Proteomics Analysis of the Estrogen Effects in the Rat Uterus Using Gel-LC and Tandem Mass Spectrometry Approaches

Paez, María D.; Callegari, Eduardo

doi:10.1007/978-1-0716-1920-9_17

Cited by 6 publications

(6 citation statements)

References 23 publications

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“…The sample analysis was performed based on previously described methods [21, 22] (also see https://red.library.usd.edu/cgi/viewcontent.cgi?article=1128&context=diss-thesis). Briefly, the proteins were separated using 12% SDS-PAGE and the gel was stained with colloidal Coomassie G250 (BioRad) and destained overnight.…”

Section: Methodsmentioning

confidence: 99%

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Impaired 26S proteasome causes learning and memory deficiency and induces neuroinflammation mediated by NF-κB in mice

Huber,

Callegari,

Paez

et al. 2024

Preprint

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Aging and many neurodegenerative disorders, including Alzheimer disease (AD), show reduced proteasome activity, loss of synapses and increased neuroinflammation in the brain. However, whether proteasome dysfunction causes neuroinflammation remains less understood. Here, we studied the effect of impaired 26S proteasome on neuroinflammation in the Psmc1 knockout (KO) mice deficient of a 19S proteasome subunit selectively in the forebrain region. We initially assessed the proteasome activity in the KO and control mouse brains and performed cognitive behavioral test to the animals. The synaptosomes were isolated from the forebrain and subjected to mass spectrometric analysis of total synaptic proteins. The identified synaptic proteins were then assessed using bioinformatic approaches. To validate identified proteins, Western blot analysis and immunofluorescent staining of specific protein were utilized. To validate NF-kB mediating neuroinflammation, we administered the KO animals at 5 weeks of age with a selective NF-kB inhibitor for three weeks and then, animal behaviors and neuroinflammation were assessed when they reached to eight weeks of age. Our results revealed that impaired 26S proteasome led to AD-like cognitive deficiency and overt neuroinflammation in the synapses of the Psmc1 KO brain at eight weeks of age. Pronounced neuroinflammation also occurred in other regions and glial cells, which was confirmed by increased levels of several key immune response-related proteins, including Stat1, Trem2 and NF-kB, and by activation of astrocytes and microglia in the KO brain. Following treating the KO mice with a selective NF-kB inhibitor, the KO mice exhibited improved behaviors and reduced neuroinflammation compared to the control animals. These data indicate that impaired 26S proteasome causes AD-like cognitive deficiency and induces neuroinflammation mediated largely by NF-kB, which will help develop effective therapeutics and aid to better understand the pathogenesis of AD and many other neurodegenerative disorders where impaired proteasome is consistently coupled with neuroinflammation.

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Section: Methodsmentioning

confidence: 99%

“…All the proteins identified in the current study were found in these domains. The comparative study was performed using ProteoIQ v2.8 (local license) [22, 23].…”

Section: Methodsmentioning

confidence: 99%

Impaired 26S proteasome causes learning and memory deficiency and induces neuroinflammation mediated by NF-κB in mice

Huber,

Callegari,

Paez

et al. 2024

Preprint

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“…For gel-dependent analysis, 5 µg of proteins were separated by SDS-PAGE in a 12% polyacrylamide gel. The gel was stained using colloidal Coomassie G-250 (Candiano et al 2004) and each lane was divided into 15 portions (Paez and Callegari 2022). The information obtained for each portion was then combined to obtain global information.…”

Section: Proteomic Analysismentioning

confidence: 99%

Light-Mediated Elicitation of Tessaria absinthioides Calli for the Production of Anthocyanins: Correlation with Protein Expression

Martinez,

Bonilla,

Aguilera

et al. 2024

Preprint

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This study explores the metabolic responses of undifferentiated cells of Tessaria absinthioides to light elicitation, with a focus on the production and accumulation of anthocyanins. We observed that anthocyanin concentrations in cell biomass were notably higher compared to those in wild flowers, albeit with slight compositional differences, according to the HPLC-DAD analysis. In a proteomic analysis of T. absinthioidescalli, light exposure induced significant changes in protein expression, with 578 proteins overexpressed and 486 uniquely identified under this condition. These proteins were predominantly clustered in key functional categories like protein processing, cellular transport, and DNA regulation, along with those involved in sesquiterpene synthesis and developmental regulation. Furthermore, proteins crucial for the phenylpropanoid pathway and the anthocyanin biosynthesis were identified in light-exposed calli, with many significantly overexpressed compared to dark-grown cultures. A similar trend was observed in enzymes associated with the de novo synthesis of arginine, a precursor to stress-responsive metabolites like polyamines and nitric oxide. These findings help elucidate the metabolic pathways underlying anthocyanin production in T. absinthioides. They also extend to the development of natural dyes, highlighting the significance of cellular culture in sustainable bioproduction.

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“…The obtained gel was subjected to staining with Coomassie R-250 0.25% w/v (Meyer and Lamberts 1965). Afterward, each line in the gel was cut in different regions following two criteria: molecular weight regions or speci c regions with high abundance proteins to resolve possible issues such as dynamic range (Gel-LC analysis) (McConnell et al 2011;Paez and Callegari 2022). The gel regions were bleached with Water/Ethanol/Acetic Acid (50:40:10), destained with 50 mM Ammonium Bicarbonate/50% Acetonitrile, reduced with Dithiothreitol (DTT, SigmaAldrich, Saint Louis, MO) at room temperature for 30 min, followed by alkylated with Iodoacetamide (Sigma-Aldrich, Saint Louis, MO) at room temperature in dark condition for 30 min and then in gel digestion with trypsin sequencing grade (Promega, Madison, WI) (Callegari 2016).…”

Section: Protein Separation and Trypsin Digestionmentioning

confidence: 99%

“…Peptide analysis was performed using nanoUPLC-ESI-MS/MS according to highly standardized protocols by Proteomics Core Facility in the Biomedical Research Infrastructure Network (BRIN)-Sanford School of Medicine, University of South Dakota (Callegari 2016;Paez and Callegari 2022). Brie y, the tryptic digested peptides coming either from the gel or in solution digestion were in-line desalted, concentrated and separated using an Easy nLC UHPLC 1200 in nano ow con guration (Thermo Scienti c, Bremen, Germany) for 110 minutes (see supplemental information X1).…”

Section: Mass Spectrometry Analysismentioning

confidence: 99%

Proteomic analysis to unravel the biochemical mechanisms triggered by Bacillus toyonensis SFC 500-1E under chromium(VI) and phenol stress

et al. 2023

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Bacillus toyonensis SFC 500-1E is a member of the consortium SFC 500-1 able to remove Cr(VI) and simultaneously tolerate high phenol concentrations. In order to elucidate mechanisms utilized by this strain during the bioremediation process, the differential expression pattern of proteins was analyzed when it grew with or without Cr(VI) (10 mg/L) and Cr(VI)+phenol (10 and 300 mg/L), through two complementary proteomic approaches: gel-based (SDS-PAGE) and gel-free (nanoUPLC-ESI-MS/MS).A total of 400 differentially expressed proteins were identi ed, out of which 152 proteins were down-regulated under Cr(VI) and 205 up-regulated in the presence of Cr(VI)+phenol, suggesting the extra effort made by the strain to adapt itself and keep growing when phenol was added. The major metabolic pathways affected include carbohydrate and energetic metabolism, followed by lipid and amino acid metabolism. Particularly interesting were also ABC transporters and the iron-siderophore transporter as well as transcriptional regulators that can bind metals. Stressassociated global response involving the expression of thioredoxins, SOS response, and chaperones appears to be crucial for the survival of this strain under treatment with both contaminants. This research not only provided a deeper understanding of B. toyonensis SFC 500-1E metabolic role in Cr(VI) and phenol bioremediation process but also allowed us to complete an overview of the consortium SFC 500-1 behavior. This may contribute to an improvement in its use as a bioremediation strategy and also provides a baseline for further research.

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Proteomics Analysis of the Estrogen Effects in the Rat Uterus Using Gel-LC and Tandem Mass Spectrometry Approaches

Cited by 6 publications

References 23 publications

Impaired 26S proteasome causes learning and memory deficiency and induces neuroinflammation mediated by NF-κB in mice

Impaired 26S proteasome causes learning and memory deficiency and induces neuroinflammation mediated by NF-κB in mice

Light-Mediated Elicitation of Tessaria absinthioides Calli for the Production of Anthocyanins: Correlation with Protein Expression

Proteomic analysis to unravel the biochemical mechanisms triggered by Bacillus toyonensis SFC 500-1E under chromium(VI) and phenol stress

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