Search Results (97)

Mitochondrial proteostasis in neurons relies on the coordinated expression, targeting, and import of a predominantly nuclear-encoded proteome to meet high metabolic demands. Here, we identify the RNA-binding protein cold shock domain containing E1 (CSDE1) as a TOM20-associated factor linked to mitochondrial protein-encoding mRNAs in sensory neurons. CSDE1 immunoprecipitation followed by sequencing from naïve dorsal root ganglion tissue revealed association with nuclear-encoded mitochondrial mRNAs enriched for inner membrane/matrix and oxidative phosphorylation pathways. A subset of CSDE1 localized to mitochondria and associated with the outer mitochondrial membrane import receptor TOM20 via its N-terminal region in an RNA-independent manner. In cultured sensory neurons, CSDE1 depletion reduced the mitochondrial-fraction abundance of representative nuclear-encoded electron transport chain mRNAs and decreased the abundance of selected mitochondrial proteins in the mitochondrial fraction. CSDE1 depletion reduced TMRM-positive mitochondrial puncta density along sensory neurites, without significantly increasing MitoSOX-detectable mitochondrial superoxide signals under either basal or oxidative challenge conditions. These findings identify CSDE1 as a TOM20-associated RNA-binding protein linked to mitochondrial protein-encoding transcripts in sensory neurons and support a model in which CSDE1 contributes to mitochondria-associated post-transcriptional regulation. Full article

Antimicrobial peptides (AMPs) are natural bioactive peptides produced by all organisms—from plants to insects, microbes and animals—and constitute a first line of defense. As they exhibit a broad spectrum of activity (antibacterial, antiviral, antifungal, antiparasitic, anticancer), strong efforts are being made to integrate AMPs into clinical use. AMPs are also being investigated as anticancer agents to overcome the side effects and/or resistance associated with current chemotherapies. In this context, we identified the natural AMP chionodracine from a new biological source: an Antarctic fish. Starting from the fragmentation of a chionodracine mutant peptide, a rational modular design approach was applied to develop three very short peptides (Pep-A, Pep-B and Pep-C), which were further modified with an N-terminal myristic acid lipid tail. The anticancer activity of the three N-myristoylated short peptides (Myr-A, Myr-B and Myr-C) was explored against the human cervical cancer HeLa cell line. The rationale behind this study is based on the previously reported antifungal activity of these myr peptides and on their ability to interact selectively with biological membrane-mimicking synthetic phospholipids without being particularly hemolytic or cytotoxic towards normal cells. We first demonstrated that myr peptides had cytotoxic activity against HeLa cells (IC₅₀ from 32 to 47 μM) but spared healthy primary human fibroblasts, whereas the corresponding non-myr peptides failed to kill cancer cells. The peptide with no hemolytic activity and a low IC₅₀, labeled Myr-B, was selected for subsequent analyses. Lactate dehydrogenase (LDH) assay and scanning electron microscopy (SEM) analysis revealed membrane damage and predominantly necrotic cell death in HeLa cells exposed to IC₅₀ doses of the Myr-B peptide, compared with cells treated with Pep-B. To thoroughly investigate the molecular effects of Myr-B in HeLa cells, we employed high-resolution label-free shotgun quantitative proteomics coupled with bioinformatics. Our results showed that exposing HeLa cells to Myr-B led to the under-expression of proteins belonging to the “apoptosis- and splicing-associated protein complex”, potentially influencing the alternative splicing process and consequently leading to a possible susceptibility to programmed cell death. These findings indicate that modifying natural AMPs may be a promising strategy for developing selective anticancer drugs and pinpoint Myr-B as an interesting target for future studies. Full article

Selective protein terminal labeling has become essential for system-wide studies of proteolytic mechanisms in disease. These methods enable precise tracking of cleavage dynamics, protease interactions, and cellular networks, offering transformative potential for proteolytic event analysis. This review explores recent advances in N-/C-terminal modification strategies, specifically for the applications in terminomics—the field focused on protein termini characterization. While protein termini provide valuable insights into functional proteome states, their low abundance in complex samples demands highly selective labeling approaches. We evaluate modern chemical and chemoenzymatic methods that leverage engineered chemical reactivity thresholds or enzymatic precision for site-specific modifications. Emerging strategies show enhanced substrate adaptability, reaction efficiency, and workflow compatibility, enabling broader applications in terminome studies. Full article

Progesterone receptor (PR) regulates gene expression through recruiting coregulators and general transcription factors by activation functions AF1 and AF2. AF1 localizes to the non-conserved and disordered N-terminal domain and is believed to facilitate tissue- and gene-specific activity. Our previous proteomic analysis identified three key residues (K464, K481 and R492) in AF1 that are monomethylated. Methylation mimic mutations KKR → FFF created hypoactive PR, whereas the KKR → QQQ mutation generated hyperactive PR in gene reporter assays. The current study used these mutants to determine the roles of AF1 in PR regulation of cellular activities and global gene regulation in breast cancer cells MCF-7. AF1-FFF mutation attenuated PR regulation of cell proliferation and apoptosis in response to progestin, whereas AF1-QQQ mutation enhanced these effects. AF1-FFF mutation attenuated gene regulation by progestin in ~60% of PR target genes, including genes involved in cell proliferation, hypoxia and TNFα signaling. However, the AF1-FFF mutation had little effect on ligand-independent gene regulation, suggesting distinct mechanisms of gene regulation by liganded and unliganded PR. Intriguingly, impaired activity of methylation mimic mutant PRB-FFF is associated with greater chromatin binding in ChIP-Seq analysis, corresponding to a stronger association between PRB-FFF and Steroid Receptor Coactivator-1 (SRC-1), a member of the p160 family of nuclear receptor coactivators, as was previously reported. In conclusion, PR AF1 is important for the core activities of liganded PR in regulating ~half of target genes and cell proliferation. AF1 monomethylation may modulate PR-chromatin interactions through stronger association with coregulators, thereby decelerating chromatin binding kinetics. This is supported by PRODIGY’s prediction of higher binding affinities of monomethylated AF1 and methylation mimic mutant with SRC-1. Full article

Objective: Emphysema is an important chronic obstructive pulmonary disease (COPD) phenotype characterized by the destruction of air spaces distal to the terminal bronchiole. Aiming to detect potential emphysema biomarkers and to assess the systemic effects of emphysema in blood plasma, we conducted a small cross-sectional shotgun proteomics study. Methods: This study included N = 40 participants divided into four subgroups (N = 10 per group): patients with emphysema and COPD (CE), patients with COPD but without emphysema (CN), healthy smokers (HS) and healthy never-smokers (HN). The participants were sampled non-probabilistically to be similar in terms of age, sex and comorbidities. Participants’ blood plasma was analyzed using liquid chromatography–mass spectrometry. Bioinformatic analysis included detection of differentially expressed proteins (DEPs) and overrepresentation analysis (ORA). Results: Across all groups, a total of 994 proteins were identified, with NADP-dependent malic enzyme (NADP-ME; encoded by ME1) being the only DEP in the CE vs. CN contrast. Proteins such as BMP1, ADAMTSL-2, -4 and IGFBP4, -5, 6 were identified to be upregulated in CE vs. HN. Fibulin-1, -3 and several immunoglobulin components were identified to be downregulated in the CE vs. HN contrast. ORA revealed several enriched processes, including serine-type endopeptidase activity, insulin-like growth factor I and II binding, and signaling receptor binding. Conclusion: We propose NADP-ME, an important enzyme of intermediary metabolism and redox homeostasis, as a potential biomarker candidate of emphysema. Notably, NADP-ME is also implicated in anoikis resistance. Additionally, changes in the expression levels of BMP1, ADAMTSL-2 and -4, and fibulin suggest potential major systemic effects of extracellular matrix perturbation. As all data was derived from LC-MS analysis, these findings need to be further evaluated with complementary methods. Full article

Glycosaminoglycans (GAGs) and their degrading enzymes have extensive applications and biotechnology and medicine, and play a crucial role in the recycling of organic matter in oceans. In this study, a potential GAG utilization gene cluster was identified in the genome of a novel marine Bacteroidetes, Roseihalotalea indica gen. nov. sp. nov. TK19036^T, through sole carbon source cultivation and differential proteomic analysis. Multiple GAG-lyases within this locus were purified and characterized. RiPL8 comprises a functionally unknown N-terminal domain and a catalytic C-terminal domain, exhibiting specificity for degrading hyaluronic acid (HA). The activity of RiPL35 is sensitive to Ca²⁺ ion concentration with an optimum at 10 mM. RiPL38 is the first reported member of the PL38 family capable of degrading HA and chondroitin sulfate (CS). In summary, our study reveals Roseihalotalea indica gen. nov. sp. nov. TK19036^T harbors an unpredicted GAG degradation gene cluster, and the encoded GAG-lyases exhibit distinct substrate specificities compared to the host organism. Full article

Accurate prediction of submitochondrial localization is fundamental to understanding mitochondrial biogenesis and cellular metabolic pathways. While deep representations from pre-trained protein language models (pLMs) have significantly advanced the field, traditional global average pooling methods often fail to capture critical, localized N-terminal targeting signals, particularly in long sequences where these motifs are mathematically diluted. To resolve this “signal dilution” bottleneck, we developed a multi-scale architecture that explicitly integrates high-resolution N-terminal features with global evolutionary context derived from ESM-2 embeddings. The proposed framework utilizes an orthogonal mixing strategy consisting of Token-mixing and Channel-mixing. Token-mixing is specifically designed to detect spatial rhythmic patterns across residue positions, while Channel-mixing refines the biochemical signatures within the latent feature space. Extensive benchmarking across diverse datasets demonstrates that our approach effectively maintains signal integrity. Compared to existing state-of-the-art methods, the model achieves a superior overall Generalized Correlation Coefficient (GCC) of 0.7443 on the SM424-18 dataset and 0.7878 on the SubMitoPred dataset, outperforming the latest benchmarks by 9.4% and 16.1%, respectively. Furthermore, on the independent M983 test set, our method maintained a high GCC of 0.6945, demonstrating a 9.9% improvement relative to the state-of-the-art methods. This robust and efficient framework provides a high-precision tool for large-scale mitochondrial proteomics. Full article

Endometrial cancer (EC) is the leading gynecological malignancy worldwide. Obesity is reported to be associated with 50% of EC cases. Significant gaps remain in investigating specific molecular mechanisms behind the obesity-driven EC. Women diagnosed with EC undergoing total hysterectomy were recruited. Patients were divided into two groups: EC-obese with BMI > 29.9 kg/m² (n = 10) and EC-Non-obese with BMI ≤ 29.9 kg/m² (n = 10). Tumor tissues were subjected to label-free quantitative proteomic analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Differentially expressed proteins were identified and subjected to pathway enrichment and network analyses to characterize obesity-associated alterations. Proteomic profiling showed a significant dysregulation of 456 proteins: 171 upregulated and 285 downregulated. Proteins involved in endoplasmic reticulum quality control particularly endoplasmic reticulum lectin 1 (ERLEC1), were reduced. Conversely, EC-obese demonstrated upregulation of hepatocyte growth factor (HGF), integrin-linked kinase (ILK), CTTNBP2 N-terminal-like protein (CTTNBP2NL), and lysyl oxidase homolog 1 (LOXL1), implicating activation of inflammatory pathways. Bioinformatic analysis showed downregulation of immune-related pathways, including neutrophil degranulation, complement activation in the EC-obese group. ROC analysis identified apolipoprotein(a), phospholipase B-like 1, CTTNBP2NL, and ILK as significant proteins that can differentiate between the obese and non-obese states. Our findings provide insights into obesity-associated proteomic changes in EC and highlight candidate proteins that can be used for molecular stratification after further validation. Full article

Periodontitis is a chronic infectious disease characterized by the destruction of the tooth-supporting tissues, including the gingiva, periodontal ligament, and alveolar bone, which may ultimately lead to tooth loss. However, blood-based biomarkers reflecting systemic inflammation in periodontitis remain poorly defined. We analyzed plasma proteomic data from the UK Biobank using Olink Explore proteomics to identify systemic protein signatures distinguishing chronic periodontitis patients (n = 90) from healthy controls (n = 2234). Among 2151 proteins passing quality control, 29 proteins showed significant differential expression (FDR < 1.0 × 10⁻⁵). Growth differentiation factor 15 (GDF15) exhibited the strongest upregulation (mean NPX: −0.183 to 0.157, effect size = 0.337, FDR = 2.82 × 10⁻¹²), followed by N-terminal pro-B-type natriuretic peptide (NT-proBNP) (effect size = 0.594), Interleukin-6 (IL-6) (effect size = 0.450), and Insulin-like growth factor binding protein-(4IGFBP4) (effect size = 0.269). Multiple TNF receptor superfamily members (TNFRSF1A/1B, TNFRSF10A/10B) and proteins involved in extracellular matrix remodeling (COL6A3, ADAM12) and vascular stress (ADM) were significantly elevated. In contrast, EGFR and DNER showed decreased expression. Protein–protein interaction network analysis revealed IL-6 as a central hub protein forming a tightly interconnected cluster with TNF receptor family members. These findings indicate systemic plasma protein profiles associated with chronic periodontitis within this population-based cohort. The identified proteins may provide a basis for future evaluation of blood-based biomarkers for chronic periodontitis, pending further validation. Full article

The tardigrade Ramazzottius varieornatus exhibits extraordinary resilience to extreme environmental stresses, yet the functional diversity of its proteome remains largely unexplored. In this study, the structural and biochemical characterization of RvCA5, an atypical α-carbonic anhydrase (CA) identified in R. varieornatus, is presented. Expression analysis in E. coli revealed the spontaneous formation of a truncated RvCA5 species, which was confirmed to be unrelated to signal peptide cleavage. RvCA5 exhibited distinct structural features, including extended intrinsically disordered regions (IDRs) at both termini. Unlike canonical α-CAs, RvCA5 exhibited negligible CO₂ hydration activity, which was partially enhanced by the removal of the N-terminal IDR, suggesting that this region acts as a dynamic entropic barrier hindering substrate diffusion. RvCA5 possesses multiple surface-exposed reactive cysteine residues, resembling the redox-sensing human CA 3. Notably, consistent with a predicted nuclear localization signal, in silico modeling predicted that RvCA5 can bind DNA via a positively charged patch near the C-terminal IDR. The DNA-binding capability of RvCA5 was experimentally demonstrated by electrophoretic mobility shift assays. Collectively, these findings suggest that RvCA5 potentially functions as a redox-responsive transcriptional regulator. Full article

The scorpion family Buthidae, renowned for its neurotoxin-rich venoms, dominates toxinology, while non-buthid venoms remain largely unexplored. Here, we present a comprehensive proteomic and biochemical characterization of the Amazonian chactid scorpion Brotheas amazonicus venom (BamazV), with emphasis on molecular complexity, proteolytic processing, and peptide diversity. Using an integrative venomics approach that combines molecular mass-based fractionation, reversed-phase chromatography, high-resolution mass spectrometry, N-terminal sequencing, and functional and immunological analyses, we reveal an unexpectedly complex venom profile enriched in high-molecular-weight components and extensively processed peptides, with more than 40 venom peptides sequenced by MS/MS and Edman degradation. The data provide evidence for non-canonical proteolytic events, including the generation of peptides from precursor regions not classically associated with mature venom components. In contrast to the venom of Tityus serrulatus, BamazV displays a “hydrolase-rich, neurotoxin-poor” profile, featuring a catalytically active Group III phospholipase A₂ (BamazPLA₂), a highly active hyaluronidase, metalloproteases, low-mass peptides, and potassium channel toxins. Our results suggest a hydrolytic prey-subjugation strategy, and limited cross-reactivity with commercial antivenom highlighted its distinct structural landscape. Overall, this study advances the understanding of venom evolution and proteolytic diversification in underexplored scorpion lineages, positioning B. amazonicus as a valuable model for investigating alternative venom strategies and identifying novel biotechnological scaffolds. Full article

Extracellular vesicles (EVs) are bilayer-membrane cellular components that deliver protected cargo to the extracellular environment and can mediate long-distance signaling. We have previously reported that EVs isolated from the virulent fungal pathogen Paracoccidioides brasiliensis Vpb18 can revert the expression, in the attenuated variant Apb18, of stress-related virulence traits. We presently show that the Vev and Aev, respectively, produced by these variants display distinct proteomes, with prevalent functional enrichment in Vev related to oxidative stress response, signal transduction, transport, and localization, in addition to richer protein–protein interaction. Proteome sequences were obtained by nanoflow liquid chromatography coupled with tandem mass spectrometry (nano LC-ESI-MS/MS). The Vev and corresponding Vpb18 proteomes also differed, suggesting a selective bias in vesicle protein cargo. Moreover, sublethal oxidative (VevOxi) and nitrosative (VevNO) stress modulated the Vev proteome and a positive correlation between VevOxi/VevNO-enriched and Vev-enriched (relative to Aev) proteins was observed. Out of 145 fungal virulence factors detected in Vev, 64% were enriched, strongly suggesting that molecules with virulence roles in Paracoccidioides are selectively concentrated in Vev. Our study significantly advanced the field by exploring protein N-terminal acetylation to a dimension rarely investigated in fungal EV proteomics. The proportion of N-terminally acetylated proteins in Vev was higher than in Vpb18 and the presence of Nt-acetylation in Vev-enriched virulence factors varied across the samples, suggesting that it might interfere with protein sorting into EVs and/or protein functionality. Our findings highlight the relevance of our fungal model to unraveling the significance of fungal EVs in pathogenesis and phenotypic transfer. Full article

Galectins, a family of glycan-binding proteins, play crucial roles in various cellular functions, acting at both intracellular and extracellular levels. Among them, Galectin-3 (Gal-3) stands out as a unique member, possessing an intrinsically unstructured N-terminal oligomerization domain and a canonical carbohydrate-recognition domain (CRD). Gal-3 binding to glycosylated plasma membrane cargo leads to its oligomerization and membrane bending, ultimately resulting in the formation of endocytic invaginations. An interactomic assay using proteomic analysis of endogenous Gal-3 immunoprecipitates identified the orphan G protein-coupled receptor GPRC5A as a novel binding partner of Gal-3. GPRC5A, also known as Retinoic Acid-Induced protein 3 (RAI3), is transcriptionally induced by retinoic acid. Our results further demonstrate that extracellular recombinant Gal-3 stimulates GPRC5A internalization. In SW480 colorectal cancer cells, glycosylated GPRC5A interacts with Gal-3. Interestingly, while GPRC5A expression was upregulated by the addition of all-trans retinoic acid (ATRA), its endogenous internalization in SW480 cells was specifically triggered by extracellular Gal-3, but not by ATRA. This study provides new insights into the endocytic mechanisms of GPRC5A, for which no specific ligand has been identified to date. Further research may uncover additional Gal-3-mediated functions in GPRC5A cellular signaling and contribute to the development of innovative therapeutic strategies. Full article

Eggshell membrane (ESM) is composed of approximately 90% protein. Our previous studies in healthy adults demonstrated that two months of daily ESM intake improved respiratory function, zigzag walking speed, and skin elasticity. The present study aims to address the knowledge gap regarding the in vivo effects of ESM in the context of inflammatory bowel disease (IBD). Proteomic analysis was performed on powdered ESM used as a dietary supplement. To investigate its pharmacokinetics in mice, tritium (³H)-labeled ESM was prepared using the ⁶Li(n,α)³H nuclear reaction. The therapeutic potential of ESM was further examined in a 2.0% dextran sulfate sodium (DSS)-induced murine model of IBD. In addition, fecal samples from both mice and healthy human subjects were analyzed using a modified terminal restriction fragment length polymorphism (T-RFLP) method. Lysozyme C (LYZ) was the most abundant protein (47%), followed by lysyl oxidase (12%) in ESM used in this study. ³H-ESM was mixed with MediGel, and orally administered to mice. Radioactivity levels were measured in blood, organs (duodenum, small intestine, large intestine, liver, kidney, lung, skin), and rectal feces at 0.5, 2, 5, 24, 48, and 72 h post-administration. Radioactivity in feces indicated excretion of undigested components, while systemic distribution suggested potential whole-body effects of ESM. Oral ESM and LYZ significantly alleviated body weight loss, diarrhea, and hematochezia in a DSS-induced murine model of IBD, leading to a significantly lower disease activity index on day 3 and showing a similar trend on day 5. Gut microbiota analysis showed increased Bacteroidales in the DSS group, while the ESM + DSS group maintained levels similar to the control. In humans, a double-blind, randomized controlled trial was conducted to evaluate the effects of ESM on gut microbiota in healthy adults. Participants received either ESM or placebo for 8 weeks. revealed a significant increase in alpha diversity at weeks 1 and 8 in the ESM group (p < 0.05), with between-group differences evident from week 1 (p < 0.01). ESM intake reduced Bacteroides and significantly increased Bifidobacterium and Lactobacillales at weeks 4 and 8. These findings suggest ESM supplementation promotes beneficial modulation of gut microbiota. These findings suggest that ESM, through its major protein components such as LYZ, may serve as a promising dietary intervention for maintaining intestinal health and mitigating inflammation in the context of IBD. Full article

In human cells, the biogenesis of membrane proteins, which account for one quarter of polypeptides and sixty percent of human drug targets, is initiated at the membrane of the endoplasmic reticulum (ER). This process involves N-terminal signal peptides or transmembrane helices in the membrane protein precursors. Over one hundred proteins enable membrane-targeting and -insertion of the precursors as well as their folding and covalent modifications. Four targeting pathways to the Sec61 channel in the ER membrane with their effectors and three cooperating or independent membrane protein–insertases have been identified. We combined knock-down of individual components of these pathways and insertases in HeLa cells with label-free quantitative mass spectrometric analysis of the proteomes. Differential protein abundance analysis in comparison to control cells was employed to identify clients of components involved in the targeting or membrane insertion of precursors. Alternatively, knock-out cells or relevant patient fibroblasts were employed. The features of the client polypeptides were characterized to identify the client types of the different components and, ideally, their rules of engagement. In this review/article-hybrid, the focus is on global lessons from and limitations of the proteomic approach in answering the cell biological question, as well as on new aspects, such as N-terminal acetylation of membrane protein precursors. Full article