Journal of Proteome Research最新文献

Enolase-phosphatase 1 (ENOPH1) is a newly identified enzyme associated with stress responses and neurodevelopmental disorders. Our previous study found that ENOPH1 mediates cerebral cell apoptosis and blood-brain barrier (BBB) dysfunction during early cerebral ischemia. Ubiquitination has been identified in neuronal damage and the neuroinflammatory response in ischemic stroke. However, whether ENOPH1 regulates ischemia-induced protein ubiquitination alteration is yet unclear. Hence, the present study explored changes in the ubiquitinomic in early ischemic brain tissues between wildtype and ENOPH1 knockout mice using a comprehensive quantitative analysis. Our results showed that 4000 ubiquitination-modified sites in 1613 proteins were quantified, with 772 ubiquitinated sites in 464 proteins significantly decreasing or increasing after ENOPH1 knockout (fold change >1.5 or <1/1.5, p < 0.05). When compared to our previous parallel proteome profiles, common differential proteins FKBP5 and Claudin-11 were observed and further validated. ENOPH1 regulates the degradation of FKBP5 and the promotion of Claudin-11 by ubiquitination mediation, leading to the activation or inhibition of nuclear-initiated steroid signaling and transendothelial migration pathways. These findings, for the first time, identified ubiquitinomic features of early ischemic brain tissues after ENOPH1 knockout, suggesting that ENOPH1 may regulate neuroinflammatory stress and barrier function by modifying FKBP5 and Claudin-11 protein ubiquitination.

Decoy-based methods are a popular choice for the statistical validation of peptide detection in tandem mass spectrometry and proteomics data. Such methods can achieve a substantial boost in statistical power when coupled with postprocessors such as Percolator that use auxiliary features to learn a better-discriminating scoring function. However, we recently showed that Percolator can struggle to control the false discovery rate (FDR) when reporting the list of discovered peptides. To address this problem, we introduce Percolator-RESET, which is an adaptation of our recently developed RESET meta-procedure to the peptide detection problem. Specifically, Percolator-RESET fuses Percolator's iterative SVM training procedure with RESET's general framework to provide valid false discovery rate control. Percolator-RESET operates in both a standard single-decoy mode and a two-decoy mode, with the latter requiring the generation of two decoys per target. We demonstrate that Percolator-RESET controls the FDR in both modes, both theoretically and empirically, while typically reporting only a marginally smaller number of discoveries than Percolator in the single-decoy mode. The two-decoy mode is marginally more powerful than both Percolator and the single-decoy mode and exhibits less variability than the latter.

Mass spectrometry has become central to identifying and quantifying histone post-translational modifications (PTMs), surpassing limitations of antibody-based methods. Histones are dynamically modified by multiple structures, especially at lysine residues on their N-terminal tails, to regulate DNA-templated processes. Reliable identification of histone PTMs remains challenging and still requires manual curation. This study focused on the Lys27-Arg40 stretch of histone H3, considered four sequence variants, an increasing number of lysine PTMs and artifacts coming from histone sample processing, which resulted in many isobaric peptides. Our analysis revealed the value of low-mass b1 and cyclic immonium fragment ions to validate identification of the distinct peptidoforms. We examined how MS/MS spectra are transformed by common identification software during the conversion of raw files into peak lists, and highlighted how some parameters may erase the informative low-mass fragments. We targeted the detection of 40 H3 K27-R40 variant × PTM combinations, including the mouse-specific variants H3mm7 and H3mm13, in histone samples extracted from mouse testis and brain via a parallel reaction monitoring analysis. We only detected very low levels of unmodified H3mm7. Our work contributes to reliably deciphering the histone code shaped by distinct sequence variants and numerous combinations of PTMs.

Intercellular communication is important for host immunity in response to bacterial infections. Nontuberculous mycobacterium (NTM), such as Mycobacterium abscessus (M. ab), is a group of environmental bacteria that can cause severe lung infections in individuals with pre-existing lung conditions, including cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). There is limited knowledge understanding the interaction between airway epithelial cells and immune cells during NTM infections. In this study, we characterized microvesicles (MVs) released from uninfected and M. ab-infected human bronchial epithelial cells and investigated the effect of these MVs on the activation and polarization of THP-1-derived macrophages in cell culture. Our results indicate that MVs released by M. ab-infected human bronchial epithelial cells stimulated the activation of M2-polarized macrophages in cell culture when compared to MVs released by uninfected cells. Additionally, the proteomic analysis for isolated MVs showed that the proteins involved in the cell adhesion pathway were enriched in MVs from M. ab-infected human bronchial epithelial cells compared to MVs from uninfected cells. Among those, the cell surface protein, intercellular adhesion molecule 1 (ICAM-1), regulated the uptake of MVs released by M. ab-infected human bronchial epithelial cells by recipient macrophages in cell culture. In conclusion, our data suggest that in response to M. ab infection, human airway epithelial cells release MVs to modulate the activation of macrophages, which are key cells for mycobacterial intracellular survival in the host.

SpecPeptidOMS directly aligns peptide fragmentation spectra to whole and undigested protein sequences. The algorithm was specifically and initially designed for peptidomics, where the aim is to identify peptides that do not result from the hydrolysis of a known protein and therefore, whose termini cannot be predicted. Thus, SpecPeptidOMS can perform alignments starting and ending anywhere in the protein sequence. The underlying computational method of SpecPeptidOMS, which is based on a dynamic programming approach, was drastically optimized. As a result, SpecPeptidOMS can process around 12,000 spectra per hour on an ordinary laptop, with alignment performed against the entire human proteome. The performance of SpecPeptidOMS was first evaluated on a publicly available data set of (nontryptic) synthetic mass spectra. Accuracy was estimated by considering the results obtained by MaxQuant on the same data set as the “ground truth”. A second series of tests on a larger, well-known proteomics data set (HEK293) highlighted SpecPeptidOMS’ additional ability to search for open modifications, a feature of interest in peptidomics but also more broadly in conventional proteomics. SpecPeptidOMS is open-source, cross-platform (written in Java), and freely available.

The Pinophyta family has long been used to protect the skin from oxidation, thanks to the action of molecules such as stilbenes, flavonoids, and lignans, which are particularly concentrated in knotwood. These molecules are of interest from a cosmetic perspective. The present study focuses on four species from larch (Larix decidua Mill.), silver fir (Abies alba Mill.), Norway spruce (Picea abies (L.) H.Karst), and Douglas fir (Pseudotsuga menziesii (Mirb.) Franco) knotwood, recovered from byproducts of the wood industry. The molecules are extracted from knotwood and used in vitro on human keratinocytes (HaCaT). Studies quantifying reactive oxygen species (ROS) have demonstrated its ability to eliminate hydroxyl radicals and superoxides. Metabolomic analyses using proton nuclear magnetic resonance (¹H NMR) and multivariate statistics (PLS-DA) demonstrated that keratinocytes modulate metabolite expression after treatment with knot extracts. Indeed, our findings indicate an increase in metabolites such as glutathione, glycine, glutamate, sarcosine, taurine, and proline, which are known to reduce intracellular oxidative stress and validate the effect on ROS levels. They also indicate that knotwood extracts may affect membrane balance, collagen formation, and oxidative stress levels. This study highlights the value of metabolomic analysis in the cosmetic industry for a detailed understanding of the mechanisms implemented in a whole cell.

Hederagenin (Hed), a natural triterpenoid, exhibits antitumor potential in cervical cancer. The present study was designed to explore Hed’s regulatory mechanisms on mitophagy in SiHa cervical cancer cells, employing tandem mass tag (TMT) proteomics and an advanced network association algorithm (NAA). Our findings revealed that Hed decreased SiHa cell viability, induced apoptosis, and altered mitochondrial membrane potential. Notably, Hed inhibited mitophagic flux under both normoxic and hypoxic conditions. Through TMT proteomics analysis and innovative NAA, we identified a close association between the HIF-1 signaling pathway and mitophagy. Network analysis further suggested that Hed acts on a target network centered on SRC, STAT3, AKT1, and HIF1A. Western blot analysis confirmed the expression and phosphorylation status of these targets in response to Hed. This study elucidates the molecular mechanisms underlying Hed’s regulation of mitophagy in SiHa cells, offering novel insights and potential therapeutic targets for cervical cancer treatment.

The metalloprotease meprin β is upregulated in neurons and astrocytes of Alzheimer’s disease patients’ brains. While the role of meprin β as the β-secretase of amyloid precursor protein (APP) has been characterized, its broader substrate profile within the brain remains largely unexplored. Hence, to identify additional substrates, we conducted N-terminomics of brain lysates from mice overexpressing meprin β in astrocytes employing the Hydrophobic Tagging-Assisted N-terminal Enrichment (HYTANE) strategy. We observed 3906 (82.2%) N-terminal peptides and identified seven new substrates that match meprin β in terms of localization and cleavage specificity. Of note, the meprin β overexpressing mice show mild cognitive impairments caused by amyloidogenic APP processing alongside hyperactivity and altered exploratory behavior seemingly independent of APP cleavage. Hence, latrophilin-3 was of particular interest, as latrophilin-3 defects are associated with hyperactivity in mice and human. In brain lysates from mice overexpressing meprin β in astrocytes as well as in cellulo, we validated the cleavage of latrophilin-3, resulting in the release of two N-terminal domains. These domains promote interactions with neuronal proteins such as fibronectin leucine-rich repeat transmembrane proteins, promoting adequate synapse formation. Thus, meprin β might affect synaptic integrity by cleaving interaction domains of latrophilin-3, potentially exacerbating the observed hyperactivity phenotype.

Metabolic perturbations of the gut microbiome have been implicated in the pathogenesis of multiple human diseases, including type-2 diabetes (T2D). However, our understanding of the global metabolic alterations of the gut microbiota in T2D and their functional roles remains limited. To address this, we conducted a high-coverage metabolomics profiling analysis of serum samples from 1282 Chinese individuals with and without T2D. Among the 220 detected microbiota-associated compounds detected, 111 were significantly altered, forming a highly interactive regulatory network associated with T2D development. Pathway enrichment and correlation analyses revealed aberrant metabolic pathways, primarily including the activation of pyrimidine metabolism, unsaturated fatty acid biosynthesis, and diverse amino acid metabolisms such as Tryptophan metabolism, Lysine metabolism, and Branched-chain amino acid biosynthesis. A microbiota-dependent biomarker panel, comprising pipecolinic acid, methoxysalicylic acid, N-acetylhistamine, and 3-hydroxybutyrylcarnitine, was defined and validated with satisfactory sensitivity (>78%) for large-scale, population-based T2D screening. The functional role of a gut microbial product, N-acetylhistamine, was further elucidated in T2D progression through its inhibition of adenosine monophosphate-activated protein kinase phosphorylation. Overall, this study expands our understanding of gut microbiota-driven metabolic dysregulation in T2D and suggests that monitoring these metabolic changes could facilitate the diagnosis and treatment of T2D.

Sperm capacitation is a critical process for fertilization. This work aims to analyze the effect in vitro capacitation had on the proteome and mitochondrial parameters of bull spermatozoa. Viability, mitochondrial membrane potential (MMP), and reactive oxygen species (mROS) were assessed by flow cytometry in noncapacitated (NC) and in vitro capacitated (IVC) sperm. Proteome was evaluated using SWATH-MS. In vitro capacitation significantly induced a decrease in sperm viability, a high MMP, and an increase in mROS production. Within the group of living spermatozoa, the capacitation significantly induced a decrease in healthy mitochondrial spermatozoa, as well as an increase in mROS production, without affecting the MMP intensity. A total number of 72 differentially abundant proteins were found of which 63 were over-represented in the NC sperm group and 9 in the IVC sperm group. It was observed that many proteins associated with the sperm membrane and acrosome were lost during the capacitation process. For the IVC sperm, the functional enrichment was found in proteins related to the oxidative phosphorylation process. Our results indicate that the capacitation process induces a significant loss of seminal plasma-derived membrane proteins and a significant increase in proteins related with the oxidative phosphorylation (OXPHOS) pathway. Data are available via ProteomeXchange with identifiers PXD056424 and PXD042286.