Faculty and Staff
Sajish Mathew, Ph.D.
|Department:||Drug Discovery & Biomedical Sciences (DDBS)
College of Pharmacy
College of Pharmacy
715 Sumter Street - CLS 506
Columbia, SC 29208
Ph.D. Biological Sciences and BioEngineering, Indian Institute of Technology Kanpur, 2009
Significance of NAD+ Metabolism and Signaling in the Maintenance of Cellular Homeostasis
NAD+ is essential to maintain energy homeostasis and redox state of the cellular environment. NAD+ is an essential coenzyme for hydride-transfer enzymes in redox reactions. It can exist in either oxidized (NAD+) or reduced (NADH) form and NAD+/NADH ratio determines the oxidative status of the cellular environment. As a coenzyme, NAD+ is also essential for the generation of cellular energy by transferring the reducing equivalents generated through the activities of glycolysis and TCA cycle in the form of NADH. These reducing equivalents are oxidized by complex I of the electron-transport chain (ETC), coupling glycolysis and the TCA cycle to ATP synthesis via oxidative phosphorylation. However, under limited availability of oxygen that compromises the function of ETC, NADH is converted to NAD+ by reduction of pyruvate into lactate and hence ensures a continuous supply of NAD+ to generate ATP through glycolysis.
NAD+ is essential for proteins that regulate major signal transduction pathways and stress
signaling. Further, NAD+ also acts as substrate for three classes of enzymes: (i) poly(ADP-ribose) polymerases
(PARPs), (ii) the SIRTuins (silent mating type information regulation), and (iii)
the cyclic ADP-ribose (cADPR) synthases (CD38 and CD157). These enzymes regulate important
biochemical reactions such as mono- and poly-ADP-ribosylation, protein deacetylation,
and ADP-ribose cyclization, respectively. Activities of PARP are essential for the
repair of DNA damages. SIRTuins are essential for gene silencing; ADP-ribose cyclization
produces mediators of calcium signaling. The activities of these three enzymes result
in the hydrolysis of the glycosidic bond between the nicotinamide (NAM) and ADP-ribosyl
moieties of NAD+ to induce stress signaling leading to changes in gene expression, modulation of post-translational
modifications, and regulation of calcium signaling. Hence NAD+ plays an indispensible role not only in the regulation of major metabolic and signal
transduction pathways, but also in the maintenance of energy homeostasis and redox
state of the cellular environment that couples with transcriptional reprogramming
to adapt to challenging environmental and cellular conditions.
Aminoacyl-tRNA synthetases (aaRSs) are novel modulators of NAD+ metabolism and signaling. aaRSs are proteins that activate L-amino acids for protein synthesis: one each for 20 standard L-amino acids. However, during evolution, aaRSs also progressively accrued ‘moonlighting’ functions that are activated under conditions of diminished protein synthesis such as cellular stress. Our previous work on the non-translational functions of Tryptophanyl-tRNA synthetase (TrpRS) demonstrated that IFN-g-mediated depletion of tryptophan, the substrate for the de novo synthesis of NAD+, is also coupled with the ability of TrpRS to activate PARP1 (1). Briefly, tryptophan bound at the active site prevents TrpRS from interacting with PARP1. However, IFN-g-mediated induction of Indoleamine 2,3-dioxygenase (IDO), the first and rate-limiting enzyme of tryptophan catabolism through the kynurenine pathway, results in the cellular depletion of tryptophan. IFN-g thus facilitates TrpRS-mediated activation of PARP1 (Fig.1) (1). Our previous work thus pioneered a unique non-translational function of aaRSs in the regulation of NAD+ metabolic signaling mediated through their interaction with PARP1, the major modulator of NAD+ metabolism and signaling (1,2).
TyrRS is a novel modulator of NAD+/stress signaling. Our previous work on the mechanism of action of resveratrol (2) brought out a novel mechanism of activation of the non-translational, nuclear function of TyrRS (Fig. 2). Briefly, under normal conditions, TyrRS transfers L-tyrosine (L-Tyr) to tRNA for protein synthesis in a two-step reaction in the cytoplasm: (1) L-Tyr + ATP ßà L-Tyr-AMP + PPi; (2) L-Tyr-AMP + Tyr-tRNA à Tyr-tRNAL-tyr + AMP. However, human TyrRS is decorated with additional insertions around the catalytic domain and with a unique EMAP II-like domain at the C-terminal. Although these unique features are not necessary for the translational function, they are indispensable for the nuclear functions of TyrRS (2). Our previous work unraveled the structural basis of the molecular mechanism through which small molecules bound at the active site of TyrRS modulate the switch between its roles in translation (cytoplasm) vs its non-translational (nuclear) functions as described below.
We exploited the structural similarities between resveratrol and L-tyrosine (resveratrol harbors a tyrosine-like phenolic ring) to decipher the structural and molecular basis of the switch that modulates the function of TyrRS (2). Once bound in cis-conformation (resveratrol has a double bond which enables it to adopt either a trans or cis-conformation), resveratrol induces a distinct conformation that promotes the interaction of TyrRS with PARP1. This TyrRS-resveratrol-PARP1-driven NAD+ signaling upregulated the expression and activation of a battery of genes, proteins and signaling cascades that elicit a protective stress response through the generation of nicotinamide (NAM) and ADP-ribose (ADPR) (2). We refer to these non-translational functions of TyrRS as the nuclear function of TyrRS (Fig. 2).
A new paradigm for the mechanism of action of PARP1: Boost cellular NAD+ levels and evoke protective stress response.
Most importantly, our work defied two existing scientific dogmas: One, that activation of PARP1 is coupled to cellular depletion of NAD+ and Two, that cellular upregulation of NAD+ levels always activates SIRTuins. Rather, our previous work for the first time demonstrated that a non-DNA damage and TyrRS- dependent activation of PARP1 upregulates cellular NAD+ levels (Fig. 3A) with the concomitant inhibition of SIRTuins through the generation of nicotinamide (NAM) (Fig. 2). However, inhibition of NAD+ synthesis
through the action of nicotinamide phosphoribosyl transferase (NAMPT), the rate-limiting enzyme in the regeneration of NAD+ through the salvage pathway, resulted in the downregulation of protective stress response (Fig. 3B). Hence, despite initial consumption of NAD+ by PARP1, induction of NAMPT ensures rapid regeneration of NAD+ and upregulation of cellular NAD+ levels (Fig. 2 and 3). However, continuous generation of NAM through activation of PARP1 would still keep SIRTuins inhibited (Fig.2). Our previous work thus brought out a new paradigm for the mechanism of action of PARP1 and also brought out a novel link between aaRS and NAD+ metabolism in the nuclear stress signaling and in the generation of protective stress response through cellular upregulation of NAD+ (1,2).
Current and Future Perspectives: Our research focus is to understand and to explore the potential of NAD+ metabolism and signaling through SIRTuins and PARPs in the regulation of the new biology of tRNA synthetases.
Sajish M, Zhou Q, Kishi S, Valdez DM Jr, Kapoor M, Guo M, Lee S, Kim S, Yang XL, Schimmel P. Trp-tRNA synthetase bridges DNA-PKcs to PARP-1 to link IFN-γ and p53 signaling. Nature Chemical Biology, 8(6): 547-54 (2012).
Sajish M and Schimmel P. A human tRNA synthetase is a potent PARP1 activating effector
target for resveratrol.
Nature, 519, 370–373 (2015).