Furan warheads for covalent trapping of weak protein-protein interactions: specific cross-linking of thymosin β4 to actin
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion Lumos
ORGANISM(S): Oryctolagus Sp. 'rabbit_od'
SUBMITTER: 
Marina Gay
LAB HEAD: Annemieke Madder
PROVIDER: PXD025066 | Pride | 2021-05-13
REPOSITORIES: Pride
Similar Datasets
Project description:Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by the absence of a functional Survival of Motor Neuron 1 gene (SMN1). The nearly identical paralog, SMN2, cannot compensate for the loss of SMN1 because exon 7 is aberrantly skipped from most SMN2 transcripts, a process mediated by synergistic activities of Sam68/KHDR1 and hnRNP A1. This results in the production of a truncated, non-functional protein that is rapidly degraded. Here we present several crystal structures of Sam68 RNA-binding domain (RBD). Sam68-RBD forms stable symmetric homodimers by antiparallel association of helices α3 from two monomers. However, the details of domain organization and the dimerization interface differ significantly from previously characterized homologs. We demonstrate that Sam68 and hnRNP A1 bind proximal but distinct motifs within the central region of SMN2(ex7). Our findings have important implications for the etiology of SMA and open new avenues for the design of novel therapeutics to treat splicing diseases.
2024-05-23 | PXD033055 | Pride
Project description:The linear ubiquitin chain assembly complex (LUBAC) is the only known ubiquitin ligase that generates linear/Met1-linked ubiquitin chains. One of the LUBAC components, HOIL-1L, was recently shown to catalyse oxyester bond formation between the C-terminus of ubiquitin and some substrates. However, oxyester bond formation in the context of LUBAC has not been directly observed. We present the first 3D reconstruction of LUBAC obtained by electron microscopy and report its generation of heterotypic ubiquitin chains containing linear linkages with oxyester-linked branches. We found that addition of the oxyester-bound branches depends on HOIL-1L catalytic activity. We suggest a coordinated ubiquitin relay mechanism between the HOIP and HOIL-1L ligases supported by cross-linking mass spectrometry data, which show proximity between the catalytic RBR domains. Mutations in the linear ubiquitin chain-binding NZF domain of HOIL-1L reduces chain branching confirming its role in the process. In cells, these heterotypic chains were induced by TNF. In conclusion, we demonstrate that LUBAC assembles heterotypic ubiquitin chains with linear and oxyester-linked branches by the concerted action of HOIP and HOIL-1L.
2021-06-28 | PXD019771 | Pride
Project description:In higher plants, a P-type proton pumping ATPase generates the proton-motive force essential for the function of all other transporters and for proper growth and development. X-ray crystallographic studies of the plant plasma membrane proton pump have provided information on amino acids involved in ATP catalysis but provided no information on the structure of the C-terminal regulatory domain. Despite progress in elucidating enzymes involved in the signaling pathways that activate or inhibit this pump, the site of interaction of the C-terminal regulatory domain with the catalytic domains remains a mystery. Genetic studies have pointed to amino acids in various parts of the protein that may be involved but direct chemical evidence for which ones are specifically interacting with the C-terminus is lacking. In this study we used in vivo cross-linking experiments with a photo-reactive unnatural amino acid, p-benzoyl-phenylalanine (BPA) and tandem mass spectrometry, to obtain direct evidence that the carboxyl terminal regulatory domain directly interacts with amino acids located within the N-terminal actuator domain. Our observations are consistent with a mechanism in which intermolecular, rather than intramolecular, interactions are involved. Our model invokes a ‘head-to-tail’ organization of ATPase monomers in which the carboxyl terminal domain of one ATPase molecule interacts with the actuator domain of another ATPase molecule. This model serves to explain why cross-linked peptides are found only in dimers and trimers, and is consistent with prior studies suggesting that within the membrane, the protein can be organized as homopolymers, including dimers, trimers and hexamers.
2018-09-24 | PXD010892 | Pride
Project description:Protein expression is regulated by production and degradation of mRNAs and proteins, but their specific relationships remain unknown. We combine measurements of protein production and degradation and mRNA dynamics to build a quantitative genomic model of the differential regulation of gene expression in LPS stimulated mouse dendritic cells. Changes in mRNA abundance play a dominant role in determining most dynamic fold changes in protein levels. Conversely, the preexisting proteome of proteins performing basic cellular functions is remodeled primarily through changes in protein production or degradation, accounting for over half of the absolute change in protein molecules in the cell. Thus, the proteome is regulated by transcriptional induction of novel cellular functions and remodeling of preexisting functions through the protein life cycle. Mouse primary dendritic cells were treated with LPS or mock stimulus and profiled over a 12-hour time course. Cells were grown in M-labeled SILAC media, which was replaced with H-labeled SILAC media at time 0. Aliquots were taken at 0, 0.5, 1, 2, 3, 4, 5, 6, 9, and 12 hours post-stimulation and added to equal volumes of a master mix of unlabeled (L) cells for the purpose of normalization. RNA-Seq was performed at 0, 1, 2, 4, 6, 9, and 12 hours post-stimulation.
2015-03-06 | E-GEOD-59792 | biostudies-arrayexpress
Project description:In order to test the effects of metastasis suppressive miR-126/126* on primary tumor microenvironment, we designed a real-time PCR-based mouse cytokine/chemokine array containing 95 cytokines/chemokines and their receptors. Considering the possibility that production of cytokines/chemokines may be dependent on the interactions among different cell types within the tumor mass, we inoculated GFP-labeled 4T1 cells with a control vector or 4T1 cells with pri-miR-126 overexpression into the fat pad of BALB/c mice. 10 days later, we harvested the primary tumors, isolated GFP-positive cancer cells, and analyzed the expression levels of different cytokines/chemokines and their receptors in the cancer cells at the mRNA level using the cytokine array. qPCR gene expression profiling. Equal amount total RNA from each cell line was pooled prior to gene expression analysis.
2012-12-13 | E-GEOD-42885 | biostudies-arrayexpress
Project description:Translation elongation factor 1A (eEF1A) is an essential and highly conserved protein required for protein synthesis in eukaryotes. In both Saccharomyces cerevisiae and human, five different methyltransferase enzymes methylate specific residues on eEF1A, making eEF1A the eukaryotic protein targeted by the highest number of dedicated methyltransferases. eEF1A methyltransferases are highly selective enzymes, only targeting eEF1A and each targeting just one or two specific residues in eEF1A. However, the mechanism of this selectivity remains poorly understood. Here we have used AlphaFold modelling in combination with crosslinking mass spectrometry (XL-MS) and enzyme mutagenesis to reveal how S. cerevisiae elongation factor methyltransferase 4 (Efm4) specifically methylates eEF1A at K316. We find that a unique beta-hairpin motif, which extends out from the core methyltransferase fold, is important for methylation of eEF1A K316 in vitro. An alanine mutation of a single residue on this beta-hairpin, F212, significantly reduces Efm4 activity in vitro and in yeast cells. We show that the equivalent residue in human eEF1A-KMT2 (METTL10), F220, is also important for its activity towards eEF1A in vitro. Lastly, we find that phosphorylation of eEF1A at S314 negatively crosstalks with Efm4-mediated methylation of K316. Our findings demonstrate how protein methyltransferases can be being highly selective towards a single residue on a single protein.
2024-01-03 | PXD042599 | Pride
Project description:Antibodies are immune glycoproteins that represent a crucial part of the adaptive immune system as they mediate broad protection against viruses, bacteria, and cancer. Moreover, antibodies may serve as a unique source of information on past and current physiological and pathological events in the human body. The dysregulation of the antibody-dependent response can lead to a variety of abnormalities and diseases. Antibodies are found in the secreted form in the blood (antibody phenotype) and membrane-bound on B cells as B-cell receptor (antibody genotype). Thus, investigating the relationship between genomic and phenotypic antibody diversity is of decisive importance for understanding antibody-driven immune protection and disease. In this project, we aim to establish a robust high-throughput framework for single-cell (SC) genomic and proteomic characterization of antibody repertoires using high-throughput genomic sequencing and mass spectrometry. The generated data will be analyzed using a variety of bioinformatics tools.
2024-06-04 | PXD046237 | Pride
Project description:Analysis of intact N-linked glycopeptides for SARS-Cov-2 S and huamn ACE2 protein by LC-MS
2020-09-09 | PXD019937 | Pride
Project description:De nove sequencing of NISTmAb and of an M-protein from an multiple myeloma patient, MM2232
2021-07-16 | PXD022784 | Pride
Project description:Broadly neutralizing antibodies (bnAbs) to the HIV envelope (Env) V2-apex region are important leads for HIV vaccine design. Most V2-apex bnAbs engage Env with an uncommonly long heavy chain complementarity-determining region 3 (HCDR3), suggesting that rarity of bnAb precursors poses a challenge for vaccine priming. We created precursor sequence definitions for V2-apex HCDR3-dependent bnAbs and searched for related precursors in human antibody heavy chain ultradeep sequencing data from 14 HIV-unexposed donors. We found potential precursors in a majority of donors for only two long-HCDR3 V2-apex bnAbs, PCT64 and PG9, identifying these bnAbs as priority vaccine targets. We then engineered ApexGT Env trimers that bind inferred germlines for PCT64 and PG9 and have higher affinities for bnAbs; determined cryo-EM structures of ApexGT trimers bound to inferred germline and bnAb forms of PCT64 and PG9; and developed an mRNA-encoded cell-surface trimer for our lead ApexGT candidate. The methods and immunogens developed here have promise to assist the development of an HIV vaccine.
2023-03-11 | PXD033766 | Pride