Human Hsp60 with Its Mitochondrial Import Signal Occurs in Solution as Heptamers and Tetradecamers Remarkably Stable over a Wide Range of Concentrations

Abstract

It has been established that Hsp60 can accumulate in the cytosol in various pathological conditions, including cancer and chronic inflammatory diseases. Part or all of the cytosolic Hsp60 could be naïve, namely, bear the mitochondrial import signal (MIS), but neither the structure nor the in solution oligomeric organization of this cytosolic molecule has still been elucidated. Here we present a detailed study of the structure and self-organization of naïve cytosolic Hsp60 in solution. Results were obtained by different biophysical methods (light and X ray scattering, single molecule spectroscopy and hydrodynamics) that all together allowed us to assay a wide range of concentrations of Hsp60. We found that Naïve Hsp60 in aqueous solution is assembled in very stable heptamers and tetradecamers at all concentrations assayed, without any trace of monomer presence.

Stability and disassembly properties of human naïve Hsp60 and bacterial GroEL chaperonins

“…The values for GroEL and mitochondrial Hsp60 (used as standard protein) are of the same order of magnitude of those reported in literature [21,22]. The chromatograph we report in the Supporting Materials is the same previously reported in the paper written by some of us [14], because we used the same stock of commercial enzymes. All the experiments were conducted by diluting naïve Hsp60 from stock solutions to reach the concentration of 4.8 μM and dissolving GroEL in 20 mM Tris pH 8.0 and 10% glycerol (w/w) at 4.8 μM, too.…”

“…Higher protein concentrations were obtained with Vivaspin concentrators with a Polyethersulfone membrane and 10 kDa molecular weight cut-offs (Sartorius, Germany). Protein concentration for each experiment was determined by the area under a peak from the corresponding High Performance Liquid Chromatography (HPLC) chromatogram recorded at 280 nm [14]. The concentration determination was performed after Dynamic Light Scattering experiments on the samples, which showed that there were only the oligomeric species considered functional for chaperonins [5,11,14].…”

“…Moreover, naïve Hsp60 form could be involved in tumor cells or during inflammatory diseases [13,7]. More recently, the structure and oligomeric state of the naïve Hsp60 have been studied in vitro in a wide range of concentrations, and results have shown that it exists in solution in a dynamic equilibrium between two conformations: heptameric single rings and double ringed tetradecamers [14].…”

Quaternary structures of GroEL and naïve-Hsp60 chaperonins in solution: a combined SAXS-MD study

“…The other two exible linkers, connecting the N-and C-ending domain to the equatorial-intermediate domains, are from residues 23 to 26 and from 548 to 552. Taking advantage of recent ndings above mentioned, 11 and considering initial attempt to tting SAXS data (see Fig. S5 †), we have considered that, unlike GroEL, naïve-Hsp60 in solution can simultaneously be present as a mixture of tetradecamers and heptamers.…”

Alzheimer's Disease and Molecular Chaperones: Current Knowledge and the Future of Chaperonotherapy

“…Various studies, published over the past several years, have demonstrated new subcellular locations and functions for Hsp60, describing it as a ubiquitous molecule with multiple roles in health and disease [42][43][44]. Moreover, detailed studies of the structure of Hsp60 have been reported that help understand the functions of this multifaceted molecule [45,46].…”