Individual Proteins Research Articles

Multiparametric analysis of the specific immune response against SARS-CoV-2

Background SARS-CoV-2, which causes COVID-19, has killed more than 7 million people worldwide. Understanding the development of postinfectious and postvaccination immune responses is necessary for effective treatment and the introduction of appropriate antipandemic measures. Objectives We analysed humoral and cell-mediated anti-SARS-CoV-2 immune responses to spike (S), nucleocapsid (N), membrane (M), and open reading frame (O) proteins in individuals collected up to 1.5 years after COVID-19 onset and evaluated immune memory. Methods Peripheral blood mononuclear cells and serum were collected from patients after COVID-19. Sampling was performed in two rounds: 3-6 months after infection and after another year. Most of the patients were vaccinated between samplings. SARS-CoV-2-seronegative donors served as controls. ELISpot assays were used to detect SARS-CoV-2-specific T and B cells using peptide pools (S, NMO) or recombinant proteins (rS, rN), respectively. A CEF peptide pool consisting of selected viral epitopes was applied to assess the antiviral T-cell response. SARS-CoV-2-specific antibodies were detected via ELISA and a surrogate virus neutralisation assay. Results We confirmed that SARS-CoV-2 infection induces the establishment of long-term memory IgG+ B cells and memory T cells. We also found that vaccination enhanced the levels of anti-S memory B and T cells. Multivariate comparison also revealed the benefit of repeated vaccination. Interestingly, the T-cell response to CEF was lower in patients than in controls. Conclusion This study supports the importance of repeated vaccination for enhancing immunity and suggests a possible long-term perturbation of the overall antiviral immune response caused by SARS-CoV-2 infection.

An Active and Versatile Electron Transport System for Cytochrome P450 Monooxygenases from the Alkane Degrading Organism Acinetobacter sp. OC4.

Cytochrome P450 monooxygenases (CYPs) are valuable biocatalysts for the oxyfunctionalization of non-activated carbon-hydrogen bonds. Most CYPs rely on electron transport proteins as redox partners. In this study, the ferredoxin reductase (FdR) and ferredoxin (FD) for a cytochrome P450 monooxygenase from Acinetobactersp.OC4 are investigated. Upon heterologous production of both proteins independently in Escherichiacoli, spectral analysis showed their reduction capability towards reporter electron acceptors, e.g., cytochrome c. The individual proteins' specific activity towards cytochrome c reduction was 25 Umg‑1. Furthermore, the possibility to enhance electron transfer by artificial fusion of the units was elucidated. FdR and FD were linked by helical linkers [EAAAK]n, flexible glycine linkers [GGGGS]n or rigid proline linkers [EPPPP]n of n=1-4 sequence repetitions. The system with a glycine linker (n=4) reached an appreciable specific activity of 19Umg‑1 towards cytochrome c. Moreover, their ability to drive different members of the CYP153A subfamily is demonstrated. By creating artificial self-sufficient P450s with FdR, FD, and a panel of four CYP153A representatives, effective hydroxylation of n-hexane in a whole-cell system was achieved. The results indicate this protein combination to constitute a functional and versatile surrogate electron transport system for this subfamily.

Proteomic reference map for sarcopenia research: mass spectrometric identification of key muscle proteins of organelles, cellular signaling, bioenergetic metabolism and molecular chaperoning.

During the natural aging process, frailty is often associated with abnormal muscular performance. Although inter-individual differences exit, in most elderly the tissue mass and physiological functionality of voluntary muscles drastically decreases. In order to study age-related contractile decline, animal model research is of central importance in the field of biogerontology. Here we have analyzed wild type mouse muscle to establish a proteomic map of crude tissue extracts. Proteomics is an advanced and large-scale biochemical method that attempts to identify all accessible proteins in a given biological sample. It is a technology-driven approach that uses mass spectrometry for the characterization of individual protein species. Total protein extracts were used in this study in order to minimize the potential introduction of artefacts due to excess subcellular fractionation procedures. In this report, the proteomic survey of aged muscles has focused on organellar marker proteins, as well as proteins that are involved in cellular signaling, the regulation of ion homeostasis, bioenergetic metabolism and molecular chaperoning. Hence, this study has establish a proteomic reference map of a highly suitable model system for future aging research.

#2062 Protein interactome and trafficking of uromodulin in HEK and MDCK cell model

Abstract Background and Aims Uromodulin (UMOD) is a kidney-specific glycoprotein and the most abundant urinary protein in healthy individuals. Genome-wide association studies have shown an association between UMOD variants, kidney diseases, and hypertension. UMOD exhibits bidirectional secretion as it releases into the renal interstitium and circulation. However, the molecular trafficking of UMOD and its interaction remains poorly defined. We aimed to establish a stable Human Embryonic Kidney (HEK293) cell line expressing human UMOD to identify the potential regulators of UMOD expression or secretion. This study will provide significant implications for conditions such as hypertension, chronic kidney diseases, acute kidney injury, or UMOD-autosomal dominant tubulointerstitial kidney disease (UMOD-ADTKD). Method A HEK-UMOD-expressing cell line was generated by introducing human UMOD transcript 1 expression and using a tetracycline-inducible expression vector (pcDNA™FRT⁄TO vector) with yellow fluorescent protein tag at the C-terminal and previously established MDCK-UMOD cell line was also used. An untargeted proteomic approach was used to identify proteins, using GFP trap co-immunoprecipitation assay followed by mass spectrometry analysis. Results We identified 1367 and 1140 UMOD interacting proteins in HEK and MDCK cell models respectively. There were 620 proteins common to both these models. Through gene ontology, we unravel the molecular mechanisms involved in the biosynthesis, folding, and transport of UMOD. We observed several proteins associated with clathrin-mediated transporting. To study the role of clathrin in UMOD transport, we treated the polarised MDCK-UMOD cells with pitstop 2. There was significantly more secretion of UMOD with pitstop 2 treatment in the apical compartment compared to untreated cells however there was no significant difference in UMOD secretion in the basolateral compartment. Conclusion These results suggested that clathrin-coated vesicles are involved in transporting and sorting UMOD. Further studies need to investigate the functional importance of these proteins in UMOD trafficking and their implications in different physiological processes.

Extracellular vesicle biomarkers in circulation for colorectal cancer detection: a systematic review and meta-analysis

We provided an overview which evaluated the diagnostic performance of circulation EV biomarkers for CRC from PubMed, Medline, and Web of Science until 21 August 2022.Weidentified 48 studies that involved 7727 participants and evaluated 162 plasma/serum individual EV biomarkers including 117 RNAs and 45 proteins, as well as 45 EV biomarker panels for CRC detection. 12 studies evaluated the diagnostic performance of EV biomarkers for early CRC. The summarized sensitivity, specificity, and AUC value of individual EV RNAs and EV RNA panels were 76%, 75%, 0.87 and 82%, 79% and 0.90, respectively. Meanwhile, those of individual EV proteins and EV protein panels were 85%, 84%, 0.92 and 87%, 83%, 0.92, respectively. These results indicated that EV biomarker panels revealed superior diagnostic performance than the corresponding individual biomarkers. In early CRC, EV biomarkers showed available diagnostic value with the sensitivity, specificity, and AUC value of 80%, 75%, and 0.89.In subgroup analyses, EV miRNAs and LncRNAs held similar diagnostic value with the sensitivity, specificity and AUC value of 75%, 78%, 0.90 and 79%, 72%, 0.83, which was highly consistent with the whole EV RNAs. Significantly, the diagnostic values of EV miRNAs in plasma were marginally higher than those based on serum. In detail, the sensitivity, specificity, and AUC values were 79%, 81%, and 0.92 in plasma, as well as 74%, 77%, and 0.88 in serum, respectively. Therefore, circulation EV biomarkers could be considered as a promising biomarker for the early detection of CRC.

Single-Cell Patch-Clamp/Proteomics of Human Alzheimer's Disease iPSC-Derived Excitatory Neurons Versus Isogenic Wild-Type Controls Suggests Novel Causation and Therapeutic Targets.

Standard single-cell (sc) proteomics of disease states inferred from multicellular organs or organoids cannot currently be related to single-cell physiology. Here, a scPatch-Clamp/Proteomics platform is developed on single neurons generated from hiPSCs bearing an Alzheimer's disease (AD) genetic mutation and compares them to isogenic wild-type controls. This approach provides both current and voltage electrophysiological data plus detailed proteomics information on single-cells. With this new method, the authors are able to observe hyperelectrical activity in the AD hiPSC-neurons, similar to that observed in the human AD brain, and correlate it to ≈1400 proteins detected at the single neuron level. Using linear regression and mediation analyses to explore the relationship between the abundance of individual proteins and the neuron's mutational and electrophysiological status, this approach yields new information on therapeutic targets in excitatory neurons not attainable by traditional methods. This combined patch-proteomics technique creates a new proteogenetic-therapeutic strategy to correlate genotypic alterations to physiology with protein expression in single-cells.

Proteomics screening after pediatric allogenic hematopoietic stem cell transplantation reveals an association between increased expression of inhibitory receptor FCRL6 on γδ Tcells and cytomegalovirus reactivation.

We studied the associations between inflammation-related proteins in circulation and complications after pediatric allogenic hematopoietic stem cell transplantation (HSCT), to reveal proteomic signatures or individual soluble proteins associated with specific complications after HSCT. We used a proteomics method called Proximity Extension Assay to repeatedly measure 180 different proteins together with clinical variables, cellular immune reconstitution and blood viral copy numbers in 27 children (1-18 years of age) during a 2-year follow-up after allogenic HSCT. Protein profile analysis was performed using unsupervised hierarchical clustering and a regression-based method, while the Bonferroni-corrected Mann-Whitney U-test was used for time point-specific comparison of individual proteins against outcome. At 6 months after allogenic HSCT, we could identify a protein profile pattern associated with occurrence of the complications such as chronic graft-versus-host disease, viral infections, relapse and death. When protein markers were analyzed separately, the plasma concentration of the inhibitory and cytotoxic T-cell surface protein FCRL6 (Fc receptor-like 6) was higher in patients with cytomegalovirus (CMV) viremia [log2-fold change 1.5 (P = 0.00099), 2.5 (P = 0.00035) and 2.2 (P = 0.045) at time points 6, 12 and 24 months]. Flow cytometry confirmed that FCRL6 expression was higher in innate-like γδ Tcells, indicating that these cells are involved in controlling CMV reactivation in HSCT recipients. In conclusion, the potentially druggable FCRL6 receptor on cytotoxic Tcells appears to have a role in controlling CMV viremia after HSCT. Furthermore, our results suggest that system-level analysis is a useful addition to the studying of single biomarkers in allogenic HSCT.

Streamlining Protein Fractional Synthesis Rates Using SP3 Beads and Stable Isotope Mass Spectrometry: A Case Study on the Plant Ribosome.

Ribosomes are an archetypal ribonucleoprotein assembly. Due to ribosomal evolution and function, r-proteins share specific physicochemical similarities, making the riboproteome particularly suited for tailored proteome profiling methods. Moreover, the structural proteome of ribonucleoprotein assemblies reflects context-dependent functional features. Thus, characterizing the state of riboproteomes provides insights to uncover the context-dependent functionality of r-protein rearrangements, as they relate to what has been termed the ribosomal code, a concept that parallels that of the histone code, in which chromatin rearrangements influence gene expression. Compared to high-resolution ribosomal structures, omics methods lag when it comes to offering customized solutions to close the knowledge gap between structure and function that currently exists in riboproteomes. Purifying the riboproteome and subsequent shot-gun proteomics typically involves protein denaturation and digestion with proteases. The results are relative abundances of r-proteins at the ribosome population level. We have previously shown that, to gain insight into the stoichiometry of individual proteins, it is necessary to measure by proteomics bound r-proteins and normalize their intensities by the sum of r-protein abundances per ribosomal complex, i.e., 40S or 60S subunits. These calculations ensure that individual r-protein stoichiometries represent the fraction of each family/paralog relative to the complex, effectively revealing which r-proteins become substoichiometric in specific physiological scenarios. Here, we present an optimized method to profile the riboproteome of any organism as well as the synthesis rates of r-proteins determined by stable isotope-assisted mass spectrometry. Our method purifies the r-proteins in a reversibly denatured state, which offers the possibility for combined top-down and bottom-up proteomics. Our method offers a milder native denaturation of the r-proteome via a chaotropic GuHCl solution as compared with previous studies that use irreversible denaturation under highly acidic conditions to dissociate rRNA and r-proteins. As such, our method is better suited to conserve post-translational modifications (PTMs). Subsequently, our method carefully considers the amino acid composition of r-proteins to select an appropriate protease for digestion. We avoid non-specific protease cleavage by increasing the pH of our standardized r-proteome dilutions that enter the digestion pipeline and by using a digestion buffer that ensures an optimal pH for a reliable protease digestion process. Finally, we provide the R package ProtSynthesis to study the fractional synthesis rates of r-proteins. The package uses physiological parameters as input to determine peptide or protein fractional synthesis rates. Once the physiological parameters are measured, our equations allow a fair comparison between treatments that alter the biological equilibrium state of the system under study. Our equations correct peptide enrichment using enrichments in soluble amino acids, growth rates, and total protein accumulation. As a means of validation, our pipeline fails to find "false" enrichments in non-labeled samples while also filtering out proteins with multiple unique peptides that have different enrichment values, which are rare in our datasets. These two aspects reflect the accuracy of our tool. Our method offers the possibility of elucidating individual r-protein family/paralog abundances, PTM status, fractional synthesis rates, and dynamic assembly into ribosomal complexes if top-down and bottom-up proteomic approaches are used concomitantly, taking one step further into mapping the native and dynamic status of the r-proteome onto high-resolution ribosome structures. In addition, our method can be used to study the proteomes of all macromolecular assemblies that can be purified, although purification is the limiting step, and the efficacy and accuracy of the proteases may be limited depending on the digestion requirements. Key features • Efficient purification of the ribosomal proteome: streamlined procedure for the specific purification of the ribosomal proteome or complex Ome. • Accurate calculation of fractional synthesis rates: robust method for calculating fractional protein synthesis rates in macromolecular complexes under different physiological steady states. • Holistic ribosome methodology focused on plants: comprehensive approach that provides insights into the ribosomes and translational control of plants, demonstrated using cold acclimation [1]. • Tailored strategies for stable isotope labeling in plants: methodology focusing on materials and labeling considerations specific to free and proteinogenic amino acid analysis [2].

Abstract PO4-24-03: Adiponectin modulates cell polarity according to ERα expression in breast cancer cells

Abstract INTRODUCTION: Tumor microenvironment (TME) is a complex and heterogeneous network, consisting of stromal and immune cells, embedded with cytokines, growth factors, soluble receptors, and exosomes. Mutual and dynamic crosstalk among cancer, stromal and immune cells predispose cancer cells to metastasis. Particularly, the main component of breast TME is represented by adipocytes, which secretory activity is compromised in obesity. Indeed, hypertrophic, and hyperplastic adipocytes undergo to a modified cytokines secretory pattern, characterized by a reduced secretion of Adiponectin, now recognized as a crucial factor in the pathogenesis of breast cancer. LKB1 is one the most important effector of Adiponectin transduction pathways, and recently it has been demonstrated its role as ERα coactivator in promoting breast cancer cell growth. Moreover, LKB1 is a key regulator in controlling cell polarity along with E-cadherin and Cdc42. Cell polarity has been considered essential for mammary epithelial integrity, and disruption of individual polarity proteins is sufficient to induce tumor progression. The aim of the present study was to investigate the effects of adiponectin on the regulation and alteration of cell polarity, essential for breast cancer cell growth and invasiveness. METHODS: Immunofluorescence analyses were done to detect protein localization in breast cancer cells. Protein expression was performed by immunoblotting in the presence or absence of ERa and LKB1 siRNA. LKB1/Cdc42 and LKB1/E-cadherin interaction was detected by Proximity ligation and immunoprecipitation assay respectively. Wound healing assay was carried out to evaluate GM130 orientation. Growth and metastatic potential of breast cancer cells were assessed in vivo by orthotopic model and metastatic study respectively. RESULTS: In Adiponectin-treated MCF-7 cells LKB1 is mostly present in the nucleus, recruited as ERa-coactivator, while in BT20 cells LKB1 is localized with E-cadherin in the cytosolic compartment, suggesting its role in polarity. Our results showed that in MCF-7 cells Adiponectin increases Cdc42 level depending on LKB1 expression. Moreover, Cdc42 and LKB1 interact and colocalized in the nucleus of Adiponectin-treated MCF-7 cells. On the contrary, LKB1 and Cdc42 are located with E-cadherin in the cytosol of BT20 cells, addressing their cytosolic cooperation in maintaining cell polarity, as confirmed by an increased Cdc42 activity. Immunofluorescence analyses demonstrated a lack of Golgi realignment toward the front of the leading edge in MCF-7 cells treated with Adiponectin, not observed in BT20 cells. All this suggest a loss of cell polarity organization that predispose to cancer growth and progression, as confirmed by an increased tumor volume mass and a major number of lung metastatic events upon tail vein injection of adiponectin-treated ERα-positive cells with respect to untreated cells. CONCLUSION: In ERα-positive breast cancer cells Adiponectin modulates the expression of the main proteins involved in cell polarity, leading to increased growth, progression, and distant metastasis. Citation Format: Giuseppina Daniela Naimo, Martina Forestiero, Alessandro Paolì, Luca Gelsomino, Rocco Malivindi, Adele Elisabetta Leonetti, Francesca Giordano, Maria Luisa Panno, Loredana Mauro, Sebastiano Andò. Adiponectin modulates cell polarity according to ERα expression in breast cancer cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-24-03.

Abstract PO5-15-06: NVIGEN X® Comprehensive Liquid Biopsy for Sensitive ctDNA, Circulating Tumor Cells (CTC), and Protein Detection in Breast Cancer: Next-Generation Sequencing, Patient Case Studies, and Clinical Implications

Abstract Background Breast cancer (BC) represents the most prevalent cancer and is a leading cause of cancer related mortality among women worldwide. Despite notable therapeutic advancements, concern for metastatic recurrence remains a significant challenge. Liquid biopsy has emerged as a promising method for monitoring cancer treatment efficacy and disease progression. However, existing technologies predominantly focus on ctDNA, and their clinical utility is hindered by limited detection sensitivity and accuracy. To address these challenges, NVIGEN has developed NVIGEN X® comprehensive liquid biopsy, a novel minimally invasive approach that integrates ctDNA, CTC, and protein biomarkers. This comprehensive method could enable longitudinal monitoring of tumor dynamics, offering valuable insights into cancer biology, treatment responses, and emerging therapeutic targets. Here, we showcase compelling patient case studies, and the potential clinical implications of our technology for an exciting and evolving field in cancer diagnostics. Method Patients with advanced breast cancer were prospectively identified and consented. 10mL blood per timepoint was collected in Streck tubes. ctDNA, CTC and proteins were captured from a single tube of blood using NVIGEN magnetic nanoparticles according to established protocols. Following DNA extraction, ctDNA and CTC samples underwent NVIGEN’s highly sensitive sample workflow, optimized for NGS data accuracy and efficiency, employing hybridization capture with a 180 gene customizable cancer panel. Sequenced data were processed with NVIGEN’s proprietary high-sensitivity data analysis pipeline. Gene alterations identified from both ctDNA and CTC samples were analyzed and integrated to provide a comprehensive view of the tumor profile. Protein biomarkers of interest were detected using NVIGEN’s on-beads ELISA assay. Correlation analysis was performed to examine the relationship between the detected gene alterations, protein biomarkers, ctDNA/CTC data, and clinicopathologic features. Treatment response and identification of potential new treatment targets were evaluated based on the integrated analysis of ctDNA, CTC, protein biomarkers, and patient information. Result We collected 94 blood samples from 85 patients. Here we will present data from initial 36 patient samples. Of these 36 patients, 18 were tested with ctDNA/protein biomarkers (duo assays), and 18 were tested with ctDNA/CTC/protein biomarkers (trio assays). Comparison between duo and trio samples revealed that trio samples provided 2.5 times the number of detected variants compared to duo samples, with the median of variant numbers in duo assays being 52 (range 22-442), and in trio assays being 130 (range 73-703). Over 50% of the detected variants fell within the 0.1-0.5% variant allele frequency (VAF) range (number 2599) with total variant number being 5123. Several patient case studies were conducted using serial blood samples. These case studies revealed notable patterns of important gene alterations, such as ESR1, ERBB2 and PIK3CA, and demonstrated trends in the presence of these genes in ctDNA, CTC, or their coexistence. Protein biomarker information was correlated with ctDNA, CTC NGS data, and patient clinical status. Conclusions The analysis of the presented data from 36 patient samples with advanced breast cancer highlight the clinical relevance and potential significance of utilizing ctDNA, CTC and protein biomarkers in monitoring disease dynamics, treatment response, and guiding personalized treatment approaches. Our results suggest that individual protein biomarker analysis may be crucial in supporting personalized diagnostics and enabling more effective personalized medicine strategies. Citation Format: Aihua Fu, Minh Ton, Kevan Wang, Weiwei Gu, Henry Jin, Nuzhat Shaikh, Sasha Madan, Shreya Perepa, Tianhong Li, Minetta Liu, Heather Parsons, George Sledge, Fauzia Riaz. NVIGEN X® Comprehensive Liquid Biopsy for Sensitive ctDNA, Circulating Tumor Cells (CTC), and Protein Detection in Breast Cancer: Next-Generation Sequencing, Patient Case Studies, and Clinical Implications [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-15-06.

Combined techniques for revealing the mechanism beneath the inhibition effects of pectin on gluten digestibility using static in vitro gastro-duodenal protocols.

In the current study, how pectin retards the digestibility of wheat gluten was investigated using a static in vitro gastric-duodenal model. The degree of protein hydrolysis was estimated using the o-phthaldialdehyde method, while the in vitro digestograms were mathematically fitted using a single first-order kinetics model. Peptides' profile, free amino acids compositions, gluten-pectin interactions and their effects on enzymatic activities of proteolytic enzymes as well as on the gluten secondary structures under digestive conditions were studied using combined techniques. Results showed that pectin could retard gluten digestibility through 1). preferential absorption to insoluble gluten aggregates by electrostatic interactions; 2). increasing the helix and reducing the β-sheet content of the solubilized gluten protein fractions in terms of their secondary molecular structures; 3). reducing pepsin activity by forming negatively charged pectin-gluten mixtures which then interacted with the positively charged pepsin molecules. The deeper insight into gluten-pectin interactions and their influences on gluten digestibility under gastrointestinal conditions provides important clues for developing effective forms of dietary fiber to improve the nutritional benefits of plant protein in individuals.

The mechanism of resveratrol stabilization and degradation by synergistic interactions between constituent proteins of whey protein

Whey protein isolate (WPI) is mainly composed of β-lactoglobulin (β-LG), α-lactalbumin (α-LA) and bovine serum albumin (BSA). The aim of this study was to compare and analyze the influence of WPI and its three main constituent proteins, as well as proportionally reconstituted WPI (R-WPI) on resveratrol. It was found that the storage stability of resveratrol was protected by WPI, not affect by R-WPI, but reduced by individual whey proteins at 45 °C for 30 days. The rank of accelerated degradation of resveratrol by individual whey proteins was BSA > α-LA > β-LG. The antioxidant activity, localization of resveratrol and oxidation of carrier proteins were determined by ABTS, H2O2 assay, synchronous fluorescence, carbonyl and circular dichroism. The non-covalent interactions and disulfide bonds between constituent proteins improved the antioxidant activity of the R-WPI-resveratrol complex, the oxidation stability of the carrier and the solvent shielding effect on resveratrol, which synergistically inhibited the degradation of resveratrol in R-WPI system. The results gave insight into elucidating the interaction mechanism of resveratrol with protein carriers.

Correlation between Cu:Zn ratio and C – Reactive protein in healthy individuals

Correlation between Cu:Zn ratio and C – Reactive protein in healthy individuals

GLUT2 regulation of p38 MAPK isoform protein expression and p38 phosphorylation in male versus female rat hypothalamic primary astrocyte Cultures

Recent studies documented regulation of hypothalamic astrocyte mitogen-activated protein kinase (MAPK) pathways, including p38, by the plasma membrane glucose carrier/sensor glucose transporter-2 (GLUT2). Sex-specific GLUT2 control of p38 phosphorylation was observed, but effects on individual p38 family protein profiles were not investigated. Current research employed an established primary astrocyte culture model, gene knockdown tools, and selective primary antisera against p38-alpha, p38-beta, p38-gamma, and p38-delta isoforms to investigate whether GLUT2 governs expression of one or more of these variants in a glucose-dependent manner. Data show that GLUT2 inhibits baseline expression of each p38 protein in male cultures, yet stimulates p38-delta profiles without affecting other p38 proteins in female. Glucose starvation caused selective up-regulation of p38-delta profiles in male versus p38-alpha and -gamma proteins in female; these positive responses were amplified by GLUT2 siRNA pretreatment. GLUT2 opposes or enhances basal p38 phosphorylation in male versus female, respectively. GLUT2 siRNA pretreatment did not affect glucoprivic patterns of phospho-p38 protein expression in either sex. Outcomes document co-expression of the four principal p38 MAPK family proteins in hypothalamic astrocytes, and implicate GLUT2 in regulation of all (male) versus one (female) variant(s). Glucoprivation up-regulated expression of distinctive p38 isoforms in each sex; these stimulatory responses are evidently blunted by GLUT2. Glucoprivic-associated loss of GLUT2 gene silencing effects on p38 phosphorylation infers either that glucose status determines whether this sensor controls phosphorylation, or that decrements in screened glucose in each instance are of sufficient magnitude to abolish GLUT2 regulation of that function.

Heat Treatment and Homogenization Influence the Gastric Digestion of Bovine Milk Protein in Growing Pigs as an Adult Human Model

BackgroundBovine milk processing influences the structure of the curd formed during gastric digestion, which may alter gastric protein hydrolysis and impact amino acid (AA) release into the small intestine. ObjectivesThis study aimed to determine the influence of heat treatment and homogenization on the gastric protein digestion and AA emptying of bovine milk. MethodsNine-wk-old pigs (n = 144) consumed either raw, pasteurized nonhomogenized (PNH), pasteurized homogenized (PH), or ultrahigh- temperature homogenized (UHT) bovine milk for 10 d. On day 11, fasted pigs received the milk treatment (500 mL) before gastric contents were collected at 0, 20, 60, 120, 180, and 300 min postprandially. The apparent degree of gastric protein hydrolysis (based on the release of free amino groups), apparent gastric disappearance of individual proteins [based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel band intensity], and the gastric emptying of digested protein and AA were determined. ResultsDuring the first 60 min, the rate of apparent gastric protein hydrolysis was fastest in pigs fed UHT milk (0.29%/min compared with on average 0.07%/min in pigs fed raw, PNH, and PH milk). Differences in the apparent degree of gastric protein hydrolysis and emptying were reflected in the rate of digested protein entering the small intestine. The AA gastric emptying half-time was generally shorter in pigs fed PH and UHT milk than pigs fed raw and PNH milk. For example, the gastric release of total essential AAs was >2-fold faster (P < 0.01) in pigs fed PH or UHT milk than that of pigs fed raw or PNH milk (i.e., homogenized compared with nonhomogenized milk). ConclusionsHeat treatment and homogenization increased the apparent gastric degree of protein hydrolysis and the release of digested protein into the small intestine. However, the rate of AAs entering the small intestine was mainly increased by homogenization.

Proteomic Changes during Human Heat Stress and Heat Acclimation

INTRODUCTION. To thrive in a warmer climate, humans will need to adapt physiologically. Improved thermoregulatory and cardiovascular responses during heat acclimation have been widely described. However, the cellular and molecular mechanisms mediating these improvements remain understudied in humans. The aim of this study was to perform a broad-spectrum, non-targeted proteomic analysis to determine how plasma protein levels change in response to moderate heat stress and heat acclimation. METHODS. Ten healthy adults (4F/6H, age: 25 ± 3 years) attended two laboratory visits performed before and after a 7-day heat acclimation protocol (hot water immersion, 60 min at a rectal temperature ≥38.5°C). During the visits, blood samples were drawn before and during passive heat stress (~1.5°C increase in esophageal temperature, water-perfused suit). Proteomic analyses were performed on 2938 plasma proteins using the Olink Explore 3072 assay. We quantified the number of proteins that changed by more than one log2(fold change) in concentration. RESULTS. Prior to acclimation, ~1% of the proteins (n=29) changed in concentration during heat stress. The change in concentration for 9 of these proteins reached the p value threshold. After acclimation, 54 proteins changed in concentration under a normothermic state. During subsequent heat stress, 175 proteins (~6%) increased in concentration and 19 (~0.6%) decreased. For 180 of these proteins, the change in concentration during heat stress was unique to the post-acclimation visit. The genes for proteins that changed in concentration during heat stress prior to acclimation are more highly expressed in brain (FGFBP3), arterial (TIMP4), skin (OBP2B), lung/salivary gland/esophageal (CXCL17) tissue. The genes for proteins that changed concentration in response to heat acclimation are highly expressed in skin (MMP7, CA6, KLK14) and brain tissue (DNER). The genes for proteins that only changed in concentration during heat stress post-acclimation are more highly expressed in brain (MOG, SMOC1, PTPRN2), and arterial (TNC, C1QTNF1, MATN2, NTF3, SMOC1) tissue. CONCLUSION. This untargeted and large-scale proteomic analysis identified novel plasma proteins that change in concentration during human heat stress and subsequent heat acclimation. The individual protein results can be utilized as external validation for other studies, as the basis of further data analysis explorations, and/or to guide targeted investigations of the biological responses and adaptations to heat exposure. Natural Sciences and Engineering Research Council of Canada. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The current use of proteomics and metabolomics in glomerulonephritis: a systematic literature review.

Glomerulonephritis inherently leads to the development of chronic kidney disease. It is the second most common diagnosis in patients requiring renal replacement therapy in the United Kingdom. Metabolomics and proteomics can characterise, identify and quantify an individual's protein and metabolite make-up. These techniques have been optimised and can be performed on samples including kidney tissue, blood and urine. Utilising omic techniques in nephrology can uncover disease pathophysiology and transform the diagnostics and treatment options for glomerulonephritis. To evaluate the utility of metabolomics and proteomics using mass spectrometry and nuclear magnetic resonance in glomerulonephritis. The systematic review was registered on PROSPERO (CRD42023442092). Standard and extensive Cochrane search methods were used. The latest search date was March 2023. Participants were of any age with a histological diagnosis of glomerulonephritis. Descriptive analysis was performed, and data presented in tabular form. An area under the curve or p-value was presented for potential biomarkers discovered. Twenty-seven studies were included (metabolomics (n = 9)), and (proteomics (n = 18)) with 1818 participants. The samples analysed were urine (n = 19) blood (n = 4) and biopsy (n = 6). The typical outcome themes were potential biomarkers, disease phenotype, risk of progression and treatment response. This review shows the potential of metabolomic and proteomic analysis to discover new disease biomarkers that may influence diagnostics and disease management. Further larger-scale research is required to establish the validity of the study outcomes, including the several proposed biomarkers.

YBX1 inhibits mitochondrial-mediated apoptosis in ischemic heart through the PI3K/AKT signaling pathway

Abstract Background Myocardial infarction (MI) is associated with higher morbidity and mortality in the world, especially in cold weather. YBX1 is an RNA-binding protein that is required for pathological growth of cardiomyocyte by regulating cell growth and protein synthesis. But YBX1, as an individual RNA-binding protein, regulates cardiomyocytes through signaling cascades during myocardial infarction remain largely unexplored. Methods In vivo, the mouse MI model was induced by ligating the left anterior descending coronary artery (LAD), and randomly divided into sham operation group, MI group, MI+ YBX1 knockdown/overexpression group and MI+ negative control (NC) group. The protective effect of YBX1 was verified by echocardiography and triphenyltetrazolium chloride staining. In vitro, mitochondrial-dependent apoptosis was investigated by using CCK8, TUNEL staining, reactive oxygen species (ROS) staining and JC-1 staining in hypoxic neonatal mouse cardiomyocytes (NMCMs). Results YBX1 expression of cardiomyocytes was downregulated in a mouse model and a cellular model on the ischemic condition. Compared to mice induced by MI, YBX1 overexpression mediated by adeno-associated virus serotype 9 (AAV9) vector reduced the infarcted size and improved cardiac function. Knockdown of endogenous YBX1 by shRNA partially aggravated ischemia-induced cardiac dysfunction. In hypoxic cardiomyocytes, YBX1 overexpression decreased lactic dehydrogenase (LDH) release, increased cell viability, and inhibited apoptosis by affecting the expression of apoptosis related proteins, while knockdown of endogenous YBX1 by siRNA had the opposite effect. Overexpression of YBX1 restored mitochondrial dysfunction in hypoxic NMCMs by increasing mitochondrial membrane potential and ATP content and decreasing ROS. In hypoxic NMCMs, YBX1 overexpression increased the expression of phosphorylated phosphatidylinositol 3 kinase (PI3K)/AKT, and the antiapoptosis effect of YBX1 was eliminated t by LY294002, PI3K/AKT inhibitor. Conclusion YBX1 protected the heart from ischemic damage by inhibiting the mitochondrial-dependent apoptosis through PI3K/AKT pathway. It is anticipated that YBX1 may serve as a novel therapeutic target for MI.

Group 3 medulloblastoma transcriptional networks collapse under domain specific EP300/CBP inhibition

Chemical discovery efforts commonly target individual protein domains. Many proteins, including the EP300/CBP histone acetyltransferases (HATs), contain several targetable domains. EP300/CBP are critical gene-regulatory targets in cancer, with existing high potency inhibitors of either the catalytic HAT domain or protein-binding bromodomain (BRD). A domain-specific inhibitory approach to multidomain-containing proteins may identify exceptional-responding tumor types, thereby expanding a therapeutic index. Here, we discover that targeting EP300/CBP using the domain-specific inhibitors, A485 (HAT) or CCS1477 (BRD) have different effects in select tumor types. Group 3 medulloblastoma (G3MB) cells are especially sensitive to BRD, compared with HAT inhibition. Structurally, these effects are mediated by the difluorophenyl group in the catalytic core of CCS1477. Mechanistically, bromodomain inhibition causes rapid disruption of genetic dependency networks that are required for G3MB growth. These studies provide a domain-specific structural foundation for drug discovery efforts targeting EP300/CBP and identify a selective role for the EP300/CBP bromodomain in maintaining genetic dependency networks in G3MB.

The expression and role of the Lem-D proteins Ankle2, Emerin, Lemd2, and TMPO in triple-negative breast cancer cell growth.

Triple-negative breast cancer (TNBC) is a sub-classification of breast carcinomas, which leads to poor survival outcomes for patients. TNBCs do not possess the hormone receptors that are frequently targeted as a therapeutic in other cancer subtypes and, therefore, chemotherapy remains the standard treatment for TNBC. Nuclear envelope proteins are frequently dysregulated in cancer cells, supporting their potential as novel cancer therapy targets. The Lem-domain (Lem-D) (LAP2, Emerin, MAN1 domain, and Lem-D) proteins are a family of inner nuclear membrane proteins, which share a ~45-residue Lem-D. The Lem-D proteins, including Ankle2, Lemd2, TMPO, and Emerin, have been shown to be associated with many of the hallmarks of cancer. This study aimed to define the association between the Lem-D proteins and TNBC and determine whether these proteins could be promising therapeutic targets. GENT2, TCGA, and KM plotter were utilized to investigate the expression and prognostic implications of several Lem-D proteins: Ankle2, TMPO, Emerin, and Lemd2 in publicly available breast cancer patient data. Immunoblotting and immunofluorescent analysis of immortalized non-cancerous breast cells and a panel of TNBC cells were utilized to establish whether protein expression of the Lem-D proteins was significantly altered in TNBC. SiRNA was used to decrease individual Lem-D protein expression, and functional assays, including proliferation assays and apoptosis assays, were conducted. The Lem-D proteins were generally overexpressed in TNBC patient samples at the mRNA level and showed variable expression at the protein level in TNBC cell lysates. Similarly, protein levels were generally negatively correlated with patient survival outcomes. siRNA-mediated depletion of the individual Lem-D proteins in TNBC cells induced aberrant nuclear morphology, decreased proliferation, and induced cell death. However, minimal effects on nuclear morphology or cell viability were observed following Lem-D depletion in non-cancerous MCF10A cells. There is evidence to suggest that Ankle2, TMPO, Emerin, and Lemd2 expressions are correlated with breast cancer patient outcomes, but larger patient sample numbers are required to confirm this. siRNA-mediated depletion of these proteins was shown to specifically impair TNBC cell growth, suggesting that the Lem-D proteins may be a specific anti-cancer target.