- by Pierson, E., Mainwaring, J. C., Patrick, W. M., Gerth, M. L.The persistence of specialised survival spores produced by microbial pathogens represents a primary bottleneck in the management of plant diseases. In oomycetes, these spores (known as oospores) are largely impervious to chemical control, allowing them to persist in soil and initiate new infection cycles over many years. A prominent example is the soil-borne pathogen Phytophthora agathidicida, the causal agent of kauri dieback disease, where long-lived oospores hinder conservation efforts in native forests. The resilience of oospores is attributed to their […]
- by Wang, G., Chen, J.-h., Qiao, Z., Guo, D., Guo, P., Wang, A., Sun, W., Lyu, J.BACKGROUNG Bisphenol A (BPA) has been linked to hypertension and disturbances in lipid metabolism; however, limited evidence is available regarding its association with hypertensive intracerebral hemorrhage (ICH). METHODS A multicenter, retrospective case-control study was conducted involving 129 participants, including individuals from an ICH group and healthy controls. Standard assays were employed to assess serum thyroid function, lipid profiles, serum fatty acid-binding protein 4 (FABP4), oxidative stress markers, gap junction proteins, Wnt/{beta}-catenin signaling pathway activity, and expression changes of S100A8-mediated inflammatory […]
- by Greenwood, M. E., Austin, S., Murciano-Martinez, P., Hollywood, K. A., Machidon, M., Spiess, R., Berrington, J., Flitsch, S., Barran, P., Stewart, C. J.Human milk contains structurally diverse glycans with key roles in shaping infant development, yet analytical constraints limit characterisation from low-volume samples. Glycosaminoglycans (GAGs), including chondroitin sulphate (CS), are understudied due to existing protocols requiring sample volumes of at least 5 mL and lengthy extraction steps prior to instrumental analysis. This study establishes a workflow for quantifying CS disaccharides from 25 L of human milk, enabling analysis of samples previously inaccessible to GAG profiling, such as those collected as salvage samples […]
- by Warkentin, R., Pyle, A. M.Ribozyme-based permuted intron-exon (PIE) systems offer a protein-independent route to circRNA production, but existing platforms require elevated temperatures that promote RNA degradation. Here we report the first application of the Candida albicans mitochondrial large subunit (C.a.mtLSU) group I intron as a PIE platform for circRNA synthesis, which we term PCanPIE (Pyle lab Candida PIE). We evaluated three peripheral stems, P5, P6b, and P8, as permutation sites and demonstrated that all three support circularization under near-physiological conditions (25 {degrees}C, 6 mM […]
- by Dudek, A., Janapatla, R. P., Chen, C. L., Chiu, C. H.Fucoidans have been widely reported to show SARS-CoV-2 antiviral activity. In this study, we observed a striking difference in the inhibitory potency between two commercially available fucoidans: Fucus vesiculosus crude (Fvc) and pure (Fvp). SEC-MALS analysis revealed two molecular weight populations for Fvc (1098 kDa, 58.58 kDa) and one for Fvp (40.48 kDa). At micromolar concentrations of fucoidans, the binding affinities (KDs) of Fvc_1098 (223 nM) and Fvc_58 (4.27 M) for the amine-biotinylated SARS-CoV-2 receptor binding domain (RBD) were higher […]
- by Gautam, A. K., umarao, P., Gourinath, S.The Rho family of small GTPases plays a critical role in regulating actin cytoskeleton dynamics during endocytic processes in E. histolytica, including phagocytosis, pinocytosis, and trogocytosis. These proteins act as molecular switches, transitioning between inactive GDP-bound and active GTP-bound states, with guanine nucleotide exchange factors (GEFs) catalyzing this transition. Among the GEFs, EhFP10, a FYVE domain-containing protein harbouring Dbl homology (DH) and pleckstrin homology (PH) domain was observed in phagocytosis along with seven functionally characterized Rho GTPases (EhRho1, EhRho2, EhRho4, […]
- by Athol, H. E., Thompson, A., O'Connor, N., Shirts, M. R., Kralj, J. M., Fox, J. M.Protein tyrosine kinases (PTKs) regulate cellular biochemistry by phosphorylating tyrosine residues that alter protein function; their substrate preferences define the topology of signaling cascades. Previous studies of PTKs have mapped their average preferences for amino acids surrounding phosphorylation sites, but their ability to discriminate between highly similar substrate sequences (i.e., their sensitivity to minor changes in sequence within different regions of a substrate, and the sequence-dependent nature of this sensitivity) remains poorly understood. Here, we use a genetically encoded biosensor […]
- by Freye, C., Miller, B. G.Multi-functionality in extant enzymes, including the ability to transform multiple substrates, is thought to arise, in part, from conformational flexibility. The hexokinase protein family represents a classic model system for investigating the relationship between substrate specificity and conformational change. Within this family, human glucokinase (hGCK) displays notable degrees of conformational heterogeneity, including an intrinsically disordered loop. The extent to which these structural features contribute to the breadth of hGCK's substrate scope is unknown. Here, we investigate the substrate specificities of […]
- by Nimkar, S., Nguyen, T., Karandur, D., Subramanian, S., O'Donnell, M. E., Kuriyan, J.DNA polymerase clamp loaders are AAA+ ATPases that load sliding clamps on DNA for high-speed replication. Using a platform for high-throughput mutagenesis of replication proteins in T4 bacteriophage, we carried out saturation mutagenesis of the AAA+ ATPase module of the T4 clamp loader bearing a mutation, Gln 118 to Asn (Q118N), that reduces fitness. We identified residues for which different mutations improve the fitness of the Q118N variant but are neutral in the wild-type background. These conditionally neutral rescue hotspots […]
- by Padhi, C., Nguyen, D. T., Zhu, L., Cha, L., Wald, J. W., Mitchell, D. A., van der Donk, W.Cytochrome P450s catalyze a diverse array of reactions including crosslinking of aromatic side chains in the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs). ApyO is a cytochrome P450 enzyme that forms a C-C bond between two tyrosines in a YLY motif in the substrate ApyA, the precursor peptide of the RiPP aminopyruvatide. We utilized cell-free translation to generate ApyA variants and probe the substrate tolerance of ApyO. Through Alphafold-based modelling and in vitro assays, we show that ApyO […]
- by Banerjee, S., Malassani, S., Banerjee, S., Lopez Vasquez, M. C., McSorley, S., Wang, Z.RAF kinases interpret signals from the three major RAS isoforms to initiate MAPK pathway activation, yet the molecular logic that governs isoform-specific RAS recruitment and the early events that relieve RAF autoinhibition are not yet fully understood. In particular, how the modular N-terminal regulatory architecture of CRAF and ARAF, anchored by the multifunctional cysteine-rich domain (CRD), discriminates among HRAS, KRAS, and NRAS has remained a central unresolved question. Here, we combine quantitative biophysical measurements with structural and dynamic analyses to […]
- by Khundoker, R., Majer, S. H., Silakov, A.O2-tolerance is a desirable property for [FeFe] hydrogenases, which are highly efficient H2-producing catalysts. While most such enzymes are highly sensitive to aerobic environments, a small number of explored representatives exhibit exceptional stability and even H2-producing activity under oxygenic conditions. However, the genetic signatures of the O2-tolerance in this class of enzymes remain largely unknown. To address this knowledge gap, we explored a close homologue of a well-characterized O2-tolerant [FeFe] hydrogenase from Clostridium beijerinckii (CbHydA1) – a hydrogenase from Terrisporobacter […]
- by Lorente Cobo, N., Goyal, S., Davidson, D., Prehna, G.The type VI secretion system (T6SS) is a dynamic nanomachine used by bacteria to compete for space and nutrients. To kill rival bacteria, the T6SS secretes toxic effector proteins directly into adjacent cells in a contact-dependent manner. Effectors have diverse biochemical functions that arise from subtle structural modifications to related enzyme folds. To protect from self-intoxication, effectors are encoded with a cognate immunity protein in effector-immunity pairs. Immunity proteins inhibit toxicity by directly binding the effector active site and are […]
- by Tan, C., Jung, J., Sugita, Y.Biomolecular condensates formed by intrinsically disordered proteins (IDPs) rely on a balance of sequenceencoded interactions and secondary-structure elements. TDP-43, a disease-associated protein, undergoes liquid-liquid phase separation (LLPS) through its low-complexity domain, whereas Hero11 has been proposed to modulate its condensate properties. However, the molecular mechanisms by which Hero11 affects the internal organization and dynamics of TDP-43 condensates remain unknown. Here, using multimicrosecond explicit-solvent all-atom simulations spanning single chains to ~100-chain condensates, we show that the TDP-43 alpha-helix, which is only […]
- by Frascogna, F., Rockwell, N. C., Layer, G., Frankenberg-Dinkel, N.Biosynthesis of the linear tetrapyrrole phycocyanobilin (PCB) by the ferredoxin-dependent bilin reductase (FDBRs) PcyA is essential for light-harvesting and regulatory processes in diverse photosynthetic organisms, yet its evolutionary origins are not fully understood. PcyA evolved from pre-PcyA proteins found in diverse bacteria. Three lineages of pre-PcyA proteins were identified: Pre-1, Pre-2 and Pre-3. Using an in vivo co-expression assay, Pre-2 and Pre-3 proteins were shown to be active FDBRs that did not synthesize PCB, whereas Pre-1 activity was apparently low. […]
- by Kinman, L. F., Grassetti, A. V., Carreira, M. V., Davis, J. H.The emergence of single-particle cryoEM as a powerful method for structure determination has in large part been fueled by its ability to resolve both single static structures and complex conformational landscapes. Indeed, modern approaches to the heterogeneous reconstruction task can resolve 100s-1,000s of different maps from a single cryoEM dataset. How accurate these algorithms are, however, has proven difficult to rigorously assess, due to a lack of suitable benchmark datasets containing both realistic noise features and ground-truth labels. To address […]
- by Milburn, A. E., Kulkami, D. S., Espejo-Serrano, C., Pachon-Penalba, M., Williams, M. E., Nicol, J. P. O., Debilio, S., Gurusaran, M., Dunce, J. M., Adams, I. R., McClurg, U. L., Hunter, N., Davies, O. R.In meiosis, crossovers between homologous chromosomes generate genetic diversity and are required for accurate chromosome segregation, ensuring fertility. In mammals, HEI10 is one of three pro-crossover RING-domain factors implicated in protein modification by ubiquitin and/or SUMO and characterised by their dynamic accumulation at future crossover sites. However, the molecular architecture and enzymatic activity of mammalian HEI10 have remained unknown. Here, we show that human HEI10 has E3-ubiquitin ligase activity that depends on its higher-order assembly. We report the crystal structure […]
- by Gulkis, M., Heidings, J. B., Huiskonen, J. T., Sawh-Gopal, A., Hsi-Bell, J., Potter, M., Song, X., Hutchinson, T. E., Bennett, A., Mietzsch, M., Bird, J. E., McKenna, R.Adeno-associated virus (AAV) capsids are important gene therapy vectors, allowing for the one-time treatment of monogenetic disorders, with durable gene expression lasting for years. However, despite the clinical success of AAV usage, low transduction efficiencies require high dosage to achieve therapeutic efficacy, resulting in prohibitive costs and rare, but life-threatening immune responses. One key knowledge gap is how the capsids traffic to the nucleus following endocytosis. Here, we identify a direct interaction between AAV2 and actin filaments. Our results show […]
- by Santiago, J. I., Freye, C., Kamalaldinezabadi, S. S., Papa, J. E., Whittington, A. C., Miller, B. G.The glucokinase regulatory protein (GKRP) derives from an ancestral etherase. Despite existing as a single locus in the metazoans, GKRP evolved multiple novel functions unrelated to etherase activity. In jawed vertebrates, a protein-protein interaction (PPI) emerged that inhibits glucokinase (GCK) activity in the liver. This PPI is critical to maintaining glucose homeostasis. In mammals, GKRP is allosterically regulated by carbohydrates, with 6-phospharylated sugars promoting inhibition of GCK by GKRP, while 1-phosphorylated sugars relieve inhibition. Here, we use a vertical evolutionary […]
- by Talbot, K. M., Su, Y.-W. N., Royster, J. B., Gohara, D. W., Firouzbakht, A., McLean, M. N., Ramalingam, B. M., Willson, T. M., Arnold, J. J., Cameron, C. E.RNA helicases encoded by positive-strand RNA viruses are essential for genome replication, yet the specific biological functions and mechanochemical basis underlying these functions remain poorly defined. Progress has been limited by the difficulty of resolving individual catalytic steps under single-turnover conditions, which are often experimentally inaccessible for viral enzymes. Alphaviruses replicate within membrane-bound spherules that may alter local metabolite concentrations, raising the possibility that the enzymatic properties of alphaviral proteins differ from those of viruses with greater cytosolic exposure. Here, […]
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