- by Jones, J. D., Ghohabi Esfahani, N., Goemann, C. L., Wiegand, T., Nemudryi, A., Ruan, M., Li, X., Tardu, M., Kennedy, R. T., Wiedenheft, B., Jain, M., Koutmou, K. S.RNA modifications contribute to the regulatory and structural complexity of RNA molecules, influencing key biological processes. Nanopore direct RNA sequencing (DRS) enables the detection of these modifications at single-molecule resolution without chemical conversion. Oxford Nanopore Technologies recently introduced RNA004 sequencing chemistry and the Dorado basecaller, which improves accuracy and enables the identification of eight RNA modifications. However, the reliability of these predictions requires careful validation using orthogonal approaches. Here, we profiled RNA modifications in the MS2 bacteriophage genome using both […]
- by zhang, x., zhang, z., zhang, x., liu, m., Li, y., zhao, p., xie, f., ma, x.Abstract Molecular hydrogen (H2) is produced by plants under hypoxia and has been implicated in stress acclimation, yet its enzymatic source in vascular plants remains unknown because canonical hydrogenases are absent from angiosperm genomes. Here, using thermodynamic modeling, kinetic analysis, pharmacological perturbation, substrate-feeding assays, metabolite profiling, and cross-species comparison, we identify a mitochondrial origin for hypoxic H2 production in plants and define its biochemical requirements. Our data reveal a hydrogenase-like activity that obligatorily couples to Complex I Fe-S/quinone branch turnover […]
- by Kent, A. D., Majumdar, C., Fitzgerald, K. A., Solivan, A. C., Boga de Teresa, M., Vance, J. T., Schepartz, A., Cate, J. H. D.Ribosomes are capable of incorporating a wide array of natural and unnatural monomers into growing polymer chains, but can be stalled by monomers with constrained or non-natural backbones. Here we evaluate whether monomer-dependent ribosome stalling can be alleviated by structure-guided mutations to 23S rRNA within the exit tunnel. Ribosomes harboring an A2062U mutation are as active as wild type (WT) ribosomes when translating non-proline sequences and up to 10-fold more active when translating sequences containing up to four consecutive proline […]
- by Martin, A., Hsieh, H.-H.Protein homeostasis depends on the 26S proteasome, the most complex ATP-dependent protease in eukaryotic cells. The proteasome base subcomplex is responsible for mechanical substrate unfolding and translocation into an internal degradation chamber. It contains three non-ATPase subunits, Rpn1, Rpn2, and Rpn13, and a heterohexameric AAA+ motor with six distinct ATPases, Rpt1-Rpt6. Correct base assembly requires four dedicated chaperones that initially form the Hsm3 module (Hsm3-Rpt1-Rpt2-Rpn1), the Rpn14/Nas6 module (Rpn14-Rpt6-Nas6-Rpt3-Rpn2-Rpn13), and the Nas2 module (Nas2-Rpt5-Rpt4). However, the mechanisms underlying module assembly […]
- by Siebeneichler, B., Liu, X., Rodriguez Cruz, P. E., Naser, D., DelMistro, G., Steckner, J., Schaefer, A., Tran, N., Holyoak, T., Meiering, E. M.Protein aggregation is of broad importance in biotechnology and disease, yet the structural heterogeneity of cellular aggregates has confounded high-resolution structural analysis. Inclusion bodies (IBs) formed in Escherichia coli are an attractive, controllable system for unravelling the complexities of protein aggregation in a cellular context. Here, a multimodal analysis integrating residue-resolved quenched amide hydrogen-deuterium exchange (qHDX), proteolysis, FTIR, Congo red binding, and chemical denaturation is applied to IBs formed by proteins encompassing stable beta- and alpha- globular folds, a partially […]
- by Aguilar-Carrillo, A. B., Garduno-Valdovinos, S. A., Nava, G. M., Sanchez-Quezada, V., Madrigal-Perez, L. A.Polyphenols are compounds synthesized by plants as part of their chemical defense system to counteract biotic and abiotic stressors. These compounds share two key chemical characteristics: their aromatic groups make them insoluble in water, while hydroxy groups provide redox properties. These characteristics may explain how polyphenols interact with mitochondrial membranes (which are lipophilic) and participate in redox (electron scavenging) reactions of the electron transport chain, ultimately affecting ATP synthesis via oxidative phosphorylation. This interaction accounts for both the beneficial and […]
- by Dunge, A., Wehlander, G., Branden, G., Kack, H.Room temperature serial crystallography offers advantages over conventional cryo-crystallography, such as simplified crystal handling and the possibility to avoid potential artefacts associated with cryo-trapping. However, to be considered as an alternative for drug discovery, where compound availability may be limited and speed of structure delivery is a key factor, it suffers from several limitations. To address these challenges, we have optimized a serial crystallography workflow for ligand soaking, data collection and data processing, significantly reducing time and reagent consumption to […]
- by Hwang, I.-J., Kim, J., Patel, A., Zhang, L., Miller, J., Piletsky, S., Clift, C. L., Hisey, C. L., Kim, Y., Kim, M.Extracellular vesicles (EVs) carry molecular signatures of their originating cells and have thus emerged as promising biomarkers. However, their clinical utility remains limited due to their low abundance and the modest sensitivity of current EV detection methods in complex biological environments. Here, we present a quantum well defect functionalized carbon nanotube sensor coupled with integrin-recognition RGD tripeptide for EV detection in human plasma. Leveraging the abundance of integrins on EV surfaces, we targeted a5b1, aVb1, and aVb3 subtypes. The nanosensor […]
- by Ramu, M., Ghanem, L., Skeens, E., Bai, L., Lolis, E., Bennett, A. M., Lisi, G. P.Dual-specificity phosphatases (DUSPs) that inactivate mitogen-activated protein kinases (MAPKs) are called MAPK phosphatases (MKPs). The stress-responsive MKP, MKP5, dephosphorylates p38 MAPK and c-Jun NH2-terminal kinase (JNK). Previously, we identified an allosteric compound that binds to the site within the phosphatase domain of MKP5 that is critical for the binding and dephosphorylation of both p38 MAPK and JNK. The allosteric site, comprised of the 4-5 loop, is an essential region for transmitting MAPK binding to the catalytic site. Here, we examine […]
- by Sun, J., Yadav, R., Catmakas, T., Fisher, L., Fitzkee, N. C., Kessl, J.A series of critical interactions within the viral core between the viral RNA (vRNA) and HIV-1 Integrase (IN) has previously been reported. In these studies, contact points between vRNA and IN were identified using RNA-seq and MS-based protein foot-printing approaches. Several IN amino acids located in its C-terminal domain (CTD) were found to be essential for vRNA binding and their alanine substitution severely impacted the correct morphogenesis of the matured viral core. Here, we have extended these studies by performing […]
- by Faisst, K. D., Sinn, L. R., Lau, K., Szyrwiel, L., Rappsilber, J., Demichev, V.Mass spectrometry-based proteomics enables in-depth investigation of protein phosphorylation, quantifying tens of thousands of phosphosites per sample following phosphopeptide enrichment. However, critical information on phosphosite occupancy (stoichiometry) is typically lost during the phospho-enrichment process. Here, we introduce SILAC-Site, a SILAC-based fractionation-free and chemical labelling-free workflow for direct phosphosite stoichiometry evaluation using stable isotope labeling and phosphatase treatment. By acquiring treated or untreated peptides together with their heavy-labelled dephosphorylated counterparts within the same LC-MS runs, this approach provides internally controlled stoichiometry […]
- by Stange, A. D., Duncan, A. L.The insulin receptor samples multiple conformational states during ligand binding and activation, but the transient structural transitions connecting experimentally resolved receptor conformations remain poorly characterised. Here, we report a spontaneous opening event of insulin receptor site 1 observed during an unbiased molecular dynamics simulation initiated from the experimentally resolved singly insulin-bound IR1 receptor structure. The transition was characterised by separation of the L1 and FnIII-2 domains, rearrangement of site 1 contacts, and bending of the CT segment on the initially […]
- by Graff, S., Stransky, S., Kraz, I., Duraivelan, K., Poppel, A. J., Chatoff, A., Snyder, N. W., Garforth, S. J., Shechter, D., Maianti, J. P., Sidoli, S.Herein we uncover a relationship between histone succinylation and Jumonji (JmjC) domain-containing histone demethylases. We used quantitative proteomics and peptide pull-down assays to identify JmjC demethylases as candidate interactors with succinylated histone peptides. Succinyl-lysine peptides bind and inhibit the catalytic activity of JmjC demethylases in a dose-dependent manner. This includes KDM4D and KDM6B, which are responsible for the removal of the silencing marks H3K9me2/3 and H3K27me2/3. Supraphysiological sodium succinate treatment of HepG2/C3A cells increased the relative abundance of histone succinylation, […]
- by Sharma, C., Sengar, S., Sen, D., Sharma, V., Ghosh, S.Oligonucleotide therapeutics such as antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) enable sequence-specific gene silencing but rely on endogenous cellular machinery and often require extensive chemical modification for stability and efficacy. DNAzymes offer a mechanistically distinct alternative through intrinsic catalytic RNA cleavage; however, their therapeutic translation has been limited by nuclease susceptibility, structural constraints, and synthetic challenges. Here, we report the development of Circular Active Nano DNAzyme (CANDe), an enzymatically synthesized circular DNAzyme platform designed to enhance stability without […]
- by Wang, J., Ren, T., Zhang, Y., Yao, L., Yu, H., Wang, Z., Zhang, Y., Li, S., Liang, S., Li, J., Jiang, B., Han, J., Liu, G., Li, Q.Energy supply is fundamental to cardiac performance, yet the mechanisms by which the heart adapts to nutrient scarcity remain incompletely understood. Here, we identify acetate as a pivotal metabolic substrate that sustains cardiac contractile function during fasting-induced energy deficiency. Fasted mice reveal significant elevation of circulating acetate derived from hepatic fatty acid catabolism. Physiological concentrations of acetate alone can maintain ex vivo beating and electrical stability in Langendorff-perfused hearts. Primary mouse cardiomyocytes preferentially utilize acetate under energy-restricted conditions, which is […]
- by Figueiredo, D., Hattori, M., Wazawa, T., Nishino, K., Zwama, M., Nagai, T.CRISPR-Cas12a underlies powerful genome-editing and nucleic-acid detection technologies, yet its performance is limited by inefficient target engagement and low catalytic turnover, particularly at low target abundance and elevated temperatures. Here, we report a modular protein-engineering strategy to enhance Cas12a trans-activity by fusing the hyperthermophilic DNA-binding protein Sso7d to the N terminus of Lachnospiraceae bacterium ND2006 Cas12a (LbaCas12a). The resulting fusion enzyme shows a twofold improvement in detection sensitivity, a 4.6-fold increase in kcat, and substantially accelerated trans cleavage kinetics compared […]
- by Muallem, H., Zemer, A., Haim, Y., Rosengarten-Levin, M., Tsitrina, A., G. Noach, Y., Yoel, U., Baraghithy, S., Tsuneki, H., Wada, T., Sasaoka, T., Tam, J., Monsonego, A., Rudich, A.Weight cycling (WC), defined as weight gain, loss, and regain, is common in obesity, but its metabolic consequences remain unclear. We tested whether WC-aggravated glucose intolerance in obesity is age-dependent and linked to circadian disruption. Young (7w) and mid-aged (12m) mice underwent a 15-week dietary intervention: Lean and Obese mice fed normal chow (NC) and high-fat diet (HFD) throughout, respectively. WC mice undergone HFD-induced weight gain, NC-induced weight loss, and a second HFD-induced weight regain. Late-onset obese (LO) mice ate […]
- by Tsai, T.-H., Aghayev, M., Ilchenko, S., Sabir, U., McMullen, M., Ghaju, S., Chen, X. L., Zhang, G., Chung, W. C. J., Nagy, L. E., Kasumov, T.Alcohol-associated liver disease (ALD) is a leading cause of liver-related morbidity and mortality. Although various omics approaches have revealed early metabolic alterations, individual datasets provide limited mechanistic insight. Here, we integrated RNA sequencing with mass spectrometry-based analyses to quantify gene expression, protein abundance, proteome and acetylome dynamics, and metabolic fluxes in livers of alcohol-fed mice. This multi-layered approach revealed extensive metabolic rewiring characterized by suppressed mitochondrial energy metabolism and compensatory upregulation of glutathione (GSH) production, utilization, and recycling, establishing a […]
- by You, Y., Kim, H., Ushakumary, M., Gritsenko, M. A., Walukiewicz, H., Li, F., Xu, J., Diaz Ludovico, I., Dakup, P., QIAN, W.-J., Clair, G. C., Many, G., Mirmira, R., Webb-Robertson, B.-J., Wolfe, A., Rao, C., Sims, E. K., Sussel, L., Nakayasu, E. S.In type 1 diabetes (T1D), insulin-producing {beta} cells are destroyed by an autoimmune response driven by pro-inflammatory cytokines, including interferons. {beta}-cell cytokine signaling is mediated in part by post-translational modifications, such as phosphorylation and acetylation. However, the role of other post-translational modifications in {beta}-cell cytokine signaling represents an important knowledge gap. In the context of autoimmune diseases, lysine carbamylation has gained attention for its role in pathogenesis. Here, we investigate the role of carbamylation in T1D. We found that pancreatic […]
- by Sanchez, A., Pla, I., Peterson, K., White, V., Fisher, T. D., Hollas, M. A. R., Van Le, N. H., Su, T., Harrington, C. R., Cravedi, P., Assis, D. N., Barrios, P., Banea, T. E., Ladner, D. P., Forte, E., Lucky, M., Wilkins, J. T., Vaughan, D. E., Caldwell, M. A., McGee, J., Kelleher, N. L.Plasma proteomics has long sought to accessibly sense human biology, precisely detect disease states, and advance diagnostics through clinical translation. In recent years, companies with defined panels such as Olink and Somascan have entered the field to complement bottom-up mass spectrometry (BUP). This study leverages a novel mass spectrometry platform to capture targeted proteoform information lost by mainline antibody-, aptamer-, and BUP-driven workflows. The Plasma Proteoform Assay (PPA) uses Individual Ion Mass Spectrometry (I2MS) to resolve mixtures of intact proteins […]
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