Systems-level analyses of protein-protein interaction network dysfunctions via epichaperomics identify cancer-specific mechanisms of stress adaptation

Abstract

Systems-level assessments of protein-protein interaction (PPI) network dysfunctions are currently out-of-reach because approaches enabling proteome-wide identification, analysis, and modulation of context-specific PPI changes in native (unengineered) cells and tissues are lacking. Herein, we take advantage of chemical binders of maladaptive scaffolding structures termed epichaperomes and develop an epichaperome-based ‘omics platform, epichaperomics, to identify PPI alterations in disease. We provide multiple lines of evidence, at both biochemical and functional levels, demonstrating the importance of these probes to identify and study PPI network dysfunctions and provide mechanistically and therapeutically relevant proteome-wide insights. As proof-of-principle, we derive systems-level insight into PPI dysfunctions of cancer cells which enabled the discovery of a context-dependent mechanism by which cancer cells enhance the fitness of mitotic protein networks. Importantly, our systems levels analyses support the use of epichaperome chemical binders as therapeutic strategies aimed at normalizing PPI networks.

Phosphorylation-Driven Epichaperome Assembly: A Critical Regulator of Cellular Adaptability and Proliferation

“…In summary, the predominant HSP90 band characteristic of epichaperomes is a 300 kDa assembly, distinctly differing from the typical ~242 kDa band observed in non-transformed cells 13,19,32 when analyzed on native PAGE gels. Mass spectrometric (MS) analysis of the ~300 kDa assembly confirmed the presence of HSP90 and HSC70 as the primary protein components of this multimeric complex (Supplementary Data 1, 300 kDa LC-MS).…”

“…Epichaperomes nucleated through enhanced interactions between HSP90 and HSP70, namely the heat shock cognate 70 (HSC70) isoform, are a distinct feature of cancer cells 13,19 . Epichaperomes containing HSP90 are detected in iPSCs (induced pluripotent stem cells) 20 , in leukemia stem cells 21,22 and in glioma cancer stem cells (CSCs) 23 .…”

“…1a-d and refs. 13,19,20 ). Notably, HSP90 immunoblotting revealed the presence of species comprising HSP90 in epichaperome assemblies in cancer cells and pluripotent stem cells, in addition to the prominent 242 kDa band, which is a characteristic of non-transformed cells 13,19,29 .…”

“…Subsequently, we labeled these homogenates with a clickable PU-probe (PU-TCO, ref. 19). After running these labeled samples on native PAGE gels, we conjugated the PU-probe with a Cy5 dye and visualized epichaperomes, confirming the presence of epichaperomes in both the ESCs and the cancer cells.…”

“…Unlike chaperones which help proteins fold or assemble, epichaperomes exert a maladaptive influence, reshaping the assembly and connectivity of proteins pivotal for sustaining pathological traits. For example, in cancer, epichaperomes take on scaffolding functions not found in normal cells, altering the assembly and connectivity of proteins important for maintaining a malignant phenotype and enhancing their activity, which provides a survival advantage to cancer cells and tumor-supporting cells 13,19 . In Alzheimer's disease epichaperomes rewire the connectivity of, and thus negatively impact, proteins integral for synaptic plasticity, brain energetics and immune response 15 .…”