- by Han, M., Frieg, B., Matthes, D., Leonov, A., Ryazanov, S., Giller, K., Nimerovsky, E., Stampolaki, M., Xue, K., Overkamp, K., Dienemann, C., Riedel, D., Giese, A., Becker, S., de Groot, B. L., Schroeder, G., Andreas, L. B., Griesinger, C.Alzheimer's disease is a specific neurodegenerative disorder, distinct from normal aging, with a growing unmet medical need. It is characterized by the accumulation of amyloid plaques in the brain, primarily consisting of amyloid beta (A{beta}) fibrils. Therapeutic antibodies can slow down the disease, but are associated with potential severe side effects, motivating the development of small molecules to halt disease progression. This study investigates the interaction between the clinical drug candidate small molecule anle138b and lipidic A{beta}40 fibrils of type […]
- by Rietjens, R. G. J., Manzato, B., van den Berg, B. M., Mahfouz, A., Giera, M., Dumas, S. J., Wang, G., Rabelink, T. J.After acute kidney injury (AKI), the persistence of failed repair proximal tubule (FR-PT) cells is postulated to hamper kidney regeneration and increase the risk of chronic kidney disease. This fibrotic shift likely depends on microenvironmental interactions, which remain largely unstudied. To investigate this, we mapped the spatial metabolic architecture of post-ischemic kidneys using an untargeted semi-quantitative spatial metabolomics (qMSI) approach, integrated with high-resolution spatial transcriptomics. Unsupervised neighborhood clustering of qMSI data revealed distinct microenvironments. Lipidome profiles identified diffusely spread areas […]
- by Maillie, C. A., Zhang, M., Goiset, N., Goldenfeld, G., Ward, A., mravic, m.Toll-like receptors (TLRs) are single-pass membrane proteins central to innate immunity, yet the role of homodimeric transmembrane (TM) domain interactions in cross-membrane signaling remains poorly understood and underexploited for therapeutic intervention. Here, we conduct a de novo computational design campaign to generate synthetic peptides that selectively target the TM domain of Toll-like receptor 4 (TLR4), a key mediator of inflammatory signaling and a clinically relevant drug target. Using in silico modeling and cell-based screening, we identified two peptides that disrupt […]
- by Caliseki, M., Borucu, U., Yadav, S., Schaffitzel, C., Kabasakal, B. V.Membrane protein quality control in Escherichia coli involves coordinated actions of the AAA+ protease FtsH, the insertase YidC, and the regulatory complex HflKC. These systems maintain proteostasis by facilitating membrane protein insertion, folding, and degradation. To gain structural insights into a putative complex formed by FtsH and YidC, we performed single-particle cryogenic electron microscopy on detergent-solubilized membrane samples, from which FtsH and YidC were purified using Ni-NTA affinity and size exclusion chromatography. Although SDS-PAGE analysis indicated a high purity of […]
- by Hsu, C., Shulman, N., Stewart, H., Petzoldt, J., Pashkova, A., Plubell, D., Denisov, E., Hagedorn, B., Damoc, E., MacLean, B. X., Remes, P. M., Canterbury, J. D., Makarov, A., Hock, C., Zabrouskov, V., Wu, C. C., MacCoss, M. J.Mass spectrometry instrumentation continues to evolve rapidly, yet quantifying these advances beyond conventional peptide and protein detections remains challenging. Here, we evaluate a modified Orbitrap Astral Zoom mass spectrometer (MS) prototype and compare its performance to the standard Orbitrap Astral MS. Across a range of acquisition methods and sample inputs, the prototype instrument outperformed the standard Orbitrap Astral MS in precursor and protein identifications, ion accumulation efficiency, and reproducibility of measurements. To enable meaningful cross-platform comparisons, we implemented an ion […]
- by Hushpulian, D. M., Ammal Kaidery, N., Soni, P., Poloznikov, A. A., Zakhariants, A. A., Razumovskaya, A. V., Silkina, M. O., Tishkov, V., Kazakov, E. H., Brown, A. M., Gaisina, I. N., Ahn, Y.-H., Kazakov, S. V., Krucher, N., Sharma, S. M., Paul, B. D., Gazaryan, I. G., Nikulin, S. V., Thomas, B.Activating Nrf2 with small molecules is a promising strategy for countering aging, oxidative stress, inflammation, and various disorders, including neurodegeneration. The primary regulator of Nrf2 protein stability is Keap1, a redox sensor protein and an adapter in the Cullin III ubiquitin ligase complex, which labels Nrf2 for proteasomal degradation. The known Nrf2 activators either chemically modify sensor thiols in Keap1 or competitively displace Nrf2 from the ubiquitin ligase complex. The latter approach is considered the most suitable for continuous administration, […]
- by Bergman, W. N., Sousa, M. C.The {beta}-barrel assembly machine (BAM) is an essential protein complex that folds and inserts outer membrane {beta}-barrel proteins (OMPs) into the bacterial outer membrane. The BAM complex contains the essential BamA OMP with five soluble polypeptide transport-associated (POTRA) domains, which scaffold the essential BamD lipoprotein and the non-essential lipoproteins BamB/C/E. The importance of each BAM component has been investigated primarily using cell-based phenotypic assays, and structural data have revealed insights into the role of the BamA {beta}-barrel in OMP folding. […]
- by Cabrera, D., Pujana Vaquerizo, M., Martinez La Osa, B., Zabala Letona, A., Manuel Falcon, J., Carracedo, A., van Liempd, S.We describe a robust, fast and accurate method for the quantification of intra-cellular concentrations of the polyamines putrescine, spermidine and spermine in cultured cells. Hydrophilic interaction liquid chromatography in combination with Time-of-Flight mass spectrometry was used to obtain high resolution data for the analytes. Assay performance was determined with respect to chromatographic resolution, quantification, analyte recovery and matrix effects. Furthermore assay variability was determined in a biological context. Based on these variability measurements, minimal detectable effects (MDEs) which would lead […]
- by Roeselova, A., Shivakumaraswamy, S., Jurkeviciute, G., He, J.-Z., Auburger, J., Schmitt, J., Kramer, G., Bukau, B., Enchev, R. I., Balchin, D.Natural proteins are structurally diverse and often form intricate multidomain, oligomeric architectures. This presents a prima facie challenge to cellular homeostasis, as topologically complex proteins seldom refold efficiently in vitro. How cells overcome sequence-intrinsic folding limitations to optimize protein biogenesis is incompletely understood. Here, we show that efficient folding and assembly of the model five-domain homotetramer {beta}-galactosidase is obligatorily coupled to its synthesis on the ribosome, and define the underlying mechanisms. During refolding of full-length protein from denaturant, maturation of […]
- by Sass, M. I., Mack, D. C., Nickles, R. A., Jones, C. A., Brunsdale, R. L., Cottam, S. L., Shen, P. S., Willardson, B. M.The Chaperonin Containing Tailless polypeptide 1 (CCT or TRiC) is an essential cytosolic chaperone that folds multiple protein substrates, including many with {beta}-propeller folds. One {beta}-propeller substrate is the G protein {beta}5 subunit (G{beta}5) of Regulator of G protein Signaling (RGS) complexes that determine the duration of G protein signals in neurons. In recent work, we used cryo-electron microscopy (cryo-EM) to visualize the complete CCT-mediated folding trajectory for G{beta}5, from an initiating electrostatic interaction of a single {beta}-strand in G{beta}5 […]
- by Dzieciatkowska, M., Hay, A., Issaian, A., Keele, G. R., Bevers, S., Nemkov, T., Reisz, J. A., Maslanka, M., Stephenson, D., Moore, A., Deng, X., Stone, M., Hansen, K., Kleinman, S., Norris, P. J., Busch, M. P., Page, G. P., Roubinian, N. H., Xia, Y., Zimring, J. C., D'Alessandro, A.Caffeine is the most widely consumed psychoactive substance globally, yet its peripheral physiological effects remain incompletely understood. Leveraging comprehensive data from 13,091 blood donors in the REDS RBC Omics study, we identify caffeine as a significant modulator of red blood cell (RBC) storage quality and transfusion outcomes. Elevated caffeine levels were reproducible across multiple donations from 643 recalled donors, selected based on their extremes in hemolytic propensity. Both in the screening and recalled cohorts, higher caffeine levels were associated with […]
- by Elgeti, M., Belyaeva, J., Helabad, M. B., Staus, D. P., Wingler, L. M."Biased" ligands of the angiotensin II type 1 receptor (AT1R) preferentially activate Gq pathways, which promote vasoconstriction, or {beta}-arrestin-mediated pathways, which have salutary cardiac effects. We previously demonstrated that pharmacologically indistinguishable {beta}-arrestin-biased ligands stabilize different AT1R conformational ensembles. Here we show these ligand-specific conformational changes determine whether {beta}-arrestin interacts with the AT1R seven-transmembrane (7TM) core or only the phosphorylated C-terminus. We developed a method to form complexes between non-phosphorylated AT1R and {beta}-arrestin1 mediated only through the 7TM core. {beta}-Arrestin-biased ligands […]
- by Brutus, M. E., Girard, C. S., Jacob-Dolan, J. W., Scheck, R. A.Glyoxal (GO) is a highly reactive 1,2 dialdehyde implicated in the formation of a set of disease-associated post-translational modifications (PTMs) known as advanced glycation end products (AGEs). While GO has been widely reported to modify lysine to form the highly studied AGE carboxymethyllysine (CML), here we demonstrate that GO alone leads to highly chemoselective arginine glycation, yielding a stable glyoxal-derived hydroimidazolidine (GH-DH) product. This near-exclusively formed AGE has the same mass change as carboxymethylarginine (CMA), which implies that it may […]
- by Munoz-Reyes, D., Aguado, L., Arroyo-Urea, S., Requena, C., Perez-Suarez, S., Sanchez-Yepes, S., Argerich, J., Miro-Rodriguez, C., Ulzurrun, E., Rodriguez, E., Garcia-Nafria, J., Campillo, N. E., Mansilla, A., Sanchez-Barrena, M. J.Dopamine D2 receptor (D2R) regulates key aspects of motor control, cognition, and reward. Its function depends not only on ligand binding and signaling efficacy, but also on the dynamic control of receptor localization at the cell surface. Neuronal Calcium Sensor 1 (NCS-1) is a calcium binding protein which directly interacts with D2R in a Ca2+-dependent manner. Here, we investigated the regulatory role of NCS-1 in D2R localization and function. We found that NCS-1 promotes the trafficking of D2R to the […]
- by Giansanti, P., Fojnica, A., Pichlmair, A.Protein degradation is critical for regulating cellular functions. Therefore, there is considerable interest in developing effective proteome-wide strategies to identify protease cleavage products to enhance our understanding of proteolytic pathways and their perturbation in diseases. Here, we present a streamlined N-termini proteome analysis leveraging N-Hydroxysuccinimide (NHS) ester chemistry. At the protein level, N-terminal amines (naturally occurring protein N-termini, lysines, or protease-generated N-termini) are blocked directly in the cell lysate, and tryptic digestion is performed straight after, without the need for […]
- by Singh, K., Pucadyil, T.Membranes are fundamental to biological systems as they serve as barriers to compartmentalize the cell and its cytoplasm. They are constantly remodeled in shape and composition during the formation and maintenance of organelles. The large GTPase dynamins are well characterized for membrane shape remodeling in eukaryotes but their functions in prokaryotes are less characterized. Here, we determine the lipid-binding, enzymatic and membrane remodeling activities of the Bacterial Dynamin-Like Protein (BDLP) from the cyanobacterium Nostoc punctiforme. We find that BDLP binds […]
- by Roberts, J. L., Ryan, M., Whiley, L., Gray, N., Gay, M., Holmes, E., Nicholson, J. K., Wist, J., Lawler, N. G.Comprehensive lipidomic profiling in diabetes has identified disease-associated lipid signatures that may support more personalised monitoring beyond routine glycaemic control. Dried blood spot (DBS) microsampling offers a minimally invasive method for collecting and storing small blood volumes ( 1.44), including HexCer(18:1;O2/24:0), HexCer(18:1;O2/22:0), LPC(16:0), PC(O-34:3), TG(18:0-18:1-20:3), and TG(18:1-18:1-22:6), were consistent with matched venous and capillary plasma samples. Lipid class and structural differences, including elevated long-chain triacylglycerols and reduced lysophosphatidylcholines reflected known dyslipidaemia in diabetes and demonstrate the capacity of DBS to […]
- by Gharabli, H., Kohler, A. M., Chiesa, C., Wagle, S., Mejia-Otalvaro, F., Orth, J. V., Bidart, G. N., Enevoldsen, A. D., Förster, J., Werner, S. J., Welner, D. H.Anthranilate (ANT) is a precursor for the synthesis of valuable compounds, including its alkyl esters (AEANTs), such as methyl anthranilate (MANT). These derivatives are industrial petrochemical products used as flavouring agents and bird repellents. Due to the mandatory green transition, their biological industrial production must be considered. However, their antimicrobial activity and physicochemical properties inhibit efficient microbial production and challenge their practical use. To overcome this, we explored enzymatic glycosylation using UDP-dependent glycosyltransferases (UGTs). Screening identified three UGTs with activity […]
- by Larson, G., Bhagi-Damodaran, A., Rama Damodaran, A.Histidine kinases (HKs) are essential bacterial signal transduction proteins and attractive drug targets due to their critical cellular functions. Direct measurement of their autophosphorylation activity is crucial for developing inhibitors and advancing disease treatments. While [{gamma}-32P]-ATP radiolabeling has long been a conventional method for kinase activity measurements, its reliance on 32P introduces inherent limitations. The short half-life of this isotope imposes time-sensitive constraints on experiments, and stringent radiation safety and compliance requirements significantly increase operational costs and hinder scalability. Several […]
- by Zhan, J., Hsieh, C.-M., Esser, L., Lang, Z. C., Morton, A. J., Robey, R. W., Zhou, F., Ambudkar, S. V., Huang, R. K., Gottesman, M. M., Xia, D.The hallmark of multidrug resistance conferred by human ABC transporter ABCB1 (hP-gp) is the recognition and efflux of diverse range of drugs, though the precise mechanism of polyspecificity remains unresolved. In aquatic animals such as zebrafish, Abcb4, a functional homolog to hP-gp, plays a vital role in surviving environmental toxicants. Here, we show that Abcb4 exhibits comparable basal and drug-stimulated ATPase activity to hP-gp. Using cryo-EM, we captured five inward-facing Abcb4 conformations with varying separations between its two lobes, illustrating […]
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