Molecular & Cellular Proteomics最新文献

Cells have many protective mechanisms against background levels of ionizing radiation orchestrated by molecular changes in expression, post-translational modifications, and subcellular localization. Radiotherapeutic treatment in oncology attempts to overwhelm such mechanisms, but radioresistance is an ongoing challenge. Here, global subcellular proteomics combined with Bayesian modeling identified 544 differentially localized proteins in A549 cells upon 6 Gy X-ray exposure, revealing subcellular-specific changes of proteins involved in ferroptosis, an iron-dependent cell death, suggestive of potential radioresistance mechanisms. These observations were independent of expression changes, emphasizing the utility of global subcellular proteomics and the promising prospect of ferroptosis-inducing therapies for combating radioresistance.

Posttranscriptional regulation of gene expression by miRNAs likely makes significant contributions to mRNA abundance at the embryo-maternal interface. In this study, we investigated how miR-26a-5p and miR-125b-5p contribute to molecular changes occurring in the uterine luminal epithelium, which serves as the first site of signal exchange between the mother and the developing embryo. To measure de novo protein synthesis after miRNA delivery to primary uterine luminal epithelial cells, we used pulsed stable isotope labeling by amino acids (pSILACs). We found that both miRNAs alter the proteome of luminal epithelial cells, impacting numerous cellular functions, immune responses, as well as intracellular and second messenger signaling pathways. Additionally, we identified several features of miRNA-mRNA interactions that may influence the targeting efficiency of miR-26a-5p and miR-125b-5p. Overall, our study suggests a complex interaction of miR-26a-5p and miR-125b-5p with their respective targets. However, both appear to cooperatively function in modulating the cellular environment of the luminal epithelium, facilitating the morphological and molecular changes that occur during the intensive communication between the embryo and uterus at pregnancy.

Once ovulated, the oocyte has to be fertilized in a short time window or it will undergo post-ovulation aging (POA), whose underlying mechanisms are still not elucidated. Here, we optimized single-cell proteomics methods and performed single-cell transcriptomic, proteomic, and phosphoproteomic analysis of fresh, POA, and melatonin-treated POA oocytes. POA oocytes showed downregulation of most differentially expressed proteins, with little correlation with mRNA expression, and the protein changes can be rescued by melatonin treatment. MG132 treatment rescued the decreased fertilization and polyspermy rates and upregulated fragmentation and parthenogenesis rates of POA oocytes. MG132-treated oocytes displayed health status at proteome, phosphoproteome, and fertilization ability similar to fresh oocytes, suggesting that protein stabilization might be the underlying mechanism for melatonin to rescue POA. The important roles of proteasome-mediated protein degradation during oocyte POA revealed by single-cell multi-omics analyses offer new perspectives for increasing oocyte quality during POA and improving assisted reproduction technologies.

Prediction of proteins and associated biological pathways from lipid analyses via matrix-assisted laser desorption/ionization (MALDI) MSI is a pressing challenge. We introduced "dry proteomics," using MALDI MSI to validate spatial localization of identified optimal clusters in lipid imaging. Consistent cluster appearance across omics images suggests association with specific lipid and protein in distinct biological pathways, forming the basis of dry proteomics. The methodology was refined using rat brain tissue as a model, then applied to human glioblastoma, a highly heterogeneous cancer. Sequential tissue sections underwent omics MALDI MSI and unsupervised clustering. Spatial omics analysis facilitated lipid and protein characterization, leading to a predictive model identifying clusters in any tissue based on unique lipid signatures and predicting associated protein pathways. Application to rat brain slices revealed diverse tissue subpopulations, including successfully predicted cerebellum areas. Similarly, the methodology was applied to a dataset from a cohort of 50 glioblastoma patients, reused from a previous study. However, among the 50 patients, only 13 lipid signatures from MALDI MSI data were available, allowing for the identification of lipid-protein associations that correlated with patient prognosis. For cases lacking lipid imaging data, a classification model based on protein data was developed from dry proteomic results to effectively categorize the remaining cohort.

Histone post-translational modifications (PTMs) regulate gene expression patterns through epigenetic mechanisms. The five histone proteins (H1, H2A, H2B, H3, and H4) are extensively modified, with over 75 distinct modification types spanning more than 200 sites. Despite strong advances in mass spectrometry (MS)-based approaches, identification and quantification of modified histone peptides remains challenging because of factors, such as isobaric peptides, pseudo-isobaric PTMs, and low stoichiometry of certain marks. Here, we describe the development of a new high-throughput method to identify and quantify over 150 modified histone peptides by LC-MS. Fast gradient microflow liquid chromatography and variable window sequential windows acquisition of all theoretical spectra data-independent acquisition on a new quadrupole time-of-flight platform is compared to a previous method using nanoflow LC-MS on an Orbitrap hybrid. Histones extracted from cells treated with either a histone deacetylase inhibitor or transforming growth factor-beta 1 were analyzed by data-independent acquisition on two mass spectrometers: an Orbitrap Exploris 240 with a 55-min nanoflow LC gradient and the SCIEX ZenoTOF 7600 with a 10-min microflow gradient. To demonstrate the reproducibility and speed advantage of the method, 100 consecutive injections of one sample were performed in less than 2 days on the quadrupole time-of-flight platform. The result is the comprehensive characterization of histone PTMs achieved in less than 20 min of total run time using only 200 ng of sample. Results for drug-treated histone samples are comparable to those produced by the previous method and can be achieved using less than one-third of the instrument time.

Phosphorylation is an indispensable regulatory mechanism in cells, with specific sites on kinases that can significantly enhance their activity. Although several such critical phosphorylation sites (phos-sites) have been experimentally identified, many more remain to be explored. To date, no computational method exists to systematically identify these critical phos-sites on kinases. In this study, we introduce PhoSiteformer, a transformer-inspired foundational model designed to generate embeddings of phos-sites using phosphorylation mass spectrometry data. Recognizing the complementary insights offered by protein sequence data and phosphorylation mass spectrometry data, we developed a classification model, CSPred, which employs a bimodal fusion strategy. CSPred combines embeddings from PhoSiteformer with those from the protein language model ProtT5. Our approach successfully identified 77 critical phos-sites on 58 human kinases. Two of these sites, T517 on PKG1 and T735 on PRKD3, have been experimentally verified. This study presents the first systematic and computational approach to identify critical phos-sites that enhance kinase activity.

The Nuclear Factor I (NFI) family of transcription factors (TFs) plays key roles in cellular differentiation, proliferation, and homeostasis. As such, NFI family members engage in a large number of interactions with other proteins and chromatin. However, despite their well-established significance, the NFIs' interactomes, their dynamics, and their functions have not been comprehensively examined. Here, we employed complementary omics-level techniques, i.e. interactomics (affinity purification mass spectrometry (AP-MS) and proximity-dependent biotinylation (BioID)), and chromatin immunoprecipitation sequencing (ChIP-Seq), to obtain a comprehensive view of the NFI proteins and their interactions in different cell lines. Our analyses included all four NFI family members, and a less-studied short isoform of NFIB (NFIB4), which lacks the DNA binding domain. We observed that, despite exhibiting redundancy, each family member had unique high-confidence interactors and target genes, suggesting distinct roles within the transcriptional regulatory networks. The study revealed that NFIs interact with other TFs to co-regulate a broad range of regulatory networks and cellular processes. Notably, time-dependent proximity-labeling unveiled a highly dynamic nature of NFI protein-protein interaction networks and hinted at the temporal modulation of NFI interactions. Furthermore, gene ontology (GO) enrichment analysis of NFI interactome and targetome revealed the involvement of NFIs in transcriptional regulation, chromatin organization, cellular signaling pathways, and pathways related to cancer. Additionally, we observed that NFIB4 engages with proteins associated with mRNA regulation, which suggests that NFIs have roles beyond traditional DNA binding and transcriptional modulation. We propose that NFIs may function as potential pioneering TFs, given their role in regulating the DNA binding ability of other TFs and their interactions with key chromatin remodeling complexes, thereby influencing a wide range of cellular processes. These insights into NFI protein-protein interactions and their dynamic, context-dependent nature provide a deeper understanding of gene regulation mechanisms and hint at the role of NFIs as master regulators.

Animal venoms, distinguished by their unique structural features and potent bioactivities, represent a vast and relatively untapped reservoir of therapeutic molecules. However, limitations associated with comprehensively constructing and expressing highly complex venom and venom-like molecule libraries have precluded their therapeutic evaluation via high-throughput screening. Here, we developed an innovative computational approach to design a highly diverse library of animal venoms and "metavenoms". We used programmable M13 hyperphage display to preserve critical disulfide-bonded structures for highly parallelized single-round biopanning with quantitation via high-throughput DNA sequencing. Our approach led to the discovery of Kunitz-type domain containing proteins that target the human itch receptor Mas-related G-protein coupled receptor member X4, which plays a crucial role in itch perception. Deep learning-based structural homology mining identified two endogenous human homologs, tissue factor pathway inhibitor (TFPI), and serine peptidase inhibitor, Kunitz type 2 (SPINT2), which exhibit agonist-dependent potentiation of Mas-related G-protein coupled receptor member X4. Highly multiplexed screening of animal venoms and metavenoms is therefore a promising approach to uncover new drug candidates.

Rheumatoid arthritis (RA) is characterized by synovial hyperplasia and cartilage/bone destruction. RA affects the synovial joints, the synovial lining, and the permeability of the synovium. As the latter is of central relevance for the distribution of systemically delivered therapeutics into synovial fluid (SF), we here assessed the protein composition of paired plasma and SF of patients diagnosed with RA at three distinct levels of depth using mass spectrometric approaches: the "total" proteome, the "total" immunoglobulin G1 (IgG1) antibody repertoire, and the RA-specific anticitrullinated protein IgG1 autoantibody repertoire. The SF proteome was found to be dominated in numbers and concentration by plasma proteins, although we additionally detected several cartilage- and neutrophil-derived proteins of lower abundance. Strikingly, the plasma proteins were not only qualitatively reflected in SF but also quantitatively, independent of their size and/or other biochemical features. Also, the synovial "total" IgG1 and autoreactive anticitrullinated protein antibody IgG1 repertoire highly resembled the IgG1 repertoires detected in plasma within the same patient. Our comprehensive multilayer data thus reveals that the proteome, including the dominant, most abundant (auto)antibody clones, present in SF of RA patients is a direct reflection of the proteome present in blood, spiked by the local (immune) processes within the RA joint. We thus conclude that proteins directly pass from blood into SF of these joints without substantial bias. These findings thereby not only exemplify the use of in-depth multilayer proteome analyses to revisit basic concepts underlying RA pathology and to monitor the local (immune) processes destructive to cartilage but also provide evidence indicating that (protein-based) therapeutics may equally enter SF of swollen joints and that pharmacokinetic analyses of such therapeutics in blood are directly relevant to the synovial compartment.

Pyrethroid pesticides have been associated with neurodevelopmental disorders including attention-deficit hyperactivity disorder (ADHD) and autism spectrum disorder (ASD). While behavioral effects of pyrethroid exposure have been previously reported, the underlying mechanisms remain unclear. Here, we hypothesized that exposure to deltamethrin (DM), a widely used pyrethroid pesticide known for its neurotoxicity during early developmental stages, induces brain dysfunction through alterations in brain-derived extracellular vesicle (BDEV) signaling. Using a well-established rodent model of early life DM exposure within the recommended no observable effect level, we isolated BDEVs from postnatal 30-day-old vehicle-exposed (control) and DM-exposed mice using a differential sucrose density gradient. Following ZetaView nanoparticle tracking and electron microscopy characterization, quantitative mass spectrometry-based proteomics revealed 89 differentially expressed proteins (DEPs) in BDEVs from DM exposed animals compared to control BDEVs. Bioinformatic analysis identified convergence of DEPs on pathways associated with mitochondrial function and synaptic plasticity. PKH67-green conjugated BDEVs derived from either control or DM-exposed mice were bilaterally injected intracerebroventricularly into naive adult mice, and the brain distribution of labeled BDEVs was verified prior to extracellular field recording experiments. Strikingly, long-term potentiation (LTP) at CA3-CA1 hippocampal synapses, a functional correlate of learning and memory, was intact in control BDEVs but absent in naive mice receiving BDEVs from DM exposed mice. Notably, exogenously delivering LRRTM1, one of the DEPs found in DM BDEVs, disrupts synaptic transmission in CA1 neurons consistent with impaired LTP. Thus, differentially regulated signaling in BDEVs represents a novel mechanism of DM neurotoxicity.