Protein Abundance Research Articles

281. PROTEOMIC, TRANSCRIPTOMIC AND GENOMIC ANALYSIS OF ESOPHAGEAL ADENOCARCINOMA UNCOVERS CANDIDATE THERAPEUTIC TARGETS AND CANCER-SELECTIVE POST-TRANSCRIPTIONAL REGULATION

Abstract Background Addressing the poor prognosis associated with esophageal adenocarcinoma (EAC) has been hindered by the absence of biomarkers for early disease detection and therapeutic targets. Despite extensive research on the somatic mutations associated with EAC, the impact of genomic aberrations on protein expression and cancer phenotype remains unclear. Methods We conducted a quantitative proteomic analysis using tumour tissue and patient-matched adjacent normal esophageal and gastric tissues from 23 patients undergoing resection for EAC. We further examined the relationship between transcript and protein levels using patient-matched whole transcriptome RNA sequencing and proteomic data from 7 patients and integrated these findings with data from a cohort of 264 EAC patients with RNA sequencing and 454 patients with whole-genome sequencing, as well as external published datasets. Results We quantified protein expression from 5897 genes in EAC and normal tissues. Several potential biomarkers showing selective expression in EAC, such as the transmembrane protein GPA33, were identified. We validated the EAC-specific expression of GPA33 in an external cohort of 115 patients, suggesting its utility as a diagnostic and therapeutic target. Additionally, integrated analysis of protein and RNA expression in EAC and normal tissues revealed genes with discordant protein and RNA levels, indicating post-transcriptional regulation of protein expression. These outlier genes, including SLC25A30, TAOK2, and AGMAT, exhibited rare somatic mutations, suggesting post-transcriptional mechanisms driving this EAC-specific phenotype. AGMAT, as an example, was found to be overexpressed at the protein level in EAC compared to adjacent normal tissues, with a proposed EAC-selective, post-transcriptional mechanism regulating protein abundance. Conclusion Through quantitative proteomic analysis, we identified GPA33 as a candidate EAC biomarker. Integrated analysis of the proteome, transcriptome, and genome in EAC uncovered genes with tumor-selective post-transcriptional regulation of protein expression, offering potential exploitable vulnerabilities for therapeutic intervention.

O48 CHRONIC EXPOSURE TO POLLUTED URBAN AIR ALTERS MOUSE SYSTEMIC AND TISSUE-SPECIFIC COMPONENTS OF THE RENIN-ANGIOTENSIN SYSTEM (RAS)

Background and Objective: Chronic exposure to air pollution induces pulmonary, cardiovascular, and renal diseases. In the current study, we investigated the hypothesis that postulates that a dysregulation of the RAS may contribute to these pathologies. Methods: Male Balb/C mice were exposed to urban air from Buenos Aires City, in whole-body exposure chambers for 14 weeks. Control animals were exposed to filtered air. The levels of main RAS components including angiotensin-converting enzyme (ACE) and ACE2; AT1, AT2, and Mas receptors (R) were determined in mouse lungs, kidneys, and heart, through western blotting (WB) and RT-qPCR, respectively. The tissue content of cytokine mARN was determined by RT-qPCR. The tissue content of nitrotyrosine and 4-hydroxynonenal (4-HNE) levels, were determined by WB. Circulating angiotensin (Ang) II levels were determined by radioimmunoassay. Systolic blood pressure was measured by the tail-cuff method. Results: Exposure to air pollution resulted in a) Increased levels of ACE and AT1R mRNA in the lungs and augmented the abundance markers of inflammation (TNF-α, IL-6, and IL-1β) and oxidative stress (nitrotyrosine and 4-HNE) in this tissue, b) increased levels of MasR mRNA (all analyzed tissues) and MasR protein levels (lungs and heart), c) upregulated mRNA and protein abundance of cardiac and renal AT2Rs, and d) Increased levels of circulating Ang II and e) augmented systolic blood pressure. Conclusions: Our findings indicate that chronic exposure to air pollution alters the expression of both tissue and systemic components of the RAS. Since both AT2R and MasRs have been shown to participate in tissue-repair mechanisms, the upregulation of these receptors detected in mouse lungs, heart, and kidney after chronic exposure to polluted urban air could represent a mechanism of tissue protection against damage induced by air pollution.

Fabrication of an IgG-imprinted monolith micro-solid phase extraction chip for IgG extraction from human plasma

Molecularly imprinted polymers (MIPs), also known as synthetic antibodies, offer a novel and promising approach for protein separation at analytical scale. In proteomics research, this approach could capture target high abundant proteins from biological fluids to reduce sample complexity. In this study, we have explored the potential to imprint human immunoglobulin G (IgG) to develop a micro-solid phase extraction chip for selective removal of IgG from human plasma. Imprinting of IgG within the polyhydroxy ethyl methacrylate monolith containing dimethyl amino ethyl methacrylate (DMAEMA) as a functional monomer was successfully achieved and characterized using FT-IR spectroscopy and protein assay kit. 0.1 M Tris buffer (pH 7) and UV-polymerization were identified as the ideal buffer and polymerization strategy to imprint IgG within 20 min. Template (IgG) elution using 10 % acetic acid containing 10 % (v/v) SDS as elution buffer was moderately successful (25 % IgG-free polymer was obtained). Protein shape has influenced its imprinting and elution from the monolith. The protocol developed to imprint one protein cannot be translated to another protein without further modifications. A 10-fold decrease of DMAEMA concentration within the monolith and a sequential elution step has resulted in >90 % IgG free-imprinted monolith. The IgG-imprinted monolith has displayed an adsorption capacity of 3.7 mg per g monolith and showed good IgG selectivity as compared to other tested human proteins. Translation of imprinted monolith protocol to develop a micro-solid phase extraction chip was successful. The chip can be re-used for 5 cycles with >95 % reusability and has selectively captured IgG from human plasma.

Optimized differential evolution and hybrid deep learning for superior drug-target binding affinity prediction

Investigating Drug-Target Interactions (DTI) is crucial for drug repositioning and discovery tasks. However, discovering DTIs through experimental approaches is time-consuming and requires substantial financial resources. To address these challenges, machine learning-based methodologies have been adopted to reduce costs and save time. Unfortunately, the effectiveness of these methods has been limited due to the binary classification approach and the lack of empirically validated negative samples. The availability of abundant DTI datasets and protein structure data has enabled the development of new approaches, such as redefining the DTI problem as a regression task. Given this context, we propose an innovative deep-learning approach to predict binding affinities between drugs and targets. Our model, named the Convolution Self-Attention Network with Attention-based Bidirectional Long Short-Term Memory Network (CSAN-BiLSTM-Att), integrates convolutional neural network (CNN) blocks with self-attention mechanisms to create an attention-based bidirectional long short-term memory (BiLSTM) model, followed by fully connected layers. Due to the model's complexity, proper hyperparameter tuning is essential. To optimize this, we employ the Differential Evolution (DE) technique to select the most suitable hyperparameters. Experimental results demonstrate that the DE-based CSAN-BiLSTM-Att model outperforms previous approaches. Specifically, the model achieved a concordance index of 0.898 and a mean square error of 0.228 on the DAVIS dataset, and a concordance value of 0.971 with a mean square error of 0.014 on the KIBA dataset.

Short-term feed restriction induces inflammation and an antioxidant response via cystathionine-β-synthase and glutathione peroxidases in ruminal epithelium from Angus steers.

Decreased intake is induced by stressors such as parturition, transportation, dietary transitions, and disease. An important function of one-carbon metabolism (OCM) is to produce the antioxidant glutathione to help reduce oxidative stress. Although various components of OCM are expressed in the bovine rumen and small intestine, the relationship among reduced feed intake, one-carbon metabolism, and antioxidant mechanisms in gut tissues is unknown. This study aimed to assess alterations in immune and antioxidant pathways in ruminal epithelium due to acute feed restriction (FR). Seven group-housed ruminally-cannulated Angus steers (663 ± 73kg body weight, 2-year old) had ad libitum access to a finishing diet (dry-rolled corn, corn silage, modified wet distiller's grains) during 15 days of a pre-FR period (PRE). Subsequently, steers were moved to a metabolism barn with tie-stalls and individually-fed at 25% of estimated intake in PRE for 3 days (FR period, FRP). This was followed by 15-days of recovery (POST) during which steers had ad libitum access to the same diet as in PRE and FRP. Plasma and ruminal tissue biopsies were collected during each period. Plasma free fatty acid and IL1-β concentrations were higher (P ≤ 0.03) in FRP than PRE or POST. The mRNA abundance of the pro-inflammatory genes TNF, TLR2, and TLR4 in ruminal epithelium peaked (P < 0.05) at FRP and remained higher at POST. These responses agreed with the higher (P < 0.05) abundance of phosphorylated (p)-MAPK (an inflammation activator) and p-EEF2 (translational repressor) in FRP than PRE and POST. Although ruminal GPX enzyme activity did not increase at FRP compared with PRE and POST, protein abundance of GPX1 and GPX3 along with the antioxidant response regulator NFE2L2 were highest (P < 0.01) and the activity of cystathione-beta synthase tended (P = 0.06) to be highest during FR. Although FR had minimal negative effects on tissue integrity-related genes (only filamin A was downregulated), it led to a systemic inflammatory response and triggered inflammation and antioxidant mechanisms within ruminal epithelium. Thus, deploying anti-inflammatory and antioxidant mechanisms via molecules that feed into OCM (e.g., dietary methyl donors such as methionine, choline, betaine, folate) could potentially counteract the stressors associated with FR.

Hyperhomocysteinemia-Induced Alterations in Protein Expression and Oxidative Stress Parameters in Rat Heart

Hyperhomocysteinemia (HHcy) is considered an independent risk factor of cardiovascular diseases. Among the proposed mechanisms underlying homocysteine toxicity are altered protein expression and induction of oxidative stress. In the present study, we explored protein abundance and parameters related to oxidative stress in heart homogenates of rats exposed to chronic mild HHcy. Using two-dimensional gel electrophoresis followed by MALDI-TOF/TOF mass spectrometry 22 altered proteins (6 upregulated and 14 downregulated) were identified. For eight proteins the altered abundances were validated by Western blot analysis. Identified proteins are primarily involved in energy metabolism (mainly enzymes of glycolysis, pyruvate dehydrogenase complex, citric acid cycle, and ATP synthase), cardiac muscle contraction (α-actin and myosin light chains), stress response (heat-shock protein β1 and αB-crystallin) and antioxidant defense (glutathione peroxidase 1). Diminished antioxidant defense was confirmed by decreases in total antioxidant capacity and GSH/GSSG ratio. Consistent with the decline in enzymatic and non-enzymatic antioxidant defense the protein oxidative modification, as determined by tyrosine nitration, was significantly increased. These findings suggest that both, altered protein expression and elevated oxidative stress contribute to cardiovascular injury caused by HHcy.

Comparative analysis of phosphorylated proteomes between plerocercoid and adult Spirometra mansoni reveals phosphoproteomic profiles of the medical tapeworm

BackgroundPlerocercoid larvae of the tapeworm Spirometra mansoni can infect both humans and animals, leading to severe parasitic zoonosis worldwide. Despite ongoing research efforts, our understanding of the developmental process of S. mansoni remains inadequate. To better characterize posttranslational regulation associated with parasite growth, development, and reproduction, a comparative phosphoproteomic study was conducted on the plerocercoid and adult stages of S. mansoni.MethodsIn this study, site-specific phosphoproteomic analysis was conducted via 4D label-free quantitative analysis technology to obtain primary information about the overall phosphorylation status of plerocercoids and adults.ResultsA total of 778 differentially abundant proteins (DAPs) were detected between adults and plerocercoids, of which 704 DAPs were upregulated and only 74 were downregulated. DAPs involved in metabolic activity were upregulated in plerocercoid larvae compared with adults, whereas DAPs associated with binding were upregulated in adults. Gene Ontology (GO) and Kyoto Encyclopedia of Genes (KEGG) analyses indicated that most DAPs involved in signal transduction and environmental information processing pathways were highly active in adults. DAPs upregulated in the plerocercoid group were enriched mainly in metabolic activities. The kinases PKACA, GSK3B, and smMLCK closely interact, suggesting potential active roles in the growth and development of S. mansoni.ConclusionsThe dataset presented in this study offers a valuable resource for forthcoming research on signaling pathways as well as new insights into functional studies on the molecular mechanisms of S. mansoni.Graphical abstract

Analyzing Porcine Corneal Xenograft Compatibility: In Silico Insights on Graft Outcomes

Background: Corneal transplantation faces significant challenges due to the shortage in donor corneas. Porcine corneas have emerged as a potential solution due to their similarities in biomechanical properties with pigs, yet xenoimmune rejection poses an obstacle to their efficacy. Methods: In this study, in silico methods were employed to analyze the compatibility of porcine corneal xenografts, focusing on two key aspects: the comparison of corneal matrix proteins and investigation of the immunological mediators and pathways involved in corneal graft rejection. The amino acid sequences of the fourteen (14) most abundant proteins in the corneal matrix were compared to determine their structural and functional differences. The primary amino acid structures and compositions, theoretical pI, and grand average of hydropathicity were determined and compared between the two species. Results: In graft performance, similarities and differences between the donor and recipient tissues influence the success of transplantation. When the proteins closely resemble each other, in terms of structural characteristics and biochemical properties, the host’s immune system is less likely to recognize the tissue as foreign. The immunological mediators and pathways involved in corneal graft rejection were investigated, elucidating the mechanisms underlying xenograft incompatibility. Based on the results generated from STRING, the specific groups of molecules that are involved in the immune-mediated rejection process are costimulatory molecules, cytokines, immune checkpoint molecules, apoptosis regulators, cell adhesion molecules, growth factors, neuropeptides and hormones, certain receptors, the cytotoxic molecule GZMA, and the chemokine CCL5. Conclusions: The results of this study establish that the porcine cornea has a high suitability for corneal xenotransplantation into humans but requires immune-based therapeutic interventions to increase graft acceptance.

Effects of Eucommia ulmoides leaf extracts and caltrate on osteoblasts proliferation and differentiation

Osteoporosis is a significant and escalating public health issue. It is a skeletal disorder in which there is a decrease in bone strength, resulting in a heightened susceptibility to fractures. Eucommia ulmoides leaves (EUL) has been traditionally employed to alleviate muscle and bone weakness. In order to investigate the induction of osteoblast proliferation and differentiation by the two EUL extracts under non-stress conditions, as well as their combined effects with Caltrate, this study examined cell proliferation, alkaline phosphatase activity, mineralization, and abundance of osteogenic differentiation-related proteins. The results demonstrated that EUL extract 1 and Caltrate intervention alone could promote osteoblast proliferation and differentiation, and their combined treatment exhibited a synergistic effect. EUL extract 2, when administered alone, was capable of enhancing osteoblast proliferation and differentiation; however, it did not exhibit a strong synergistic effect when combined with Caltrate. Therefore, administering EUL extract 1 either individually or in combination with Caltrate serve as a potential dietary supplement for osteoporosis.

One day of environment-induced heat stress damages the murine myocardium.

The physiological consequences of environment-induced heat stress (EIHS), caused by prolonged exposure to excess heat and humidity, are largely unknown. The purpose of this investigation was to determine the extent to which EIHS alters cardiac health. We hypothesized that 24 h of EIHS would cause cardiac injury and cellular dysfunction in a murine EIHS model. To test this hypothesis, 7 wk old female mice were housed under thermoneutral (TN) conditions (n=12, 31.2 ± 1.01 °C, 35 ± 0.7% humidity) or EIHS conditions (n=14; 37.6 ± 0.01 °C, 42.0 ± 0.06 % humidity) for 24 h. Environment-induced heat stress increased rectal temperature by 2.1 °C (P< 0.01) and increased subcutaneous temperature by 1.8 °C (P< 0.01). Body weight was decreased by 10% (P=0.03), heart weight/body weight was increased by 26% (P <0.01), and tissue water content was increased by 11% (P<0.05) in EIHS compared to TN. In comparison to TN, EIHS increased protein abundance of heat shock protein (HSP) 27 by 84% (P=0.01); however, HSPs 90, 60, 70, and phosphorylated HSP 27 were similar between groups. Histological inspection of the heart revealed EIHS animals had increased myocyte vacuolation in left ventricle (P=0.01), right ventricle (P<0.01), and septum (P=0.01) compared to TN animals. Biochemical indices are suggestive of mitochondrial remodeling, increased autophagic flux, and robust activation of endoplasmic reticulum stress in hearts from EIHS mice compared to TN mice. These data demonstrate that 1 d of EIHS is sufficient to induce myocardial injury and biochemical dysregulation.

EIF4G2 Promotes Hepatocellular Carcinoma Progression via IRES-dependent PLEKHA1 Translation Regulation.

Hepatocellular carcinoma (HCC) is a highly lethal cancer, and proteomic studies have shown increased protein diversity and abundance in HCC tissues, whereas the role of protein translation has not been extensively explored in HCC. Our research focused on key molecules in the translation process to identify a potential contributor in HCC. We discovered that EIF4G2, a crucial translation initiation factor, is significantly upregulated in HCC tissues and associated with poor prognosis. This study uniquely highlights the impact of EIF4G2 deletion, which suppresses tumor growth and metastasis both in vitro and in vivo. Furthermore, polysome analysis and nascent protein synthesis assays revealed EIF4G2's role in regulating protein translation, specifically identifying PLEKHA1 as a key translational product. This represents a novel mechanistic insight into HCC malignancy. RNA immunoprecipitation (RIP) and Dual-luciferase reporter assays further revealed that EIF4G2 facilitates PLEKHA1 translation via an IRES-dependent manner. Importantly, the synergistic effects of EIF4G2 depletion and PLEKHA1 reduction in inhibiting cell migration and invasion underscore the therapeutic potential of targeting this axis. This study not only advances our understanding of translational regulation in HCC but also identifies the EIF4G2-PLEKHA1 axis as a promising therapeutic target, offering new avenues for intervention in HCC treatment.

Proteomic profiles revealed enzymatic activities associated with the flavor formation of salted shrimp paste influenced by Bacillus subtilis K-C3 inoculation.

Enzymatic proteomic profiles were examined to comprehend the predominant enzymes involved in the flavor development of salted shrimp paste influenced by Bacillus subtilis K-C3 inoculation (Inoc), compared to those without inoculation (CON). Inoc showed greater proteolytic, lipolytic, and chitinolytic activities than CON (P < 0.05) throughout 30 days of fermentation, indicating B. subtilis's ability to accelerate the fermentation rate and render distinctive flavor profiles to shrimp paste. Among 50 differential abundance proteins (DAPs), 24 DAPs were identified as potential key regulating enzymes, with a P-value < 0.05 and |FC| > 0.50, indicating their significance and regulating capacity within specific metabolic pathways. Notably, 27 and 23 DAPs were up-regulated in Inoc and CON, respectively. Moreover, gene ontology (GO) enrichment analysis revealed that hydrolases, involved in carbohydrate metabolic processes and proteolysis, were the most differentiating pathways between Inoc and CON. Both samples exhibited different flavor profiles. A greater abundance of N-containing volatile compounds with a lower total abundance of aldehydes, ketones, alcohols, and acids could suggest a more favorable flavor in Inoc, compared to CON. Principal component analysis (PCA) revealed a positive correlation between L-ascorbate peroxidase, carboxypeptidase, and tripeptidyl peptidase sed2, with proteolytic and lipolytic activities in Inoc (P < 0.05). Meanwhile, acids and alcohols were positively correlated with CON. Therefore, B. subtilis inoculation could produce a distinctive flavor with a desirable sensory perception of shrimp paste regarding its ability to release extracellular enzymes/proteins. B. subtilis K-C3 inoculation could be suggested in the production of shrimp paste to improve its flavor characteristics.

A Multi-Omics Study of Epigenetic Changes in Type II Alveolar Cells of A/J Mice Exposed to Environmental Tobacco Smoke.

Lung cancer remains a major contributor to cancer fatalities, with cigarette smoking known to be responsible for up to 80% of cases. Based on the ability of cigarette smoke to induce inflammation in the lungs and increased lung cancer incidence in smokers with inflammatory conditions such as COPD, we hypothesized that inflammation plays an important role in the carcinogenicity of cigarette smoke. To test this hypothesis, we performed multi-omic analyses of Type II pneumocytes of A/J mice exposed to cigarette smoke for various time periods. We found that cigarette smoke exposure resulted in significant changes in DNA methylation and hydroxymethylation, gene expression patterns, and protein abundance that were partially reversible and contributed to an inflammatory and potentially oncogenic phenotype.

Detection of TurboID fusion proteins by fluorescent streptavidin outcompetes antibody signals and visualises targets not accessible to antibodies.

Immunofluorescence localises proteins via fluorophore-labelled antibodies. However, some proteins evade detection due to antibody-accessibility issues or because they are naturally low abundant or antigen density is reduced by the imaging method. Here, we show that the fusion of the target protein to the biotin ligase TurboID and subsequent detection of biotinylation by fluorescent streptavidin offers an 'all in one' solution to these restrictions. For all proteins tested, the streptavidin signal was significantly stronger than an antibody signal, markedly improving the sensitivity of expansion microscopy and correlative light and electron microscopy. Importantly, proteins within phase-separated regions, such as the central channel of the nuclear pores, the nucleolus, or RNA granules, were readily detected with streptavidin, while most antibodies failed. When TurboID is used in tandem with an HA epitope tag, co-probing with streptavidin and anti-HA can map antibody-accessibility and we created such a map for the trypanosome nuclear pore. Lastly, we show that streptavidin imaging resolves dynamic, temporally, and spatially distinct sub-complexes and, in specific cases, reveals a history of dynamic protein interaction. In conclusion, streptavidin imaging has major advantages for the detection of lowly abundant or inaccessible proteins and in addition, provides information on protein interactions and biophysical environment.

Unravelling the physicochemical and antimicrobial mechanisms of human serum albumin/tannic acid coatings for medical-grade polycaprolactone scaffolds

Biofilm-related biomaterial infections are notoriously challenging to treat and can lead to chronic infection and persisting inflammation. To date, a large body of research can be reviewed for coatings which potentially prevent bacterial infection while promoting implant integration. Yet only a very small number has been translated from bench to bedside. This study provides an in-depth analysis of the stability, antibacterial mechanism, and biocompatibility of medical grade polycaprolactone (mPCL), coated with human serum albumin (HSA), the most abundant protein in blood plasma, and tannic acid (TA), a natural polyphenol with antibacterial properties. Molecular docking studies demonstrated that HSA and TA interact mainly through hydrogen-bonding, ionic and hydrophobic interactions, leading to smooth and regular assemblies. In vitro bacteria adhesion testing showed that coated scaffolds maintained their antimicrobial properties over 3 days by significantly reducing S. aureus colonization and biofilm formation. Notably, amplitude modulation-frequency modulation (AMFM) based viscoelasticity mapping and transmission electron microscopy (TEM) data suggested that HSA/TA-coatings cause morphological and mechanical changes on the outer cell membrane of S. aureus leading to membrane disruption and cell death while proving non-toxic to human primary cells. These results support this antibiotic-free approach as an effective and biocompatible strategy to prevent biofilm-related biomaterial infections.

Evaluating stability and bioactivity of Rehmannia-derived nanovesicles during storage

Plant-derived nanovesicles (PDNVs) have garnered growing attention in the biomedical field owing to their abundance in plant-derived ribonucleic acids (RNA), proteins, lipids and metabolites. The question about the preservation of PDNVs is a crucial and unavoidable concern in both experiments’ settings and their potential clinical application. The objective of this research was to examine the impact of varying storage temperatures on the stability and bioactivity of Rehmannia-derived nanovesicles (RDNVs). The results showed that RDNVs aggregated after 2 weeks of storage period at 4 °C, and the particle size of some RDNVs gradually increased with time, along with the increase of solution potential. After 2 months of storage, all RDNVs exhibited varying levels of aggregation irrespective of storage temperature. The bioactivities of nanovesicles under different temperature storage conditions revealed a gradual decline in cell proliferation inhibition bioactivity over time, significantly lower than that of freshly prepared RDNVs. In contrast, the preservation of anti-migratory activity in RDNVs was found to be more effective when subjected to rapid freezing in liquid nitrogen followed by storage at − 80 °C, as opposed to direct storage at − 80 °C. These findings suggest that temperature alone may not be sufficient in safeguarding the activity and stability of RDNVs, highlighting the necessity for the development of novel protective agents for PDNVs.

Loss of the alpha subunit distal furin cleavage site blunts ENaC activation following Na+ restriction.

Epithelial Na+ channels (ENaCs) are activated by proteolysis of the α and γ subunits at specific sites flanking embedded inhibitory tracts. To examine the role of α subunit proteolysis in channel activation in vivo, we generated mice lacking the distal furin cleavage site in the α subunit (αF2M mice). On a normal Na+ control diet, no differences in ENaC protein abundance in kidney or distal colon were noted between wild-type (WT) and αF2M mice. Patch-clamp analyses revealed similar levels of ENaC activity in kidney tubules, while no physiologically relevant differences in blood chemistry or aldosterone levels were detected. Male αF2M mice did exhibit diminished ENaC activity in the distal colon, as measured by amiloride-sensitive short-circuit current (ISC). Following dietary Na+ restriction, WT and αF2M mice had similar natriuretic and colonic ISC responses to amiloride. However, single-channel activity was significantly lower in kidney tubules from Na+-restricted αF2M mice compared with WT littermates. ENaC α and γ subunit expression in kidney and distal colon were also enhanced in Na+-restricted αF2M vs. WT mice, in association with higher aldosterone levels. These data provide evidence that disrupting α subunit proteolysis impairs ENaC activity in vivo, requiring compensation in response to Na+ restriction. KEY POINTS: The epithelial Na+ channel (ENaC) is activated by proteolytic cleavage in vitro, but key questions regarding the role of ENaC proteolysis in terms of whole-animal physiology remain to be addressed. We studied the in vivo importance of this mechanism by generating a mouse model with a genetic disruption to a key cleavage site in the ENaC's α subunit (αF2M mice). We found that αF2M mice did not exhibit a physiologically relevant phenotype under normal dietary conditions, but have impaired ENaC activation (channel open probability) in the kidney during salt restriction. ENaC function at the organ level was preserved in salt-restricted αF2M mice, but this was associated with higher aldosterone levels and increased expression of ENaC subunits, suggesting compensation was required to maintain homeostasis. These results provide the first evidence that ENaC α subunit proteolysis is a key regulator of channel activity in vivo.

Optimized workflow of EV enrichment from human plasma samples for downstream mass spectrometry analysis

To improve the prognosis of bladder and prostate cancer, highly specific and sensitive biomarkers are needed for early detection, prognosis prediction, and therapeutic stratification. Extracellular vesicles (EV) from plasma could fill this gap due to their potential to serve as cancer biomarkers. However, the enrichment of EV is a major challenge, because the highly abundant plasma proteins are interfering with analytical downstream applications like mass spectrometry (MS). Therefore, the purity requirements of the EV samples must be carefully considered when selecting or developing a suitable EV enrichment method. The aim of this study was to compare a self-designed EV enrichment method based on density cushion centrifugation (DCC) combined with size exclusion chromatography (SEC) and concentration (method 1) with the exoRNeasy midi kit from Qiagen (method 2) and with unprocessed plasma. Furthermore, the single steps of method 1 were evaluated for their effectiveness to enrich EV from plasma. The results showed that the EV samples enriched with method 1 contained the highest levels of EV and exosome markers with simultaneously low levels of highly abundant plasma proteins. In summary, the combination of DCC, SEC and concentration proved to be a promising approach to discover EV-based biomarkers from plasma of cancer patients.

DNAJA1 regulates protein ubiquitination and is essential for spermatogenesis in the testes of mice and rats

DNAJA1 is a member of type I DnaJ proteins, which is essential for spermatogenesis and male fertility. However, its expression pattern in the testes and its impact on spermatogenesis remains unclear. Our study aimed to elucidate the mechanism of action of DNAJA1. We employed DNAJA1 knockout mice in this study. Western blotting and immunofluorescence analysis were conducted to determine the protein abundance of DNAJA1 in testes at various developmental stages. Our results revealed that DNAJA1 is predominantly expressed in the testes, and its knockout leads to complete infertility in male mice. We observed that DNAJA1 protein levels increased on postnatal days 14, 21, and 28, peaking on postnatal day 35 in mice. Immunofluorescence staining indicated that DNAJA1 expression varies across different stages of the spermatogenesis cycle. Additionally, DNAJA1 was absent in epididymal sperm. In early- and mid-stage tubules, DNAJA1 protein distribution was co-localized with residual bodies in elongating spermatids. Furthermore, we found that DNAJA1 knockout significantly reduced protein polyubiquitination in the testis. Analysis of the GEO database showed that DNAJA1 levels were significantly decreased in semen samples from subjects with teratozoospermia, asthenozoospermia, and impaired spermatogenesis. Our findings suggest that DNAJA1 is an essential protein for spermatogenesis, and its deletion reduces protein polyubiquitination in the testis, ultimately resulting in infertility and spermatogenesis defects.

Tandem mass tag (TMT)-based quantitative proteomics analysis reveals the different responses of contrasting alfalfa varieties to drought stress

BackgroundDrought stress restricts the growth, distribution and productivity of alfalfa (Medicago sativa L.). In order to study the response differences of alfalfa cultivars to drought stress, we previously carried out physiological and molecular comparative analysis on two alfalfa varieties with contrasting drought resistance (relatively drought-tolerant Longdong and drought-sensitive Algonquin). However, the differences in proteomic factors of the two varieties in response to drought stress still need to be further studied. Therefore, TMT-based quantitative proteomic analysis was performed using leaf tissues of the two alfalfa cultivars to identify and uncover differentially abundant proteins (DAPs).ResultsIn total, 677 DAPs were identified in Algonquin and 277 in Longdong under drought stress. Subsequently, we conducted various bioinformatics analysis on these DAPs, including subcellular location, functional classification and biological pathway enrichment. The first two main COG functional categories of DAPs in both alfalfa varieties after drought stress were ‘Translation, ribosomal structure and biogenesis’ and ‘Posttranslational modification, protein turnover, chaperones’. According to KEGG database, the DAPs of the two alfalfa cultivars after drought treatment were differentially enriched in different biological pathways. The DAPs from Algonquin were enriched in ‘photosynthesis’ and ‘ribosome’. The pathways of ‘linoleic acid metabolism’, ‘protein processing in endoplasmic reticulum’ and ‘RNA transport’ in Longdong were significantly enriched. Finally, we found significant differences in DAP enrichment and expression patterns between Longdong and Algonquin in glycolysis/glycogenesis, TCA cycle, photosynthesis, protein biosynthesis, flavonoid and isoflavonoid biosynthesis, and plant-pathogen interaction pathway after drought treatment.ConclusionsThe differences of DAPs involved in various metabolic pathways may explain the differences in the resistance of the two varieties to drought stress. These DAPs can be used as candidate proteins for molecular breeding of alfalfa to cultivate new germplasm with more drought tolerance to adapt to unfavorable environments.