Abstract
The molecular chaperone Hsp90-dependent proteome represents a complex protein network of critical biological and medical relevance. Known to associate with proteins with a broad variety of functions termed clients, Hsp90 maintains key essential and oncogenic signalling pathways. Consequently, Hsp90 inhibitors are being tested as anti-cancer drugs. Using an integrated systematic approach to analyse the effects of Hsp90 inhibition in T-cells, we quantified differential changes in the Hsp90-dependent proteome, Hsp90 interactome, and a selection of the transcriptome. Kinetic behaviours in the Hsp90-dependent proteome were assessed using a novel pulse-chase strategy (Fierro-Monti et al., accompanying article), detecting effects on both protein stability and synthesis. Global and specific dynamic impacts, including proteostatic responses, are due to direct inhibition of Hsp90 as well as indirect effects. As a result, a decrease was detected in most proteins that changed their levels, including known Hsp90 clients. Most likely, consequences of the role of Hsp90 in gene expression determined a global reduction in net de novo protein synthesis. This decrease appeared to be greater in magnitude than a concomitantly observed global increase in protein decay rates. Several novel putative Hsp90 clients were validated, and interestingly, protein families with critical functions, particularly the Hsp90 family and cofactors themselves as well as protein kinases, displayed strongly increased decay rates due to Hsp90 inhibitor treatment. Remarkably, an upsurge in survival pathways, involving molecular chaperones and several oncoproteins, and decreased levels of some tumour suppressors, have implications for anti-cancer therapy with Hsp90 inhibitors. The diversity of global effects may represent a paradigm of mechanisms that are operating to shield cells from proteotoxic stress, by promoting pro-survival and anti-proliferative functions. Data are available via ProteomeXchange with identifier PXD000537.
Highlights
Molecular chaperones are central to cellular proteostasis
Data from standard SILAC (stSILAC) were integrated with protein-protein interactions to build a network and with synthesis and decay rates from pulse-chase SILAC (pcSILAC) and mRNA data for a multi-parameter analysis of the system (Figure 1B)
To correlate changes in net protein levels with changes in synthesis and decay, we considered 12 protein categories defined by Gene Ontology (GO) annotation terms enriched in the four main clusters 2,6,7, or 11 of the stSILAC dataset, together with 4 other categories of interest identified from the network analysis
Summary
Molecular chaperones are central to cellular proteostasis. They are closely involved in essential biological processes such as translation, folding, complex assembly and disassembly, translocation across membranes and protein degradation [1,2]. Hsp inhibitors, such as geldanamycin (GA) are competitive inhibitors of ATP-binding These inhibit chaperone function, and as a consequence, they may exert anti-tumour activity by decreasing the levels of oncogenic clients [12,13,14]. Recent efforts have been directed to identify and to quantify the portion of the proteome that is dependent on Hsp, most commonly using standard SILAC (Stable Isotope Labelling by Amino acids in cell Culture, stSILAC)-based quantitative proteomics [11,16,17,18,19] Results from these and previous studies using different proteomic approaches have improved our understanding of the role of Hsp in cancer, as well as a target of promising anticancer drugs [20]. Our analysis of Hsp inhibition allowed an integrated assessment of the dynamics of the T-cell Hsp90-dependent proteome, giving further insights on the mechanism of action of an inhibitor of Hsp
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