Readme for: Supplementary data to the Article: Proteasomal Activity and Disease-Outcome in Patients with Phenylketonuria, Carriers of a Structural SLC7A5 Variant. Ersteller*in Readme: Bik-Multanowski; Miroslaw Erstellungsdatum Readme: 2025-10-15 ______________________________ Titel des Forschungsdatensatzes: Supplementary data to the Article: Proteasomal Activity and Disease-Outcome in Patients with Phenylketonuria, Carriers of a Structural SLC7A5 Variant Ort der Datenerhebung: Germany, Munich, LMU, Poland, Krakow, Jagiellonian University; Version: 1.0 ______________________________ Ersteller*in 1 des Datensatzes: Bik-Multanowski; Miroslaw Ersteller*in 1 Identifier: ORCID: 0000-0003-1542-7050 Ersteller*in 1 Affiliation: Institute of Human Genetics, University Hospital, LMU Munich, Germany Ersteller*in 2 des Datensatzes: Piejko; Marcin Ersteller*in 2 Identifier: ORCID: 0000-0003-3251-5984 Ersteller*in 2 Affiliation: Jagiellonian University Medical College, Krakow, Poland Beteiligte[*] 1: Jankowska; Urszula Beteiligte 1 Identifier: ORCID: 0000-0002-7165-8964 Beteiligte 1 Affiliation : Jagiellonian University Medical College, Krakow, Poland Beteiligte 1.Typ Researcher Beteiligte 2: Skupien-Rabian; Bozena Beteiligte 2 Identifier: ORCID: 0000-0002-1131-2582 Beteiligte 2 Affiliation : Jagiellonian University Medical College, Krakow, Poland Beteiligte 2.Typ Researcher [*] An der Sammlung, Verarbeitung oder Erstellung von Forschungsdaten beteiligte Personen. ______________________________ Projektname: Proteasomal Activity and Disease-Outcome in Patients with Phenylketonuria, Carriers of a Structural SLC7A5 Variant ______________________________ Abstract: Treatment in phenylketonuria is aimed at protecting the brain from uncontrolled hyperphenylalaninemia. Although treatment methods are well established, clinical outcomes vary among patients. This may be due to alterations of phenylalanine transport mediated by the amino acid transporter LAT1, which also regulates the function of the mTORC1/proteasomal pathway. We explored the cellular mechanisms underlying potential phenotypic differences between carriers and noncarriers of the rs113883650 polymorphism – a marker of a structural variant of the SLC7A5 gene. For our experiments we used a model of hyperphenylalaninemia based on induced pluripotent stem cells (hiPSCs). We conducted a targeted analysis of the expression of peptides involved in the FOXO/proteasomal signaling pathway. We confirmed a statistically significant difference in FOXO pathway expression between the two groups of carriers and non carriers of the investigated variant (p = 0.005, t-test) under high Phe conditions. Notably, an increased abundance of CDK2 and ERK1 (MAPK1)—both known to downregulate FOXO1—was observed in carriers of rs113883650. We did not detect statistically significant differences between the tested groups under low Phe conditions. The files Proteome Expression high Phe and Proteome expression low Phe contain data on expression of cytoplasmic proteins detected in both experimental settings. Statistical differentes (t test) between the tested groups (N=5+5) are shown in column K. Schlagwörter: FOXO, Proteasome, Phenylketonuria, LAT1 Methode der Datenerhebung: To quantitatively assess the differential response of carrier and noncarrier hiPSC to treatment with various concentrations of Phe, we employed the stable isotope labeling by amino acids in cell culture (SILAC) technique. For the higher Phe concentration (0.6 mmol/l), cells were maintained in a "heavy" medium comprising SILAC DMEM/F12 (Thermo-Fisher, Ref. A2494301) supplemented with isotopically labeled amino acids, including 13C615N2 L-Lys (Thermo-Fisher, ref. 88209), 13C615N4 L-Arg (Thermo-Fisher, ref. 89990). Parallel cultures were maintained under physiological Phe conditions using a "light" medium formulated with SILAC DMEM/F12 supplemented with unlabeled L-Lysine (Thermo-Fisher, Ref. 89987) and L-Arginine (Thermo-Fisher, Ref. 89989). Both cell culture media were supplemented with the Essential8 supplement (Gibco/Thermo Fisher Scientific). Cells were passaged for 5-6 cycles to ensure that heavy isotope incorporation exceeded 95%. Following this incorporation period, cells in the heavy medium were directly exposed to a higher Phe concentration (0.6 mM), while cells in the light medium were maintained at the lower Phe concentration (0.215 mM). After 48 hours of exposure, all cells were lysed in lysis buffer composed of 1M Tris with 1% SDS, and lysates were stored at -80°C for subsequent analysis by liquid chromatography-mass spectrometry (LC-MS). ______________________________ Dateiliste: Files: Proteome Expression high Phe.csv; Proteome expression low Phe.csv Dateiformate: .csv ______________________________