- by Mierzejewski, V., Mathis, C., Kempes, C. P., Walker, S.The predictability of biochemical evolution remains highly debated, as it is unknown if biological function can be predicted a priori. Here, we explore predictability of function not in individual reactions, but in ensembles. Sampling from 13,777 bacterial, 371 archaeal, and 203 eukaryotic genomes, we use Enzyme Commission (EC) classification to hierarchically group enzyme-catalyzed reactions that perform similar transformations into functional equivalence classes. We show that organisms partition their reaction diversity among these functional equivalence classes in a predictable way, identifying […]
- by Movassaghi, C. S., Meyer, J. G.Antibiotics are routinely added to mammalian cell culture media to prevent bacterial growth. However, the use of antibiotics in cell culture can confound downstream experimental results. While genomic and transcriptomic differences between cell cultures treated with and without antibiotics are well-documented, far fewer, if any, comprehensive proteomic comparisons on the use of antibiotics in cell culture have been performed. Here, we present a study on the proteome-wide differences of culturing HepG2 cells in antibiotic (i.e., penicillin/streptomycin) and non-antibiotic-containing media. Using […]
- by Yuan, S., Abdelrahman, S., Garcia-Vazquez, N., Nada, H., Calvo Barreiro, L., Kuncewicz, K., Gabr, M.The SLIT2/ROBO1 signaling axis plays a critical role in neural development, immune regulation, and tumor progression, including glioblastoma. However, small molecule inhibitors targeting this protein-protein interaction remain unexplored. Herein, we report the discovery and validation of DEL-S1, a first-in-class small molecule that binds to SLIT2 and disrupts its interaction with ROBO1. Using a DNA-encoded library (DEL) screen of 4.2 billion compounds, DEL-S1 was identified and confirmed to bind SLIT2 via temperature related intensity change (TRIC) assay. Functional inhibition of the […]
- by LaBrant, E. W., Smith, C. N., Torres-Gerena, A. D., Ishimwe, J., Huang, F., Tullis, A., Litterer, L., Modi, B., Naldrett, M., Altartouri, B., Roston, R. L.Biogenesis and maintenance of the photosynthetic thylakoid membrane requires transport of lipids from their site of synthesis in the chloroplast envelopes to their destination in the thylakoid. While vesicle trafficking is likely involved, we hypothesized a complementary mechanism involving direct membrane interactions. Using domain homology and proteomic profiling of chloroplast membrane fractions, we identified candidate lipid transport proteins enriched in a distinct, intermediate-density membrane population. This fraction was enriched in lipid metabolic enzymes and proteins homologous to known membrane organization […]
- by Ward, A., Baeza-Ballesteros, L. J., Schuck, R. J., Garcia-Murria, M. J., Lamichhane, R., Mingarro, I., Barrera, F. N.ROR1 is a member of the receptor tyrosine kinase (RTK) family that plays a crucial role during organogenesis of bone and neural systems by regulating non-canonical Wnt signaling. Misregulation of ROR1 is additionally a causative factor for carcinogenesis in solid and liquid tumors. However, we have a poor understanding of how ROR1 activity is regulated. We employed a recently developed single-molecule method termed SiMPull-POP to study the oligomeric state of ROR1. RTK function is typically triggered by ligand binding, which […]
- by Lin, H., Wang, J.Covalent inhibitors are an emerging class of therapeutics, but methods to comprehensively profile their binding kinetics and selectivity across the proteome have been limited. Here we introduce COOKIE-Pro (COvalent Occupancy KInetic Enrichment via Proteomics), an unbiased method for quantifying irreversible covalent inhibitor binding kinetics on a proteome-wide scale. COOKIE-Pro uses a two-step incubation process with mass spectrometry-based proteomics to determine kinact and KI values for covalent inhibitors against both on-target and off-target proteins. We validated COOKIE-Pro using the BTK inhibitors […]
- by Block, S., Chi, F., Rosen, P. C., Pineda, S. S., Darnell, A. M., Abbott, K. L., Pena, I. A., Heiman, M., Yilmaz, O. H., Kory, N., Vander Heiden, M. G.Mitochondria contribute to compartmentalized metabolism in eukaryotic cells, supporting key enzymatic reactions for cell function and energy homeostasis. However, this compartmentalization necessitates regulated metabolite transport across mitochondrial membranes. Although many transport proteins have been identified, several mitochondrial amino acid transporters remain largely uncharacterized. Using CRISPR-Cas9-mediated candidate transporter knockouts coupled with assessment of metabolite transport via a mitochondrial swelling assay, we identify SFXN1, previously characterized for its role in mitochondrial serine transport, as a protein that mediates mitochondrial transport of a […]
- by D'Andrea, S., Schmidt, E. N., Bui, D., Singh, O., Han, L., Mahal, L. K., Klassen, J. S., Macauley, M. S., fadda, e.Sialic acid-binding immunoglobulin-type lectins (Siglecs) regulate immune response through interactions with sialylated glycan epitopes on glycoproteins and glycolipids. Human Siglecs count 14 unique proteins allocated to two subsets; group 1 includes four evolutionarily conserved proteins with clear orthologues in all mammals with low sequence similarity and group 2 includes ten rapidly evolving proteins with high sequence similarity and species specificity. In all Siglecs, the recognition and binding of the sialic acid on the glycan target involves a conserved, or canonical, […]
- by Lemos, P., Beckwith, Z., Bandi, S., Van Damme, M., Crivelli-Decker, J., Shields, B. J., Merth, T., Jha, P. K., De Mitri, N., Callahan, T. J., Nish, A., Abruzzo, P., Salomon-Ferrer, R., Ganahl, M.Accurate prediction of protein-ligand binding affinities remains a cornerstone problem in drug discovery. While binding affinity is inherently dictated by the 3D structure and dynamics of protein-ligand complexes, current deep learning approaches are limited by the lack of high-quality experimental structures with annotated binding affinities. To address this limitation, we introduce the Structurally Augmented IC50 Repository (SAIR), the largest publicly available dataset of protein-ligand 3D structures with associated activity data. The dataset comprises 5,244,285 structures across 1,048,857 unique protein-ligand systems, […]
- by Ladke, J. S., Khan, A., Singh, A., Singh, J., Steck, N., Jessen, H., Kolthur-Seetharam, U., Jaiswal, M., Bhandari, R.Inorganic polyphosphate (polyP) is a linear polymer of varying length composed of phosphate residues linked by phosphoanhydride bonds. PolyP remains poorly understood in mammals due to its low abundance and lack of information on its synthesis and regulation. Using synthetic polyP8 as a bait, we identified polyP interacting proteins involved in RNA splicing, transcription, and mitochondrial function. We developed a DAPI fluorescence-based assay to quantify the low levels of polyP present in mammalian cell lines and tissues, and detected a […]
- by Padilla-Garfias, F., Poot-Hernandez, A. C., Araiza-Villanueva, M., Calahorra, M., Sanchez, N. S., Pena, A.The environmental accumulation of polycyclic aromatic hydrocarbons (PAHs), such as benzo(a)pyrene (BaP), poses significant threats to ecosystems and public health due to their persistent nature, mutagenic potential, and well-documented carcinogenicity. In this study, we investigated the ability of the extremophilic yeast Debaryomyces hansenii to activate specialized detoxification mechanisms for BaP degradation, even under nutrient-deprived conditions. When exposed to 100 ppm BaP, D. hansenii eliminated over 70% of the contaminant within three days while maintaining normal growth dynamics. RNA-Seq analysis revealed […]
- by Aburto, C., Ruminot, I., San Martin, A.Oxidative stress reprograms metabolic flux from glycolysis to the pentose phosphate pathway. Recently, it has been proposed that NADPH acts as a key molecule in pentose phosphate pathway regulation by exerting negative feedback through tonic inhibition of glucose-6-phosphate dehydrogenase. Interestingly, recent studies show that NADPH levels remain stable during acute exposure to hydrogen peroxide in the presence of glucose, ruling out NADPH-dependent feedback inhibition. We hypothesize that hydrogen peroxide triggers a feedforward activation mechanism, increasing NADPH production even before any […]
- by Cabana, V. C., Bouchard, A. Y., Senecal, A. M., Cappadocia, L., Lussier, M. P.Mucopolysaccharidosis type II, also known as Hunter syndrome, is a rare and fatal disease caused by mutations in the iduronate 2-sulfatase (IDS) encoding gene. The enzymatically inactive variant proteins lead to pathological accumulation of glycosaminoglycans in the lysosomes, causing dysfunction in multiple organs. IDS is expressed as a precursor protein, and its processing and lysosomal targeting are crucial for proper enzymatic activity. However, IDS intracellular dynamic is poorly understood and a better understanding of its processing mechanisms would benefit the […]
- by Munoz-Estrada, J., Mostafania, A., Halwatura, L., Haghani, A., Jiang, Y., Meyer, J. G.iPSC-derived neuronal cultures can provide valuable insights into the pathogenesis of neurological disease. However, single-cell iPSC clones expressing NGN2 and mCherry exhibit spontaneous loss of mCherry fluorescence, raising questions about the homogeneity of neurons derived from what appear to be heterogeneous iPSCs. We find that mCherry silencing does not influence iNeurons with two lines of evidence. First, using single-cell proteomics, we found that spontaneous mCherry silencing does not drive heterogeneity in iPSCs. Second, bulk proteomics and immunofluorescence analysis indicated that […]
- by Samanta, A., Malle, M. G., Tsang, E., Omer, M., Skaanning, M. K., Kjems, J., Gothelf, K. V.Inspired by the natural ability of bacteriophages to deliver genetic material directly into host cells, we employed a bottom-up approach to construct a multifunctional synthetic DNA origami needle-like structure. This origami is functionalized with trastuzumab antibodies, cholesterol, protective polymers, and two dyes, which together enable selective targeting and insertion into SKBR3 cancer cells. A disulfide-linked dye payload was attached to the apex of the needle, allowing controlled release in the cytoplasm triggered by the high intracellular glutathione concentration. Real-time tracking […]
- by Stuteley, S. M., Chen, J., Wang, J., Dawes, S. S., Baker, E. N., Squire, C. J., Pandelia, M.-E., Bashiri, G.Iron-sulfur (Fe-S) clusters are ubiquitous cofactors in biological systems. Given their central role in bacterial metabolism and pathogenesis, the biogenesis of Fe-S clusters is tightly controlled. We reveal a feedback regulatory mechanism involving the sulfide producing SufS/SufU complex within the sulfur utilization (SUF) system of Mycobacterium tuberculosis, the bacterium that causes tuberculosis. In this mechanism, [2Fe-2S] clusters compete with zinc ions for binding to the sulfide transfer protein SufU. Cluster binding induces SufU tetramerization, which prevents its interaction with the […]
- by Liu, Y., Paulson, J. C., Hoskins, A. A.Precise recognition of the boundaries between exons and introns (splice sites, SS) is essential for the fidelity of gene expression. In contrast with the 5'SS, the consensus 3'SS sequence in both S. cerevisiae and humans is just three nucleotides long: YAG. How the correct 3'SS is chosen among many possible alternates by the spliceosome is often unclear but likely involves proofreading by the Prp22 ATPase. In cryo-EM structures of spliceosome product (P) complexes, glutamine 1594 in the highly conserved -finger […]
- by Omini, J., Krassovskaya, I., Dele-Osibanjo, T., Pedersen, C., Obata, T.The tricarboxylic acid (TCA) cycle enzymes, malate dehydrogenase (MDH1) and citrate synthase (CIT1), form a multienzyme complex called metabolon that channels intermediate, oxaloacetate, between the reaction centers of the enzymes. Since the MDH1-CIT1 metabolon enhances the pathway reactions in vitro, it is postulated to regulate the TCA cycle flux through dynamic assembly in response to cellular metabolic demands. Here, we demonstrated that yeast mitochondrial MDH1 and CIT1 dissociated when aerobic respiration was suppressed by the Crabtree effect and associated when […]
- by Crook, O.Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful technique to probe changes in protein structural dynamics. In differential settings, HDX-MS compares dynamics between protein states, such as conformational changes resulting from antibody-antigen binding or the effects of mutations. As the method becomes more high-throughput, the number of comparisons between peptides and states grows, creating a multiple hypothesis testing challenge where some observed changes may result from statistical randomness rather than biological differences. While this problem can be addressed by […]
- by Torres Ortega, L. R., Dietrich, J., Wandy, J., Mol, H., van der Hooft, J. J. J.Untargeted mass spectrometry measures many spectra of unknown molecules. Tandem mass spectrometry (MS/MS) generates fragmentation patterns representing common substructures termed Mass2Motifs. However, MS/MS-based substructure identification is limited by computational efficiency and interpretability. Here, we introduce a complete overhaul of MS2LDA, overcoming limitations of its predecessor. We report an up to 14x speed improvement for unsupervised Mass2Motif discovery, allowing it to process larger datasets. Furthermore, the new automated Mass2Motif Annotation Guidance (MAG) aids in structurally identifying Mass2Motifs. Using three curated MotifDB-MotifSets […]
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