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Epichaperomes as a Gateway to Understanding, Diagnosing, and Treating Disease Through Rebalancing Protein-Protein Interaction Networks

Chander S. Digwal1, Sahil Sharma1, Anand R. Santhaseela1, Stephen D. Ginsberg2,3, and Gabriela Chiosis1,4

1Program in Chemical Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA

2Center for Dementia Research, Nathan Kline Institute, Orangeburg, NY, USA

3Departments of Psychiatry, Neuroscience & Physiology and the NYU Neuroscience Institute, New York University Grossman School of Medicine, New York, NY, USA

4Department of Medicine, Memorial Sloan Kettering Cancer Center and Weill Cornell Medical College, New York, NY, USA

1.1 Introduction

Proteostasis, or protein homeostasis, the process that regulates proteins within the cell, involves an extensive network of components that control protein biogenesis, folding, trafficking, and degradation [1]. In this context, proteostasis depends on the proper function of several cellular machineries, including molecular chaperones, autophagy regulators, ubiquitin-proteasomal system, and the unfolded protein response network. These work together to govern the fate of a protein from synthesis to degradation, and act to maintain proteins in the correct concentration, conformation, and subcellular location [2]. The impact that these machineries exert on proteostasis, as well as the decline of their function that results in proteostasis defects, has been studied and reviewed in great detail [1-5], illustrating their importance for proper cellular function, and in turn, disease biology.

Paradoxically, this widely used definition of proteostasis overlooks the fact that proteins rarely act alone. It is the interaction of each protein with other proteins and biomolecules that impacts and defines the function of each individual protein and its homeostasis, as well as the proteome as a whole [6-8]. Even though proteins have historically been examined in isolation, it is now clear that proteomes are defined by complex protein-protein interactions (PPIs) shaped by protein posttranscriptional modifications (PTMs), protein abundance, and other factors, most of which manifest in a cell-, tissue-, and disease-specific manner [9-11]. In this context, proteostasis changes that lead to disease represent perturbations of the underlying tissue- and cell-specific PPI networks arising from both internal and external stressors (e.g. genetic mutations, proteotoxic stress, aging, chemical or other environmental exposures, and/or lifestyle choices, among others) [11-13]. It is the severity of perturbation to the complex network of PPIs, i.e. interactomes, that reproducibly captures proteostasis alterations and thus define phenotypes [8, 14-16]. Therefore, understanding proteostasis requires a mindset that goes beyond biogenesis, folding, trafficking, and degradation of proteins, and incorporates the study of the interactome.

Herein, we provide a brief overview of how the discipline of chemical biology has introduced an additional enriching layer in our appreciation of proteostasis and, in the process, has reshaped our understanding of interactomes in disease. We start by providing a short historical overview on the discovery of epichaperomes, long-lived assemblies, and disease-associated pathologic scaffolds composed of tightly bound chaperones, co-chaperones, and other factors. We highlight our current knowledge of the effect epichaperomes have on reshaping PPIs at a proteome-wide level in cancer and neurodegenerative disorders. Finally, we discuss novel opportunities for drug discovery by detecting and rebalancing proteome-wide PPI defects through epichaperomes.

1.2 Epichaperomes Explored Through Chemical Biology

1.2.1 What are Epichaperomes?

The term "epichaperome" was coined by Rodina et al. in 2016 to describe multimeric, long-lived chaperone assemblies identified in cancer cells and primary tumor specimens [17]. These structures were discovered when the authors applied a variety of chemical biology tools, including heat-shock protein 90 (HSP90) inhibitor PU-H71 (Zelavespib) [18-20], and solid support immobilized [19-21] and fluorescently labeled [19, 20, 22] variants, Figure 1.1. These molecules were used in combination with chemoproteomics and analytic biochemical methods that retain native protein conformations and complexes to investigate factors that determine the sensitivity of tumors to HSP90-targeted therapy [17]. The authors found that the apoptotic response of cancer cells caused by PU-H71 was dependent on the presence and abundance of epichaperomes, but independent of levels of HSP90, other chaperones, and HSP90 client proteins (Figure 1.2a,b) [17, 23, 30]. These preclinical findings were later tested in the clinic in the context of metastatic breast cancer, demonstrating that patients with highest epichaperome levels in their tumors at baseline receive the greatest therapeutic benefit of PU-H71 therapy, as evidenced by longer time to progression [31].

Figure 1.1 Chemical structure of epichaperome chemical probes ranging from epichaperome disrupters to epichaperome detection and quantification probes to tools for affinity purification. Note a control chemical probe that contains the purine core but has little to no affinity for epichaperomes is also shown.

Independent of tissue of origin, tumor subtype, or genetic background, approximately 50-60% of tumors were found to express variable epichaperome levels, with ~10-15% being high expressors [12, 17]. Unlike individual chaperones (e.g. HSP90, heat shock cognate 70 (HSC70)) found at high levels throughout the body [32-35], epichaperomes are found specifically in cells exposed to chronic stressors, such as those that contribute to cancer and Alzheimer's disease (AD) [17, 24, 25, 32] (Figure 1.2a). Additionally, epichaperomes function as pathologic scaffolds, and cause thousands of proteins to improperly interact and organize inside cells [12] (Figure 1.2a). Thus, the higher the epichaperome levels, the higher the number of proteins being negatively impacted, i.e. the higher of number of aberrant PPIs [17, 24, 25, 36] (Figure 1.2a,b).

Figure 1.2 (a) Stressors associated with disease remodel interactomes through the switch of chaperones into epichaperomes, long-lived assemblies, and disease-associated pathologic scaffolds composed of tightly bound chaperones, co-chaperones, and other factors. Not to be confused with chaperones, ubiquitous proteins which fold and act through one-on-one dynamic complexes, epichaperomes act as pathologic scaffolds that form specifically in disease. They cause thousands of proteins to improperly interact and organize inside cells. Major chaperones such as HSP90 and HSC70 play a central role in the formation of epichaperome structures, yet these chaperones become functionally and biochemically distinct entities when part of epichaperomes. (b) The higher the epichaperome levels, the higher the number of proteins being negatively impacted, i.e. the higher of number of aberrant protein-protein interactions (PPIs), the more vulnerable the cell is to epichaperome therapy. (c) Epichaperome formation, constituency, and function are context dependent and shaped by stressors intrinsic to each pathologic phenotype.

The constituency of epichaperome scaffolding platforms is context dependent [13, 21, 23-29] (Figure 1.2c). For example, Rodina et al. discovered that HSP90 and HSC70 play a central role in the formation of epichaperome structures in tumors [17], where these structures dysregulate PPI networks to provide a survival advantage to cancer cells and tumor-supporting cells in the microenvironment [12, 17, 32]. Glucose-regulated protein 94 (GRP94), an endoplasmic reticulum (ER) HSP90 paralog, participates in epichaperomes in HER2- and EGFR-overexpressing breast cancers where it is found to be translocated to the plasma membrane and involved in rewiring signaling pathways [29, 37, 38]. Interestingly, HSC70 is also an epichaperome constituent along with HSP90 in AD brains where epichaperomes negatively impact the connectivity of proteins integral for synaptic plasticity and metabolic rewiring [25]. HSP60 becomes an epichaperome component in neurons exposed to mitochondrial toxins, such as rotenone, to produce defects in dopamine pathways relevant to understanding the pathogenesis of Parkinson's disease (PD), whereas HSC70 is co-opted in conditions of genetic stress (e.g. PARKIN mutation) to activate inflammatory pathways [24].

Although specific triggers for epichaperome formation are not understood, PTMs may play an important role...