- by Henzeler, B., Hofmeister, O., Kögel, K., Qi, Y., Hagelskamp, F., Heiss, M., Schelter, F., Xu, F., Daumann, L. J., Carell, T., Kaiser, S., Schneider, S.AlkB homolog 1 (ALKBH1) is a member of the AlkB family of Fe(II) and -ketoglutarate (-KG)-dependent dioxygenases, known for its enzymatic activity on various nucleic acid substrates. Its known targets include N1-methyladenosine (m1A), N6-methyladenosine (m6A), N3-methylcytidine (m3C), and 5-methylcytosine (m5C) in RNA, as well as N6-methyladenine (N6-mA, 6mA) in DNA and the histone protein H2A. Moreover, dysregulation or dysfunction of ALKBH1 has been implicated in a broad spectrum of human diseases. In order to shed further light on the substrate […]
- by Sargent, R. A., Bradford, C., Hughes, L. M., Ta, N., Limpiado, M. A., Vattepu, R., Brungardt, J. G., Beck, M. R.Interdomain linkers in multidomain proteins influence spatial arrangement, flexibility, and cooperative binding, yet their functional roles remain underexplored. Palladin, an actin scaffold protein essential for cytoskeletal organization, contains tandem immunoglobulin domains (Ig3-4) separated by an unusually long (41-residue), arginine-rich, intrinsically disordered linker. While Ig3 mediates direct F-actin binding, the adjacent Ig4 domain and linker enhance binding and bundling, suggesting active roles beyond passive connection. Using targeted mutagenesis, actin co-sedimentation, bundling assays, and SAXS, we show that linker length, charge, and […]
- by Chen, H., Guzman, U., Olsen, J. V.Formalin-fixed paraffin-embedded (FFPE) samples are essential for clinical research and spatial proteomics (SP) but are technically challenging to analyze by liquid-chromatography coupled mass-spectrometry (LC-MS) due to losses during protein extraction, evaporation during decrosslinking and low-throughput sample preparation methods. Here, we present a streamlined micro-FFPE proteomics sample preparation protocol for laser-capture micro-dissection (LMD) that enables scalable and high-throughput processing in 96-well format, completing the entire process from tissue lysis to tryptic peptide mixtures on LC-MS-ready Evotips in just 2-hours. Our method […]
- by Santos, E. S., Granato, D. C., Carnielli, C. M., de Figueiredo, D., Trino, L. D., Patroni, F. M. S., Pauletti, B. A., Domingues, R. R., Sa, J., Normando, A. G. C., Daher, N., Kravchenko-Balasha, N., Debasis, P., Minghim, R., Kowalski, L. P., Santos-Silva, A. R., Lopes, M. A., Brandao, T. B., Prado-Ribeiro, A. C., Leme, A. F. P.Oral leukoplakias (OLs) are premalignant lesions that can progress into oral squamous cell carcinoma (OSCC). This study hypothesized that tear fluid, as a noninvasive biofluid, reflects proteomic alterations associated with malignant transformation. The tear proteome of 44 individuals, including healthy controls, OL/PVL (proliferative verrucous leukoplakia), and OSCC patients, was deeply profiled, revealing 828 protein groups clustered according to histopathological alterations. N-glycoproteome analysis identified immune-related proteins, while public RNA-seq integration indicated immune imbalance marked by increased B-cell and decreased macrophage signatures […]
- Discovery and chemical biology of CDK8 inhibitors reveals insights for kinase inhibitor development.by Le Bihan, Y. V., Foll-Josselin, B., Robert, T., Duez, J., Angevin, L., Simboeck, E., Prieto, S., Hodimont, E., Ferrer, J.-L., Krasinska, L., Ruchaud, S., Fisher, D.Protein kinase inhibitors are a key class of targeted cancer therapies, but finding compounds that show on-target efficacy with minimal toxicity remains challenging. CDK8 and CDK19 are paralogous kinases that regulate Mediator complex to promote transcriptional responses to extracellular signals and have also been reported to facilitate genome replication. Preclinical studies show that CDK8/19 inhibitors may have therapeutic benefit in several cancers, but whether effective inhibition of Mediator kinases is cytotoxic is debated. Here, we present a multi-modal approach for […]
- by Choudhary, C., Walter, J., Weisser, M., Masternak, M., WIld, T., Garcia-Martin, R., Liebner, T., Bragado-Nilsson, E., Narita, T., Zavogianni, N., Montoya, G.The ribosome is a universally conserved and essential protein complex, but its biogenesis in mammals is more complex than in single-celled eukaryotes. To explore this added complexity, we conducted a protein-protein interaction screen in human cells. This led to the identification of the eumetazoan-specific SPATA5-SPATA5L1-CINP-C1ORF109 (55LCC) complex as a key regulator of ribosome biogenesis. Structural analyses using cryo-EM and X-ray crystallography defined the architecture of 55LCC. Functional studies following acute depletion revealed that each component is essential for pre-60S maturation. […]
- by Lopez-Tavera, E., Stepnov, A. A., Ersdal, N. S., Barros-Reguera, M., Sandholm, R. M., La Rosa, S. L., Sorlie, M., Eijsink, V. G. H., Vaaje-Kolstad, G.Unspecific peroxygenases (UPOs, EC. 1.11.2.1) are promising biocatalysts for the oxyfunctionalization of organic molecules and the synthesis of industrially relevant compounds due to their vast repertoire of catalyzed reactions. To date, thousands of putative UPO genes have been identified in eukaryotic genomes, most of them in the Ascomycota and Basidiomycota phyla, and several UPOs have been characterized. Remarkably, no related enzymes have ever been reported in prokaryotic organisms. Here, we describe the discovery of a novel family of diverse bacterial […]
- by Kiri, T., Ram, J., Reis, N., Maniv, I., Ordureau, A., Glickman, M. H., Sulkshane, P.Mitochondria are essential organelles responsible for cellular energy production and metabolism. Hypoxia, a pathophysiological condition, impairs the electron transport chain, disrupts mitochondrial function, and produces harmful reactive oxygen species (ROS). Ubiquitin signaling regulates mitochondrial health through several mechanisms, including protein degradation and mitophagy. Here, we show that hypoxia-induced mitophagy occurs independently of ubiquitination. However, mitochondria are heavily ubiquitinated under hypoxic stress. A significant portion of these hypoxia-induced ubiquitin chains constitute a specific type: linear head-to-tail fusions (M1), which are known […]
- by Buchanan, J. A., Lavidis, O., Shields, N., Harrison, K., Nguyen, H. T., Payne, R. J., Passioura, T., Steain, M., Sunde, M.Receptor Interacting Protein Kinase 3 (RIPK3) is a central regulator of necroptosis and a key mediator of inflammatory signaling. Its function is orchestrated through RIP Homotypic Interaction Motif (RHIM)-dependent interactions with other RHIM-containing adapter proteins, forming amyloid-structured necrosomes that trigger kinase activation and subsequently lead to immunogenic cell lysis. Necroptosis eliminates infected or damaged cells and provokes a strong inflammatory response. Dysregulated necroptosis is implicated in chronic inflammatory diseases and associated with ischaemic injury. Despite separate structural elucidation of the […]
- by Yang, X., Taghavi, A., Akahori, Y., Pedrini, M., Ishii, T., Disney, M. D.Disease-associated RNAs are increasingly recognized as promising therapeutic targets for small-molecule intervention. While DNA-encoded libraries (DELs) have long been established for protein ligand discovery, recent studies have demonstrated their feasibility for identifying RNA-binding small molecules. To further advance RNA-targeted ligand discovery, a diverse, solid-phase DEL enriched in privileged RNA-binding scaffolds was constructed and applied to identify ligands of r(G4C2)exp, a toxic RNA repeat expansion implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). DEL selection outcomes were analyzed through […]
- by Michlmayr, H., Wiesenberger, G., Rehak, K., Funtak, K., Sopel, M., Malachova, A., Fruhmann, P., Weber, J., Krska, R., Dufresne, M., Berthiller, F., Adam, G.The Fusarium metabolite culmorin (CUL) frequently co-occurs with the mycotoxin deoxynivalenol (DON) on cereals. While DON is recognized as a major Fusarium virulence factor on plants, the function of CUL is still unclear. Herein, we show that CUL-deficient F. graminearum mutants created by CLM1 deletion are less aggressive on wheat than the wild-type, accompanied by increased DON-3-glucoside/DON ratios in infected wheat ears. In root elongation assays with wheat and Brachypodium distachyon, CUL had no effect alone but significantly increased the […]
- by Oeo-Santos, C., Knüpling, E., Davis, C., Meng, X., Maslen, S., Kunzelmann, S., D'Antuono, R., Olerinyova, A., Auchynnikava, T., Skehel, M., Schreiber, A.Autophagy is a complex intracellular degradation pathway that depends on the coordinated interplay between the core autophagy machinery and diverse membrane sources to drive the de novo formation of double-membrane vesicles, known as autophagosomes. Golgi-derived Atg9-containing vesicles are essential for this process, delivering membranes to the pre-autophagosomal structure (PAS). These vesicles contain the transmembrane proteins Atg9 and Atg27 and the peripheral membrane protein Atg23; however, the nature, function, and regulation of their interactions remain poorly understood. Here, we systematically dissect […]
- by Causemann, J., Schmidt, B., Granz, D., Mosler, T., Jung, M., Dikic, I., Rieger, H., Ernst, R.Cellular membranes are dynamic platforms whose composition and biophysical properties are surveyed by sensor proteins to maintain homeostasis. How these sensors convert weak physical cues into robust biochemical outputs remains unclear. Failure to preserve membrane homeostasis, however, results in cellular stress and organelle dysfunction. Here, we investigate the prototypical yeast lipid saturation sensor Mga2 to reveal how lipid-controlled rotational movements in the transmembrane region are decoded into robust transcriptional responses. Using a fully reconstituted in vitro ubiquitylation system with quantitative […]
- by Lin, J., Gerlits, O., Kneller, D. W., Weiss, K. L., Coates, L., Hix, M. A., Effah, S. Y., Kovalevsky, A., Walker, A. R., Wilson, M. A.Hydrogen bonds (H-bonds) are central to biomolecular structure and dynamics. Although H-bonds are typically characterized by well-defined proton positions, proton delocalization can play a key role in facilitating enzyme catalysis and allostery in some systems. Experimentally locating protons is difficult, hampering the study of the proton mobility in H-bonds. We used neutron crystallography and large quantum mechanics/molecular mechanics-Born Oppenheimer molecular dynamics (QM/MM-BOMD) simulations of human DJ-1 and its bacterial homolog YajL to validate atomic resolution X-ray crystallographic bond length analysis, […]
- by Medvedeva, A., Kolomeisky, K., Kolomeisky, A. B.Understanding how specific molecular substructures control chemical behavior is central to rational molecular design and the development of new materials. However, most current predictive models offer limited mechanistic resolution at the fragmental level. We present a novel physical-chemical computational framework, which is based on systematically perturbing a parent molecule, to quantify fragment-level contributions to both a specific mechanistic action and a broader functional outcome. To test our theoretical approach, we investigated local structural contributions to pyronaridine (PY), a clinically used […]
- by Mizuno, Y., Pan, S., Zhou, T., Kehoe, P. G., Feng Earley, Y.Alzheimers disease (AD) is a neurodegenerative disorder characterized by cognitive decline, synaptic dysfunction, and the accumulation of amyloid plaques and neurofibrillary tangles. While prior research has focused mainly on protein aggregation and neuroinflammation, emerging evidence suggests that ionic imbalances, particularly involving sodium (Na+) and potassium (K+), may contribute to AD progression. Na+ and K+ are critical for maintaining neuronal membrane potential, regulating action potential firing, and supporting neurotransmitter function. Although studies primarily focused on absolute Na+ concentrations, the Na+/K+ ratio […]
- by Xiao, L., Grzemska, M., Pi, X., Chen, L., Turner, M., Peddada, N., Calvache, S., Jia, J., Beutler, B., Wu, H., Nair-Gill, E.Phosphofurin acidic cluster sorting protein 1 (Pacs1) is a multidomain adaptor proposed to bind transmembrane cargo proteins to facilitate their intracellular trafficking. Pacs1 also forms a complex with WD-repeat protein 37 (Wdr37), which is essential for lymphocyte homeostasis. Despite numerous proposed binding partners, a validated structure for Pacs1-containing protein complexes is lacking. Here, we present the cryo-electron microscopy structure of the Pacs1-Wdr37 complex. Pacs1 binds Wdr37 through a conserved interface within its furin-binding region (FBR), the domain previously linked to […]
- by Kehl, A. J., Taylor-Kearney, L., Jaffe, A. L., Pereira, J. H., Lee, J., Hammel, M., Waldburger, L., Yeow, C., Alvarado, L. V., Adams, P. D., Banfield, J., Siegel, J. B., Prywes, N., Shih, P. M.Rubisco is the entry point of nearly all organic carbon into the biosphere and is present in all domains of life. Despite its global importance, biochemical studies of this enzyme superfamily have been limited to a relatively narrow set of subclades. Recent advances in metagenomics have dramatically reshaped our understanding of both microbial and rubisco diversity; however, biochemical characterization of these sequences has not kept pace with the exponential growth in sequence data. To better survey the functional and structural […]
- by Shatouri, M. M., Pirozzi, I., Soker, P. D., Ali, Z., Amirkhani, A., Haynes, P. A.Commercial feed formulations are increasingly being evaluated for their nutritional impacts on aquaculture species, yet the molecular consequences of commonly used commercial diets remain underexplored. This study investigated the effects of two commercial diets, diet A (higher land animal protein) and diet B (higher fish meal content), on protein expression in the brain, liver, and intestine of barramundi (Lates calcarifer). A 12-week feeding trial was conducted with controlled water quality, and proteomic profiling was performed using data-independent acquisition. Differential analysis […]
- by Hackney, C. M., Koch, T. L., Ryding, N. L., Rogalski, A., Chase, K., Giglio, M. L., Espino, S. S., Acyatan, Z. G., Watkins, M., Olivera, B. M., Safavi-Hemami, H., Teilum, K., Ellgaard, L.Marine cone snails produce a diverse array of bioactive peptides, known as conotoxins, in their venom. Given their high target potency and specificity, conotoxins are attractive compounds for the development of precision research tools and pharmacological agents. Here, we provide the first experimental characterization of a conotoxin from the MKAVA superfamily, conkazal-M1, from Conus magus. Using NMR spectroscopy, we show that conkazal-M1 adopts a fold characteristic of the Kazal-type protease inhibitor family, featuring a Glu residue at the inhibitory P1 […]
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