- by Komatsu, T., Minoda, M., Uchida, T., Hata, M., Kanai, S., Hiraide, H., Kagami, Y., Honda, K., Urano, Y.Carboxypeptidases play diverse roles in physiological and pathological processes, yet comprehensive analysis of their activities in complex biological samples remains challenging. Here we report a solid-phase synthesis strategy for fluorogenic ProTide-based probes that enables systematic profiling of carboxypeptidase activities based on defined C-terminal amino acid motifs. By modular synthesis of dipeptide-fluorophore conjugates, we generated a focused probe set that revealed distinct substrate preferences among carboxypeptidases, including carboxypeptidase A and B family enzymes. Integration of these probes with a single-molecule enzyme […]
- by Böhm, M., Land, H.Carbon monoxide dehydrogenases (CODHs) are metalloenzymes central to microbial CO metabolism and CO2 fixation. We report the heterologous production and characterisation of Clostridium pasteurianum BC1 CODH-III (CpBC1CODH-III), from the phylogenetic clade E, co-expressed with its maturation machinery CooCTJ. CpBC1CODH-III shows moderate CO oxidation (150 U/mg) and CO2 reduction (0.568 U/mg) activities. Electron paramagnetic resonance (EPR) spectroscopy under varying redox conditions identified a rhombic signal at g {approx} 2.0, characteristic of reduced B-clusters, and a C-clusters at different stages (g {approx} […]
- by Dirvelyte-Valauske, E., Mazerimas, M., Pavliukeviciene, B., Daugelaviciene, N., Kutanovas, S., Kao, C.-Y., Chen, Y.-T., Neniskyte, U., Budvytyte, R.Efficient intracellular delivery of nucleic acids, proteins, and other biomolecules is critical to advancing therapeutic strategies and genome-editing technologies. Lipid nanoparticles (LNPs) have emerged as highly promising delivery vehicles owing to their self-assembly properties, biocompatibility, and capacity to encapsulate large molecular cargos. Their biological performance is determined largely by lipid composition, which influences particle stability, cellular uptake, membrane fusion, and intracellular trafficking. In this study, we designed and optimized LNP formulations inspired by the lipid architecture of enveloped viruses. Four […]
- by Duong, M. T. H., Parviainen, T. A. O., Thiruvaiyaru, A., Ahola, T., Heiskanen, J. P., Lehtiö, L.The chikungunya virus (CHIKV) outbreak imposes a significant burden on healthcare systems and raises an urgent need for effective antiviral therapies. So far there are no specific drugs against CHIKV. A CHIKV macrodomain is critical for virulence and counteracts the host immune response, representing a promising antiviral drug target. Here, we describe small molecule inhibitors targeting the CHIKV macrodomain. Compound 1 (MDOLL-0273) was identified through a high-throughput screening using a fluorescence resonance energy transfer (FRET)-based assay, and its inhibitory activity […]
- by Capra, N., Bourgery, C., Parks, J. M., Carper, D. L., Cahill, J. F., Michener, J. K., Meilleur, F.Enzymatic degradation of synthetic polymers has attracted broad interest because it offers environmental and manufacturing advantages compared to traditional mechanical and chemical breakdown approaches. Enzymes are highly specific and reaction conditions are generally aqueous and require low pressure and temperature, resulting in lower energy consumption and lower chemical waste production. Here we report the biochemical and structural characterization of three newly discovered enzymes capable of Nylon hydrolysis: Nyl10, Nyl12 and Nyl50. Using solution characterization techniques, we found that the enzymes […]
- by Barreto, L. V., Lourenco, E. M. G., da Silva, E. B., de Godoy, M. O., Martins, L. C., Laureano de Souza, M., Almeida, R. G., Cunha, V. L. S., Pires, M. C., Lavorato, S. N., de Souza, T. B., Bretas, A. C. O., Ottoni, F. M., Junior, E. N. S., Oliva, G., Alves, R. J., de Oliveira, R. B., Guido, R. V. C., Ferreira, R. S.Despite the development of vaccines and antivirals, coronavirus disease 2019 (COVID-19) continues to affect populations worldwide. Given the high mutation rate of the SARS-CoV-2 virus and reports of drug resistance, there is a continued need for new therapeutic options. SARS-CoV-2 main protease (Mpro) is essential for viral replication and is a conserved target among coronaviruses. Most known Mpro inhibitors target the active site, although allosteric sites have already been identified. In this study, we conducted a virtual screening of 2,060 […]
- by Plekhova, V., Van de Velde, N., VandenBerghe, A., Diana Di Mavungu, J., Vanhaecke, L.Ambient metabolomics techniques such as laser-assisted rapid evaporative ionization mass spectrometry (LA-REIMS) enable fast, preparation-free fingerprinting of biological samples but are inherently limited by spectral congestion in the absence of chromatographic separation. While ion mobility spectrometry provides additional gas-phase separation, maintaining ion transmission under the transient signals characteristic of laser desorption remains analytically challenging. Here, we define operating conditions for cyclic traveling-wave ion mobility spectrometry (cIMS) that preserve transmission under LA-REIMS duty-cycle constraints and systematically evaluate how mobility integration reshapes […]
- by Tutol, J., Pathiranage, V., Walker, A. R. C., Dodani, S. C.Chloride transport across cellular membranes is fundamental to physiology. Yet, this dynamic process remains difficult to capture with existing methods that rely on electrophysiology or indirect iodide-quenching assays, leaving real-time imaging of chloride transport a largely unexplored frontier. To address this gap, here, we upgrade our first-generation fluorescent protein indicator ChlorON-1 into ChlorON-1-PRO through targeted mutagenesis of an evolutionarily conserved gatepost residue. A single mutation (C139N) preserves the turn-on response (13.9-fold) while boosting affinity (Kd = 47.4 mM) and bound-state […]
- by MESNAGE, S., Yue, Z., Alrafaie, A., Robertson, M., Smith, E., Evans, C., Jinquan, L., Rafferty, J., Stafford, G.Bacteriophages rely on breaching the bacterial cell wall as the first step of infection. We characterise ORF11, a putative tail-spike lysin from the 19 kbp Minhovirus SHEF14, a podovirus infecting Enterococcus faecium. Bioinformatic analyses indicate that ORF11 comprises four domains: a predicted glycosyl hydrolase (D1) a cysteine, histidine-dependent amidohydrolases/peptidases (CHAP, D4), separated by a helical linker (D2) and a CHAP like domain (D3). This modular organisation is conserved among Enterococcus minhoviruses but differs markedly from analogous proteins in Copernicusvirus phage […]
- by Monge-Loria, M., Brady, C., Wu, H., Aron, A., Garg, N.Iron is an essential component of cellular biology. Thus, iron's low bioavailability is a key evolutionary pressure guiding microbial dynamics in the marine environment. Among marine bacteria, Microbulbifer is an underexplored and functionally versatile bacterial genus, which is commonly associated with sponges, algae, corals and sediments. Previously, genome analyses have revealed that Microbulbifer spp. can degrade polymers and synthesize natural products. Despite their recognized potential to produce secondary metabolites, siderophores are yet to be identified in Microbulbifer , and their […]
- by McDonald, I., Wilms, J., Cardi, N., Engstrom, A., Miao, J., Willbold, D., Lin, Y.-S., Lokey, S., Weiergraber, O., Kritzer, J.The LC3/GABARAP protein family is a promising target for selective inhibition of autophagy and for targeted protein degradation. LC3/GABARAP proteins are challenging targets for small-molecule drug development due to their long, shallow binding grooves. In this work, we evaluate multiple approaches to stabilizing the extended structure of the native binding motif, producing N-methylated peptides and stapled peptides with low nanomolar affinity. A crystal structure and molecular dynamics simulations support a model where the N-methylation pre-organizes the motif into an extended, […]
- by Banerjee, B., Chatterjee, D., Dasgupta, P., Kamale, C. K., Bhaumik, P.The hydrolytic breakdown of cellobiose into glucose, catalysed by {beta}-glucosidases, is the last and rate-limiting step in cellulose saccharification for producing fermentable glucose in the bioethanol industry. This limitation arises because {beta}-glucosidase activity is inhibited by factors such as temperature, pH, and glucose accumulation in reactors. Enzyme inactivation leads to the buildup of cello-oligosaccharides, which, in turn, inhibit upstream cellulases. Therefore, glucose-tolerant {beta}-glucosidases are preferred for the formulation of industrial cellulase cocktails. In this study, we have recombinantly expressed, purified, […]
- by Seidel, D. C., Wagner, A. G., Pezzullo, J. L., Thayer, K. A., Beadle, S., Olejarczyk, M. L., Giner, J.-L., Callahan, B. P.Extracellular secretion of the oncogenic sonic hedgehog signaling ligand is contingent on its release from a precursor protein through peptide bond cholesterolysis, mediated by the hedgehog C-terminal domain, SHhC. In this work, we describe the in vitro reconstitution of cholesterolysis activity for SHhC domains from vertebrate model organisms, Xenopus laevis (Xla) and Danio rerio (Dre). Cholesterolysis is assayed continuously in multi-well plates by monitoring changes in fluorescence resonance energy transfer (FRET) from an engineered precursor construct, expressed in E. coli […]
- by Puri, A., Hembram, D., Ravichandran, A., Das, R.Varicella Zoster Virus (VZV) is a dsDNA virus that infects dermal cells and causes characteristic cutaneous lesions. The virus undergoes neurotropism and later causes secondary cycles of infection. In the host nucleus, Promyelocytic Leukaemia Nuclear Bodies (PML-NBs) spontaneously form around the VZV genome to repress viral gene expression. VZV encodes for a ubiquitin E3 ligase ORF61 to disperse PML-NBs and alleviate repression. ORF61 functions as a ubiquitin E3 ligase with a conserved RING domain at the N-terminal end. It carries […]
- by Tsao, L. H., Brackney, D. E., Pyle, A. M.The RNA genome of West Nile Virus (WNV) folds into an elaborate series of RNA structural elements that are crucial for viral function. Among these elements, four pseudoknots (PKs) at the viral 3'-terminus, designated as SLII, SLIV, DBI, and DBII, are among the most crucial players in the overall flaviviral lifecycle. While many studies have focused on exploring the behavior of individual PKs, we investigated the collective role of all four PKs in viral growth and small flaviviral RNA (sfRNA) […]
- by Mallet, M., Martin, Y., Carvalho, J., Guchen, E., Betous, R., Bechara, C., Lespine, A., Schubert, M., Bourguet, W., le Maire, A.Parasitic nematodes infect billions of humans and livestock worldwide, causing major health and economic burdens, while the spread of anthelmintic resistance threatens current control strategies. A critical step in parasite infection is the resumption of development of infective third-stage larvae (iL3) upon host entry, a process controlled by the nuclear receptor DAF-12. Activation of DAF-12 by dafachronic acids promotes developmental progression and reproductive maturation, making this receptor an attractive therapeutic target. However, the molecular mechanisms governing DAF-12 activation, particularly transcriptional […]
- by Masubuchi, T., Wen, G., Song, X., Gaddam, K., Shao, H., Wu, C., HUI, E.The formation of microclusters is a hallmark of PD1 engagement with its ligands, yet the physical basis and functional significance of this phenomenon remain unclear. Here we show that ligand-bound PD1 licenses Shp2 self-association and liquid-liquid phase separation (LLPS), producing dynamic PD1:Shp2 condensates whose liquidity depends on Shp2 catalytic activity. Mutations that selectively disrupt Shp2 self-association weaken PD1 microcluster formation and impair PD1 inhibitory function. Mechanistically, PD1-induced Shp2 LLPS promotes the co-compartmentalization of signaling substrates such as CD3{zeta} and CD28, […]
- by ROY, R., Chidambaram, S., Arunachalam, J. P., Rajendran, R.Photoreceptor integrity depends on the precise coordination of membrane trafficking and signal transmission. Despite their well-known roles in germline biology, the functions of PIWI family proteins in post-mitotic neuronal cells remain unclear. We investigated the role of HIWI2 in photoreceptor-derived 661W cells. Silencing of HIWI2 resulted in a significant decrease in the early endosomal marker, Rab5, and its effector EEA1, and reduced expression of the recycling endosome marker Rab11, indicating poor endosomal sorting and receptor recycling. In contrast, the marker […]
- by Moritz, C., Lutz, L., Baumschabl, M., Glinsner, D., Gassler, T., Mattanovich, D., Ata, O.The efficient production of food and biochemicals using microorganisms that utilize single-carbon feedstocks presents a promising approach for advancing a circular bioeconomy. Komagataella phaffii (formerly Pichia pastoris) is a methylotrophic yeast already widely used in industry, making it an attractive host for such applications. Recently, K. phaffii was converted into an autotrophic strain capable of assimilating CO2 into both biomass and secreted organic acids, using energy derived from dissimilation of methanol to CO2. In these strains, methanol oxidation is catalysed […]
- by Rodriguez-Rangel, S., Gutierrez-Coronado, O., Mata-Ortega, B., Sun, Y., El-Saadi, S., Brugarolas, P., Sanchez-Rodriguez, J. E.Aminopyridines, including 4-aminopyridine (4AP), 3,4-diaminopyridine, and [18F]3-fluoro-4-aminopyridine, are voltage-gated potassium (KV) channel blockers used clinically to enhance conduction in neurological disorders and to image demyelination by PET. Developing new aminopyridines may yield improved therapeutics or imaging agents. Here, we characterized the physicochemical properties (pKa, logD), KV channel-blocking activity, toxicity (LD50), and pharmacokinetics of a novel compound, 4-methyl-3-aminopyridine (4Me3AP). 4Me3AP was less basic and more lipophilic than 4AP and showed greater blocking potency across multiple KV channels expressed in Xenopus oocytes. […]
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