CYB5R3: a key player in aerobic metabolism and aging?

Aging results from a complex and not completely understood chain of processes that are associated with various negative metabolic consequences and ultimately leads to senescence and death. The intracellular ratio of pyridine nucleotides (NAD+/NADH), has been proposed to be at the center stage of age-related biochemical changes in organisms, and may help to explain the observed influence of calorie restriction and energy-sensitive proteins on lifespan in model organisms. Indeed, the NAD+/NADH ratios affect the activity of a number of proteins, including sirtuins, which have gained prominence in the aging field as potential mediators of the beneficial effects of calorie restriction and mediating lifespan. Here we review the activities of a redox enzyme (NQR1 in yeast and CYB5R3 in mammals) that also influences the NAD+/NADH ratio and may play a regulatory role that connects aerobic metabolism with aging.

elevates SIRT1 expression in a number of tissues [11], and transgenic mice that overexpress SIRT1 exhibit a phenotype mirroring some aspects of CR [12]. SIRT1 has also been shown to improve insulin sensitivity [13], another consequence of a CR diet [14].
CR promotes a healthy aging phenotype through a myriad of mechanisms, one of which is thought to be its ability to increase mitochondrial efficiency and biogenesis. Increases in mitochondrial biogenesis are driven by eNOS and PGC-1α expression and activation. Furthermore, these changes in mitochondria following CR are accompanied by a decrease in production of reactive oxygen species (ROS) without a net reduction of ATP biosynthesis, which indicates a higher bioenergetics efficiency [15,16]. There are several reports on how CR induces the deacetylation of PGC-1α by SIRT1 [17]. Sirtuins are NAD + -dependent deacetylases [18], and this dependence has led researchers to propose that sirtuins are at the center of the regulatory nexus between energy metabolism and aging because NAD + is a primary marker for intracellular energy status. It has also been demonstrated that CR activates sirtuins and thereby increases both the stability of chromatin [19] and cell survival [11]. Given the dependence of sirtuins on NAD + and the published activities of sirtuins under CR conditions, it has been hypothesized that NAD + levels and its metabolism are at the center of the regulatory mechanisms behind the beneficial effects of CR [17]. Furthermore, the conversion of NADH to its reduced form NAD + in mitochondria, a reaction that is supported by coenzyme Q (CoQ), is also thought to protect mitochondria during aging [20]. We propose, therefore, mechanisms that affect the NAD + /NADH ratio and thereby modulate sirtuins and other NAD +dependent enzymes are key players in the regulation of the aging process.
We have recently described the role of NQR1, a gene that encodes cytochrome b 5 reductase, a protein that uses both NADH and CoQ as substrates, in chronological and replicative lifespan in Saccharomyces cerevisiae [21]. This enzyme is located at the plasma membrane and is homologous to the mammalian enzyme encoded by CYB5R3, which can also be found in plasma membranes and uses exclusively NADH and CoQ as substrates [22]. This enzyme is a key component of the trans-plasma membrane redox system (PMRS). The PMRS provides both protection against extracellular oxidants [23] and prevention of apoptosis initiated by the activation of the neutral sphingomyelinase at the plasma membrane [24]. CR induces the expression of NQR1 in yeast, increasing the cytosolic NADH oxidation rate [21]. Similarly, CR increases the presence of this enzyme in the plasma membranes of both the liver and brain of rats, improving the antioxidant protection of phospholipids in these membranes [25,26]. This antioxidant system is also activated in mitochondrial DNA-deficient (ρ°) mammalian cells [27,28], and in vitamin E-deficient rat livers [29]. In the case of mammalian ρ° cells, cell survival is dependent on the redox homeostasis maintained by NADH oxidation by the PMRS. As indicated above, the increase of aerobic metabolism induced by CR also requires the cytosolic cooperation of CYB5R3 to maintain the NAD + /NADH ratio. Thus, any intervention that induces membrane instability or alters respiratory metabolism will evoke the transcription of CYB5R3 and activation of its enzymatic product.
Similar to the case in mammals, yeast NQR1 is upregulated by CR in parallel with an activation of respiration. Given that the same conditions activate the CoQ biosynthesis pathway [30], this may indicate a connection between CoQ biosynthesis and respiration. Interestingly, over-expression of NQR1 in yeast requires respiration to maintain cell survival. The mitochondrial mutant strains ΔATP2 and ΔCOR1 cannot grow under anaerobic conditions when NQR1 is overexpressed. The ΔATP2 strain has a defective ATP synthase complex and the ΔCOR1 strain is defective in the bc 1 complex. Similar results are obtained when the ΔCOQ2 strain, in which the CoQ biosynthesis pathway is inoperable, is used to overexpress NQR1. However, the addition of external CoQ 6 restored both respiration and growth in the latest strain. These results indicate that NQR1 effect acts through the respiratory metabolism in yeast [21].
Over-expression of NQR1 extends chronological lifespan in the absence of SIR2, perhaps acting through a pathway dependent on NAD + /NADH balance that requires respiration [31], but not SIR2 [32]. NQR1 overexpression also extends replicative lifespan in a SIR2dependent manner that mimics CR [8]. NQR1 promotes oxygen consumption while inhibiting ethanol production and this shift occurs alongside an increase in respiratory chain enzyme activities. NQR1 thus causes a shift from fermentative to respiratory metabolism that may help explain its role in longevity. Yeast growing in low glucose (CR) media also shows the increase of both chronological and replicative lifespan through the activation of respiration [8,31].
We can hypothesize then that NQR1 in yeast and CYB5R3 in mammals play a regulatory role connecting aerobic metabolism and aging processes through their ability to alter the NAD + /NADH ratio. Cytosolic NAD + / NADH must be balanced with that of mitochondria. We expect that NQR1 would partially prevent the de novo www.impactaging.com biosynthesis of NAD + most likely by increasing the recycling of the redox state of nucleotides and maintaining the availability of NAD + to consumer enzymes.
It is assumed that sirtuins connect metabolism to aging because they use NAD + as substrate [18]. This rationale can also be applied to CYB5R3 because the enzyme consumes NADH as an obligatory substrate. This enzyme would then be an essential component of the NAD + /NADH-dependent metabolic pathways in cooperation with the mitochondrial respiratory chain (Figure 1), which both contribute to the maintenance of the NAD + /NADH ratio and, as a consequence, regulate the function of sirtuins and other downstream NAD + consumers. The NADH consumers and NAD + consumers may participate in a regulatory loop, as a decrease of NAD + availability will activate NAD + biosynthesis as has been shown to occur under stress such as in nutrient-dependent survival mechanisms [33].
Mammalian CYB5R3 may also connect aerobic metabolism and aging. CYB5R3 encodes for a membrane-bound form of cytochrome b 5 reductase in somatic cells that is N-myristoylated and thereby anchored to the plasma membrane, mitochondrial outer membrane and endoplasmic reticulum. This isoform participates in cholesterol biosynthesis [34], fatty acid elongation and desaturation [35], P-450 mediated hydro-xylation of drugs and steroid hormones [36] and the PMRS [22]. There is also a soluble isoform, which lacks the N-terminal binding domain and exists in the cytoplasm of erythrocytes where its main function is to reduce methaemoglobin [37]. Both isoforms come from alternative splicing of the same CYB5R3 gene. Deficiencies of cytochrome b 5 reductase cause recessive congenital methaemoglobulinemia (RCM), which presents with two distinct clinical forms. RCM type I is benign and limited to red blood cells. RCM type II is severe, affects all cells in the organism, and can lead to neurological dysfunction (for review see [38]).
Recently, the proteomic profile of metabolic proteins in the invasive glioblastoma phenotype has been studied by applying a functional analysis using the Ingenuity Pathway Knowledge Base (Ingenuity Systems, Redwood City, CA) [39]. The results identified oxidative phosphorylation, mitochondrial dysfunction and ubiquinone biosynthesis as canonical pathways of the cancerous phenotype and CYB5R3 is identified as a protein associated with the mitochondrial dysfunction pathway. Furthermore, the relationship between mitochondrial dysfunction and CYB5R3 has also reported in a study carried out to analyze gene expression induced by bromide exposure using the Ingenuity Pathway Analysis [40]. www.impactaging.com Data from our laboratory seem to indicate a positive role for mammalian CYB5R3 in mitochondrial respiration. We have used siRNA technology to silence CYB5R3 in cultured human cells (Figure 2). Preliminary results indicate that CYB5R3 KO cells exhibit an apparent senescent phenotype based on the accumulation of βgalactosidase. These cells also show a reduction in the mitochondrial respiration rate based on analysis of oxygen consumption. Biochemical analysis of these cells also revealed an increase in the expression of PGC-1α that indicates increased recycling or de novo biogenesis of mitochondria. In a recently-reported global analysis of lysine-acetylated proteins, a posttranslational modification of CYB5R3-encoded protein by lysine acetylation in its FAD-binding domain has been identified [41]. Lysine acetylation is necessary for the interaction between SIRT1 and other sirtuins their targets before deacetylation can occur. Though conclusive experimental data still need to be shown, we hypothesize that SIRT1 regulates the cytosolic NAD + /NADH ratio by influencing CYB5R3 activity (Figure 3). Conditions of high NADH would lead to partial inactivation of SIRT1, leading to an accumulation of the acetylated form of CYB5R3. This active form of CYB5R3 would increase NADH oxidation and release NAD + that, in turn, would activate SIRT1. CYB5R3 would be then deacetylated, causing a decrease in its activity and thereby maintaining the NAD + /NADH ratio in proper balance. PGC-1α activity will be also affected by this cycle through its interaction with SIRT1. Taken together, our preliminary data indicate CYB5R3 could play an essential role in the mitochondrial metabolism by its contribution to cellular redox homeostasis. A coordination of the redox balance in both the cytosol and mitochondria appears to be necessary for optimum cellular health, and may be of consequence to healthy aging as well.