- by Dicksion, C. A., Chao, D. N., Rickmeyer, J. D., Bess, E. N.The human gut microbiome is home to numerous small molecules that impact health. Three prominent classes of molecules in this environment include phenolic acids, lignans, and enterolignans, which have been linked to anti-inflammatory and anti-oxidant effects as well as protection from cancer, cardiovascular disease, and neurodegeneration. The abundance of these molecules in the gut microbiome as well as their biological significance motivated the development of the LC-MS/MS method reported herein, which provides a simple, robust, and high-throughput approach to simultaneously […]
- by Come, J., Muianga, J., Fumo, W. C., Bila, S., Simbine-Ribisse, E., Changule, A., Tomo, O., Garcia-Herreros, M., Bila, C. G.Honey is a natural product made by bees and is widely valued as a healthy food. Its quality can deteriorate due to microbial activity and physical or chemical changes. This study assessed honey from formal (n=12) and informal (n=12) markets in three Mozambican provinces: Maputo, Sofala, and Inhambane, to evaluate its quality and safety. Physicochemical parameters such as viscosity, water activity (Aw), pH, ash content, total soluble solids, and diastase activity were measured using standard AOAC methods. Microbiological quality was […]
- by Nouri, S., Niemela, A., Nencini, R., Kolypetris, G., Niemi-Aro, T., Virtanen, S. I., Ollila, S. O. H., Koivuniemi, A.Peptide-based nanodiscs are promising anti-atherosclerosis biologics, drug delivery particles and structural biology tools. However, the structural and dynamic properties of these peptide-based nanodiscs remains unknown, hindering their development for the aforementioned purposes. Here we integrated molecular dynamics (MD) simulations with nuclear magnetic resonance (NMR), small-angle x-ray scattering (SAXS), and small-angle neutron scattering (SANS) experiments to investigate the structure and dynamics of therapeutic peptide-based nanodiscs constituted of DMPC phospholipids and the amphiphilic apolipoprotein A-I mimetic peptide 22A. This multi-technique approach reveals […]
- by Karpel, R. L., Guei, J., Chapman, M. P., Brothers, P. N., Desai, S.The single-stranded DNA binding protein of bacteriophage T4, gp32, has important roles in replication, recombination, and repair. gp32 possesses three domains: the central (core) domain which contains the binding trough for single-stranded DNA, the N-terminal domain, which interacts with the core domain of an adjacent ssDNA-bound protein, bringing about binding cooperativity, and the C-terminal domain, which interacts with other proteins involved in replication, recombination, and repair. The essential residues within the N-domain for the association with the adjacent DNA bound […]
- by Vito, A. F., Ling, J. A., Ferrara, J. C., Jacques, C. S., Gillet, N., Gonzalez-Aleman, R., Qiu, Y., Hashemian, M., Trasvina-Arenas, C. H., David, S. S., Delaney, S., Bignon, E., Freudenthal, B. D.Eukaryotic genomic DNA is packaged into chromatin through a repeating unit known as the nucleosome. In this chromatin environment, DNA is constantly exposed to several sources of DNA damage, such as reactive oxygen species (ROS), which can lead to the formation of 8-oxo-7,8-dihydroguanine (8oxoG). 8oxoG can base pair with cytosine (8oxoG:C) or form a mutagenic base pair with adenine (8oxoG:A), which can lead to single base transversions if left unrepaired. To date, the structure and dynamics of these two possible […]
- by Arsene, C., Gates, A., Roemmert, A.-K., Maertens, A., Faustinelli, V., Luckau, L., OConnor, G.We present a novel method for analyzing the folding of intrinsically disordered proteins (IDPs), such as Tau and phosphorylated Tau (pTau), in solution. Using cross linking mass spectrometry combined with a new downstream analysis framework, we construct weighted interaction networks from cross-link derived residue pairs without relying on predefined secondary structure assumptions. Structural differences between protein conformations are quantified by comparing the organization of loop structures within their cross-link networks. Validation with bovine serum albumin (BSA) in native and denatured […]
- by Agrawal, N., Parisini, E.Semen-derived enhancer of viral infection (SEVI) fibrils, assembled from the peptide fragment PAP248-286, enhance HIV transmission by promoting viral attachment to host cells. However, the molecular basis of SEVI nucleation and early aggregation remains unclear. Here, we conducted 80 independent all-atom molecular dynamics (MD) simulations spanning a total of 40 s, together with 100 independent steered MD and umbrella sampling runs, to explore the dimerization and dissociation of PAP248-286 Our results show that hydrogen bonding is the dominant stabilizing force […]
- by Virdee, S., Fletcher, A. J., Squair, D. R., Rivers, E., Sowar, H., Balci, A., Harmo, R., Wright, D. J., Beniwal, G., Soetens, M., Mathur, S., Tollervey, A., Stanton, C.The detection of viral RNA inside cells triggers a diverse range of antiviral responses, including global translation inhibition, interferon secretion and RNA sequestration. Mutations in the gene ZNFX1 cause severe paediatric immunodeficiencies, including chronic viral infection and autoinflammation. Here, we show that ZNFX1 is an RNA helicase with cryptic and unusual bifurcating E3 ubiquitin ligase activity. Nucleotide-dependent RNA binding stimulates ZNFX1 to generate complex ubiquitin chains via a two-component ubiquitin circuit wired in parallel, with ubiquitin flux occurring via either […]
- by Schlösser, L., Kutzner, M., Hellmann, N., Kiesewetter, D., Bieber, J., Quarta, N., Ge, X., Goetze, T., Junglas, B., Matsumura, F., Bonn, M., Gräter, F., Sachse, C., Liu, L.-N., Schmidt, C., Aponte-Santamaria, C., Schneider, D.IM30, the inner membrane-associated protein of 30 kDa (also known as Vipp1) is essential for thylakoid membrane biogenesis and/or maintenance in chloroplasts and cyanobacteria. IM30 and its bacterial homolog PspA belong to the ESCRT-III superfamily, proteins previously thought to be restricted to eukaryotes and archaea. Despite low sequence similarity, IM30 shares key structural and functional features with eukaryotic ESCRT-IIIs, including a conserved 1-2 helical hairpin core and the ability to form oligomeric barrel- or rod assemblies that mediate membrane remodeling. […]
- by Pratihar, S., Zhong, W., Feng, S., Chatterjee, S., Kool, E. T.Installing functional groups at specific sites in existing RNA molecules remains a challenge for modification, labeling, and therapeutic strategies. Here we describe the use of DNA oligonucleotides carrying a catalytic amine group to effect the aqueous SNAr arylation of 2'-OH groups at sequence-complementary sites in RNAs. Chloro-pyrimidine electrophiles are shown to react with amino-DNA conjugates, resulting in a proposed transient ammonium aryl intermediate that can react with RNA near the DNA binding site, delivering the heterocycle to the RNA in […]
- by Lenz, C., Elson, L., Dopfer, J., Farges, F., Kraemer, A., Loehr, F., Mueller, S., Gueret, S. M., Waldmann, H., Doetsch, V., Saxena, K., Knapp, S.Developing new E3 ligase ligands for the design of heterobivalent molecules, such as PROteolysis TArgeting Chimeras (PROTACs), requires careful evaluation of target engagement (TE). Characterizing protein-protein interactions (PPIs) is therefore essential in drug discovery, as it enables the assessment of ligand binding to sites that are often difficult to target. Degrons, peptide motifs recognized by E3 ligases, may serve as valuable starting points for designing E3 ligands. However, many degrons are highly polar and lack intrinsic membrane permeability, requiring alternative […]
- by Ahn, S., Oh, T.-S., Suh, S., Jeon, J.-y., Lah, S., Ryu, K., Kim, H., Lee, E. G., Lee, H., Lee, J., Kim, D.-K., Ku, B. M., Jung, W., Ahn, M.-J., Jung, J. U., Kim, Y.-S., Oh, B.-H., Jeong, B.-S.Antibodies that recognize peptide-loaded class I major histocompatibility complex (pMHC I) molecules could enable therapeutic targeting of intracellular oncogenic proteins, yet their discovery has been hampered by the small size of peptide antigens and allele-specificity. We describe an integrated in silico-in vitro workflow for generating high-affinity, selective antibodies to KRAS(G12D)10 presented by HLA-C*08:02, a clinically validated cancer neoantigen. In silico, multiple human antibody-derived variable fragments (Fvs) plausibly docked to the target pMHC were generated, followed by limited complementarity-determining region (CDR) […]
- by Hiki, K., Pham, T., Yamamoto, M., Hayashi, T. I., Shimizu, S.Statistical causal discovery (SCD) has the potential to advance the development and evaluation of Adverse Outcome Pathways (AOPs) by inferring causal relationships directly from data. However, ecotoxicology data often has challenges for SCD applications, such as missing data and violation of SCD algorithm assumptions. As a proof-of-concept, we applied a linear non-Gaussian acyclic model (LiNGAM), a representative SCD method, to three types of ecotoxicology datasets: (1) bivariate dose-response relationships, (2) bivariate response-response relationships, and (3) a multivariate dataset with a […]
- by Xia, S., Zhang, D., Shang, X., Xu, J.Accurate biomolecular simulations are essential for understanding chemical processes and advancing applications such as protein engineering and drug design. While quantum mechanical (QM) methods can provide chemical accuracy, their computational cost limits their scalability. Machine learning force fields (MLFFs) offer a promising alternative by achieving similar accuracy with vastly improved efficiency, but their effectiveness is often constrained by limited conformational and chemical diversity in training. We introduce QuantaMind, a robust MLFF workflow designed to extend chemical and conformational coverage, particularly […]
- by Das, K., Gordon, C., Oliva, M., Lee, H., Goovaerts, Q., De Wijngaert, B., Gotte, M.Nucleoside analogs (NAs) have been successfully used to treat viral infections. dNTP analogs are primarily DNA chain terminators, while NTP analogs, such as remdesivir, can inhibit as delayed chain terminators or when in the template strand. Determining the frequency of remdesivir triphosphate (RTP) incorporation in the presence of the competing ATP can help in understanding different modes of viral RNA-dependent RNA polymerase (RdRp) inhibition by NTP analogs. We employed enzymatic assays, mass spectrometry, and cryo-EM to show that SARS-CoV-2 RdRp […]
- by Lee, H. K., Kim, S., Hong, J., Bae, T., An, Y., Sohn, C. H., Hwang, W. C., Park, C.-K., Lee, S. H., Koh, H. R., Hur, J. K.The CRISPR-Cas9 system is a powerful genome editing tool capable of precisely recognizing and cleaving specific DNA sequences, and has been extensively investigated as a strategy for correcting mutations associated with genetic diseases and cancer. However, when single-nucleotide mutations do not occur within or near the protospacer adjacent motif (PAM) sequence, conventional CRISPR designs often fail to discriminate between mutant and wild-type alleles. To address this limitation, we developed a guide RNA (gRNA) engineering approach that introduces an intentional mismatch […]
- by Feng, Z., Shen, J., Li, Y., Gonzalez-Gutierrez,, G., Wang, Q., Niu, H.Telomeres protect chromosome ends from degradation and inappropriate DNA repair. In budding yeast, the telomeric protein Cdc13 binds G-rich overhangs to maintain telomere integrity. Here, we show that Cdc13 also weakly interacts with adjacent duplex DNA at the ss/dsDNA junction – a property essential for inhibiting Exo1- and Sgs1 – Dna2 – mediated resection. Mutation of a conserved residue (K504E) disrupts this junction recognition and impairs resection inhibition, RPA displacement, and cooperation with Ku. cdc13-K504E cells are hypersensitive to Exo1 […]
- by Boulton, A., Kabra, A., Achille, N., Adelman, E. R., Anastasakis, D. G., Beckedorff, F., Cingaram, P., Zhang, Y., Leach, B., Shiekhattar, R., Yokoyama, A., Hafner, M., Figueroa, M., Zeleznik-Le, N., Bushweller, J.The protein MLLT3 (AF9) is a critical regulator of hematopoiesis. The N-terminal YEATS domain of MLLT3 is an epigenetic reader that binds to acetylated as well as crotonylated lysine. Using PAR-CLIP, biochemical assays, and NMR based mapping of binding, we demonstrate that the YEATS domain of MLLT3 binds to a specific stem-loop region of the noncoding RNA 7SK. 7SK is a noncoding RNA with a well-documented function in transcriptional elongation. We developed point mutations in the YEATS domain that disrupt […]
- by Sun, Y., Yuan, C., Long, L., Ding, S.The single-domain auxiliary activity family 12 (AA12) pyrroloquinoline quinone-dependent oxidoreductases and free AA8 modules are prevalent in cellulolytic fungi, however, their function in polysaccharide biodegradation is still confused. Here, we characterized three single-domain AA12 oxidoreductases and one free AA8 module from Thermothelomyces thermophilus and Thermothielavioides terrestris. All three single-domain AA12 oxidoreductases are restrict dehydrogenases with trace oxidase activity. All three single-domain AA12 enzymes could directly transfer electrons to lytic polysaccharide monooxygenase (LPMO) and drive NcLPMO9C activity. Furthermore, inter-protein electron transfer […]
- by Gao, A., Wassarman, D. R., Kranzusch, P. J.QueC proteins are nucleoside biosynthesis enzymes required for production of the 7-deazaguanine derivative queuosine. Recently, QueC-family proteins were also shown to catalyze a deazaguanylation protein-nucleobase conjugation reaction in type IV CBASS bacterial anti-phage defense. Here we determine the structural basis of QueC-family protein function in a distinct bacterial immunity system named qatABCD. We demonstrate that the QueC-family protein QatC forms a specific complex with the immunity protein QatB and that this complex is minimally required for qatABCD defense. Crystal structures […]
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