Human Hsp70-escort protein 1 (hHep1) is a critical cochaperone responsible for maintaining the solubility and functional integrity of mitochondrial Hsp70 (HSPA9), also known as mortalin. Under thermal stress, HSPA9 and its cytoplasmic counterpart HSPA1A readily undergo self-assembly into supramolecular aggregates (SMA), which can disrupt proteostasis and contribute to pathological conditions such as neurodegeneration and cancer. This study provides comprehensive insights into how hHep1 modulates the structural dynamics and functional activity of these HSPAs during stress-induced aggregation.
Using light scattering assays, we demonstrated that hHep1 effectively inhibits the formation of thermally induced SMA in both HSPA9 and HSPA1A in a dose-dependent manner. At a molar ratio of 1:6, hHep1 suppressed HSPA9 aggregation by 90%, while a higher ratio of 1:16 was required for 65% inhibition of HSPA1A. These results highlight hHep1’s superior efficacy against HSPA9, consistent with its known role in mitochondrial maintenance. Additionally, hHep1 displayed intrinsic chaperone activity by preventing thermal denaturation of model substrates—malate dehydrogenase (MDH) and luciferase—with maximal protection reaching 85%. Notably, it failed to inhibit citrate synthase aggregation, underscoring substrate selectivity and suggesting a specific recognition mechanism.
Functional analysis revealed that hHep1 significantly enhances ATP hydrolysis rates in both monomeric and preformed SMA forms of HSPA9 and HSPA1A. For HSPA9, ATPase activity increased up to 330% in monomers and 170% in SMA, while HSPA1A showed 150% and 220% stimulation, respectively. Kinetic modeling using Michaelis-Menten analysis indicated no change in KM values but a substantial increase in Vmax and kcat, confirming that hHep1 acts catalytically by lowering the activation barrier for ATP hydrolysis rather than altering substrate binding affinity. The maximum stimulation occurred at ~20 μM hHep1, indicating saturation of functional sites.
Isothermal titration calorimetry (ITC) confirmed direct, high-affinity interactions between hHep1 and both monomeric and aggregated forms of HSPA9 and HSPA1A. The binding stoichiometry approached 1:1 in all cases, suggesting that each hHep1 molecule engages a single protomer within the assembly. The dissociation constants (KD) were 0.9 μM for HSPA9 monomer, 1.4 μM for HSPA9 SMA, and 2.4 μM for HSPA1A SMA. Thermodynamic profiling indicated that interactions with SMA complexes are predominantly enthalpically driven, likely due to strong electrostatic and hydrogen-bonding networks formed upon complexation, whereas monomer interactions involve favorable entropy changes from dehydration at the interface.
Transmission electron microscopy (TEM) with negative staining provided visual evidence of hHep1-mediated remodeling. Pre-formed HSPA9 and HSPA1A SMAs exhibited heterogeneous particle sizes ranging from 200 to 20,000 nm². Upon incubation with hHep1 (20-fold excess), particle size distribution narrowed dramatically, with average areas reduced to approximately 90 nm² for HSPA9 and ~800 nm² for HSPA1A. Particle morphology became more uniform, indicating disaggregation and reorganization into smaller, stable units. Control samples without hHep1 retained large, irregular structures.
Filter retardation assays further validated these findings. SMA samples were retained on cellulose acetate membranes due to their high molecular weight and size. However, increasing concentrations of hHep1 progressively displaced these aggregates, resulting in dose-dependent membrane clearance. EC50 values were determined at 110 nM for HSPA9 SMA and 160 nM for HSPA1A SMA, reinforcing the higher affinity and specificity of hHep1 for HSPA9. In contrast, lysozyme and luciferase aggregates remained unaffected, confirming that hHep1’s remodeling activity is specific to Hsp70 assemblies.
Subcellular localization studies using confocal microscopy revealed that hHep1 localizes in mitochondria and nucleus when overexpressed in U2OS cells. Nuclear accumulation occurred in distinct dot-like foci, which were absent in endogenous hHep1, suggesting overexpression-induced redistribution.PPME1 Antibody Purity & Documentation Co-staining with Cox IV confirmed mitochondrial targeting, while DAPI labeling highlighted nuclear presence.Glutaminase C Antibody custom synthesis These dual localizations imply potential roles beyond mitochondrial function, possibly in regulating nuclear Hsp70 activity or DNA repair processes.PMID:34951982
Liposome interaction experiments demonstrated that hHep1 binds preferentially to anionic lipid bilayers composed of cardiolipin (CL) and phosphatidylserine (POPS), mimicking inner mitochondrial membrane composition. It also interacted with outer membrane (OMM)-like liposomes containing POPC and POPE, though with lower affinity. This suggests that hHep1 may facilitate Hsp70 recruitment during mitochondrial protein import and help stabilize membrane-associated complexes.
In summary, hHep1 functions not only as a stabilizing factor for HSPA9 but also as a dynamic remodeler of dysfunctional Hsp70 assemblies. Its ability to prevent aggregation, stimulate ATPase activity, disassemble SMA, and interact with lipid membranes underscores its central role in cellular proteostasis. These findings position hHep1 as a versatile molecular chaperone with implications for understanding disease mechanisms involving Hsp70 dysfunction and offer new avenues for therapeutic intervention in protein misfolding disorders.MedChemExpress (MCE) offers a wide range of high-quality research chemicals and biochemicals (novel life-science reagents, reference compounds and natural compounds) for scientific use. We have professionally experienced and friendly staff to meet your needs. We are a competent and trustworthy partner for your research and scientific projects.Related websites: https://www.medchemexpress.com