MESH1 is a cytosolic NADPH phosphatase that regulates ferroptosis

Abstract

S tringent response is the main strategy used by bacteria to cope with fluctuating nutrient supplies and metabolic and oxidative stresses 1,2 . This process rapidly redirects energy from cell proliferation toward stress survival by reduction of biosynthesis, conservation of ATP and blockage of GTP production 3 . The stringent response is triggered by the accumulation of the bacterial 'alarmone' (p)ppGpp (guanosine tetra-or penta-phosphate, shortened as ppGpp below) through the regulation of ppGpp synthetases and hydrolases in the RelA and SpoT homologue family 2 .Recent studies suggest that the stringent response may also function in metazoans, as metazoan genomes encode a homologue of bacterial SpoT-MESH1 (Metazoan SpoT Homologue 1, encoded by HDDC3)-that can hydrolyse ppGpp in vitro and functionally complement SpoT in Escherichia coli 4 . Furthermore, Mesh1 deletion in Drosophila displays impaired starvation resistance and extensive transcriptional reprogramming 4 . Despite these supporting lines of evidence, neither ppGpp nor its synthetase has been discovered in metazoans, thus obscuring the genuine function and the relevant substrate(s) of MESH1 in mammalian cells. Here, we have identified NADPH as an efficient substrate of MESH1. MESH1 is a cytosolic NADPH phosphatase that is induced under stress conditions, leading to the NADPH depletion and ferroptosis-a novel form of iron-dependent regulated cell death characterized by lipid peroxidation 5 . Accordingly, MESH1 removal preserves the NADPH level in stressed cells and promotes their ferroptotic survival.Critical to the bacterial stringent response is the rapid relocation of resources from proliferation toward stress survival through the respective accumulation and degradation of (p)ppGpp by RelA and SpoT homologues. While mammalian genomes encode MESH1, a homologue of the bacterial (p)ppGpp hydrolase SpoT, neither (p)ppGpp nor its synthetase has been identified in mammalian cells. Here, we show that human MESH1 is an efficient cytosolic NADPH phosphatase that facilitates ferroptosis. Visualization of the MESH1-NADPH crystal structure revealed a bona fide affinity for the NADPH substrate. Ferroptosisinducing erastin or cystine deprivation elevates MESH1, whose overexpression depletes NADPH and sensitizes cells to ferroptosis, whereas MESH1 depletion promotes ferroptosis survival by sustaining the levels of NADPH and GSH and by reducing lipid peroxidation. The ferroptotic protection by MESH1 depletion is ablated by suppression of the cytosolic NAD(H) kinase, NADK, but not its mitochondrial counterpart NADK2. Collectively, these data shed light on the importance of cytosolic NADPH levels and their regulation under ferroptosis-inducing conditions in mammalian cells.

Ferroptosis of epithelial ovarian cancer: genetic determinants and therapeutic potential

“…In addition, pharmacogenomic analyses identified NAPDH as a robust determinant of ferroptosis [48], probably by regenerating GSH. Consistently, we have found that MESH1, the metazoa homolog of SpoT, is the first cytosolic NADPH phosphatase [49] whose induction is responsible for the NAPDH depletion during ferroptosis [50].…”

Possible Roles for Basal Levels of (p)ppGpp: Growth Efficiency Vs. Surviving Stress

“…No synthetases of (p)ppGpp have been yet found in animal cells, but an enzyme capable of hydrolysis of (p)ppGpp, called Mesh1 has been detected in worms, flies, and humans ( Sun et al, 2010 ). The human version of Mesh1 has been found to hydrolyze (p)ppApp and NADPH as well as (p)ppGpp ( Ding et al, 2020 ; Jimmy et al, 2020 ). Whether NADPH is the bona fide substrate of Mesh1 is still a matter of debate, considering that the Drosophila version did not reduce the cellular pools of NADPH when expressed in Escherichia coli ( Zhu and Dai, 2019 ).…”

Estimates of RelSeq, Mesh1, and SAHMex Hydrolysis of (p)ppGpp and (p)ppApp by Thin Layer Chromatography and NADP/NADH Coupled Assays

“…Recently, is has been suggested that that the human MESH1 enzyme is an NADPH phosphatase that degrades NADPH to NADH (Ding et al, 2020). Since in the coupled assay for pppNpp hydrolysis NADPH accumulation is being followed (see Figure 2), this could have a potential impact on the obtained results.…”