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Cryptosporidium parvum mitochondrial-type HSP70 targets homologous and heterologous mitochondria.

ABSTRACT: A mitochondrial HSP70 gene (Cp-mtHSP70) is described for the apicomplexan Cryptosporidium parvum, an agent of diarrhea in humans and animals. Mitochondrial HSP70 is known to have been acquired from the proto-mitochondrial endosymbiont. The amino acid sequence of Cp-mtHSP70 shares common domains with mitochondrial and proteobacterial homologues, including 34 amino acids of an NH2-terminal mitochondrion-like targeting presequence. Phylogenetic reconstruction places Cp-mtHSP70 within the mitochondrial clade of HSP70 homologues. Using reverse transcription-PCR, Cp-mtHSP70 mRNA was observed in C. parvum intracellular stages cultured in HCT-8 cells. Polyclonal antibodies to Cp-mtHSP70 recognize a approximately 70-kDa protein in Western blot analysis of sporozoite extracts. Both fluorescein- and immunogold-labeled anti-Cp-mtHSP70 localize to a single mitochondrial compartment in close apposition to the nucleus. Furthermore, the NH2-terminal presequence of Cp-mtHSP70 can correctly target green fluorescent protein to the single mitochondrion of the apicomplexan Toxoplasma gondii and the mitochondrial network of the yeast Saccharomyces cerevisiae. When this presequence was truncated, the predicted amphiphilic alpha-helix was shown to be essential for import into the yeast mitochondrion. These data further support the presence of a secondarily reduced relict mitochondrion in C. parvum.

SUBMITTER: Slapeta J

PROVIDER: S-EPMC387664 | biostudies-literature | 2004 Apr

REPOSITORIES: biostudies-literature

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Cryptosporidium parvum mitochondrial-type HSP70 targets homologous and heterologous mitochondria.

Slapeta Jan J Keithly Janet S JS

Eukaryotic cell 20040401 2

A mitochondrial HSP70 gene (Cp-mtHSP70) is described for the apicomplexan Cryptosporidium parvum, an agent of diarrhea in humans and animals. Mitochondrial HSP70 is known to have been acquired from the proto-mitochondrial endosymbiont. The amino acid sequence of Cp-mtHSP70 shares common domains with mitochondrial and proteobacterial homologues, including 34 amino acids of an NH2-terminal mitochondrion-like targeting presequence. Phylogenetic reconstruction places Cp-mtHSP70 within the mitochondr ...[more]

PMID: 15075277

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Project description:BackgroundHundreds of millions of people are infected with cryptosporidiosis annually, with immunocompromised individuals suffering debilitating symptoms and children in socioeconomically challenged regions at risk of repeated infections. There is currently no effective drug available. In order to facilitate the pursuit of anti-cryptosporidiosis targets and compounds, our study spans the classification of the Cryptosporidium parvum kinome and the structural and biochemical characterization of representatives from the CDPK family and a MAP kinase.ResultsThe C. parvum kinome comprises over 70 members, some of which may be promising drug targets. These C. parvum protein kinases include members in the AGC, Atypical, CaMK, CK1, CMGC, and TKL groups; however, almost 35% could only be classified as OPK (other protein kinases). In addition, about 25% of the kinases identified did not have any known orthologues outside of Cryptosporidium spp. Comparison of specific kinases with their Plasmodium falciparum and Toxoplasma gondii orthologues revealed some distinct characteristics within the C. parvum kinome, including potential targets and opportunities for drug design. Structural and biochemical analysis of 4 representatives of the CaMK group and a MAP kinase confirms features that may be exploited in inhibitor design. Indeed, screening CpCDPK1 against a library of kinase inhibitors yielded a set of the pyrazolopyrimidine derivatives (PP1-derivatives) with IC₅₀ values of < 10 nM. The binding of a PP1-derivative is further described by an inhibitor-bound crystal structure of CpCDPK1. In addition, structural analysis of CpCDPK4 identified an unprecedented Zn-finger within the CDPK kinase domain that may have implications for its regulation.ConclusionsIdentification and comparison of the C. parvum protein kinases against other parasitic kinases shows how orthologue- and family-based research can be used to facilitate characterization of promising drug targets and the search for new drugs.

Dataset Information

Cryptosporidium parvum mitochondrial-type HSP70 targets homologous and heterologous mitochondria.

Publications

Cryptosporidium parvum mitochondrial-type HSP70 targets homologous and heterologous mitochondria.

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