The interaction network of the proteasome assembly chaperone PSMD9 regulates proteostasis.

Functional networks in cells are created by physical, genetic, and regulatory interactions. Mapping them and annotating their functions by available methods remains a challenge. We use affinity purification mass spectrometry (AP-MS) coupled with SLiMFinder to discern such a network involving 26S proteasome non-ATPase regulatory subunit 9 (PSMD9), a chaperone of proteasome assembly. Approximately 20% of proteins within the PSMD9 interactome carry a short linear motif (SLiM) of the type 'EXKK'. The binding of purified PSMD9 with the peptide sequence ERKK, proteins heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNPA2B1; containing ERKK), and peroxiredoxin-6 (PRDX6; containing EAKK) provided proof of principle for this motif-driven network. The EXKK motif in the peptide primarily interacts with the coiled-coil N domain of PSMD9, a unique interaction not reported for any coiled-coil domain. PSMD9 knockout (KO) HEK293 cells experience endoplasmic reticulum (ER) stress and respond by increasing the unfolded protein response (UPR) and reducing the formation of aggresomes and lipid droplets. Trans-expression of PSMD9 in the KO cells rescues lipid droplet formation. Overexpression of PSMD9 in HEK293 cells results in reduced UPR, and increased lipid droplet and aggresome formation. The outcome argues for the prominent role of PSMD9 in maintaining proteostasis. Probable mechanisms involve the binding of PSMD9 to binding immunoglobulin protein (BIP/GRP78; containing EDKK), an endoplasmic reticulum chaperone and key regulator of the UPR, and fatty acid synthase (FASN; containing ELKK), involved in fatty acid synthesis/lipid biogenesis. We propose that PSMD9 acts as a buffer in the cellular milieu by moderating the UPR and enhancing aggresome formation to reduce stress-induced proteotoxicity. Akin to waves created in ponds that perpetuate to a distance, perturbing the levels of PSMD9 would cause ripples down the networks, affecting final reactions in the pathway, one of which is altered proteostasis.

PSMD9 ribosomal protein network maintains nucleolar architecture and WT p53 levels.

Capitalizing on an unexpected observation that multiple free ribosomal proteins co-purify/pull-down with PSMD9, we report here for the first time that PSMD9 is necessary to maintain the morphology and integrity of the nucleolus. As seen by NPM1 immunofluorescence and electron microscopy, the nucleolar structure is clearly disrupted in PSMD9 null MCF7 breast cancer cells. The resultant stress is pronounced leading to the accumulation of WT p53 and slow growth. A dual insult with Actinomycin D exasperates the nucleolar stress in these cells which fail to recover in stipulated time. This double insult in the WT cells enhances the interaction of PSMD9 with ribosomal subunits. Our data also reveals that in PSMD9 null cells, ribosomal proteins RPS25 and RPL15 fail to localise in the nucleolus. We speculate that the interaction of PSMD9 with multiple free ribosome subunits has at least two important implications: a) PSMD9 plays a role in trafficking of ribosomal proteins into the nucleolus, therefore contributing to the maintenance of structural and morphological organization of the membrane-less nucleolar compartment; b) under conditions that induce nucleolar stress, PSMD9-Ribosomal Protein interaction protects WT MCF7 breast cancer cells from slow growth and eventual death. This possibility renders the domains of PSMD9 to be attractive drug targets in the context of cancer and other multiple ribosome-associated disorders.

Enhanced proteasomal activity is essential for long term survival and recurrence of innately radiation resistant residual glioblastoma cells.

Therapy resistance and recurrence in Glioblastoma is due to the presence of residual radiation resistant cells. However, because of their inaccessibility from patient biopsies, the molecular mechanisms driving their survival remain unexplored. Residual Radiation Resistant (RR) and Relapse (R) cells were captured using cellular radiation resistant model generated from patient derived primary cultures and cell lines. iTRAQ based quantitative proteomics was performed to identify pathways unique to RR cells followed by in vitro and in vivo experiments showing their role in radio-resistance. 2720 proteins were identified across Parent (P), RR and R population with 824 and 874 differential proteins in RR and R cells. Unsupervised clustering showed proteasome pathway as the most significantly deregulated pathway in RR cells. Concordantly, the RR cells displayed enhanced expression and activity of proteasome subunits, which triggered NFkB signalling. Pharmacological inhibition of proteasome activity led to impeded NFkB transcriptional activity, radio-sensitization of RR cells in vitro, and significantly reduced capacity to form orthotopic tumours in vivo. We demonstrate that combination of proteasome inhibitor with radio-therapy abolish the inaccessible residual resistant cells thereby preventing GBM recurrence. Furthermore, we identified first proteomic signature of RR cells that can be exploited for GBM therapeutics.