Glyoxalate reductase/hydroxypyruvate reductase interacts with the sodium-dependent vitamin C transporter-1 to regulate cellular vitamin C homeostasis.

The human sodium-dependent vitamin C transporter 1 (hSVCT1) contributes to cellular uptake of ascorbic acid (AA). Although different aspects of hSVCT1 cell biology have been extensively studied, nothing is currently known about the broader hSVCT1 interactome that modulates its role in cellular physiology. Here, we identify the enzyme human glyoxalate reductase/hydroxypyruvate reductase (hGR/HPR) as an hSVCT1 associated protein by yeast two-hybrid (Y2H) screening of a human liver cDNA library. The interaction between hSVCT1 and hGR/HPR was further confirmed by in vitro GST pull-down assay, in vivo coimmunoprecipitation and mammalian two-hybrid firefly luciferase assays. This interaction had functional significance as coexpression of hGR/HPR with hSVCT1 led to an increase in AA uptake. Reciprocally, siRNA-mediated knockdown of endogenous hGR/HPR led to an inhibition of AA uptake. Given that oxalate is a degradation product of vitamin C and hGR/HPR acts to limit cellular oxalate levels, this association physically couples two independent regulators of cellular oxalate production. Furthermore, confocal imaging of human liver HepG2 cells coexpressing GFP-hSVCT1 and hGR/HPR-mCherry demonstrated that these two proteins colocalize within a subpopulation of intracellular organelles. This provides a possible molecular basis for organellar AA transport and regulation of local glyoxylate/glycolate concentration in the vicinity of organelle membranes.

Type 5 G protein beta subunit (Gbeta5) controls the interaction of regulator of G protein signaling 9 (RGS9) with membrane anchors.

The R7 family of regulators of G protein signaling (RGS) proteins, comprising RGS6, RGS7, RGS9, and RGS11, regulate neuronal G protein signaling pathways. All members of the R7 RGS form trimeric complexes with the atypical G protein ß subunit, Gß5, and membrane anchor R7BP or R9AP. Association with Gß5 and membrane anchors has been shown to be critical for maintaining proteolytic stability of the R7 RGS proteins. However, despite its functional importance, the mechanism of how R7 RGS forms complexes with Gß5 and membrane anchors remains poorly understood. Here, we used protein-protein interaction, co-localization, and protein stability assays to show that association of RGS9 with membrane anchors requires Gß5. We further establish that the recruitment of R7BP to the complex requires an intact interface between the N-terminal lobe of RGS9 and protein interaction surface of Gß5. Site-directed mutational analysis reveals that distinct molecular determinants in the interface between Gß5 and N-terminal Dishevelled, EGL-10, Pleckstrin/DEP Helical Extension (DEP/DHEY) domains are differentially involved in R7BP binding and proteolytic stabilization. On the basis of these findings, we conclude that Gß5 contributes to the formation of the binding site to the membrane anchors and thus is playing a central role in the assembly of the proteolytically stable trimeric complex and its correct localization in the cell.

Interrelationships between negative energy balance (NEB) and IGF regulation in liver of lactating dairy cows.

In dairy cows, negative energy balance (NEB) during the early post-partum period is associated with major alterations in the growth hormone-insulin-like growth factor (GH-IGF) axis. Since the liver mediates nutrient partitioning during lactation, we aimed to determine how NEB alters the endocrine regulation of the insulin-like growth factor (IGF) system by investigating the expression of IGF family members and related steroid receptors. On the second day of lactation, cows were allocated to one of two treatments designed to produce mild (MNEB) or severe NEB (SNEB). MNEB cows (n=5) were fed ad lib grass silage supplemented with concentrate and milked x1 daily and SNEB cows (n=6) were restricted in dietary intake and milked x3 daily. Energy balance (EB) status was monitored until the second week of lactation when plasma and liver samples revealed a markedly divergent metabolic profile. At this time, plasma protein and hepatic mRNA for IGF-I was reduced in SNEB cows compared with MNEB cows. Both levels of expression correlated highly when data from all animals was pooled (r=0.963; P<0.01). SNEB cows also exhibited reduced hepatic expression for transcripts encoding IGF-1R, IGF-2R, IGF binding proteins (IGFBPs) -3, -4, -5, -6, acid labile subunit, and receptors for oestrogen (ERalpha) and growth hormone (total GHR and 1A variant), while the expression of IGFBP-2 was elevated. Expression of mRNA for IGF-II, IGFBP-1 and receptors for insulin (A/B) and glucocorticoid (alpha) was unaffected by EB. Results demonstrate that SNEB affects hepatic synthesis of IGF-I, and other components known to modulate the bioavailability and stability of circulating IGF-I.

Induction of acid phosphatase transcripts, protein and enzymatic activity by simulated herbivory of hybrid poplar.

Herbivory and wounding upregulate a large suite of defense genes in hybrid poplar leaves. A strongly wound- and herbivore-induced gene with high similarity to Arabidopsis vegetative storage proteins (VSPs) and acid phosphatase (AP) was identified among genes strongly expressed during the poplar herbivore defense response. Phylogenetic analysis showed that the putative poplar acid phosphatase (PtdAP1) gene is part of an eight-member AP gene family in poplar, and is most closely related to a functionally characterized soybean nodule AP. Unlike the other poplar APs, PtdAP1 is expressed in variety of tissues, as observed in an analysis of EST data. Following wounding, the gene shows an expression profile similar to other known poplar defense genes such as protease inhibitors, chitinase, and polyphenol oxidase. Significantly, we show for the first time that subsequent to the wound-induction of PtdAP1 transcripts, AP protein and activity increase in extracts of leaves and other tissues. Although its mechanism of action is as yet unknown, these results suggest in hybrid poplar PtdAP1 is likely a component of the defense response against leaf-eating herbivores.