A database search with Inhibitor 1 and Inhibitor 2, known to be powerful regulators of PP1c, identified one open reading frame in the P. falciparum genome encoding a potential protein with identity to known I2. Inhibitor 2 is a thermo and acid stable regulator initially purified from rabbit skeletal muscle and is conserved among all eukaryotes. The potency of the inhibition by re combinant I2 of different species measured in parallel seems to be species specific in terms of inhibitory effect. It is interesting to note that the peptide sequences of I2 orthologs vary in length, from 164 amino acids in plants up to 205 amino acids in humans. This may ac count for specificities mentioned above.

The comparison of I2 sequences of different species along with in vitro functional studies revealed that two main regions par ticipate in the interaction with PP1c and the inhibition of its activity one binding region containing a KSQKW motif suggested to fulfill the role of the canonical RV F motif and a second region containing a HYNE motif. In addition, a third region present in the N terminal moiety of human I2 and containing a KGILK motif has also been shown to be involved in the inter action with PP1c. The resolution of the rodent PP1c I2 crystal structure confirmed the implication of these regions in the binding process. In vivo, the overe pression of GLC8 or the knockdown of human I2 by RNA interference showed its direct role in the cell cycle. For instance, human I2 knockdown produced multinucleated cells during anaphase and blocked cyto kinesis.

Moreover, e ploration of the role of I2 in Drosophila development evidenced that an I2 loss of function in mothers leads to a dramatic reduction in the viability of progeny as measured by a decrease in embryonic hatch rates and larval lethality. However, I2 gain of function by transgenic e pression of I2 in mu tant mothers reversed this effect. Altogether, these observations indicate that I2 plays a critical role in achieving successful mitosis and it is apparent that interfering with I2 functions represents an attractive approach for pharmacological intervention. Here, we re port the structure function relationship of inhibitor 2 of P. falciparum and e plore its role and the means to affect its function in the parasite. Results Cloning and bioinformatics analysis of Pf Inhibitor 2 Analysis of PlasmoDB using the human Inhibitor 2 sequence AV-951 identified a gene encoding a potential P.

falciparum Inhibitor 2 homolog. To establish the identity of the PfI2 sequence, we determined the nucleotide sequence by RT PCR using cDNA from total RNA of blood stage par asites and primers specified in Additional file 1 Table S1. The amplification showed a PCR product of the predicted size, confirming its transcription and the microarray data available in PlasmoDB.