Comprehending Meningioma Signaling Cascades Using Multipronged Proteomics Approaches & Targeted Validation of Potential Markers.

Meningiomas are one of the most prevalent primary brain tumors. Our study aims to obtain mechanistic insights of meningioma pathobiology using mass spectrometry-based label-free quantitative proteome analysis to identifying druggable targets and perturbed pathways for therapeutic intervention. Label-free based proteomics study was done from peptide samples of 21 patients and 8 non-tumor controls which were followed up with Phosphoproteomics to identify the kinases and phosphorylated components of the perturbed pathways. In silico approaches revealed perturbations in extracellular matrix remodeling and associated cascades. To assess the extent of influence of Integrin and PI3K-Akt pathways, we used an Integrin Linked Kinase inhibitor on patient-derived meningioma cell line and performed a transcriptomic analysis of the components. Furthermore, we designed a Targeted proteomics assay which to the best of our knowledge for very first-time enables identification of peptides from 54 meningioma patients via SRM assay to validate the key proteins emerging from our study. This resulted in the identification of peptides from CLIC1, ES8L2, and AHNK many of which are receptors and kinases and are difficult to be characterized using conventional approaches. Furthermore, we were also able to monitor transitions for proteins like NEK9 and CKAP4 which have been reported to be associated with meningioma pathobiology. We believe, this study can aid in designing peptide-based validation assays for meningioma patients as well as IHC studies for clinical applications.

A novel role for the proteasomal chaperone PSMD9 and hnRNPA1 in enhancing IκBα degradation and NF-κB activation - functional relevance of predicted PDZ domain-motif interaction.

PSMD9 is a PDZ domain containing chaperone of proteasome assembly. Based on the ability of PDZ-like domains to recognize C-terminal residues in their interactors, we recently predicted and identified heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) as one of the novel interacting partners of PSMD9. Contingent on the reported role of hnRNPA1 in nuclear factor κB (NF-κB) activation, we tested the role of human PSMD9 and hnRNPA1 in NF-κB signaling. We demonstrated in human embryonic kidney 293 cells that PSMD9 influences both basal and tumor necrosis factor α (TNF-α) mediated NF-κB activation through inhibitor of nuclear factor κB α (IκBα) proteasomal degradation. PSMD9 mediates IκBα degradation through a specific domain-motif interaction involving its PDZ domain and a short linear sequence motif in the C-terminus of hnRNPA1. Point mutations in the PDZ domain or deletion of C-terminal residues in hnRNPA1 disrupt interaction between the two proteins which has a direct influence on NF-κB activity. hnRNPA1 interacts with IκBα directly, whereas PSMD9 interacts only through hnRNPA1. Furthermore, hnRNPA1 shows increased association with the proteasome upon TNF-α treatment which has no such effect in the absence of PSMD9. On the other hand endogenous and trans-expressed PSMD9 are found associated with the proteasome complex. This association is unaffected by PDZ mutations or TNF-α treatment. Collectively, these interactions between IκBα, hnRNPA1 and proteasome bound PSMD9 illustrate a potential mechanism by which ubiquitinated IκBα is recruited on the proteasome for degradation. In this process, hnRNPA1 may act as a shuttle receptor and PSMD9 as a subunit acceptor. The interaction sites of PSMD9 and hnRNPA1 may emerge as a vulnerable drug target in cancer cells which require consistent NF-κB activity for survival.