The citric acid cycle is central to the regulation of energy homeostasis and cell metabolism 1 . Mutations in enzymes that catalyse steps in the citric acid cycle result in human diseases with various clinical presentations 2 . The intermediates of the citric acid cycle are present at micromolar concentration in blood and are regulated by respiration, metabolism and renal reabsorption/ extrusion. Here we show that GPR91 (ref.3), a previously orphan G-protein-coupled receptor (GPCR), functions as a receptor for the citric acid cycle intermediate succinate. We also report that GPR99 (ref. 4), a close relative of GPR91, responds to a-ketoglutarate, another intermediate in the citric acid cycle. Thus by acting as ligands for GPCRs, succinate and a-ketoglutarate are found to have unexpected signalling functions beyond their traditional roles. Furthermore, we show that succinate increases blood pressure in animals. The succinate-induced hypertensive effect involves the renin-angiotensin system and is abolished in GPR91-deficient mice. Our results indicate a possible role for GPR91 in renovascular hypertension, a disease closely linked to atherosclerosis, diabetes and renal failure 5,6 .In a search for natural ligands for orphan GPCRs, we tested extracts from various animal tissues for their ability to evoke an increase in intracellular Ca 2þ concentration ([Ca 2þ ] i ) using the aequorin assay 7 . We found that fractions from pig kidney extracts specifically activated cells expressing GPR91 (Fig. 1a). GPR91 is an orphan GPCR highly expressed in the kidney and shares 33% amino acid identity with GPR99/GPR80 (refs 4, 8). On the basis of their homology with the purinergic receptor P2Y1, nucleotide ligands were predicted for GPR91 and GPR99 (ref. 4). However, the GPR91 ligand activity in pig kidney extracts was resistant to various stringent treatments including alkaline phosphatase, peptidase, and hydrolysis in 6 M HCl at 100 8C. Accordingly, the supposition that GPR91 might be activated by a nucleotide or peptide ligand was unlikely. We purified the natural ligand for GPR91 by ion-exchange, size-exclusion and reversed-phase fast performance liquid chromatography/high-performance liquid chromatography (Fig. 1a).A major molecular ion [M þ H] þ at m/z 119.2 was observed by mass spectrometry (Fig. 1b). 1 H NMR analysis revealed a single type of proton in the highly purified GPR91 ligand (Fig. 1c). 13 C NMR analysis further suggested the presence of -CH 2 -(methylene) and ¼C¼O (carbonyl) groups (Fig. 1d). Combined with mass spectrometry results and the biochemical properties of the ligand, the purified GPR91 ligand was predicted and confirmed to be succinic acid (Fig. 1c, d).Commercially obtained succinate (the physiological form of succinic acid) increased [Ca 2þ ] i dose-dependently in the aequorin assay (Fig. 2a). Succinate also activated mouse and rat orthologues of GPR91 (Fig. 2a). The succinate-induced increase in [Ca 2þ ] i was further confirmed with a fluorimetric imaging plate reader (FLIPR) system in the 293-hGP...

Astragalus-based Chinese herbal medicine may increase effectiveness of platinum-based chemotherapy when combined with chemotherapy. These results require confirmation with rigorously controlled trials.

Methionine, folate, vitamin B(6), vitamin B(12), niacin, and riboflavin intakes may be related to breast carcinogenesis. These associations may vary by breast cancer type. Using the prospective cohort Shanghai Women's Health Study (1997-2008) including 718 Chinese breast cancer cases, the authors evaluated baseline dietary intake of these factors and breast cancer risk and whether the associations varied by menopausal status and estrogen receptor (ER) and progesterone receptor (PR) status. They estimated associations using hazard ratios and 95% confidence intervals from Cox proportional hazards regression models and stratified analyses by menopausal status and ER/PR status. Lowest quantile of intake was used as the comparison group. For postmenopausal women, dietary intakes of methionine and B vitamins were not associated with breast cancer risk. For premenopausal women, higher intake of folate was associated with decreased breast cancer risk (hazard ratio = 0.58, 95% confidence interval: 0.34, 0.99 for the highest vs. lowest quintile of intake). Only niacin intake was associated with ER+/PR+ breast cancer risk (hazard ratio = 1.62, 95% confidence interval: 1.07, 2.46; P for trend = 0.04 for the highest vs. lowest quartile of intake). Findings support the hypothesis that high folate intake may reduce breast cancer risk and that the association may vary by menopausal and ER/PR status.

Because the folate pathway is essential for microorganisms but absent from mammals, HPPK, like other enzymes in the pathway, is an important target for developing antimicrobial agents.HPPK belongs to a class of enzymes that catalyze the pyrophosphoryl transfer reaction (2). Although the mechanisms of many kinases that catalyze monophosphoryl transfer have been extensively characterized, little is known about the mechanisms of pyrophosphokinases. As a small (158 residues, ϳ18 kDa), stable, monomeric protein, Escherichia coli HPPK is an excellent model system for studying the mechanisms of enzymatic pyrophosphoryl transfer.We have recently determined the crystal structures of ligand-free (apo-) E. coli HPPK (1) and its complex with HP, ␣,␤-methyleneadenosine 5Ј-triphosphate (AMPCPP), and two Mg 2ϩ ions (3) at 1.5 and 1.25 Å resolution, respectively. Hennig and co-workers (4) have determined the crystal structure of Haemophilus influenzae HPPK in complex with an HP analog at 2.05-Å resolution. Stammers and co-workers (5) have determined the crystal structure of E. coli HPPK in complex with an HP analog, ATP, and two Mg 2ϩ ions at 2.00-Å resolution. Comparative analysis of the crystal structures of the apo-HPPK and its ternary complex has revealed the interactions of the enzyme with the substrates at the atomic resolution and the dramatic substrate-induced conformational changes involving three catalytic loops (3). It appears that the complete active center of HPPK is assembled only after both substrates bind to the enzyme. However, how the catalytic center is assembled is not known. It appears that some catalytic residues cannot move into their catalytic positions because of steric constraints.In this paper, we present the crystal structure of HPPK⅐MgADP at 1.5-Å resolution and the NMR solution structure of HPPK⅐MgAMPPCP. The two structures reveal a dramatic, unusual movement of loop 3 and other significant changes in the conformation and dynamical property of HPPK. The dramatic substrate-induced movement of loop 3 is unusual because it moves away from the active center. Comparative structural analysis suggests that the structures reported here may represent an intermediate conformation required for both substrate binding and product release in the catalytic cycle. EXPERIMENTAL PROCEDURESCrystal Structure Determination of HPPK⅐MgADP-E. coli HPPK was purified as previously described (6). Crystals of HPPK⅐MgADP were grown in hanging drops at 19 Ϯ 1°C. The protein solution contained 4 mg/ml HPPK, 5 mM MgATP in 10 mM Tris buffer (pH 8.0), and the reservoir contained 30% polyethylene glycol 4000, 0.2 M NaAc in 0.2 M Tris buffer (pH 8.5). The 4-l hanging drops contained equal volumes (2 l) of protein solution and reservoir solution. Two rounds of seeding produced diffraction-quality crystals. * This work was supported in part by National Institutes of Health Grant GM51901 (to H. Y.). This study made use of a Varian INOVA-600 NMR spectrometer at Michigan State University funded in part by National Science Foundation Grant...