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FGF2 binds to the allosteric site (site 2) and activates integrin αIIbβ3 and FGF1 binds to site 2 but suppresses integrin activation by FGF2: A potential mechanism of anti-thrombotic action of FGF1. bioRxiv. Biochem. Pub Date : 2024-04-18 Yoko K Takada, Xueson Wu, David Wei, Samuel Hwang, Yoshikazu Takada
It has been believed that platelet integrin αIIbβ3 recognizes fibrinogen and several ECM proteins, and we recently showed that αIIbβ3 binds to several inflammatory cytokines (e.g., CCL5, and CXCL12), which are stored in platelet granules. These ligands bind to the classical ligand (RGD)-binding site (site 1) of integrin αIIbβ3. Also, they bind to the allosteric site (site 2) of αIIbβ3, which is distinct
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Effect of different odors on the rat urine proteome bioRxiv. Biochem. Pub Date : 2024-04-18 Yuqing Liu, Haitong Wang, Youhe Gao
Do rats have corresponding changes in their urinary proteome when smelling different odors? In this study, urine samples were collected from six rats after smelling sesame oil and essential balm for three days. And samples were collected before and on the third and fourth days. Comparing the urinary protein groups of Day0 and Day4 of the sesame oil group, 143 differential proteins were identified,
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VASP phase separation with priming proteins of fast endophilin mediated endocytosis modulates actin polymerization bioRxiv. Biochem. Pub Date : 2024-04-18 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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Delineation of the Complete Reaction Cycle of a Natural Diels-Alderase bioRxiv. Biochem. Pub Date : 2024-04-18 Laurence Maschio, Catherine R Back, Jawaher Alnawah, James I Bowen, Samuel T Johns, Sbusisiwe Z Mbatha, Li-Chen Han, Nicholas R Lees, Katja Zorn, James E M Stach, Martin A Hayes, Marc W van der Kamp, Christopher R Pudney, Steven G Burston, Christine L Willis, Paul R Race
The Diels-Alder reaction is one of the most effective methods for the synthesis of substituted cyclohexenes. The development of protein catalysts for this reaction remains a major priority, affording new sustainable routes to high value target molecules. Whilst a small number of natural enzymes have been shown capable of catalysing [4+2] cycloadditions, there is a need for significant mechanistic understanding
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SLC17 transporters mediate renal excretion of Lac-Phe in mice and humans bioRxiv. Biochem. Pub Date : 2024-04-18 Veronica Lynn Li, Shuke Xiao, Pascal Schlosser, Nora Scherer, Amanda Lauren Wiggenhorn, Jan Spaas, Alan Sheng-Hwa Tung, Edward D. Karoly, Anna Kottgen, Jonathan Z Long
N-lactoyl-phenylalanine (Lac-Phe) is a lactate-derived metabolite that suppresses food intake and body weight. Little is known about the mechanisms that mediate Lac-Phe transport across cell membranes. Here we identify SLC17A1 and SLC17A3, two kidney-restricted plasma membrane-localized solute carriers, as physiologic urine Lac-Phe transporters. In cell culture, SLC17A1/3 exhibit high Lac-Phe efflux
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De novo design of protein minibinder agonists of TLR3 bioRxiv. Biochem. Pub Date : 2024-04-18 Chloe S Adams, Hyojin Kim, Abigail E Burtner, Dong Sun Lee, Craig Dobbins, Cameron Criswell, Brian Coventry, Ho Min Kim, Neil P King
Toll-like Receptor 3 (TLR3) is a pattern recognition receptor that initiates antiviral immune responses upon binding double-stranded RNA (dsRNA). Several nucleic acid-based TLR3 agonists have been explored clinically as vaccine adjuvants in cancer and infectious disease, but present substantial manufacturing and formulation challenges. Here, we use computational protein design to create novel miniproteins
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FGF2 binds to the allosteric site (site 2) and activates αvβ3 integrin and FGF1 binds to site 2 but suppresses integrin activation by FGF2: a potential mechanism of anti-inflammatory action of FGF1 bioRxiv. Biochem. Pub Date : 2024-04-18 Yoko K Takada, Xueson Wu, David Wei, Samuel Hwang, Yoshikazu Takada
FGF1 is known as an anti-inflammatory and has suppresses insulin resistance. Its homologue FGF2 is pro-inflammatory. Mechanism of FGF1 anti-inflammatory action and of FGF1 and pro-inflammatory action of FGF2 are unknown. Several inflammatory cytokines (e.g., CX3CL1, CCL5, and CXCL12, and CD40L) bind to the classical ligand (RGD)-binding site (site 1) of integrin αvβ3. In addition, they bind to the
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Toxicity assessment of 3–O–[6–deoxy–3–O–methyl–β–D–allopyranosyl–(1→4)–β–D–oleandropyranosyl]–17β–marsdenin isolated from Gongronema latifolium leaf on selected brain and kidney function indices in mice bioRxiv. Biochem. Pub Date : 2024-04-18 Onyedika G Ani, Oluwatobi A Medayedupin, Aminat A Azeez, Gideon A Gyebi, Isaac D Boateng, Joseph O Adebayo
The safety of bioactive compounds, especially those isolated from medicinal plants, is a major concern for health authorities, pharmaceutical industries, and the public. Of recent, anti–tumor pregnane glycosides were isolated from Gongronema latifolium leaf, of which the toxicity of one,3–O–[6–deoxy–3–O–methyl–β–D–allopyranosyl–(1→4)–β–D–oleandropyranosyl]–17β–marsdenin (3DMAOM), has not been evaluated
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A Sequential Binding Mechanism for 5' Splice Site Recognition and Modulation for the Human U1 snRNP bioRxiv. Biochem. Pub Date : 2024-04-18 David S. White, Bryan M. Dunyak, Frédéric H. Vaillancourt, Aaron A. Hoskins
Splice site recognition is essential for defining the transcriptome. Drugs like risdiplam and branaplam change how U1 snRNP recognizes particular 5' splice sites (5'SS) and promote U1 snRNP binding and splicing at these locations. Despite the therapeutic potential of 5'SS modulators, the complexity of their interactions and snRNP substrates have precluded defining a mechanism for 5'SS modulation. We
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nP-collabs: Investigating counterion mediated bridges in the multiply phosphorylated tau-R2 repeat bioRxiv. Biochem. Pub Date : 2024-04-18 Jules Marien, Chantal Prevost, Sophie Sacquin-Mora
Tau is an instrinsically disordered (IDP), microtubule-associated protein (MAP) that plays a key part in microtubule assembly and organization. The function of tau can be regulated via multiple phosphorylation sites. These post-translational modifications are known to decrease the binding affinity of tau for microtubules, and abnormal tau phosphorylation patterns are involved in Alzheimer's disease
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Multiresolution molecular dynamics simulations reveal the interplay between conformational variability and functional interactions in membrane-bound cytochrome 2B4 bioRxiv. Biochem. Pub Date : 2024-04-18 Sungho Bosco Han, Jonathan Teuffel, Goutam Mukherjee, Rebecca C. Wade
Cytochrome P450 2B4 (CYP 2B4) is one of the best characterized CYPs and serves as a key model system for understanding the mechanisms of microsomal class II CYPs, which metabolize most known drugs. The highly flexible nature of CYP 2B4 is apparent from crystal structures that show the active site with either a wide open or a closed heme binding cavity. Here, we investigated the conformational ensemble
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Molecular basis of proton-sensing by G protein-coupled receptors bioRxiv. Biochem. Pub Date : 2024-04-18 Matthew K Howard, Nicholas Hoppe, Xi-Ping Huang, Christian B Macdonald, Eshan Mehrotra, Patrick Rockefeller Grimes, Adam M Zahm, Donovan D Trinidad, Justin G English, Willow Coyote-Maestas, Aashish Manglik
Three proton-sensing G protein-coupled receptors (GPCRs), GPR4, GPR65, and GPR68, respond to changes in extracellular pH to regulate diverse physiology and are implicated in a wide range of diseases. A central challenge in determining how protons activate these receptors is identifying the set of residues that bind protons. Here, we determine structures of each receptor to understand the spatial arrangement
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Spirolactone, an unprecedented antifungal β-lactone spiroketal macrolide from Streptomyces iranensis bioRxiv. Biochem. Pub Date : 2024-04-18 Zhijie Yang, Yijun Qiao, Emil Strøbech, Jens Preben Morth, Grit Walther, Tue Sparholt Jørgensen, Gundela Peschel, Miriam Agler Rosenbaum, Viola Previtali, Mads Hartvig Clausen, Marie Vestgaard Lukassen, Charlotte Held Gotfredsen, Oliver Kurzai, tilmann weber, Ling Ding
Fungal infections pose a great threat to public health. There are only four classes of antifungals that have limitations due to high toxicity, drug-drug interactions, and emerging drug-resistance. Streptomyces spp. represent an important source of antimicrobial substances, notably including the antifungal agent amphotericin B. The rapamycin-producer Streptomyces iranensis displayed strong antifungal
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Structure and evolution of Alanine/Serine Decarboxylases and the engineering of theanine production bioRxiv. Biochem. Pub Date : 2024-04-18 Hao Wang, Biying Zhu, Siming Qiao, Chunxia Dong, Xiaochun Wan, Weimin Gong, Zhaoliang Zhang
Ethylamine (EA), the precursor of theanine biosynthesis, is synthesized from alanine decarboxylation by alanine decarboxylase (AlaDC) in tea plants. AlaDC evolves from serine decarboxylase (SerDC) through neofunctionalization and has lower catalytic activity. However, lacking structure information hinders the understanding of the evolution of substrate specificity and catalytic activity. In this study
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Elucidation of chalkophomycin biosynthesis reveals N-hydroxypyrrole-forming enzymes bioRxiv. Biochem. Pub Date : 2024-04-18 Anne Marie Crooke, Anika K. Chand, Zheng Cui, Emily P. Balskus
Reactive functional groups, such as N-nitrosamines, impart unique bioactivities to the natural products in which they are found. Recent work has illuminated enzymatic N-nitrosation reactions in microbial natural product biosynthesis, motivating an interest in discovering additional metabolites constructed using such reactivity. Here, we use a genome mining approach to identify over 400 cryptic biosynthetic
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Biological and Genetic Determinants of Glycolysis: Phosphofructokinase Isoforms Boost Energy Status of Stored Red Blood Cells and Transfusion Outcomes bioRxiv. Biochem. Pub Date : 2024-04-17 Travis Nemkov, Daniel Stephenson, Eric Jay Earley, Gregory R. Keele, Ariel Hay, Alicia M Key, Zachary Haiman, Christopher Erickson, Monika Dzieciatkowska, Julie A Reisz, Amy Moore, Mars Stone, Xutao Deng, Steven Kleinman, Steven L Spitalnik, Eldad A Hod, Krystalyn E Hudson, Kirk C Hansen, Bernhard O. Palsson, Gary A Churchill, Nareg Roubinian, Philip J Norris, Michael P Busch, James C Zimring, Grier
Mature red blood cells (RBCs) lack mitochondria, and thus exclusively rely on glycolysis to generate adenosine triphosphate (ATP) during aging in vivo and during storage in vitro in the blood bank. Here we identify an association between blood donor age, sex, ethnicity and end-of-storage levels of glycolytic metabolites in 13,029 volunteers from the Recipient Epidemiology and Donor Evaluation Study
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Protein design of two-component tubular assemblies like cytoskeletons bioRxiv. Biochem. Pub Date : 2024-04-17 Masahiro Noji, Yukihiko Sugita, Yosuke Yamazaki, Makito Miyazaki, Yuta Suzuki
Recent advances in protein design have ushered in an era of constructing intricate higher-order structures. Despite this progress, orchestrating the assembly of diverse protein units into cohesive artificial structures akin to biological assembly systems, especially in tubular forms, remains an elusive goal. To address this, we introduce the Nature-Inspired Protein Assembly Design (NIPAD), a novel
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Chemical codes promote selective compartmentalization of proteins bioRxiv. Biochem. Pub Date : 2024-04-17 Henry Kilgore, Itamar Chinn, Peter Mikhael, Ilan Mitnikov, Catherine Van Dongen, Guy Zylberberg, Lena Afeyan, Salman Banani, Susana Wilson-Hawken, Tony Lee, Regina Barzialy, Richard Young
Cells have evolved mechanisms to distribute ~10 billion protein molecules to subcellular compartments where diverse proteins involved in shared functions must efficiently assemble. Such assembly is presumed to unfold as a result of specific interactions between biomolecules; however, recent evidence suggests that distinctive chemical environments within subcellular compartments may also play an important
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Structural and dynamic changes in P-Rex1 upon activation by PIP3 and inhibition by IP4 bioRxiv. Biochem. Pub Date : 2024-04-17 Sandeep K. Ravala, Sendi Rafael Adame-Garcia, Sheng Li, Chun-Liang Chen, Michael A. Cianfrocco, J. Silvio Gutkind, Jennifer N. Cash, John J.G. Tesmer
PIP3-dependent Rac exchanger 1 (P–Rex1) is abundantly expressed in neutrophils and plays central roles in chemotaxis and cancer metastasis by serving as a guanine nucleotide exchange factor (GEF) for Rac. The enzyme is synergistically activated by PIP3 and the heterotrimeric Gβγ subunits, but mechanistic details remain poorly understood. While investigating the regulation of P–Rex1 by PIP3, we discovered
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Proteome of human glioblastoma and meningioma tissue small extracellular vesicles bioRxiv. Biochem. Pub Date : 2024-04-16 Huaqi Su, Adityas Purnianto, Andrew Kaye, Andrew Morokoff, Katherine j Drummond, Stanley Stylli, Laura J Vella
Small extracellular vesicles have gained attention in neuroscience due to their role in cell-to-cell communication and their potential diagnostic and therapeutic applications. Despite progress in the field, there remains a gap in our understanding of the composition and function of extracellular vesicles with regards to brain tumours. Previous studies have primarily evaluated extracellular vesicles
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Aldehydic load as an objective imaging biomarker of mild traumatic brain injury bioRxiv. Biochem. Pub Date : 2024-04-16 Alexia Kirby, Cian Ward, Nicholas D Calvert, Ryan Daniel, Joseph Wai-Hin Leung, Ashwin Shawma, Mojmir Suchy, Cassandra Donatelli, Jing Wang, Emily Standen, Adam J Shuhendler
Concussion is a mild traumatic brain injury (mTBI) defined as complex neurological impairment induced by biomechanical forces without structural brain damage. There does not yet exist an objective diagnostic tool for concussion. Downstream injury from mTBI stems from oxidative damage leading to the production of neurotoxic aldehydes. A collagen-based 3D corticomimetic scaffold was developed affording
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Utility of Cellular Measurements of Non-Specific Endocytosis to Assess the Target-Independent Clearance of Monoclonal Antibodies bioRxiv. Biochem. Pub Date : 2024-04-16 Mark A Bryniarski, Md Tariqul Haque Tuhin, Carolyn D Shomin, Fatemeh Nasrollahi, Clare Eunkyung Ko, Marcus Soto, Kyu Chung, Carrie Poon-Andersen, Ronya Primack, Diana Wong, Esperanza Ojeda, John Chung, Kevin D Cook, Kip P Conner
Past studies have demonstrated higher clearance for monoclonal antibodies possessing increased rates of non-specific endocytosis. However, this metric is oftentimes evaluated indirectly using biophysical techniques or cell surface binding studies that may not provide insight into the specific rates of cellular turnover. Furthermore, few examples evaluating non-specific endocytosis have been reported
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The Natural Material Evolution and Stage-wise Assembly of Silk Along the Silk Gland bioRxiv. Biochem. Pub Date : 2024-04-16 Ori Brookstein, Eyal Shimoni, Dror Eliaz, Nili Dezorella, Idan Biran, Katya Rechav, Ehud Sivan, Anna Kozell, Ulyana Shimanovich
Silk fibers, with their highly ordered structure and mechanically superb properties, are produced in arthropod glands at minimal energy input and ambient conditions, a remarkable feat yet to be achieved synthetically. Due to the high instability and shear sensitivity of the silk protein feedstock, understanding silk fiber formation has been largely limited to in-vitro studies of certain gland sections
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Validation of plasmonic-based biosensors for rapid and sensitive detection of rabbit hemorrhagic and foot-and-mouth disease viruses in biological samples bioRxiv. Biochem. Pub Date : 2024-04-16 Chiara Urbinati, Vittoria di Giovanni, Giulia Pezzoni, Lorenzo Capucci, Marco Rusnati
Biosensing technologies and monoclonal antibodies (MAbs) are gaining increasing importance as powerful tools in the field of virology. Surface plasmon resonance (SPR) is an optical biosensing technology already used in virus detection and in the screening of MAbs of diagnostic and therapeutic value. Rabbit haemorrhagic disease virus 2 (RHDV) and foot-and-mouth disease virus (FMDV) are top veterinary
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luxA gene from Enhygromyxa salina encodes a functional homodimeric luciferase bioRxiv. Biochem. Pub Date : 2024-04-16 Anna Yudenko, Sergey V. Bazhenov, Vladimir A. Aleksenko, Ivan M. Goncharov, Oleg Semenov, Alina Remeeva, Vera V. Nazarenko, Elizaveta Kuznetsova, Vadim V. Fomin, Maria N. Konopleva, Rahaf Al Ebrahim, Nikolai N. Sluchanko, Yury Ryzhykau, Yury S. Semenov, Alexander Kuklin, Ilya V. Manukhov, Ivan Gushchin
Several clades of luminescent bacteria are known currently. They all contain similar lux operons, which include the genes luxA and luxB encoding a heterodimeric luciferase. The aldehyde oxygenation reaction is catalyzed by the subunit LuxA, while LuxB is inactive. Recently, genomic analysis identified a subset of bacterial species with rearranged lux operons lacking luxB. Here, we show that the product
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Elucidating microRNA-34a organisation within Human Argonaute-2 by DNP MAS NMR bioRxiv. Biochem. Pub Date : 2024-04-16 Rubin Dasgupta, Walter Becker, Katja Petzold
Understanding mRNA regulation by microRNA (miR) relies on the structural understanding of the RNA-induced silencing complex (RISC). Here, we elucidate the structural organisation of miR-34a, de-regulated in various cancers, in hAgo2, effector protein in RISC, using guanosine-specific isotopic labelling and dynamic nuclear polarisation (DNP)-enhanced solid-state NMR. Homonuclear correlation experiments
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Structure and function of the ROR2 cysteine-rich domain in vertebrate noncanonical WNT5A signaling bioRxiv. Biochem. Pub Date : 2024-04-15 Samuel C Griffiths, Jia Tan, Armin Wagner, Levi L Blazer, Jarret J Adams, Srisathya Srinivasan, Shayan Moghisaei, Sachdev S Sidhu, Christian Siebold, Hsin-Yi Henry Ho
The receptor tyrosine kinase ROR2 mediates noncanonical WNT5A signaling to orchestrate tissue morphogenetic processes, and dysfunction of the pathway causes Robinow syndrome, Brachydactyly B and metastatic diseases. The domain(s) and mechanisms required for ROR2 function, however, remain unclear. We solved the crystal structure of the extracellular cysteine-rich (CRD) and Kringle (Kr) domains of ROR2
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Subcellular, biochemical and biophysical alterations in two glial cell models of ARSACS bioRxiv. Biochem. Pub Date : 2024-04-15 Fernanda Murtinheira, Ana Sofia Boasinha, Joao Belo, Luana Macedo, Elisa Farsetti, Tiago T. Robalo, Vukosava M. Torres, Francisco R. Pinto, Adelaide Fernandes, Patricia Nascimento, Mario S. Rodrigues, Federico Herrera
Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a developmental and degenerative disorder caused by loss-of-function mutations in the gene that codifies for the sacsin chaperone. Sacsin was initially described as a neuronal protein but is found in various cell types, including astroglial, microglial, kidney, and skin cell lines. We and others have shown that virtually all cell
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Alternate recognition by dengue protease: Proteolytic and binding assays provide functional evidence beyond an induced-fit bioRxiv. Biochem. Pub Date : 2024-04-15 Mira A.M. Behnam, Christian D Klein
Proteases are key enzymes in viral replication, and interfering with these targets is the basis for therapeutic interventions. We previously introduced a hypothesis about conformational selection in the protease of dengue virus and related flaviviruses, based on conformational plasticity noted in X-ray structures. The present work presents the first functional evidence for alternate recognition by
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Cryo-EM analysis of complement C3 reveals a reversible major opening of the macroglobulin ring bioRxiv. Biochem. Pub Date : 2024-04-15 Trine AF Gadeberg, Martin H Joergensen, Heidi G Olesen, Josefine Lorentzen, Seandean L Harwood, Ana V Almeida, Marlene U Fruergaard, Rasmus K Jensen, Philipp Kanis, Henrik Pedersen, Emil Tranchant, Steen V Petersen, Ida B Thoegersen, Joseph A Lyons, Jan J Enghild, Gregers Rom R Andersen
The C3 protein is the central molecule within the complement system and undergoes pattern-recognition-dependent proteolytic activation to C3b in the presence of pathogens and damage-associated patterns. Spontaneous pattern-independent activation of C3 occurs via hydrolysis, resulting in C3(H2O). However, the structural details of C3 hydrolysis remain elusive. Here, we show that the conformation of
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Nuclear receptor interdomain communication is mediated by the hinge with ligand specificity bioRxiv. Biochem. Pub Date : 2024-04-15 Saurov Hazarika, Tracy Yu, Arumay D Biswas, Namita Dube, Priscilla Villalona, C. Denise Okafor
Nuclear receptors are ligand-induced transcription factors that bind directly to target genes and regulate their expression. Ligand binding initiates conformational changes that propagate to other domains, allosterically regulating their activity. The nature of this interdomain communication in nuclear receptors is poorly understood, largely owing to the difficulty of experimentally characterizing
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Multicomponent depolymerization of actin filament pointed ends by cofilin and cyclase-associated protein depends upon filament age bioRxiv. Biochem. Pub Date : 2024-04-15 Ekram M Towsif, Blake Andrew Miller, Heidi Ulrichs, Shashank Shekhar
Intracellular actin networks assemble through the addition of ATP actin subunits at the growing barbed ends of actin filaments. This is followed by aging of the filament via ATP hydrolysis and subsequent phosphate release. Aged ADP–actin subunits thus treadmill through the filament before being released back into the cytoplasmic monomer pool as a result of depolymerization at filament pointed ends
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S-Wipe: stool sample collection for metabolomic gut health tracking bioRxiv. Biochem. Pub Date : 2024-04-15 Alexey V. Melnik, Konstantin Pobozhev, Ali Lotfi, Dana Moradi, Hannah Monahan, Evguenia Kopylova, Alexander A Aksenov
Microbiome is increasingly recognized as a key factor in health. Intestinal microbiota modulates gut homeostasis via a range of diverse metabolites. Molecules such as short chain fatty acids (SCFAs), the microbial fermentation products of dietary fiber, have been established to be reflective of microbiome and/or dietary shifts and have been linked to multiple gastrointestinal disorders from cancer
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Characterizing the monomer-dimer equilibrium of UbcH8/Ube2L6: A combined SAXS and NMR study bioRxiv. Biochem. Pub Date : 2024-04-14 Kerem Kahraman, Scott A. Robson, Oktay Gocenler, Cansu M. Yenici, Cansu D. Tozkoparan, Jennifer M. Klein, Volker Dötsch, Emine S. Elgin, Arthur L. Haas, Joshua J. Ziarek, Çağdaş Dağ
Interferon-stimulated gene-15 (ISG15) is an interferon-induced protein with two ubiquitin-like (Ubl) domains linked by a short peptide chain, and the conjugated protein of the ISGylation system. Similar to ubiquitin and other Ubls, ISG15 is ligated to its target proteins with a series of E1, E2, and E3 enzymes known as Uba7, Ube2L6/UbcH8, and HERC5, respectively. Ube2L6/UbcH8 plays a literal central
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Top-down Mass Spectrometric Analysis of a Lipooligosaccharide from the Human Commensal Bacteroides fragilis bioRxiv. Biochem. Pub Date : 2024-04-14 Tiandi Yang, Jason Daugherty, Dennis L Kasper
Lipopolysaccharides (LPS) and lipooligosaccharides (LOS) are ubiquitous structures found on the outer membrane of gram-negative bacteria. Bacteroides fragilis, is a gram-negative anaerobe commonly inhabiting the human colon. The LOS of this organism is known to trigger a type I interferon response in dendritic cells. However, detailed structural analysis of this LOS has been largely elusive. Using
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Human transmembrane protein 68 links triacylglycerol synthesis to membrane lipid homeostasis bioRxiv. Biochem. Pub Date : 2024-04-14 Fansi Zeng, Christoph Heier, Qing Yu, Huimin Pang, Feifei Huang, Zheng Zhao, Pingan Chang
Transmembrane protein 68 (TMEM68) is a recently identified mammalian triacylglycerol (TAG) synthase with high expression in the brain. How TMEM68 regulates cellular lipid metabolism in concert with other enzymatic pathways remains poorly understood. In this study, we assessed TMEM68 function in neuro- and gliablastoma cells by combining genetic gain- and loss-of-function approaches with lipidomics
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A Stapled Peptide Inhibitor of METTL3-METTL14 for Cancer Therapy bioRxiv. Biochem. Pub Date : 2024-04-14 Zenghui Li, Yuqing Feng, Hong Han, Xingyue Jiang, Weiyu Chen, Xuezhen Ma, Yang Mei, Xuhui Yan, Ping Yin, Dan Yuan, Dingxiao Zhang, Junfeng Shi
METTL3, a primary methyltransferase catalyzing RNA N6-methyladenosine (m6A) modification, has been identified as an oncogene in several cancer types and thus nominated as a potentially effective target for therapeutic inhibition, although current options using this strategy are limited. In this study, we targeted protein-protein interactions at the METTL3-METTL14 binding interface to inhibit complex
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Inferring residue level hydrogen deuterium exchange with ReX bioRxiv. Biochem. Pub Date : 2024-04-13 Oliver Crook, Nathan Gittens, Chun-wa Chung, Charlotte Deane
Hydrogen-Deuterium Exchange Mass-Spectrometry (HDX-MS) has emerged as a powerful technique to explore the conformational dynamics of proteins and protein complexes in solution. The bottom-up approach to MS uses peptides to represent an average of residues, leading to reduced resolution of deuterium exchange and complicates the interpretation of the data. Here, we introduce ReX, a method to infer residue-level
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A framework for quality control in quantitative proteomics bioRxiv. Biochem. Pub Date : 2024-04-13 Kristine A Tsantilas, Gennifer E Merrihew, Julia E Robbins, Richard S Johnson, Jea Park, Deanna L Plubell, Eric Huang, Michael Riffle, Vagisha Sharma, Brendan X MacLean, Josh Eckels, Michael B Bereman, Sandra E Spencer, Andrew N Hoofnagle, Michael J MacCoss
A thorough evaluation of the quality, reproducibility, and variability of bottom-up proteomics data is necessary at every stage of a workflow from planning to analysis. We share real-world case studies applying adaptable quality control (QC) measures to assess sample preparation, system function, and quantitative analysis. System suitability samples are repeatedly measured longitudinally with targeted
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Conformational dynamics associated with remote residues regulate the kinetic properties of homologous glutamate dehydrogenases (GDHs) bioRxiv. Biochem. Pub Date : 2024-04-13 Barsa Kanchan Jyotshna Godsora, Parijat Das, Anjali Sairaman, Prasoon Kumar Mishra, Sandip Kaledhonkar, Narayan S. Punekar, Prasenjit Bhaumik
Glutamate dehydrogenase (GDH) is a key enzyme in all living organisms and some of the GDHs exhibit substrate-dependent homotropic cooperativity. However, the mode of allosteric communication during the homotropic effect in GDHs remains poorly understood. In this study, we examined two homologous GDHs, Aspergillus niger GDH (AnGDH) and Aspergillus terreus GDH (AtGDH), with differing substrate utilization
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Essential and virulence-related protein interactions of pathogens revealed through deep learning bioRxiv. Biochem. Pub Date : 2024-04-12 Ian R Humphreys, Jing Zhang, Minkyung Baek, Yaxi Wang, Aditya Krishnakumar, Jimin Pei, Ivan Anishchenko, Catherine A Tower, Blake A Jackson, Thulasi Warrier, Deborah T Hung, S Brook Peterson, Joseph D Mougous, Qian Cong, David Baker
Identification of bacterial protein protein interactions and predicting the structures of the complexes could aid in the understanding of pathogenicity mechanisms and developing treatments for infectious diseases. Here, we developed a deep learning based pipeline that leverages residue-residue coevolution and protein structure prediction to systematically identify and structurally characterize protein
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Toxin-based screening of C-terminal tags in Escherichia coli reveals the exceptional potency of ssrA-like degrons bioRxiv. Biochem. Pub Date : 2024-04-12 Patrick C Beardslee, Karl R Schmitz
All bacteria possess ATP-dependent proteases that destroy cytosolic proteins. These enzymes help cells mitigate proteotoxic stress, adapt to changing nutrient availability, regulate virulence phenotypes, and transition to pathogenic lifestyles. Moreover, ATP-dependent proteases have emerged as promising antibacterial and antivirulence targets in a variety of pathogens. The physiological roles of these
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A Small Multidrug Resistance Transporter in Pseudomonas aeruginosa Confers Substrate-Specific Resistance or Susceptibility bioRxiv. Biochem. Pub Date : 2024-04-12 Andrea Killian Wegrzynowicz, William J Heelan, Sydnye P Demas, Maxwell S McLean, Jason M Peters, Katherine A Henzler-Wildman
Small Multidrug Resistance (SMR) transporters are key players in the defense of multidrug-resistant pathogens to toxins and other homeostasis-perturbing compounds. However, recent evidence demonstrates that EmrE, an SMR from Escherichia coli and a model for understanding transport, can also induce susceptibility to some compounds by drug-gated proton leak. This runs down the ∆pH component of the Proton
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A map of the rubisco biochemical landscape bioRxiv. Biochem. Pub Date : 2024-04-11 Noam Prywes, Naiya R. Philips, Luke M. Oltrogge, Sebastian Lindner, Yi-Chin Candace Tsai, Benoit de Pins, Aidan E. Cowan, Leah J. Taylor-Kearney, Hana A. Chang, Laina N. Hall, Daniel Bellieny-Rabelo, Hunter M. Nisonoff, Rachel F. Weissman, Avi I. Flamholz, David Ding, Abhishek Y. Bhatt, Patrick M. Shih, Oliver Mueller-Cajar, Ron Milo, David F. Savage
Rubisco is the primary CO2 fixing enzyme of the biosphere yet has slow kinetics. The roles of evolution and chemical mechanism in constraining the sequence landscape of rubisco remain debated. In order to map sequence to function, we developed a massively parallel assay for rubisco using an engineered E. coli where enzyme function is coupled to growth. By assaying >99% of single amino acid mutants
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Elaboration of the Homer1 Recognition Landscape Reveals Incomplete Divergence of Paralogous EVH1 Domains bioRxiv. Biochem. Pub Date : 2024-04-11 Avinoam Singer, Alejandra Ramos, Amy E. Keating
Short sequences that mediate interactions with modular binding domains are ubiquitous throughout eukaryotic proteomes. Networks of Short Linear Motifs (SLiMs) and their corresponding binding domains orchestrate many cellular processes, and the low mutational barrier to evolving novel interactions provides a way for biological systems to rapidly sample selectable phenotypes. Mapping SLiM binding specificity
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Heterologous expression and purification of glutamate decarboxylase-1 from the model plant Arabidopsis thaliana: characterization of the enzyme’s in vitro truncation by thiol endopeptidase activity bioRxiv. Biochem. Pub Date : 2024-04-11 Brittany S. Menard, Kirsten H. Benidickson, Lee Marie Raytek, Wayne A. Snedden, William C. Plaxton
Plant glutamate decarboxylase (GAD) is a Ca2+-calmodulin activated cytosolic enzyme that produces γ-aminobutyrate (GABA) as the first committed step of the GABA shunt. This pathway circumvents the 2-oxoglutarate to succinate reactions of the mitochondrial tricarboxylic acid cycle. Our prior research established that in vivo phosphorylation of the root-specific AtGAD1 isozyme (AT5G17330) occurs at multiple
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Evolution of the substrate specificity of an RNA ligase ribozyme from phosphorimidazole- to triphosphate-activation bioRxiv. Biochem. Pub Date : 2024-04-11 Saurja DasGupta, Zoe Weiss, Collin Nisler, Jack W. Szostak
The acquisition of new RNA functions through evolutionary processes would have been essential for the diversification of RNA-based primordial biology and its subsequent transition to modern biology. However, the mechanisms by which RNAs access new functions remain unclear. Do ribozymes need completely new folds to support new but related functions, or is re-optimization of the active site sufficient
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Identification of drug-like molecules targeting the ATPase activity of dynamin-like EHD4 bioRxiv. Biochem. Pub Date : 2024-04-11 Saif Mohd, Andreas Oder, Edgar Specker, Martin Neuenschwander, Jens Peter Von Kries, Oliver Daumke
Eps15 (epidermal growth factor receptor pathway substrate 15) homology domain-containing proteins (EHDs) comprise a family of eukaryotic dynamin-related ATPases that participate in various endocytic membrane trafficking pathways. Dysregulation of EHDs function has been implicated in various diseases, including cancer. The lack of small molecule inhibitors which acutely target individual EHD members
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Reconstitution of human DNA licensing and the structural and functional analysis of key intermediates bioRxiv. Biochem. Pub Date : 2024-04-11 Jennifer N. Wells, Vera Leber, Lucy V Edwardes, Shenaz Allyjaun, Matthew Peach, Joshua Tomkins, Antonia Kefala-Stavridi, Sarah V Faull, Ricardo Aramayo, Carolina M. Pestana, Lepakshi Ranjha, Christian Speck
Human DNA licensing initiates the process of replication fork assembly. Specifically, this reaction leads to the loading of hMCM2-7 on DNA, which represents the core of the replicative helicase that unwinds DNA during S-phase. Here, we report the biochemical reconstitution of human DNA licensing using purified proteins, the structural and functional analysis of the process and reveal the impact of
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Quantification of histone H1 subtypes using targeted proteomics bioRxiv. Biochem. Pub Date : 2024-04-11 J López-Gómez, L Villarreal, M Andrés, I Ponte, B Xicoy, L Zamora, M Vilaseca, A Roque
Histone H1 is involved in the regulation of chromatin structure. Human somatic cells express up to seven subtypes: H1.0, H1.1-H15, and H1X. The variability in the proportions of somatic H1s (H1 complement) is one evidence supporting their functional specificity. Alterations in the protein levels of different H1 subtypes have been observed in cancer, suggesting their potential as biomarkers and that
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A narrow ratio of nucleic acid to SARS-CoV-2 N-protein enables phase separation bioRxiv. Biochem. Pub Date : 2024-04-11 Patrick M. Laughlin, Kimberly Young, Giovanni Gonzalez-Gutierrez, Joseph C.Y. Wang, Adam Zlotnick
SARS-CoV-2 Nucleocapsid protein (N) is a viral structural protein that packages the 30kb genomic RNA inside virions and forms condensates within infected cells through liquid-liquid phase separation (LLPS). N, in both soluble and condensed forms, has accessory roles in the viral life cycle including genome replication and immunosuppression. The ability to perform these tasks depends on phase separation
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Ancestral versus Modern Substrate Scope in Family-1 Glycosidases bioRxiv. Biochem. Pub Date : 2024-04-11 Luis I. Gutierrez-Rus, Dušan Petrović, Pascal Schneider, Katja Zorn, Gloria Gamiz-Arco, Leonardo De Maria, Francesco Falcioni, Valeria A. Risso, Martin A. Hayes, Jose M. Sanchez-Ruiz
Promiscuity, the capability of catalyzing a diversity of chemical reactions, is a desirable feature in many enzymes intended for biotechnological applications. Promiscuous activities, however, are typically depressed in specialized modern enzymes. On the other hand, several ancestral reconstruction studies have reported enzymes with enhanced promiscuity. Glycosidases catalyze the hydrolysis of glycosidic
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A Ratiometric Catalog of Protein Isoform Shifts in the Cardiac Fetal Gene Program bioRxiv. Biochem. Pub Date : 2024-04-10 Yu Han, Sara A. Wennersten, Boomathi P. Pandi, Dominic C. M. Ng, Edward Lau, Maggie P. Y. Lam
The fetal genetic program orchestrates cardiac development and the re-expression of fetal genes is thought to underlie cardiac disease and adaptation. Here, a proteomics ratio test using mass spectrometry is applied to find protein isoforms with statistically significant usage differences in the fetal vs. postnatal mouse heart. Changes in isoform usage ratios are pervasive at the protein level, with
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Active state structures of a bistable visual opsin bound to G proteins bioRxiv. Biochem. Pub Date : 2024-04-10 Oliver Tejero, Filip Pamula, Mitsumasa Koyanagi, Takashi Nagata, Pavel Afanasyev, Ishita Das, Xavier Deupi, Mordechai Sheves, Akihisa Terakita, Gebhard F.X. Schertler, Matthew J. Rodrigues, Ching-Ju Tsai
Opsins are G protein-coupled receptors (GPCRs) that have evolved to detect light stimuli and initiate intracellular signaling cascades. Their role as signal transducers is critical to light perception across the animal kingdom. Opsins covalently bind to the chromophore 11-cis retinal, which isomerizes to the all-trans isomer upon photon absorption, causing conformational changes that result in receptor
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Phage-Encoded Bismuth Bicycles: Instant Access to Targeted Bioactive Peptides bioRxiv. Biochem. Pub Date : 2024-04-10 Sven Ullrich, Upamali Somathilake, Minghao Shang, Christoph Nitsche
Genetically encoded libraries play a crucial role in discovering structurally rigid, high-affinity macrocyclic peptide ligands for therapeutic applications. This study represents the first genetic encoding of peptide-bismuth and peptide-arsenic bicyclic peptides in phage display. We introduce bismuth tripotassium dicitrate (gastrodenol) as a water-soluble Bi(III) reagent for phage library modification
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Ovoselenol, a Selenium-containing Antioxidant Derived from Convergent Evolution bioRxiv. Biochem. Pub Date : 2024-04-10 Chase M. Kayrouz, Kendra A. Ireland, Vanessa Ying, Katherine M. Davis, Mohammad R. Seyedsayamdost
Selenium is an essential micronutrient, but its presence in biology has been limited to protein and nucleic acid biopolymers. The recent identification of the first biosynthetic pathway for selenium-containing small molecules suggests that there is a larger family of selenometabolites that remains to be discovered. Using a bioinformatic search strategy that relies on mapping of composite active site
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Ligand Response of Guanidine-IV riboswitch at Single-molecule Level bioRxiv. Biochem. Pub Date : 2024-04-10 Lingzhi Gao, Dian Chen, Yu Liu
Riboswitches represent a class of non-coding RNA that possess the unique ability to specifically bind ligands and, in response, regulate gene expression. A recent report unveiled a type of riboswitch, known as the guanidine-IV riboswitch, which responds to guanidine levels to regulate downstream genetic transcription. However, the precise molecular mechanism through which the riboswitch senses its
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Architectonic Principles of Polyproline II Helix Bundle Protein Domains bioRxiv. Biochem. Pub Date : 2024-04-10 Cristian Segura Rodríguez, Douglas V. Laurents
Glycine rich polyproline II helix assemblies are an emerging class of natural domains found in several proteins with different functions and diverse origins. The distinct properties of these domains relative to those composed of α-helices and β-sheets could make glycine-rich polyproline II helix assemblies a useful building block for protein design. Whereas the high population of polyproline II conformers
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Multiple pathways for licensing human replication origins bioRxiv. Biochem. Pub Date : 2024-04-10 Ran Yang, Olivia Hunker, Marleigh Wise, Franziska Bleichert
The loading of replicative helicases constitutes an obligatory step in the assembly of DNA replication machineries. In eukaryotes, the MCM2-7 replicative helicase motor is deposited onto DNA by the origin recognition complex (ORC) and co-loader proteins as a head-to-head MCM double hexamer to license replication origins. Although extensively studied in the budding yeast model system, the mechanisms
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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 : 2024-04-10 George N. 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