MODY mutations in transcription factors HNF1A and HNF1B affect the production of interferon signaling proteins: evidence at the proteome level

MODY mutations in transcription factors HNF1A and HNF1B affect the production of interferon signaling proteins: evidence at the proteome level Ksenia G Kuznetsova1,2,3,*, Jakub Vašíček1,2, Dafni Skiadopoulou1,2, Lucas Unger1, Rachel Anand Nethala1, Michael Wierer4, Luiza Ghila1, Stefan Johansson1,5, Pål Rasmus Njølstad1,6, Simona Chera1, Bente Johansson1, Alisa Manning3,7,8, and Marc Vaudel1,2,9 1Mohn Center for Diabetes Precision Medicine Department of Clinical Science, University of Bergen, Norway 2Computational Biology Unit, Department of Informatics, University of Bergen, Bergen, Norway 3Broad Institute of MIT and Harvard, Cambridge, MA, USA 4Proteomics Research Infrastructure, Center for Core Facilities, University of Copenhagen, Denmark 5Department of Medical Genetics, Haukeland University Hospital, Bergen, Norway 6Department of Pediatrics and Adolescents, Haukeland University Hospital, Bergen, Norway 7Department of Medicine, Massachusetts General Hospital, Boston, MA, USA 8Department of Medicine, Harvard Medical School, Boston, MA, USA 9Department of Genetics and Bioinformatics, Norwegian Institute of Public Health, Bergen, Norway Supplementary figures 465 6987013 All identified proteins Stage4 Stage4 Stage0 Stage0 213 77102 DDA DDA DIA DIA DDA DDA DIA DIA Differentially abundant proteins 1465 1796281 All identified proteins 144 599 Differentially abundant proteins A. B. C. D. Figure S.1: Venn diagram of identified and differentially abundant proteins in hiPSC datasets A. Comparison of all identified proteins in stage 4 pancreatic progenitor cells using DDA and DIA mass spectrometry methods. B. Differentially abundant proteins in stage 4 cells identified by DDA and DIA. C. Comparison of all identified proteins in stage 0 stem cells using DDA and DIA. D. Differentially abundant proteins in stage 0 cells identified by DDA and DIA. 6 4 2 0 2 4 Log2 Fold Change 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 -Log10 adj.P-value COX7A2L NDUFB4 RIGI LDHA OAS1 ISG15 IFIT1 COX5B NCAM1 COX7A2 MX1 CACNA2D1 B2M IRF9 IFITM3 H1-1 IFIT5 PDE12 NDUFS8 NDUFS4 NDUFA3 GSR HLA-B HLA-H MT-ND1 MT-ND4 MT-ND5 MT-ND6 HLA-A COX6A1 TUBA1B BST2 IFIH1 NDUFA12 NDUFAF1 Differentially expressed proteins Pathways interferon signaling energy exchange 8 6 4 2 0 2 4 Log2 Fold Change 0 1 2 3 4 -Log10 adj.P-value SLC25A12 RIGI OAS1 ISG15 HSPA1L CACNA2D1IRF9 IFITM3 H1-1 BST2 IFIT5 PDE12 NDUFB9 IFIT3 UQCR11 NDUFB3 NDUFA7 GSR MT-CO3 HLA-B MT-ND5 MT-ND6 IFIT1 SLC25A6 TOMM20 IFIH1 OAS3 Differentially expressed proteins Pathways interferon signaling energy exchange A. Stage 4 DDA B. Stage 4 DIA WT mut WT mut Figure S.2: Volcano plots of differentially abundant proteins in stage 4 pancreatic progenitor cells Comparison of cells with the HNF1A frameshift mutation to control cells. Colors highlight proteins related to interferon signaling and energy exchange pathways identified by Qiagen IPA. A. DDA mass spectrometry dataset. B. DIA mass spectrometry dataset. 6 4 2 0 2 4 Log2 Fold Change 0.0 0.5 1.0 1.5 2.0 -Log10 adj.P-value UQCR11 TIMM17B NDUFB8 MT-CYB MT-CO1 MT-ND5 MT-ND6 COX6A1 NDUFB7 PSEN2 ATP5MF NDUFV3 H1-1 ITPR2 PDE12 CPT1C TOMM6 CMC2 TOMM7 NDUFB9 COX11 Differentially expressed proteins Pathways interferon signaling energy exchange 6 4 2 0 2 4 Log2 Fold Change 0 1 2 3 4 -Log10 adj.P-value NDUFS8 UQCR11 MRPS12 OGG1 CYB5B MT-CO3 HLA-H MT2A MT-ND1 MT-ND5 H1-0 COX6B1 PSEN2 MRPS25 H1-1 PDE12 NDUFA12 Differentially expressed proteins Pathways interferon signaling energy exchange A. Stage 0 DDA B. Stage 0 DIA WT mut WT mut Figure S.3: Volcano plots of differentially abundant proteins in stage 0 stem cells Comparison of cells with the HNF1A frameshift mutation to control cells. Colors highlight proteins related to interferon signaling and energy exchange pathways identified by Qiagen IPA. A. DDA mass spectrometry dataset. B. DIA mass spectrometry dataset. 2 4 6 8 10 12 14 -log(p-value) Oxidative Phosphorylation Granzyme A Signaling Mitochondrial Dysfunction Interferon alpha/beta signaling Hematoma Resolution Signaling Pathway Parkinson's Signaling Pathway Post-translational protein phosphorylation Interferon Signaling Sirtuin Signaling Pathway OAS antiviral response Regulation of Insulin-like Growth Factor (IGF) transport and uptake by IGFBPs Cytoprotection by HMOX1 significance Number of Proteins 1 proteins 2 proteins 3 proteins 3 2 1 0 1 2 3 4 z-score z-score 4 2 0 2 4 Top 12 pathways with z-score no less than 2 significance Interferon gamma signaling Synthesis, secretion, and deacylation of Ghrelin Interferon Signaling Cytoprotection by HMOX1 Interferon alpha/beta signaling Neutrophil Extracellular Trap Signaling Pathway Sirtuin Signaling Pathway Parkinson's Signaling Pathway Hematoma Resolution Signaling Pathway Mitochondrial Dysfunction Oxidative Phosphorylation Granzyme A Signaling -log(p-value) 2015105 Number of Proteins 1 proteins 2 proteins 3 proteins 2 4 6 8 10 -log(p-value) Oxidative Phosphorylation Respiratory electron transport Mitochondrial Dysfunction Complex I biogenesis Granzyme A Signaling Neutrophil Extracellular Trap Signaling Pathway Mitochondrial RNA degradation Mitochondrial protein import tRNA processing in the mitochondrion Complex IV assembly Parkinson's Signaling Pathway Mitochondrial protein degradation significance Number of Proteins 1 proteins 2 proteins 3 proteins 3 2 1 0 1 2 3 z-score 2 4 6 8 -log(p-value) Oxidative Phosphorylation Respiratory electron transport Granzyme A Signaling Mitochondrial Dysfunction Complex I biogenesis Hematoma Resolution Signaling Pathway Parkinson's Signaling Pathway Neutrophil Extracellular Trap Signaling Pathway Molecular Mechanisms of Cancer significance Number of Proteins 1 proteins 2 proteins 3 proteins 2 1 0 1 2 3 z-score A. Stage 4 DDA B. Stage 4 DIA C. Stage 0 DDA D. Stage 0 DIA Figure S.4: Most significantly enriched pathways predicted by Qiagen IPA Based on differentially abundant proteins with at least 2-fold change and BH-adjusted p-values < 0.05. Z-scores indicate the direction of regulation (≥2). Plots show the top 12 pathways selected by z-scores. S4_DDA S4_DIA S0_DDA S0_DIA Mitochondrial Dysfunction Oxidative Phosphorylation Granzyme A Signaling Neutrophil Extracellular Trap Signaling Pathway Hematoma Resolution Signaling Pathway Sirtuin Signaling Pathway Parkinson's Signaling Pathway Neutrophil degranulation Oxytocin Signaling Pathway HER-2 Signaling in Breast Cancer Serotonin Receptor Signaling Eicosanoid Signaling Post-translational protein phosphorylation G Beta Gamma Signaling Ephrin Receptor Signaling Sertoli Cell-Sertoli Cell Junction Signaling DHCR24 Signaling Pathway Regulation of Insulin-like Growth Factor (IGF) transport and uptake by IGFBPs Mitochondrial protein import ISG15 antiviral mechanism Interferon Signaling Interferon gamma signaling Complex I biogenesis Role of Hypercytokinemia/hyperchemokinemia in the Pathogenesis of Influenza Cytoprotection by HMOX1 Respiratory electron transport Electron transport, ATP synthesis, and heat production by uncoupling proteins Interferon alpha/beta signaling Coronavirus Pathogenesis Pathway Extra-nuclear estrogen signaling Platelet homeostasis Complex IV assembly Mitochondrial RNA degradation G alpha (12/13) signalling events DNA Damage/Telomere Stress Induced Senescence OAS antiviral response tRNA processing in the mitochondrion Necroptosis Signaling Pathway DDX58/IFIH1-mediated induction of interferon-alpha/beta G alpha (s) signalling events Class B/2 (Secretin family receptors) NAD Signaling Pathway Mitochondrial protein degradation Synthesis, secretion, and deacylation of Ghrelin Meiotic synapsis 4 2 0 2 4 Figure S.5: Heat map of the most regulated pathways predicted by Qiagen IPA This heat map illustrates the most regulated pathways predicted by Qiagen IPA software, comparing pathways enriched across all datasets. The color indicates z-scores, showing the direction and magnitude of regulation. The figure demonstrates the reproducibility of the most regulated pathways among the datasets. Energy exchangerelated pathways show consistent regulation in both stage 0 and stage 4, while interferon signalingrelated pathways are only downregulated in samples at stage 4. Mtx IS Mtx Mtx Mtx Mtx Mtx Mtx IS IS IS IS IS IS IS IS Mtx Mtx Mtx Mtx Mtx Mtx Mtx A. Stage 4 DDA B. Stage 4 DIA C. Stage 0 DDA D. Stage 0 DIA Figure S.6: Hallmark gene set enrichment of downregulated proteins Analysis performed by FragPipeAnalyst tool across all hiPSC datasets. 1234567 -log10(Adjusted.P.value) Oxidative Phosphorylation Pancreas Beta Cells Interferon Gamma Response Interferon Alpha Response Epithelial Mesenchymal Transition Pperoxisome UV Response Dn Xenobiotic Metabolism IL-2/STAT5 Signaling Apoptosis significance Number of Proteins 1 proteins 2 proteins 3 proteins 4 6 8 10 12 14 16 18 Odds Ratio 1 2 3 4 5 6 7 8 -log10(Adjusted.P.value) Interferon Alpha Response Oxidative Phosphorylation Interferon Gamma Response Pancreas Beta Cells Coagulation Estrogen Response Late Apical Junction Epithelial Mesenchymal Transition KRAS Signaling Up UV Response Dn significance Number of Proteins 1 proteins 2 proteins 3 proteins 4 6 8 10 12 14 16 18 Odds Ratio 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 -log10(Adjusted.P.value) Oxidative Phosphorylation PI3K/AKT/mTOR Signaling Pperoxisome Adipogenesis Estrogen Response Early Estrogen Response Late Notch Signaling Spermatogenesis Wnt-beta Catenin Signaling Reactive Oxygen Species Pathway significance Number of Proteins 1 proteins 2 proteins 3 proteins 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 Odds Ratio 0.4 0.6 0.8 1.0 1.2 1.4 1.6 -log10(Adjusted.P.value) Oxidative Phosphorylation Apoptosis Adipogenesis mTORC1 Signaling UV Response Up Hypoxia Myc Targets V1 Cholesterol Homeostasis Androgen Response Pperoxisome significance Number of Proteins 1 proteins 2 proteins 3 proteins 5 6 7 8 9 Odds Ratio C18orf32C18orf32 COX5BCOX5B COX6A1COX6A1 COX7A2COX7A2 COX7CCOX7C ECSITECSIT FILIP1LFILIP1L GGNBP2GGNBP2 MT-ATP6MT-ATP6 MT-ATP8MT-ATP8 MT-CO3MT-CO3 MT-ND1MT-ND1 MT-ND2MT-ND2 MT-ND4MT-ND4 MT-ND5MT-ND5 MT-ND6MT-ND6 NDUFA10NDUFA10 NDUFA12NDUFA12 NDUFA13NDUFA13 NDUFA3NDUFA3 NDUFA4NDUFA4 NDUFA7NDUFA7 NDUFAF1NDUFAF1 NDUFB1NDUFB1 NDUFB11NDUFB11 NDUFB3NDUFB3 NDUFB4NDUFB4 NDUFB7NDUFB7 NDUFB8NDUFB8 NDUFB9NDUFB9 NDUFS4NDUFS4 NDUFS8NDUFS8 TMEM126BTMEM126B UQCR11UQCR11 BST2BST2 CMPK2CMPK2 DDX58DDX58 DTX3LDTX3L EPSTI1EPSTI1 HELZ2HELZ2 HERC5HERC5 IFI16IFI16 IFIH1IFIH1 IFIT1IFIT1 IFIT3IFIT3 IFIT5IFIT5 IFITM3IFITM3 IRF9IRF9 ISG15ISG15 MX1MX1 OAS1OAS1 OAS3OAS3 PARP14PARP14 PARP9PARP9 PGGHGPGGHG RALBRALB A. Stage 4 STRING clustering B. Oxidative phosphorylation C. Antiviral defense Figure S.7: Protein interaction analysis in stage 4 pancreatic progenitor cells Performed by STRING on differentially abundant proteins. MCL clustering with inflation parameter 3. A. Network of all proteins with highlighted clusters. B. Oxidative Phosphorylation/Respiratory Electron Transport cluster. C. Negative Regulation of Viral Genome Replication/Interferon Alpha/Beta Signaling/ Antiviral Defense cluster. ATP5IF1ATP5IF1 ATP5MFATP5MF ATP6V0CATP6V0C COX11COX11 COX6A1COX6A1 COX6B1COX6B1 DECR2DECR2 FDX2FDX2 GHITMGHITM HIGD2AHIGD2A MPC2MPC2 MT-CO1MT-CO1 MT-CO3MT-CO3 MT-CYBMT-CYB MT-ND1MT-ND1 MT-ND5MT-ND5 MT-ND6MT-ND6 NDUFA12NDUFA12 NDUFB7NDUFB7 NDUFB8NDUFB8 NDUFB9NDUFB9 NDUFS8NDUFS8 NDUFV3NDUFV3 TIMM17BTIMM17B UQCR11UQCR11 A. Stage 0 STRING clustering B. Oxidative phosphorylation Figure S.8: Protein interaction analysis in stage 0 stem cells Performed by STRING on differentially abundant proteins. MCL clustering with inflation parameter 3. A. Network of all proteins with highlighted clusters. B. Oxidative Phosphorylation/Mitochondrial ATP Synthesis Coupled Electron Transport cluster.