RESUMO
Post-translational modifications such as protein N-glycosylation, significantly influence cellular processes. Dysregulated N-glycosylation, exemplified in Grp94, a member of the Hsp90 family, leads to structural changes and the formation of epichaperomes, contributing to pathologies. Targeting N-glycosylation-induced conformations offers opportunities for developing selective chemical tools and drugs for these pathologic forms of chaperones. We here demonstrate how a specific Grp94 conformation induced by N-glycosylation, identified previously via molecular dynamics simulations, rationalizes the distinct behavior of similar ligands. Integrating dynamic ligand unbinding information with SAR development, we differentiate ligands productively engaging the pathologic Grp94 conformers from those that are not. Additionally, analyzing binding site stereoelectronic properties and QSAR models using cytotoxicity data unveils relationships between chemical, conformational properties, and biological activities. These findings facilitate the design of ligands targeting specific Grp94 conformations induced by abnormal glycosylation, selectively disrupting pathogenic protein networks while sparing normal mechanisms.
RESUMO
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.
RESUMO
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.