- by Weicksel, S. E., Thomas, C., Hall, E., Ray, S., Towle-Weicksel, J. B.DNA polymerases are important for maintaining genomic stability by protecting against mutagenic lesions caused by external and internal factors. If left unrepaired, DNA damage can lead to replication errors resulting in changes in DNA sequences that could impact peptide sequences and gene expression, as well as lead to chromosomal breaks. Highlighting the importance of DNA polymerase repair function, variant DNA polymerases and cofactors of DNA repair pathways have been identified in many different cancer types. Recently, variant forms of DNA […]
- by Ziti, A., Aksakal, O., Vitsupakorn, D., Rizkallah, P., Makolajeck, H., Platts, J., Menzies, G., Jones, D.The red fluorescent protein mCherry is one of the most utilised fluorescent proteins in biology. Here, we have changed the chromophore chemistry by converting the thioether group of M66 to a thiol group through mutation to cysteine. The new variant termed mCoral due to its orange fluorescence hue has similar brightness to mCherry but has improved resistance to hydrogen peroxide. The variant is also responsive to pH with a low and high pKa forms that have distinct spectral properties, which […]
- by Gupta, P., Sahu, R., Nandi, I., Setty, S. R. G., Das, S., Singh, M.The nucleocapsid (N) protein of SARS-CoV-2 is central to viral assembly and replication. It binds viral RNA to form a helical nucleocapsid and enabling genome packaging and release into host cells. Human hnRNPA1, one of the most abundant RNA-binding proteins in eukaryotes, regulates key aspects of RNA metabolism, including splicing, transcription, localisation, and transport. Here, we report a direct physical interaction between SARS-CoV-2 N protein and hnRNPA1 with moderate affinity (Kd ~ 0.18 M), primarily mediated through their intrinsically disordered […]
- by Chao, E. H. P., Largy, E., Kaiyum, Y. A., Nguyen, M.-D., Dauphin-Ducharme, P., Johnson, P. E., Mackereth, C. D.Aptamers are oligonucleotides that have been selected to bind a particular target. Despite the growing popularity of functional DNA aptamers, there remains limited knowledge of their binding mechanisms as few have been characterized at the atomic level. Here we use NMR spectroscopy to obtain structural details of RKEC1, a shortened version of a DNA aptamer previously reported to bind dopamine. We find that RKEC1 forms a compact structure upon ligand binding that lacks any Watson-Crick duplex regions or G-quadruplex core, […]
- by Nocker, C., Pecak, M., Nocker, T., Fahim, A., Susac, L., Tampe, R.ATP-binding cassette (ABC) transporters are molecular machines involved in diverse physiological processes, including antigen processing by TAP, a key component of adaptive immunity. TAP and its bacterial homolog TmrAB use ATP to translocate peptides across membranes, yet the precise mechanism linking ATP binding to substrate movement remains unclear. Here, we employ a single-molecule FRET sensor to visualize single translocation events by individual ABC transporters, overcoming the limitations of ensemble averaging. This approach reveals that substrate transport is driven by a […]
- by Bourgery, C., Zhang, L., Carper, D. L., Zhou, M., Sacci, R., Korey, M., Wang, L., Hemond, J., Cahill, J. F., Bocharova, V., Michener, J. K.Plastics such as polyamides (PAs) possess unique physicochemical properties that make them indispensable in modern society. However, their energy-intensive production and challenging end-of-life management highlight the urgent need for efficient recycling or remanufacturing solutions. Enzymatic depolymerization offers a promising route toward circular recycling, yet remains constrained by limited enzyme characterization, lack of validation under industrially relevant conditions, and overall performance. Here we optimized the reaction conditions for three recently discovered nylon-degrading enzymes. One of them, Nyl12, achieved product titers with […]
- by Broadrup, R. L., Schwartz, A. C., Smith, J., von Trentini, D., Snyder, N. W.Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a branched very long chain fatty acid (VLCFA) derived in humans from the diet and catabolized via peroxisomal metabolism. Catabolism of phytanic acid is important in humans as certain inborn errors of peroxisomal metabolism, including Refsum Disease, manifest when catabolism of diet derived phytanic acid is impaired. We developed a novel, linear, unified, unoptimized synthesis of all-racemic phytanic acid along with its 2,3,6,7,10,11,14,15-d8, 1,2-13C2, and 1,2-13C2-2,3,6,7,10,11,14,15-d8 stable isotope labeled analogs from commercially available materials including […]
- by Wang, Q., Zhao, Y., Zhao, S., Wu, A., Hao, L., Hong, T., Hong, L.Aptamers are single-stranded nucleic acids with ligand-binding capacity as cost-effective alternatives to antibodies. However, their utility is often undermined by the inherent conformational instability of single-stranded nucleic acids, which can compromise binding kinetics and final affinity. To overcome this critical limitation, we synthesized a novel primary library incorporating multiple inlaid stem-loop structures to enhance molecular stability and rigidity. This structural constraint dramatically accelerated the selection process, allowing us to isolate aptamer candidates with superior affinity in just three rounds of […]
- by Rosa, R. S., Mendonca, A. P. M., Oliveira, M. P., Carvalho, D. B., Vazquez, L., Oliveira, M.The fruit fly Drosophila melanogaster is a valuable model for studying cellular and metabolic processes conserved with mammals. Using its embryonic Schneider S2 cell line, commonly used for heterologous protein expression, we investigated how culture duration and slight temperature changes affect mitochondrial physiology via high-resolution respirometry. We assessed five mitochondrial bioenergetic parameters and two coupling parameters at early and late growth phases, and at two temperatures (26{degrees}C and 28{degrees}C). We observed that a moderate temperature increase coupled with late growth […]
- by Kornilov, D., Bukhdruker, S., Astashkin, R., Tsybrov, F., Shevtsov, M., Bukhalovich, S., Ivashchenko, S., Mikhailov, A., Zinovev, E., Golybev, V., Ryzhykau, Y., Vlasov, A., Rulev, M., Borshchevskiy, V., Kuzmichev, P., Bamberg, E., Gordeliy, V.Heliorhodopsins (HeRs), the third rhodopsin family, are characterized by inverted membrane topology and confinement to monoderm organisms, yet their biological meaning has so far remained a mystery. We report the first crystal structure of a eukaryotic HeR, supported by structural modeling and comparative analyses across all domains of life. A conserved carotenoid-binding site, reminiscent of secondary antennae in some microbial rhodopsins, is identified and found to be common among HeRs. We show that inverted topology allows recruitment of exogenous xanthophylls, […]
- by Cascarina, S. M., Paul, K. R., Ford, L. L., Ross, E. D.Intrinsically disordered regions (IDRs) play important molecular roles in cells even though they do not adopt a stable structure. Relative to structured regions, IDRs have skewed amino acid compositions favoring polar and charged amino acids. This feature is a major contributor to the biophysical behavior and in vivo activity of IDRs, but the relationship between composition and activity depends strongly on which amino acids are enriched within the IDRs. Here, we present a new search algorithm, MatchIDR, that takes as […]
- by Georgiou, I., O'Neill, S., Robinson, C., Cunningham, F., O'Byrne, S. N., Gray, D., Scott, D. E., Gilbert, I. H.Different biochemical assays yield different rates of false positives than others either due to the nature of the enzyme, the technology associated with the assay, or properties of the compounds being screened. Ensuring that the right counter-screens are in place to identify false positives without wasting time and resources on them is of great importance. Herein we describe the results of a high throughput screen (HTS) against non-structural protein 3 (nsp3) protease PLPro, which resulted exclusively in false-positive hits. By […]
- by Kandanur, S. P. S., Dalhus, B., Emsley, J., Kanse, S. M.Factor VII activating protease (FSAP) is a circulating serine protease involved in stroke pathophysiology. This effect is likely mediated by the cleavage of substrates such as fibrinogen and extracellular histones. Our goal was to modify FSAP properties through mutagenesis to explore its different functions. Therefore, we expressed the serine protease domain (SPD) of FSAP with mutations and measured the cleavage of chromogenic substrate (S-2288), pro-urokinase (uPA), histones, and fibrinogen, as well as their inhibition by aprotinin, tissue factor pathway inhibitor […]
- by Heher, P., Pfeiffer-Vogl, J., Ganassi, M., Fulea, R.-C., McGuire, L., Engquist, E. N., Tyszkiewicz, M., Prueller, J., Grillari, J., Tasca, G., Banerji, C. R., Zammit, P. S.Facioscapulohumeral muscular dystrophy (FSHD) is driven by DUX4-induced toxicity, yet the pathomechanisms remain unclear. Here, we identify persistent transcriptional suppression of the mitochondrial respiratory chain and metabolic rewiring in FSHD muscle biopsies and myotubes. Using DUX4-inducible human myogenic cells, we show that DUX4 target gene activation is accompanied by mitochondrial function impairment and Caspase 9-mediated apoptosis. Reverse electron transfer (RET) at complex I is the dominant oxidative stress generating mechanism in FSHD muscle cells. RET-driven mitochondrial reactive oxygen species (mitoROS) […]
- by Chang, V., Lian, I., Mahoney, K. E., Romano, J., Afshari, A. R., Chen, R. J., Chen, X., Fineide, F., Khan, A. Z., Utheim, T. P., Karlsson, N. G., Laurie, G. W., Malaker, S. A.Galectin-3 (Gal-3) is a carbohydrate-binding protein which plays crucial roles in inflammation, immune response, cell migration, autophagy, and signaling. At the ocular surface, Gal-3 is also known to crosslink transmembrane mucins across the epithelial cell glycocalyx, forming lattice structures important for barrier function. However, the biological role of Gal-3 in circulating tear fluid remains largely unexplored. Similarly, whether Gal-3 engages extracellular glycoproteins in tears to affect downstream biological processes has yet to be investigated. As increased Gal-3 levels in tears […]
- by Cho, H., Olsen, J. V.Glucocorticoids (GCs) are steroid hormones that bind to glucocorticoid receptor (GR) as ligands to initiate systemic anti-inflammatory effects. GCs are commonly administered alongside chemotherapy to reduce treatment-related side effects in breast cancer patients. However, GC administration has been shown to promote metastasis in breast cancer. In this study, we used quantitative mass spectrometry-based approaches to analyze proteome and phosphoproteome of three breast cancer cell lines following GC treatments. By comparing MCF7, MDA-MB-231, and MDA-MB-436 cells, we suggest that the level […]
- by Sneddon, M. S., Tsou, C.-J., Fu, X., Shelat, A. A., Pomerantz, W. C. K.Epigenetic reader proteins, such as bromo-domains, are often associated with diseases such as cancer and inflammation. BET bromodomain inhibitors have been studied extensively; however, non-BET bromodomains are understudied. Moreover, available high-throughput biological assays to assess inhibitors are limited. One non-BET bromodomain-containing protein, BPTF, has a recently reported inhibitor, BZ1, with an in vitro affinity of 6.3 nM. Additionally, BZ1 is known to be non-selective towards other class I bromodomains PCAF, GCN5, and CECR2. Here, we use a BZ1 analog, BZ1-THQ, […]
- by Arendt, D., Tamilarasan, S., Golbik, R. P., Thondorf, I., Bender, J., Lilie, H., Gursinsky, T., Parthier, C., Schmidt, C., Stubbs, M. T., Behrens, S.-E.Viral suppressors of RNA silencing (RNAi) expressed by plant viruses, VSRs, are exceptional proteins. Although not conserved, even within virus families, most VSRs bind small interfering RNAs, siRNAs, thereby blocking antiviral RNAi. Turnip crinkle virus, a member of the Tombusviridae family, encodes a VSR, TCV P38, which also forms the viral capsid. Biochemical studies of the purified protein revealed that the TCV P38 VSR functions as a metastable dimer that binds double-stranded (ds) RNA with high affinity via an induced-fit […]
- by Cingolani, G., Li, F., Bellis, N., Lokareddy, R. K., Hou, C.-F. D., Yang, R., Branston, S., Kovach, Z., Geiger, R., Soriaga, A., Sim, L., Kyme, P., Birx, D., Lemire, S.Bacteriophages of the Pakpunavirus genus exhibit broad host range and potent bacteriolytic activity, making them promising candidates for clinical use. Here, we present a structural atlas of the therapeutic phage Pakpunavirus P7-1, a component of a phage cocktail targeting Pseudomonas aeruginosa that has undergone Phase 1/2 clinical trials. We determined the near-atomic structure of the extended virion and obtained a medium-resolution reconstruction of the contracted tail. Atomic models were built for 20 structural proteins comprising the icosahedral capsid, neck, contractile […]
- by Palumbo, R. M., Kasaju, M., Hershey, S. C., McCann, M. E., Woon, Z. H., Heisler, D. B., Mihailescu, M. R.Multiple sclerosis (MS) is a fatal neurodegenerative disease that progresses by eroding the myelin sheath and exposing the neuron, leading to neuronal degradation and death. While MS remains without an effective treatment or cure, studies have identified genes that are dysregulated in MS patients and predicted to be involved with disease progression. These genes are primarily involved in controlling DNA methylation: a process required for regulating gene expression, which is critical for cellular health. Having identified potential genetic risk factors, […]
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