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Zeraik, A. E.

Publications and source records attributed to Zeraik, A. E..

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Mechanisms determining Schistosoma mansoni CRAC channel activation

Schistosoma mansoni and its schistosome relatives are parasitic worms that impose a substantial disease burden on human populations and livestock. On the rationale that calcium signalling is a critical process in multicellular organisms, we have examined wildtype and engineered S. mansoni STIM and ORAI-- orthologues of STIM and ORAI known in mammals and other species for their central role in cellular calcium signalling-- by imaging their localization, interactions, and contribution to ion currents and calcium influx in living cells. The ER membrane protein S. mansoni STIM recapitulates the essential functions of mammalian STIM1, namely, calcium-sensing by its ER-luminal domain, targeting to ER-plasma membrane junctions through interactions with the plasma membrane and with plasma membrane S. mansoni ORAI channels, and an ability to gate the S. mansoni ORAI channel. S. mansoni ORAI is a plasma membrane calcium channel that exhibits striking parallels with mammalian ORAI1 in its pore architecture and gating mechanism. The schistosome and human proteins are not completely interchangeable, however, and schistosome-human ORAI chimeras point to a special role of the ORAI N terminus in channel gating. Importantly, we demonstrate pharmacological differences between the schistosome and human channels that may offer an opportunity for selective therapeutic targeting of schistosome STIM-ORAI-dependent calcium entry. Author SummaryCalcium channels represent potential targets to parasitic helminths. We investigated Schistosoma mansoni CRAC channel activation through the expression of its proteins. We have established that the fundamental protein conformational changes and protein-protein interactions underlying STIM-ORAI signaling are shared between humans and schistosome proteins. Importantly, a key finding is that evolutionary divergence in residues that are not implicated in the basic mechanisms of STIM-ORAI activation appears to offer a window for pharmacological inhibitors that would be selective for the schistosome ORAI channel. We identified pharmacological differences for two compounds tested. These differences open avenues for the development of selective drugs that can target the S. mansoni CRAC channel without affecting human physiology, thus offering the prospect of new treatments for schistosomiasis.

biochemistry↗

The molecular structure of Schistosoma mansoni PNP isoform 2 provides insights into the nucleotide selectivity of PNPs

Purine nucleoside phosphorylases (PNPs) play an important role in the blood fluke parasite Schistosoma mansoni as a key enzyme of the purine salvage pathway. Here we present the structural and kinetic characterization of a new PNP isoform from S. mansoni, named as SmPNP2. Screening of different ligands using a thermofluorescence approach indicated cytidine and cytosine as potential ligands. The binding of cytosine was confirmed by isothermal titration calorimetry, with a KD of 27 M, and kinetic parameters for cytidine catalysis were obtained by ITC resulting in a KM of 76.3 M. SmPNP2 also displays catalytic activity against inosine and adenosine, making it the first described PNP with robust catalytic activity towards both pyrimidines and purines. Crystallographic structures of SmPNP2 with different ligands were obtained and comparison of these structures with the previously described S. mansoni PNP (SmPNP1) provided clues for the unique capability of SmPNP2 to bind pyrimidines. When compared with the structure of SmPNP1, substitutions in the vicinity of SmPNP2 active site alter the architecture of the nucleoside base binding site allowing an alternative binding mode for nucleosides, with a 180{degrees} rotation from the canonical binding mode. The remarkable plasticity of this binding site deepens the understanding of the correlation between structure and nucleotide selectivity, offering new ways to analyses PNP activity.\n\nAuthor SummarySchistosoma mansoni is a human parasite dependent on purine salvage for purine bases supply. Purine nucleoside phosphorylase (PNP) is a key enzyme in this pathway. It carries two PNP isoforms, one previously characterized (SmPNP1) and one unknown (SmPNP2). Here we present the crystallographic structure of SmPNP2 and its complex with cytosine, cytidine, ribose-l-phosphate, adenine, hypoxanthine, and tubercidin. Cytidine and cytosine were identified as ligands of SmPNP2 using a thermofluorescence approach. Binding of cytosine was proven by Isothermal Titration Calorimetry (ITC) and cytidine, inosine, and adenosine kinetic parameters were also obtained. Purine bases showed different binding in the active site, rotated 180{degrees} from the canonical binding mode. Its the first report showing a Low Molecular Mass PNP capable of catalyzing both types of nucleotide bases. The SmPNP2 odd behavior sheds a new light on the Schistosoma mansonis life cycle metabolic adaptation.

biochemistry↗