Inhibition of dipeptidyl peptidase IV (DPP-IV), the main glucagon-like peptide 1 (GLP1)-degrading enzyme, has been proposed for the treatment of type II diabetes. We expressed and purified the ectodomain of human DPP-IV in Pichia pastoris and determined the X-ray structure at 2.1 A resolution. The enzyme consists of two domains, the catalytic domain, with an alpha/beta hydrolase fold, and a beta propeller domain with an 8-fold repeat of a four-strand beta sheet motif. The beta propeller domain contributes two important functions to the molecule that have not been reported for such structures, an extra beta sheet motif that forms part of the dimerization interface and an additional short helix with a double Glu sequence motif. The Glu motif provides recognition and a binding site for the N terminus of the substrates, as revealed by the complex structure with diprotin A, a substrate with low turnover that is trapped in the tetrahedral intermediate of the reaction in the crystal.

Endothelin-converting enzyme 1 (ECE-1) is a membrane-bound metalloprotease that catalyses the conversion of inactive big endothelins into active endothelins. Two different isoforms (ECE-1a and ECE-1b) have previously been identified for human ECE-1. In the present study we have cloned a novel human ECE-1 isoform, termed ECE-1c, and have thus shown for the first time the existence of three distinct ECE-1 isoforms. The three isoforms differ only in their N-terminal regions and are derived from a single gene through the use of alternative promoters. Ribonuclease protection experiments revealed that, although the relative levels of the three isoform mRNA species vary between human tissues, ECE-1c mRNA is generally the predominant isoform messenger. Immunofluorescence microscopy analysis showed distinct subcellular localizations for the three isoforms: whereas ECE-1a and ECE-1c are localized at the cell surface, ECE-1b was found to be intracellular and showed significant co-localization with a marker protein for the trans-Golgi network. We determined that the three isoforms have similar kinetic rate constants (Km, kcat and Vmax) for the processing of big endothelin 1 and that the big endothelin isoforms 1, 2 and 3 are cleaved with similar relative velocities of 1.0:0.1:0.1 by the three isoenzymes.

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Background-An activated endothelin (ET) system may be of pathophysiological relevance in human heart failure. We characterized the functional effects of ET-1, ET receptors, and ET-1 peptide concentration in left ventricular myocardium from 10 nonfailing hearts (NF) and 27 hearts in end-stage failure due to idiopathic dilative cardiomyopathy (DCM).

Abstract-Reports on the effectiveness of endothelin receptor blockers in angiotensin (Ang) II-induced end-organ damage are conflicting, and the mechanisms involved are uncertain. We tested the hypothesis that endothelin (ET) A/B receptor blockade with bosentan (100 mg/kg by gavage after age 4 weeks) ameliorates cardiac and renal damage by decreasing inflammation in rats harboring both human renin and angiotensinogen genes (dTGR). Furthermore, we elucidated the effect of bosentan on tissue factor (TF), which is a key regulator of the extrinsic coagulation cascade. We compared bosentan with hydralazine (80 mg/L in the drinking water for 3 weeks) as a blood pressure control. Untreated dTGR featured hypertension, focal necrosis in heart and kidney, and a 45% mortality rate (9 of 20) at age 7 weeks. Compared with Sprague-Dawley controls, both systolic blood pressure and 24-hour albuminuria were increased in untreated dTGR (203Ϯ8 versus 111Ϯ2 mm Hg and 67.1Ϯ8.6 versus 0.3Ϯ0.06 mg/d at week 7, respectively). Bosentan and hydralazine both reduced blood pressure and cardiac hypertrophy. Mortality rate was markedly reduced by bosentan (1/15) and partially by hydralazine (4/15). However, only bosentan decreased albuminuria and renal injury. Untreated and hydralazine-treated dTGR showed increased nuclear factor (NF)-B and AP-1 expression in the kidney and heart; the p65 NF-B subunit was increased in the endothelium, vascular smooth muscles cells, infiltrating cells, glomeruli, and tubules. In the heart and kidney, ET A/B receptor blockade inhibited NF-B and AP-1 activation compared with hydralazine treatment. Macrophage infiltration, ICAM-1 expression, and the integrin expression on infiltrating cells were markedly reduced. Renal vasculopathy was accompanied by increased tissue factor expression on macrophages and vessels of untreated and hydralazine-treated dTGR, which was markedly reduced by bosentan. Thus, ET A/B receptor blockade inhibits NF-B and AP-1 activation and the NF-B-and/or AP-1-regulated genes ICAM-1, VCAM-1, and TF, independent of blood pressure-related effects. We conclude that Ang II-induced NF-B and AP-1 activation and subsequent inflammation and coagulation involve at least in part the ET A/B receptors. (Hypertension. 2000;36:282-290.)

These experiments demonstrate that inhibition of NO synthesis unmasks a tonic pressor influence of endothelin, suggesting that this peptide could play a major role in pathophysiological situations associated with an impaired formation of NO.

Cancer results from the expansion of cell clones that progressively lose control of proliferation, differentiation, and death, owing to accumulation of mutational events in genes that control the cell cycle and apoptosis. Nuclear protein p53 is thought to play a major role in malignancy, since it induces genes that determine apoptosis and cell-cycle arrest, interacts with proteins employed in DNA repair, and binds to DNA strand breaks. As expected, somatic mutations in p53 are found in a variety of human cancers. Mutations are predominantly inactivating, thus eliminating the "guardian of the genome" from the proliferating cells. Germ-line mutations

These findings suggest that in HAPE-susceptible mountaineers, an augmented release of the potent pulmonary vasoconstrictor peptide endothelin-1 and/or its reduced pulmonary clearance could represent one of the mechanisms contributing to exaggerated pulmonary hypertension at high altitude.