The Protein Society 38th Annual Symposium

HSP60 is translocated to mitochondria by a 26 amino acid mitochondrial targeting sequence (MTS) where, following its cleavage, HSP60 forms a functional toroidal oligomer. Mis-regulation of HSP60, characterized by over-expression and cytosolic accumulation, is prevalent in many cancers and is associated with tumor survival, aggressiveness, and poor prognosis through the modulation of several oncogenic pathways. Developing drugs that selectively target aberrant HSP60 in the cytosol (ctHSP60) of cancer cells could provide a therapeutic approach that minimizes off-target effects on mitochondrial HSP60 (mtHSP60) in healthy cells. Such selectivity could be possible due to ctHSP60’s distinct sub-cellular localization, differences in client proteins, and potential structural and functional divergence from mtHSP60. However, therapeutic development has been hindered by the poorly understood characteristics of aberrant ctHSP60, which putatively retains the MTS. To address this deficiency and gain a better understanding of ctHSP60’s biophysical and biochemical properties, we used analytical size exclusion chromatography and found that ctHSP60 eluted as a mix of double and single-ring complexes. Using established ATPase and client protein folding assays, we found that ctHSP60 exhibited comparable ATPase activity to mtHSP60, with stimulation in the presence of HSP10; however, ctHSP60 did not facilitate client protein refolding in an HSP10-mediated manner. To further examine this defect, we employed cryoEM and found that, unlike typical double-ring structures of mtHSP60 and E. coli GroEL, ctHSP60 alone associated as an inverted complex with its apical domains forming the ring-ring interface. However, in the presence of ATP, ctHSP60 formed ‘football’ complexes similar to mtHSP60-HSP10, with HSP10 bound to either end of the properly oriented HSP60 double-ring. These perplexing findings prompt our continued exploration for the cause of ctHSP60's inability to facilitate client protein folding, the results of which will help elucidate ctHSP60’s role in cancer progression and its viability as a chemotherapeutic target.