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.

Evolution of biophysical tools for quantitative protein interactions and drug discovery.

With millions of signalling events occurring simultaneously, cells process a continuous flux of information. The genesis, processing, and regulation of information are dictated by a huge network of protein interactions. This is proven by the fact that alterations in the levels of proteins, single amino acid changes, post-translational modifications, protein products arising out of gene fusions alter the interaction landscape leading to diseases such as congenital disorders, deleterious syndromes like cancer, and crippling diseases like the neurodegenerative disorders which are often fatal. Needless to say, there is an immense effort to understand the biophysical basis of such direct interactions between any two proteins, the structure, domains, and sequence motifs involved in tethering them, their spatio-temporal regulation in cells, the structure of the network, and their eventual manipulation for intervention in diseases. In this chapter, we will deliberate on a few techniques that allow us to dissect the thermodynamic and kinetic aspects of protein interaction, how innovation has rendered some of the traditional techniques applicable for rapid analysis of multiple samples using small amounts of material. These advances coupled with automation are catching up with the genome-wide or proteome-wide studies aimed at identifying new therapeutic targets. The chapter will also summarize how some of these techniques are suited either in the standalone mode or in combination with other biophysical techniques for the drug discovery process.

Novel nucleolar localization of clusterin and its associated functions in human oral cancers: An in vitro and in silico analysis.

Clusterin (CLU), a multifunctional chaperonic glycoprotein associated with diverse cellular functions has been shown to act as an oncogene or tumour suppressor gene in different cancers, implying a dual role in tumorigenesis. Here, we investigated the expression of CLU isoforms, their subcellular localization and functional significance in oral cancer cells. Significant downregulation of secretory CLU (sCLU) transcripts was observed in oral cancer cell lines and tumours versus normal cells while the nuclear CLU (nCLU) transcripts were undetectable. We demonstrated for the first time the nucleolar localization of sCLU, its response to different nucleolar stresses and association with cajal bodies post nucleolar stress. Functionally, knockdown of CLU revealed its negative association with ribosome biogenesis implying a possible tumour suppressor like role in oral cancers. Further, loss of sCLU in these cells also resulted in altered nuclear morphology and shrunken tubulin filaments. In addition, the levels of nucleolar Nucleophosmin 1(NPM1) and Fibrillarin, known to regulate nuclear morphology were downregulated indicating a possible role of sCLU in their stabilization. Further, an in silico docking approach to gain insights into the interaction of sCLU with nucleolar proteins NPM1, Fibrillarin, UBF and Nucleolin, revealed the involvement of a conserved region comprising of amino acid residues 140-155 of sCLU ß-chain, specifically via the Phe152 residue in hydrophobic interactions with these client nucleolar proteins indicating a possible stabilizing or regulatory role of sCLU. SIGNIFICANCE OF THE STUDY: This is the first study to demonstrate the nucleolar localization of sCLU and its associated functions in oral cancer cells. Downregulation of sCLU in oral cancer tissues and cell lines, and its negative association with ribogenesis suggest its tumour suppressor like role in oral cancers. The possible role of sCLU in stabilization or regulation of different nucleolar proteins thereby impacting their functions is also implicated.

A Novel Determinant of PSMD9 PDZ Binding Guides the Evolution of the First Generation of Super Binding Peptides.

The PDZ domain is one of the most widespread protein interaction domains found in nature. Due to their integral role in numerous biological functions, their ability to act as scaffolds for signal amplification, and the occurrence of mutations linked to human diseases, PDZ domains are attractive therapeutic targets. On the basis of the differential binding affinities of selected C-terminal peptides of the human proteome for one such PDZ domain (PSMD9) and by exploring structure-activity relationships, we design and convert a low-affinity tetrapeptide (∼439 µM) to a tight binding sequence (∼5 µM). The peptide inhibits PSMD9-hnRNPA1 interactions that are critical in basal and stimulus-induced NF-κB signaling and a potential therapeutic target in cancers, including chemotherapy or radiation-induced therapy resistance. Extensive application of computer modeling, including ligand mapping and all-atom molecular dynamics simulations, helps us to rationalize the structural basis for the huge differences in binding affinity and inform us about the residue-wise contributions to the binding energy. Our findings are in accord with the classical preference of the (PSMD9) PDZ domain for C-terminal sequences that contain hydrophobic residues at the P0 (C-terminal) position. In addition, for the first time, we identify a hitherto unknown occupancy for cysteine at the P-2 position that drives high-affinity interaction in a PDZ domain.