Inhibition of the tumor necrosis factor-alpha-converting enzyme by its pro domain

Tumor necrosis factor-alpha-converting enzyme (TACE) is a disintegrin metalloproteinase that processes tumor necrosis factor and a host of other ectodomains. TACE is biosynthesized as a zymogen, and activation requires the removal of an inhibitory pro domain. Little is known about how the pro domain exerts inhibition for this class of enzymes. To study the inhibitory properties of the pro domain of TACE, we have expressed it in isolation from the rest of the protease. Here we show that the TACE pro domain (TACE Pro) is a stably folded protein that is able to inhibit this enzyme. TACE Pro inhibited the catalytic domain of TACE with an IC(50) of 70 nm. In contrast, this inhibitory potency decreased over 30-fold against a TACE form containing the catalytic plus disintegrin/cysteine-rich domains (IC(50) greater that 2 microm). The disintegrin/cysteine-rich region in isolation also decreases the interaction of TACE Pro with the catalytic domain. Surprisingly, we found that the cysteine switch motif located in TACE Pro was not essential for inhibition of the enzymatic activity of TACE; the pro domain variant C184A showed the same inhibitory potency against both TACE forms as wild type TACE Pro. X-ray absorption spectroscopy experiments indicate that binding of TACE Pro to the catalytic domain does include ligation of the catalytic zinc ion via the sulfur atom of its conserved Cys(184) residue. Moreover, the binding of TACE Pro to the catalytic zinc ion partially oxidizes the catalytic zinc ion of the enzyme. Despite this, the nature of the interaction between the pro and catalytic domains of TACE is not consistent with a simple competitive model of inhibition based on cysteine switch ligation of the zinc ion within the active site of TACE.     

Human heparanase is localized within lysosomes in a stable form

Heparanase is an endo-beta-D-glucuronidase involved in degradation of heparan sulfate (HS) and extracellular matrix (ECM) of a wide range of cells of vertebrate and invertebrate tissues. The enzymatic activity of heparanase is characterized by specific intrachain cleavage of glycosidic bonds with a hydrolase mechanism. This enzyme facilitates cell invasion and hence plays a role in tumor metastasis, angiogenesis, inflammation, and autoimmunity. Although the expression pattern and molecular properties of heparanase have been characterized, its subcellular localization has not been unequivocally determined. We have previously suggested that heparanase subcellular localization is a major determinant in regulating the enzyme's biological functions. In the present study we examined heparanase localization in three different cell types, utilizing immunofluorescent staining and electron microscopy. Our results indicate that heparanase is localized primarily within lysosomes and the Golgi apparatus. A construct composed of heparanase cDNA fused to green fluorescent protein, utilized in order to visualize the enzyme within living cells, confirmed its localization in acidic vesicles. We suggest that following synthesis, heparanase is transported into the Golgi apparatus and subsequently accumulates in a stable form within the lysosomes, where it functions in HS turnover. The lysosomal compartment may also serve as a site for heparanase confinement within the cells, limiting its secretion and uncontrolled extracellular activities associated with tumor metastasis and angiogenesis.     

The docking protein FRS2alpha controls a MAP kinase-mediated negative feedback mechanism for signaling by FGF receptors

The docking protein FRS2alpha functions as a major mediator of signaling by FGF and NGF receptors. Here we demonstrate that, in addition to tyrosine phosphorylation, FRS2alpha is phosphorylated by MAP kinase on multiple threonine residues in response to FGF stimulation or by insulin, EGF, and PDGF, extracellular stimuli that do not induce tyrosine phosphorylation of FRS2alpha. Prevention of FRS2alpha threonine phosphorylation results in constitutive tyrosine phosphorylation of FRS2alpha in unstimulated cells and enhanced tyrosine phosphorylation of FRS2alpha, MAPK stimulation, cell migration, and proliferation in FGF-stimulated cells. Expression of an FRS2alpha mutant deficient in MAPK phosphorylation sites induces anchorage-independent cell growth and colony formation in soft agar. These experiments reveal a novel MAPK-mediated, negative feedback mechanism for control of signaling pathways that are dependent on FRS2 and a mechanism for heterologous control of signaling via FGF receptors.     

Activation of human neutrophil NADPH oxidase and lateral mobility of membrane proteins: A study with crosslinkers

Fluorescence photobleaching recovery was employed to investigate the relationship between the activation of neutrophil NADPH oxidase and lateral mobility of membrane proteins. Treatment of neutrophils with the crosslinking reagent disuccinimidyl suberate (DSS) blocked activation of the respiratory burst without affecting the lateral motion of concanavalin A receptors. Neutrophils treated with DSS after prestimulation with concanavalin A generated superoxide in response to another stimulator, phorbol myristate acetate, in spite of the lateral immobilization of concanavalin A receptors. The apparent lack of correlation between the activation of NADPH oxidase and the lateral motion of membrane proteins is discussed.     

Tn554: Isolation and characterization of plasmid insertions

Tn554, a transposon in Staphylococcus aureus that carries determinants of spectinomycin resistance and inducible macrolide-lincosamide resistance, is characterized by a highly efficient transposition, exceptional site specificity for insertion, and inhibition of transposition by a copy of the transposon inserted at its preferred chromosomal site. In this communication we describe the characteristics of a number of rare, secondary-site insertions of Tn554 into several related penicillinase plasmids. These plasmid insertions display considerable variation in the frequencies with which they can act as transposon donors, as well as in the frequencies at which they undergo apparently precise excision. Transposition from the plasmid transposon donors is ordinarily a duplicative process and these subsequent transposition events always return Tn554 to its preferred site in the S. aureus chromosome; such derivatives are indistinguishable from the primary chromosomal insertion from which they were originally derived. We also report an unusual relationship between Tn554 and the transducing phage, φ11, in which Tn554 is frequently transferred independently of its plasmid carrier. We suggest that the bacteriophage may play an important role in the mobility of Tn554, in addition to the usual transduction mechanism, in a process that we have referred to as “hitchhiking.”     

Rationally designed neuropeptide antagonists: A novel approach for generation of environmentally friendly insecticides

We report our approach for the generation of a novel type of putative insecticides based on backbone cyclic peptidomimetic antagonists of insect neuropeptides using pheromone biosynthesis activating neuropeptide (PBAN) as a model. This approach, called the backbone cyclic neuropeptide based antagonist (BBC-NBA), includes the following steps: (i) elucidation of the active sequence of the chosen insect neuropeptide; (ii) disclosure of a lead antagonist based on the sequence found in step (i); (iii) design and synthesis of backbone cyclic peptide libraries (cycloscan) based on the sequence of the lead antagonist; and (iv) design and synthesis of a peptidomimetic prototype insecticide. The BBC-NBA approach was applied to PBAN and led to the discovery of a potent linear lead antagonist and a potent backbone cyclic antagonist devoid of agnoistic activity which inhibited sex pheromone biosynthesis inHeliothis peltigera female moths.     

Circadian variations in plasma growth hormone and prolactin in the infant rat: Comparison with the adult pattern

Radioimmunoassayable (RIA) plasma growth hormone (GH) and prolactin (PRL) levels were determined at 3 hr intervals during a controlled 24-hr light-dark cycle in 10-day-old male and female rats; parallel measurements were made of brain monoamines (MA's), dopamine (DA), norepinephrine (NE) and serotonin (5-HT) concentration. Plasma GH and PRL and brain MA levels found in infant rats were compared to the same determinations made during the 24-hr cycle in 50-day-old male rats. GH levels were rather uniform and did not show circadian periodicity in the plasma of infant rats, while PRL levels showed a diurnal surge in the late afternoon hr (1800). In adult rats, GH levels exhibited wide fluctuations during the 24-hr cycle and no circadian periodicity, while PRL levels showed one diurnal (1500–1800) and one nocturnal (2400) surge. A pulsatile GH secretion was found in adult rats sampled at 15 min intervals over a period of 2 hr, which seemed to be lacking in infant rats. In the brain of infant rats, DA and NE levels exhibited circadian patterns which resembled the ones present in the brain of adult rats, whereas no circadian variations were present in 5-HT levels.     

Lacrimo-auriculo-dento-digital syndrome is caused by reduced activity of the fibroblast growth factor 10 (FGF10)-FGF receptor 2 signaling pathway

Lacrimo-auriculo-dento-digital (LADD) syndrome is characterized by abnormalities in lacrimal and salivary glands, in teeth, and in the distal limbs. Genetic studies have implicated heterozygous mutations in fibroblast growth factor 10 (FGF10) and in FGF receptor 2 (FGFR2) in LADD syndrome. However, it is not clear whether LADD syndrome mutations (LADD mutations) are gain- or loss-of-function mutations. In order to reveal the molecular mechanism underlying LADD syndrome, we have compared the biological properties of FGF10 LADD and FGFR2 LADD mutants to the activities of their normal counterparts. These experiments show that the biological activities of three different FGF10 LADD mutants are severely impaired by different mechanisms. Moreover, haploinsufficiency caused by defective FGF10 mutants leads to LADD syndrome. We also demonstrate that the tyrosine kinase activities of FGFR2 LADD mutants expressed in transfected cells are strongly compromised. Since tyrosine kinase activity is stimulated by ligand-induced receptor dimerization, FGFR2 LADD mutants may also exert a dominant inhibitory effect on signaling via wild-type FGFR2 expressed in the same cell. These experiments underscore the importance of signal strength in mediating biological responses and that relatively small changes in receptor signaling may influence the outcome of developmental processes in cells or organs that do not possess redundant signaling pathway.     

Transgenic expression of human C-reactive protein suppresses endothelial nitric oxide synthase expression and bioactivity after vascular injury

C-reactive protein (CRP) is a risk marker and a potential modulator of vascular disease. Whether CRP modulates nitric oxide (NO) synthase (NOS) activity and NO metabolism remains unclear. We studied the effect of CRP on NO metabolism in transgenic mice that express human CRP (CRPtg). CRPtg and wild-type mice were subjected to controlled femoral artery wire injury. CRP serum levels at baseline and 6 and 24 h after injury were 12.4 +/- 9, 18.6 +/- 6.9, and 58.4 +/- 13 mg/l, respectively, in CRPtg mice but were undetectable at all time points in wild-type mice. Endothelial NOS protein and mRNA expression were significantly suppressed in the injured arteries of CRPtg mice (n = 5, P < 0.05). A similar reduction in eNOS expression was observed in the distant lung and heart. NO release after injury was significantly lower in CRPtg mice, as measured by nitrate and nitrite breakdown products, with a concomitant suppression of cGMP NO signaling after injury. Endothelial NOS and NO expression after vascular injury are locally and systemically suppressed in mice that express human CRP. These in vivo observations support the hypothesis that CRP modulates NO metabolism and may have implications regarding the mechanisms by which CRP modulates vascular disease.