- by Thomson, B. D., Marquez, M. D., Rawson, S., dos Santos, T. M. A., Harrison, S. C., Kahne, D.The outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria contain {beta}-barrel membrane proteins that are assembled by conserved multi-subunit machines. In bacteria, the {beta}-barrel assembly machine (BAM) folds over a hundred compositionally different substrates into barrels that vary greatly in size. Some larger barrels require globular proteins to plug the barrel lumen. How a single machine can assemble such different barrels is unknown. Here we report three structures representing progressively folded stages of a 16-stranded barrel engaged with BAM, as […]
- by Mohona, S., Shakya, A. K., Singh, S., Kearns, F. L., Jemison, K., Erramilli, S. K., Dey, D., Qing, E., Jennings, B. C., Doray, B., Kossiakoff, A. A., Amaro, R. E., Klose, T., Gallagher, T., Hasan, S. S.Assembly of infectious coronaviruses requires spike (S) protein trafficking by host coatomer, typically via a dibasic signal in the S cytoplasmic tail. However, the human embecoviruses HKU1 and OC43, as well as the model virus MHV, lack this motif. Here we identify a conserved His-x-Asp (HxD) sequence that functions as an unconventional coatomer-binding signal. Structural and biochemical analyses show that the MHV HxD motif engages coatomer subunits through distinct conformations, while cellular imaging demonstrates its role in directing S to […]
- by Barlow-Busch, I., Walsh, E. E., Nyvall, H. G., Burke, J. E.Class IA phosophoinositide kinases (PI3Ks) are master regulators of growth, metabolism, and immunity. The class IA PI3Ks are a heterodimer composed of a p110 catalytic subunit and one of five possible regulatory subunits (p85, p85{beta}, p55{gamma}, p55, p50). The regulatory subunit plays critical roles in stability, inhibition, and activation of the p110 catalytic subunit. The p110 catalytic subunit frequently contains activating mutations in human cancer, with many of these mutations altering the interaction between catalytic and regulatory subunits. It has […]
- by Kulakman, C., Mathews, I., Yoshikun, Y., Wakatsuki, S., DeMirci, H.Enoyl-CoA carboxylases/reductases (ECRs) are enzymes with the fastest carbon dioxide (CO2) fixation capabilities, yet the precise mechanisms behind their assembly and catalytic activity are structurally not yet fully understood. Here, we employed cryo X-ray crystallography to reveal the dimeric structural organization of a novel ECR, isolated from mesophilic Mesorhizobium metallidurans (M. metallidurans). We examined the interactions in silico and compared oligomerization of our dimeric ECR from M. metallidurans (ECRMm_Dim) with tetrameric ECR from Burkholderia ambifaria (ECRBa_Tet)by using size exclusion chromatography […]
- by Kim, J., Franchini, L., Yudin, Y., Denissiouk, A., Rohacs, T., Orlandi, C., Das, A.Lipid mediators derived from omega 6 polyunsaturated fatty acids (PUFAs) support neurological health in part through their oxidative and non oxidative transformation into a diverse array of bioactive molecules. Among these are lipidated neurotransmitters, formed via conjugation of neurotransmitters with fatty acids such as arachidonic acid (AA) or docosahexaenoic acid (DHA). Previous studies links these lipidated neurotransmitters to beneficial outcomes in neurological diseases. Here, we focus on two such endogenous lipidated neurotransmitters, arachidonoyl glycine (NAGly) and docosahexaenoyl glycine (DHAGly) and […]
- by Brandenberg, O. F., Janssen, E. M.- L., Schubert, O. T.Analyte detection through aptamer-induced signal generation by CRISPR-Cas enzymes has rapidly emerged as a popular biosensing approach. Here, we investigated the implementability and analytical performance of this setup for the detection of diverse small molecule analytes. We selected nine aptamers from the literature targeting seven analytes and tested a commonly used assay design whereby analyte binding by the aptamer liberates a short complementary DNA strand, which in turn activates Cas12a to generate a fluorescence signal. After extensive optimization, the assay […]
- by Zimanyi, M., Hulce, K. R., Bohn, M. F., Norman, J., Rohweder, P. J., Cheng, Y., Craik, C.Human herpesviruses (HHVs) are widespread pathogens that cause severe disease. Their replication depends on the HHV protease (HHV Pr), an enzyme essential for capsid maturation. Because HHV Pr must dimerize to become catalytically active, disrupting dimer formation is a promising strategy for antiviral therapeutic development. We isolated a conformationally selective antibody inhibitor, Fab5, from a fully human naive Fab-phage library that recognizes monomeric human cytomegalovirus protease (HCMV Pr). A 2.6 [A] cryoelectron microscopy (cryo-EM) structure of the Fab5-HCMV Pr complex […]
- by Kaydanov, N., Olesinska-Mönch, M., Leite, M., Prevedel, R., Deo, C.Fluorescence and photoacoustic imaging are complementary modalities that provide distinct advantages for biological imaging: fluorescence microscopy offers high sensitivity and resolution, while photoacoustic imaging enables deeper penetration in complex tissue. Leveraging the strengths of both modalities through optically switchable contrast agents can offer enhanced imaging contrast and facilitate dual-modality imaging. Here, we report a photoswitchable probe capable of toggling between high fluorescence and high photoacoustic signal upon illumination, exploiting Forster Resonance Energy Transfer (FRET). We engineer novel spironaphtopyran photoswitches which […]
- by Amaral, B. C., Brodie, N. I., Michael, A. R., Crowder, D. A., Douglas, P., Khan, M. F., Schriemer, D. C.Crosslinking mass spectrometry (XL-MS) could replace traditional techniques for sampling the cellular interactome, such as affinity pulldown MS. It generates superior interaction data that can be used to better model cellular structure and function. However, the sampling depth of current XL-MS techniques is disappointing. Poor sampling is often blamed on a low-yielding crosslinking reaction, estimated to be ~0.1% based on relative ion abundance in the mass spectrometer. Here, using a new two-step crosslinker installation process, we demonstrate that the yield […]
- by Huang, S., Liu, H., Wu, Y., Braz, J. M., Kranthi, D., Hall, B. W., ZHANG, X., Radchenko, D., Moroz, Y. S., Irwin, J. J., Basbaum, A., Xu, H. E., Wetsel, W. C., Shoichet, B. K.Schizophrenia is a severe mental illness whose current treatments primarily target dopamine and serotonin receptors. These drugs often cause side effects and vary in effectiveness across patients. The trace amine-associated receptor 1 (TAAR1), which modulates monoamine signaling, has emerged as a promising alternative target. To discover new TAAR1 ligands, we computationally docked 65 million molecules against the active state of TAAR1 and experimentally tested 55 of those highly ranked. Fourteen molecules active against TAAR1 with potencies ranging from mid-nanomolar to […]
- by Valentine, Y., Jamil, M., Kovilakath, A. P., Brown, R. D. R., Dail, J., Farooq, S., Spiegel, S., Cowart, L. A.Background: As of 2023, approximately 100.1 million adults and 14.7 million children in the USA are obese. Many comorbidities develop with obesity, which impairs quality of life and burdens the health care system. Consequently, there is an urgent need for interventions and treatments to reverse obesity and its comorbidities and restore health. Sphingosine Kinase 1 (SphK1), a key enzyme in sphingolipid metabolism, produces sphingosine-1-phosphate (S1P), a bioactive lipid implicated in obesity and metabolic dysfunction. While global deletion of Sphk1 protects […]
- by Tao, Z., Thakare, R., cheung, M., Zhang, J., Chen, X., Hu, X., Pierce, B., Wu, X., Mao, J.GSDMA, the primary member of the gasdermin family found in the skin, is critical for pathogen-induced pyroptosis during infection. Recent studies revealed that another gasdermin, GSDMD, undergoes palmitoylation during pyroptosis. However, whether and how the other gasdermin members undergo lipid modification remains poorly understood. Here, we demonstrate that GSDMA is S-acylated at the conserved cysteine residues in its N-terminal domain. We show that S-acylation of GSDMA promotes pyroptosis by facilitating its membrane anchoring and protein oligomerization, a mechanism distinct from […]
- by Bailey, B., Manis, J. R. Z., Seth, G., Rajpurohit, S., Oxenrider, A., Martin, P. M., Jadeja, R. N., Thounaojam, M.BackgroundPrevious studies have shown the metabolic and regulatory significance of CYP46A1 in the adult retina; however, its role in the developing retina is unknown. Here, we evaluate CYP46A1 expression and the impact of its activation in the developing mouse retina under normal and pathological conditions. Materials and MethodsSeven-day-old (P7) C57BL/6J mice maintained in room air (controls) or subjected to oxygen-induced retinopathy (OIR) were treated with/without 20 mg/kg of the CYP46A1 activator efavirenz (EFV) from P7 to P17. Retinal cross sections […]
- by Calcinoni, A., Casazza, A. P., Viola, S., Agostini, A., Carbonera, D., Santabarbara, S.The energetic limits of Photosystem II (PSII) photochemical reactivity required reconsideration after the discovery of far-red light acclimation responses in cyanobacteria. Insights on PSII functionality following the inclusion of the red shifted Chlorophylls d and f can be obtained by extending the current knowledge on spectroscopic and structural properties of its reaction center (RC). The photo-induced triplet states, that represent selective endogenous probes, were therefore investigated in far-red adapted PSII by magnetic resonance techniques. Zero-field splitting tensor analysis combined with […]
- by Suaini, N. B., Narvekar, A., Chundawat, S. P. S.N-glycosylation is a post-translational modification of proteins that represents a critical quality attribute (CQA) for therapeutics like monoclonal antibodies (mAbs), directly affecting drug efficacy, safety, and stability. Real-time CQA monitoring analytical platforms depend on rapid N-glycan release and fluorophore labeling chemistries to support automated bioprocess analytics during mAb manufacturing. Procainamide is a well-known fluorophore used for released N-glycans reducing sugar aldehydes labeling that offers both high fluorescence and mass spectrometry detection sensitivity comparable to several commercial reagents available in the […]
- by Dillon, O., Benner, I., Zaliznyak, T., Cisternas-Novoa, C., Reineccius, J., Taylor, G. T., Passow, U.Understanding the main sources of microplastic pollution is key towards developing efficient measures to reduce microplastic loadings to marine waters. Yet identifying the main sources of marine microplastic is challenging. Source tracking should be easier in marine bays where inputs are limited. In 2021 we determined the concentrations and characteristics of microplastics > 300 m in surface waters of Placentia Bay, Newfoundland; a bay with negligible river input in an area of low population density, and no plastic processing plants […]
- by Casas Mao, D., Carrington, G., Peckham, M.Determining the high-resolution structure of the widely expressed non-muscle myosin 2A (NM2A), in its dephosphorylated shutdown state, is important in understanding its regulation and disease roles. In shutdown molecules, the coiled-coil tail wraps around the myosin heads preventing them from forming filaments and binding to actin. We have solved the shutdown structure of NM2A to a global resolution of 3 angstroms in the heads region, and 6.3 angstroms for the whole molecule. This reveals specific ionic interactions that explain why […]
- by Hu, J., Moraga, D., Xu, A., Peysakhova, L., Crickard, J. B.Homologous recombination (HR) is a DNA repair pathway that utilizes a template-based approach to repair double-strand breaks within the genome. Template utilization requires the exchange of individual DNA strands, which members of the RecA family of recombinases facilitate. Rad51 is a primary strand exchange factor in eukaryotes. During regular mitotic DNA repair, Rad51 is aided by the DNA translocase Rad54, which acts as a motor to remodel the template DNA and stabilize primary-strand exchange intermediates. The regulation of this activity […]
- by Jain, P., Yehorova, D., Febri, R. R., Clifton, B. E., Demkiv, A., Uechi, G.-i., Robinson, M., Kamerlin, S. C. L., Okada, M., Imamoto, A., Laurino, P.Enzymes are central to life, with their catalytic activity often shaped by the dynamic conformations of regulatory loops. In hub enzymes such as tyrosine kinases, the activation loop critically controls substrate specificity, catalytic efficiency, and downstream signaling, shaping cellular fate. Yet, the molecular mechanisms by which loop dynamics encode these functions remain incompletely understood. Here, we used SRC kinase as a model to dissect how minimal perturbations of the activation loop reprogram kinase behavior. By generating and characterizing multiple variants, […]
- by Padgett-Pagliai, K. A., Grant, J. D., Petrides, M. J., Newcomb, E., Lovett, S. T., Bloom, L.Faithful DNA replication in Escherichia coli requires the DNA polymerase III holoenzyme (DNA pol III HE) and its clamp loader complex, which couples processive DNA synthesis with {beta}2 clamp loading. The clamp loader accessory subunits {chi} and {psi} link single-stranded DNA-binding protein (SSB) to the replisome, stabilizing replication on SSB-coated templates and coordinating access for other SSB-interacting proteins (SIPs). Chi has also been implicated in tolerance to the chain-terminating nucleotide analog azidothymidine (AZT), though whether this function depends on {chi} […]
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