CYB5R3: a key player in aerobic metabolism and aging?

Aging involves multiple processes that render cells, tissues and organs vulnerable to stress, damage and ultimately death. Aging itself is not a disease, but there are a number of diseases that become exponentially more prevalent with advancing age such as cancer, cardiovascular disease, metabolic syndrome and neurodegenerative diseases. Energy sensing, food intake and caloric utilization must be kept in equilibrium to preserve appropriate fat stores to prevent the deregulation of glucose homeostasis and other obesity-related disorders.

Calorie restriction (CR) is an intervention aimed to produce undernutrition without malnutrition. CR increases healthspan and lifespan in almost all species tested such as yeast, insects, nematodes and mammals [1], including nonhuman primates [2]. CR has been studied extensively with consistent results showing its beneficial effects on longevity, age-associated diseases, attenuation of functional decline, and carcinogenesis across a variety of species and diet formulations [3]. Among mammals mice have been the most heavily-researched model with CR eliciting myriad behavioral, physiological, and metabolic changes that include decreased body temperature, blood glucose, insulin and fat mass, and increased physical activity, glucose tolerance and insulin sensitivity [4]. Studies in Saccharomyces cerevisiae and Drosophila melanogaster have demonstrated that SIR2, which encodes for a NAD⁺-dependent histone deacetylase, plays a central role in mediating the increase in longevity associated with CR in these species [5-7]. The involvement of SIR2 in lifespan extension by CR may relate to its responsiveness to nicotinamide levels and the NAD⁺/NADH ratio, both indicators of cellular energy status [8-10]. A growing body of evidence indicates that the mammalian homologue of SIR2, SIRT1, also plays a significant role in responding to CR. For example, CR 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 b₅ 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 sphingo-myelinase 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 ΔATP2strain has a defective ATP synthase complex and the ΔCOR1 strain is defective in the bc₁ 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 CoQ₆ 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 over-expression also extends replicative lifespan in a SIR2-dependent 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 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 b₅reductase 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 hydroxylation 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 b₅reductase 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].

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 β-galactosidase. 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 modifica-tion 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.