- by Williams, J. D., Tesmer, V. M., Kannoly, S., Shibuya, H., Nandakumar, J.Telomeres are nucleoprotein structures at the ends of eukaryotic chromosomes that safeguard them from triggering inappropriate DNA damage signaling. POT1, a member of the mammalian shelterin complex, binds single-stranded (ss) telomeric DNA and blocks the activation of the ATR kinase-mediated DNA damage response at telomeres. Yet until recently, it was poorly understood how the double-stranded (ds)-ss telomeric junction was protected from DNA damage response factors. An initial study of the DNA-binding activity of human POT1 (hPOT1) using systematic evolution of […]
- by Modi, A., Toriki, E. S., Stieger, C. E., Lau, E. A., Song, C., Chew, A., Tsao, A., Nishikawa, K., McKenna, J., Nomura, D. K.Molecular glue degraders represent a powerful modality for targeting proteins that are refractory to traditional inhibition. However, rational design principles for molecular glue degraders remain poorly defined. Previously, we reported a chemistry-centric strategy to identify covalent degradative handles that, when appended to established ligands, convert non-degradative inhibitors into molecular glue degraders by engaging permissive E3 ligases. This effort identified a fumarate-based electrophilic handle that covalently modified the E3 ligase RNF126, enabling degradation of multiple protein targets when transplanted across diverse […]
- by Iradukunda, C., Salter, E. A., Uredi, D., Wang, X., Wierzbicki, A., Rameh, L. E.mTORC1 integrates growth factor and nutrient signals to regulate cellular metabolism, yet there are no metabolites known to directly regulate mTORC1 activity in cells. Cryo-EM studies revealed that inositol hexakisphosphate (IP6) associates with the FAT domain of mTOR, suggesting that inositol phosphates may directly modulate mTOR activity. We previously showed that higher-order inositol phosphates enhance mTORC1 kinase activity and stability in vitro. Here, we investigated whether inositol phosphate metabolism regulates mTORC1 signaling in pancreatic {beta}-cells. Suppression or acute inhibition of […]
- by Biswas, A., Saha, S., Maiti, T. K.The global industrialization and rapid urbanization elevated the risk of toxic pollutant exposure, which affects human health specially during pregnancy. Pregnant mothers are daily exposed to bisphenol-A (BPA), which is a common plastic leachate and a prominent toxic pollutant present in our environment. BPA act as an endocrine disrupting chemical (EDCs) by altering feto-placental homeostasis. This persistent and potent exposure of BPA during gestation can trigger placental damage affecting trophoblast cell function and survival. BPA even disrupts specific signalling cascades […]
- by Shaw, A. L., Doerr, S., Nyvall, H. G., Jenkins, M. L., Suresh, S., Yip, C. K., Hansen, S. D., Burke, J. E.Phosphatidylinositol 4-kinase alpha (PI4KA) is an essential lipid kinase that generates phosphatidylinositol 4-phosphate (PI4P) from phosphatidylinositol (PI) at the plasma membrane (PM). PI4P is a precursor for PIP2 and PIP3 lipid signalling, with PI4P serving a critical role in maintaining PM identity and asymmetry. Given the important roles of PI4KA in myriad processes, understanding how it is regulated is of immense importance. Here, we have identified that PI4KA can be phosphorylated in its dimerization domain (pY1154) and kinase domain (pY2090) […]
- by Buzuk, A., Ho, J. V., Marquez, M. D., Wang, B., Perlstein, D. L.The cytosolic iron-sulfur cluster assembly (CIA) pathway maturates essential nuclear and cytosolic Fe-S proteins required for genome maintenance and cellular metabolism. Nar1 (also called CIAO3 or IOP1) is a conserved Fe-S protein that connects the early and late steps of the CIA pathway, yet the molecular basis for its proposed function as a metallocluster carrier remains poorly defined. In particular, the interactions responsible for Nar1 recruitment to the CIA targeting complex (CTC) during cluster delivery remain unknown. Here, we define […]
- by Achat, Y., Prevost, M. S., Mechaly, A., Genera, M., Colcombet-Cazenave, B., Bezault, A., Winter, J.-M., Venien-Bryan, C., Raynal, B., Lafaye, P., England, P., Ayme, G., Bonomi, M., Prezeau, L., Wolff, N.Adhesion G protein-coupled receptors (aGPCRs) are involved in numerous physiological processes, including cell-cell and cell-matrix interactions, and are associated with several human diseases. ADGRV1 is a member of the aGPCR family and plays a significant role in the sensorineural systems. Mutations of ADGRV1 are linked to the Usher syndrome, a genetic disorder causing deafness and blindness in human. However, the molecular mechanisms that control the activity of ADGRV1 remain unclear. In this study, we present the high-resolution cryo-electron microscopy structure […]
- by Johnsson, K., Nasufovic, V., Pispek, A., Kuehn, S., Wilhelm, J., Bibrowski, M., Fischer, J., Koch, B., Kompa, J., Mao, R., Tarnawski, M., Hiblot, J.CLIP-tag is a self-labeling protein tag used for the specific fluorescence labeling of proteins. However, its low labeling speed and the poor cell permeability of its substrates result in low labeling efficiencies in live-cell applications. Here, we introduce a substrate optimized for live cell applications, as well as an engineered CLIP-tag variant, CLIP-tag2, which reacts with the new substrate almost 1000-fold faster than the original CLIP-tag-substrate pair. CLIP-tag2 fusion proteins can be specifically and efficiently fluorescently labeled in cells within […]
- by Guffick, C., Rincon Pabon, J. P., Griffiths, D., Inaba-Inoue, S., Beis, K., Politis, A.The structural study of membrane proteins has traditionally relied on detergent-based extraction from cellular membranes. Although native-like reconstitution approaches have advanced, fully understanding membrane protein dynamics requires examining them within their native membrane environment. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful method for probing structural dynamics in reconstituted systems, but the presence of the lipid bilayer introduces considerable complexity, limiting broader adoption under physiological conditions. Here, we present the first fully automated HDX-MS platform incorporating a two-stage delipidation workflow. […]
- by Wang, C., Ostergaard, O., Malero, R., Nagy-Davidescu, G., Eibauer, M., Olsen, J. V., Carazo, J. M., Plueckthun, A., Medalia, O.The structural and functional characteristics of membrane proteins can be influenced by the composition of the membrane. Consequently, native membranes are most relevant for the study of receptors and other membrane proteins. In this study, we investigated two types of cell-derived vesicles: natively shed extracellular vesicles (EVs) and mechanically derived vesicles (MVs). To this end, we utilized the human breast cancer cell line SKBR3, which strongly overexpresses the receptor HER2. We designed a protocol based on designed ankyrin repeat proteins […]
- by Chauhan, M., Ivanova, I., Sudnick, E. G., Steere, R. W., Tennant, J. R., Hensley, J. A., Arede, P., Jensen, G. M., Hatin, I., Namy, O., Bouloc, P., Carroll, R. K., Granneman, S.Methicillin-resistant Staphylococcus aureus (MRSA) is a formidable human pathogen responsible for life-threatening infections worldwide. Central to its pathogenic success is the tightly coordinated regulation of virulence factors, including the phenol soluble modulins (PSM), short amphipathic toxins that drive cytolysis, immune evasion and biofilm maturation. We previously identified the conserved non-coding RNA RsaE as a putative regulator of the psm operon, but the biological significance of this interaction remained unclear. Here we show that RsaE and endoribonuclease Y jointly regulate psm […]
- by Kazemzadeh, K., Faivre, B., Chobert, S.-C., Abby, S. S., Michaud, J., Dinh, T. A., Alexandre, C., Olivier, L., Fontecave, M., Fabien, P., Lombard, M., Pelosi, L.Polyisoprenoid quinones such as ubiquinone (UQ) play an essential role in cellular physiology, acting as membrane-bound electron and proton carriers in respiratory chains and other biological processes across all domains of life. In Escherichia coli, the canonical UQ biosynthesis pathway is well characterized. It involves twelve proteins (UbiA-UbiK and UbiX), most of which catalyzing one of the eight modifications of the aromatic ring derived from 4-hydroxybenzoic acid (4-HB), while others (UbiB, UbiJ, UbiK) act as accessory factors ensuring efficient UQ […]
- by Ness, M., Wendt, K., Peramuna, T., Tillery, D. I., Murray, J. E., Cichewicz, R. H., McCall, L.-I.Natural products are a rich source of bioactive molecules and undiscovered chemical scaffolds with significant potential for novel drug discovery. Among these, fungi are particularly promising, offering diverse metabolites and undiscovered structural motifs. Large, well-curated collections of crude extracts, or libraries, are central to fungal natural product discovery, serving as starting material for bioassay-guided isolation of new compounds. However, the systematic influence of fungal selection strategies, culturing methods, and environmental factors on chemical diversity remains underexplored. In this study, we […]
- by Grundler, J., Chertok, B., Nilam, A., Edmundson, A., Song, M., Newton, M., Scholfield, M. R., Padilla, A. M., Payton, N. M.mRNA-lipid nanoparticles (LNP) have proven their potential as a rapidly adaptable vaccine platform and promise to revolutionize numerous therapeutic areas. A major hurdle towards the widespread adoption of mRNA-LNP vaccines and therapeutics is their limited liquid shelf-life compared to more established modalities currently necessitating an ultralow temperature cold-chain to enable their distribution and storage. While ongoing efforts aim to improve liquid stability through chemical modification of mRNA and lipid components, complementary strategies that are broadly applicable across chemistries may further […]
- by Rahim, A., Zhan, X., Han, Q., O'Donnell, A., Jeong, A., Madugundu, G.-S., Pujari, S., Kruk, M., Luo, X., Li, L., Wu, T. P., Tretyakova, N. Y.N6-methyldeoxyadenosine (N6medA) is a recently identified endogenous DNA modification widely found in bacteria, plants, and eukaryotes. In mammals, N6medA has been implicated in brain function, immunity, and response to environmental stress, but its relevance to gene regulation and mammalian aging remains controversial due to its extremely low abundance (< 1 per 10 million adenines) and an uncertainty regarding its genomic origin. We have developed and validated an ultrasensitive isotope dilution nano liquid chromatography-nanospray ionization Orbitrap mass spectrometry methodology to quantify […]
- by Yu, J., Wang, J.-J., Li, H.-Z., Liu, Y.-L., Guo, Z.-Y.The newly identified signaling system comprising C-X-C motif chemokine ligand 17 (CXCL17) and G protein-coupled receptor 25 (GPR25) is involved in immune regulation and tumor development. However, the evolutionary origin of this pair has remained unclear because CXCL17 orthologs in lower vertebrates exhibit extreme sequence variation and cannot be identified through conventional homology-based searches. In this study, we identified seven possible CXCL17 orthologs in primitive cartilaginous fishes, including sharks and rays, using an integrated approach based on key amino acid […]
- by Risbud, M., Ramteke, P., Watson, B., Jagannath, S., Bell, S. E.Aging is one of the most important risk factors for Intervertebral disc degeneration, a major contributor to chronic low back and neck pain. Recently, we demonstrated a critical role for SIRT6, a nuclear NAD – dependent deacetylase and defatty acylase, in maintaining intervertebral disc health with aging. We therefore investigated whether pharmacological activation of SIRT6 improves disc health by examining the spinal phenotype of 24-month-old mice treated with the well-studied agonist MDL-800 for 6 months. Histological studies revealed healthy disc […]
- by Zhou, Y., Kang, Y., Liu, Y., Li, R., Wang, D., Yi, C., Li, Y., Zhang, Y., Tian, Z., Liu, J.Benzoylphenylurea (BPU) insecticides disrupt insect chitin formation, yet their molecular target is debated. Although resistance-associated mutations map to the Chitin Synthase (CHS) gene, direct inhibition of CHS has not been demonstrated. Given the structural similarity of BPUs to mammalian glycogen phosphorylase (GP) inhibitors, GP has been proposed as a potential target controlling chitin precursor flux. We characterized Plutella xylostella GP (PxGP) and found that the human GP inhibitor GPI potently inhibits both recombinant PxGP (IC50 = 2.96 nM) and native […]
- by Ye, X., Sokol, J., Kofoed, C., Dheer, A., Zhang, J., Muir, T. W.Chemistry-driven approaches that allow proteins to be manipulated in ways not permitted by standard genetics are indispensable in the basic and applied biomedical sciences. Of the various platform technologies in common use, those that exploit intein-mediated protein splicing have proven especially powerful owing to compatibility with both in vitro and in vivo applications. Here, we provide detailed biochemical characterization of the split intein, NrdJ-1. We demonstrate that the rapid splicing kinetics associated with this system are largely independent of splice […]
- by Anokye, R., Boadu, K. B., Boateng, K. O.The production of petroleum-based plastics used for packaging has led to significant environmental challenges in both aquatic and terrestrial ecosystems. Consequently, there is a growing need to explore viable alternatives to the usage of these conventional plastics. This study investigates the utilization of cellulose powder for producing of biodegradable plastics as a more sustainable substitute for petroleum-based materials. Bioplastic films were formulated with varying glycerol contents ranging from 0.5ml – 2.0ml. The glycerol served as a plasticizer to improve the […]
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