- by Granados, A. J., Pratap, P. P., Alamo, K. A. E., Susa, K. J.Norrin is an atypical ligand that regulates retinal angiogenesis through the Wnt/beta-catenin pathway by triggering heterodimerization of Frizzled-4 (FZD4) with Low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6), leading to downstream beta-catenin stabilization. Unlike Wnt ligands, Norrin requires the tetraspanin Tspan12 for signaling amplification, but it is not understood why. Here, we report a 3.4-angstrom structure of Tspan12 in complex with FZD4 determined by cryo-electron microscopy. The structure reveals that FZD4 and Tspan12 form a direct complex in the absence […]
- by Garcia, E. M., Lavidor, O. R., Raval, S., Lue, N. Z., Liang, J. K., McFadden, M., Hwang, D. D., Zepeda, M. U., Chen, L. W., Carr, S. A., Papanastasiou, M., Liau, B. B.DNA methyltransferase 3A (DNMT3A) is a de novo DNA methyltransferase that is recurrently mutated in hematological malignancies and developmental disorders. The most prevalent mutation, R882H, compromises DNMT3A activity in a dominant-negative manner, but its precise biochemical mechanism has been debated. Here, we use paired deep mutational scanning of the wild-type and R882H-mutant proteins to systematically identify mutations on a massively parallel scale that modify DNMT3A activity by suppressing, phenocopying, or selectively rescuing the dominant-negative effect of R882H. By leveraging the […]
- by Garcia, F. C.The monosaccharide N-acetyl-glucosamine (GlcNAc), which dynamically modifies serine and threonine residues of nuclear and cytoplasmic proteins, is a key regulator of numerous biological processes. Investigating O-GlcNAc modification in vivo and in vitro remains challenging, making animal models essential for gaining powerful insights into this post-translational modification. In this study, we conducted a proteomic analysis for identifying O-GlcNAc-modified proteins in both early and adult larval stages of N2 wild-type Caenorhabditis elegans and in aex-3p::tau(V337M), a nematode model of ageing and Alzheimer's […]
- by Granat, J., Liu, S., Popoca, L., Oksuz, O., Reinberg, D.Polycomb Repressive Complex 2 (PRC2) facilitates the formation of facultative heterochromatin, instrumental to tissue specific gene expression. PRC2 catalyzes tri-methylation of lysine 27 of histone H3 (H3K27me3), which is targeted for chromatin compaction by PRC1. Importantly, PRC2-associated cofactors regulate its distinct activities, as in the case of MTF2 and JARID2 that direct PRC2 to specific chromatin nucleation sites based on preferred DNA-binding motifs. Here, we investigated EPOP whose role in regulating PRC2 was not well-defined. We find that both EPOP […]
- by Lall, S., Balaram, P., Mathew, M. K., Gosavi, S.The viral envelope-resident viral fusion proteins (VFPs) fuse the envelope with the host cell membrane, which allows virus entry into host cells. The homotrimeric class I VFPs (cI-VFPs) are anchored to the envelope by three single-pass transmembrane helices (spTMHs). It is generally accepted that the hydrophobic length of a TMH matches the thickness of the hydrophobic membrane core, and this reduces TMH dynamics by stabilizing both the TMHs and the membrane. However, to enable fusion, a cI-VFP undergoes a large […]
- by Hartley, A. D., Upadhyay, V., Boorla, V. S., Maranas, C. D.Despite the importance of understanding the step-by-step mechanism of enzymatically catalyzed reactions, fewer than one thousand cataloged mechanistic annotations can be found in the open literature and databases. Herein, we introduce MechFind, a computational tool that generates elementally and charge-balanced putative enzyme mechanisms. Unlike previous methods that require structural data or user-supplied active site residues, MechFind uses only the overall reaction stoichiometry as input, abstracting individual reaction steps as the gain or loss of chemical moieties. An optimization framework then […]
- by Wulf, A. M., Lutimba, S., Cameron, D., Desjardins, J., Shaw, T. J., Rossetti, L., Mansour, M., Liu, K. J.Mutations in anaplastic lymphoma kinase (ALK) are associated with high-risk neuroblastoma, a childhood cancer arising in trunk neural crest cells. The role of ALK in undifferentiated NC is still unknown; however, the presence of activating mutations in ALK correlates with migratory and invasive cell behaviours in neuroblastoma cell lines. Here, we show the functional consequences of ALK overexpression on neural crest cells, by comparing wildtype ALK (ALKWT) protein to ALK gain-of-function variants ALKF1174L and ALKR1275Q. Elevated ALK activity, independent of […]
- by Shin, J., Jaruga, P., Dizdaroglu, M., Kool, E. T.DNA in foods is a source of nucleotides that are salvaged by tissues as building blocks for chromosomal and mitochondrial DNA. A recent study provided preliminary evidence that high-temperature cooking damages the DNA in foods and suggested that certain forms of DNA damage can be taken up as nucleotides via metabolic salvage in cells and animals, directly incorporating genotoxic and mutagenic species into the host DNA. To assess potential risks, we surveyed DNA in 21 food ingredients, including plant- and […]
- by Mohammed, I., Mijatovic, E., Philipp, T. M., Janickova, L., Ascencao, K., Asturias, F. J., Szabo, C., Stahlberg, H., Majtan, T.Human cystathionine beta-synthase (CBS) is a vital enzyme that regulates sulfur amino acid metabolism, hydrogen sulfide production, and cellular redox balance. Using a multidisciplinary approach, we demonstrate that CBS functions as a filamentous morpheein, with its stability, turnover, and activity governed by dynamic quaternary structural transitions. Three distinct filamentous assemblies were resolved by cryo-EM and are mediated by the oligomerization loop (residues 516-525): (i) ligand-free trans-dimers that form trans-basal filaments with basal stability and activity, (ii) adenosylornithine-bound cis-dimers that assemble […]
- by Patel, K., Mohanty, B., Norman, A., Franck, C., Pachl, P., Yang, L., Jing, X., Reid, X. J., Walshe, J. L., Solomon, P., Tran, D. H., Ford, D., Low, J. K. K., Wilkinson-White, L., Passioura, T., Payne, R. J., Suga, H., Walport, L. J., Mackay, J. P.Achieving selective target inhibition is critical for minimising drug side effects. This can be especially challenging when targeting individual members of protein families with high sequence similarity. A well-recognised example is the Bromodomain and Extraterminal domain (BET) family of proteins. Chemical inhibition of the acetyllysine (AcK)-binding bromodomains (BDs) of BET proteins has shown considerable promise in a range of disease models. However, despite over a decade of medicinal chemistry efforts, it has proven challenging to develop BET BD inhibitors that […]
- by Oliveira-Filho, E. R., Van Gelder, K., Obe, D., Voiniciuc, C. R., Wilson, M. A., Hanson, A. D.Suicide Thi4 thiazole synthases are mononuclear metal enzymes that form the thiazole moiety of thiamin from NAD+, glycine, and a sulfur atom that is stripped from an active-site cysteine residue, causing enzyme inactivation. However, many prokaryotic Thi4 genes cluster on the chromosomal regions with genes encoding ThiS, ThiF, and other proteins that produce, relay, or use persulfide or thiocarboxylate sulfur. Such clusters include DUF6775 proteins, with domains of unknown function that feature a metal-binding motif. This comparative genomic evidence suggests […]
- by Krolak, P., Ribeiro, O., Gehl-Vaisanen, B., Hiltunen, M., Goldman, A., Vidilaseris, K.Acidocalcisomes are evolutionarily conserved acidic organelles that are rich in cations and inorganic phosphate, primarily polyphosphates. In kinetoplastid parasites, acidocalcisomes and their polyphosphate content are essential for osmoregulation and environmental adaptation during host switching. In this organelle, polyphosphate is synthesised and transported to the lumen by the vacuolar transporter chaperone (VTC) complex. Interestingly, unlike yeast VTC, which has five components, only two have been observed in kinetoplastids: Vtc1, which contains only a transmembrane domain and Vtc4, which, in addition to […]
- by Wu, Q., Cheng, L., Assmus, A. M., Zheng, X., Esteva Font, C., Petersen, C. C., Ding, X., Paludan, S. R., Fenton, R. A.Background Technological advancements in protein mass spectrometry have significantly enhanced analytical sensitivity and throughput, enabling single-cell proteomics by mass spectrometry (SCP-MS) to become reality. SCP-MS allows high-resolution analysis of cellular heterogeneity and function, bypassing bulk analysis limitations. Here, we used SCP-MS to document at the protein level the cellular diversity of mouse kidney cells. We further focused SCP-MS on low abundance distal convoluted tubule (DCT) cells that are essential for body electrolyte homeostasis and blood pressure control. Methods Mouse kidney […]
- by Oh, H., Bae, E.QatABCD is a widespread anti-phage defense system in prokaryotes comprising four protein components. QatC, a signature component, is homologous to QueC, an enzyme involved in nucleobase modification during queuosine biosynthesis. QatA and QatD are predicted to function as an ATPase and a nuclease, respectively, while QatB lacks identifiable sequence motifs. Here, we report the structural and functional characterization of QatB and QatC. We determined the structure of QatC bound to the ATP analog AMPPNP and performed structure-guided functional assays. We […]
- by Ma, Z., Bethel, N. P.Protein language models (pLMs) have the capacity to infer structural information from amino acid sequences. Evaluating the extent to which structural information they truly encode is crucial for assessing their generalizability and the interpretability of their latent representations, yet current approaches lack a model-free, quantitative framework to evaluate these encodings. We introduce RemoteFoldSet, a curated collection of protein sequence sets stratified by high structural similarity but minimal sequence identity. We also define the Structural Awareness (SA) score, a novel metric […]
- by Schnacke, P., Fottner, M., Kvasha, D., Fabian, W., Lang, K.Deciphering the ubiquitin code requires homogenous, site-specifically ubiquitylated proteins, yet their access remains difficult. Current chemical and enzymatic methods are often limited by harsh conditions, low yields, or the introduction of non-native scars. Here, we present UbyW (Ubiquitylation by UBE2W), a chemoenzymatic platform that overcomes these hurdles. By repurposing the E2 enzyme UBE2W to target a genetically encoded non-canonical amino acid, a near-native Ub-protein conjugate is formed with high efficiency and site-specificity. The method is broadly applicable to diverse proteins, […]
- by Zarges, C., Fieler, H., Rothemann, R. A., Poepsel, S., Jae, L. T., Riemer, J.The mitochondrial disulphide relay is the key machinery for import and oxidative protein folding in the mitochondrial intermembrane space. Among IMS proteins with unknown function, we identified FAM136A as a new substrate of the mitochondrial disulphide relay. We demonstrate a transient interaction between FAM136A and MIA40, and that MIA40 introduces four disulphide bonds in two twin-CX3C motifs of FAM136A. Consequently, IMS import of FAM136A requires these cysteines and its steady state levels in intact cells are strongly dependent on MIA40 […]
- by Konstantinou, A., Cordova-Perez, A., Varga, J. K., Madhu, P., Simonetti, L., Vieler, M., Ishimura, R., Lamoliatte, F., Schueler-Furman, O., Davey, N. E., Kulathu, Y., Ivarsson, Y.The ubiquitin-specific proteases (USPs) family is the largest family of human deubiquitinating enzymes (DUBs). While most USPs are agnostic to polyubiquitin linkage-type, their substrate specificity is thought to be mediated by the recognition of the ubiquitylated protein itself. In addition to their catalytic domain, USPs have one or more auxiliary domains (ADs) with key functions in regulating DUB activity and subcellular localization. We hypothesize that some ADs bind short linear motifs (SLiMs) typically found in intrinsically disordered regions of proteins […]
- by Ruan, M., Engels, S. M., Burroughs, M. R., Li, X., Stower, R., Tzadikario, T., Powell, C., Bloch, D., Fanari, O., Akeson, S., Eyler, D. E., Weidmann, C., Rouhanifard, S. H., Jain, M., Contreras, L. M., Koutmou, K. S.Pseudouridine ({Psi}) is an abundant post-transcriptional modification found across all classes of RNA. It has been widely speculated that {Psi} inclusion in mRNAs might provide an avenue for cells to control gene expression post-transcriptionally. Here we demonstrate that one of the principal mRNA pseudouridylating enzymes, pseudouridine synthase 7 (PUS7), exhibits a stress-induced accumulation in the cytoplasm of yeast and human epithelial lung cells. Stress-induced and cytoplasmic localization of PUS7 promote {Psi}-incorporation into hundreds of mRNA targets. Furthermore, engineered PUS7 cytoplasmic […]
- by Wu, L., Choi, Y.-M., Omrane, M., Chai, J., Gao, S., Thiam, A. R., Canals, D., Airola, M. V.Hereditary spastic paraplegia subtype SPG54 is a genetic neurological disorder caused by mutations in the DDHD2 gene. Excessive lipid droplet accumulation is observed in the brains of SPG54 patients and DDHD2 knockout mice, consistent with DDHD2s reported neutral lipase activity. Here, we find recombinant human DDHD2 preferentially hydrolyzes diacylglycerol (DAG) over phospholipids, with a slight preference for DAG over triacylglycerol (TAG). DDHD2 also exhibits transacylase activity, which enables transfer of acyl chains from triacylglycerols to diacylglycerols and monoacylglycerols to remodel […]
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