A slow-tight binding inhibitor of dopamine beta-monooxygenase: a transition state analogue for the product release step

The steady-state kinetic data show that 3-hydroxy-4-phenylthiazole-2(3H)-thione (3H4PTT) is a potent tight-binding inhibitor for dopamine beta-monooxygenase (DbetaM) with a dissociation constant of 0.9 nM. Ackermann-Potter plots of the enzyme dependence of the inhibition revealed that the stoichiometry of the enzyme inhibition by 3H4PTT is 1:1. Pre-steady-state progress curves at varying inhibitor with fixed reductant and enzyme concentrations clearly show the slow binding behavior of the inhibitor. The observed kinetic behavior is consistent with the apparent direct formation of the tightly bound E x I* complex. The k(on) and k(off) for 3H4PTT which were determined under pre-steady-state conditions at variable inhibitor concentrations were found to be (1.85 +/- 0.07) x 10(6) M(-1) s(-1) and (1.9 +/- 0.6) x 10(-3) s(-1), respectively. The dissociation constant calculated from these rates was similar to that determined under steady-state conditions, confirming that 3H4PTT is a kinetically well-behaved inhibitor. The steady-state as well as pre-steady-state kinetic studies at variable DMPD concentrations show that the inhibition is competitive with respect to the reductant, demonstrating the exclusive interaction of 3H4PTT with the oxidized form of the enzyme. The kinetic behavior and the structural properties of 3H4PTT are consistent with the proposal that the E x 3H4PTT complex may mimic the transition state for the product (protonated) release step of the enzyme. Therefore, 3H4PTT could be used as a convenient probe to examine the properties of the E x P complex of the DbetaM reaction and also as an active site titrant for the oxidized enzyme.     

Nuclear matrix: isolation and characterization of a framework structure from rat liver nuclei

A nuclear framework structure termed the nuclear matrix has been isolated and characterized. This matrix forms the major residual structure of isolated nuclei and consists largely of protein with smaller amounts of RNA, DNA, carbohydrate, and phospholipid. The nuclear matrix can be further resolved by combined treatment with DNase and RNase. The remaining nuclear protein structure, after extraction of 90 percent of the nuclear protein, 99.9 percent of the DNA, and 98 percent of the RNA and phospholipid, is termed the nuclear protein matrix. Electron microscopy of this final nuclear protein matrix reveals an interior framework structure composed of residual nucleolar structures associated with a granular and fibrous internal matrix structure. The internal matrix framework is derived from the interchromatinic structures of the nucleus, and is connected to a surrounding residual nuclear envelope layer containing residual nuclear pore complex structures. Sodium dodecyl sulfate-acrylamide gel electrophoresis of the nuclear matrix proteins demonstrates three major polypeptide fractions, P-1, P-2, and P-3, with average molecular weights of approximately 69,000, 66,000 and 62,000, as well as several minor polypeptides which migrate at approximately 50,000 and at higher molecular weights (>100,000). Polypeptides with molecular weights identical to those of P-1, P-2 and P-3 are also components of isolated nuclear envelopes and nucleoli, whereas isolated chromatin contains no detectable matrix polypeptides. This suggests that the major matrix polypeptides are localized in specific structural regions of the nucleus, i.e., nuclear envelope, nucleoli, and interchromatinic structures. The presence of cytochrome oxidase activity in the isolated nuclear matrix indicates that at least some integral proteins of the nuclear membrane are associated with the matrix.     

Copper ions disrupt dopamine metabolism via inhibition of V-H+-ATPase: a possible contributing factor to neurotoxicity

The involvement of copper in the pathophysiology of neurodegeneration has been well documented but is not fully understood. Commonly, the effects are attributed to increased reactive oxygen species (ROS) production due to inherent redox properties of copper ions. Here we show copper can have physiological effects distinct from direct ROS production. First, we show that extragranular free copper inhibits the vesicular H(+)-ATPase of resealed chromaffin granule ghosts. Extragranular ascorbate potentiates this inhibition. The inhibition is mixed type with K(is) = 6.8 +/- 2.8 micromol/L and K(ii) = 3.8 +/- 0.6 micromol/L, with respect to ATP. Second, extracellular copper causes an inhibition of the generation of a pH-gradient and rapid dissipation of pre-generated pH and catecholamine gradients. Copper chelators and the ss-amyloid peptide 1-42 were found to effectively prevent the inhibition. The inhibition is reversible and time-independent suggesting the effects of extracellular copper on H(+)-ATPase is direct, and not due to ROS. The physiological significance of these observations was shown by the demonstration that extracellular copper causes a dramatic perturbation of dopamine metabolism in SH-SY5Y cells. Thus, we propose that the direct inhibition of the vesicular H(+)-ATPase may also contribute to the neurotoxic effects of copper.     

Effect of 2-fluorohistidine labeling of the anthrax protective antigen on stability, pore formation, and translocation

The action of anthrax toxin relies in part upon the ability of the protective antigen (PA) moiety to form a heptameric pore in the endosomal membrane, providing a portal for entry of the enzymic moieties of the toxin into the cytosol. Pore formation is dependent on a conformational change in the heptameric prepore that occurs in the neutral to mildly acidic pH range, and it has been hypothesized that protonation of one or more histidine residues triggers this transition. To test this hypothesis, we used biosynthetic methods to incorporate the unnatural amino acid analogue 2-fluorohistidine (2-FHis) into PA. 2-FHis is isosteric with histidine but resists protonation at physiological pH values due to a dramatically reduced side-chain pKa ( approximately 1). We found that 2-FHis-labeled PA was biologically inactive, as judged by its inability to deliver a model intracellular effector, LFN-DTA, to the cytosol of CHO-K1 cells. However, whereas 2-FHis blocked a conformational transition in the full-length PA83 protein in the pH 5-6 range, the pH dependence of prepore-to-pore conversion of (PA63)7 was unchanged from the wild-type protein, implying that this conversion is not dependent on His protonation. Consistent with this result, the labeled, trypsin-activated PA was able to permeabilize liposomes to K+ and retained pore-forming activity in planar phospholipid bilayers. The pores in planar bilayers were incapable, however, of translocating a model ligand in response to a transmembrane pH gradient or elevated voltage. The results indicate that protonation of residues other than His, presumably Glu and/or Asp side chains, triggers pore formation in vitro, but His residues are nonetheless important for PA functioning in vivo.     

Inhibition of PARP activation by enalapril is crucial for its renoprotective effect in cisplatin-induced nephrotoxicity in rats

Oxidative stress-induced PARP activation has been recognized to be a main factor in the pathogenesis of cisplatin-induced nephrotoxicity. Accumulating literature has revealed that ACE inhibitors may exert beneficial effect in several disease models via preventing PARP activation. Based on this hypothesis, we have evaluated the renoprotective effect of enalapril, an ACE inhibitor, and its underlying mechanism(s) in cisplatin-induced renal injury in rats. Male Albino Wistar rats were orally administered normal saline or enalapril (10, 20 and 40?mg/kg) for 10 days. Nephrotoxicity was induced by a single dose of cisplatin (8?mg/kg; i.p.) on the 7th day. The animals were thereafter sacrificed on the 11th day and both the kidneys were excised and processed for biochemical, histopathological, molecular, and immunohistochemical studies. Enalapril (40?mg/kg) significantly prevented cisplatin-induced renal dysfunction. In comparison to cisplatin-treated group, the elevation of BUN and creatinine levels was significantly less in this group. This improvement in kidney injury markers was well substantiated with reduced PARP expression along with phosphorylation of MAPKs including JNK/ERK/p38. Enalapril, in a dose-dependent fashion, attenuated cisplatin-induced oxidative stress as evidenced by augmented GSH, SOD and catalase activities, reduced TBARS and oxidative DNA damage (8-OHDG), and Nox-4 protein expression. Moreover, enalapril dose dependently inhibited cisplatin-induced inflammation (NF-κB/IKK-β/IL-6/Cox-2/TNF-α expressions), apoptosis (increased Bcl-2 and reduced p53, cytochrome c, Bax and caspase-3 expressions, and TUNEL/DAPI positivity) and preserved the structural integrity of the kidney. Thus, enalapril attenuated cisplatin-induced renal injury via inhibiting PARP activation and subsequent MAPKs/TNF-α/NF-κB mediated inflammatory and apoptotic response.     

Mode of substrate interaction and energetics of carbon-oxygen bond formation of the dopamine beta-monooxygenase reaction.

Previous studies have shown that the dopamine beta-monooxygenase (DbetaM; E.C. 1.14.17.1)/1-(2-aminoethyl)-1,4-cyclohexadiene (CHDEA) reaction partitions between side chain and ring H-abstraction to produce the side-chain-hydroxylated product, 2-amino-1-(1, 4-cyclohexadienyl)ethanol, and the aromatized product, phenylethylamine, and that the two pathways do not crossover. [Wimalasena, K., and May, S. W. (1989) J. Am. Chem. Soc. 111, 2729-2731; Wimalasena, K., and Alliston, K. R. (1995) J. Am. Chem. Soc. 117, 1220-1224]. We now report that the ring H-abstraction pathway of the reaction further partitions to produce the ring hydroxylated product, CHDEA-6OH, and the aromatized product, PEA, at the carbon-oxygen bond formation step. The ring hydroxylation is shown to be stereospecific, exclusively producing the (S) product. The absolute stereospecificity of the ring and side-chain hydroxylations of the DbetaM/CHDEA reaction suggests that the side-chain pro-R hydrogen of the enzyme-bound substrate is close to perpendicular to the aromatic ring of the phenylethylamine substrate or cyclohexadiene ring of CHDEA. The relative activation energy parameters suggest that the partitioning of the ring H abstraction pathway between aromatized and ring hydroxylated products is due to the partitioning of the high-energy intermediates, the cyclohexadienyl radical and the Cu(II)-O(*) species, between carbon-oxygen bond formation and direct electron transfer. The relatively high activation enthalpic favorability and entropic unfavorability for the carbon-oxygen bond formation strongly suggest that the critical balancing of these two opposing forces is mandatory for the desired product formation.     

Genetic variation in TIMP1 but not MMPs predict excess FEV1 decline in two general population-based cohorts

Background

An imbalance in Matrix MetalloProteases (MMPs) and Tissue Inhibitors of MMPs (TIMPs) contributes to Chronic Obstructive Pulmonary Disease (COPD) development. Longitudinal studies investigating Single Nucleotide Polymorphisms (SNPs) in MMPs and TIMPs with respect to COPD development and lung function decline in the general population are lacking.

Methods

We genotyped SNPs in MMP1 (G-1607GG), MMP2 (-1306 C/T), MMP9 (3 tagging SNPs), MMP12 (A-82G and Asn357Ser) and TIMP1 (Phe124Phe and Ile158Ile) in 1390 Caucasians with multiple FEV₁ measurements from a prospective cohort study in the general population. FEV₁ decline was analyzed using linear mixed effect models adjusted for confounders. Analyses of the X-chromosomal TIMP1 gene were stratified according to sex. All significant associations were repeated in an independent general population cohort (n = 1152).

Results

MMP2 -1306 TT genotype carriers had excess FEV₁ decline (-4.0 ml/yr, p = 0.03) compared to wild type carriers. TIMP1 Ile158Ile predicted significant excess FEV₁ decline in both males and females. TIMP1 Phe124Phe predicted significant excess FEV₁ decline in males only, which was replicated (p = 0.10) in the second cohort. The MMP2 and TIMP1 Ile158Ile associations were not replicated. Although power was limited, we did not find associations with COPD development.

Conclusions

We for the first time show that TIMP1 Phe124Phe contributes to excess FEV₁ decline in two independent prospective cohorts, albeit not quite reaching conventional statistical significance in the replication cohort. SNPs in MMPs evidently do not contribute to FEV₁ decline in the general population.