Hydrolase Controls Cellular NAD, Sirtuin, and Secondary Metabolites

Abstract

Cellular levels of NAD؉ and NADH are thought to be controlled by de novo and salvage mechanisms, although evidence has not yet indicated that they are regulated by NAD ؉ degradation. Here we show that the conserved nudix hydrolase isozyme NdxA hydrolyzes and decreases cellular NAD ؉ and NADH in Aspergillus nidulans. The NdxA-deficient fungus accumulated more NAD ؉ during the stationary growth phase, indicating that NdxA maintains cellular NAD ؉ /NADH homeostasis. The deficient strain also generated less of the secondary metabolites sterigmatocystin and penicillin G and of their gene transcripts than did the wild type. These defects were associated with a reduction in acetylated histone H4 on the gene promoters of aflR and ipnA that are involved in synthesizing secondary metabolites. Thus, NdxA increases acetylation levels of histone H4. We discovered that the novel fungal sirtuin isozyme SirA uses NAD ؉ as a cosubstrate to deacetylate the lysine 16 residue of histone H4 on the gene promoter and represses gene expression. The impaired acetylation of histone and secondary metabolite synthesis in the NdxA-deficient strain were restored by eliminating functional SirA, indicating that SirA mediates NdxA-dependent regulation. These results indicated that NdxA controls total levels of NAD ؉ /NADH and negatively regulates sirtuin function and chromatin structure. N udix (nucleoside diphosphates linked to moiety X) hydrolases are ubiquitous in viruses, bacteria, and eukaryotes, and they hydrolyze NADH, NAD ϩ , ADP-ribose, and other nucleotide sugars, which allows their classification into subfamilies (6,22). One important function of nudix hydrolases is the hydrolytic degradation of oxidatively damaged nucleotides to prevent spontaneous mutations (39). Saccharomyces cerevisiae Ysa1p and Arabidopsis thaliana AtNUDX2 and AtNUDX7 hydrolyze ADP-ribose and respond to oxidative stress (17,43). Nudix hydrolases that hydrolyze NAD ϩ and NADH [NAD(H)] exist throughout the biological kingdom and include A. thaliana AtNUDX1 (12) and yeast peroxisomal Npy1p (1). The physiological role of NAD(H) hydrolases is unknown, especially that in epigenetic gene regulation.Gene expression is regulated by specific transcription regulators and by posttranslational modifications of nucleosomal histones. Acetylation is this type of modification that correlates with conformational changes in chromatin. Two groups of histone deacetylases (HDAC) deacetylate acetylated histones. One is classical HDAC, and the other is sirtuin, which deacetylates lysine residues of histones H3 and/or H4 using NAD ϩ as a cosubstrate (10,16,45). Yeast Sir2p is the prototype sirtuin, and it silences genes at mating type, ribosomal DNA (rDNA), and subtelomeric loci (14, 37). Its mammalian counterparts control aging, stress responses, and circadian rhythms (15, 25). Sirtuin activity is controlled by cellular NAD ϩ production (4, 19) that links cellular metabolic status and gene regulation, since NAD ϩ is a crucial coenzyme for biological redox reactions and energy conservat...

Sirtuin A regulates secondary metabolite production by <i>Aspergillus nidulans</i>

“…These clusters included genes for synthesizing ST,PN,EAS,XPT,MDP,SID,citreoisocoumarin,6,-isocoumarin and alternariol (COU/ALT), and 6-hydroxy-7-methyl-3-nonylisoquinoline-5,8-dione (HMN) (Ahuja and Chiang, 2012), indicating that SirA represses the expression of these SM gene clusters. The repression of ST and PN agrees with our findings that SirA deacetylates histone on their gene promoters and consequently represses their gene expression and production (Shimizu et al, 2012). SirAD also expressed genes in the predicted SM gene clusters involving non-ribosomal pep-S. cerevisiae Sir2p and Hst1p, as well as the A. oryzae sirtuin isozyme AoHstD, up-regulate multiple genes (Bedalov et al, 2003;Kawauchi et al, 2013).…”

“…Sirtuin-dependent global transcriptional repression of the genes in Sets 2 and 3 is consistent with the notion that the fungus produces SirA and histone deacetylase activity during the stationary growth phase (Shimizu et al, 2012).…”

“…In addition to ST and PN synthesis (Shimizu et al, 2012), gene regulation of ausA and other genes for AUS/DAUS synthesis is a novel function of SirA that was noted in the present study. Culture in liquid and solid media affected SM gene expression, which is often found during the production of other SM (Andersen et al, 2013), and the present findings indicated that A. nidulans SirA discriminates culture conditions upon expression of the AUS/ DAUS synthetic genes.…”

“…An increase in cellular NAD + , which is a substrate for sirtuin-dependent histone deacetylation, stimulates the activity of mammalian and yeast sirtuins (Haigis and Guarente, 2006;Kato and Lin, 2014). However, we previously found that the amount of NAD + in cultured A. nidulans remains unchanged during the late growth phase for up to 72 h (Shimizu et al, 2012). This implies that SirA is not activated by the amount of NAD + at the growth stage.…”

“…Aspergillus nidulans strains A26 and SirAD (biA1, argB2, DsirA::argB) (Shimizu et al, 2012) were cultured in 200 mL of GMM at 30∞C for 24, 48 and 72 h, and then the total RNA was purified using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Transcriptomes were analyzed using a custom-synthesized A. nidulans GeneChip (Masuo et al, 2010).…”