- by Kuehn, T., Tumova, S., Zacharewski, N., Averdung, P., Berdel, B., Kellner, K.-H., Pusch, S., Jindra, M., Opitz, C. A., Prentzell, M. T.The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that enables cellular adaptation to environmental, nutritional and metabolic cues. Upon ligand binding, AHR translocates to the nucleus, heterodimerizes with the AHR nuclear translocator (ARNT) and regulates gene expression. Current approaches to measure AHR activity rely on transcriptional readouts, which vary depending on cell type and ligand. Here, we introduce two complementary protein-protein interaction-based assays that detect AHR activation by monitoring AHR-ARNT complex formation. Split-luciferase (NanoBiT) and bimolecular fluorescence complementation […]
- by Zanon, P. R. A., Shashikadze, B., Winkler, D., Scheller, I., Bednarz, A., Bartoschek, D., Machata, S., Graef, T., Ohmayer, U., Schwalb, B., Steger, M., Daub, H.Molecular glue degraders (MGDs) are proximity-inducing molecules that promote the destruction of disease-causing proteins by stabilizing novel interfaces between E3 ubiquitin ligases and target proteins. The rational design of MGDs remains exceptionally challenging, historically relying on serendipitous discoveries. Here, we deployed a high-throughput, mass spectrometry (MS)-based screen evaluating thousands of cereblon (CRBN)-directed compounds to expedite the identification of novel neosubstrates. This workflow led to the discovery of NE26394, a first-in-class MGD that selectively eliminates cyclin-dependent kinase 4 (CDK4), a critical […]
- Nucleotide-driven KaiC dynamics coordinate the core properties of the cyanobacterial circadian clockby Ito-Miwa, K., Muranaka, T., Kondo, T., Terauchi, K.Circadian clocks generate stable ~24-h rhythms with a defined period, temperature compensation, and entrainment to external cues that set phase. However, the molecular reactions that generate these features are not fully understood. In cyanobacteria, timekeeping is driven by the hexameric ATPase KaiC, which consists of two homologous domains, CI and CII, and whose enzymatic turnover underlies rhythmic phosphorylation. Here we identify ADP release as the rate-limiting step in the KaiC ATPase cycle and demonstrate that the KaiC ATPase nucleotide cycle […]
- by Pereira-Antonio, A. C., Oliveira, F. G. d. C., Costa-Lima, M. M., Coelho, A. F., Rodrigues, E. M., Franco, G. R., de Barros, M. H., Bleicher, L., Tahara, E. B.Protein homeostasis (i.e., proteostasis) is the biological process by which the qualitative and quantitative balance of the proteome is conducted, either by preserving functionally relevant proteins or by degrading unnecessary ones. Stress conditions can modulate cellular proteostasis in order to promote cytoprotection and preserve the viability of living organisms. Among the cellular pathways already described that can play an important role in preserving biological functions by modulating proteostasis are the heat shock response and the ribosome quality control pathways. In […]
- by Scherlo, M., Wippermann, E., Fuertges, T., Kuenne, R., Yelboga, A., Ruetten, F., Boeckmann, M., Hoeweler, U., Rudack, T.Molecular interactions govern cellular function, making them essential to discover biomolecular mechanisms by unravelling structure-function relationships. The rapid growth of AI-based prediction, experimental determination, and molecular dynamics simulations generates structural data at an unprecedented scale. However, structural information is typically represented as Cartesian coordinates, leaving chemical interactions and conformational relationships largely implicit. We introduce a high-throughput framework transforming structural geometry into a standardized, compact contact space. Moving beyond simple distance cutoffs, it provides a chemically and geometrically informed representation of […]
- by Torii, K., Gerasimaite, R., Lukinavicius, G.Inorganic polyphosphate (polyP) is a ubiquitous phosphate biopolymer involved in diverse cellular processes. Despite its significance, selective detection of polyP remains challenging because of its simple and highly charged structure. Here, we report a near-infrared (NIR) fluorogenic turn-on chemosensor for selective polyP detection and imaging, SiX-DPA-Zn. The probe combines a silicon-xanthene (SiX) fluorophore with a zinc(II)-coordinated 2,2'-dipicolylamine (DPA-Zn2+) recognition unit and shows more than 100-fold selectivity for inorganic polyP over ADP and ATP. SiX-DPA-Zn enables quantitative detection of polyP at […]
- by Towler, A. G., Wang, F., Bi, Y., Bandura, L. J., Zhu, Y., Zhu, J., Perciaccante, A. J., Aballo, T. J., Ji, Q. C., Jin, L., Buck, W., Phillips, L., Kadoya, K., Schnapp, L. M., He, Y., Tian, Y., Ge, Y.The lung extracellular matrix (ECM) governs tissue architecture, mechanics, and function, yet how it remodels with age across sex and anatomical regions remains poorly understood. Here, we performed a systematic multi-factor proteomic analysis of rat lungs to define age-, sex-, and region-dependent remodeling across the tissue landscape. Age emerged as the dominant source of variation, with a conserved aging signature modified by region- and sex-specific effects. Young lungs showed coordinated ECM assembly, balanced proteolysis, and active biosynthetic programs consistent with […]
- by Estevez-Gallego, J., Filipcik, P., Munoz-Hernandez, H., Matinyan, S., Wieczorek, M., Steinmetz, M. O.Microtubules are cytoskeletal filaments that dynamically grow and shrink to support vital biological functions. Their behavior is regulated by GTP hydrolysis in tubulin incorporated into the lattice, but the underlying chemical transitions and their impact on microtubule stability remain unclear. Using cryo-electron microscopy, we resolved microtubule structures at 1.9 – 2.2 [A] in four different nucleotide states, revealing the roles of amino acids, ions, and hundreds of water molecules in the hydrolysis process. We found that compaction of the GTP-bound […]
- by Clement, J., Lkhagvajargal, T., Hoare, B. L., Myint, T., Fox, D. R., Wang, C., Knott, G. J., Bathgate, R. A., Grinter, R.Relaxin family peptide receptor 1 (RXFP1) is a multi-domain GPCR with compelling therapeutic potential, yet uncertainty surrounding the mechanism of its activation by the hormone H2 relaxin has hindered the development of selective modulators. Here, we combine deep learning based structural modelling with de novo protein design to overcome this barrier. We generate a high-confidence structural model of the RXFP1-relaxin complex that is strongly supported by existing biochemical and functional evidence. This model reveals that relaxin engagement stabilises the RXFP1 […]
- by Nguyen, S., Pinner, I., Wang, C. R., Pukala, T. L., Jovcevski, B., Bruning, J. B.Infections caused by the opportunistic fungal pathogen Aspergillus fumigatus pose a serious public health system burden. The inherent limitations in existing antifungal drugs in conjunction with a rising emergence of antifungal resistance emphasizes an urgent need to identify and target alternative pathways crucial to survival and virulence. Targeting the fungal mannitol biosynthesis enzymes provides a promising avenue in the development of new antifungals due to the multifaceted roles mannitol fulfils in the fungal life cycle. However, a distinct lack of […]
- by Mureddu, L. G., Brooksbank, E. J., Vuister, G. W., Muskett, F. W.Nuclear Magnetic Resonance (NMR) relaxation experiments provide a powerful residue-resolved access to biomolecular dynamics across a wide range of timescales. Unfortunately, the quantitative analysis of the relaxation data remains distributed across specialised and often disconnected tools. Here, we present CcpNmr AnalysisDynamics, the latest addition to the CcpNmr Analysis program suite, providing an integrated framework for relaxation analysis, exchange-aware interpretation and dynamical modelling. The platform unifies relaxation-rate extraction, diagnostic validation, model-based analysis and structural visualisation within reproducible workflows, while supporting future […]
- by Shukla, A., Busch, M., Haener, M., Classen, A.-K., Jessen, H.Inositol phosphates (InsPs) and inositol pyrophosphates (PP-InsPs) are key regulators of cellular signaling and metabolism, yet developmental changes in the InsP/PP-InsP network in eukaryotic systems remain poorly understood. Here, we combine isomer-resolved capillary electrophoresis-mass spectrometry (CE-MS) with genetic perturbations to characterize InsP metabolism during the Drosophila melanogaster development. Our analyses reveal extensive developmental reprogramming of the InsP pathway, with early developmental stages exhibiting broad lower-order isomer diversity that progressively narrows into a more restricted metabolic state enriched in Ins(1,3,4,5,6)P5, InsP6, […]
- by Clifton, B. E., Akdavletov, B., Jain, P., Laurino, P.Post-translational modifications usually occur under enzymatic control as a mechanism for regulation of protein activity or localization, but can also occur spontaneously during protein aging and degradation. In contrast, there are few examples of spontaneous post-translational modifications with a significant role in the stability or biochemical function of a bacterial protein. Here we show that a spontaneous post-translational modification is structurally encoded and functionally important in a bacterial solute-binding protein. We show that the solute-binding protein SAR11_0655 from the abundant […]
- by Correa-Olivares, A., Lahera Champagne, A. d. l. C., Bertadillo-Jilote, A. D., Lira-de Leon, K. I., Garcia-Gutierrez, D. G., Nava, G. M., Sanchez-Quezada, V., Madrigal-Perez, L. A.Cancer, one of the worlds leading causes of death, is characterized by a complex metabolic reprogramming that features the Warburg effect as one of its hallmarks. The Warburg effect involves increased glucose and amino acid metabolism, which promotes tumor proliferation and progression. Although cancer has historically been attributed to genetic mutations, recent studies suggest a possible metabolic origin. However, a key characteristic of cancer cells is their greater adaptability than normal cells, as evidenced by their resistance to chemotherapy, which […]
- by Bushhouse, D. Z., Fu, J., Lucks, J. B.Riboswitches are compact regulatory RNAs that modulate gene expression by switching between mutually exclusive structures in response to binding diverse signalling molecules. Riboswitches that regulate transcription have been shown to function through sequence overlap between their ligand binding aptamer domain and their regulatory expression platform, a constraint that limits their engineering for biotechnological applications. Here we describe a new, modular, transcriptional riboswitch architecture, termed "tug-of-war" (TOW), that removes this constraint, based on the discovery that ligand-stabilized RNA structures positioned immediately […]
- by Abdella, R. H., Belica, C. A., Chen, Y., Brown, W. L., Carpenter, M. A., Ibrahim, M. A., de la Pena Avalos, B., Mullally, C. D., York, A. J., Harris, R. S., Aihara, H.Human APOBEC3B (A3B) restricts virus infections by catalyzing the deamination of cytosines to uracils in single-stranded DNA. A3B also contributes to mutagenesis and genome instability in cancer cells, driving tumor evolution and detrimental outcomes including therapy resistance and metastasis. A3B comprises tandem globular deaminase domains, with a multifunctional amino-terminal domain (NTD) and a catalytically active carboxy-terminal domain (CTD). Although individual domain structures have been studied, the structure of full-length A3B has remained elusive. Here, we report the cryoEM structure of […]
- by Khal, S. K., Linhart, N. A., Jain, S., Rosario Acevedo, G., Boyce, M.Glycosylation depends on tightly regulated pools of nucleotide-sugars (NS), yet the mechanisms controlling mammalian NS homeostasis and their downstream effects on glycoprotein biosynthesis remain poorly understood. UDP-galactose 4'-epimerase (GALE) catalyzes the reversible interconversion of UDP-galactose/UDP-glucose and UDP-N-acetylgalactosamine/UDP-N-acetylglucosamine, making it a central regulator of glycan precursor pools and an excellent model enzyme for studying NS metabolism. Here, we report the discovery of a cell-active small molecule inhibitor of human GALE through a high-throughput chemical screening strategy. Using a coupled luminescence-based assay, […]
- by Xin, Y., Qiu, X., Urner, L., Duerr, K., Liko, I., Robinson, C. V.Melanocortin 2 receptor (MC2R) is a G protein-coupled receptor (GPCR) for adrenocorticotropic hormone (ACTH), and its trafficking and signalling are associated with the melanocortin receptor accessory protein (MRAP). Mutations in either MC2R or MRAP disrupt this signalling and cause familial glucocorticoid deficiency. Here, we combine native mass spectrometry (MS) and hydrogen deuterium exchange mass spectrometry (HDX MS) to uncover how MRAP association and post-translational modification status shape the conformations of MC2R. Using native MS, we demonstrate that MC2R associates with […]
- by Frongia Mancini, D., Alabed, H. B. R., Pellegrino, R. M.LC/MS-based food lipidomics provides detailed information on intact lipid species, but the resulting datasets are often difficult to translate into concepts directly useful for food quality, processing, nutritional profiling and authenticity assessment. Here, we present Lipid Food Profile (LFP), a module of the LipidOne platform designed to convert annotated LC/MS lipidomics data into interpretable food-relevant lipid indices. LFP applies an in silico hydrolysis strategy to reconstruct acyl, alkyl and alkenyl chains from intact lipid species while preserving their lipid-class origin. […]
- by Lang Harman, R. M., Blackstone, H. G., Reynes, J.-P., Parviainen, A., Figueredo, D., Nochebuena, J., Mori, S.Natural product (NPs) and their derivatives are a major source of small-molecule drugs, and the building blocks of these NPs are often amino acids. These include both proteinogenic and nonproteinogenic amino acids (NPAAs), the latter of which expand the structural diversity of NPs. Homologation, or the addition of a methylene group to the amino acid side chain, is one modification that generates NPAAs. If the natural homologation pathway can be characterized and engineered, it could be used to diversify NPs. […]
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