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Molecular Cell Jul 2023RNA-binding proteins (RBPs) control RNA metabolism to orchestrate gene expression and, when dysfunctional, underlie human diseases. Proteome-wide discovery efforts...
RNA-binding proteins (RBPs) control RNA metabolism to orchestrate gene expression and, when dysfunctional, underlie human diseases. Proteome-wide discovery efforts predict thousands of RBP candidates, many of which lack canonical RNA-binding domains (RBDs). Here, we present a hybrid ensemble RBP classifier (HydRA), which leverages information from both intermolecular protein interactions and internal protein sequence patterns to predict RNA-binding capacity with unparalleled specificity and sensitivity using support vector machines (SVMs), convolutional neural networks (CNNs), and Transformer-based protein language models. Occlusion mapping by HydRA robustly detects known RBDs and predicts hundreds of uncharacterized RNA-binding associated domains. Enhanced CLIP (eCLIP) for HydRA-predicted RBP candidates reveals transcriptome-wide RNA targets and confirms RNA-binding activity for HydRA-predicted RNA-binding associated domains. HydRA accelerates construction of a comprehensive RBP catalog and expands the diversity of RNA-binding associated domains.
Topics: Animals; Humans; RNA; Protein Binding; Binding Sites; Hydra; Deep Learning
PubMed: 37421941
DOI: 10.1016/j.molcel.2023.06.019 -
International Journal of Molecular... Jun 2023Protein self-association is a biologically remarkable event that involves and affects the structural and functional properties of proteins [...].
Protein self-association is a biologically remarkable event that involves and affects the structural and functional properties of proteins [...].
Topics: Proteins; Protein Binding; Protein Multimerization; Protein Conformation
PubMed: 37445826
DOI: 10.3390/ijms241310648 -
Journal of Nuclear Medicine : Official... Sep 2023Radiolabeled fibroblast activation protein (FAP) inhibitors (FAPIs) have shown promise as cancer diagnostic agents; however, the relatively short tumor retention of...
Radiolabeled fibroblast activation protein (FAP) inhibitors (FAPIs) have shown promise as cancer diagnostic agents; however, the relatively short tumor retention of FAPIs may limit their application in radioligand therapy. In this paper, we report the design, synthesis, and evaluation of a FAPI tetramer. The aim of the study was to evaluate the tumor-targeting characteristics of radiolabeled FAPI multimers in vitro and in vivo, thereby providing information for the design of FAP-targeted radiopharmaceuticals based on the polyvalency principle. FAPI tetramers were synthesized on the basis of FAPI-46 and radiolabeled with Ga, Cu, and Lu. In vitro FAP-binding characteristics were identified using a competitive cell-binding experiment. To evaluate their pharmacokinetics, small-animal PET, SPECT, and ex vivo biodistribution analyses were performed on HT-1080-FAP and U87MG tumor-bearing mice. In addition, the 2 tumor xenografts received radioligand therapy with Lu-DOTA-4P(FAPI), and the antitumor efficacy of the Lu-FAPI tetramer was evaluated and compared with that of the Lu-FAPI dimer and monomer. Ga-DOTA-4P(FAPI) and Lu-DOTA-4P(FAPI) were highly stable in phosphate-buffered saline and fetal bovine serum. The FAPI tetramer exhibited high FAP-binding affinity and specificity both in vitro and in vivo. Ga-, Cu-, and Lu-labeled FAPI tetramers exhibited higher tumor uptake, longer tumor retention, and slower clearance than FAPI dimers and FAPI-46 in HT-1080-FAP tumors. The uptake (percentage injected dose per gram) of Lu-DOTA-4P(FAPI), Lu-DOTA-2P(FAPI), and Lu-FAPI-46 in HT-1080-FAP tumors at 24 h was 21.4 ± 1.7, 17.1 ± 3.9, and 3.4 ± 0.7, respectively. Moreover, Ga-DOTA-4P(FAPI) uptake in U87MG tumors was approximately 2-fold the uptake of Ga-DOTA-2P(FAPI) (SUV, 0.72 ± 0.02 vs. 0.42 ± 0.03, < 0.001) and more than 4-fold the uptake of Ga-FAPI-46 (0.16 ± 0.01, < 0.001). In the radioligand therapy study, remarkable tumor suppression was observed with the Lu-FAPI tetramer in both HT-1080-FAP and U87MG tumor-bearing mice. The satisfactory FAP-binding affinity and specificity, as well as the favorable in vivo pharmacokinetics of the FAPI tetramer, make it a promising radiopharmaceutical for theranostic applications. Improved tumor uptake and prolonged retention of the Lu-FAPI tetramer resulted in excellent characteristics for FAPI imaging and radioligand therapy.
Topics: Humans; Animals; Mice; Tissue Distribution; Gallium Radioisotopes; Neoplasms; Protein Binding; Biological Transport; Positron Emission Tomography Computed Tomography
PubMed: 37321827
DOI: 10.2967/jnumed.123.265599 -
Biomolecules Apr 2024Tumor necrosis factor receptor-associated factor (TRAF) proteins play pivotal roles in a multitude of cellular signaling pathways, encompassing immune response, cell... (Review)
Review
Tumor necrosis factor receptor-associated factor (TRAF) proteins play pivotal roles in a multitude of cellular signaling pathways, encompassing immune response, cell fate determination, development, and thrombosis. Their involvement in these processes hinges largely on their ability to interact directly with diverse receptors via the TRAF domain. Given the limited binding interface, understanding how specific TRAF domains engage with various receptors and how structurally similar binding interfaces of TRAF family members adapt their distinct binding partners has been the subject of extensive structural investigations over several decades. This review presents an in-depth exploration of the current insights into the structural and molecular diversity exhibited by the TRAF domain and TRAF-binding motifs across a range of receptors, with a specific focus on TRAF1.
Topics: Humans; TNF Receptor-Associated Factor 1; Animals; Protein Binding; Signal Transduction; Protein Domains; Models, Molecular
PubMed: 38785916
DOI: 10.3390/biom14050510 -
Biomedicine & Pharmacotherapy =... Sep 2023Glucocorticoids (GCs), steroid hormones that depend on glucocorticoid receptor (GR) binding for their action, are essential for regulating numerous homeostatic functions... (Review)
Review
Glucocorticoids (GCs), steroid hormones that depend on glucocorticoid receptor (GR) binding for their action, are essential for regulating numerous homeostatic functions in the body.GR signals are biased, that is, GR signals are various in different tissue cells, disease states and ligands. This biased regulation of GR signaling appears to depend on ligand-induced metameric regulation, protein post-translational modifications, assembly at response elements, context-specific assembly (recruitment of co-regulators) and intercellular differences. Based on the bias regulation of GR, selective GR agonists and modulators (SEGRAMs) were developed to bias therapeutic outcomes toward expected outcomes (e.g., anti-inflammation and immunoregulation) by influencing GR-mediated gene expression. This paper provides a review of the bias regulation and mechanism of GR and the research progress of drugs.
Topics: Receptors, Glucocorticoid; Glucocorticoids; Anti-Inflammatory Agents; Signal Transduction; Protein Binding
PubMed: 37454592
DOI: 10.1016/j.biopha.2023.115145 -
Genes Nov 2023Translation initiation in eukaryotes is regulated at several steps, one of which involves the availability of the cap binding protein to participate in cap-dependent...
Translation initiation in eukaryotes is regulated at several steps, one of which involves the availability of the cap binding protein to participate in cap-dependent protein synthesis. Binding of eIF4E to translational repressors (eIF4E-binding proteins [4E-BPs]) suppresses translation and is used by cells to link extra- and intracellular cues to protein synthetic rates. The best studied of these interactions involves repression of translation by 4E-BP1 upon inhibition of the PI3K/mTOR signaling pathway. Herein, we characterize a novel 4E-BP, C8ORF88, whose expression is predominantly restricted to early spermatids. C8ORF88:eIF4E interaction is dependent on the canonical eIF4E binding motif (4E-BM) present in other 4E-BPs. Whereas 4E-BP1:eIF4E interaction is dependent on the phosphorylation of 4E-BP1, these sites are not conserved in C8ORF88 indicating a different mode of regulation.
Topics: Carrier Proteins; Eukaryotic Initiation Factor-4E; Phosphoproteins; Protein Binding; Phosphorylation
PubMed: 38003019
DOI: 10.3390/genes14112076 -
Journal of Chemical Theory and... Nov 2023Membrane proteins have diverse functions within cells and are well-established drug targets. The advances in membrane protein structural biology have revealed drug and... (Review)
Review
Membrane proteins have diverse functions within cells and are well-established drug targets. The advances in membrane protein structural biology have revealed drug and lipid binding sites on membrane proteins, while computational methods such as molecular simulations can resolve the thermodynamic basis of these interactions. Particularly, alchemical free energy calculations have shown promise in the calculation of reliable and reproducible binding free energies of protein-ligand and protein-lipid complexes in membrane-associated systems. In this review, we present an overview of representative alchemical free energy studies on G-protein-coupled receptors, ion channels, transporters as well as protein-lipid interactions, with emphasis on best practices and critical aspects of running these simulations. Additionally, we analyze challenges and successes when running alchemical free energy calculations on membrane-associated proteins. Finally, we highlight the value of alchemical free energy calculations calculations in drug discovery and their applicability in the pharmaceutical industry.
Topics: Membrane Proteins; Molecular Dynamics Simulation; Entropy; Thermodynamics; Ligands; Lipids; Protein Binding
PubMed: 37902715
DOI: 10.1021/acs.jctc.3c00365 -
Science (New York, N.Y.) Jun 2023The barbed and pointed ends of the actin filament (F-actin) are the sites of growth and shrinkage and the targets of capping proteins that block subunit exchange,...
The barbed and pointed ends of the actin filament (F-actin) are the sites of growth and shrinkage and the targets of capping proteins that block subunit exchange, including CapZ at the barbed end and tropomodulin at the pointed end. We describe cryo-electron microscopy structures of the free and capped ends of F-actin. Terminal subunits at the free barbed end adopt a "flat" F-actin conformation. CapZ binds with minor changes to the barbed end but with major changes to itself. By contrast, subunits at the free pointed end adopt a "twisted" monomeric actin (G-actin) conformation. Tropomodulin binding forces the second subunit into an F-actin conformation. The structures reveal how the ends differ from the middle in F-actin and how these differences control subunit addition, dissociation, capping, and interactions with end-binding proteins.
Topics: Actin Cytoskeleton; Actins; Cryoelectron Microscopy; Tropomodulin; CapZ Actin Capping Protein; Protein Binding; Single Molecule Imaging; Protein Conformation
PubMed: 37228182
DOI: 10.1126/science.adg6812 -
Nature Communications Jun 2023Altered protein phosphorylation in cancer cells often leads to surface presentation of phosphopeptide neoantigens. However, their role in cancer immunogenicity remains...
Altered protein phosphorylation in cancer cells often leads to surface presentation of phosphopeptide neoantigens. However, their role in cancer immunogenicity remains unclear. Here we describe a mechanism by which an HLA-B*0702-specific acute myeloid leukemia phosphoneoantigen, pMLL (EPR(pS)PSHSM), is recognized by a cognate T cell receptor named TCR27, a candidate for cancer immunotherapy. We show that the replacement of phosphoserine P with serine or phosphomimetics does not affect pMHC conformation or peptide-MHC affinity but abrogates TCR27-dependent T cell activation and weakens binding between TCR27 and pMHC. Here we describe the crystal structures for TCR27 and cognate pMHC, map of the interface produced by nuclear magnetic resonance, and a ternary complex generated using information-driven protein docking. Our data show that non-covalent interactions between the epitope phosphate group and TCR27 are crucial for TCR specificity. This study supports development of new treatment options for cancer patients through target expansion and TCR optimization.
Topics: Humans; Phosphopeptides; Receptors, Antigen, T-Cell; Protein Binding
PubMed: 37353482
DOI: 10.1038/s41467-023-39425-1 -
Journal of Muscle Research and Cell... Sep 2023The techniques of X-ray protein crystallography, NMR and high-resolution cryo-electron microscopy have all been used to determine the high-resolution structure of... (Review)
Review
The techniques of X-ray protein crystallography, NMR and high-resolution cryo-electron microscopy have all been used to determine the high-resolution structure of proteins. The most-commonly used method, however, remains X-ray crystallography but it does rely heavily on the production of suitable crystals. Indeed, the production of diffraction quality crystals remains the rate-limiting step for most protein systems. This mini-review highlights the crystallisation trials that used existing and newly developed crystallisation methods on two muscle protein targets - the actin binding domain (ABD) of α-actinin and the C0-C1 domain of human cardiac myosin binding protein C (cMyBP-C). Furthermore, using heterogenous nucleating agents the crystallisation of the C1 domain of cMyBP-C was successfully achieved in house along with preliminary actin binding studies using electron microscopy and co-sedimentation assays .
Topics: Humans; Actins; Muscle Proteins; Cryoelectron Microscopy; Protein Binding; Actinin
PubMed: 37133758
DOI: 10.1007/s10974-023-09648-2