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J. Biol. Chem., Vol. 283, Issue 23, 15694-15700, June 6, 2008

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The Hsp60-(p.V98I) Mutation Associated with Hereditary Spastic Paraplegia SPG13 Compromises Chaperonin Function Both in Vitro and in Vivo^*

Peter Bross¹, Søren Naundrup, Jakob Hansen, Marit Nyholm Nielsen, Jane Hvarregaard Christensen, Mogens Kruhøffer^¶, Johan Palmfeldt, Thomas Juhl Corydon^||, Niels Gregersen, Debbie Ang^**2, Costa Georgopoulos^**2, and Kåre Lehmann Nielsen

From the Research Unit for Molecular Medicine, Aarhus University Hospital, Skejby, Brendstrupgaardsvej 100, Århus 8200, Denmark, the Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, Sohngaardsholmsvej 49, Aalborg 9000, Denmark, the ^¶Molecular Diagnostic Laboratory, Aarhus University Hospital, Skejby, Brendstrupgaardsvej 100, Århus 8200, Denmark, the ^||Institute of Human Genetics, University of Aarhus, Wilhelm Meyers Allé, Århus 8000, Denmark, and the ^**Department of Microbiology and Molecular Medicine, Centre Médical Universitaire, 1 Rue Michel-Servet, Geneva 1211, Switzerland

We have previously reported the association of a mutation (c.292G > A/p.V98I) in the human HSPD1 gene that encodes the mitochondrial Hsp60 chaperonin with a dominantly inherited form of hereditary spastic paraplegia. Here, we show that the purified Hsp60-(p.V98I) chaperonin displays decreased ATPase activity and exhibits a strongly reduced capacity to promote folding of denatured malate dehydrogenase in vitro. To test its in vivo functions, we engineered a bacterial model system that lacks the endogenous chaperonin genes and harbors two plasmids carrying differentially inducible operons with human Hsp10 and wild-type Hsp60 or Hsp10 and Hsp60-(p.V98I), respectively. Ten hours after shutdown of the wild-type chaperonin operon and induction of the Hsp60-(p.V98I)/Hsp10 mutant operon, bacterial cell growth was strongly inhibited. No globally increased protein aggregation was observed, and microarray analyses showed that a number of genes involved in metabolic pathways, some of which are essential for robust aerobic growth, were strongly up-regulated in Hsp60-(p.V98I)-expressing bacteria, suggesting that the growth arrest was caused by defective folding of some essential proteins. Co-expression of Hsp60-(p.V98I) and wild-type Hsp60 exerted a dominant negative effect only when the chaperonin genes were expressed at relatively low levels. Based on our in vivo and in vitro data, we propose that the major effect of heterozygosity for the Hsp60-(p.V98I) mutation is a moderately decreased activity of chaperonin complexes composed of mixed wild-type and Hsp60-(p.V98I) mutant subunits.

Received for publication, January 23, 2008 , and in revised form, April 2, 2008.

^* This work was supported by grants from the Ludvig og Sara Elsass Fond, the Lundbeck Foundation, the EU 6th Framework Program, the NOVO Nordisk Foundation, and the Swiss National Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3, Tables S1 and S2, and additional text and references.

² Present address: Dept. of Biochemistry, University of Utah, 15 N. Medical Drive East, Rm. 4100, Salt Lake City, UT 84112-5650.

¹ To whom correspondence should be addressed. Tel.: 45-89-495-147; Fax: 45-89-496-018; E-mail: peter.bross{at}KI.au.dk.

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