Mitochondrial NADP(H) generation is essential for proline biosynthesis

Abstract

The coenzyme nicotinamide adenine dinucleotide phosphate (NADP+) and its reduced form (NADPH) regulate reductive metabolism in a subcellularly compartmentalized manner. Mitochondrial NADP(H) production depends on the phosphorylation of NAD(H) by NAD kinase 2 (NADK2). Deletion of NADK2 in human cell lines did not alter mitochondrial folate pathway activity, tricarboxylic acid cycle activity, or mitochondrial oxidative stress, but rather led to impaired cell proliferation in minimal medium. This growth defect was rescued by proline supplementation. NADK2-mediated mitochondrial NADP(H) generation was required for the reduction of glutamate and hence proline biosynthesis. Furthermore, mitochondrial NADP(H) availability determined the production of collagen proteins by cells of mesenchymal lineage. Thus, a primary function of the mitochondrial NADP(H) pool is to support proline biosynthesis for use in cytosolic protein synthesis.

Disruption of the TCA cycle reveals an ATF4-dependent integration of redox and amino acid metabolism

“…This data suggests that TCAi places cells under conditions of oxidative stress and that the drop in NAD(P)H is due in part to an increased requirement of the cell to convert oxidized cystine to reduced cysteine to support GSH synthesis. Recently, NAD(P)H has been implicated as the major cofactor supporting mitochondrial proline biosynthesis and this could partly explain the decrease in proline (Tran et al, 2021; Zhu et al, 2021). However, neither BSO or eGSH could rescue proline synthesis despite increasing intracellular glutamate or re-establishing NAD(P)H levels (Figure 4B and C).…”

“…We also observed an increase in γ -glutamylcysteine (Figure 4H) and no significant change in the NADH/NAD + or NAD(P)H/NADP + ratio (Figure 4G), although a trend towards a decrease in the NAD(P)H/NADP + ratio was observed. Given the requirement of ATP for NADK2-mediated phosphorylation of NAD + to generate NADP + in mitochondria (Tran et al ., 2021; Zhu et al ., 2021), it’s likely that a combined reduction of mitochondrial ATP and NAD(P)H give rise to the defect in proline synthesis. Further support for mitochondrial respiration involvement in regulating de novo proline synthesis came from the inhibition of complex III with antimycin A, which significantly decreased aspartate, asparagine and proline, while promoting GSH synthesis (Figure 4I).…”

“…Our analysis implicated decreased α-KG siphoning and diminished mitochondrial ATP levels as the mechanism by which the TCA cycle impairs proline synthesis. Recently, two elegant studies revealed that contrary to previous reports, NAD(P)H and not NADH is the primary redox cofactor required for mitochondrial proline synthesis (Tran et al ., 2021; Zhu et al ., 2021). Given the drop in the NAD(P)H/NADP + ratio we observe and the reliance of mitochondrial NADP + pools on ATP and the NAD kinase NADK2 (Tran et al ., 2021; Zhu et al ., 2021), it is tempting to speculate that a combined bioenergetic and reductive power defect plays a role upon TCAi.…”

“…Recently, two elegant studies revealed that contrary to previous reports, NAD(P)H and not NADH is the primary redox cofactor required for mitochondrial proline synthesis (Tran et al ., 2021; Zhu et al ., 2021). Given the drop in the NAD(P)H/NADP + ratio we observe and the reliance of mitochondrial NADP + pools on ATP and the NAD kinase NADK2 (Tran et al ., 2021; Zhu et al ., 2021), it is tempting to speculate that a combined bioenergetic and reductive power defect plays a role upon TCAi. Furthermore, studies show that Pycr1 is a redox sensitive enzyme that can be modified by reactive electrophilic species (RES) (Timblin et al, 2021), while proline synthetic enzymes are oxidative stress-inducible (Krishnan et al, 2008) and are also targets of reactive oxygen specie (ROS)-mediated cysteine oxidation, as determined by quantitative redox proteomics of murine tissues in vivo (OxiMouse) (Xiao et al, 2020).…”

“…The use of this medium enables careful control of our in vitro experiments and is commonly used for most molecular biology studies. Recently, it has also proved important in revealing specific metabolic dependencies relating to proline (Tran et al, 2021; Zhu et al ., 2021). However, it will be important to assess this response under nutrient scarcity or with different nutrient compositions in the future to reveal other facets of the metabolic response.…”