Hsp70:CHIP Ubiquitinates Dysfunctional but Not Native Neuronal NO Synthase

Area of Science:

• Molecular Biology
• Protein Degradation Pathways
• Chaperone-Mediated Protein Homeostasis

Background:

• Heat shock protein 70 (Hsp70) modulators are investigated for treating protein misfolding diseases by promoting toxic protein clearance.
• Neuronal nitric oxide synthase (nNOS) inactivation, due to heme or tetrahydrobiopterin (BH4) loss, leads to its ubiquitination and degradation via the Hsp70:c-terminus of Hsp70-interacting protein (CHIP) complex.
• Understanding the selectivity of the Hsp70:CHIP complex for inactivated client proteins is crucial for therapeutic development.

Purpose of the Study:

• To develop and validate a sensitive assay for measuring Hsp70:CHIP-dependent nNOS ubiquitination.
• To assess the selectivity of the Hsp70:CHIP complex for inactivated versus functional nNOS.
• To evaluate the potential of pharmacological Hsp70 activation as a therapeutic strategy for protein misfolding diseases.

Main Methods:

• Development of a highly sensitive ELISA to quantify Hsp70:CHIP-mediated nNOS ubiquitination, validated using Bcl-2 associated athanogene 1-M.
• Utilized JG-98, an Hsp70 activator, to demonstrate enhanced nNOS ubiquitination in vitro.
• Compared ubiquitination of heme-deficient (apo-nNOS) versus heme-containing (holo-nNOS) and BH4-depleted holo-nNOS.

Main Results:

• The Hsp70:CHIP complex preferentially ubiquitinates heme-deficient nNOS (apo-nNOS) over heme-containing nNOS (holo-nNOS).
• Depletion of nNOS-bound BH4 triggers ubiquitination of holo-nNOS by the Hsp70:CHIP complex.
• Pharmacological activation of Hsp70 with JG-98 selectively enhances ubiquitination of dysfunctional nNOS, leaving native functional nNOS unaffected.

Conclusions:

• The Hsp70:CHIP complex exhibits selectivity for ubiquitinating dysfunctional nNOS.
• Pharmacological activation of Hsp70 enhances the degradation of misfolded or inactivated proteins without affecting native functional proteins.
• This targeted approach holds significant promise for developing selective therapeutics for protein misfolding diseases.