Journal: Analytical Chemistry

Article Title: Label-Free Quantitative Thermal Proteome Profiling Reveals Target Transcription Factors with Activities Modulated by MC3R Signaling

doi: 10.1021/acs.analchem.3c03643

Figure Lengend Snippet: Overview of the preparatory and analytical workflows. (A) POMC derived ligands and their downstream signaling cascades. (B) Schematic overview of the thermal proteome profiling (TPP) workflow. MC3R-expressing HEK293 cells were treated with ACTH, α-MSH, or γ-MSH at concentrations of 20, 100, and 500 nM or with DMSO as a vehicle-only negative control. (C) Schematic overview of the TPP data analysis workflow. Protein identification and relative quantification were achieved by direct analysis of the raw LC–MS data, after which various bioinformatics tools were used to infer changes in transcription factor (TF) activity, perform enriched pathway analysis, and identify thermally affected proteins.

Article Snippet: A human embryonic kidney 293 cell line transfected with a tetracycline-regulated expression system to overexpress MC3R (HEK-TREx-MC3R) was provided by Astra Zeneca.

Techniques: Derivative Assay, Expressing, Negative Control, Quantitative Proteomics, Liquid Chromatography with Mass Spectroscopy, Activity Assay

Journal: Analytical Chemistry

Article Title: Label-Free Quantitative Thermal Proteome Profiling Reveals Target Transcription Factors with Activities Modulated by MC3R Signaling

doi: 10.1021/acs.analchem.3c03643

Figure Lengend Snippet: Overview of identified proteins and thermally stabilized or destabilized proteins. (A) Venn diagrams showing the numbers of proteins exhibiting altered melting points, associations with enriched pathways, and phosphorylation in MC3R-expressing HEK293 cells incubated with ACTH, α-MSH, and γ-MSH. (B) Venn diagrams showing the numbers of stabilized, destabilized, and phosphorylated proteins after incubation with ACTH, α-MSH, and γ-MSH. (C) Upset plot representing individual numbers of stabilized and destabilized proteins for each ligand and those common between various combinations of ligands.

Article Snippet: A human embryonic kidney 293 cell line transfected with a tetracycline-regulated expression system to overexpress MC3R (HEK-TREx-MC3R) was provided by Astra Zeneca.

Techniques: Phospho-proteomics, Expressing, Incubation

Journal: Analytical Chemistry

Article Title: Label-Free Quantitative Thermal Proteome Profiling Reveals Target Transcription Factors with Activities Modulated by MC3R Signaling

doi: 10.1021/acs.analchem.3c03643

Figure Lengend Snippet: Characterization of transcription factors. (A) Heat map showing the relative abundance (compared to vehicle-only controls) of the transcription factors CCAR2, HMGB2, DDX21, SRSF7, and TET2 in MC3R-expressing HEK293 cells incubated with ACTH, α-MSH, and γ-MSH at different ligand concentrations and temperatures. (B) Phosphorylation of tryptic peptides derived from the thermally stabilized and destabilized transcription factors shown in panel A whose activity was inferred to change following stimulation with ACTH, α-MSH, or γ-MSH. Phosphorylation sites are indicated by asterisks next to the modified amino acid (shown in parentheses when the exact amino acid is unknown). (C) Transcription factor activities and relational networks inferred from differential expression data using BITFAM. The heatmap shows fold changes in transcription factor activities (relative to vehicle-only treatments) in MC3R-expressing HEK293 cells incubated with ACTH, α-MSH, or γ-MSH. (D) Network showing the interconnectivity of the transcription factors identified within our experimental LC–MS data set.

Article Snippet: A human embryonic kidney 293 cell line transfected with a tetracycline-regulated expression system to overexpress MC3R (HEK-TREx-MC3R) was provided by Astra Zeneca.

Techniques: Expressing, Incubation, Phospho-proteomics, Derivative Assay, Activity Assay, Modification, Quantitative Proteomics, Liquid Chromatography with Mass Spectroscopy

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Selective Cannabinoid 2 Receptor Stimulation Reduces Tubular Epithelial Cell Damage after Renal Ischemia-Reperfusion Injury

doi: 10.1124/jpet.117.245522

Figure Lengend Snippet: (A) Functional activities of SMM-295 in the CB2 ACTOne assay (open circles), CB1 ACTOne assay (open triangles), and parental ACTOne cells containing only the CNG ion channel (filled squares). The functional activation of CB2 by the internal control (CP 55,940; filled diamonds) is shown. (B) Western blot analysis of signaling proteins after brief exposure (15 minutes) to SMM-295 in rat NRK-52E proximal tubule epithelial cells. Activation of prosurvival Akt/PKB and MAPK (ERK1/2 and p38) was detected using specific antibodies. β-actin was used as a loading control. ERK1/2, extracellular signal–regulated kinase 1/2.

Article Snippet: Human embryonic kidney (HEK)–cyclic nucleotide gated (CNG), HEK-CNG+CB1, and HEK-CNG+CB2 cells were obtained from Codex BioSolutions.

Techniques: Functional Assay, Activation Assay, Western Blot