Phosphorylation and stabilization of ATP binding cassette transporter A1 by synthetic amphiphilic helical peptides

To investigate structural requirement of helical apolipoprotein to phosphorylate and stabilize ATP-binding cassette transporter A1 (ABCA1), synthetic peptides (Remaley, A. T., Thomas, F., Stonik, J. A., Demosky, S. J., Bark, S. E., Neufeld, E. B., Bocharov, A. V., Vishnyakova, T. G., Patterson, A. P., Eggerman, T. L., Santamarina-Fojo, S., and Brewer, H. B. (2003) J. Lipid Res. 44, 828-836) were examined for these activities. L37pA, an L amino acid peptide that contains two class-A amphiphilic helices, and D37pA, the same peptide with all D amino acids, both removed cholesterol and phospholipid from differentiated THP-1 cells more than apolipoproteins (apos) A-I, A-II, and E. Both peptides also mediated lipid release from human fibroblasts WI-38 similar to apoA-I. L2D37pA, an L-peptide whose valine and tyrosine were replaced with D amino acids also promoted lipid release from WI-38 but less so with THP-1, whereas L3D37pA, in which alanine, lysine, and asparatic acid were replaced with D amino acids was ineffective in lipid release for both cell lines. ABCA1 protein in THP-1 and WT-38 was stabilized against proteolytic degradation by apoA-I, apoA-II, and apoE and by all the peptides tested except for L3D37pA, and ABCA1 phosphorylation closely correlated with its stabilization. The analysis of the relationship among these parameters indicated that removal of phospholipid triggers signals for phosphorylation and stabilization of ABCA1. We thus concluded that an amphiphilic helical motif is the minimum structural requirement for a protein to stabilize ABCA1 against proteolytic degradation.     

Inhibitory effects of trientine, a copper-chelating agent, on induction of DNA strand breaks in hepatic cells of Long-Evans Cinnamon rats

Effects of treatment with trientine, a specific copper-chelating agent, on accumulation of copper and induction of DNA strand breaks were investigated in Long-Evans Cinnamon (LEC) rats, an animal model for human Wilson's disease. Copper accumulated in the livers of LEC rats in an age-dependent manner from 4 to 13 weeks of age. When LEC rats were treated with trientine from 10 weeks of age, hepatic copper contents did not increase and were maintained at the same levels as those in 10-week-old LEC rats. When the amounts of DNA single-strand breaks (SSBs) were estimated by a comet assay, SSBs of DNA were induced in a substantial population of LEC rat hepatic cells around 8 weeks of age and the amounts of SSBs increased in an age-dependent manner from 8 to 15 weeks of age. When LEC rats were treated with trientine from 10 weeks of age, the observed number of cells with DNA damage decreased dramatically, suggesting that induction of SSBs of DNA was inhibited and/or SSBs were repaired during the period of treatment with trientine. The results show that treatment of LEC rats with trientine decreases the number of DNA strand breaks observed, although copper contents remain high in the liver.     

Singlet oxygen inhibits the repair of photosystem II by suppressing the translation elongation of the D1 protein in Synechocystis sp. PCC 6803

Singlet oxygen, generated during photosynthesis, is a strong oxidant that can, potentially, damage various molecules of biological importance. We investigated the effects in vivo of singlet oxygen on the photodamage to photosystem II (PSII) in the cyanobacterium Synechocystis sp. PCC 6803. Increases in intracellular concentrations of singlet oxygen, caused by the presence of photosensitizers, such as rose bengal and ethyl eosin, stimulated the apparent photodamage to PSII. However, actual photodamage to PSII, as assessed in the presence of chloramphenicol, was unaffected by the production of singlet oxygen. These observations suggest that singlet oxygen produced by added photosensitizers acts by inhibiting the repair of photodamaged PSII. Labeling of proteins in vivo revealed that singlet oxygen inhibited the synthesis of proteins de novo and, in particular, the synthesis of the D1 protein. Northern blotting analysis indicated that the accumulation of psbA mRNAs, which encode the D1 protein, was unaffected by the production of singlet oxygen. Subcellular localization of polysomes with bound psbA mRNAs suggested that the primary target of singlet oxygen might be the elongation step of translation.     

Purification and characterization of human uroporphyrinogen III synthase expressed in Escherichia coli

The side-chain asymmetry of physiological porphyrins is produced by the cooperative action of hydroxymethylbilane synthase and uroporphyrinogen (uro'gen) III synthase. Although the role of uro'gen III synthase is essential for the chemistry of porphyrin biosynthesis, many aspects, structural as well as mechanical, of uro'gen III synthase have yet to be studied. We report here an expression system in Escherichia coli and a purification procedure for human uro'gen III synthase. The enzyme in the lysate was unstable, but we found that glycerol prevents the activity loss in the lysate. The purified enzyme showed remarkable thermostability, particularly when kept in phosphate buffer containing DTT or EDTA, indicating that the enzyme activity may depend on its oxidation state. Examination of the relationship between the number of Cys residues that are accessible to 5,5'-dithiobis(2-nitrobenzoic acid) and the remaining activity during heat inactivation showed that a particular Cys residue is involved in activity loss. From the crystal structure of human uro'gen III synthase [Mathews et al. (2001) EMBO J. 20, 5832-5839], this Cys residue was considered to be Cys73, which is buried deep inside the enzyme, suggesting that Cys73 of human uro'gen III synthase plays an important role in enzyme activity.     

Interaction of the Escherichia coli lipoprotein NlpI with periplasmic Prc (Tsp) protease

Escherichia coli spr (suppressor of prc) mutants and nlpI mutants show thermosensitive growth. The thermosensitivity of the spr mutants was suppressed by the nlpI mutations. Expression of the fusion genes encoding hexa-histidine-tagged NlpI (NlpI-His) and purification of the tagged NlpI showed that NlpI-His bound with Prc protease and IbpB chaperone. NlpI-His with the amino acid substitution of G103D did not bind with either of these proteins, while NlpI-His variants (NlpI-284-His, NlpI-Q283-His, and NlpI-G282-His) lacking 10 to 12 residues from the carboxy terminus bound with both proteins. The tagged NlpI lacking 11 amino acid residues from the carboxy terminus was processed by Prc, but that lacking 12 residues was not. The thermosensitivity of the nlpI mutant was corrected by the production of the former NlpI variant, but not by production of the latter. Expression of the truncated NlpI that lacked 10 or 11 residues from the carboxy terminus corrected the thermosensitivity of the prc nlpI double mutant, while expression of the full-length NlpI did not. Thus, it was suggested that NlpI was activated by Prc protease processing.     

Functional identification of SLC5A8, a tumor suppressor down-regulated in colon cancer, as a Na(+)-coupled transporter for short-chain fatty acids

SLC5A8, a tumor suppressor gene down-regulated in human colon cancer, codes for a transporter in the Na(+)/glucose cotransporter gene family, but the definitive functional identity of the transporter protein is not known. Since this gene is expressed abundantly in the colon where short-chain fatty acids are generated by bacterial fermentation, we tested the hypothesis that it codes for a Na(+)-coupled transporter for these fatty acids. The coding region of SLC5A8 mRNA was amplified from human intestine and expressed heterologously in Xenopus laevis oocytes. Transport function was monitored by uptake of radiolabeled substrates and by substrate-induced currents under voltage-clamp conditions. Uptake of short-chain fatty acids (lactate, pyruvate, acetate, propionate, and butyrate) in oocytes expressing SLC5A8 was severalfold higher than in uninjected oocytes. Exposure of SLC5A8-expressing oocytes to these fatty acids induced inward currents under voltage-clamp conditions in a Na(+)-dependent manner. These currents were saturable and the substrate concentrations needed for half-maximal induction of the current were in the range of 0.08-2.5 mm. The substrate-induced currents decreased as the carbon chain length of the substrates increased. The Na(+)-activation kinetics indicated involvement of more than one Na(+) ion in the activation process. Direct measurements of substrate (propionate) and charge transfer showed that three positive charges are transferred into oocytes per substrate molecule. These studies establish the functional identity of SLC5A8 as a Na(+)-coupled transporter for short-chain fatty acids.     

Insulin receptor substrate-1/SHP-2 interaction, a phenotype-dependent switching machinery of insulin-like growth factor-I signaling in vascular smooth muscle cells

Insulin-like growth factor-I (IGF-I) plays a role in mutually exclusive processes such as proliferation and differentiation in a variety of cell types. IGF-I is a potent mitogen and motogen for dedifferentiated vascular smooth muscle cells (VSMCs) in vivo and in vitro. However, in differentiated VSMCs, IGF-I is only required for maintaining the differentiated phenotype. Here we investigated the VSMC phenotype-dependent signaling and biological processes triggered by IGF-I. In differentiated VSMCs, IGF-I activated a protein-tyrosine phosphatase, SHP-2, recruited by insulin receptor substrate-1 (IRS-1). The activated SHP-2 then dephosphorylated IRS-1 Tyr(P)-895, resulting in blockade of the pathways from IRS-1/Grb2/Sos to the ERK and p38 MAPK. Conversely, such negative regulation was silent in dedifferentiated VSMCs, where IGF-I activated both MAPKs via IRS-1/Grb2/Sos interaction-linked Ras activation, leading to proliferation and migration. Thus, our present results demonstrate that the IRS-1/SHP-2 interaction acts as a switch controlling VSMC phenotype-dependent IGF-I-induced signaling pathways and biological processes, and this mechanism is likely to be applicable to other cells.     

Monitoring for dynamic biological processing by intramolecular bioluminescence resonance energy transfer system using secreted luciferase

Proteolytic processing plays crucial roles in physiological and pathophysiological cellular functions such as peptide generation, cell cycle, and apoptosis. We developed a novel biophysical bioluminescence resonance energy transfer (BRET) system between a secreted Vargula luciferase (Vluc) and an enhanced yellow fluorescent protein (EYFP) for visualization of cell biological processes. The bioluminescence spectrum of the fusion protein (Vluc-EYFP) is bimodal (lambdamax = 460 nm (Vluc) and 525nm (EYFP)), indicating that the excited-state energy of Vluc transfers to EYFP (in short, BRET). The BRET signal can be measured in the culture medium and pursue quantitative production of two neuropeptides, nocistatin (NST) and nociceptin/orphanin FQ (N/OFQ) in living cells. NST and N/OFQ are located in tandem on the same precursor, but NST exhibits antagonistic action against N/OFQ-induced central functions. Insertion of a portion of the NST-N/OFQ precursor (Glu-Gln-Lys-Gln-Leu-Gln-Lys-Arg-Phe-Gly-Gly-Phe-Tyr-Gly) in Vluc-EYFP makes the fusion protein cleavable at Lys-Arg in NG108-15 cells, and proprotein convertase 1 enhances this digestion. The change in BRET signals quantifies the processing of the fusion protein. Our novel intramolecular BRET system using a secreted luciferase is useful for investigating peptide processing in living cells.