Structural Insights into Nox4 and Nox2: Motifs Involved in Function and Cellular Localization

Regulated generation of reactive oxygen species (ROS) is primarily accomplished by NADPH oxidases (Nox). Nox1 to Nox4 form a membrane-associated heterodimer with p22^phox, creating the docking site for assembly of the activated oxidase. Signaling specificity is achieved by interaction with a complex network of cytosolic components. Nox4, an oxidase linked to cardiovascular disease, carcinogenesis, and pulmonary fibrosis, deviates from this model by displaying constitutive H₂O₂ production without requiring known regulators. Extensive Nox4/Nox2 chimera screening was initiated to pinpoint structural motifs essential for ROS generation and Nox subcellular localization. In summary, a matching B loop was crucial for catalytic activity of both Nox enzymes. Substitution of the carboxyl terminus was sufficient for converting Nox4 into a phorbol myristate acetate (PMA)-inducible phenotype, while Nox2-based chimeras never gained constitutive activity. Changing the Nox2 but not the Nox4 amino terminus abolished ROS generation. The unique heterodimerization of a functional Nox4/p22^phox Y121H complex was dependent on the D loop. Nox4, Nox2, and functional Nox chimeras translocated to the plasma membrane. Cell surface localization of Nox4 or PMA-inducible Nox4 did not correlate with O₂⁻ generation. In contrast, Nox4 released H₂O₂ and promoted cell migration. Our work provides insights into Nox structure, regulation, and ROS output that will aid inhibitor design.The family of NADPH oxidases consists of seven members termed Nox/Duox that differ in their tissue expression profiles, modes of activation, reactive oxygen species (ROS) outputs, and physiological functions. Understanding their distinguishing features is a prerequisite for rational inhibitor design and thus targeted intervention in ROS-mediated pathophysiologies (4). The coexpression of different Nox isoforms, each with potentially distinct functional profiles, in the same cell type necessitates a more discriminating approach than application of pan-Nox inhibitors. Detailed structure-function studies are necessary to identify unique regions and their impact with respect to catalytic function or localization of the enzyme. All Nox/Duox enzymes share a Nox backbone with six predicted transmembrane domains and an intracellular carboxyl-terminal domain which harbors FAD and NADPH binding sites. Nox5 and Duox1/2 enzymes contain additional structural elements such as amino terminal EF-hand motifs, a hallmark of their regulation by the intracellular calcium concentration (13, 30).The founding member of the NADPH oxidase family, the phagocyte oxidase, consists of membrane-bound Nox2 in a complex with the smaller subunit p22^phox (3). Heterodimerization of these two proteins is required for maturation and translocation of the enzyme complex to the plasma membrane or to intracellular vesicles. The Nox family members Nox1, Nox3, and Nox4 follow this paradigm (1, 14, 21, 25, 31). Heterodimer formation and association of the Nox/p22^phox complex at particular cellular membranes is essential for catalytic activity, i.e., for ROS generation. Nox2, and to a lesser degree Nox1 and Nox3, remain dormant under resting conditions and rely on stimulus-dependent translocation and assembly of oxidase components such as p47^phox and p67^phox, or NoxO1 and NoxA1 in the case of Nox1 and Nox3 (16). These steps, together with activation and translocation of the GTPase Rac, ultimately lead to the assembled, catalytically active oxidase and to ROS generation.Nox4 differs from the usual theme of multimeric assembly of active NADPH oxidases found in Nox1 to Nox3 (21, 22, 28, 32). Constitutive H₂O₂ production by Nox4 localized at perinuclear vesicles has been reported (1, 21, 28). Since NADPH oxidases catalyze the one-electron reduction of molecular oxygen to superoxide anion, the current dogma suggests that Nox4 generates intracellular superoxide. The superoxide produced will then dismutate rapidly to H₂O₂, diffusing from the cell into the extracellular milieu. Cytosolic proteins, which regulate the activity of Nox1 to Nox3 by binding to the carboxyl-terminal domains of Nox1 to Nox3, seem to be irrelevant for Nox4 function. The membrane-bound subunit p22^phox is to date the only known protein associated with Nox1 to Nox4. Heterodimerization, translocation, and enzymatic function of these oxidases require p22^phox. Recent structure-function analyses of complexes between Nox2 or Nox4 and the subunit p22^phox documented specific regions and amino acid residues in p22^phox necessary for complex formation and oxidase activity (35, 37). Interestingly, a p22^phox mutant (p22^phox Y121H) is capable of distinguishing between Nox1 to Nox3 and Nox4 by forming a functional complex only with Nox4, further suggesting unique structural features in Nox4 (35).In this study, we expand structure-function analysis of the oxidase complex by comparing Nox4/Nox2 chimeric enzymes with respect to NADPH oxidase activity, type of reactive oxygen species produced, requirement for additional oxidase components, and detailed subcellular localization.     

A Simple Device for Staining Many Single-Hole Grids Without Individual Manipulation

In serial sectioning for electron microscopy one of the greatest problems encountered is that the Formvar support film may break when grids are being mounted in or removed from a holder used for staining, or during staining. The latter is particularly troublesome when grids are stained individually. We describe here a device that conveniently eliminates this problem.     

Impaired substance P release from renal sensory nerves in SHR involves a pertussis toxin-sensitive mechanism

Stretching the renal pelvic wall activates renal mechanosensory nerves by a PGE2-mediated release of substance P via activation of the cAMP-PKA pathway. Renal pelvic ANG II modulates the responsiveness of renal sensory nerves by suppressing the PGE2-mediated activation of adenylyl cyclase via a pertussis toxin (PTX)-sensitive mechanism. In SHR, activation of renal mechanosensory nerves is impaired. This is due to suppressed release of substance P in response to increased pelvic pressure. The present study was performed to investigate whether the PGE2-mediated release of substance P was suppressed in SHR vs. WKY and, if so, whether the impaired PGE2-mediated release of substance P was due to ANG II activating a PTX-sensitive mechanism. In an isolated renal pelvic wall preparation, PGE2, 0.14 microM, increased substance P release from 9 +/- 3 to 22 +/- 3 pg/min (P < 0.01) in Wistar-Kyoto rats (WKY), but had no effect in spontaneously hypertensive rats (SHR). A tenfold higher concentration of PGE2, 1.4 microM, was required to increase substance P release in SHR, from 7 +/- 1 to 22 +/- 3 pg/min (P < 0.01). In SHR, treating renal pelvises with losartan enhanced the release of substance P produced by subthreshold concentration of PGE2, 0.3 microM, from 16 +/- 2 to 26 +/- 3 pg/min (P < 0.01). Likewise, treating renal pelvises with PTX enhanced the PGE2-mediated release of substance P from 10 +/- 1 to 33 +/- 3 pg/min (P < 0.01) in SHR. In WKY, neither losartan nor PTX had an effect on the release of substance P produced by subthreshold concentrations of PGE2, 0.03 microM. In conclusion, the impaired responsiveness of renal sensory nerves in SHR involves endogenous ANG II suppressing the PGE2-mediated release of substance P via a PTX-sensitive mechanism.     

M protein, a classical bacterial virulence determinant, forms complexes with fibrinogen that induce vascular leakage

Increased vascular permeability is a key feature of inflammatory conditions. In severe infections, leakage of plasma from the vasculature induces a life-threatening hypotension. Streptococcus pyogenes, a major human bacterial pathogen, causes a toxic shock syndrome (STSS) characterized by excessive plasma leakage and multi-organ failure. Here we find that M protein, released from the streptococcal surface, forms complexes with fibrinogen, which by binding to beta2 integrins of neutrophils, activate these cells. As a result, neutrophils release heparin binding protein, an inflammatory mediator inducing vascular leakage. In mice, injection of M protein or subcutaneous infection with S. pyogenes causes severe pulmonary damage characterized by leakage of plasma and blood cells. These lesions were prevented by treatment with a beta2 integrin antagonist. In addition, M protein/fibrinogen complexes were identified in tissue biopsies from a patient with necrotizing fasciitis and STSS, further underlining the pathogenic significance of such complexes in severe streptococcal infections.     

PGE(2) increases release of substance P from renal sensory nerves by activating the cAMP-PKA transduction cascade

Increasing renal pelvic pressure increases afferent renal nerve activity (ARNA) by a PGE(2)-mediated release of substance P (SP) from renal pelvic nerves. The role of cAMP activation in the PGE(2)-mediated release of SP was studied by examining the effects of the adenylyl cyclase (AC) activator forskolin and AC inhibitor dideoxyadenosine (DDA). Forskolin enhanced the bradykinin-mediated release of SP from an isolated rat renal pelvic wall preparation, from 7.3 +/- 1.3 to 15.6 +/- 3.0 pg/min. PGE(2) at a subthreshold concentration for SP release mimicked the effects of forskolin. The EP(2) receptor agonist butaprost, 15 microM, and PGE(2), 0.14 microM, produced similar increases in SP release, from 5.8 +/- 0.8 to 17.0 +/- 2.3 pg/min and from 8.0 +/- 1.3 to 21.6 +/- 2.7 pg/min. DDA blocked the SP release produced by butaprost and PGE(2). The PGE(2)-induced release of SP was also blocked by the PKA inhibitors PKI(14-22) and H-89. Studies in anesthetized rats showed that renal pelvic administration of butaprost, 10 microM, and PGE(2), 0.14 microM, resulted in similar ARNA responses, 1,520 +/- 390 and 1,170 +/- 270%. s (area under the curve of ARNA vs. time) that were blocked by DDA. Likewise, the ARNA response to increased renal pelvic pressure, 7,180 +/- 710%. s, was blocked by DDA. In conclusion, PGE(2) activates the cAMP-PKA pathway leading to a release of SP and activation of renal pelvic mechanosensory nerve fibers.     

Primary sequence determinants responsible for site-selective dephosphorylation of the PDGF beta-receptor by the receptor-like protein tyrosine phosphatase DEP-1

Site-selective dephosphorylation of receptor tyrosine kinases contributes to receptor regulation. The receptor-like protein tyrosine phosphatase DEP-1 site-selectively dephosphorylates the PDGF beta-receptor. DEP-1 dephosphorylation of original and chimeric phospho-peptides spanning the preferred pY1021 and the less preferred pY857 and pY562 sites was analyzed. Double substitutions of basic residues at -4 and +3 of pY857 and pY562 peptides improved affinity. Substitutions of single amino acids indicated preference for an acidic residue at position -1 and a preference against a basic residue at position +3. DEP-1 site-selective dephosphorylation of PDGF beta-receptor is thus determined by the primary sequence surrounding phosphorylation sites and involves interactions with residues spanning at least between positions -1 and +3.     

FGFR1 is required for the development of the auditory sensory epithelium

The mammalian auditory sensory epithelium, the organ of Corti, comprises the hair cells and supporting cells that are pivotal for hearing function. The origin and development of their precursors are poorly understood. Here we show that loss-of-function mutations in mouse fibroblast growth factor receptor 1 (Fgfr1) cause a dose-dependent disruption of the organ of Corti. Full inactivation of Fgfr1 in the inner ear epithelium by Foxg1-Cre-mediated deletion leads to an 85% reduction in the number of auditory hair cells. The primary cause appears to be reduced precursor cell proliferation in the early cochlear duct. Thus, during development, FGFR1 is required for the generation of the precursor pool, which gives rise to the auditory sensory epithelium. Our data also suggest that FGFR1 might have a distinct later role in intercellular signaling within the differentiating auditory sensory epithelium.     

Life cycle assessment in road infrastructure planning using spatial geological data

Purpose

The purpose of the study was to outline and demonstrate a new geographic information system (GIS)-based approach for utilising spatial geological data in three dimensions (i.e. length, width and depth) to improve estimates on earthworks during early stages of road infrastructure planning.

Methods

This was undertaken by using three main methodological steps: mass balance calculation, life cycle inventory analysis and spatial mapping of greenhouse gas (GHG) emissions and energy use. The mass balance calculation was undertaken in a GIS environment using two assumptions of geological stratigraphy for two proposed alternative road corridors in Sweden. The estimated volumes of excavated soil, blasted rock and filling material were later multiplied with the GHG emission and energy use factors for these processes, to create spatial data and maps in order to show potential impacts of the studied road corridors. The proposed GIS-based approach was evaluated by comparing with actual values received after one alternative was constructed.

Results and discussion

The results showed that the estimate of filling material was the most accurate (about 9 % deviation from actual values), while the estimate for excavated soil and blasted rock resulted in about 38 and 80 % deviation, respectively, from the actual values. It was also found that the total volume of excavated and ripped soils did not change when accounting for stratigraphy.

Conclusions

The conclusion of this study was that more information regarding embankment height and actual soil thickness would further improve the model, but the proposed GIS-based approach shows promising results for usage in LCA at an early stage of road infrastructure planning. Thus, by providing better data quality, GIS in combination with LCA can enable planning for a more sustainable transport infrastructure.