Mitochondrial chaperonins are necessary for the folding of newly imported and stress-denatured mitochondrial proteins. The goal of this study was to investigate the structure and function of the mammalian mitochondrial chaperonin system. We present evidence that the 60 kDa chaperonin (mt-cpn60) exists in solution in dynamic equilibrium between monomers, heptameric single rings and doubleringed tetradecamers. In the presence of ATP and the 10 kDa cochaperonin (mt-cpn10), the formation of a double ring is favored. ADP at very high concentrations does not inhibit malate dehydrogenase refolding or ATP hydrolysis by mt-cpn60 in the presence of mt-cpn10. We propose that the cis (mt-cpn60) 14´n uleotide´(mt-cpn10) 7 complex is not a stable species and does not bind ADP effectively at its trans binding site.Keywords: chaperonin; mitochondrial folding; cpn60; cpn10; hsp60.The 60 kDa chaperonins constitute a highly conserved family of proteins found in chloroplasts, mitochondria and eubacteria, which plays an important role in the folding of nascent, translocating and stress-denatured proteins [1]. In yeast mitochondria, chaperonins mediate the folding of both newly imported and stress-denatured proteins, and are essential for the viability of yeast under all conditions [2±5]. Consistent with such an important role, numerous studies have found altered levels of mammalian mitochondrial chaperonin 60 (mt-cpn60) associated with various pathological states [6±11]. Despite the importance of this molecule, few studies have been carried out to understand the details of mammalian mt-cpn60 structure and function. Due to the high level of homology at both the sequence level and functional level, it was commonly assumed that mt-cpn60 was mechanistically similar to its well-studied bacterial counterpart, GroEL. Indeed, the mitochondrial chaperonin system can fold denatured proteins in vitro, with a similar efficiency to GroEL [12,13], and is able to replace the bacterial GroEL in Escherichia coli in vivo [14]. However, a number of significant differences have been reported in the structure of mt-cpn60, which suggest that this eucaryotic chaperonin may have developed a different mechanism of action. While all other chaperonin homologs exist as tetradecamers composed of two seven-membered rings, the mammalian mt-cpn60 has been consistently isolated as a single ring [12,13]. Moreover, mt-cpn60 readily dissociates to monomers in the presence of ATP, low temperatures, and the nonphysiological concentrations that are routinely used for in vitro studies [15]. The functional significance of such instability is unclear. One other interesting difference lies in the fact that mitochondrial cpn60s are functional only with their own 10 kDa cofactor, mitochondrial chaperonin 10 (mt-cpn10) [12,15], whereas the bacterial and chloroplast cpn60 can fold proteins with cpn10 from any source [12,13,16±18].Numerous mechanistic studies over the past decade have resulted in a putative mechanism of action for the GroEL chaperonin [19]. Central to this mechan...

SummaryTight regulation of the production of the key pro-inflammatory cytokine tumour necrosis factor-a (TNF-a) is essential for the prevention of chronic inflammatory diseases. In vivo administration of a synthetic phospholipid, named hereafter phospho-ceramide analogue-1 (PCERA-1), was previously found to suppress lipopolysaccharide (LPS)-induced TNF-a blood levels. We therefore investigated the in vitro anti-inflammatory effects of PCERA-1. Here, we show that extracellular PCERA-1 potently suppresses production of the pro-inflammatory cytokine TNF-a in RAW264.7 macrophages, and in addition, independently and reciprocally regulates the production of the anti-inflammatory cytokine interleukin-10 (IL-10). Specificity is demonstrated by the inability of the phospholipids ceramide-1-phosphate (C1P), sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) to perform these activities. Similar TNF-a suppression and IL-10 induction by PCERA-1 were observed in macrophages when activated by Toll-like receptor 4 (TLR4), TLR2 and TLR7 agonists. Regulation of cytokine production is demonstrated at the mRNA and protein levels. Finally, we show that, while PCERA-1 does not block activation of nuclear factor (NF)-jB and mitogen-activated protein kinases by LPS, it elevates the intracellular cAMP level. In conclusion, the antiinflammatory activity of PCERA-1 seems to be mediated by a cell membrane receptor, upstream of cAMP production, and eventually TNF-a suppression and IL-10 induction. Thus, identification of the PCERA-1 receptor may provide new pharmacological means to block inflammation.

Type I chaperonins play an essential role in the folding of newly translated and stress-denatured proteins in eubacteria, mitochondria and chloroplasts. Since their discovery, the bacterial chaperonins have provided an excellent model system for investigating the mechanism by which chaperonins mediate protein folding. Due to the high conservation of the primary sequence among Type I chaperonins, it is generally accepted that organellar chaperonins function similar to the bacterial ones. However, recent studies indicate that the chloroplast and mitochondrial chaperonins possess unique structural and functional properties that distinguish them from their bacterial homologs. This review focuses on the unique properties of organellar chaperonins. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

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Type I chaperonins (cpn60/Hsp60) are essential proteins that mediate the folding of proteins in bacteria, chloroplast and mitochondria. Despite the high sequence homology among chaperonins, the mitochondrial chaperonin system has developed unique properties that distinguish it from the widely-studied bacterial system (GroEL and GroES). The most relevant difference to this study is that mitochondrial chaperonins are able to refold denatured proteins only with the assistance of the mitochondrial co-chaperonin. This is in contrast to the bacterial chaperonin, which is able to function with the help of co-chaperonin from any source. The goal of our work was to determine structural elements that govern the specificity between chaperonin and co-chaperonin pairs using mitochondrial Hsp60 as model system. We used a mutagenesis approach to obtain human mitochondrial Hsp60 mutants that are able to function with the bacterial co-chaperonin, GroES. We isolated two mutants, a single mutant (E321K) and a double mutant (R264K/E358K) that, together with GroES, were able to rescue an E. coli strain, in which the endogenous chaperonin system was silenced. Although the mutations are located in the apical domain of the chaperonin, where the interaction with co-chaperonin takes place, none of the residues are located in positions that are directly responsible for co-chaperonin binding. Moreover, while both mutants were able to function with GroES, they showed distinct functional and structural properties. Our results indicate that the phenotype of the E321K mutant is caused mainly by a profound increase in the binding affinity to all co-chaperonins, while the phenotype of R264K/E358K is caused by a slight increase in affinity toward co-chaperonins that is accompanied by an alteration in the allosteric signal transmitted upon nucleotide binding. The latter changes lead to a great increase in affinity for GroES, with only a minor increase in affinity toward the mammalian mitochondrial co-chaperonin.