Journal: eLife

Article Title: Deep mutational scanning reveals pharmacologically relevant insights into TYK2 signaling and disease

doi: 10.7554/eLife.110149

Figure Lengend Snippet: ( A ) Schematic showing the reporter design for TYK2 deep mutational scanning (DMS) assays on interferon alpha (IFN-α) signaling (left) and protein abundance (right). BC, unique DNA barcode; BFP, blue flourescent protein; GFP, green fluorescent protein; ISRE, interferon-stimulated response element. ( B ) Heatmaps showing the functional effects of each TYK2 variant, represented as z-scores, for IFN-α signaling (left, 100 U/mL IFN-α condition) and protein abundance (right). Rows and columns of the heatmap correspond to amino acid position and variant identity, respectively. TYK2 protein domains are shown on the far right. GOF, gain-of-function; LOF, loss-of-function; WT, wild-type. ( C ) Structures of the TYK2 kinase and pseudokinase domains (PDB: 4OLI) with residues colored blue to indicate positions that have at least two significant LOF variants at that position in the IFN-α signaling (left) or protein abundance (right) assay (FDR<0.01). Panel A was created with BioRender.com .

Article Snippet: For experiments involving TYK2 inhibitors (MedChemExpress), a pre-drugging phase was introduced after overnight doxycycline induction where media was replaced with DMEM+0.5% dialyzed FBS, ±drug for 1 hr, followed by an exchange into DMEM+0.5% dialyzed FBS, ±cytokine, ±drug.

Techniques: Quantitative Proteomics, Functional Assay, Variant Assay

Journal: eLife

Article Title: Deep mutational scanning reveals pharmacologically relevant insights into TYK2 signaling and disease

doi: 10.7554/eLife.110149

Figure Lengend Snippet: ( A ) IFN-α reporter assay dose-response curves for wild-type (WT) and catalytically inactive (K930E variant) TYK2. RLU, relative luciferase units; WT, wild-type. ( B ) Flow cytometry data showing that the abundance DMS reporter distinguishes between WT and two strongly destabilizing variants (E154X and K930E). BFP, blue fluorescent protein; GFP, green fluorescent protein.

Article Snippet: For experiments involving TYK2 inhibitors (MedChemExpress), a pre-drugging phase was introduced after overnight doxycycline induction where media was replaced with DMEM+0.5% dialyzed FBS, ±drug for 1 hr, followed by an exchange into DMEM+0.5% dialyzed FBS, ±cytokine, ±drug.

Techniques: Reporter Assay, Variant Assay, Luciferase, Flow Cytometry

Journal: eLife

Article Title: Deep mutational scanning reveals pharmacologically relevant insights into TYK2 signaling and disease

doi: 10.7554/eLife.110149

Figure Lengend Snippet: ( A ) Bar plots quantifying the frequency of loss-of-function (LOF) (blue), neutral (gray), and gain-of-function (GOF) (red) variant effects across the interferon alpha (IFN-α) and abundance DMS datasets. ( B ) Histograms of stop (red) versus nonstop (gray) variant effects, where greater separation of these two distributions indicates greater dynamic range and power to detect intermediate variant effects. The x-axis represents variant effect measured as Z-statistic of the variant normalized to wild-type (WT) (i.e. WT = 0), and this convention is carried through all subsequent panels. ( C ) Violin plots of IFN-α (left) and abundance (right) DMS variant effects (Z-statistic, y-axis), grouped and colored by AlphaMissense annotation. The boxplots show the median, the interquartile range (IQR), and 1.5 x IQR whiskers. Brackets indicate pairwise two-sided Kolmogorov–Smirnov tests comparing the Z-statistic distributions between annotation groups (*** p < 0.001, ** p < 0.01, * p < 0.05, ns = not significant). ( D ) DMS variant effects (log 2 (fold-change)) for a panel of 32 previously characterized TYK2 variants, grouped by whether they were predicted to be GOF (top), neutral (middle), or LOF (bottom). Points show the log₂ fold change (IFNα conditions) or midpoint shift (Abundance). Error bars span ±2 standard errors (≈95% confidence interval), with errors propagated from the variant and control estimates. Points are colored black where the variant is significant at FDR < 1% (Benjamini–Hochberg) and gray otherwise. ( E ) Violin plots of IFN-α (left) and abundance (right) DMS variant effects (Z-statistic, x-axis), grouped and colored by ClinVar annotation.

Article Snippet: For experiments involving TYK2 inhibitors (MedChemExpress), a pre-drugging phase was introduced after overnight doxycycline induction where media was replaced with DMEM+0.5% dialyzed FBS, ±drug for 1 hr, followed by an exchange into DMEM+0.5% dialyzed FBS, ±cytokine, ±drug.

Techniques: Variant Assay, Control

Journal: eLife

Article Title: Deep mutational scanning reveals pharmacologically relevant insights into TYK2 signaling and disease

doi: 10.7554/eLife.110149

Figure Lengend Snippet: ( A ) Schematic showing approach to identify key functional sites by pinpointing TYK2 variants that impact interferon alpha (IFN-α) signaling but not protein abundance. LOF, loss-of-function. ( B ) Scatterplot of individual TYK2 variant effects on IFN-α signaling activity (x-axis) and TYK2 protein abundance (y-axis). Variants highlighted in blue were classified as signaling-only LOF (see Materials and methods for details). GOF, gain-of-function; WT, wild-type. ( C ) Count of signaling-only LOF variants (horizontal axis) at each residue of TYK2 (vertical axis), shown alongside the functional domains of the protein (left). Regions important for TYK2 function are highlighted in red, including known catalytic, known allosteric, and novel allosteric sites predicted by this analysis. ( D ) Structure of the TYK2 kinase and pseudokinase domains (PDB: 4OLI) with amino acid positions that have at least two significant signaling-only LOF variants colored blue. Novel allosteric sites predicted by this analysis are labeled with an asterisk (*). ( E ) Structural model of inactive full-length TYK2 (colored as in D ) bound to the intracellular tail of the IFNAR1 receptor (yellow). See Materials and methods for details on how this composite model was constructed. Panel E was created with BioRender.com .

Article Snippet: For experiments involving TYK2 inhibitors (MedChemExpress), a pre-drugging phase was introduced after overnight doxycycline induction where media was replaced with DMEM+0.5% dialyzed FBS, ±drug for 1 hr, followed by an exchange into DMEM+0.5% dialyzed FBS, ±cytokine, ±drug.

Techniques: Functional Assay, Quantitative Proteomics, Variant Assay, Activity Assay, Residue, Labeling, Construct

Journal: eLife

Article Title: Deep mutational scanning reveals pharmacologically relevant insights into TYK2 signaling and disease

doi: 10.7554/eLife.110149

Figure Lengend Snippet: ( A ) Histogram quantifying the number of amino acid positions that have X signaling-only loss-of-function (LOF) variants (‘count’). ( B ) TYK2 kinase-pseudokinase structure (PDB: 4OLI) with amino acid positions that have at least one significant signaling-only LOF variant colored blue.

Article Snippet: For experiments involving TYK2 inhibitors (MedChemExpress), a pre-drugging phase was introduced after overnight doxycycline induction where media was replaced with DMEM+0.5% dialyzed FBS, ±drug for 1 hr, followed by an exchange into DMEM+0.5% dialyzed FBS, ±cytokine, ±drug.

Techniques: Variant Assay

Journal: eLife

Article Title: Deep mutational scanning reveals pharmacologically relevant insights into TYK2 signaling and disease

doi: 10.7554/eLife.110149

Figure Lengend Snippet: ( A ) Conceptual schematic of how inhibitor dose influences variant effect interpretation. ( B ) Volcano plot of variant effects under very high dose (>IC 99 ) of BMS-986202, with significant drug-resistant variants colored pink (FDR<0.01 used as significance threshold throughout figure). ( C ) Surface representation of the TYK2 pseudokinase domain (PDB: 6NZP) colored as in ( B ). ( D ) Allosteric binding site of the TYK2 pseudokinase domain with inhibitor BMS-986202 bound (dark gray) and positions with significant drug resistance colored pink (as in C) for increasing concentrations of BMS-986202. ( E ) Scatter plot of variant effects (Z-statistic) in the presence of inhibitor (IC 99 concentration), with variants colored by whether they exhibit resistance to BMS-986202 (pink), NDI-034858 (cyan), or both (orange). ( F ) Heatmap of drug-resistant positions (x-axis) and variants (y-axis) colored as in ( E, G ). Structure (PDB: 6NZP) of TYK2 pseudokinase domain (colored as in E ). ( H, I, J ) Variants that exhibit drug-potentiation to each inhibitor (at IC 75 concentrations) displayed as in ( E , F , and G ), respectively.

Article Snippet: For experiments involving TYK2 inhibitors (MedChemExpress), a pre-drugging phase was introduced after overnight doxycycline induction where media was replaced with DMEM+0.5% dialyzed FBS, ±drug for 1 hr, followed by an exchange into DMEM+0.5% dialyzed FBS, ±cytokine, ±drug.

Techniques: Variant Assay, Binding Assay, Concentration Assay

Journal: eLife

Article Title: Deep mutational scanning reveals pharmacologically relevant insights into TYK2 signaling and disease

doi: 10.7554/eLife.110149

Figure Lengend Snippet: ( A ) Odds ratios from comprehensive phenome-wide association study (PheWAS) for the TYK2 P1104A allele (see Materials and methods), which confirm protective effect on many autoimmune phenotypes (see for list of phenotype abbreviations). ( B ) Deep mutational scanning (DMS) results for the interferon alpha (IFN-α) (x-axis) and protein abundance (y-axis) assays, as in . Each point represents a unique missense or nonsense variant, and P1104A is highlighted in blue. GOF, gain-of-function; LOF, loss-of-function; WT, wild-type. ( C ) TYK2 protein abundance measured using western blotting of HEK293T and Kit225 cells harboring the wild-type (WT, gray) and P1104A (blue) alleles of TYK2. Data are shown normalized by the abundance of the WT allele for each cell type. Bars represent means, with error bars reporting standard error, and p-values determined by unpaired t-test. ( D ) Effect sizes ( beta ) of association between P1104A allele and abundance of three proteins in human plasma from UK Biobank. Error bars are 95% confidence intervals. ( E ) Rare variant dose-response curve for TYK2 variants found in the UK Biobank shows that TYK2 protein abundance (x-axis, from DMS assay) predicts the probability of autoimmune disease diagnosis (y-axis). See Materials and methods for details. Gray shading indicates 95% confidence intervals.

Article Snippet: For experiments involving TYK2 inhibitors (MedChemExpress), a pre-drugging phase was introduced after overnight doxycycline induction where media was replaced with DMEM+0.5% dialyzed FBS, ±drug for 1 hr, followed by an exchange into DMEM+0.5% dialyzed FBS, ±cytokine, ±drug.

Techniques: Quantitative Proteomics, Variant Assay, Western Blot, Clinical Proteomics, Biomarker Discovery

Journal: eLife

Article Title: Deep mutational scanning reveals pharmacologically relevant insights into TYK2 signaling and disease

doi: 10.7554/eLife.110149

Figure Lengend Snippet: ( A ) Effects (as z-scores) of two common partial loss-of-function missense variants of TYK2 on relatively well-powered autoimmune traits in UK Biobank (UKBB). (**) denotes that the variant is the most likely causal variant for the trait at the 5E-08 threshold, while (*) denotes that the variant is the most likely causal variant for the trait at the 5E-05 significance threshold. PsO, psoriasis; RA, rheumatoid arthritis; HT, hypothyroidism; UC, ulcerative colitis; IBD, inflammatory bowel disease; pooled, autoimmune phenotypes combined; pooled (no IBD), same as pooled but excluding IBD cases. ( B ) Example flow cytometry data showing variants with intermediate abundance (P1104A, L1014P) relative to wild-type (WT) and an early stop variant (E154X). ( C ) Sanger sequencing traces confirming P1104A mutation in Kit225 cells. ( D ) Representative western blot of WT and P1104A TYK2 in HEK293T and Kit225 cells. ( E ) Effect sizes ( beta ) of association between TYK2 alleles (P1104A, blue; and I684S, pink) and abundance of 12 proteins in human plasma from UKBB. All 12 proteins show a significant association with P1104A, while only the top 3 show a significant association with I684S. Error bars are 95% confidence intervals. ( F ) Deep mutational scanning results for the interferon alpha (IFN-α) (x-axis) and protein abundance (y-axis) assays, as in . Each point represents a unique missense or nonsense variant, with P1104A and I684S variants colored blue and pink, respectively. Figure 4—figure supplement 1—source data 1. Uncropped, labeled image of western blot for . Figure 4—figure supplement 1—source data 2. Uncropped, unlabeled original image of western blot for .

Article Snippet: For experiments involving TYK2 inhibitors (MedChemExpress), a pre-drugging phase was introduced after overnight doxycycline induction where media was replaced with DMEM+0.5% dialyzed FBS, ±drug for 1 hr, followed by an exchange into DMEM+0.5% dialyzed FBS, ±cytokine, ±drug.

Techniques: Variant Assay, Flow Cytometry, Sequencing, Mutagenesis, Western Blot, Clinical Proteomics, Quantitative Proteomics, Labeling

Journal: eLife

Article Title: Deep mutational scanning reveals pharmacologically relevant insights into TYK2 signaling and disease

doi: 10.7554/eLife.110149

Figure Lengend Snippet: ( A ) Cumulative density plots quantifying the frequency of autoimmune disease in patient variants against the respective variant effect on protein abundance (left) or interferon alpha (IFN-α) signaling (right). Variants with very low abundance scores are uniquely associated with no autoimmune disease. ( B ) Rare variant association test for TYK2 variants found in the UK Biobank, comparing probability of autoimmune disease (y-axis) against variant effects on TYK2 abundance (left, reproduced from main ) or IFN-α signaling (right), showing that reduced TYK2 protein abundance (left) is correlated with a reduced probability of autoimmune disease. Effects on IFN-α signaling (right) show no correlation with autoimmune disease. Gray shading indicates 95% confidence intervals.

Article Snippet: For experiments involving TYK2 inhibitors (MedChemExpress), a pre-drugging phase was introduced after overnight doxycycline induction where media was replaced with DMEM+0.5% dialyzed FBS, ±drug for 1 hr, followed by an exchange into DMEM+0.5% dialyzed FBS, ±cytokine, ±drug.

Techniques: Variant Assay, Quantitative Proteomics

Journal: eLife

Article Title: Deep mutational scanning reveals pharmacologically relevant insights into TYK2 signaling and disease

doi: 10.7554/eLife.110149

Figure Lengend Snippet: ( A-G ) Images of all blots stained for TYK2 (top) and GAPDH loading control (bottom). For each lane, TYK2 band density was normalized to GAPDH band density. Lanes that were excluded are marked with a red X, determined by whether either band was outside of the linear detection range or contained blotting artifacts. Lanes are labeled by allele (wild-type [WT] or P1104A), with the cell background (HEK or Kit225) labeled above the alleles. Protein molecular weights (in kDa) are indicated along the left side of each image. See Materials and methods for more details on how bands were quantified and normalized for analysis. Figure 4—figure supplement 3—source data 1. Uncropped, labeled images of western blots for (all panels). Figure 4—figure supplement 3—source data 2. Uncropped, unlabeled original images of western blots for (all panels).

Article Snippet: For experiments involving TYK2 inhibitors (MedChemExpress), a pre-drugging phase was introduced after overnight doxycycline induction where media was replaced with DMEM+0.5% dialyzed FBS, ±drug for 1 hr, followed by an exchange into DMEM+0.5% dialyzed FBS, ±cytokine, ±drug.

Techniques: Staining, Control, Labeling, Western Blot