Hiro Yasukawa

Silent information regulator 2 proteins encoded by Cryptosporidium parasites.

Screening in a database has revealed that Cryptosporidium hominis encodes a silent information regulator 2 (Sir2), a nicotinamide adenine dinucleotide (NAD)-dependent protein deacetylase. Cellular localization of the protein, ChSir2, was analyzed by the use of the social amoeba Dictyostelium discoideum as a model system. Fluorescent microscopic analysis showed that ChSir2 fused with green fluorescent protein was localized in the D. discoideum nucleus. D. discoideum expressing ChSir2 grew faster and reached higher cell density than did D. discoideum harboring a control vector. These results suggest that ChSir2 is a nucleus-localizing protein that plays an important role in the growth of C. hominis. We cloned and sequenced the genes for Sir2 orthologs encoded by three isolates of C. hominis, two isolates of Cryptosporidium parvum and one isolate of Cryptosporidium meleagridis. The orthologs conserve critical catalytic or NAD-binding residues but do not have similarity with human Sir2 proteins (SIRTs). Cryptosporidium Sir2 orthologs would therefore be attractive therapeutic targets. The Cryptosporidium orthologs were classified into four variants based on their nucleotide sequences. Each of the four variants produces its own unique restriction fragment length polymorphism pattern by digestion with TfiI.

Analysis of Sir2E in the cellular slime mold Dictyostelium discoideum: Cellular localization, spatial expression and overexpression

It has been reported that Dictyostelium discoideum encodes four silent information regulator 2 (Sir2) proteins (Sir2A–D) showing sequence similarity to human homologues of Sir2 (SIRT1–3). Further screening in a database revealed that D. discoideum encodes an additional Sir2 homologue (Sir2E). The amino acid sequence of Sir2E is not similar to those of SIRTs but is similar to those of proteins encoded by Giardia lamblia, Cryptosporidium hominis and Cryptosporidium parvum. Fluorescence of Sir2E‐green fluorescent protein fusion protein was detected in the D. discoideum nucleus, indicating that Sir2E is a nuclear localizing protein. Reverse transcription–polymerase chain reaction and whole‐mount in situ hybridization analyses showed that D. discoideum expressed sir2E in amoebae in the growth phase and in prestalk cells in the developmental phase. D. discoideum overexpressing sir2E grew faster than the wild type. These results indicate that Sir2E plays important roles both in the growth phase and developmental phase of D. discoideum.