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: bioRxiv

Article Title: Synaptic plasticity via receptor tyrosine kinase/G protein-coupled receptor crosstalk

doi: 10.1101/2023.08.28.555210

Figure Lengend Snippet: (A-C) Representative traces showing intracellular Ca 2+ responses to TrkB activation by BDNF (A), mGluR5 activation by glutamate (B) and TrkB activation by BDNF in mGluR5 co-expressing cells (C). (D) Distribution of BDNF responses in the absence or presence of mGluR5 co-expression. Only cells showing a response to glutamate are analyzed in the TrkB + mGluR5 condition. (E-G) Representative traces showing Ca 2+ responses to extended 30 min BDNF application in cells expressing TrkB (E) or TrkB and mGluR5 (F), with summary histogram (G) showing the distribution of response durations. (H-I) Co-application of the mGluR5 PAM VU-29 enhances the response to low dose BDNF, as seen in a representative cell (H) and a summary bar graph of the percentage of cells responding to BDNF (I). Only cells responding to glutamate were including in the bar graph in (I). (J-K) Representative western blot (J) and quantification (K) of BDNF-induced ERK activation (p-ERK/ERK ratio at 15 min) in HEK 293-TrkB cells co-expressing mGluR5, showing an enhanced response in the presence of 500 nM VU-29. For (D) and (I), points represent values from individual movies taken from distinct coverslips. For (K), individual points represent value from individual blots. For all conditions, data comes from at least 3 separate cell preparations. Unpaired t-test for (I); One-way ANOVA with Tukey’s multiple comparisons for (K). All data shown as mean ± SEM; ** P < 0.01, *** P < 0.001. See also Figure S3 and S4.

Article Snippet: HEK 293 cells were purchased from ATCC (CRL-1573) and authenticated by Bio-Synthesis, Inc. HEK 293-TrkB stable cell line is as previously described .

Techniques: Activation Assay, Expressing, Western Blot

Journal: bioRxiv

Article Title: Synaptic plasticity via receptor tyrosine kinase/G protein-coupled receptor crosstalk

doi: 10.1101/2023.08.28.555210

Figure Lengend Snippet: (A) Summary bar graph showing that co-expression of Gα q enhances Ca 2+ responses upon low dose BDNF-induced TrkB activation in HEK 293-TrkB cells. (B) Representative trace showing Ca 2+ response upon co-application of BDNF and subthreshold dose of 5-HT in HEK 293 cells co-expressing TrkB-ΔPLC and 5-HT 2A R. (C) Summary bar graph showing percentage of cells responding to BDNF in HEK 293 cells co-expressing TrkBΔPLC and mGluR1 or 5-HT 2A R or mGluR2 with or without co-application of 5-HT or glutamate. (D-E) Representative traces showing intracellular Ca 2+ responses to endogenous EGFR activation by EGF in the absence (D) or presence (E) of mGluR5. (F) Distribution of EGF responses in the absence or presence of mGluR5 co-expression. (G-H) Representative traces showing intracellular Ca 2+ responses to TrkA activation by NGF in the absence (G) or presence (H) of mGluR5. (I) Distribution of NGF responses in the absence or presence of mGluR5 co-expression. (J-K) Representative traces showing intracellular Ca 2+ responses to IGF1R activation by IGF in the absence (J) or presence (K) of mGluR5. (L) Distribution of IGF responses in the absence or presence of mGluR5 co-expression. For (A) (C), (F), (I) and (L), points represent values from individual movies taken from distinct coverslips. For all conditions, data comes from at least 3 separate cell preparations. Only cells showing a response to glutamate are analyzed in the conditions with mGluR5. One-way ANOVA with Tukey’s multiple comparisons is used for (A) and (C). All data shown as mean ± SEM; *** P < 0.01, *** P < 0.001. See also Figure S8.

Article Snippet: HEK 293 cells were purchased from ATCC (CRL-1573) and authenticated by Bio-Synthesis, Inc. HEK 293-TrkB stable cell line is as previously described .

Techniques: Expressing, Activation Assay