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.

Research on SARS-CoV-2, the viral pathogen that causes COVID-19, has identified angiotensin converting enzyme 2 (ACE2) as the primary viral receptor. Several genes that encode viral cofactors, such as TMPRSS2, NRP1, CTSL, and possibly KIM1, have since been discovered. Glutamyl aminopeptidase (APA), encoded by the gene ENPEP, is another cofactor candidate due to similarities in its biological role and high correlation with ACE2 and other human coronavirus receptors, such as aminopeptidase N (APN) and dipeptidyl peptidase 4 (DPP4). Recent studies have proposed a role for ENPEP as a viral receptor in humans, and ENPEP and ACE2 are both closely involved in the renin-angiotensin-aldosterone system proposed to play an important role in SARS-CoV-2 pathophysiology. We performed bioinformatic analyses using publicly available bulk (>17,000 samples from 49 distinct tissues) and single-cell (>2.5 million cells) RNA-Seq gene expression datasets to evaluate the expression and function of the ENPEP gene. We also investigated age- and sex-related changes in ENPEP expression. Overall, expression of ENPEP was highest in the small intestine enterocyte brush border and the kidney cortex. ENPEP is widely expressed in a subset of vascular smooth muscle cells (likely pericytes) in systemic vasculature, the heart, and the brain. ENPEP is expressed at low levels in the lower respiratory epithelium. In the lung, ENPEP is most highly expressed in para-alveolar fibroblasts. Single-cell data revealed ENPEP expression in a substantial fraction of ependymal cells, a finding not reported before in humans. Age increases ENPEP expression in skeletal muscle and the prostate, while decreasing it in the heart and aorta. Angiogenesis was found to be a central biological function associated with the ENPEP gene. Tissue-specific roles, such as protein digestion and fat metabolism, were also identified in the intestine. In the liver, the gene is linked to the complement system, a connection that has not yet been thoroughly investigated. Expression of ENPEP and ACE2 is strongly correlated in the small intestine and renal cortex. Both overall and in blood vessels, ENPEP and ACE2 have a stronger correlation than many other genes associated with SARS-CoV-2, such as TMPRSS2, CTSL, and NRP1. Possible interaction between glutamyl aminopeptidase and SARS-CoV-2 should be investigated experimentally.

In-feed antibiotic growth promoters (AGPs) have been a cornerstone in the livestock industry due to their role in enhancing growth and feed efficiency. However, concerns over antibiotic resistance have driven a shift away from AGPs toward natural alternatives. Despite the widespread use, the exact mechanisms of AGPs and alternatives are not fully understood. This necessitates holistic studies that investigate microbiota dynamics, host responses, and the interactions between these elements in the context of AGPs and alternative feed additives.

In this study, we conducted a multifaceted investigation of how Bacitracin, a common AGP, and a natural alternative impact both cecum microbiota and host expression in chickens. In addition to univariate and static differential abundance and expression analyses, we employed multivariate and time-course analyses to study this problem. To reveal host-microbe interactions, we assessed their overall correspondence and identified treatment-specific pairs of species and host expressed genes that showed significant correlations over time.

Our analysis revealed that factors such as developmental age substantially impacted the cecum ecosystem more than feed additives. While feed additives significantly altered microbial compositions in the later stages, they did not significantly affect overall host gene expression. The differential expression indicated that with AGP administration, host transmembrane transporters and metallopeptidase activities were upregulated around day 21. Together with the modulated kininogen binding and phenylpyruvate tautomerase activity over time, this likely contributes to the growth-promoting effects of AGPs. The difference in responses between AGP and PFA supplementation suggests that these additives operate through distinct mechanisms.

We investigated the impact of a common AGP and its natural alternative on poultry cecum ecosystem through an integrated analysis of both the microbiota and host responses. We found that AGP appears to enhance host nutrient utilization and modulate immune responses. The insights we gained are critical for identifying and developing effective AGP alternatives to advance sustainable livestock farming practices.

ERAP2 is an aminopeptidase involved in antigen processing and presentation, and harbor genetic variants linked to several inflammatory diseases such as Inflammatory Bowel Disease (IBD). The lack of an ERAP2 gene homologue in mice has hampered functional studies, and most human studies have focused on cells of hematopoietic origin. Using an IBD biobank as vantage point, this study explores how genetic variation in ERAP2 affects gene expression in human-derived epithelial organoids upon proinflammatory stimulation.

An IBD patient cohort was genotyped with regards to two single nucleotide polymorphisms (SNP) (rs2910686/rs2248374) associated with ERAP2 expression levels, and we examined the correlation between colon gene expression and genotype, specifically aiming to establish a relationship with ERAP2 expression proficiency. Human-derived colon organoids (colonoids) with known ERAP2 genotype were established and used to explore differences in whole genome gene expression between ERAP2-deficient (n = 4) and -proficient (n = 4) donors upon pro-inflammatory encounter.

When taking rs2910686 genotype into account, ERAP2 gene expression is upregulated in the inflamed colon of IBD patients. Colonoids upregulate ERAP2 upon IFNɣ stimulation, and ERAP2 expression proficiency is dependent on rs2910686 genotype. Colonoid genotyping confirms that mechanisms independent of the frequently studied SNP rs2248374 can cause ERAP2-deficiency. A total of 586 genes involved in various molecular mechanisms are differentially expressed between ERAP2 proficient- and deficient colonoids upon proinflammatory stimulation, including genes encoding proteins with the following molecular function: catalytic activity (AOC1, CPE, ANPEP and MEP1A), regulator activity (TNFSF9, MDK, GDF15, ILR6A, LGALS3 and FLNA), transmembrane transporter activity (SLC40A1 and SLC5A1), and extracellular matrix structural constituents (FGL2, HMCN2, and MUC17).

ERAP2 is upregulated in the inflamed IBD colon mucosa, and expression proficiency is highly correlated with genotype of rs2910686. While the SNP rs2248374 is commonly used to determine ERAP2 expressional proficiency, our data confirms that mechanisms independent of this SNP can lead to ERAP2 deficiency. Our data demonstrates that epithelial ERAP2 presence affects the inflammatory response in colonoids, suggesting a pleiotropic role of ERAP2 beyond MHC class I antigen processing.

The accurate estimation of basal endogenous losses (BEL) of amino acids at the ileum is indispensable to improve nutrient utilization efficiency. This study used a quantitative proteomic approach to identify variations in BEL in the ileal digesta of growing pigs fed a nitrogen-free diet (NFD) or a casein diet (CAS). Eight barrow pigs (39.8 ± 6.3 kg initial body weight (BW)) were randomly assigned to a 2 × 2 crossover design. A total of 348 proteins were identified and quantified in both treatments, of which 101 showed a significant differential abundance between the treatments (p < 0.05). Functional and pathway enrichment analyses revealed that the endogenous proteins were associated with intestinal metabolic function. Furthermore, differentially abundant proteins (DAPs) in the digesta of pigs fed the NFD enriched terms and pathways that suggest intestinal inflammation, the activation of innate antimicrobial host defense, an increase in cellular autophagy and epithelial turnover, and reduced synthesis of pancreatic and intestinal secretions. These findings suggest that casein diets may provide a more accurate estimation of BEL because they promote normal gastrointestinal secretions. Overall, proteomic and bioinformatic analyses provided valuable insights into the composition of endogenous proteins in the ileal digesta and their relationship with the functions, processes, and pathways modified by diet composition.

Background Pulmonary arterial hypertension (PAH) is a complex, fatal disease where disease severity has been associated with the single nucleotide polymorphism (SNP) rs2856830, located near the human leukocyte antigen DPA1 (HLA-DPA1) gene. We aimed to define the genetic architecture of functional variants associated with PAH disease severity by identifying allele-specific binding transcription factors and downstream targets that control endothelial pathophenotypes and PAH. Methods and Results Electrophoretic mobility shift assays of oligonucleotides containing SNP rs2856830 and 8 SNPs in linkage disequilibrium revealed functional SNPs via allele-imbalanced binding to human pulmonary arterial endothelial cell nuclear proteins. DNA pulldown proteomics identified SNP-binding proteins. SNP genotyping and clinical correlation analysis were performed in 84 patients with PAH at University of Pittsburgh Medical Center and in 679 patients with PAH in the All of Us database. SNP rs9277336 was identified as a functional SNP in linkage disequilibrium (r2>0.8) defined by rs2856830, and the minor allele was associated with decreased hospitalizations and improved cardiac output in patients with PAH, an index of disease severity. SNP pulldown proteomics showed allele-specific binding of nuclear ACTN4 (alpha actinin 4) protein to rs9277336 minor allele. Both ACTN4 and HLA-DPA1 were downregulated in pulmonary endothelium in human patients and rodent models of PAH. Via transcriptomic and phenotypic analyses, knockdown of HLA-DPA1 phenocopied knockdown of ACTN4, both similarly controlling cell structure pathways, immune pathways, and endothelial dysfunction. Conclusions We defined the pathogenic activity of functional SNP rs9277336, entailing the allele-specific binding of ACTN4 and controlling expression of the neighboring HLA-DPA1 gene. Through inflammatory or genetic means, downregulation of this ACTN4-HLA-DPA1 regulatory axis promotes endothelial pathophenotypes, providing a mechanistic explanation for the association between this SNP and PAH outcomes.

The gut mucosa of human is covered by mucus, functioning as a crucial defense line for the intestine against external stimuli and pathogens. Mucin2 (MUC2) is a subtype of secretory mucins generated by goblet cells and is the major macromolecular component of mucus. Currently, there is an increasing interest on the investigations of MUC2, noting that its function is far beyond a maintainer of the mucus barrier. Moreover, numerous gut diseases are associated with dysregulated MUC2 production. Appropriate production level of MUC2 and mucus contributes to gut barrier function and homeostasis. The production of MUC2 is regulated by a series of physiological processes, which are orchestrated by various bioactive molecules, signaling pathways and gut microbiota, etc., forming a complex regulatory network. Incorporating the latest findings, this review provided a comprehensive summary of MUC2, including its structure, significance and secretory process. Furthermore, we also summarized the molecular mechanisms of the regulation of MUC2 production aiming to provide developmental directions for future researches on MUC2, which can act as a potential prognostic indicator and targeted therapeutic manipulation for diseases. Collectively, we elucidated the micro-level mechanisms underlying MUC2-related phenotypes, hoping to offer some constructive guidance for intestinal and overall health of mankind.

The astacins are a family of metallopeptidases (MPs) that has been extensively described from animals. They are multidomain extracellular proteins, which have a conserved core architecture encompassing a signal peptide for secretion, a prodomain or prosegment and a zinc-dependent catalytic domain (CD). This constellation is found in the archetypal name-giving digestive enzyme astacin from the European crayfish Astacus astacus. Astacin catalytic domains span ∼200 residues and consist of two subdomains that flank an extended active-site cleft. They share several structural elements including a long zinc-binding consensus sequence (HEXXHXXGXXH) immediately followed by an EXXRXDRD motif, which features a family-specific glutamate. In addition, a downstream SIMHY-motif encompasses a "Met-turn" methionine and a zinc-binding tyrosine. The overall architecture and some structural features of astacin catalytic domains match those of other more distantly related MPs, which together constitute the metzincin clan of metallopeptidases. We further analysed the structures of PRO-, MAM, TRAF, CUB and EGF-like domains, and described their essential molecular determinants. In addition, we investigated the distribution of astacins across kingdoms and their phylogenetic origin. Through extensive sequence searches we found astacin CDs in > 25,000 sequences down the tree of life from humans beyond Metazoa, including Choanoflagellata, Filasterea and Ichtyosporea. We also found < 400 sequences scattered across non-holozoan eukaryotes including some fungi and one virus, as well as in selected taxa of archaea and bacteria that are pathogens or colonizers of animal hosts, but not in plants. Overall, we propose that astacins originate in the root of Holozoa consistent with Darwinian descent and that the latter genes might be the result of horizontal gene transfer from holozoan donors.

Membrane-type-I matrix metalloproteinase (MT1-MMP) is one of six human membrane-bound MMPs and is responsible for extracellular matrix remodelling by degrading several substrates like fibrillar collagens, including types I-III, or fibronectin. Moreover, MT1-MMP was described as a key player in cancer progression and it is involved in various inflammatory processes, as well as in the pathogenesis of Alzheimer's disease (AD). The membrane-tethered metalloprotease meprin β as well as a disintegrin and metalloproteinase 10 (ADAM10) and ADAM17 are also associated with these diseases. Interestingly, meprin β, ADAM10/17 and MT1-MMP also have a shared substrate pool including the interleukin-6 receptor and the amyloid precursor protein. We investigated the interaction of these proteases, focusing on a possible connection between MT1-MMP and meprin β, to elucidate the potential mutual regulations of both enzymes. Herein, we show that besides ADAM10/17, MT1-MMP is also able to shed meprin β from the plasma membrane, leading to the release of soluble meprin β. Mass spectrometry-based cleavage site analysis revealed that the cleavage of meprin β by all three proteases occurs between Pro₆₀₂ and Ser₆₀₃ , N-terminal of the EGF-like domain. Furthermore, only inactive human pro-meprin β is shed by MT1-MMP, which is again in accordance with the shedding capability observed for ADAM10/17. Vice versa, meprin β also appears to shed MT1-MMP, indicating a complex regulatory network. Further studies will elucidate this well-orchestrated proteolytic web under distinct conditions in health and disease and will possibly show whether the loss of one of the above-mentioned sheddases can be compensated by the other enzymes.

Pesticides are extensively applied globally. Pesticide residues induce calamitous effects on the environment and untargeted organisms. Public concerns for the safety of freshwater organisms and the challenges posed by aquatic contaminants remain high. In the present study, the acute toxicity of avermectins (AVMs) to the crayfish, Procambarus clarkii was evaluated. We also evaluated the potential effects of AVM on the biochemical and transcriptomic status of the hepatopancreas and gastrointestinal tract in P. clarkii.

The 24, 48, 72, 96 h median lethal concentrations (LC₅₀ ) of AVM on crayfish were 2.626, 1.162, 0.723, 0.566 mg L^-1 , respectively. The crayfish were then exposed to 0.65 mg L^-1 of AVM for 96 h. AVM significantly altered biochemical parameters including AChE and CAT activities in the hepatopancreas, and AChE, SOD and Na + -K + -ATPase activities in the gastrointestinal tract at several time points. Furthermore, transcriptomic analysis identified 953 and 1851 differentially-expressed genes (DEGs) in the hepatopancreas and gastrointestinal tract, respectively. KEGG enrichment showed that the gene expression profiles of the hepatopancreas and gastrointestinal tract were distinct from each other. The DEGs in the hepatopancreas were mostly enriched with stress-response pathways, while the majority of the DEGs in the gastrointestinal tract belonged to metabolism-related pathways.

We demonstrated that the AVM induced acute toxicity, oxidative stress, osmoregulation disturbance, neurotoxicity and transcriptome imbalance in crayfish. These findings unraveled the detrimental effects of AVMs exposure on crayfish. © 2022 Society of Chemical Industry.

Consisting of a defined set of extracellular proteins secreted from resident cells and with minor contributions from serum proteins, the extracellular matrix (ECM) is an essential component of all tissues. Maintaining tissue homeostasis, structural support and cellular control through cell-ECM communication, the ECM has come to be viewed as not just a passive structural entity but rather as a dynamic signaling conduit between cells and the extracellular compartment. Proteins and their cleavage products mediate this communication, and aberrant signaling, either directly or indirectly distorting the ECM, results in pathological conditions including cancer, inflammation, fibrosis, and neurodegenerative diseases. Characterization of ECM components, the matrisome, the extracellular environment and their changes in disease is therefore of importance to understand and mitigate by developing novel therapeutics. Liquid chromatography-mass spectrometry (LC-MS) proteomics has been integral to protein and proteome research for decades and long superseded the obsolescent gel-based approaches. A continuous effort has ensured progress with increased sensitivity and throughput as more advanced equipment has been developed hand in hand with specialized enrichment, detection, and identification methods. Part of this effort lies in the field of degradomics, a branch of proteomics focused on discovering novel protease substrates by identification of protease-generated neo-N termini, the N-terminome, and characterizing the responsible protease networks. Various methods to do so have been developed, some specialized for specific tissue types, others for particular proteases, throughput, or ease of use. This review aims to provide an overview of the state-of-the-art proteomics techniques that have successfully been recently utilized to characterize proteolytic cleavages in the ECM and thereby guided new research and understanding of the ECM and matrisome biology.

The purpose of this review is to provide an update regarding the gut barrier and its involvement with chronic diseases, as well as to review biomarkers for identification of gut barrier integrity. This review is timely and relevant as our knowledge is increasing regarding the role of the gut microbiome and the gut barrier in health and disease.

This review provides an overview of: the gut barrier, which is complex and comprised of the mucus layer and the intestinal apical junctional protein complex; the gut microbiome in its relation to regulating the integrity of the gut barrier; select acute and chronic conditions that are known to be associated with gut dysbiosis and impaired gut integrity or 'leaky gut'; and current means for identifying loss in gut barrier integrity.

Many chronic conditions are associated with gut dysbiosis and systemic inflammation. Identifying whether the gut barrier is compromised in these conditions could help to inform potential therapeutics as a means to correct losses in gut barrier integrity and mitigate associated medical conditions.