SPG13, an autosomal dominant form of pure hereditary spastic paraplegia, was recently mapped to chromosome 2q24-34 in a French family. Here we present genetic data indicating that SPG13 is associated with a mutation, in the gene encoding the human mitochondrial chaperonin Hsp60, that results in the V72I substitution. A complementation assay showed that wild-type HSP60 (also known as "HSPD1"), but not HSP60 (V72I), together with the co-chaperonin HSP10 (also known as "HSPE1"), can support growth of Escherichia coli cells in which the homologous chromosomal groESgroEL chaperonin genes have been deleted. Taken together, our data strongly indicate that the V72I variation is the first disease-causing mutation that has been identified in HSP60.
Although the mitochondrial chaperonin Hsp60 and its co-chaperonin Hsp10 have received great attention in the last decade, and it has been proposed that mutations and variations in these genes may be implicated in genetic diseases, the genome structure of the human HSP60 and HSP10 genes (also known as HSPD1 and HSPE1, respectively) has not been firmly established. The picture has been confused by the presence of many pseudogenes of both HSP60 and HSP10 and the long surviving assumption that the HSP60 gene is intron-less. An earlier report on the partial sequence of the human HSP60 gene and the presence of introns has largely been overlooked. We present the full sequence of the human HSP60 and HSP10 genes. The two genes are linked head to head comprising approximately 17 kb and consist of 12 and 4 exons, respectively. The first exon of the human HSP60 gene is non-coding and the first exon of the human HSP10 gene ends with the start codon. Analysis of human and mouse expressed sequence tag sequences in GenBank indicates that alternative splicing occurs resulting in HSP60 gene transcripts with different exon-1 sequences. By sequencing of the exons, the exon/intron boundaries and the region between the two genes in 10 Danish individuals (five couples), nine nucleotide variations and one intronic deletion have been detected that, by subsequent typing of one child from each couple, have been assigned to five haplotypes. The human HSP60 gene has been localised, by radiation hybrid mapping, between markers AFMA121YH1 and WI-10756 on chromosome 2. This location and the position of two homologous fragments in the Human Genome Assembly are consistent with cytogenetic position 2q33.1. Using a luciferase-reporter assay, we demonstrate that the region between the two genes functions as a bi-directional promoter. The transcriptional activity of the promoter fragment in the HSP60 direction is approximately twice that in the HSP10 direction under normal growth conditions and, upon heat-shock, promoter activity in either direction increased by a factor of approximately 12. One of the nucleotide variations detected is localised in a putative SP1-transcription-factor-binding site in the bidirectional promoter region and analysis of the transcriptional activity of the promoter fragment with this variation has shown that it does not affect transcription levels both with and without heat-shock.
The mitochondrial Hsp60 chaperonin plays an important role in sustaining cellular viability. Its dysfunction is related to inherited forms of the human diseases spastic paraplegia and hypomyelinating leukodystrophy. However, it is unknown whether the requirement for Hsp60 is neuron specific or whether a complete loss of the protein will impair mammalian development and postnatal survival. In this study, we describe the generation and characterization of a mutant mouse line bearing an inactivating genetrap insertion in the Hspd1 gene encoding Hsp60. We found that heterozygous mice were born at the expected ratio compared to wild-type mice and displayed no obvious phenotype deficits. Using quantitative reverse transcription PCR, we found significantly decreased levels of the Hspd1 transcript in all of the tissues examined, demonstrating that the inactivation of the Hspd1 gene is efficient. By Western blot analysis, we found that the amount of Hsp60 protein, compared to either cytosolic tubulin or mitochondrial voltage-dependent anion-selective channel protein 1/porin, was decreased as well. The expression of the nearby Hspe1 gene, which encodes the Hsp10 co-chaperonin, was concomitantly down regulated in the liver, and the protein levels in all tissues except the brain were reduced. Homozygous Hspd1 mutant embryos, however, died shortly after implantation (day 6.5 to 7.5 of gestation, Theiler stages 9-10). Our results demonstrate that Hspd1 is an essential gene for early embryonic development in mice, while reducing the amount of Hsp60 by inactivation of one allele of the gene is compatible with survival to term as well as postnatal life.
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.
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