085 | Cytosolic and mitochondrial chaperonins (CCT/TRIC and HSPD1/HSP60) in human dermal fibroblasts cells under environmental stressors: heat, oxidative and cold paradigms

Nima Shadan¹, Alessandra Maria Vitale¹, Federica Scalia^1|2, Rosario Barone¹ | ¹Department of Biomedicine, Neurosciences and Advanced Diagnostics BiND, University of Palermo, Italy; ²Department of Medicine and Surgery, Kore University of Enna, Italy.

Cellular stress disrupts protein folding homeostasis and challenges the capacity of chaperone systems to preserve proteome integrity across distinct intracellular compartments [1]. While molecular chaperones are well recognized as central regulators of stress adaptation, a direct comparison between cytosolic and mitochondrial chaperonin systems under different environmental stress conditions in human cells remains limited. In this study, a unified experimental framework was applied to human dermal fibroblasts (HDFs) to characterize the coordinated stress response of the cytosolic CCT/TRiC chaperonin complex, monitored through its CCT5 subunit, and the mitochondrial chaperonin HSPD1/HSP60 under acute heat, oxidative, and cold stress conditions. Cells were exposed to heat stress (44°C, 2h), oxidative stress (H₂O₂, 200 µM, 2h), and cold stress (4°C, 2h), using a harmonized control setup consisting of a 24h baseline control, a 48h time-matched control, and a 24h post-stress recovery, allowing strict cross-paradigm comparability. Protein-level modulation of CCT5 and HSP60 was quantified by Western blot (RIPA lysis, Bradford quantification, SDS-PAGE/PVDF transfer, ECL detection, ImageJ densitometry, GAPDH normalization), while transcriptional responses were assessed by RT-qPCR (TRIzol RNA extraction, NanoDrop QC, cDNA synthesis, SYBR Green amplification, 2⁻ΔΔCt analysis with GAPDH as reference). In parallel, immunofluorescence analyses were performed to capture stress-associated spatial remodeling (MitoTracker-based mitochondrial labeling, CCT5/HSP60 immunostaining, DAPI nuclear counterstaining, confocal microscopy under constant acquisition settings). For each stress paradigm and each analytical layer (WB, RT-qPCR, IF), data were derived from three independent biological replicates (n=3). Across all stress conditions, the integrated dataset supports a dual-compartment proteostasis model characterized by rapid chaperonin mobilization at the protein level, coupled to more temporary, stress-dependent transcriptional adjustments that only partially converge within the 24h recovery window. Notably, mitochondrial HSP60 displayed a broader dynamic range and more persistent elevation compared with cytosolic CCT5, consistent with the higher sensitivity of mitochondrial proteostasis to thermal and oxidative challenges [2]. Cold stress produced a milder response profile, indicating a distinct adaptive approach in which folding demand is modulated without extensive proteotoxic damage. Overall, these findings demonstrate that cytosolic and mitochondrial chaperonins are activated in a coordinated yet asymmetric manner during cellular stress, reflecting compartment-specific priorities within a shared proteostasis network (1,2). This integrated cellular stress model provides a robust framework for interpreting how human cells balance folding capacity, energy allocation, and recovery dynamics under diverse environmental challenges and offers a molecular baseline for future studies aimed at targeted modulation of stress-responsive chaperone systems.