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Molecular & Cellular Proteomics : MCP Jun 2024Microglia are resident immune cells of the brain and regulate its inflammatory state. In neurodegenerative diseases, microglia transition from a homeostatic state to a...
Microglia are resident immune cells of the brain and regulate its inflammatory state. In neurodegenerative diseases, microglia transition from a homeostatic state to a state referred to as disease associated microglia (DAM). DAM express higher levels of proinflammatory signaling molecules, like STAT1 and TLR2, and show transitions in mitochondrial activity toward a more glycolytic response. Inhibition of Kv1.3 decreases the proinflammatory signature of DAM, though how Kv1.3 influences the response is unknown. Our goal was to identify the potential proteins interacting with Kv1.3 during transition to DAM. We utilized TurboID, a biotin ligase, fused to Kv1.3 to evaluate potential interacting proteins with Kv1.3 via mass spectrometry in BV-2 microglia following TLR4-mediated activation. Electrophysiology, western blotting, and flow cytometry were used to evaluate Kv1.3 channel presence and TurboID biotinylation activity. We hypothesized that Kv1.3 contains domain-specific interactors that vary during a TLR4-induced inflammatory response, some of which are dependent on the PDZ-binding domain on the C-terminus. We determined that the N-terminus of Kv1.3 is responsible for trafficking Kv1.3 to the cell surface and mitochondria (e.g. NUDC, TIMM50). Whereas, the C-terminus interacts with immune signaling proteins in an LPS-induced inflammatory response (e.g. STAT1, TLR2, and C3). There are 70 proteins that rely on the C-terminal PDZ-binding domain to interact with Kv1.3 (e.g. ND3, Snx3, and Sun1). Furthermore, we used Kv1.3 blockade to verify functional coupling between Kv1.3 and interferon-mediated STAT1 activation. Overall, we highlight that the Kv1.3 potassium channel functions beyond conducting the outward flux of potassium ions in an inflammatory context and that Kv1.3 modulates the activity of key immune signaling proteins, such as STAT1 and C3.
PubMed: 38936775
DOI: 10.1016/j.mcpro.2024.100809 -
Redox Biology Jun 2024The effects of low energy availability (LEA) on the immune system are poorly understood. This study examined the effects of 14 days of LEA on immune cell redox balance...
INTRODUCTION
The effects of low energy availability (LEA) on the immune system are poorly understood. This study examined the effects of 14 days of LEA on immune cell redox balance and inflammation at rest and in response to acute exercise, and exercise performance in female athletes.
METHODS
Twelve female endurance athletes (age: 26.8 ± 3.4 yrs, maximum oxygen uptake (V˙O): 55.2 ± 5.1 mL × min × kg) were included in a randomized, single-blinded crossover study. They were allocated to begin with either 14 days of optimal energy availability diet (OEA, 52 ± 2 kcal × kg fat free mass (FFM) × day) or LEA diet (22 ± 2 kcal × kg FFM × day), followed by 3 days of refueling (OEA) with maintained training volume. Peripheral blood mononuclear cells (PBMCs) were isolated, and plasma obtained at rest before and after each dietary period. The PBMCs were used for analysis of mitochondrial respiration and HO emission and specific proteins. Exercise performance was assessed on cycle by a 20-min time trial and time to exhaustion at an intensity corresponding to ∼110 % V˙O).
RESULTS
LEA was associated with a 94 % (P = 0.003) increase in PBMC NADPH oxidase 2 protein content, and a 22 % (P = 0.013) increase in systemic cortisol. LEA also caused an alteration of several inflammatory related proteins (P < 0.05). Acute exercise augmented HO emission in PBMCs (P < 0.001) following both OEA and LEA, but to a greater extent following LEA. LEA also reduced the mobilization of white blood cells with acute exercise. After LEA, performance was reduced in both exercise tests (P < 0.001), and the reduced time trial performance remained after the 3 days of refueling (P < 0.001).
CONCLUSION
14 days of LEA in female athletes increased cortisol levels and had a pronounced effect on the immune system, including increased capacity for ROS production, altered plasma inflammatory proteome and lowered exercise induced mobilization of leukocytes. Furthermore, LEA resulted in a sustained impairment in exercise performance.
PubMed: 38936255
DOI: 10.1016/j.redox.2024.103250 -
PLoS Pathogens Jun 2024The cGMP-dependent protein kinase (PKG) is the sole cGMP sensor in malaria parasites, acting as an essential signalling hub to govern key developmental processes...
The cGMP-dependent protein kinase (PKG) is the sole cGMP sensor in malaria parasites, acting as an essential signalling hub to govern key developmental processes throughout the parasite life cycle. Despite the importance of PKG in the clinically relevant asexual blood stages, many aspects of malarial PKG regulation, including the importance of phosphorylation, remain poorly understood. Here we use genetic and biochemical approaches to show that reduced cGMP binding to cyclic nucleotide binding domain B does not affect in vitro kinase activity but prevents parasite egress. Similarly, we show that phosphorylation of a key threonine residue (T695) in the activation loop is dispensable for kinase activity in vitro but is essential for in vivo PKG function, with loss of T695 phosphorylation leading to aberrant phosphorylation events across the parasite proteome and changes to the substrate specificity of PKG. Our findings indicate that Plasmodium PKG is uniquely regulated to transduce signals crucial for malaria parasite development.
PubMed: 38935780
DOI: 10.1371/journal.ppat.1012360 -
MSphere Jun 2024Bacterial ribonuclease E (RNase E) is vital for posttranscriptional regulation by degrading and processing RNA. The RraA protein inhibits RNase E activity through...
Bacterial ribonuclease E (RNase E) is vital for posttranscriptional regulation by degrading and processing RNA. The RraA protein inhibits RNase E activity through protein-protein interactions, exerting a global regulatory effect on gene expression. However, the specific role of RraA remains unclear. In this study, we investigated expression in ZJ-T and identified three promoters responsible for its expression, resulting in transcripts with varying 5'-UTR lengths. During the stationary phase, was significantly posttranscriptionally inhibited. Deletion of had no impact on bacterial growth in rich medium Luria-Bertani broth with salt (LBS) but resulted in decreased biofilm formation and increased resistance to polymyxin B. Transcriptome analysis revealed 350 differentially expressed genes (DEGs) between the wild type and the mutant, while proteome analysis identified 267 differentially expressed proteins (DEPs). Integrative analysis identified 55 genes common to both DEGs and DEPs, suggesting that RraA primarily affects gene expression at the posttranscriptional level. KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis demonstrated that RraA facilitates the conversion of fatty acids, propionic acid, and branched-chain amino acids to acetyl-CoA while enhancing amino acid and peptide uptake. Notably, RraA positively regulates the expression of virulence-associated genes, including those involved in biofilm formation and the type VI secretion system. This study expands the understanding of the regulatory network of RraA through transcriptome analysis, emphasizing the importance of proteomic analysis in investigating posttranscriptional regulation.IMPORTANCERraA is an inhibitor protein of ribonuclease E that interacts with and suppresses its endonucleolytic activity, thereby playing a widespread regulatory role in the degradation and maturation of diverse mRNAs and noncoding small RNAs. However, the physiological functions and associated regulon of RraA in have not been fully elucidated. Here, we report that RraA impacts virulence-associated physiological processes, namely, antibiotic resistance and biofilm formation, in . By conducting an integrative analysis of both the transcriptome and proteome, we revealed the involvement of RraA in carbon metabolism, amino acid catabolism, and transport, as well as in the type VI secretion system. Collectively, these findings elucidate the regulatory influence of RraA on multiple pathways associated with metabolism and pathogenesis in .
PubMed: 38934599
DOI: 10.1128/msphere.00020-24 -
MSystems Jun 2024Airway microbiota are known to contribute to lung diseases, such as cystic fibrosis (CF), but their contributions to pathogenesis are still unclear. To improve our...
Airway microbiota are known to contribute to lung diseases, such as cystic fibrosis (CF), but their contributions to pathogenesis are still unclear. To improve our understanding of host-microbe interactions, we have developed an integrated analytical and bioinformatic mass spectrometry (MS)-based metaproteomics workflow to analyze clinical bronchoalveolar lavage (BAL) samples from people with airway disease. Proteins from BAL cellular pellets were processed and pooled together in groups categorized by disease status (CF vs. non-CF) and bacterial diversity, based on previously performed small subunit rRNA sequencing data. Proteins from each pooled sample group were digested and subjected to liquid chromatography tandem mass spectrometry (MS/MS). MS/MS spectra were matched to human and bacterial peptide sequences leveraging a bioinformatic workflow using a metagenomics-guided protein sequence database and rigorous evaluation. Label-free quantification revealed differentially abundant human peptides from proteins with known roles in CF, like neutrophil elastase and collagenase, and proteins with lesser-known roles in CF, including apolipoproteins. Differentially abundant bacterial peptides were identified from known CF pathogens (e.g., ), as well as other taxa with potentially novel roles in CF. We used this host-microbe peptide panel for targeted parallel-reaction monitoring validation, demonstrating for the first time an MS-based assay effective for quantifying host-microbe protein dynamics within BAL cells from individual CF patients. Our integrated bioinformatic and analytical workflow combining discovery, verification, and validation should prove useful for diverse studies to characterize microbial contributors in airway diseases. Furthermore, we describe a promising preliminary panel of differentially abundant microbe and host peptide sequences for further study as potential markers of host-microbe relationships in CF disease pathogenesis.IMPORTANCEIdentifying microbial pathogenic contributors and dysregulated human responses in airway disease, such as CF, is critical to understanding disease progression and developing more effective treatments. To this end, characterizing the proteins expressed from bacterial microbes and human host cells during disease progression can provide valuable new insights. We describe here a new method to confidently detect and monitor abundance changes of both microbe and host proteins from challenging BAL samples commonly collected from CF patients. Our method uses both state-of-the art mass spectrometry-based instrumentation to detect proteins present in these samples and customized bioinformatic software tools to analyze the data and characterize detected proteins and their association with CF. We demonstrate the use of this method to characterize microbe and host proteins from individual BAL samples, paving the way for a new approach to understand molecular contributors to CF and other diseases of the airway.
PubMed: 38934598
DOI: 10.1128/msystems.00929-23 -
MSystems Jun 2024The application of fecal metaproteomics to large-scale studies of the gut microbiota requires high-throughput analysis and standardized experimental protocols. Although...
The application of fecal metaproteomics to large-scale studies of the gut microbiota requires high-throughput analysis and standardized experimental protocols. Although high-throughput protein cleanup and digestion methods are increasingly used in shotgun proteomics, no studies have yet critically compared such protocols using human fecal samples. In this study, human fecal protein extracts were processed using several different protocols based on three main approaches: filter-aided sample preparation (FASP), solid-phase-enhanced sample preparation (SP3), and suspension trapping (S-Trap). These protocols were applied in both low-throughput (i.e., microtube-based) and high-throughput (i.e., microplate-based) formats, and the final peptide mixtures were analyzed by liquid chromatography coupled to high-resolution tandem mass spectrometry. The FASP-based methods and the combination of SP3 with in-StageTips (iST) yielded the best results in terms of the number of peptides identified through a database search against gut microbiome and human sequences. The efficiency of protein digestion, the ability to preserve hydrophobic peptides and high molecular weight proteins, and the reproducibility of the methods were also evaluated for the different protocols. Other relevant variables, including interindividual variability of stool, duration of protocols, and total costs, were considered and discussed. In conclusion, the data presented here can significantly contribute to the optimization and standardization of sample preparation protocols in human fecal metaproteomics. Furthermore, the promising results obtained with the high-throughput methods are expected to encourage the development of automated workflows and their application to large-scale gut microbiome studies.IMPORTANCEFecal metaproteomics is an experimental approach that allows the investigation of gut microbial functions, which are involved in many different physiological and pathological processes. Standardization and automation of sample preparation protocols in fecal metaproteomics are essential for its application in large-scale studies. Here, we comparatively evaluated different methods, available also in a high-throughput format, enabling two key steps of the metaproteomics analytical workflow (namely, protein cleanup and digestion). The results of our study provide critical information that may be useful for the optimization of metaproteomics experimental pipelines and their implementation in laboratory automation systems.
PubMed: 38934547
DOI: 10.1128/msystems.00661-24 -
MSystems Jun 2024The alarming rise of antibiotic-resistant bacterial infections is driving efforts to develop alternatives to conventional antibiotics. In this context, antimicrobial...
UNLABELLED
The alarming rise of antibiotic-resistant bacterial infections is driving efforts to develop alternatives to conventional antibiotics. In this context, antimicrobial peptides (AMPs) have emerged as promising candidates for their ability to target a broad range of microorganisms. However, the development of AMPs with optimal potency, selectivity, and/or stability profiles remains a challenge. To address it, computational tools for predicting AMP properties and designing novel peptides have gained increasing attention. PyAMPA is a novel platform for AMP discovery. It consists of five modules, namely AMPScreen, AMPValidate, AMPSolve, AMPMutate, and AMPOptimize, that allow high-throughput proteome inspection, candidate screening, and optimization through point-mutation and genetic algorithms. The platform also offers additional tools for predicting and evaluating AMP properties, including antimicrobial and cytotoxic activity, and peptide half-life. By providing innovative and accessible inroads into AMP motifs in proteomes, PyAMPA will enable advances in AMP development and potential translation into clinically useful molecules. PyAMPA is available at: https://github.com/SysBioUAB/PyAMPA.
IMPORTANCE
This paper introduces PyAMPA, a new bioinformatics platform designed for the discovery and optimization of antimicrobial peptides (AMPs). It addresses the urgent need for new antimicrobials due to the rise of antibiotic-resistant infections. PyAMPA, with its five predictive modules -AMPScreen, AMPValidate, AMPSolve, AMPMutate and AMPOptimize, enables high-throughput screening of proteomes to identify potential AMP motifs and optimize them for clinical use. Its unique approach, combining prediction, design, and optimization tools, makes PyAMPA a robust solution for developing new AMP-based therapies, offering a significant advance in combatting antibiotic resistance.
PubMed: 38934543
DOI: 10.1128/msystems.01358-23 -
Neural Regeneration Research Jun 2024Mature oligodendrocytes form myelin sheaths that are crucial for the Insulation of axons and efficient signal transmission in the central nervous system. Recent evidence...
Mature oligodendrocytes form myelin sheaths that are crucial for the Insulation of axons and efficient signal transmission in the central nervous system. Recent evidence has challenged the classical view of the functionally static mature oligodendrocyte and revealed a gamut of dynamic functions such as the ability to modulate neuronal circuitry and provide metabolic support to axons. Despite the recognition of potential heterogeneity in mature oligodendrocyte function, a comprehensive summary of mature oligodendrocyte diversity is lacking. We delve into early 20th-century studies by Robertson and Río-Hortega that laid the foundation for the modern identification of regional and morphological heterogeneity in mature oligodendrocytes. Indeed, recent morphologic and functional studies call into question the long-assumed homogeneity of mature oligodendrocyte function through the identification of distinct subtypes with varying myelination preferences. Furthermore, modern molecular investigations, employing techniques such as single cell/nucleus RNA sequencing, consistently unveil at least six mature oligodendrocyte subpopulations in the human central nervous system that are highly transcriptomically diverse and vary with central nervous system region. Age and disease related mature oligodendrocyte variation denotes the impact of pathological conditions such as multiple sclerosis, Alzheimer's disease, and psychiatric disorders. Nevertheless, caution is warranted when subclassifying mature oligodendrocytes because of the simplification needed to make conclusions about cell identity from temporally confined investigations. Future studies leveraging advanced techniques like spatial transcriptomics and single-cell proteomics promise a more nuanced understanding of mature oligodendrocyte heterogeneity. Such research avenues that precisely evaluate mature oligodendrocyte heterogeneity with care to understand the mitigating influence of species, sex, central nervous system region, age, and disease, hold promise for the development of therapeutic interventions targeting varied central nervous system pathology.
PubMed: 38934385
DOI: 10.4103/NRR.NRR-D-24-00055 -
Frontiers in Genetics 2024The single nucleotide polymorphism (SNP) rs4644 at codon 64 of galectin-3 (gal-3, gene name: ), specifying the variant proline (P64) to histidine (H64), is known to...
INTRODUCTION
The single nucleotide polymorphism (SNP) rs4644 at codon 64 of galectin-3 (gal-3, gene name: ), specifying the variant proline (P64) to histidine (H64), is known to affect the protein's functions and has been associated with the risk of several types of cancer, including differentiated thyroid carcinoma (DTC).
MATERIALS AND METHODS
To deepen our understanding of the biological effects of this SNP, we analyzed the proteome of two isogenic cell lines (NC-P64 vs. NA-H64) derived from the immortalized non-malignant thyrocyte cell line Nthy-Ori, generated through the CRISPR-Cas9 technique to differ by rs4644 genotype. We compared the proteome of these cells to detect differentially expressed proteins and studied their proteome in relation to their transcriptome.
RESULTS
Firstly, we found, consistently with previous studies, that gal-3-H64 could be detected as a monomer, homodimer, and heterodimer composed of one cleaved and one uncleaved monomer, whereas gal-3-P64 could be found only as a monomer or uncleaved homodimer. Moreover, results indicate that rs4644 influences the expression of several proteins, predominantly upregulated in NA-H64 cells. Overall, the differential protein expression could be attributed to the altered mRNA expression, suggesting that rs4644 shapes the function of gal-3 as a transcriptional co-regulator. However, this SNP also appeared to affect post-transcriptional regulatory mechanisms for proteins whose expression was oppositely regulated compared to mRNA expression. It is conceivable that the rs4644-dependent activities of gal-3 could be ascribed to the different modalities of self-dimerization.
CONCLUSION
Our study provided further evidence that rs4644 could affect the gal-3 functions through several routes, which could be at the base of differential susceptibility to diseases, as reported in case-control association studies.
PubMed: 38933925
DOI: 10.3389/fgene.2024.1380495 -
Frontiers in Plant Science 2024leaves are highly valued in traditional Chinese medicine for their substantial concentration of flavonoids, which play a crucial role in manifesting the plant's...
leaves are highly valued in traditional Chinese medicine for their substantial concentration of flavonoids, which play a crucial role in manifesting the plant's therapeutic properties. This study investigated the metabolomic, transcriptomic and proteomic profiles of leaves from two cultivars, (J) and (R), at three different developmental stages. Metabolite identification and analysis revealed a total of 1,412 and 1,421 metabolites with known structures were found. Flavonoids made up of 33%, including 10 significant accumulated icariin analogues. Transcriptomic analysis unveiled totally 41,644 differentially expressed genes (DEGs) containing five encoded genes participated in icariin biosynthesis pathways. Totally, 9,745 differentially expressed proteins (DEPs) were found, including Cluster-47248.2.p1 (UDP-glucuronosy/UDP-glucosyltransferase), Cluster-30441.2.p1 (O-glucosyltransferase), and Cluster-28344.9.p1 (anthocyanidin 3-O-glucoside 2 "-O-glucosyltransferase-like) through proteomics analysis which are involved to icariin biosynthesis. Protein-protein interaction (PPI) assay exhibited, totally 12 proteins showing a strong relationship of false discovery rate (FDR) <0.05 with these three proteins containing 2 leucine-rich repeat receptor kinase-like protein SRF7, and 5 methyl jasmonate esterase 1. Multi-omics connection networks uncovered 237 DEGs and 72 DEPs exhibited significant associations with the 10 icariin analogues. Overall, our integrated omics approach provides comprehensive insights into the regulatory network underlying icariin synthesis in , offering valuable resources for further research and development in medicinal plant cultivation and pharmaceutical applications.
PubMed: 38933461
DOI: 10.3389/fpls.2024.1409601