 with covalent small molecules bioRxiv. Biochem. Pub Date : 2024-03-21 Sang Joon Won, Yuxiang Zhang, Christopher J. Reinhardt, Nicole S. MacRae, Kristen E. DeMeester, Evert Njomen, Lauren M. Hargis, Jarrett R. Remsberg, Bruno Melillo, Benjamin F. Cravatt, Michael A. Erb
Pioneer transcription factors (TFs) exhibit a specialized ability to bind to and open closed chromatin, facilitating engagement by other regulatory factors involved in gene activation or repression. Chemical probes are lacking for pioneer TFs, which has hindered their mechanistic investigation in cells. Here, we report the chemical proteomic discovery of electrophilic small molecules that stereoselectively
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Discovery and chemical optimisation of a Potent, Bi-cyclic (Bicycle®) Antimicrobial Inhibitor of Escherichia coli PBP3 bioRxiv. Biochem. Pub Date : 2024-03-21 Catherine E Rowland, Hector Newman, Tazmin T. Martin, Rachel Dods, Nikolaos Bournakas, James M Wagstaff, Nick Lewis, Steven J Stanway, Matthew Balmforth, Celia Kessler, Katerine van Rietschoten, Dom Bellini, David I Roper, Adrian J Lloyd, Christopher G Dowson, Michael J Skynner, Paul Beswick, Michael J Dawson
Penicillin binding proteins (PBPs) are well validated antimicrobial targets, but the prevalence of β-lactamase driven resistance and, more rarely, target-based mutations, necessitates new classes of PBP-targeting drugs. Here we describe the discovery and optimisation of novel, bicyclic peptide (Bicycle®) inhibitors of E. coli PBP3 (EcPBP3) using a proprietary phage display platform, and their conjugation
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Asynchronous microexon splicing of LSD1 and PHF21A during neurodevelopment bioRxiv. Biochem. Pub Date : 2024-03-21 Masayoshi Nagai, Robert S Porter, Elizabeth Hughes, Thomas Saunders, Shigeki Iwase
LSD1 histone H3K4 demethylase and its binding partner PHF21A, a reader protein for unmethylated H3K4, both undergo neuron-specific microexon splicing. The LSD1 neuronal microexon weakens H3K4 demethylation activity and can alter the substrate specificity to H3K9 or H4K20. Meanwhile, the PHF21A neuronal microexon interferes with nucleosome binding. However, the temporal expression patterns of LSD1 and
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PLD3 and PLD4 synthesize S,S-BMP, a key phospholipid enabling lipid degradation in lysosomes bioRxiv. Biochem. Pub Date : 2024-03-21 Shubham Singh, Ulrich Dransfeld, Yohannes Ambaw, Joshua Lopez-Scarim, Robert V. Farese, Tobias C. Walther
Bis(monoacylglycero)phosphate (BMP) is an abundant lysosomal phospholipid required for degradation of lipids, in particular gangliosides. Alterations in BMP levels are associated with neurodegenerative diseases. Unlike typical glycerophospholipids, lysosomal BMP has two chiral glycerol carbons in the S (rather than the R) stereo-conformation, protecting it from lysosomal degradation. How this unusual
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Delineating redox cooperativity in water-soluble and membrane multiheme cytochromes through protein design bioRxiv. Biochem. Pub Date : 2024-03-21 Benjamin J. Hardy, Paulina Dubiel, Ethan L. Bungay, May Rudin, Christopher Williams, Christopher J. Arthur, Matthew J. Guberman-Pfeffer, A. Sofia F. Oliveira, Paul Curnow, J. L. Ross Anderson
Nature has evolved diverse electron transport proteins and multiprotein assemblies essential to the generation and transduction of biological energy. However, substantially modifying or adapting these proteins for user-defined applications or to gain fundamental mechanistic insight can be hindered by their inherent complexity. De novo protein design offers an attractive route to stripping away this
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Phase separation of polyubiquitinated proteins in UBQLN2 condensates controls substrate fate bioRxiv. Biochem. Pub Date : 2024-03-21 Isabella M Valentino, Jeniffer G Llivicota-Guaman, Thuy P Dao, Erin O Mulvey, Andrew M Lehman, Sarasi K. K. Galagedera, Erica L Mallon, Carlos A Castañeda, Daniel A Kraut
Ubiquitination is one of the most common post-translational modifications in eukaryotic cells. Depending on the architecture of polyubiquitin chains, substrate proteins can meet different cellular fates, but our understanding of how chain linkage controls protein fate remains limited. UBL-UBA shuttle proteins, such as UBQLN2, bind to ubiquitinated proteins and to the proteasome or other protein quality
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A yeast two-hybrid system to obtain triple-helical ligands from combinatorial random peptide libraries bioRxiv. Biochem. Pub Date : 2024-03-20 Ryo Masuda, Khine Phyu Phyu Thant, Kazuki Kawahara, Hiroya Oki, Tetsuya Kadonosono, Yuji Kobayashi, Takaki Koide
Many bioactive proteins interact with collagen, recognizing amino acid sequences displayed on the triple helix. We report here a selection strategy to obtain triple-helical peptides that interact with the proteins from a combinatorial random library constructed in yeast cells. This system enables us to select them using the standard two-hybrid protocol, detecting interactions between triple-helical
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Effects of protein G-quadruplex interactions on phase transitions and protein aggregation bioRxiv. Biochem. Pub Date : 2024-03-20 Bikash Sahoo, Vojċ Kocman, Nathan Clark, Nikhil Myers, Xiexiong Deng, Ee L Wong, Harry J Yang, Anita Kotar, Bryan B Guzman, Daniel CA Dominguez, Janez Plavec, James CA Bardwell
The SERF family of proteins were originally discovered for their ability to accelerate amyloid formation. Znf706 is an uncharacterized protein whose N-terminus is homologous to SERF proteins. We show here that human Znf706 can promote protein aggregation and amyloid formation. Unexpectedly, Znf706 specifically interacts with stable, non-canonical nucleic acid structures known as G-quadruplexes. G-quadruplexes
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High-throughput Kinetics using Capillary Electrophoresis and Robotics (HiKER) platform used to Study T7, T3, and Sp6 RNA Polymerase Misincorporation bioRxiv. Biochem. Pub Date : 2024-03-20 Andrew F. Gardner, Zachariah Ingram Carter, Sean Robert Lund, William O'Brien
T7 RNA Polymerase (RNAP) is a well-studied and widely used enzyme with recent applications in the production of RNA vaccines. For over 50 years denaturing sequencing gels have been used as a key analysis tool for probing the kinetic mechanism of T7 RNAP nucleotide addition. However, sequencing gels are both slow and low throughput limiting their utility for comprehensive enzyme analysis. Here, we report
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Baraitser–Winter Syndrome Hotspot Mutation R196H in Cytoskeletal β–actin Reduces F–actin Stability and Perturbs Interaction with the Arp2/3 Complex bioRxiv. Biochem. Pub Date : 2024-03-20 Johannes N. Greve, Dietmar J. Manstein
Baraitser-Winter cerebrofrontofacial syndrome (BWCFF) is the most common and best-defined clinical entity associated with heterozygous single-point missense mutations in cytoskeletal β-actin. Patients present with distinct craniofacial anomalies and neurodevelopmental disabilities of variable severity. To date, the most frequently observed variants affect residue R196 of cytoskeletal β-actin, with
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Designing a Novel 3D Scaffold for Multiepitope Vaccine Development: Engineering Ag85a Protein for Enhanced Stability and Antigenicity bioRxiv. Biochem. Pub Date : 2024-03-20 Torsha Mondal, Shakilur Rahman, Amit Kumar Das, Ditipriya Hazra, Amlan Roychowdhury
Designing multi epitope vaccine (MEV) by reverse vaccinology has become immensely important in the area of vaccine research due to the emergence of new pathogens as well as rise of multi drug resistant old evils like tuberculosis. Administering a vaccine may have the best possibility to save mankind from these unforeseeable events. The strategy of designing a MEV in-silico lies in a few basic steps
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Chemoproteomics reveals immunogenic and tumor-associated cell surface substrates of ectokinase CK2α bioRxiv. Biochem. Pub Date : 2024-03-20 Corleone S Delaveris, Sophie Kong, Jeff Glasgow, Rita P Loudermilk, Lisa L Kirkemo, Fangzhu Zhao, Fernando Salangsang, Paul Phojanakong, Juan Antonio Camara Serrano, Veronica Steri, James A Wells
New epitopes for immune recognition provide the basis of anticancer immunity. Due to the high concentration of extracellular adenosine triphosphate in the tumor microenvironment, we hypothesized that extracellular kinases (ectokinases) could have dysregulated activity and introduce aberrant phosphorylation sites on cell surface proteins. We engineered a cell-tethered version of the extracellular kinase
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mRNA display in cell lysates enables identification of cyclic peptides targeting the BRD3 extraterminal domain bioRxiv. Biochem. Pub Date : 2024-03-20 Catherine A Hurd, Jacob Bush, Andrew J Powell, Louise Jane Walport
mRNA display is a powerful technology to screen libraries of >1012 cyclic peptides against a protein target, enabling the rapid discovery of high affinity ligands. These cyclic peptides are particularly well suited to challenging protein targets that have been difficult to drug with small molecules. However, target choice can still be limited as screens are typically performed against purified proteins
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Senescent cells inhibit muscle differentiation via the SASP-lipid 15d-PGJ2 mediated modification and control of HRas bioRxiv. Biochem. Pub Date : 2024-03-19 Swarang Sachin Pundlik, Alok Barik, Ashwin Venkatesvaran, Snehasudha Subhadarshini Sahoo, Mahapatra Anshuman Jaysingh, Raviswamy G H Math, Arvind Ramanathan
Senescent cells, which are characterized by multiple features such as increased expression of Senescence-Associated β-galactosidase activity (SA β-gal) and cell cycle inhibitors such as p21 or p16, accumulate with tissue damage and dysregulate tissue homeostasis. In the context of skeletal muscle, it is known that agents used for chemotherapy such as Doxorubicin cause buildup of senescent cells, leading
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Structures of wild-type and a constitutively closed mutant of connexin26 shed light on channel regulation by CO2 bioRxiv. Biochem. Pub Date : 2024-03-19 Deborah H Brotherton, Sarbjit Nijjar, Christos G Savva, Nicholas Dale, Alexander D Cameron
Connexins allow intercellular communication by forming gap junction channels (GJCs) between juxtaposed cells. Connexin26 (Cx26) can be regulated directly by CO2. This is proposed to be mediated through carbamylation of K125. We show that mutating K125 to glutamate, mimicking the negative charge of carbamylation, causes Cx26 GJCs to be constitutively closed. Through cryo-EM we observe that the K125E
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Three Prime Repair Exonuclease 1 preferentially degrades the integration-incompetent HIV-1 DNA through favorable kinetics, thermodynamic, structural and conformational properties. bioRxiv. Biochem. Pub Date : 2024-03-19 Prem Prakash, Purva Khodke, Muthukumar Balasubramaniam, Benem-Orom Davids, Thomas Hollis, Jamaine Davis, Jui Pandhare, Bajarang Kumbhar, Chandravanu Dash
HIV-1 integration into the human genome is dependent on 3 prime-processing of the reverse transcribed viral DNA. Recently, we reported that the cellular Three Prime Repair Exonuclease 1 (TREX1) enhances HIV-1 integration by degrading the unprocessed viral DNA, while the integration-competent 3 prime-processed DNA remained resistant. Here, we describe the mechanism by which the 3 prime-processed HIV-1
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Molecular basis of mRNA delivery to the bacterial ribosome bioRxiv. Biochem. Pub Date : 2024-03-19 Michael W Webster, Adrien Chauvier, Huma Rahil, Andrea Graziadei, Kristine Charles, Maria Takacs, Charlotte Saint-Andre, Juri Rappsilber, Nils G Walter, Albert Weixlbaumer
Protein synthesis begins with the formation of a ribosome-mRNA complex. In bacteria, the 30S ribosomal subunit is recruited to many mRNAs through base pairing with the Shine Dalgarno (SD) sequence and RNA binding by ribosomal protein bS1. Translation can initiate on nascent mRNAs and RNA polymerase (RNAP) can promote recruitment of the pioneering 30S subunit. Here we examined ribosome recruitment to
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Molecular Mechanism of Condensin I Activation by KIF4A bioRxiv. Biochem. Pub Date : 2024-03-19 Erin E Cutts, Damla Tetiker, Eugene Kim, Luis Aragon
During mitosis, the condensin I and II complexes compact chromatin into chromosomes. Loss of the chromokinesin, KIF4A, results in reduced condensin I chromosome association. However, the molecular mechanism behind this phenotype is unknown. Here, we show that KIF4A binds directly to condensin I HAWK subunit, NCAPG, via a conserved disordered short linear motif (SLiM) found in its C-terminal tail. KIF4A
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Multi-substrate specificity shaped the complex evolution of the aminotransferase family across the tree of life bioRxiv. Biochem. Pub Date : 2024-03-19 Kaan Koper, Sang-Woo Han, Ramani Kothadia, Hugh Salamon, Yasuo Yoshikuni, Hiroshi A. Maeda
Aminotransferases (ATs) are an ancient enzyme family that play central roles in core nitrogen metabolism essential to all organisms. However, many of the AT enzyme functions remain poorly defined, limiting our fundamental understanding of the nitrogen metabolic networks that exist in different organisms. Here we traced the deep evolutionary history of the AT family by analyzing AT enzymes from 90 species