Cost-effective selective deuteration of aromatic amino acid residues produces long-lived solution 1H NMR magnetization in proteins.

Solution NMR studies of large proteins are hampered by rapid signal decay due to short-range dipolar 1H-1H and 1H-13C interactions. These are attenuated by rapid rotation in methyl groups and by deuteration (2H), so selective 1H,13C-isotope labelling of methyl groups in otherwise perdeuterated proteins, combined with methyl transverse relaxation optimized spectroscopy (methyl-TROSY), is now standard for solution NMR of large protein systems > 25 kDa. For non-methyl positions, long-lived magnetization can be introduced as isolated 1H-12C groups. We have developed a cost-effective chemical synthesis for producing selectively deuterated phenylpyruvate and hydroxyphenylpyruvate. Feeding these amino acid precursors to E. coli in D2O, along with selectively deuterated anthranilate and unlabeled histidine, results in isolated and long-lived 1H magnetization in the aromatic rings of Phe (HD, HZ), Tyr (HD), Trp (HH2, HE3) and His (HD2 and HE1). We are additionally able to obtain stereoselective deuteration of Asp, Asn, and Lys amino acid residues using unlabeled glucose and fumarate as carbon sources and oxalate and malonate as metabolic inhibitors. Combining these approaches produces isolated 1H-12C groups in Phe, Tyr, Trp, His, Asp, Asn, and Lys in a perdeuterated background, which is compatible with standard 1H-13C labeling of methyl groups in Ala, Ile, Leu, Val, Thr, Met. We show that isotope labeling of Ala is improved using the transaminase inhibitor L-cycloserine, and labeling of Thr is improved through addition of Cys and Met, which are known inhibitors of homoserine dehydrogenase. We demonstrate the creation of long-lived 1H NMR signals in most amino acid residues using our model system, the WW domain of human Pin1, as well as the bacterial outer membrane protein PagP.

A role for phosphatidylcholine and phosphatidylethanolamine in hepatic insulin signaling.

Phosphatidylethanolamine N-methyltransferase (PEMT) is an important enzyme in hepatic phosphatidylcholine (PC) biosynthesis. Pemt-/- mice fed a high-fat diet are protected from obesity and whole-body insulin resistance. However, Pemt-/- mice develop severe nonalcoholic steatohepatitis (NASH). Because NASH is often associated with hepatic insulin resistance, we investigated whether the increased insulin sensitivity in Pemt-/- mice was restricted to nonhepatic tissues or whether the liver was also insulin sensitive. Strikingly, the livers of Pemt-/- mice compared with those of Pemt+/+ mice were not insulin resistant, despite elevated levels of hepatic triacylglycerols and diacylglycerols, as well as increased hepatic inflammation and fibrosis. Endogenous glucose production was lower in Pemt-/- mice under both basal and hyperinsulinemic conditions. Experiments in primary hepatocytes and hepatoma cells revealed improved insulin signaling in the absence of PEMT, which was not due to changes in diacylglycerols, ceramides, or gangliosides. On the other hand, the phospholipid composition in hepatocytes seems critically important for insulin signaling such that lowering the PC:phosphatidylethanolamine (PE) ratio improves insulin signaling. Thus, treatments to reduce the PC:PE ratio in liver may protect against the development of hepatic insulin resistance.-Van der Veen, J. N., Lingrell, S., McCloskey, N., LeBlond, N. D., Galleguillos, D., Zhao, Y. Y., Curtis, J. M., Sipione, S., Fullerton, M. D., Vance, D. E., Jacobs, R. L. A role for phosphatidylcholine and phosphatidylethanolamine in hepatic insulin signaling.

Doxycycline attenuates breast cancer related inflammation by decreasing plasma lysophosphatidate concentrations and inhibiting NF-κB activation.

BACKGROUND: We previously discovered that tetracyclines increase the expression of lipid phosphate phosphatases at the surface of cells. These enzymes degrade circulating lysophosphatidate and therefore doxycycline increases the turnover of plasma lysophosphatidate and decreases its concentration. Extracellular lysophosphatidate signals through six G protein-coupled receptors and it is a potent promoter of tumor growth, metastasis and chemo-resistance. These effects depend partly on the stimulation of inflammation that lysophosphatidate produces. METHODS: In this work, we used a syngeneic orthotopic mouse model of breast cancer to determine the impact of doxycycline on circulating lysophosphatidate concentrations and tumor growth. Cytokine/chemokine concentrations in tumor tissue and plasma were measured by multiplexing laser bead technology. Leukocyte infiltration in tumors was analyzed by immunohistochemistry. The expression of IL-6 in breast cancer cell lines was determined by RT-PCR. Cell growth was measured in Matrigel™ 3D culture. The effects of doxycycline on NF-κB-dependent signaling were analyzed by Western blotting. RESULTS: Doxycycline decreased plasma lysophosphatidate concentrations, delayed tumor growth and decreased the concentrations of several cytokines/chemokines (IL-1ß, IL-6, IL-9, CCL2, CCL11, CXCL1, CXCL2, CXCL9, G-CSF, LIF, VEGF) in the tumor. These results were compatible with the effects of doxycycline in decreasing the numbers of F4/80+ macrophages and CD31+ blood vessel endothelial cells in the tumor. Doxycycline also decreased the lysophosphatidate-induced growth of breast cancer cells in three-dimensional culture. Lysophosphatidate-induced Ki-67 expression was inhibited by doxycycline. NF-κB activity in HEK293 cells transiently expressing a NF-κB-luciferase reporter vectors was also inhibited by doxycycline. Treatment of breast cancer cells with doxycycline also decreased the translocation of NF-κB to the nucleus and the mRNA levels for IL-6 in the presence or absence of lysophosphatidate. CONCLUSION: These results contribute a new dimension for understanding the anti-inflammatory effects of tetracyclines, which make them potential candidates for adjuvant therapy of cancers and other inflammatory diseases.

Tetracyclines increase lipid phosphate phosphatase expression on plasma membranes and turnover of plasma lysophosphatidate.

Extracellular lysophosphatidate and sphingosine 1-phosphate (S1P) are important bioactive lipids, which signal through G-protein-coupled receptors to stimulate cell growth and survival. The lysophosphatidate and S1P signals are terminated partly by degradation through three broad-specificity lipid phosphate phosphatases (LPPs) on the cell surface. Significantly, the expression of LPP1 and LPP3 is decreased in many cancers, and this increases the impact of lysophosphatidate and S1P signaling. However, relatively little is known about the physiological or pharmacological regulation of the expression of the different LPPs. We now show that treating several malignant and nonmalignant cell lines with 1 µg/ml tetracycline, doxycycline, or minocycline significantly increased the extracellular degradation of lysophosphatidate. S1P degradation was also increased in cells that expressed high LPP3 activity. These results depended on an increase in the stabilities of the three LPPs and increased expression on the plasma membrane. We tested the physiological significance of these results and showed that treating rats with doxycycline accelerated the clearance of lysophosphatidate, but not S1P, from the circulation. However, administering 100 mg/kg/day doxycycline to mice decreased plasma concentrations of lysophosphatidate and S1P. This study demonstrates a completely new property of tetracyclines in increasing the plasma membrane expression of the LPPs.

Autotaxin is an inflammatory mediator and therapeutic target in thyroid cancer.

Autotaxin is a secreted enzyme that converts extracellular lysophosphatidylcholine to lysophosphatidate (LPA). In cancers, LPA increases tumour growth, metastasis and chemoresistance by activating six G-protein coupled receptors. We examined >200 human thyroid biopsies. Autotaxin expression in metastatic deposits and primary carcinomas was four- to tenfold higher than in benign neoplasms or normal thyroid tissue. Autotaxin immunohistochemical staining was also increased in benign neoplasms with leukocytic infiltrations. Malignant tumours were distinguished from benign tumours by high tumour autotaxin, LPA levels and inflammatory mediators including IL1ß, IL6, IL8, GMCSF, TNFα, CCL2, CXCL10 and platelet-derived growth factor (PDGF)-AA. We determined the mechanistic explanation for these results and revealed a vicious regulatory cycle in which LPA increased the secretion of 16 inflammatory modulators in papillary thyroid cancer cultures. Conversely, treating cancer cells with ten inflammatory cytokines and chemokines or PDGF-AA and PDGF-BB increased autotaxin secretion. We confirmed that this autotaxin/inflammatory cycle occurs in two SCID mouse models of papillary thyroid cancer by blocking LPA signalling using the autotaxin inhibitor ONO-8430506. This decreased the levels of 16 inflammatory mediators in the tumours and was accompanied by a 50-60% decrease in tumour volume. This resulted from a decreased mitotic index for the cancer cells and decreased levels of vascular endothelial growth factor and angiogenesis in the tumours. Our results demonstrate that the autotaxin/inflammatory cycle is a focal point for driving malignant thyroid tumour progression and possibly treatment resistance. Inhibiting autotaxin activity provides an effective and novel strategy for decreasing the inflammatory phenotype in thyroid carcinomas, which should complement other treatment modalities.

Regulation of autotaxin expression and secretion by lysophosphatidate and sphingosine 1-phosphate.

Autotaxin (ATX) is a secreted enzyme, which produces extracellular lysophosphatidate (LPA) from lysophosphatidylcholine (LPC). LPA activates six G protein-coupled receptors and this is essential for vasculogenesis during embryonic development. ATX is also involved in wound healing and inflammation, and in tumor growth, metastasis, and chemo-resistance. It is, therefore, important to understand how ATX is regulated. It was proposed that ATX activity is inhibited by its product LPA, or a related lipid called sphingosine 1-phosphate (S1P). We now show that this apparent inhibition is ineffective at the high concentrations of LPC that occur in vivo. Instead, feedback regulation by LPA and S1P is mediated by inhibition of ATX expression resulting from phosphatidylinositol-3-kinase activation. Inhibiting ATX activity in mice with ONO-8430506 severely decreased plasma LPA concentrations and increased ATX mRNA in adipose tissue, which is a major site of ATX production. Consequently, the amount of inhibitor-bound ATX protein in the plasma increased. We, therefore, demonstrate the concept that accumulation of LPA in the circulation decreases ATX production. However, this feedback regulation can be overcome by the inflammatory cytokines, TNF-α or interleukin 1ß. This enables high LPA and ATX levels to coexist in inflammatory conditions. The results are discussed in terms of ATX regulation in wound healing and cancer.

Common variant rs7597774 in ADD2 is associated with dilated cardiomyopathy in Chinese Han population.

Two polymorphisms, rs7597774 and rs1739843 in ADD2 and HSPB7 respectively, were found to be associated with dilated cardiomyopathy (DCM) in European cohorts but the results were not validated in the Chinese Han population. We aimed to test the association of the two variants with DCM in a cohort of Chinese Han population. DCM (399) and control (1384) individuals were identified from the GeneID database in China, and DNA was isolated from peripheral blood lymphocytes for genotyping. Alleles were amplified by PCR, and amplicons harboring polymorphisms rs1739843 and rs7597774 were directly genotyped using high-resolution melting analysis. Statistical analysis was subsequently performed to evaluate the association of the variants with DCM. Allelic analysis demonstrated that rs7597774 was significantly related to DCM (P -adj = 0.0157), and an increased risk of DCM was specifically associated with the minor allele A (OR = 1.582). High-grade cardiac dysfunction (NYHA III/IV) was a clinical parameter significantly associated with the rs7597774 genotypes AA + AC relative to genotype CC (P = 0.021). Furthermore, DCM patients with the rs7597774 genotype AA tended to undergo more invasive medical interventions than those with the genotype CC (P = 0.008). No association was detected between rs1739843 and DCM under any allelic (P -adj = 0.407, OR = 0.920) or genotypic model. In the Chinese Han population, rs7597774 but not rs1739843 was found to be associated with DCM. This study is the first to demonstrate that underlying genotypes of rs7597774 may assist in assessing the heart functional status of DCM patients and also in the prediction of the benefit of particular therapies for these patients.

Lipid phosphate phosphatase-1 expression in cancer cells attenuates tumor growth and metastasis in mice.

Lipid phosphate phosphatase-1 (LPP1) degrades lysophosphatidate (LPA) and attenuates receptor-mediated signaling. LPP1 expression is low in many cancer cells and tumors compared with normal tissues. It was hypothesized from studies with cultured cells that increasing LPP1 activity would decrease tumor growth and metastasis. This hypothesis has never been tested in vivo. To do this, we inducibly expressed LPP1 or a catalytically inactive mutant in cancer cells. Expressing active LPP1 increased extracellular LPA degradation by 5-fold. It also decreased the stimulation of Ca(2+) transients by LPA, a nondephosphorylatable LPA1/2 receptor agonist and a protease-activated receptor-1 peptide. The latter results demonstrate that LPP1 has effects downstream of receptor activation. Decreased Ca(2+) mobilization and Rho activation contributed to the effects of LPP1 in attenuating the LPA-induced migration of MDA-MB-231 breast cancer cells and their growth in 3D culture. Increasing LPP1 expression in breast and thyroid cancer cells decreased tumor growth and the metastasis by up to 80% compared with expression of inactive LPP1 or green fluorescent protein in syngeneic and xenograft mouse models. The present work demonstrates for the first time that increasing the LPP1 activity in three lines of aggressive cancer cells decreases their abilities to produce tumors and metastases in mice.

Inhibition of autotaxin delays breast tumor growth and lung metastasis in mice.

Autotaxin is a secreted enzyme that produces most extracellular lysophosphatidate, which stimulates 6 G-protein-coupled receptors. Lysophosphatidate promotes cancer cell survival, growth, migration, invasion, metastasis, and resistance to chemotherapy and radiotherapy. The present work investigated whether inhibiting autotaxin could decrease breast tumor growth and metastasis. We used a new autotaxin inhibitor (ONO-8430506; IC90=100 nM), which decreased plasma autotaxin activity by >60% and concentrations of unsaturated lysophosphatidates by >75% for 24 h compared with vehicle-treated mice. The effects of ONO-8430506 on tumor growth were determined in a syngeneic orthotopic mouse model of breast cancer following injection of 20,000 BALB/c mouse 4T1 or 4T1-12B cancer cells. We show for the first time that inhibiting autotaxin decreases initial tumor growth and subsequent lung metastatic nodules both by 60% compared with vehicle-treated mice. Significantly, 4T1 cells express negligible autotaxin compared with the mammary fat pad. Autotaxin activity in the fat pad of nontreated mice was increased 2-fold by tumor growth. Our results emphasize the importance of tumor interaction with its environment and the role of autotaxin in promoting breast cancer growth and metastasis. We also established that autotaxin inhibition could provide a novel therapeutic approach to blocking the adverse effects of lysophosphatidate in cancer.