Stability and disassembly properties of human naïve Hsp60 and bacterial GroEL chaperonins
Graphical abstract
Introduction
Molecular chaperones are a class of proteins, present in all organisms from bacteria to humans, able to prevent non-specific aggregation of other proteins and to facilitate their folding. Recent findings have shown that they inhibit the formation of amyloid fibrils associated with neurodegenerative disorders, such as Alzheimer's disease [1], [2]. These pathologies are characterized by conformational changes in proteins, which result in misfolding, aggregation and intra- or extra-neuronal accumulation of amyloid fibrils. A complete characterization of the chaperones' structure may lead to the understanding of their function and of their inhibition role and is therefore relevant for the comprehension of the mechanism underlying the onset of the disease. One of the most exhaustively characterized molecular chaperones is GroEL, present in prokaryotic cells. GroEL is known to interact with nonfolded proteins and to facilitate both their correct folding and their assembly in a ATP-dependent manner. In this respect, ATP binding and hydrolysis induce GroEL conformational changes determining cycles of substrate binding and release. For proper functioning, GroEL requires the ring shaped co-chaperonin GroES protein complex, forming the lid on a folding cage where polypeptides are enclosed during folding. GroEL structure can be described as a large oligomeric protein containing 14 identical 57-kDa subunits, each one constituted by three domains: an apical domain (191–376 residues), an equatorial domain (1–133 and 409–548) and an intermediate domain (134–190 and 377–408) [3]. Several GroEL crystallographic structures have been released in the form of a homotetradecamer, composed of two back-to-back seven member rings [4], [5], and a new GroEL structure has been recently redefined by some of us [6].
The human GroEL homolog is Hsp60 (also called Cpn60). It is encoded and transcribed by a nuclear gene and translated in the cytosol. The newly translated (naïve) polypeptide has a mitochondrial import signal (MIS), i.e., a sequence of 26 amino acids at the N-terminus that drives Hsp60 to the inside of mitochondria where the MIS is cleaved and the protein reaches the final conformation (mtHsp60 or Cpn60). Hsp60, together with its co-chaperone Hsp10 (Cpn10) represents the mitochondrial protein folding machine. The process in which they are involved has been investigated by studying their bacterial homologs GroEL and GroES, respectively [7]. Mitochondrial human Hsp60, similarly to bacterial GroEL, forms a ring-shaped homo-oligomer of seven subunits, and, in the presence of ATP and/or its own 10 kDa co-chaperonin Hsp10, two rings associate to form a tetradecamer shaped like a barrel with a central cavity, which can accommodate polypeptide substrates of up to 50 kDa or so [8], [9]. However, differently from bacterial homologs that exist only as tetradecamers, the human mtHsp60 seems to exist also as a homo-oligomer of seven subunits and a single heptameric ring seems to be sufficient for productive chaperonin-mediated folding in vivo [10], [11]. Although much more attention has been directed in order to investigate and study the structure, function and oligomeric equilibrium of mtHsp60, less than a decade ago Chandra et al. pointed out some important features concerning also naïve Hsp60's role [12]. In fact, they suggested that Hsp60 has a pro-survival function if, once imported into mitochondria, it is released to the cytosol, and a pro-death function if it accumulates in the cytoplasm without mitochondrial secretion (naïve condition) and bearing its MIS 26 amino acid signal sequence at the N terminus. 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].
In the present study we focus our attention on the stability characteristics of naïve Hsp60 in order to further elucidate the structural basis underlying Hsp60 functions, and providing information relevant to support, in general, a relationship between stability and function [15].
In this light, we have conducted a study of the effect of guanidinium hydrochloride in solution to provide information on naïve Hsp60 stability and on the related oligomeric equilibria. We have performed a parallel study with GroEL, whose chemical unfolding mechanism by denaturing agents has been previously extensively characterized by several biophysical techniques [3], [16], [17], [18], [19]. Small Angle X-ray Scattering (SAXS) experiments performed with Synchrotron Radiation evidenced different stabilities of Hsp60 and GroEL, both in oligomeric dissociation from tetradecamer to heptamer until monomer, and in the unfolding stages. Circular dichroism (CD) measurements have provided information on the change in the secondary structure due to protein unfolding, confirming that Hsp60 shows up less stability and lower cooperativity in the chemical denaturation transition with respect to GroEL.
Section snippets
Sample preparation
The recombinant naïve Hsp60 was purchased from ATGen (Seongnam, South Korea) in stock solution at 16.0 μM (1 g L− 1) (buffer 20 mM Tris pH 8.0 and 10% glycerol (w/w)). Lyophilized GroEL was obtained from SIGMA (St. Louis, MO, USA). As a step prior to the experiments, we verified that the chaperonins under analysis were able to properly work. Hence, ATP activities were measured by using the ImageJ Software [20], and they are (16.48 ± 0.02) nmol ATP min− 1 mg− 1 for GroEL; (4.7 ± 0.1) nmol ATP min− 1 mg− 1 for naïve
Results
Far-UV CD spectra at different denaturant concentrations are reported in Fig. 1. The CD values corresponding to the two minima in the spectra (located at 212 nm and at 223 nm and hereafter referred to as CD212 and CD223) are reported as a function of denaturant concentration in the middle panel of Fig. 2. In the simple two-state approximation [24], the experimental CD spectrum is considered a linear combination of the CD spectra of a unique folded (F) state and a unique unfolded (U) state and the
Discussion
In the present study, we have assessed the stability by chemical denaturation of the human chaperonin Hsp60 and compared it to that of its bacterial homolog, GroEL. We have used one of the most frequently employed protein denaturants, GdnHCl, that leads to a complete unfolding [34]. The overall results have shown that Hsp60 is significantly less stable than its bacterial homolog. First, the protein conformational changes monitored by circular dichroism at increasing amounts of GdnHCl present a
Acknowledgment
We gratefully acknowledge the financial support from the Italian MIUR within the “FIRB — Futuro in Ricerca 2012” Program, grant number RBFR12SIPT.
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