Isocitrate Dehydrogenase Alpha-1 Modulates Lifespan and Oxidative Stress Tolerance in Caenorhabditis elegans.

Altered metabolism is a hallmark of aging. The tricarboxylic acid cycle (TCA cycle) is an essential metabolic pathway and plays an important role in lifespan regulation. Supplementation of α-ketoglutarate, a metabolite converted by isocitrate dehydrogenase alpha-1 (idha-1) in the TCA cycle, increases lifespan in C. elegans. However, whether idha-1 can regulate lifespan in C. elegans remains unknown. Here, we reported that the expression of idha-1 modulates lifespan and oxidative stress tolerance in C. elegans. Transgenic overexpression of idha-1 extends lifespan, increases the levels of NADPH/NADP+ ratio, and elevates the tolerance to oxidative stress. Conversely, RNAi knockdown of idha-1 exhibits the opposite effects. In addition, the longevity of eat-2 (ad1116) mutant via dietary restriction (DR) was reduced by idha-1 knockdown, indicating that idha-1 may play a role in DR-mediated longevity. Furthermore, idha-1 mediated lifespan may depend on the target of rapamycin (TOR) signaling. Moreover, the phosphorylation levels of S6 kinase (p-S6K) inversely correlate with idha-1 expression, supporting that the idha-1-mediated lifespan regulation may involve the TOR signaling pathway. Together, our data provide new insights into the understanding of idha-1 new function in lifespan regulation probably via DR and TOR signaling and in oxidative stress tolerance in C. elegans.

Experimentally validated novel inhibitors of Helicobacter pylori phosphopantetheine adenylyltransferase discovered by virtual high-throughput screening.

Helicobacter pylori is a major etiologic agent associated with the development and maintenance of human gastritis. The goal of this study was to develop novel antibiotics against H. pylori, and we thus targeted H. pylori phosphopantetheine adenylyltransferase (HpPPAT). PPAT catalyzes the penultimate step in coenzyme A biosynthesis. Its inactivation effectively prevents bacterial viability, making it an attractive target for antibacterial drug discovery. We employed virtual high-throughput screening and the HpPPAT crystal structure to identify compounds in the PubChem database that might act as inhibitors of HpPPAT. d-amethopterin is a potential inhibitor for blocking HpPPAT activity and suppressing H. pylori viability. Following treatment with d-amethopterin, H. pylori exhibited morphological characteristics associated with cell death. d-amethopterin is a mixed inhibitor of HpPPAT activity; it simultaneously occupies the HpPPAT 4'-phosphopantetheine- and ATP-binding sites. Its binding affinity is in the micromolar range, implying that it is sufficiently potent to serve as a lead compound in subsequent drug development. Characterization of the d-amethopterin and HpPPAT interaction network in a docked model will allow us to initiate rational drug optimization to improve the inhibitory efficacy of d-amethopterin. We anticipate that novel, potent, and selective HpPPAT inhibitors will emerge for the treatment of H. pylori infection.

Crystal structure and biophysical characterisation of Helicobacter pylori phosphopantetheine adenylyltransferase.

Helicobacter pylori is a bacterium that causes chronic active gastritis and peptic ulcers. Drugs targeting H. pylori phosphopantetheine adenylyltransferase (HpPPAT), which is involved in CoA biosynthesis, may be useful. Herein, we report the expression in Escherichia coli and purification of recombinant HpPPAT and describe a crystal structure for an HpPPAT/CoA complex. As is the case for E. coli PPAT (EcPPAT), HpPPAT is hexameric in solution and as a crystal. Each protomer has a well-packed dinucleotide-binding fold in which CoA binds. Structural characterisation demonstrated that CoA derived from the E. coli expression system bound tightly to HpPPAT, presumably to initiate feedback inhibition. However, the interactions between the active-site residues of HpPPAT and CoA are not identical to those of other PPATs. Finally, CoA binding affects HpPPAT thermal denaturation.

Structural and functional comparison of cytokine interleukin-1 beta from chicken and human.

Interleukin-1 beta (IL-1ß) is an important cytokine in the immune system. The properties of avian IL-1ßs are less well understood than the mammalian IL-1ßs, and there is no available structure of avian IL-1ßs in the Protein Data Bank. Here, we report the crystal structures of wild-type and Y157F mutant IL-1ßs from chicken. Both the wild-type and mutant IL-1ßs share a beta-trefoil conformation similar to that of human IL-1ß and also have an internal hydrophobic cavity. However, the cavity sizes clearly differ from that of human IL-1ß due to the packing of hydrophobic residues. Our studies also reveal that the relative thermal stability of IL-1ßs does not correlate with cavity size but rather is dependent on the amino acid residues present around the cavity. This cavity serves as a scaffold for maintaining the structure of the IL-1ß core region but does not have a biological function per se. Moreover, we found that human IL-1ß cannot induce chemokine expression in chicken fibroblasts or elevate plasma cortisol levels in chickens, implying a lack of cross-species bioactivity. Close examination reveals that significant structural and sequence differences occur in the terminal and some loop regions between human and chicken IL-1ßs. These variable regions have been shown to be critical for receptor binding, thus resulting in a lack of species cross-reactivity between human and chicken IL-1ß.