mapping-dysfunctional-protein-protein-interactions-in-disease

Mapping Dysfunctional Protein-Protein Interactions in Disease

Protein-protein interaction (PPI) networks are dynamically remodeled in disease, yet most systems biology approaches focus on changes in protein ...

Memorial Sloan Kettering Cancer Center

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.

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

Epichaperomes are disease-associated pathologic scaffolds, composed of tightly bound chaperones, co-chaperones, and other factors. They mediate anomalous protein-protein interactions inside cells, which aberrantly affects the function of protein networks, and in turn, cellular phenotypes. Epichaperome study necessitates the implementation of methods that retain these protein complexes in their native cellular states for analysis. Here we describe a protocol for detection and composition analysis of epichaperomes in cell homogenates through native polyacrylamide gel electrophoresis.

Use of Native-PAGE for the Identification of Epichaperomes in Cell Lines.

Glycosylation, a prevalent post-translational modification, plays a pivotal role in regulating intricate cellular processes by covalently attaching glycans to macromolecules. Dysregulated glycosylation is linked to a spectrum of diseases, encompassing cancer, neurodegenerative disorders, congenital disorders, infections, and inflammation. This review delves into the intricate interplay between glycosylation and protein conformation, with a specific focus on the profound impact of N-glycans on the selection of distinct protein conformations characterized by distinct interactomes-namely, protein assemblies-under normal and pathological conditions across various diseases. We begin by examining the spike protein of the SARS virus, illustrating how N-glycans regulate the infectivity of pathogenic agents. Subsequently, we utilize the prion protein and the chaperone glucose-regulated protein 94 as examples, exploring instances where N-glycosylation transforms physiological protein structures into disease-associated forms. Unraveling these connections provides valuable insights into potential therapeutic avenues and a deeper comprehension of the molecular intricacies that underlie disease conditions. This exploration of glycosylation's influence on protein conformation effectively bridges the gap between the glycome and disease, offering a comprehensive perspective on the therapeutic implications of targeting conformational mutants and their pathologic assemblies in various diseases. The goal is to unravel the nuances of these post-translational modifications, shedding light on how they contribute to the intricate interplay between protein conformation, assembly, and disease.

N-Glycosylation as a Modulator of Protein Conformation and Assembly in Disease.

The intricate protein-chaperone network is vital for cellular function. Recent discoveries have unveiled the existence of specialized chaperone complexes called epichaperomes, protein assemblies orchestrating the reconfiguration of protein-protein interaction networks, enhancing cellular adaptability and proliferation. This study delves into the structural and regulatory aspects of epichaperomes, with a particular emphasis on the significance of post-translational modifications in shaping their formation and function. A central finding of this investigation is the identification of specific PTMs on HSP90, particularly at residues Ser226 and Ser255 situated within an intrinsically disordered region, as critical determinants in epichaperome assembly. Our data demonstrate that the phosphorylation of these serine residues enhances HSP90's interaction with other chaperones and co-chaperones, creating a microenvironment conducive to epichaperome formation. Furthermore, this study establishes a direct link between epichaperome function and cellular physiology, especially in contexts where robust proliferation and adaptive behavior are essential, such as cancer and stem cell maintenance. These findings not only provide mechanistic insights but also hold promise for the development of novel therapeutic strategies targeting chaperone complexes in diseases characterized by epichaperome dysregulation, bridging the gap between fundamental research and precision medicine.

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

Neurodegenerative disorders, including Alzheimer's disease (AD) and Parkinson's disease (PD), represent debilitating conditions with complex, poorly understood pathologies. Epichaperomes, pathologic protein assemblies nucleated on key chaperones, have emerged as critical players in the molecular dysfunction underlying these disorders. In this study, we introduce the synthesis and characterization of clickable epichaperome probes, PU-TCO, positive control, and PU-NTCO, negative control. Through comprehensive in vitro assays and cell-based investigations, we establish the specificity of the PU-TCO probe for epichaperomes. Furthermore, we demonstrate the efficacy of PU-TCO in detecting epichaperomes in brain tissue with a cellular resolution, underscoring its potential as a valuable tool for dissecting single-cell responses in neurodegenerative diseases. This clickable probe is therefore poised to address a critical need in the field, offering unprecedented precision and versatility in studying epichaperomes and opening avenues for novel insights into their role in disease pathology.

Synthesis and Characterization of Click Chemical Probes for Single-Cell Resolution Detection of Epichaperomes in Neurodegenerative Disorders.

Most of the human cancers are dependent on telomerase to extend the telomeres. But ∼10% of all cancers use a telomerase-independent, homologous recombination mediated pathway called alternative lengthening of telomeres (ALT). Due to the poor prognosis, ALT status is not being considered yet in the diagnosis of cancer. No such specific treatment is available to date for ALT positive cancers. ALT positive cancers are dependent on replication stress to deploy DNA repair pathways to the telomeres to execute homologous recombination mediated telomere extension. SMARCAL1 (SWI/SNF related, matrix-associated, actin-dependent regulator of chromatin, subfamily A-like 1) is associated with the ALT telomeres to resolve replication stress thus providing telomere stability. Thus, the dependency on replication stress regulatory factors like SMARCAL1 made it a suitable therapeutic target for the treatment of ALT positive cancers. In this study, we found a significant downregulation of SMARCAL1 expression by stabilizing the G-quadruplex (G4) motif found in the promoter of  by potent G4 stabilizers, like TMPyP4 and BRACO-19. SMARCAL1 downregulation led toward the increased localization of PML (promyelocytic leukemia) bodies in ALT telomeres and triggered the formation of APBs (ALT-associated promyelocytic leukemia bodies) in ALT positive cell lines, increasing telomere replication stress and DNA damage at a genomic level. Induction of replication stress and hyper-recombinogenic phenotype in ALT positive cells mediated by G4 stabilizing molecules already highlighted their possible application as a new therapeutic window to target ALT positive tumors. In accordance with this, our study will also provide a valuable insight toward the development of G4-based ALT therapeutics targeting 

ALTering Cancer by Triggering Telomere Replication Stress through the Stabilization of Promoter G-Quadruplex in .

Protein-protein interactions (PPIs) play a crucial role in cellular function and disease manifestation, with dysfunctions in PPI networks providing a direct link between stressors and phenotype. The dysfunctional Protein-Protein Interactome (dfPPI) platform, formerly known as epichaperomics, is a newly developed chemoproteomic method aimed at detecting dynamic changes at the systems level in PPI networks under stressor-induced cellular perturbations within disease states. This review provides an overview of dfPPIs, emphasizing the novel methodology, data analytics, and applications in disease research. dfPPI has applications in cancer research, where it identifies dysfunctions integral to maintaining malignant phenotypes and discovers strategies to enhance the efficacy of current therapies. In neurodegenerative disorders, dfPPI uncovers critical dysfunctions in cellular processes and stressor-specific vulnerabilities. Challenges, including data complexity and the potential for integration with other omics datasets are discussed. The dfPPI platform is a potent tool for dissecting disease systems biology by directly informing on dysfunctions in PPI networks and holds promise for advancing disease identification and therapeutics.

Introducing dysfunctional Protein-Protein Interactome (dfPPI) - A platform for systems-level protein-protein interaction (PPI) dysfunction investigation in disease.

The intricate network of protein-chaperone interactions is crucial for maintaining cellular function. Recent discoveries have unveiled the existence of specialized chaperone assemblies, known as epichaperomes, which serve as scaffolding platforms that orchestrate the reconfiguration of protein-protein interaction networks, thereby enhancing cellular adaptability and proliferation. This study explores the structural and regulatory aspects of epichaperomes, with a particular focus on the role of post-translational modifications (PTMs) in their formation and function. A key finding is the identification of specific PTMs on HSP90, particularly at residues Ser226 and Ser255 within an intrinsically disordered region, as critical determinants of epichaperome assembly. Our data demonstrate that phosphorylation of these serine residues enhances HSP90's interactions with other chaperones and co-chaperones, creating a microenvironment conducive to epichaperome formation. Moreover, we establish a direct link between epichaperome function and cellular physiology, particularly in contexts where robust proliferation and adaptive behavior are essential, such as in cancer and pluripotent stem cell maintenance. These findings not only provide mechanistic insights but also hold promise for the development of novel therapeutic strategies targeting chaperone assemblies in diseases characterized by epichaperome dysregulation, thereby bridging the gap between fundamental research and precision medicine.

Phosphorylation-driven epichaperome assembly is a regulator of cellular adaptability and proliferation.

Vascular endothelial growth factor-A (VEGF-A) is a growth factor and pluripotent cytokine that promotes angiogenesis in cancer cells, transitioning to an angiogenic phenotype. The binding of VEGF-A protein to VEGF receptors (VEGFR-1 and VEGFR-2) initiates a cascade of events that stimulates angiogenesis by facilitating the migration and enhancing the permeability of endothelial cells. The proximal promoter of the VEGF gene encompasses a 36-base pair region (from -85 to -50) that can form a stable G-quadruplex (G4) structure in specific conditions. The activity of the VEGF promoter is reliant on this structure. During cancer progression, the VEGF-A G4 succumbs to cellular pressure and fails to maintain a stable structure. This shifts the balance to form a duplex structure, increasing the transcription rate. Earlier research has tried to develop small-molecule ligands to target and stabilise G4, demonstrating the possibility of suppressing VEGF expression. However, they either lack specificity or toxic. Peptides, on the other hand, are significantly less studied as G4 binders. Here, we designed a peptide that successfully binds and stabilises the VEGF-A G4 while reducing its gene expression. This further alters the expression fate of the VEGF-A signalling cascade and blocks angiogenesis in cancer cells. We employed high-resolution nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulation to elucidate the chemical details of G4-peptide interaction. In addition, we used qPCR and western blot techniques to investigate the expression pattern of the molecules implicated in the VEGF-A signalling cascade. The study explores the intricate relationship between peptides and quadruplex structures, revealing valuable insights that can improve the design of pharmacophores targeting the dynamic quadruplex structure. The results of our study are encouraging, opening possibilities for advancements in, the characterisation and optimisation of peptides as G-quadruplex ligands in view of their potential therapeutic uses.

In Silico-Designed G-Quadruplex Targeting Peptide Attenuates VEGF-A Expression, Preventing Angiogenesis in Cancer Cells.

Alzheimer's disease (AD) progresses as a continuum, from preclinical stages to late-stage cognitive decline, yet the molecular mechanisms driving this progression remain poorly understood. Here, we provide a systems-level map of protein-protein interaction (PPI) network dysfunction across the AD spectrum and uncover epichaperomes-stable scaffolding platforms formed by chaperones and co-factors-as central drivers of this process. Using over 100 human brain specimens, mouse models, and human neurons, we show that epichaperomes emerge early, even in preclinical AD, and progressively disrupt multiple PPI networks critical for synaptic function and neuroplasticity. Glutamatergic neurons, essential for learning and memory, exhibit heightened vulnerability, with their dysfunction driven by protein sequestration into epichaperome scaffolds, independent of changes in protein expression. Notably, pharmacological disruption of epichaperomes with PU-AD restores PPI network integrity and reverses synaptic and cognitive deficits, directly linking epichaperome-driven network dysfunction to AD pathology. These findings establish epichaperomes as key mediators of molecular collapse in AD and identify network-centric intervention strategies as a promising avenue for disease-modifying therapies.

Article	Year
Systems-level analyses of protein-protein interaction network dysfunctions via epichaperomics identify cancer-specific mechanisms of stress adaptation. Nature communications\| PubMed ID: 37353488	2023
Use of Native-PAGE for the Identification of Epichaperomes in Cell Lines. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 37540435	2023
N-Glycosylation as a Modulator of Protein Conformation and Assembly in Disease. Biomolecules\| PubMed ID: 38540703	2024
Phosphorylation-Driven Epichaperome Assembly: A Critical Regulator of Cellular Adaptability and Proliferation. Research square\| PubMed ID: 38645031	2024
Synthesis and Characterization of Click Chemical Probes for Single-Cell Resolution Detection of Epichaperomes in Neurodegenerative Disorders. Biomedicines\| PubMed ID: 38927459	2024
ALTering Cancer by Triggering Telomere Replication Stress through the Stabilization of Promoter G-Quadruplex in . ACS chemical biology\| PubMed ID: 38959478	2024
Introducing dysfunctional Protein-Protein Interactome (dfPPI) - A platform for systems-level protein-protein interaction (PPI) dysfunction investigation in disease. Current opinion in structural biology\| PubMed ID: 39003916	2024
Phosphorylation-driven epichaperome assembly is a regulator of cellular adaptability and proliferation. Nature communications\| PubMed ID: 39414766	2024
In Silico-Designed G-Quadruplex Targeting Peptide Attenuates VEGF-A Expression, Preventing Angiogenesis in Cancer Cells. Chemical biology & drug design\| PubMed ID: 39704035	2024
Systems-Level Interactome Mapping Reveals Actionable Protein Network Dysregulation Across the Alzheimer's Disease Spectrum. Research square\| PubMed ID: 39989971	2025

Tanaya Roychowdhury

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