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: Physiological Reports

Article Title: Multiple mechanisms underlie reduced potassium conductance in the p.T1019PfsX38 variant of hERG

doi: 10.14814/phy2.15341

Figure Lengend Snippet: Step‐ramp voltage clamp protocol on cells expressing WT‐ or T1019PfsX38‐hERG. (ai and bi) Example current recordings from HEK293 cells that were transfected with WT‐ (ai) or T1019PfsX38‐hERG (bi) channels, as indicated. The currents were elicited using the protocol shown above A (V hold = –80 mV). The grey arrows mark the level where V C was 50 mV. (aii and bii) Current recordings from (ai) and (bi) presented as functions of membrane voltage. The ramp durations in (aii) and (bii) were colour‐coded as indicated in the box. The dotted lines represent ramp slope lines between 50 and –80 mV. (c) Average densities of peak current amplitudes plotted as functions of ramp durations. (d) Average values of area under each curve of current densities (I integral ) at different ramp durations. (e) Average values of membrane voltages at the time of occurance of peak current amplitudes. Data are mean ± SEM, * p < 0.05; WT‐hERG, n = 12, grey circles; T1019PfsX38‐hERG, n = 11 (except in E where n = 7 at 10 ms ramp duration), blue circles. At 20 ms in d and 40 ms in c and d the individual data points obtained from T1019PfsX38‐hERG did not meet normal statistical distribution criteria (Shapiro–Wilk test), in which case Mann–Whitney rank sum test was used for statistical comparison only at these three data points. Other details as in Materials and Methods and Results sections.

Article Snippet: For patch‐clamp recording, we used human embryonic kidney 293 (HEK‐293) cell line (passage <40) (ECACC, MERCK, Catalog # 85120602) grown in Dulbecco's Modified Eagle Medium (DMEM) high glucose (Sigma‐Aldrich, St. Louis, MO, USA).

Techniques: Expressing, Transfection, MANN-WHITNEY

Journal: Physiological Reports

Article Title: Multiple mechanisms underlie reduced potassium conductance in the p.T1019PfsX38 variant of hERG

doi: 10.14814/phy2.15341

Figure Lengend Snippet: The current densities and the voltage dependence of activation of WT‐ and T1019PfsX38‐hERG channels. (a, b) Representative whole‐cell currents recorded from WT‐ (a) and T1019PfsX38‐hERG (b) channels expressed in HEK293 cells. The currents were elicited using the protocol shown above A (V hold = −80 mV). The grey arrows mark the level where V C was −80 mV. (c) Quantification of step currents measured as average current densities in the last 50 ms of the depolarizing test potentials. (d) Quantification of peak tail currents measured after stepping to −40 mV. The continuous lines in (d) are best fits to the average data using the Boltzmann equation. (e, f) Whisker plots of the V mid and k values predicted by fitting the data in (d). Data in (c) and (d) are mean ± SEM; WT‐hERG, n = 5, grey circles and triangles; T1019PfsX38‐hERG, n = 6, blue circles and triangles. Other details as in Materials and Methods and Results sections.

Article Snippet: For patch‐clamp recording, we used human embryonic kidney 293 (HEK‐293) cell line (passage <40) (ECACC, MERCK, Catalog # 85120602) grown in Dulbecco's Modified Eagle Medium (DMEM) high glucose (Sigma‐Aldrich, St. Louis, MO, USA).

Techniques: Activation Assay, Whisker Assay

Journal: Physiological Reports

Article Title: Multiple mechanisms underlie reduced potassium conductance in the p.T1019PfsX38 variant of hERG

doi: 10.14814/phy2.15341

Figure Lengend Snippet: Kinetics of activation of WT‐ and T1019PfsX38‐hERG channels. (a, b) Example whole cell currents recorded from WT‐ (a) and T1019PfsX38‐hERG (b) channels expressed in HEK293 cells. The currents were elicited using the protocol shown above (a). (V hold = −100 mV). The example recordings in (a) and (b) were from cells activated at 80 mV. For better visualization, the x‐axes in (a) and (b) were presented in logarithmic scales. The grey and black arrows mark the levels where V C was −100 mV and 80 mV, respectively. (c–e) Quantification of the negative peak tail currents obtained after activating the channels at 80 mV (c), 40 mV (d) or 0 mV (e) and presented as fractions of the peak inward tail currents after depolarization to 80 mV for 5.12 s in the same cell. The continuous lines in (c–e) are best fits to the average data using an exponential function. (f) Whisker plots and individual data points of the time constants of activation of WT‐ and T1019PfsX38‐hERG channels. Data are mean ± SEM; WT‐hERG, grey circles/lines, n = 7–8 (c), n = 5–6 (d), n = 5 (e) and n = 4–7 (f); T1019PfsX38‐hERG, blue circles / lines, n = 7–9 (c), n = 6–7 (d), n = 6 (e) and n = 4–8 (f). Other details as in Materials and Methods, and Results sections.

Article Snippet: For patch‐clamp recording, we used human embryonic kidney 293 (HEK‐293) cell line (passage <40) (ECACC, MERCK, Catalog # 85120602) grown in Dulbecco's Modified Eagle Medium (DMEM) high glucose (Sigma‐Aldrich, St. Louis, MO, USA).

Techniques: Activation Assay, Whisker Assay

Journal: Physiological Reports

Article Title: Multiple mechanisms underlie reduced potassium conductance in the p.T1019PfsX38 variant of hERG

doi: 10.14814/phy2.15341

Figure Lengend Snippet: The voltage dependence of recovery from inactivation of WT‐ and T1019PfsX38‐hERG channels. (a, b) Representative whole cell currents recorded from WT‐ (a) and T1019PfsX38‐hERG (b) channels expressed in HEK293 cells. The currents were elicited using the protocol shown above A (V hold = −80 mV). The grey arrows mark the level where V C was 50 mV. For better representation, isolated segments from (a) and (b) at different test potentials are shown separately. (c) Quantification of peak current amplitudes measured after stepping to the test potential and normalized to the maximum inward current amplitudes. Data are mean ± SEM, * p < 0.05; WT‐hERG, n = 6, grey; T1019PfsX38‐hERG, n = 7, blue. Other details as in Materials and Methods and Results sections.

Article Snippet: For patch‐clamp recording, we used human embryonic kidney 293 (HEK‐293) cell line (passage <40) (ECACC, MERCK, Catalog # 85120602) grown in Dulbecco's Modified Eagle Medium (DMEM) high glucose (Sigma‐Aldrich, St. Louis, MO, USA).

Techniques: Isolation

Journal: Physiological Reports

Article Title: Multiple mechanisms underlie reduced potassium conductance in the p.T1019PfsX38 variant of hERG

doi: 10.14814/phy2.15341

Figure Lengend Snippet: The time dependence of WT‐ and T1019PfsX38‐hERG recovery from inactivation and deactivation. (a, b) Representative whole‐cell currents recorded from WT‐ (a) and T1019PfsX38‐hERG (b) channels expressed in HEK293 cells. The currents were elicited using the protocol shown above A (V hold = −80 mV). The example recordings in (a) and (b) were from cells activated at 80 mV. The grey arrows mark the level where V C was 80 mV. (c–e) Quantification of the negative peak tail currents obtained after 80 mV (c) 40 mV (d) and 0 mV (e) test potentials. The values in (c) to (e) are shown after normalization, the normalization range was from the lowest peak current amplitudes (at 0 mV) to the highest peak current amplitudes (at 80 mV) in the same cell. The continuous lines in (c–e) are best fits to the average data using a 2‐component exponential function, τ 1 is the time constant for recovery from inactivation and τ 2 is the time constant for deactivation. Data are mean ± SEM, WT‐hERG, n = 4, grey circles; T1019PfsX38‐hERG, n = 4, blue circles. Other details as in Materials and Methods and Results sections.

Article Snippet: For patch‐clamp recording, we used human embryonic kidney 293 (HEK‐293) cell line (passage <40) (ECACC, MERCK, Catalog # 85120602) grown in Dulbecco's Modified Eagle Medium (DMEM) high glucose (Sigma‐Aldrich, St. Louis, MO, USA).

Techniques:

Journal: Physiological Reports

Article Title: Multiple mechanisms underlie reduced potassium conductance in the p.T1019PfsX38 variant of hERG

doi: 10.14814/phy2.15341

Figure Lengend Snippet: The voltage dependence of inactivation of WT‐ and T1019PfsX38‐hERG channels. (a, b) Representative whole‐cell currents recorded from WT‐ (a) and T1019PfsX38‐hERG (b) channels expressed in HEK293 cells. The currents were elicited using the protocol shown above A (V hold = −80 mV). The grey arrows mark the level where V C was 40 mV. (c) Quantification of currents measured at 1 ms before the end of the test potential and normalized to the maximal inward peak current amplitude. (d) Quantification of normalized tail currents measured at 5 ms after stepping to 20 mV. The grey and blue continuous lines in d are best fits to the average data after correction for deactivation. (e, f) Whisker plots of the V mid and the k values as predicted by fitting the raw (open boxes) and the corrected (dashed boxes) data in d. Data are mean ± SEM; WT‐hERG, n = 5, grey boxes, squares, circles, and lines; T1019PfsX38‐hERG, n = 5–6, blue boxes, squares, circles, and lines. Other details as in Materials and Methods and Results sections.

Article Snippet: For patch‐clamp recording, we used human embryonic kidney 293 (HEK‐293) cell line (passage <40) (ECACC, MERCK, Catalog # 85120602) grown in Dulbecco's Modified Eagle Medium (DMEM) high glucose (Sigma‐Aldrich, St. Louis, MO, USA).

Techniques: Whisker Assay

Journal: Physiological Reports

Article Title: Multiple mechanisms underlie reduced potassium conductance in the p.T1019PfsX38 variant of hERG

doi: 10.14814/phy2.15341

Figure Lengend Snippet: The inactivation rates of WT‐ and T1019PfsX38‐hERG channels. (a, b) Representative whole‐cell currents recorded from WT‐ (a) and T1019PfsX38‐hERG (b) channels expressed in HEK293 cells. The currents were elicited using the protocol shown above A (V hold = −80 mV). The grey arrows mark the level where V C was 50 mV. (c) Quantification of the rate constants of channel inactivation measured by fitting the tail currents at the different tested potentials using a standard exponential function. Data are mean ± SEM, * p < 0.05; WT‐hERG, n = 7 except at −10 mV ( n = 6), grey circles; T1019PfsX38‐hERG, n = 5, blue circles. Other details as in Materials and Methods and Results sections.

Article Snippet: For patch‐clamp recording, we used human embryonic kidney 293 (HEK‐293) cell line (passage <40) (ECACC, MERCK, Catalog # 85120602) grown in Dulbecco's Modified Eagle Medium (DMEM) high glucose (Sigma‐Aldrich, St. Louis, MO, USA).

Techniques: