Activation of pro-caspase-3 is a central event in the execution phase of apoptosis and appears to serve as the convergence point of different apoptotic signaling pathways. Recently, mitochondria were found to play a central role in apoptosis through release of cytochrome c and activation of caspases. Moreover, a sub-population of pro-caspase-3 has been found to be localized to this organelle. In the present study, we demonstrate that pro-caspase-3 is present in the mitochondrial fraction of Jurkat T cells in a complex with the chaperone proteins Hsp60 and Hsp10. Induction of apoptosis with staurosporine led to the activation of mitochondrial pro-caspase-3 and its dissociation from the Hsps which were released from mitochondria. The release of Hsps occurred simultaneously with the release of other mitochondrial intermembrane space proteins including cytochrome c and adenylate kinase, prior to a loss of mitochondrial transmembrane potential. In in vitro systems, recombinant Hsp60 and Hsp10 accelerated the activation of pro-caspase-3 by cytochrome c and dATP in an ATP-dependent manner, consistent with their function as chaperones. This finding suggests that the release of mitochondrial Hsps may also accelerate caspase activation in the cytoplasm of intact cells.

MEN1 is a tumor suppressor gene that encodes a 610 amino acid nuclear protein (menin) of previously unknown function. Using a yeast two-hybrid screen with menin as the bait, we have identified the transcription factor JunD as a direct menin-interacting partner. Menin did not interact directly with other Jun and Fos family members. The menin-JunD interaction was confirmed in vitro and in vivo. Menin repressed transcriptional activation mediated by JunD fused to the Gal4 DNA-binding domain from a Gal4 responsive reporter, or by JunD from an AP1-responsive reporter. Several naturally occurring and clustered MEN1 missense mutations disrupted menin interaction with JunD. These observations suggest that menin's tumor suppressor function involves direct binding to JunD and inhibition of JunD activated transcription.

Fhit protein is lost in most cancers, its restoration suppresses tumorigenicity, and virus-mediated FHIT gene therapy induces apoptosis and suppresses tumors in preclinical models. We have used protein cross-linking and proteomics methods to characterize a Fhit protein complex involved in triggering Fhit-mediated apoptosis. The complex includes Hsp60 and Hsp10 that mediate Fhit stability and may affect import into mitochondria, where it interacts with ferredoxin reductase, responsible for transferring electrons from NADPH to cytochrome P450 via ferredoxin. Viral-mediated Fhit restoration increases production of intracellular reactive oxygen species, followed by increased apoptosis of lung cancer cells under oxidative stress conditions; conversely, Fhit-negative cells escape apoptosis, carrying serious oxidative DNA damage that may contribute to an increased mutation rate. Characterization of Fhit interacting proteins has identified direct effectors of the Fhit-mediated apoptotic pathway that is lost in most cancers through loss of Fhit.

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.

A full-length cDNA clone encoding chaperonin 10 (cpn10) from a HeLa cell cDNA library was isolated. The cDNA is 538 bp in length, contains an ATG codon and a putative polyadenylation signal, and specifies a protein of 102 amino acids. Immunoprecipitation experiment showed that this human cpn10 has an apparent molecular mass of 11 kDa in sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Activation of pro-caspase-3 is a central event in the execution phase of apoptosis and appears to serve as the convergence point of different apoptotic signaling pathways. Recently, mitochondria were found to play a central role in apoptosis through release of cytochrome c and activation of caspases. Moreover, a sub-population of pro-caspase-3 has been found to be localized to this organelle. In the present study, we demonstrate that pro-caspase-3 is present in the mitochondrial fraction of Jurkat T cells in a complex with the chaperone proteins Hsp60 and Hsp10. Induction of apoptosis with staurosporine led to the activation of mitochondrial pro-caspase-3 and its dissociation from the Hsps which were released from mitochondria. The release of Hsps occurred simultaneously with the release of other mitochondrial intermembrane space proteins including cytochrome c and adenylate kinase, prior to a loss of mitochondrial transmembrane potential. In in vitro systems, recombinant Hsp60 and Hsp10 accelerated the activation of pro-caspase-3 by cytochrome c and dATP in an ATP-dependent manner, consistent with their function as chaperones. This finding suggests that the release of mitochondrial Hsps may also accelerate caspase activation in the cytoplasm of intact cells.

We have recently reported the cloning of a cDNA coding for a stress inducible human chaperonin 10. The protein was shown to possess 100% identity with the bovine homologue and a single amino acid replacement (glycine to serine at position 52) compared to rat chaperonin 10. Here we report the heterologous expression of human chaperonin 10 in Escherichia coli, its purification and its functional characterization. The recombinant protein was purified to homogeneity as judged by different analytical techniques, and mass spectrometry analysis showed a MW of 10,801 Da in agreement with the predicted sequence. This molecular weight accounts for a protein which is not modified post-translationally. In fact, natural rat chaperonin 10 has been shown to be acetylated at the N-terminus, a feature suggested to be important for targeting and functional activity. Here we show that recombinant human chaperonin 10 is fully active in assisting the chaperonin 60 GroEL in the refolding of denatured yeast enolase, thereby showing that, at least in the present system, post-translational acetylation is not necessary for its activity.