Integrated in silico MS-based phosphoproteomics and network enrichment analysis of RASopathy proteins

Abstract

Background RASopathies are a group of syndromes showing clinical overlap caused by mutations in genes affecting the RAS-MAPK pathway. Consequent disruption on cellular signaling leads and is driven by phosphoproteome remodeling. However, we still lack a comprehensive picture of the different key players and altered downstream effectors. Methods An in silico interactome of RASopathy proteins was generated using pathway enrichment analysis/STRING tool, including identification of main hub proteins. We also integrated phosphoproteomic and immunoblotting studies using previous published information on RASopathy proteins and their neighbors in the context of RASopathy syndromes. Data from Phosphosite database (www.phosphosite.org) was collected in order to obtain the potential phosphosites subjected to regulation in the 27 causative RASopathy proteins. We compiled a dataset of dysregulated phosphosites in RASopathies, searched for commonalities between syndromes in harmonized data, and analyzed the role of phosphorylation in the syndromes by the identification of key players between the causative RASopathy proteins and the associated interactome. Results In this study, we provide a curated data set of 27 causative RASopathy genes, identify up to 511 protein–protein associations using pathway enrichment analysis/STRING tool, and identify 12 nodes as main hub proteins. We found that a large group of proteins contain tyrosine residues and their biological processes include but are not limited to the nervous system. Harmonizing published RASopathy phosphoproteomic and immunoblotting studies we identified a total of 147 phosphosites with increased phosphorylation, whereas 47 have reduced phosphorylation. The PKB signaling pathway is the most represented among the dysregulated phosphoproteins within the RASopathy proteins and their neighbors, followed by phosphoproteins implicated in the regulation of cell proliferation and the MAPK pathway. Conclusions This work illustrates the complex network underlying the RASopathies and the potential of phosphoproteomics for dissecting the molecular mechanisms in these syndromes. A combined study of associated genes, their interactome and phosphorylation events in RASopathies, elucidates key players and mechanisms to direct future research, diagnosis and therapeutic windows.

Changes of RAS Pathway Phosphorylation in Lymphoblastoid Cell Lines from Noonan Syndrome Patients Carrying Hypomorphic Variants in Two NS Genes

“…The number of large-scale mass spectrometry-based phosphoproteome studies has swelled over the past decade, initiating its application to RASopathies. However, only a few studies focused on dysregulated phosphoproteins and their biological implications [ 23 , 24 ]. Phosphoproteomics data in RASopathies are still scarce and their potential to decipher crucial signaling pathways involved in this family of disorders needs to be considered.…”

Non-Mammalian Models for Understanding Neurological Defects in RASopathies

“…The core of this cascade comprises the membrane-bound RAS-GTPase, the protein kinases RAF and MEK, and the transcription factor ERK (extracellular signal-related kinase), which control numerous cellular and physiological processes, including organism development, cell cycle control, cell proliferation and differentiation, cell survival, and death [1,2]. These disorders manifest as multisystem syndromes, including, but not limited to, neurofibromatosis type 1 (NF1), Noonan syndrome (NS), Legius syndrome (LS), Costello syndrome (CS), cardio-facio-cutaneous (CFC) syndrome and SYNGAP1 encephalopathy (SE) [3]. Most RASopathies are associated Biomedicines 2024, 12, 841 2 of 20 with an autosomal dominant mode of inheritance, one exception being Noonan syndrome, where recessive phenotypes caused by mutations in LZTR1 and SPRED2 have been described [4,5].…”