Calcium-dependent NADPH oxidase that generates superoxide. Also functions as a calcium-dependent proton channel and may regulate redox-dependent processes in lymphocytes and spermatozoa. May play a role in cell growth and apoptosis.
Superoxide and its derivatives are increasingly implicated in the regulation of physiological functions from oxygen sensing and blood pressure regulation to lymphocyte activation and sperm-oocyte fusion. Here we describe a novel superoxide-generating NADPH oxidase referred to as NADPH oxidase 5 (NOX5). NOX5 is distantly related to the gp91(phox) subunit of the phagocyte NADPH oxidase with conserved regions crucial for the electron transport (NADPH, FAD and heme binding sites). However, NOX5 has a unique N-terminal extension that contains three EF hand motifs. The mRNA of NOX5 is expressed in pachytene spermatocytes of testis and in B- and T-lymphocyte-rich areas of spleen and lymph nodes. When heterologously expressed, NOX5 was quiescent in unstimulated cells. However, in response to elevations of the cytosolic Ca(2+) concentration it generated large amounts of superoxide. Upon Ca(2+) activation, NOX5 also displayed a second function: it became a proton channel, presumably to compensate charge and pH alterations due to electron export. In summary, we have identified a novel NADPH oxidase that generates superoxide and functions as a H(+) channel in a Ca(2+)-dependent manner. NOX5 is likely to be involved in Ca(2+)-activated, redox-dependent processes of spermatozoa and lymphocytes such as sperm-oocyte fusion, cell proliferation, and cytokine secretion.
Reactive oxygen species (ROS) appear to play an important role in regulating growth and survival of prostate cancer. However, the sources for ROS production in prostate cancer cells have not been determined. We report that ROS are generated by intact American Type Culture Collection DU 145 cells and by their membranes through a mechanism blocked by NAD(P)H oxidase inhibitors. ROS are critical for growth in these cells, because NAD(P)H oxidase inhibitors and antioxidants blocked proliferation. Components of the human phagocyte NAD(P)H oxidase, p22phox and gp91phox, as well as the Ca2+ concentration-responsive gp91phox homolog NOX5 were demonstrated in DU 145 cells by RT-PCR and sequencing. Although the protein product for p22phox was not detectable, both gp91phox and NOX5 were identified throughout the cell by immunostaining and confocal microscopy and NOX5 immunostaining was enhanced in a perinuclear location, corresponding to enhanced ROS production adjacent to the nuclear membrane imaged by 2',7'-dichlorofluorescin diacetate oxidation. The calcium ionophore ionomycin dramatically stimulated ferricytochrome c reduction in cell media, further supporting the importance of NOX5 for ROS production. Antisense oligonucleotides for NOX5 inhibited ROS production and cell proliferation in DU 145 cells. In contrast, antisense oligonucleotides to p22phox or gp91phox did not impair cell growth. Inhibition of ROS generation with antioxidants or NAD(P)H oxidase inhibitors increased apoptosis in cells. These results indicate that ROS generated by the newly described NOX5 oxidase are essential for prostate cancer growth, possibly by providing trophic intracellular oxidant tone that retards programmed cell death.
NADPH oxidases have been identified as sources of reactive oxygen species (ROS) in vascular cells. In addition to the initially described enzyme containing gp91phox (NOX2), several homologues to NOX2 have been identified. Whereas NOX1, NOX2, and NOX4 are expressed in endothelial cells, a functional role of NOX5 containing additional N-terminal calcium-binding domains of varying sequences has not been reported in these cells. NOX5 protein was found in the endoplasmic reticulum of human microvascular endothelial cells (HMEC-1) and in the vascular wall. HMEC-1 cells expressed NOX5beta and NOX5delta as well as a variant lacking calcium-binding domains (NOX5S). NOX5beta and NOX5S increased basal ROS levels. Ionomycin exclusively enhanced NOX5beta-mediated ROS production. Although p22phox, when overexpressed, interacted with both NOX5 proteins, it was not essential for NOX5-mediated ROS production. NOX5 proteins stimulated endothelial cell proliferation and the formation of capillary-like structures whereas depletion of NOX5 by siRNA prevented these responses to thrombin. These data show that endothelial cells express different NOX5 variants including NOX5S lacking calcium-binding domains. NOX5 proteins are functional, promoting endothelial ROS production, proliferation, and the formation of capillary-like structures and contribute to the endothelial response to thrombin. These findings suggest that NOX5 variants play a novel role in controlling ROS-dependent processes in the vasculature.
Isoforms
v2
and
v5
Are involved in endothelial generation of reactive oxygen species (ROS), proliferation and angiogenesis and contribute to endothelial response to thrombin.
Superoxide and its derivatives are increasingly implicated in the regulation of physiological functions from oxygen sensing and blood pressure regulation to lymphocyte activation and sperm-oocyte fusion. Here we describe a novel superoxide-generating NADPH oxidase referred to as NADPH oxidase 5 (NOX5). NOX5 is distantly related to the gp91(phox) subunit of the phagocyte NADPH oxidase with conserved regions crucial for the electron transport (NADPH, FAD and heme binding sites). However, NOX5 has a unique N-terminal extension that contains three EF hand motifs. The mRNA of NOX5 is expressed in pachytene spermatocytes of testis and in B- and T-lymphocyte-rich areas of spleen and lymph nodes. When heterologously expressed, NOX5 was quiescent in unstimulated cells. However, in response to elevations of the cytosolic Ca(2+) concentration it generated large amounts of superoxide. Upon Ca(2+) activation, NOX5 also displayed a second function: it became a proton channel, presumably to compensate charge and pH alterations due to electron export. In summary, we have identified a novel NADPH oxidase that generates superoxide and functions as a H(+) channel in a Ca(2+)-dependent manner. NOX5 is likely to be involved in Ca(2+)-activated, redox-dependent processes of spermatozoa and lymphocytes such as sperm-oocyte fusion, cell proliferation, and cytokine secretion.
NADPH oxidases have been identified as sources of reactive oxygen species (ROS) in vascular cells. In addition to the initially described enzyme containing gp91phox (NOX2), several homologues to NOX2 have been identified. Whereas NOX1, NOX2, and NOX4 are expressed in endothelial cells, a functional role of NOX5 containing additional N-terminal calcium-binding domains of varying sequences has not been reported in these cells. NOX5 protein was found in the endoplasmic reticulum of human microvascular endothelial cells (HMEC-1) and in the vascular wall. HMEC-1 cells expressed NOX5beta and NOX5delta as well as a variant lacking calcium-binding domains (NOX5S). NOX5beta and NOX5S increased basal ROS levels. Ionomycin exclusively enhanced NOX5beta-mediated ROS production. Although p22phox, when overexpressed, interacted with both NOX5 proteins, it was not essential for NOX5-mediated ROS production. NOX5 proteins stimulated endothelial cell proliferation and the formation of capillary-like structures whereas depletion of NOX5 by siRNA prevented these responses to thrombin. These data show that endothelial cells express different NOX5 variants including NOX5S lacking calcium-binding domains. NOX5 proteins are functional, promoting endothelial ROS production, proliferation, and the formation of capillary-like structures and contribute to the endothelial response to thrombin. These findings suggest that NOX5 variants play a novel role in controlling ROS-dependent processes in the vasculature.
Reactive oxygen species (ROS) appear to play an important role in regulating growth and survival of prostate cancer. However, the sources for ROS production in prostate cancer cells have not been determined. We report that ROS are generated by intact American Type Culture Collection DU 145 cells and by their membranes through a mechanism blocked by NAD(P)H oxidase inhibitors. ROS are critical for growth in these cells, because NAD(P)H oxidase inhibitors and antioxidants blocked proliferation. Components of the human phagocyte NAD(P)H oxidase, p22phox and gp91phox, as well as the Ca2+ concentration-responsive gp91phox homolog NOX5 were demonstrated in DU 145 cells by RT-PCR and sequencing. Although the protein product for p22phox was not detectable, both gp91phox and NOX5 were identified throughout the cell by immunostaining and confocal microscopy and NOX5 immunostaining was enhanced in a perinuclear location, corresponding to enhanced ROS production adjacent to the nuclear membrane imaged by 2',7'-dichlorofluorescin diacetate oxidation. The calcium ionophore ionomycin dramatically stimulated ferricytochrome c reduction in cell media, further supporting the importance of NOX5 for ROS production. Antisense oligonucleotides for NOX5 inhibited ROS production and cell proliferation in DU 145 cells. In contrast, antisense oligonucleotides to p22phox or gp91phox did not impair cell growth. Inhibition of ROS generation with antioxidants or NAD(P)H oxidase inhibitors increased apoptosis in cells. These results indicate that ROS generated by the newly described NOX5 oxidase are essential for prostate cancer growth, possibly by providing trophic intracellular oxidant tone that retards programmed cell death.
Interacting selectively and non-covalently with FAD, flavin-adenine dinucleotide, the coenzyme or the prosthetic group of various flavoprotein oxidoreductase enzymes, in either the oxidized form, FAD, or the reduced form, FADH2.
Superoxide and its derivatives are increasingly implicated in the regulation of physiological functions from oxygen sensing and blood pressure regulation to lymphocyte activation and sperm-oocyte fusion. Here we describe a novel superoxide-generating NADPH oxidase referred to as NADPH oxidase 5 (NOX5). NOX5 is distantly related to the gp91(phox) subunit of the phagocyte NADPH oxidase with conserved regions crucial for the electron transport (NADPH, FAD and heme binding sites). However, NOX5 has a unique N-terminal extension that contains three EF hand motifs. The mRNA of NOX5 is expressed in pachytene spermatocytes of testis and in B- and T-lymphocyte-rich areas of spleen and lymph nodes. When heterologously expressed, NOX5 was quiescent in unstimulated cells. However, in response to elevations of the cytosolic Ca(2+) concentration it generated large amounts of superoxide. Upon Ca(2+) activation, NOX5 also displayed a second function: it became a proton channel, presumably to compensate charge and pH alterations due to electron export. In summary, we have identified a novel NADPH oxidase that generates superoxide and functions as a H(+) channel in a Ca(2+)-dependent manner. NOX5 is likely to be involved in Ca(2+)-activated, redox-dependent processes of spermatozoa and lymphocytes such as sperm-oocyte fusion, cell proliferation, and cytokine secretion.
Superoxide and its derivatives are increasingly implicated in the regulation of physiological functions from oxygen sensing and blood pressure regulation to lymphocyte activation and sperm-oocyte fusion. Here we describe a novel superoxide-generating NADPH oxidase referred to as NADPH oxidase 5 (NOX5). NOX5 is distantly related to the gp91(phox) subunit of the phagocyte NADPH oxidase with conserved regions crucial for the electron transport (NADPH, FAD and heme binding sites). However, NOX5 has a unique N-terminal extension that contains three EF hand motifs. The mRNA of NOX5 is expressed in pachytene spermatocytes of testis and in B- and T-lymphocyte-rich areas of spleen and lymph nodes. When heterologously expressed, NOX5 was quiescent in unstimulated cells. However, in response to elevations of the cytosolic Ca(2+) concentration it generated large amounts of superoxide. Upon Ca(2+) activation, NOX5 also displayed a second function: it became a proton channel, presumably to compensate charge and pH alterations due to electron export. In summary, we have identified a novel NADPH oxidase that generates superoxide and functions as a H(+) channel in a Ca(2+)-dependent manner. NOX5 is likely to be involved in Ca(2+)-activated, redox-dependent processes of spermatozoa and lymphocytes such as sperm-oocyte fusion, cell proliferation, and cytokine secretion.
Catalysis of facilitated diffusion of a hydrogen ion (by an energy-independent process) involving passage through a transmembrane aqueous pore or channel without evidence for a carrier-mediated mechanism.
Superoxide and its derivatives are increasingly implicated in the regulation of physiological functions from oxygen sensing and blood pressure regulation to lymphocyte activation and sperm-oocyte fusion. Here we describe a novel superoxide-generating NADPH oxidase referred to as NADPH oxidase 5 (NOX5). NOX5 is distantly related to the gp91(phox) subunit of the phagocyte NADPH oxidase with conserved regions crucial for the electron transport (NADPH, FAD and heme binding sites). However, NOX5 has a unique N-terminal extension that contains three EF hand motifs. The mRNA of NOX5 is expressed in pachytene spermatocytes of testis and in B- and T-lymphocyte-rich areas of spleen and lymph nodes. When heterologously expressed, NOX5 was quiescent in unstimulated cells. However, in response to elevations of the cytosolic Ca(2+) concentration it generated large amounts of superoxide. Upon Ca(2+) activation, NOX5 also displayed a second function: it became a proton channel, presumably to compensate charge and pH alterations due to electron export. In summary, we have identified a novel NADPH oxidase that generates superoxide and functions as a H(+) channel in a Ca(2+)-dependent manner. NOX5 is likely to be involved in Ca(2+)-activated, redox-dependent processes of spermatozoa and lymphocytes such as sperm-oocyte fusion, cell proliferation, and cytokine secretion.
Interacting selectively and non-covalently with nicotinamide-adenine dinucleotide phosphate, a coenzyme involved in many redox and biosynthetic reactions; binding may be to either the oxidized form, NADP+, or the reduced form, NADPH.
Superoxide and its derivatives are increasingly implicated in the regulation of physiological functions from oxygen sensing and blood pressure regulation to lymphocyte activation and sperm-oocyte fusion. Here we describe a novel superoxide-generating NADPH oxidase referred to as NADPH oxidase 5 (NOX5). NOX5 is distantly related to the gp91(phox) subunit of the phagocyte NADPH oxidase with conserved regions crucial for the electron transport (NADPH, FAD and heme binding sites). However, NOX5 has a unique N-terminal extension that contains three EF hand motifs. The mRNA of NOX5 is expressed in pachytene spermatocytes of testis and in B- and T-lymphocyte-rich areas of spleen and lymph nodes. When heterologously expressed, NOX5 was quiescent in unstimulated cells. However, in response to elevations of the cytosolic Ca(2+) concentration it generated large amounts of superoxide. Upon Ca(2+) activation, NOX5 also displayed a second function: it became a proton channel, presumably to compensate charge and pH alterations due to electron export. In summary, we have identified a novel NADPH oxidase that generates superoxide and functions as a H(+) channel in a Ca(2+)-dependent manner. NOX5 is likely to be involved in Ca(2+)-activated, redox-dependent processes of spermatozoa and lymphocytes such as sperm-oocyte fusion, cell proliferation, and cytokine secretion.
Superoxide and its derivatives are increasingly implicated in the regulation of physiological functions from oxygen sensing and blood pressure regulation to lymphocyte activation and sperm-oocyte fusion. Here we describe a novel superoxide-generating NADPH oxidase referred to as NADPH oxidase 5 (NOX5). NOX5 is distantly related to the gp91(phox) subunit of the phagocyte NADPH oxidase with conserved regions crucial for the electron transport (NADPH, FAD and heme binding sites). However, NOX5 has a unique N-terminal extension that contains three EF hand motifs. The mRNA of NOX5 is expressed in pachytene spermatocytes of testis and in B- and T-lymphocyte-rich areas of spleen and lymph nodes. When heterologously expressed, NOX5 was quiescent in unstimulated cells. However, in response to elevations of the cytosolic Ca(2+) concentration it generated large amounts of superoxide. Upon Ca(2+) activation, NOX5 also displayed a second function: it became a proton channel, presumably to compensate charge and pH alterations due to electron export. In summary, we have identified a novel NADPH oxidase that generates superoxide and functions as a H(+) channel in a Ca(2+)-dependent manner. NOX5 is likely to be involved in Ca(2+)-activated, redox-dependent processes of spermatozoa and lymphocytes such as sperm-oocyte fusion, cell proliferation, and cytokine secretion.
NADPH oxidases have been identified as sources of reactive oxygen species (ROS) in vascular cells. In addition to the initially described enzyme containing gp91phox (NOX2), several homologues to NOX2 have been identified. Whereas NOX1, NOX2, and NOX4 are expressed in endothelial cells, a functional role of NOX5 containing additional N-terminal calcium-binding domains of varying sequences has not been reported in these cells. NOX5 protein was found in the endoplasmic reticulum of human microvascular endothelial cells (HMEC-1) and in the vascular wall. HMEC-1 cells expressed NOX5beta and NOX5delta as well as a variant lacking calcium-binding domains (NOX5S). NOX5beta and NOX5S increased basal ROS levels. Ionomycin exclusively enhanced NOX5beta-mediated ROS production. Although p22phox, when overexpressed, interacted with both NOX5 proteins, it was not essential for NOX5-mediated ROS production. NOX5 proteins stimulated endothelial cell proliferation and the formation of capillary-like structures whereas depletion of NOX5 by siRNA prevented these responses to thrombin. These data show that endothelial cells express different NOX5 variants including NOX5S lacking calcium-binding domains. NOX5 proteins are functional, promoting endothelial ROS production, proliferation, and the formation of capillary-like structures and contribute to the endothelial response to thrombin. These findings suggest that NOX5 variants play a novel role in controlling ROS-dependent processes in the vasculature.
Superoxide and its derivatives are increasingly implicated in the regulation of physiological functions from oxygen sensing and blood pressure regulation to lymphocyte activation and sperm-oocyte fusion. Here we describe a novel superoxide-generating NADPH oxidase referred to as NADPH oxidase 5 (NOX5). NOX5 is distantly related to the gp91(phox) subunit of the phagocyte NADPH oxidase with conserved regions crucial for the electron transport (NADPH, FAD and heme binding sites). However, NOX5 has a unique N-terminal extension that contains three EF hand motifs. The mRNA of NOX5 is expressed in pachytene spermatocytes of testis and in B- and T-lymphocyte-rich areas of spleen and lymph nodes. When heterologously expressed, NOX5 was quiescent in unstimulated cells. However, in response to elevations of the cytosolic Ca(2+) concentration it generated large amounts of superoxide. Upon Ca(2+) activation, NOX5 also displayed a second function: it became a proton channel, presumably to compensate charge and pH alterations due to electron export. In summary, we have identified a novel NADPH oxidase that generates superoxide and functions as a H(+) channel in a Ca(2+)-dependent manner. NOX5 is likely to be involved in Ca(2+)-activated, redox-dependent processes of spermatozoa and lymphocytes such as sperm-oocyte fusion, cell proliferation, and cytokine secretion.
The regulated release of cytokines from a cell or group of cells. Cytokines are any of a group of proteins that function to control the survival, growth and differentiation of tissues and cells, and which have autocrine and paracrine activity.
Superoxide and its derivatives are increasingly implicated in the regulation of physiological functions from oxygen sensing and blood pressure regulation to lymphocyte activation and sperm-oocyte fusion. Here we describe a novel superoxide-generating NADPH oxidase referred to as NADPH oxidase 5 (NOX5). NOX5 is distantly related to the gp91(phox) subunit of the phagocyte NADPH oxidase with conserved regions crucial for the electron transport (NADPH, FAD and heme binding sites). However, NOX5 has a unique N-terminal extension that contains three EF hand motifs. The mRNA of NOX5 is expressed in pachytene spermatocytes of testis and in B- and T-lymphocyte-rich areas of spleen and lymph nodes. When heterologously expressed, NOX5 was quiescent in unstimulated cells. However, in response to elevations of the cytosolic Ca(2+) concentration it generated large amounts of superoxide. Upon Ca(2+) activation, NOX5 also displayed a second function: it became a proton channel, presumably to compensate charge and pH alterations due to electron export. In summary, we have identified a novel NADPH oxidase that generates superoxide and functions as a H(+) channel in a Ca(2+)-dependent manner. NOX5 is likely to be involved in Ca(2+)-activated, redox-dependent processes of spermatozoa and lymphocytes such as sperm-oocyte fusion, cell proliferation, and cytokine secretion.
The division of the cytoplasm and the plasma membrane of a cell and its separation into two daughter cells. Cytokinesis usually occurs after growth, replication, and segregation of cellular components, and occurs after division of the nucleus.
Superoxide and its derivatives are increasingly implicated in the regulation of physiological functions from oxygen sensing and blood pressure regulation to lymphocyte activation and sperm-oocyte fusion. Here we describe a novel superoxide-generating NADPH oxidase referred to as NADPH oxidase 5 (NOX5). NOX5 is distantly related to the gp91(phox) subunit of the phagocyte NADPH oxidase with conserved regions crucial for the electron transport (NADPH, FAD and heme binding sites). However, NOX5 has a unique N-terminal extension that contains three EF hand motifs. The mRNA of NOX5 is expressed in pachytene spermatocytes of testis and in B- and T-lymphocyte-rich areas of spleen and lymph nodes. When heterologously expressed, NOX5 was quiescent in unstimulated cells. However, in response to elevations of the cytosolic Ca(2+) concentration it generated large amounts of superoxide. Upon Ca(2+) activation, NOX5 also displayed a second function: it became a proton channel, presumably to compensate charge and pH alterations due to electron export. In summary, we have identified a novel NADPH oxidase that generates superoxide and functions as a H(+) channel in a Ca(2+)-dependent manner. NOX5 is likely to be involved in Ca(2+)-activated, redox-dependent processes of spermatozoa and lymphocytes such as sperm-oocyte fusion, cell proliferation, and cytokine secretion.
A process in which a series of electron carriers operate together to transfer electrons from donors to any of several different terminal electron acceptors to generate a transmembrane electrochemical gradient.
The multiplication or reproduction of endothelial cells, resulting in the expansion of a cell population. Endothelial cells are thin flattened cells which line the inside surfaces of body cavities, blood vessels, and lymph vessels, making up the endothelium.
NADPH oxidases have been identified as sources of reactive oxygen species (ROS) in vascular cells. In addition to the initially described enzyme containing gp91phox (NOX2), several homologues to NOX2 have been identified. Whereas NOX1, NOX2, and NOX4 are expressed in endothelial cells, a functional role of NOX5 containing additional N-terminal calcium-binding domains of varying sequences has not been reported in these cells. NOX5 protein was found in the endoplasmic reticulum of human microvascular endothelial cells (HMEC-1) and in the vascular wall. HMEC-1 cells expressed NOX5beta and NOX5delta as well as a variant lacking calcium-binding domains (NOX5S). NOX5beta and NOX5S increased basal ROS levels. Ionomycin exclusively enhanced NOX5beta-mediated ROS production. Although p22phox, when overexpressed, interacted with both NOX5 proteins, it was not essential for NOX5-mediated ROS production. NOX5 proteins stimulated endothelial cell proliferation and the formation of capillary-like structures whereas depletion of NOX5 by siRNA prevented these responses to thrombin. These data show that endothelial cells express different NOX5 variants including NOX5S lacking calcium-binding domains. NOX5 proteins are functional, promoting endothelial ROS production, proliferation, and the formation of capillary-like structures and contribute to the endothelial response to thrombin. These findings suggest that NOX5 variants play a novel role in controlling ROS-dependent processes in the vasculature.
Superoxide and its derivatives are increasingly implicated in the regulation of physiological functions from oxygen sensing and blood pressure regulation to lymphocyte activation and sperm-oocyte fusion. Here we describe a novel superoxide-generating NADPH oxidase referred to as NADPH oxidase 5 (NOX5). NOX5 is distantly related to the gp91(phox) subunit of the phagocyte NADPH oxidase with conserved regions crucial for the electron transport (NADPH, FAD and heme binding sites). However, NOX5 has a unique N-terminal extension that contains three EF hand motifs. The mRNA of NOX5 is expressed in pachytene spermatocytes of testis and in B- and T-lymphocyte-rich areas of spleen and lymph nodes. When heterologously expressed, NOX5 was quiescent in unstimulated cells. However, in response to elevations of the cytosolic Ca(2+) concentration it generated large amounts of superoxide. Upon Ca(2+) activation, NOX5 also displayed a second function: it became a proton channel, presumably to compensate charge and pH alterations due to electron export. In summary, we have identified a novel NADPH oxidase that generates superoxide and functions as a H(+) channel in a Ca(2+)-dependent manner. NOX5 is likely to be involved in Ca(2+)-activated, redox-dependent processes of spermatozoa and lymphocytes such as sperm-oocyte fusion, cell proliferation, and cytokine secretion.
A metabolic process that results in the removal or addition of one or more electrons to or from a substance, with or without the concomitant removal or addition of a proton or protons.
Superoxide and its derivatives are increasingly implicated in the regulation of physiological functions from oxygen sensing and blood pressure regulation to lymphocyte activation and sperm-oocyte fusion. Here we describe a novel superoxide-generating NADPH oxidase referred to as NADPH oxidase 5 (NOX5). NOX5 is distantly related to the gp91(phox) subunit of the phagocyte NADPH oxidase with conserved regions crucial for the electron transport (NADPH, FAD and heme binding sites). However, NOX5 has a unique N-terminal extension that contains three EF hand motifs. The mRNA of NOX5 is expressed in pachytene spermatocytes of testis and in B- and T-lymphocyte-rich areas of spleen and lymph nodes. When heterologously expressed, NOX5 was quiescent in unstimulated cells. However, in response to elevations of the cytosolic Ca(2+) concentration it generated large amounts of superoxide. Upon Ca(2+) activation, NOX5 also displayed a second function: it became a proton channel, presumably to compensate charge and pH alterations due to electron export. In summary, we have identified a novel NADPH oxidase that generates superoxide and functions as a H(+) channel in a Ca(2+)-dependent manner. NOX5 is likely to be involved in Ca(2+)-activated, redox-dependent processes of spermatozoa and lymphocytes such as sperm-oocyte fusion, cell proliferation, and cytokine secretion.
NADPH oxidases have been identified as sources of reactive oxygen species (ROS) in vascular cells. In addition to the initially described enzyme containing gp91phox (NOX2), several homologues to NOX2 have been identified. Whereas NOX1, NOX2, and NOX4 are expressed in endothelial cells, a functional role of NOX5 containing additional N-terminal calcium-binding domains of varying sequences has not been reported in these cells. NOX5 protein was found in the endoplasmic reticulum of human microvascular endothelial cells (HMEC-1) and in the vascular wall. HMEC-1 cells expressed NOX5beta and NOX5delta as well as a variant lacking calcium-binding domains (NOX5S). NOX5beta and NOX5S increased basal ROS levels. Ionomycin exclusively enhanced NOX5beta-mediated ROS production. Although p22phox, when overexpressed, interacted with both NOX5 proteins, it was not essential for NOX5-mediated ROS production. NOX5 proteins stimulated endothelial cell proliferation and the formation of capillary-like structures whereas depletion of NOX5 by siRNA prevented these responses to thrombin. These data show that endothelial cells express different NOX5 variants including NOX5S lacking calcium-binding domains. NOX5 proteins are functional, promoting endothelial ROS production, proliferation, and the formation of capillary-like structures and contribute to the endothelial response to thrombin. These findings suggest that NOX5 variants play a novel role in controlling ROS-dependent processes in the vasculature.
The directed movement of protons (hydrogen ions) into, out of or within a cell, or between cells, by means of some agent such as a transporter or pore.
Superoxide and its derivatives are increasingly implicated in the regulation of physiological functions from oxygen sensing and blood pressure regulation to lymphocyte activation and sperm-oocyte fusion. Here we describe a novel superoxide-generating NADPH oxidase referred to as NADPH oxidase 5 (NOX5). NOX5 is distantly related to the gp91(phox) subunit of the phagocyte NADPH oxidase with conserved regions crucial for the electron transport (NADPH, FAD and heme binding sites). However, NOX5 has a unique N-terminal extension that contains three EF hand motifs. The mRNA of NOX5 is expressed in pachytene spermatocytes of testis and in B- and T-lymphocyte-rich areas of spleen and lymph nodes. When heterologously expressed, NOX5 was quiescent in unstimulated cells. However, in response to elevations of the cytosolic Ca(2+) concentration it generated large amounts of superoxide. Upon Ca(2+) activation, NOX5 also displayed a second function: it became a proton channel, presumably to compensate charge and pH alterations due to electron export. In summary, we have identified a novel NADPH oxidase that generates superoxide and functions as a H(+) channel in a Ca(2+)-dependent manner. NOX5 is likely to be involved in Ca(2+)-activated, redox-dependent processes of spermatozoa and lymphocytes such as sperm-oocyte fusion, cell proliferation, and cytokine secretion.
Superoxide and its derivatives are increasingly implicated in the regulation of physiological functions from oxygen sensing and blood pressure regulation to lymphocyte activation and sperm-oocyte fusion. Here we describe a novel superoxide-generating NADPH oxidase referred to as NADPH oxidase 5 (NOX5). NOX5 is distantly related to the gp91(phox) subunit of the phagocyte NADPH oxidase with conserved regions crucial for the electron transport (NADPH, FAD and heme binding sites). However, NOX5 has a unique N-terminal extension that contains three EF hand motifs. The mRNA of NOX5 is expressed in pachytene spermatocytes of testis and in B- and T-lymphocyte-rich areas of spleen and lymph nodes. When heterologously expressed, NOX5 was quiescent in unstimulated cells. However, in response to elevations of the cytosolic Ca(2+) concentration it generated large amounts of superoxide. Upon Ca(2+) activation, NOX5 also displayed a second function: it became a proton channel, presumably to compensate charge and pH alterations due to electron export. In summary, we have identified a novel NADPH oxidase that generates superoxide and functions as a H(+) channel in a Ca(2+)-dependent manner. NOX5 is likely to be involved in Ca(2+)-activated, redox-dependent processes of spermatozoa and lymphocytes such as sperm-oocyte fusion, cell proliferation, and cytokine secretion.
Any process that modulates the frequency, rate or extent of proton transport into, out of or within a cell, or between cells, by means of some agent such as a transporter or pore.
Superoxide and its derivatives are increasingly implicated in the regulation of physiological functions from oxygen sensing and blood pressure regulation to lymphocyte activation and sperm-oocyte fusion. Here we describe a novel superoxide-generating NADPH oxidase referred to as NADPH oxidase 5 (NOX5). NOX5 is distantly related to the gp91(phox) subunit of the phagocyte NADPH oxidase with conserved regions crucial for the electron transport (NADPH, FAD and heme binding sites). However, NOX5 has a unique N-terminal extension that contains three EF hand motifs. The mRNA of NOX5 is expressed in pachytene spermatocytes of testis and in B- and T-lymphocyte-rich areas of spleen and lymph nodes. When heterologously expressed, NOX5 was quiescent in unstimulated cells. However, in response to elevations of the cytosolic Ca(2+) concentration it generated large amounts of superoxide. Upon Ca(2+) activation, NOX5 also displayed a second function: it became a proton channel, presumably to compensate charge and pH alterations due to electron export. In summary, we have identified a novel NADPH oxidase that generates superoxide and functions as a H(+) channel in a Ca(2+)-dependent manner. NOX5 is likely to be involved in Ca(2+)-activated, redox-dependent processes of spermatozoa and lymphocytes such as sperm-oocyte fusion, cell proliferation, and cytokine secretion.
The enzymatic generation of superoxide, the superoxide anion O2- (superoxide free radical), or any compound containing this species, by a cell in response to environmental stress, thereby mediating the activation of various stress-inducible signaling pathways.
Superoxide and its derivatives are increasingly implicated in the regulation of physiological functions from oxygen sensing and blood pressure regulation to lymphocyte activation and sperm-oocyte fusion. Here we describe a novel superoxide-generating NADPH oxidase referred to as NADPH oxidase 5 (NOX5). NOX5 is distantly related to the gp91(phox) subunit of the phagocyte NADPH oxidase with conserved regions crucial for the electron transport (NADPH, FAD and heme binding sites). However, NOX5 has a unique N-terminal extension that contains three EF hand motifs. The mRNA of NOX5 is expressed in pachytene spermatocytes of testis and in B- and T-lymphocyte-rich areas of spleen and lymph nodes. When heterologously expressed, NOX5 was quiescent in unstimulated cells. However, in response to elevations of the cytosolic Ca(2+) concentration it generated large amounts of superoxide. Upon Ca(2+) activation, NOX5 also displayed a second function: it became a proton channel, presumably to compensate charge and pH alterations due to electron export. In summary, we have identified a novel NADPH oxidase that generates superoxide and functions as a H(+) channel in a Ca(2+)-dependent manner. NOX5 is likely to be involved in Ca(2+)-activated, redox-dependent processes of spermatozoa and lymphocytes such as sperm-oocyte fusion, cell proliferation, and cytokine secretion.
NADPH oxidase 5 (NOX5) is a homologue of the gp91(phox) subunit of the phagocyte NADPH oxidase. NOX5 is expressed in lymphoid organs and testis and distinguished from the other NADPH oxidases by its unique N terminus, which contains three canonical EF-hands, Ca(2+)-binding domains. Upon heterologous expression, NOX5 was shown to generate superoxide in response to intracellular Ca(2+) elevations. In this study, we have analyzed the mechanism of Ca(2+) activation of NOX5. In a cell-free system, Ca(2+) elevations triggered superoxide production by NOX5 (K(m) = 1.06 microm) in an NADPH- and FAD-dependent but cytosol-independent manner. That result indicated a role for the N-terminal EF-hands in NOX5 activation. Therefore, we generated recombinant proteins of NOX5 N terminus and investigated their interactions with Ca(2+). Flow dialysis experiments showed that NOX5 N terminus contained four Ca(2+)-binding sites and allowed us to define the hitherto unidentified fourth, non-canonical EF-hand. The EF-hands of NOX5 formed two pairs: the very N-terminal pair had relatively low affinity for Ca(2+), whereas the more C-terminal pair bound Ca(2+) with high affinity. Ca(2+) binding caused a marked conformation change in the N terminus, which exposed its hydrophobic core, and became able to bind melittin, a model peptide for calmodulin targets. Using a pull-down assay, we demonstrate that the regulatory N terminus and the catalytic C terminus of NOX5 interact in a Ca(2+)-dependent way. Our results indicate that the Ca(2+)-induced conformation change of NOX5 N terminus led to enzyme activation through an intra-molecular interaction. That represents a novel mechanism of activation among NAD(P)H oxidases and Ca(2+)-activated enzymes.
NADPH oxidase 5 (NOX5) is a homologue of the gp91(phox) subunit of the phagocyte NADPH oxidase. NOX5 is expressed in lymphoid organs and testis and distinguished from the other NADPH oxidases by its unique N terminus, which contains three canonical EF-hands, Ca(2+)-binding domains. Upon heterologous expression, NOX5 was shown to generate superoxide in response to intracellular Ca(2+) elevations. In this study, we have analyzed the mechanism of Ca(2+) activation of NOX5. In a cell-free system, Ca(2+) elevations triggered superoxide production by NOX5 (K(m) = 1.06 microm) in an NADPH- and FAD-dependent but cytosol-independent manner. That result indicated a role for the N-terminal EF-hands in NOX5 activation. Therefore, we generated recombinant proteins of NOX5 N terminus and investigated their interactions with Ca(2+). Flow dialysis experiments showed that NOX5 N terminus contained four Ca(2+)-binding sites and allowed us to define the hitherto unidentified fourth, non-canonical EF-hand. The EF-hands of NOX5 formed two pairs: the very N-terminal pair had relatively low affinity for Ca(2+), whereas the more C-terminal pair bound Ca(2+) with high affinity. Ca(2+) binding caused a marked conformation change in the N terminus, which exposed its hydrophobic core, and became able to bind melittin, a model peptide for calmodulin targets. Using a pull-down assay, we demonstrate that the regulatory N terminus and the catalytic C terminus of NOX5 interact in a Ca(2+)-dependent way. Our results indicate that the Ca(2+)-induced conformation change of NOX5 N terminus led to enzyme activation through an intra-molecular interaction. That represents a novel mechanism of activation among NAD(P)H oxidases and Ca(2+)-activated enzymes.
Activated by calcium which induces conformational changes and interaction between the N-terminal regulatory region and the C-terminal catalytic region. Inhibited by diphenylene iodonium.
NADPH oxidase 5 (NOX5) is a homologue of the gp91(phox) subunit of the phagocyte NADPH oxidase. NOX5 is expressed in lymphoid organs and testis and distinguished from the other NADPH oxidases by its unique N terminus, which contains three canonical EF-hands, Ca(2+)-binding domains. Upon heterologous expression, NOX5 was shown to generate superoxide in response to intracellular Ca(2+) elevations. In this study, we have analyzed the mechanism of Ca(2+) activation of NOX5. In a cell-free system, Ca(2+) elevations triggered superoxide production by NOX5 (K(m) = 1.06 microm) in an NADPH- and FAD-dependent but cytosol-independent manner. That result indicated a role for the N-terminal EF-hands in NOX5 activation. Therefore, we generated recombinant proteins of NOX5 N terminus and investigated their interactions with Ca(2+). Flow dialysis experiments showed that NOX5 N terminus contained four Ca(2+)-binding sites and allowed us to define the hitherto unidentified fourth, non-canonical EF-hand. The EF-hands of NOX5 formed two pairs: the very N-terminal pair had relatively low affinity for Ca(2+), whereas the more C-terminal pair bound Ca(2+) with high affinity. Ca(2+) binding caused a marked conformation change in the N terminus, which exposed its hydrophobic core, and became able to bind melittin, a model peptide for calmodulin targets. Using a pull-down assay, we demonstrate that the regulatory N terminus and the catalytic C terminus of NOX5 interact in a Ca(2+)-dependent way. Our results indicate that the Ca(2+)-induced conformation change of NOX5 N terminus led to enzyme activation through an intra-molecular interaction. That represents a novel mechanism of activation among NAD(P)H oxidases and Ca(2+)-activated enzymes.
Protein involved in angiogenesis, the sprouting or splitting of capillaries from pre-existing vasculature. Angiogenesis plays an important role for example during embryonic development, normal growth of tissues and maintenance of the normal vasculature, wound healing, tumor growth and metastasis.
Protein involved in the transport of electrons, a process by which electrons are transported through a series of reactions from the reductant, or electron donor, to the oxidant, or electron acceptor, with concomitant energy conversion. Necessary for both photosynthesis and aerobic respiration.
Protein involved in the transport of ions. Such proteins are usually transmembrane and mediate a movement of ions across cell membranes. Transport may be passive (facilitated diffusion; down the electrochemical gradient), or active (against the electrochemical gradient). Active transport requires energy which may come from light, oxidation reactions, ATP hydrolysis, or cotransport of other ions or molecules.
Protein involved in the transport of a molecule (metabolite, protein, etc), a ion or an electron across cell membranes, inside the cell or in a tissue fluid.
Protein which is part of a transmembrane protein complex that forms a hydrophilic channel across the lipid bilayer through which specific inorganic ions can diffuse down their electrochemical gradients. The channels are usually gated and only open in response to a specific stimulus, such as a change in membrane potential (voltage-gated) or the binding of a ligand (ligand-gated channel).
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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