Hsp27 suppresses the Cu(2+)-induced amyloidogenicity, redox activity, and cytotoxicity of α-synuclein by metal ion stripping.

Aberrant copper homeostasis and oxidative stress have critical roles in several neurodegenerative diseases. Expression of heat-shock protein 27 (Hsp27) is elevated under oxidative stress as well as upon treatment with Cu(2+), and elevated levels of Hsp27 are found in the brains of patients with Alzheimer and Parkinson diseases. We demonstrate, using steady-state and time-resolved fluorescence spectroscopy as well as isothermal titration calorimetry studies, that Hsp27 binds Cu(2+) with high affinity (Kd ~10(-11) M). Treating IMR-32 human neuroblastoma cells with Cu(2+) leads to upregulation of endogenous Hsp27. Further, overexpression of Hsp27 in IMR-32 human neuroblastoma cells confers cytoprotection against Cu(2+)-induced cell death. Hsp27 prevents the deleterious interaction of Cu(2+) with α-synuclein, the protein involved in Parkinson disease and synucleinopathies. Hsp27 attenuates Cu(2+)- or Cu(2+)-α-synuclein-mediated generation of reactive oxygen species and confers cytoprotection on IMR-32 cells as well as on mouse primary neural precursor cells. Hsp27 prevents Cu(2+)-ascorbate or Cu(2+)-α-synuclein-ascorbate treatment-induced increase in mitochondrial superoxide level and mitochondrial disorganization in IMR-32 cells. Hsp27 dislodges the α-synuclein-bound Cu(2+) and prevents the Cu(2+)-mediated amyloidogenesis of α-synuclein. Our findings that Hsp27 binds Cu(2+) with high affinity leading to beneficial effects and that Hsp27 can dislodge Cu(2+) from α-synuclein, preventing amyloid fibril formation, indicate potential therapeutic strategies for neurodegenerative diseases involving aberrant Cu(2+) homeostasis.

Heat stress-induced localization of small heat shock proteins in mouse myoblasts: intranuclear lamin A/C speckles as target for alphaB-crystallin and Hsp25.

We examined the effect of heat stress on localization of two sHsps, alphaB-crystallin and Hsp25, and of Hsc70, a member of a different class of heat shock proteins (Hsps), in both undifferentiated and differentiated mouse C2C12 cells. Under normal conditions, alphaB-crystallin and Hsp25 are found in the cytoplasm; only alphaB-crystallin is also found in the nucleus, distributed in a speckled pattern. Hsc70 is found to be homogeneously distributed throughout the cell. On heat stress, all these proteins translocate almost entirely into the nucleus and upon recovery relocate to the cytoplasm. Dual staining experiments using C2C12 myoblasts show that alphaB-crystallin and Hsp25, but not Hsc70, colocalize with the intranuclear lamin A/C and the splicing factor SC-35, suggesting interactions of sHsps and intranuclear lamin A/C. Interestingly, none of these proteins are found in the myotube nuclei. Upon heat stress, only Hsc70 translocates into the myotube nuclei. This differential entry of alphaB-crystallin and Hsp25 into the nuclei of myoblasts and myotubes upon heat stress may have functional role in the development and/or in the maintenance of muscle cells. Our study therefore suggests that these sHsps may be a part of the intranuclear lamin A/C network or stabilizing this specific network.

Oxidative refolding of lysozyme in trifluoroethanol (TFE) and ethylene glycol: interfering role of preexisting alpha-helical structure and intermolecular hydrophobic interactions.

The oxidative refolding of equilibrium intermediates of lysozyme stabilized in trifluoroethanol (TFE) and ethylene glycol was monitored. Equilibrium intermediates of disulfide reduced lysozyme in TFE are known to contain considerable amounts of alpha-helical structure and resemble the early intermediate in the oxidative refolding of lysozyme. We find that the intermediates in TFE do not proceed to folding; they form aggregates. However, interestingly, intermediates in ethylene glycol refold to the native state with improved folding yield. Secondary structure of these intermediates was monitored by far-UV circular dichroism. Our results indicate that formation of alpha-helical structure prior to oxidative refolding does not help the process in the case of lysozyme. Interfering with intermolecular hydrophobic interactions in the unfolded state is more productive.