Age-related NADH oxidase (arNOX = ENOX3) proteins are superoxide-generating cell surface oxidases that increase in activity with age beginning at about 30 y. A soluble and truncated exfoliated form of the activity is ...Age-related NADH oxidase (arNOX = ENOX3) proteins are superoxide-generating cell surface oxidases that increase in activity with age beginning at about 30 y. A soluble and truncated exfoliated form of the activity is present in blood and other body fluids. The activity was purified to apparent homogeneity from human urine and resolved by 2-D gel electrophoresis into a series of 24 to 32 kDa components of low isoelectric point. The purified proteins were resistant both to N-terminal sequencing and trypsin cleavage. Cleavage with pepsin revealed peptides corresponding to the TM9 family of transmembrane proteins. Peptide antisera raised to all five members of the human TM9 family sequentially blocked the arNOX activity of human saliva and sera. The soluble truncated N-terminus of the human homolog TM9SF4 was expressed in bacteria. The recombinant protein was characterized biochemically and exhibited ar-NOX activity. The findings identify five arNOX isoforms each of which correspond to one of the five known TM9 family members. The exfoliated soluble arNOX forms are derived from the 24 to 32 kDa N-termini exposed to the cell’s exterior at the cell surface. Each of the shed forms contain putative functional motifs characteristic of ECTO-NOX (ENOX) proteins despite only minimal sequence identity. Our findings identify arNOX as having functional characteristics of ENOX proteins and the TM9 superfamily of proteins as the genetic origins of the five known arNOX isoforms present in human sera, plasma and other body fluids1.展开更多
Age-related Ecto-Nicotinamide Adenine Dinucleotide Oxidase Disulfide Thiol Exchangers 3 (ENOX3) or age-related NADH oxidases (arNOX) are expressed at the cell surface as five members of the TM-9 superfamily, initially...Age-related Ecto-Nicotinamide Adenine Dinucleotide Oxidase Disulfide Thiol Exchangers 3 (ENOX3) or age-related NADH oxidases (arNOX) are expressed at the cell surface as five members of the TM-9 superfamily, initially membrane anchored, all functionally similar, with the N-termini exposed at the cell’s exterior. ECTO-NOXes are cell surface proteins with both time-keeping CoQH2 [NAD(P)H] oxidase and protein disulfidethiol interchange activities. They are designated as ECTO-NOX proteins because of their localization on the outer surface of the plasma membrane and to distinguish them from the phox-NOXes of host defense. A ca. 30 kDa N-terminal fragment is cleaved and accumulates in body fluids (serum, saliva, urine, perspiration). arNOXes appear around age 30 and increase steadily thereafter. Reduced quinones, i.e., reduced coenzyme Q, of the plasma membrane are natural substrates. NAD(P)H is oxidized as an artificial substrate. In one phase of the arNOX cycle electrons are transferred to oxygen to generate superoxide. Substrates for the shed forms of arNOX appear to be proteins of body fluids. Circulating lipoproteins and skin matrix proteins emerge as potentially important health-related targets. Through oxidation of collagen, elastin and other proteins of the skin matrix, arNOXes are major contributors to skin aging through tyrosine and thiol oxidation and subsequent cross linking. The main destructive action of arNOX, however, may be to directly oxidize circulating lipoproteins. arNOX in the blood is structured as an integral component of the LDL particle through site-specific binding. As such, arNOXes are implicated as major risk factors for cardiovascular disease due to specific oxidation of LDLs. The superoxide produced and its conversion to hydrogen peroxide would be one part of the potentially destructive properties by contribution to lipid oxidation. Inhibition of arNOX proteins provides a rational basis for anti-aging interventions and their elimination as a major risk factor of atherogenesis.展开更多
文摘Age-related NADH oxidase (arNOX = ENOX3) proteins are superoxide-generating cell surface oxidases that increase in activity with age beginning at about 30 y. A soluble and truncated exfoliated form of the activity is present in blood and other body fluids. The activity was purified to apparent homogeneity from human urine and resolved by 2-D gel electrophoresis into a series of 24 to 32 kDa components of low isoelectric point. The purified proteins were resistant both to N-terminal sequencing and trypsin cleavage. Cleavage with pepsin revealed peptides corresponding to the TM9 family of transmembrane proteins. Peptide antisera raised to all five members of the human TM9 family sequentially blocked the arNOX activity of human saliva and sera. The soluble truncated N-terminus of the human homolog TM9SF4 was expressed in bacteria. The recombinant protein was characterized biochemically and exhibited ar-NOX activity. The findings identify five arNOX isoforms each of which correspond to one of the five known TM9 family members. The exfoliated soluble arNOX forms are derived from the 24 to 32 kDa N-termini exposed to the cell’s exterior at the cell surface. Each of the shed forms contain putative functional motifs characteristic of ECTO-NOX (ENOX) proteins despite only minimal sequence identity. Our findings identify arNOX as having functional characteristics of ENOX proteins and the TM9 superfamily of proteins as the genetic origins of the five known arNOX isoforms present in human sera, plasma and other body fluids1.
文摘Age-related Ecto-Nicotinamide Adenine Dinucleotide Oxidase Disulfide Thiol Exchangers 3 (ENOX3) or age-related NADH oxidases (arNOX) are expressed at the cell surface as five members of the TM-9 superfamily, initially membrane anchored, all functionally similar, with the N-termini exposed at the cell’s exterior. ECTO-NOXes are cell surface proteins with both time-keeping CoQH2 [NAD(P)H] oxidase and protein disulfidethiol interchange activities. They are designated as ECTO-NOX proteins because of their localization on the outer surface of the plasma membrane and to distinguish them from the phox-NOXes of host defense. A ca. 30 kDa N-terminal fragment is cleaved and accumulates in body fluids (serum, saliva, urine, perspiration). arNOXes appear around age 30 and increase steadily thereafter. Reduced quinones, i.e., reduced coenzyme Q, of the plasma membrane are natural substrates. NAD(P)H is oxidized as an artificial substrate. In one phase of the arNOX cycle electrons are transferred to oxygen to generate superoxide. Substrates for the shed forms of arNOX appear to be proteins of body fluids. Circulating lipoproteins and skin matrix proteins emerge as potentially important health-related targets. Through oxidation of collagen, elastin and other proteins of the skin matrix, arNOXes are major contributors to skin aging through tyrosine and thiol oxidation and subsequent cross linking. The main destructive action of arNOX, however, may be to directly oxidize circulating lipoproteins. arNOX in the blood is structured as an integral component of the LDL particle through site-specific binding. As such, arNOXes are implicated as major risk factors for cardiovascular disease due to specific oxidation of LDLs. The superoxide produced and its conversion to hydrogen peroxide would be one part of the potentially destructive properties by contribution to lipid oxidation. Inhibition of arNOX proteins provides a rational basis for anti-aging interventions and their elimination as a major risk factor of atherogenesis.
