Molecular basis for the lack of enantioselectivity of human 3-phosphoglycerate kinase

Non-natural l-nucleoside analogues are increasingly used as therapeutic agents to treat cancer and viral infections. To be active, l-nucleosides need to be phosphorylated to their respective triphosphate metabolites. This stepwise phosphorylation relies on human enzymes capable of processing l-nucleoside enantiomers. We used crystallographic analysis to reveal the molecular basis for the low enantioselectivity and the broad specificity of human 3-phosphoglycerate kinase (hPGK), an enzyme responsible for the last step of phosphorylation of many nucleotide derivatives. Based on structures of hPGK in the absence of nucleotides, and bound to l and d forms of MgADP and MgCDP, we show that a non-specific hydrophobic clamp to the nucleotide base, as well as a water-filled cavity behind it, allows high flexibility in the interaction between PGK and the bases. This, combined with the dispensability of hydrogen bonds to the sugar moiety, and ionic interactions with the phosphate groups, results in the positioning of different nucleotides so to expose their diphosphate group in a position competent for catalysis. Since the third phosphorylation step is often rate limiting, our results are expected to alleviate in silico tailoring of l-type prodrugs to assure their efficient metabolic processing

Molecular basis for the lack of enantioselectivity of human 3-phosphoglycerate kinase

Non-natural l-nucleoside analogues are increasingly used as therapeutic agents to treat cancer and viral infections. To be active, l-nucleosides need to be phosphorylated to their respective triphosphate metabolites. This stepwise phosphorylation relies on human enzymes capable of processing l-nucleoside enantiomers. We used crystallographic analysis to reveal the molecular basis for the low enantioselectivity and the broad specificity of human 3-phosphoglycerate kinase (hPGK), an enzyme responsible for the last step of phosphorylation of many nucleotide derivatives. Based on structures of hPGK in the absence of nucleotides, and bound to l and d forms of MgADP and MgCDP, we show that a non-specific hydrophobic clamp to the nucleotide base, as well as a water-filled cavity behind it, allows high flexibility in the interaction between PGK and the bases. This, combined with the dispensability of hydrogen bonds to the sugar moiety, and ionic interactions with the phosphate groups, results in the positioning of different nucleotides so to expose their diphosphate group in a position competent for catalysis. Since the third phosphorylation step is often rate limiting, our results are expected to alleviate in silico tailoring of l-type prodrugs to assure their efficient metabolic processing

P113 is a merozoite surface protein that binds the N terminus of Plasmodium falciparum RH5.

Invasion of erythrocytes by Plasmodium falciparum merozoites is necessary for malaria pathogenesis and is therefore a primary target for vaccine development. RH5 is a leading subunit vaccine candidate because anti-RH5 antibodies inhibit parasite growth and the interaction with its erythrocyte receptor basigin is essential for invasion. RH5 is secreted, complexes with other parasite proteins including CyRPA and RIPR, and contains a conserved N-terminal region (RH5Nt) of unknown function that is cleaved from the native protein. Here, we identify P113 as a merozoite surface protein that directly interacts with RH5Nt. Using recombinant proteins and a sensitive protein interaction assay, we establish the binding interdependencies of all the other known RH5 complex components and conclude that the RH5Nt-P113 interaction provides a releasable mechanism for anchoring RH5 to the merozoite surface. We exploit these findings to design a chemically synthesized peptide corresponding to RH5Nt, which could contribute to a cost-effective malaria vaccine

RGK GTPase-dependent CaV2.1 Ca2+ channel inhibition is independent of CaVbeta-subunit-induced current potentiation.

International audienceRGK (Rad-Gem-Rem) GTPases have been described as potent negative regulators of the Ca(2+) influx via high-threshold voltage-activated Ca(2+) channels. Recent work, mostly performed on Ca(V)1.2 Ca(2+) channels, has highlighted the crucial role played by the channel auxiliary Ca(V)beta subunits and identified several GTPase and beta-subunit protein domains involved in this regulation. We now extend these conclusions by producing the first complete characterization of the effects of Gem, Rem, and Rem2 on the neuronal Ca(V)2.1 Ca(2+) channels expressed with Ca(V)beta(1) or Ca(V)beta(2) subunits. Current inhibition is limited to a decrease in amplitude with no modification in the voltage dependence or kinetics of the current. We demonstrate that this inhibition can occur for Ca(V)beta constructs with impaired capacity to induce current potentiation, but that it is lost for Ca(V)beta constructs deleted for their beta-interaction domain. The RGK C-terminal last approximately 80 amino acids are sufficient to allow potent current inhibition and in vivo beta-subunit/Gem interaction. Interestingly, although Gem and Gem carboxy-terminus induce a completely different pattern of beta-subunit cellular localization, they both potently inhibit Ca(V)2.1 channels. These data therefore set the status of neuronal Ca(V)2.1 Ca(2+) channel inhibition by RGK GTPases, emphasizing the role of short amino acid sequences of both proteins in beta-subunit binding and channel inhibition and revealing a new mechanism for channel inhibition

HCV core-mediated activation of latent TGF-β via thrombospondin drives the crosstalk between hepatocytes and stromal environment

The mechanisms by which fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) develop during chronic hepatitis C virus (HCV) infection are not fully understood. We previously observed that HCV core protein induced a TGF-β-dependent epithelial mesenchymal transition, a process contributing to the promotion of cell invasion and metastasis by impacting TGF-β1 signalling. Here we investigated HCV core capacity to drive increased expression of the active form of TGF-β1n transgenic mice and hepatoma cell lines