Molecular mechanisms underlying the interaction between malarial sporozoites and putative receptor(s) on the salivary glands of Anopheles gambiae remain largely unknown. In previous studies, a salivary gland protein of ~100 kDa was identified as a putative target based on recognition of the protein by a monoclonal antibody (mAb) 2A3 that caused a >/= 70% reduction in the average number of sporozoites per infected salivary gland when fed to mosquitoes. Using affinity purification we purified the target of this mAb from extracts of female A. gambiae salivary glands and it was found to be a novel protein by tandem mass spectrometric analysis. Biochemical and molecular characterization of the 100 kDa protein showed that this molecule, designated Saglin, exists as a disulphide-bonded homodimer of 50 kDa subunits. The ability to form homodimers was retained even in the recombinant Saglin expressed in mammalian cells (HEK293). The amino acid sequence of Saglin contains a signal peptide suggesting that Saglin is a secreted protein. If Saglin is indeed involved in the process of invasion of A. gambiae salivary glands by sporozoites of Plasmodium, it could provide a novel target for future investigations aimed at interruption of malaria transmission.
In recent years, Hsp90 is found to interact with several telomeric proteins at various phases of cell cycle. The Hsp90 chaperone system controls assembly and disassembly of telomere structures and thus maintains the dynamic state of telomere. Here, for the first time we report that the activity of another telomeric protein Sir2p is modulated by Hsp82, the ortholog of Hsp90 from budding yeast (Saccharomyces cerevisiae). In a temperature sensitive Hsp90 deficient yeast strain (iG170Dhsp82), less abundant Sir2p is observed, resulting in de-repression of telomere silencing and a complete loss of mating type silencing. Intriguingly, over expression of Hsp90, either by exposing cells to heat shock or by introducing HSP82 overexpression plasmid also yields reduced level of Sir2p, with a consequential loss of telomere silencing. Thus, Hsp90 homeostasis maintains the cellular pool of Sir2p and thereby controls the reversible nature of telomere silencing. Interestingly, such regulation is independent of one of its major co-chaperones Sba1 (human ortholog of p23).
The inhibition of Hsp90 in cancerous cells has been correlated with the reduction in double-strand break (DSB repair) activity. However, the precise effect of Hsp90 on the DSB repair pathway in normal cells has remained enigmatic. Our results show that the Hsp82 chaperone, the ortholog of mammalian Hsp90, is indispensable for homologous-recombination (
bRadicicol, an antifungal antibiotic, was previously identified as a compound having antimalarial activity. However, its mechanism of action in Plasmodium falciparum was not elucidated. While characterizing its antimalarial function, we observed that radicicol manifested two distinct developmental defects in cultured P. falciparum in a concentration-dependent manner. At a low concentration of radicicol, a significant percentage of drug-treated parasites were arrested at the schizont stage, while at a higher concentration, the parasites were unable to multiply from schizont to ring. Also, the newly formed rings and trophozoites were extremely delayed in development, eventually leading to cell death. We intended to characterize the potential molecular target of radicicol at its sublethal doses. Our results demonstrated that radicicol specifically impaired mitochondrial replication. This decrement was associated with a severalfold increment of the topoisomerase VIB transcript as well as protein in treated cells over that of untreated parasites. Topoisomerase VIB was found to be localized in the organelle fraction. Our docking study revealed that radicicol fits into the Bergerat fold of Pf topoisomerase VIB present in its ATPase domain. Altogether, these data allow us to conclude that P. falciparum topoisomerase VIB might be one of the targets of radicicol causing inhibition of mitochondrial replication. Hence, radicicol can be suitably employed to explore the mitochondrial physiology of malaria parasites.
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