- by Ng, M. Y., Nix, M. N., Du, G., Davidek, I., Burger, N., Shin, S., Toenjes, S., Xiao, H., Wei, S., Seo, H.-S., Dhe-Paganon, S., Wales, T. E., Engen, J., Mills, E., Che, J., Zhang, T., Gray, N., Chouchani, E.Immunological proteins are major disease targets, and the majority remain undrugged. Post-translational redox modification of protein cysteine residues has emerged as a mode of immune cell regulation, especially in the context of the macrophage cytokine response1,2. Here, we develop a strategy for systematic discovery and small molecule functionalization of redox regulated cysteines on immunological proteins. Using deep redox proteomics, we annotate 788 cysteines across diverse functional domains of immune-relevant proteins that are redox regulated in vivo. We demonstrate how these […]
- by Mahajan, S., Demirer, K., Clemons, W. M., Rees, D. C.Bacteria resist toxic arsenite [As(III)] in their environments by actively pumping the metalloid out of the cell via efflux pumps such as ArsB. However, the mechanism of extrusion remains poorly understood, which hinders the development of engineered bioremediation strategies. We report high-resolution cryo-EM structures of ArsB from the arsenic-tolerant bacterium Leptospirillum ferriphilum. ArsB adopts an inverted two-fold repeat architecture resembling that of other ion transporter (IT) superfamily proteins. Structures determined in the presence of As(III) and antimonite [Sb(III)] reveal that […]
- by Mestre, A. A., Oh, Y., Wu, J., Dunn, D., Setayeshpour, Y., Chen, S.-Y., Lin, C.-C., Cochrane, C. S., Jeong, P., Nam, G., Markey, C., Reker, D., Floyd, S. R., Hong, J., Zhou, P., Chi, J.-T. A.Ferroptosis is a regulated form of cell death driven by iron-dependent lipid peroxidation and contributes to diverse pathologies including ischemia-reperfusion injury and neurodegenerative disorders. Current ferroptosis inhibitors largely function as nonspecific radical-trapping antioxidants, limiting their clinical utility. We previously identified MESH1 as a key regulator of ferroptosis through its NADPH phosphatase activity. Here, we identify 4,5,6,7-tetrabromo-1H-benzotriazole (TBB) as a small molecule inhibitor of MESH1 with an IC50 value of 4.7 {+/-}0.3 M. X-ray crystallography revealed the molecular determinants of TBB […]
- by Bhattacharya, S., Sutter, B. M., Tu, B.Adaptive transcriptional rewiring underlies the metabolic flexibility of Saccharomyces cerevisiae. We demonstrate that the histone deacetylase Rpd3 mediates nutrient-dependent chromatin reprogramming that coordinates transcriptional shutdown and global acetylation balance during metabolic transitions. Genome-wide analyses reveal that Rpd3 complexes drive rapid, reversible histone deacetylation across promoters and gene bodies, fine-tuning transcriptional output. Rpd3, primarily through the large complex (Rpd3L), localizes at promoters of active genes enriched in H3K9ac and the acetyltransferase Gcn5. Upon nutrient shift, Gcn5 disengages while Rpd3-mediated H3K9 deacetylation […]
- by Perciaccante, A. J., Rogers, H. T., Zhu, Y., Barnwal, A., Inman, D., Wang, M.-D., Jin, S., Ponik, S. M., Ge, Y.Small extracellular vesicles (sEVs) are membrane-bound particles whose protein, lipid, and metabolite cargo reflects the molecular state of their cells of origin, making them attractive targets for biomarker discovery and therapeutic development. However, comprehensive characterization of sEVs remains challenging due to the extremely limited material available. Here, we present an integrated mass spectrometry-based multi-omics platform for simultaneous characterization of lipids, metabolites, and proteins from a single sEV sample enabled by sequential extraction, maximizing sample utilization. To enhance molecular coverage and […]
- by Fairchild, D., Semenova, I. V., Geddes-Buehre, D., Li, Y., Szczepaniak, R., Weller, S. K., Hao, B., Bezsonova, I.The promyelocytic leukemia protein (PML) is a stress-response factor that assembles into PML nuclear bodies, dynamic subnuclear compartments involved in tumor suppression and antiviral defense. The most abundant isoform, PML-1, has been linked to transcriptional regulation, genome stability, and antiviral responses, yet the molecular basis of these functions remains unclear. Here, we report that PML-1 contains a unique nucleic acid-binding module, PXL, and determine its three-dimensional structure by X-ray crystallography. Further biochemical, mutational, and cellular analyses, including RNA-seq, demonstrate that […]
- by Li, C. Y., Grob, A., Repa, L., Huxley, O., Brotherton, D. H., Becker, P., Dadzie, R., Beckstein, O., Cameron, A. D.ASBT and NTCP are sodium-coupled secondary transporters of the SLC10 family that play critical roles in the enterohepatic recycling of bile acids. Secondary transporters generally function through the alternating access mechanism. However, in outward-facing structures of NTCP this mechanism is violated with a pore running through the protein. No such pore is observed in previously reported structures of bacterial ASBT homologues, but these proteins have an additional transmembrane-helix adjacent to the binding site. Here we investigate another homologue of ASBT […]
- by Jiang, H., Song, Y., Hao, Y., Latacz, M., Cykowiak, M., Kirylczuk, J., Quan, X., Palomba, F., Ni, S., Liu, L., Hu, J., Shi, B., Posey, A., Li, Q., Yuan, H., Sun, J., Pappu, R., Digman, M., Huang, K.The pathological mechanisms and significance for the prevalent hotspot mutations in disordered protein regions are poorly understood. ASXL1 is an obligate co-factor for BAP1 in H2AK119 deubiquitination. ASXL1 mutations are very frequent in myeloid malignancies, and are mostly C-terminal truncating mutations concentrated in a specific disordered region of ASXL1. ASXL1 truncations are gain-of-function mutations that promote myeloid malignancies, but the underlying mechanisms remain poorly understood. Here we show that the frequently truncated mutants of ASXL1 possess an intrinsic property of […]
- by Ernst, L., Lumbantobing, T., Barlow, C., Todd, J., Orsi, W. D., Greening, C.Dimethyl sulfide (DMS) and dimethyl sulfoxide (DMSO) have important microbial-driven roles in global nutrient cycling, chemotaxis and/or climate regulation. Microbial DMSO and DMS assimilation were thought to require their enzymatic reduction to methanethiol. However, reactive oxygen species were recently shown to mediate methane production from DMS and DMSO. Here, we show that this oxidative mechanism can sustain microbial growth without the need for enzymes to directly reduce DMS or oxidize DMSO. Both, light and heat were effective geochemical drivers of […]
- by Palmer, D., Chauvier, A., Silva, T. F. D., Ontiveros, A., Bussi, G., Walter, N. G., Mishanina, T. V.The widespread yybP-ykoY riboswitches control bacterial manganese (Mn) homeostasis by activating exporter expression in response to intracellular Mn2+ levels. The E. coli alx riboswitch distinctively couples Mn2+ sensing to cytoplasmic alkalinity, but the mechanism is unknown. We show that pH tunes the alx aptamer's conformational sampling to modulate Mn2+ sensitivity. Single-molecule FRET reveals that Mn2+ stabilizes a docked three-way-junction conformation, and alkaline pH shifts this equilibrium to sensitize metal-dependent folding. Molecular dynamics simulations identify a loop whose low-pH-induced base pairing […]
- Reconstitution of Ras-PI3Kγ membrane communication and feedback using light-induced signaling inputsby Doerr, S., Olavarrieta-Colasurdo, A., Hansen, S. D.Biochemical reactions involving phosphatidylinositol phosphate (PIP) lipids and small GTPases serve essential roles in signal transduction immediately downstream of cell surface receptor activation. Interconnected positive and negative feedback loops link these distinct classes of reactions and allow for the rapid establishment of the steep and precisely oriented intracellular activity gradients that are hallmarks of cell polarity. Although genetic approaches have identified numerous molecules that potentially regulate feedback between GTPases and PIP lipids in cells, it is unclear how different types […]
- by Pattani Ameerjan, A. B., Dabirmanesh, B., Hungerland, J., Kasahara, T., Bartoelke, R., Dautaj, G., Saberamoli, G., Schmidt, J., Xu, J., Solov'yov, I., Koch, K.-W., Mouritsen, H.Cryptochromes and photolyases are blue-light-sensitive flavoproteins that generally bind flavin adenine dinucleotide (FAD) and have distinct functions. Cryptochrome 4a (CRY4a) is a protein expressed in the double-cone photoreceptors of the retina in migratory songbirds like European robin (Erithacus rubecula) and is hypothesized as the primary sensor for avian magnetoreception. In addition to FAD, most photolyases and some cryptochromes bind antenna chromophores such as 8-hydroxy-5-deazaflavin (8-HDF) or 5,10-methenyltetrahydrofolate (MTHF) to enhance light absorption. Here, we investigated whether Erithacus rubecula Cryptochrome 4a […]
- by van Dinther, M., Schwartze, T., Zhang, J., Fan, K., van der Zon, G., Power, L., Hinck, C., Cianca, C., Mukundan, A., Gonzalez Prieto, R., van Veelen, P. A., Maizels, R. M., Hinck, A. P., ten Dijke, P.Heligmosomoides polygyrus, a mouse parasite, modulates host immunity by secreting modular transforming growth factor-{beta} (TGF{beta}) mimics (TGMs). The agonist TGM1 interacts with TGFBR1, TGFBR2, and the co-receptor CD44 through domains D1/2, D3, and D4/5, respectively. In contrast, the antagonist TGM6, which lacks D1/2, but retains TGFBR2 binding through D3, targets different subsets of cells compared to TGM1. The TGM6 co-receptor is unknown. Using X-ray crystallography and binding studies, we show that TGM6 preferentially binds mouse TGFBR2 over human TGFBR2, and […]
- by Wake, N., Alcalde, J., Jutzi, D., Bajaj, A., Kour, S., Barai, M., Weng, S.-L., Cummings, S., Zheng, T., Anderson, E. N., Wang, S.-H., Puterbaugh, R. Z., Bosco, D. A., Schuster, B. S., Mittal, J., Pandey, U. B., Ruepp, M.-D., Fawzi, N. L.The RNA-binding protein Fused in Sarcoma (FUS) undergoes phase separation associated with RNA processing. However, the prion-like low complexity (LC) domain of FUS forms solid-like aggregates in neurodegenerative diseases. Whether the formation of {beta}-sheet structure associated with pathology is also physiologically/functionally relevant is debated. Similarly, if mislocalization alone or concomitant aggregation is responsible for FUS gain-of-function toxicity remains to be probed. Here, we introduce {beta}-sheet breaking proline residues into FUS LC with the goal of preventing cross-{beta}-driven aggregation without disrupting […]
- by Perry, B. C., Kim, J., Romero, P. A.AlphaFold 3 (AF3) enables accurate biomolecular modeling but is limited by slow, CPU-bound multiple sequence alignment (MSA) generation. We introduce AlphaFast, a drop-in framework that integrates GPU-accelerated MMseqs2 sequence search to remove this bottleneck. AlphaFast achieves a 68.5x speedup in MSA construction and a 22.8x reduction in end-to-end runtime on a single GPU, and delivers predictions in 8 seconds per input on four GPUs while maintaining indistinguishable structural accuracy. A serverless deployment enables structure prediction for as little as $0.035 […]
- by Yao, X., He, L., Yoo, S., Sun, H., Pathakota, V., Kaur, M., Li, P., Alba, B.Six-transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) has emerged as a promising therapeutic target for prostate cancer. We have optimized the expression and purification conditions of human STEAP1 to maximize the production of its homotrimeric form, which is crucial for metal ion reduction and maintaining cellular redox balance. Proteins obtained from these optimized conditions were complexed with both heme and flavin-adenine dinucleotide (FAD), two cofactors that are fundamental to STEAP functionality, suggesting native folding and interactions of the protein. […]
- by Vazquez Nunez, R. J., Kesha, S., Vos, S. M.Promoter proximal pausing by RNA polymerase II is critical for regulating gene expression in multicellular eukaryotes. How nucleic acid sequence and protein factors contribute to pausing remains incompletely understood. We developed Gene-specific Analysis of Transcriptional Output (GATO)-seq, which for the first time enables massively parallel, temporally resolved, reconstituted transcription in an assay that uses direct RNA sequencing to map 3' ends of nascent transcripts from a library of human genes. GATO-seq identified a "super pause" sequence that potently induces RNA […]
- by Chisholm, T. S.Fibrillar protein aggregates are a defining feature of neurodegenerative diseases and are attractive biomarkers and therapeutic targets. However, rational ligand design is limited by a poor mechanistic understanding of fibril binding. This work demonstrates that high-affinity binding to amyloid fibrils occurs via two topologically distinct binding modes, informing design changes that enhance ligand binding. Mathematical models were outlined that demonstrate these binding modes can be distinguished using diagnostic features from standard binding assays. Reanalysis of published binding data indicates that […]
- by Schurman, C. A., Chandler, W., Hu, D., Taylor, H., Tao, N., Miclau, T., Angel, P., Marcucio, R., Schilling, B.Age-related delays in fracture healing are prevalent and contribute to morbidity and mortality in elderly populations. Clinical and preclinical studies demonstrate that aging is associated with slower and less complete fracture repair characterized by delayed cartilage and bone formation, impaired matrix resorption, and an increased risk of delayed union or non-union. Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI MSI) enables spatially resolved, in situ molecular analysis of proteins directly from murine fracture tissues. We applied collagenase type III (MMP-13) mediated […]
- by Mouannes, N., Burrows, A. C., Horak, A. J., Venkateshwari, V., Dutta, S., Mahen, K., Banerjee, R., Massey, W. J., Ye, X., Mrdjen, M., Brown, A. L., Awoniyi, M., Kitao, K., Sandhu, A., Tomimoto, C., Tsuji, K., Yonejima, Y., Ampong, I., Zhang, R., Qiu, Y., Willard, B., Hajjar, A. M., Dwidar, M., Sangwan, N., Walker, M. E., Spite, M., Cheng, F., Brown, J. M.Following a meal, our gut microbiome and human cells collaborate via metaorganismal metabolic circuits to produce diverse nutrient metabolites that systemically circulate to influence health and disease. Although there are now several examples of bacterial fiber-, amino acid-, and micronutrient-derived metabolites impacting cardiometabolic disease, very little is known in regards to how diet-microbe-host interactions impact lipid homeostasis. Here we address this by defining dietary fatty acid substrate availability in germ-free versus conventionally-raised mice coupled to deep multi-omic metabolic phenotyping. Our […]
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