N-Myc downstream regulated gene 3 (NDRG3) is a unique pro-tumorigenic member among NDRG family genes, mediating growth signals. Here, we investigated the pathophysiological roles of NDRG3 in relation to cell metabolism by disrupting its functions in liver. Mice with liver-specific KO of NDRG3 (Ndrg3 LKO) exhibited glycogen storage disease (GSD) phenotypes including excessive hepatic glycogen accumulation, hypoglycemia, elevated liver triglyceride content, and several signs of liver injury. They suffered from impaired hepatic glucose homeostasis, due to the suppression of fasting-associated glycogenolysis and gluconeogenesis. Consistently, the expression of glycogen phosphorylase (PYGL) and glucose-6-phosphate transporter (G6PT) was significantly down-regulated in an Ndrg3 LKO-dependent manner. Transcriptomic and metabolomic analyses revealed that NDRG3 depletion significantly perturbed the methionine cycle, redirecting its flux towards branch pathways to upregulate several metabolites known to have hepatoprotective functions. Mechanistically, Ndrg3 LKO-dependent downregulation of glycine N-methyltransferase in the methionine cycle and the resultant elevation of the S-adenosylmethionine level appears to play a critical role in the restructuring of the methionine metabolism, eventually leading to the manifestation of GSD phenotypes in Ndrg3 LKO mice. Our results indicate that NDRG3 is required for the homeostasis of liver cell metabolism upstream of the glucose-glycogen flux and methionine cycle and suggest therapeutic values for regulating NDRG3 in disorders with malfunctions in these pathways.
The N-Myc downstream-regulated gene (NDRG) family belongs to the alpha/beta-hydrolase fold and is known to exert various physiologic functions in cell proliferation, dierentiation, and hypoxia-induced cancer metabolism. In particular, NDRG3 is closely related to proliferation and migration of prostate cancer cells, and recent studies reported its implication in lactate-triggered hypoxia responses or tumorigenesis. However, the underlying mechanism for the functions of NDRG3 remains unclear. Here, we report the crystal structure of human NDRG3 at 2.2 resolution, with six molecules in an asymmetric unit. While NDRG3 adopts the alpha/beta-hydrolase fold, complete substitution of the canonical catalytic triad residues to non-reactive residues and steric hindrance around the pseudo-active site seem to disable the alpha/beta-hydrolase activity. While NDRG3 shares a high similarity to NDRG2 in terms of amino acid sequence and structure, NDRG3 exhibited remarkable structural differences in a flexible loop corresponding to helix alpha6 of NDRG2 that is responsible for tumor suppression. Thus, this flexible loop region seems to play a distinct role in oncogenic progression induced by NDRG3. Collectively, our studies could provide structural and biophysical insights into the molecular characteristics of NDRG3.
Organisms must be able to respond to low oxygen in a number of homeostatic and pathological contexts. Regulation of hypoxic responses via the hypoxia-inducible factor (HIF) is well established, but evidence indicates that other, HIF-independent mechanisms are also involved. Here, we report a hypoxic response that depends on the accumulation of lactate, a metabolite whose production increases in hypoxic conditions. We find that the NDRG3 protein is degraded in a PHD2/VHL-dependent manner in normoxia but is protected from destruction by binding to lactate that accumulates under hypoxia. The stabilized NDRG3 protein binds c-Raf to mediate hypoxia-induced activation of Raf-ERK pathway, promoting angiogenesis and cell growth. Inhibiting cellular lactate production abolishes the NDRG3-mediated hypoxia responses. Our study, therefore, elucidates the molecular basis for lactate-induced hypoxia signaling, which can be exploited for the development of therapies targeting hypoxia-induced diseases.
Title: NDRG3-mediated lactate signaling in hypoxia Park KC, Lee DC, Yeom YI Ref: BMB Rep, 48:301, 2015 : PubMed
Hypoxia is associated with many pathological conditions as well as the normal physiology of metazoans. We identified a lactate-dependent signaling pathway in hypoxia, mediated by the oxygen- and lactate-regulated protein NDRG family member 3 (NDRG3). Oxygen negatively regulates NDRG3 expression at the protein level via the PHD2/VHL system, whereas lactate, produced in excess under prolonged hypoxia, blocks its proteasomal degradation by binding to NDRG3. We also found that the stabilized NDRG3 protein promotes angiogenesis and cell growth under hypoxia by activating the Raf-ERK pathway. Inhibiting cellular lactate production abolishes NDRG3-mediated hypoxia responses. The NDRG3-Raf-ERK axis therefore provides the genetic basis for lactate-induced hypoxia signaling, which can be exploited for the development of therapies targeting hypoxia-induced diseases in addition to advancing our understanding of the normal physiology of hypoxia responses.
BACKGROUND: N-myc downstream regulated gene 2 (NDRG2) belongs to the NDRG family, which is comprised of 4 members, NDRG1-4. Recently, NDRG2 was reported as a new candidate for a tumor suppressor gene. We developed a reverse-phase protein microarray assay to access NDRG2 levels in human tissue specimens and cell lines. METHODS: We synthesized recombinant NDRG2 protein and produced monoclonal antibodies (mAb) to the NDRG2 protein. We selected 2 hybridomas producing mAb that specifically recognize the NDRG2 protein. To determine the NDRG2 concentration, the samples of serially-diluted NDRG2 protein, cell lysate, or tissue lysate were spotted onto a nitrocellulose membrane-coated slide glass and allowed to react with the mAb to the NDRG2 protein. The reaction was followed by additional incubation with biotin-linked anti-mouse IgG and horseradish peroxidase (HRP)-conjugated streptavidin, subsequently. The addition of dimethylaminobenzidine induced color development, which was measured using the GenePix program. We determined the NDRG2 concentration in various tissue specimens and cell lines using the new protein microarray technique. RESULTS: The dose-response relationship between NDRG2 and color intensity showed linearity in a range 0-10 ng/ml and a sensitivity of 50 pg/ml. The NDRG2 concentrations in the liver tissue lysates of patients with hepatocellular carcinoma (52.0+21.5 ng/mg) were significantly diminished as compared with those in the normal liver tissues (549.6+94.6 ng/mg). The results of the assay showed good agreement with those of Western blot analysis. CONCLUSIONS: The protein microarray is a highly sensitive and accurate method, and can adopted to assess specific proteins in human tissues or cell lines, particularly in the field of cancer and pathological research.
