Ming-Cheh Liu

Sirt1 protects against thrombomodulin down-regulation and lung coagulation after particulate matter exposure

Exposure to ambient particulate matter (PM) air pollution has been reported to trigger inflammation and thrombosis. However, molecular mechanisms underlying the modulation of coagulation pathways in PM-induced thrombosis remain largely unknown. We report here that Sirt1, a member of class III histone deacetylase, controls lung inflammation and coagulation after PM exposure. Sirt1 knock-out mice exhibited aggravated lung vascular leakage and inflammation after PM exposure, which was correlated with increased NF-κB acetylation and activation. Furthermore, Sirt1 knock-out mice were highly susceptible to PM-induced lung coagulation as demonstrated by increased fibrin formation. The increased fibrin formation was associated with reduced tissue factor pathway inhibitor (TFPI) expression and increased plasminogen activator inhibitor-1 (PAI-1) activity in the lungs, thus favoring elevated coagulation and disrupted fibrinolysis responses. Thrombomodulin (TM), a central player of the anticoagulant protein C system, is regulated by Kruppel-like factor 2 (KLF2) at the transcriptional level. Our data show that PM exposure led to decreased lung KLF2 and TM expression in wild-type mice, and lung KLF2 and TM protein levels were further decreased in Sirt1 knock-out mice. Importantly, Sirt1 gene delivery inhibited TM and KLF2 down-regulation and reduced lung coagulation after PM exposure. Collectively, our studies indicate that Sirt1 functions as a suppressor of coagulation after particulate matter exposure.

Crystal structure of human tyrosylprotein sulfotransferase-2 reveals the mechanism of protein tyrosine sulfation reaction

Post-translational protein modification by tyrosine-sulfation plays an important role in extracellular protein-protein interactions. The protein tyrosine sulfation reaction is catalyzed by the Golgi-enzyme called the tyrosylprotein sulfotransferase (TPST). To date, no crystal structure is available for TPST. Detailed mechanism of protein tyrosine sulfation reaction has thus remained unclear. Here we present the first crystal structure of the human TPST isoform 2 (TPST2) complexed with a substrate peptide (C4P5Y3) derived from complement C4 and 3’-phosphoadenosine-5’-phosphate (PAP) at 1.9Å resolution. Structural and complementary mutational analyses revealed the molecular basis for catalysis being an SN2-like in-line displacement mechanism. TPST2 appeared to recognize the C4 peptide in a deep cleft by using a short parallel β-sheet type interaction, and the bound C4P5Y3 forms an L-shaped structure. Surprisingly, the mode of substrate peptide recognition observed in the TPST2 structure resembles that observed for the receptor type tyrosine kinases.

Urokinase Expression by Tumor Suppressor Protein p53: A Novel Role in mRNA Turnover

Lung carcinoma (H1299) cells deficient in p53 (p53(-/-)) express large amounts of urokinase-type plasminogen activator (uPA) protein and uPA mRNA, and exhibit slower degradation of uPA mRNA than that of p53-expressing nonmalignant Beas2B human airway epithelial cells. Expression of p53 protein in H1299 cells, upon transfection with p53 cDNA, suppressed basal as well as uPA-induced expression of uPA protein in both conditioned media and cell lysates, and decreased the level of steady-state uPA mRNA primarily due to increased uPA mRNA turnover. Inhibition of p53 expression by RNA silencing (SiRNA) in Beas2B cells enhanced basal and uPA-mediated uPA protein and mRNA expression with stabilization of uPA mRNA. Purified p53 binds to the uPA mRNA 3 untranslated region (UTR) in a sequence-specific manner and endogenous uPA mRNA associates with p53 protein isolated from Beas2B cytosolic extracts. p53 binds to a 35-nucleotide uPA 3UTR sequence and insertion of this sequence into beta-globin mRNA accelerates degradation of otherwise stable beta-globin mRNA. These observations confirm a new role for p53 as a uPA mRNA binding protein that down-regulates uPA mRNA stability and decreases cellular uPA expression.

Identification, characterization, and ontogenic study of a catechol O-methyltransferase from zebrafish

To establish the zebrafish as a model for investigating the methylation pathway of drug metabolism, we embarked on the molecular cloning of the zebrafish catechol O-methyltransferase (COMT). By searching the GenBank database, a zebrafish nucleotide sequence encoding a putative COMT was identified. Based on the sequence information, we designed and synthesized oligonucleotides corresponding to its 5- and 3-coding regions of this zebrafish COMT. Using the first-strand cDNA reverse-transcribed from the total RNA isolated from a 3-month-old adult female zebrafish as the template, the cDNA encoding the zebrafish COMT was PCR-amplified. The recombinant zebrafish COMT protein was subsequently expressed in and purified from BL21 (DE3) Escherichia coli cells transformed with the pGEX-2TK expression vector harboring the zebrafish COMT cDNA. Upon enzymatic characterization, purified COMT displayed methylating activity toward dopamine, dopa, and catecholestrogens, as well as three representative catechol drugs, methyldopa, dobutamine, and isoproterenol. A reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed developmental stage-dependent expression of the zebrafish COMT during embryonic development and throughout the larval stage onto maturity. These results provide a foundation for investigating the involvement of COMT-mediated methylation in protection against the adverse effects of catechol drugs and other xenobiotic catechols during the developmental process.

Molecular cloning, expression and characterization of a novel mouse SULT6 cytosolic sulfotransferase.

By searching the mouse EST database, we identified a novel mouse cytosolic sulfotransferase (SULT) cDNA (RIKEN cDNA 2410078J06). Sequence analysis revealed that this new SULT belongs to the cytosolic SULT6 gene family. The recombinant form of this newly identified SULT, designated SULT6B1, was expressed using the pGEX-4T-1 glutathione S-transferase fusion system and purified from transformed BL21 Escherichia coli cells. Purified mouse SULT6B1 exhibited sulfonating activity toward thyroxine and bithionol among a variety of endogenous and xenobiotic compounds tested as substrates. pH optimum of purified mouse SULT6B1 was determined to be 8.0. Tissue-specific expression of mouse and human SULT6B1 was examined by RT-PCR. While human SULT6B1 was specifically expressed in kidney and testis, mouse SULT6B1 was detected in brain, heart, kidney, thymus, lung, liver and testis. Further studies are needed in order to clarify the role of SULT6B1 in the metabolism of thyroxine and possibly some xenobiotics in mouse.

A Novel Hydroxysteroid-Sulfating Cytosolic Sulfotransferase, SULT3 ST3, from Zebrafish: Identification, Characterization, and Ontogenic Study

To establish the zebrafish as a model for investigating the drug metabolism through sulfation, we had embarked on establishing a complete repertoire of the zebrafish Phase II cytosolic sulfotransferases (SULTs). By searching the expressed sequence tag database, a zebrafish cDNA encoding a putative cytosolic sulfotransferase (SULT) was identified. Based on the sequence analysis, this zebrafish SULT was found to belong to the SULT3 gene family. The recombinant protein of the zebrafish SULT, designated SULT3 ST3, was expressed in and purified from BL21 (DE3) Escherichia coli cells transformed with the pGEX-2TK expression vector harboring SULT3 ST3 cDNA. Upon enzymatic characterization, purified SULT3 ST3 displayed sulfating activity toward hydroxysteroids, particularly pregnenolone and dehydroepiandrosterone (DHEA), as well as several drugs among various endogenous and xenobiotic compounds tested as substrates. The pH-dependence and kinetic constants of this enzyme with DHEA were determined. The regulatory effects of various divalent metal cations on the DHEA-sulfating activity of SULT3 ST3 were quantitatively evaluated. A reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed developmental stage-dependent expression of SULT3 ST3 during embryonic development and throughout the larval stage onto maturity. Collectively, these results suggest a possible involvement of the newly discovered SULT3 ST3 in the metabolism of hydroxysteroids and xenobiotics including drugs in zebrafish.

Snapshot of a Michaelis complex in a sulfuryl transfer reaction: Crystal structure of a mouse sulfotransferase, mSULT1D1, complexed with donor substrate and accepter substrate

We report the crystal structure of mouse sulfotransferase, mSULT1D1, complexed with donor substrate 3-phosphoadenosine 5-phosphosulfate and accepter substrate p-nitrophenol. The structure is the first report of the native Michaelis complex of sulfotransferase. In the structure, three proposed catalytic residues (Lys48, Lys106, and His108) were in proper positions for engaging in the sulfuryl transfer reaction. The data strongly support that the sulfuryl transfer reaction proceeds through an S(N)2-like in-line displacement mechanism.

Concerted action of the cytosolic sulfotransferase, SULT1A3, and catechol-O-methyltransferase in the metabolism of dopamine in SK-N-MC human neuroblastoma cells

Conjugation reactions catalyzed by the cytosolic sulfotransferase, SULT1A3, or catechol-O-methyltransferase (COMT) are known to be involved in the regulation and homeostasis of dopamine and other monoamine neurotransmitters. Whether different conjugation reactions may act in a concerted manner, however, remains unclear. The current study aimed to investigate the concerted action of SULT1A3 and COMT in dopamine metabolism. Analysis of the medium of SK-N-MC cells, metabolically labeled with [(35)S]sulfate in the presence of dopamine, revealed the generation and release of predominantly [(35)S]sulfated 3-methyldopamine and, to a lesser extent [(35)S]sulfated dopamine. Addition to the labeling medium of tropolone, a COMT inhibitor, enhanced the production of [(35)S]sulfated dopamine, with a concomitant decrease of [(35)S]sulfated 3-methyldopamine. Enzymatic assays using the eleven known human cytosolic SULTs revealed SULT1A3 as the major enzyme responsible for the sulfation of both dopamine and 3-methyldopamine. Kinetic analysis showed that the catalytic efficiency of SULT1A3 with 3-methyldopamine was 1.6 times than that with dopamine. Using subcellular fractions prepared from SK-N-MC cells, the majority of COMT dopamine-methylating activity was found to be present in the cytosol. Collectively, these results imply a concerted action of sulfation and methylation in the irreversible inactivation and disposal of excess dopamine in SK-N-MC cells.

Identification and characterization of a novel kaempferol sulfotransferase from Arabidopsis thaliana.

In plants, flavonoids have been shown to be subjected to conjugation modifications such as glycosylation, methylation, and sulfation. Among these modifications, sulfation is known as an important pathway in the regulation of the levels of endogenous compounds such as steroids. Although a large variety of flavonoid sulfates also exist in plants, the detailed biochemical characterization of Arabidopsis thaliana sulfotransferases (AtSULTs) remains to be fully clarified. We report here that uncharacterized AtSULT202E1 (AGI code: At2g03770), a SULT202E subfamily member, shows the sulfating activity toward flavonoids. The general characteristics of the enzyme were studied on the optimum temperature and pH, the effect of divalent cations, and the thermal stability with kaempferol as substrate. A comparative analysis of the sulfation of flavonoids by AtSULT202E1, AtSULT202B1 and AtSULT202A1 revealed that three AtSULTs have differential substrate specificities. Surprisingly, 3-hydroxyflavone was sulfated only by AtSULT202A1 while 7-hydroxyflavone was highly sulfated by AtSULT202E1 and AtSULT202B1. These results indicate that flavonols might be sulfated in a position specific manner. In conclusion, our studies indicate that a novel AtSULT202E1 has the sulfating activity toward flavonoids together with AtSULT202B1 and AtSULT202A1. The existence of three flavonoid sulfotransferases in A. thaliana suggests that sulfation of flavonoids have an important role in regulation of their functions.

Blockade of LOX-1 Prevents Endotoxin-Induced Acute Lung Inflammation and Injury in Mice

Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1), a cell surface receptor expressed in endothelial cells, is known to mediate oxidized LDL-induced vascular inflammation and atherogenesis. Although the role of LOX-1 in vascular inflammation has been well established, its involvement in acute lung inflammation and injury remains unclear. In the present study, we examined the effects of a LOX-1-blocking antibody on lung inflammation in a mouse endotoxin lipopolysaccharide (LPS)-induced acute lung injury model. We demonstrated that intraperitoneal challenge with LPS induced a rapid and robust increase in LOX-1 expression in mouse lung. Pre-treatment of mice with anti-LOX-1-blocking antibody significantly inhibited LPS-induced lung inflammation as indicated by decreased neutrophil accumulation in the lung. Furthermore, anti-LOX-1 was capable of inhibiting LPS-induced inflammatory responses, including NF-κB activation, ICAM-1 expression and apoptotic signaling, in mouse lung. Collectively, these results indicate that LOX-1 may serve as a valuable therapeutic target in the prevention of acute lung inflammation and injury in sepsis.