NAD-consuming enzymes: Sirtuins, PARPs, and CD38
The goal of our research program is to discover new nicotinamide adenine dinucleotide (NAD)-consuming reactions and new biological pathways that are regulated by known or new NAD-consuming reactions. Cells carry out numerous chemical reactions to achieve diverse biological functions. For example, phosphorylation of proteins is involved in many cell signaling processes, and histone acetylation and methylation provide epigenetic control. Of all the reactions that posttranslationally modify proteins, NAD-consuming reactions stand out as they not only have diverse and important biological functions but also display very interesting chemistry. Figure 1 summarizes some of the known NAD-consuming reactions, including three that modify proteins: NAD-dependent deacetylation, mono(ADP-ribosyl)ation, and poly(ADP-ribosyl)ation.
Figure 1. Known cellular reactions that consume NAD. These reactions include: (1) Poly(ADP-ribosyl)ation, catalyzed by poly(ADP-ribose) polymerases (PARPs); (2) mono(ADP-ribosyl)ation, catalyzed by both sirtuins and ADP-ribosyltransferases (ARTs); (3) NAD-dependent deacetylation, catalyzed by sirtuins; (4) removal of 2′-phosphate from tRNA splicing intermediates, catalyzed by RNA 2′-phosphotransferases; (5) formation of cyclic ADP-ribose, catalyzed by both CD38 and CD157 in mammals. Some of the biological functions of these NAD-consuming reactions are also indicated in the figure.
Sirtuins and HDACs. The Sir2 (silencing information regulator 2) family of enzymes, or sirtuins, were originally known as NAD-dependent protein lysine deacetylases (Figure 2). They are present in all domains of life and have been shown to be important in regulating numerous biological pathways, including genome stability, metabolism, and longevity. Mammals have seven sirtuin enzymes, Sirt1-7. They are considered promising targets for treating several human diseases, including cancer, diabetes, and neurodegeneration. Among the seven mammalian sirtuins, only Sirt1-3 have efficient deacetylase activity. Sirt4-7 have very weak and sometimes undetectable deacetylase activity. We have recently demonstrated that Sirt5 can remove succinyl and malonyl groups while Sirt6 can remove myristoyl and palmitoyl groups very efficiently (Figure 2). We demonstrated that protein lysine succinylation, malonylation, and long-chain fatty acylation are common protein posttranslational modifications (PTMs) that were previously unknown or under-recognized. We are currently working towards understanding the enzymatic activity of Sirt4 and Sirt7 and developing tools to study these new PTMs. We are also utilizing the information about the enzymatic activity of sirtuins to develop small molecule inhibitors for therapeutic applications.
Another class of enzymes closely related to sirtuins are the zinc-dependent histone deacetylases or HDACs. There are 11 HDACs in humans and 5 of them have weak deacetylase activity (HDAC4, 5, 7, 8, 9). We are also interested in studying the enzymatic function of these HDACs.
Representative publications on sirtuins:
- J. Hu, B. He, S. Bhargava, H. Lin, “A fluorogenic assay for screening Sirt6 modulators”, Org. Biomol. Chem.,11, 5213-5216 (2013).
- H. Jiang et al., “Sirt6 regulates TNFa secretion via hydrolysis of long chain fatty acyl lysine”, Nature, 496, 110-113 (2013).
- H. Lin, X. Su, B. He, “Protein lysine acylation and cysteine succination from intermediates of energy metabolism”, ACS Chem. Biol., 7, 947-960 (2012).
- B. He, J. Du, H. Lin, “Thiosuccinyl Peptides as Sirt5-Specific Inhibitors”, J. Am. Chem. Soc., 134, 1922-1925 (2012).
- A. Y. Zhu, Y. Zhou, S. Khan, K. W. Deitsch, Q. Hao, H. Lin, “Plasmodium falciparum Sir2A preferentially hydrolyzes medium and long chain fatty acyl lysine”, ACS Chem. Biol., 7, 155-159 (2012).
- J. Du, Y. Zhou, X. Su, J. Yu, Khan S., H. Jiang, J. Kim, J. Woo, J.H. Kim, B.H. Choi, B. He, W. Chen, S. Zhang, R.A. Cerione, J. Auwerx, Q. Hao, H. Lin, “Sirt5 is a NAD-dependent protein lysine demalonylase and desuccinylase”, Science, 334, 806-809 (2011).
PARPs. PARPs or poly(ADP-ribose) polymerases catalyze the poly(ADP-ribosyl)ation, or PARylation, of various substrate proteins. The first ADP-ribosyl group is added to Glu or Asp residues of substrate proteins, followed by addition of more ADP-ribosyl groups to the 2’-OH groups of adenosine, leading to long poly(ADP-ribose) chains (PAR) that can contain hundreds of ADP-ribosyl units. The size and negative charge of PAR chains can affect protein structure and function and thus regulate various biological processes. There are 17 PARPs in humans, of which only two (PARP1 and Tankyrase-1) are relatively well studied. PARP1 is required for DNA repair/genome maintenance and transcriptional regulation of certain genes. It is also responsible for the induction of cell death under extreme stress (e.g. excessive DNA damage) or pathological conditions (stroke, ischemia, diabetes). Tankyrase-1 is known to be required for mitosis and telomere length maintenance. However, the molecular mechanisms underlying their function are unclear. For most other PARPs, their biological functions are still unknown. Many PARP inhibitors are in clinical trial for treating cancers, especially triple-negative breast cancers. To better realize the potential of PARP inhibitors as therapeutics, it is important to discover the biological functions of various PARPs and understand the functions at molecular levels.
To discover and understand the biological function of different PARPs, it is necessary to find out what substrate proteins are modified and regulated by PARPs. We have developed clickable NAD analogs to identify PARP substrate proteins (Figure 3). The clickable NAD analog has an alkyne functional group, which allows the conjugation (via click chemistry) of different tags, such as fluorescent tags for in-gel visualization and affinity tags for purification. Furthermore, we demonstrate that by identifying the substrate proteins of PARP1, novel insights into its biological functions can be obtained.
Figure 3. Labeling the substrate proteins of PARPs using clickable NAD analogs. (A) Labeling of PARylated proteins with 6-alkyne NAD. An affinity tag can be added using click chemistry after the substrate protein is labeled. The labeled protein can then be affinity purified, separated on 1D/2D protein gel, and then the sequence identified by MS. (B) Structures of compounds used in labeling reactions.
CD38. CD38, originally identified as a T cell surface antigen, is a type II membrane protein with a very short cytoplasmic N-terminal tail (~20 amino acids), a single transmembrane domain, and an extracellular C-terminal domain that has NAD-consuming enzymatic activity. The enzymatic activity of CD38 (Figure 4) converts nicotinamide adenine dinucleotide (NAD) to adenosine diphosphate-ribose (ADPR) or cyclic ADPR (cADPR). It is reported that cADPR is a potent second messengers that can trigger Ca2+ release from internal Ca2+ stores. It is reported to be associated with many physiological and pathophysiological conditions. Despite the large amount of data accumulated on CD38, a molecular understanding of its physiological function is still lacking. Our lab has been focusing developing small molecule probes to investigate the function of CD38 (Figure 4). We previously reported a Rh-6-(F-araNAD) probe and are currently develop more cell permeable probes for CD38.
Figure 4. (A) The enzymatic activity of CD38; (B) mechanism-based labeling of CD38 with F-araNAD; (C) the structure of impermeable CD38 probe, Rh-6-(F-araNAD).
Representative publications on PARPs and CD38:
- H. Jiang et al., “Identification of ADP-ribosylation sites of CD38 mutants by precursor ion scanning mass spectrometry”, Anal. Biochem. 433, 218-226 (2013).
- H. Jiang, J. H. Kim, K. Frizzell, W. L. Kraus, H. Lin, “Clickable NAD analogs for labeling substrate proteins of PARPs”, J. Am. Chem. Soc., 132, 9363-9372 (2010).
- H. Jiang, J. Congleton, Q. Liu, P. Merchant, F. Malavasi, H.C. Lee, Q. Hao, A. Yen, H. Lin, “Mechanism-based fluorescent labeling of human CD38”, J. Am. Chem. Soc., 131, 1658-1659 (2009).
Dipthamide: biosynthesis and biological function
Diphtheria was once a deadly disease causing many deaths before modern vaccination was available. The disease is caused by Corynebacterium diphtheriae, a bacterium that secrets a toxin called diphtheria toxin. Diphtheria toxin catalyzes the ADP-ribosylation of a unique posttranslationally-modified His residue, termed diphthamide, in eukaryotic and archaeal translation elongation factor 2 (EF2). EF2 is a GTPase that catalyzes the translocation of the peptidyl-tRNA and mRNA from the ribosome A site to the P site, and therefore is essential for protein biosynthesis. The biological function of diphthamide is not understood yet, although it has been shown that knock out dph1 or dph2 is lethal in mouse, and dph1 heterozygote mouse are prone to tumor formation. The biosynthesis of the diphthamide residue has been a long time puzzle. A recent progress is the identification of the genes (Dph1, Dph2, Dph3, Dph4, Dph5, Dph6, and Dph7) required for the biosynthesis. Our lab contributed to the discovery of Dph6 and Dph7.
The proposed biosynthesis pathway is shown in Figure 5. Our goal is to use in vitro biochemistry to figure out the molecular functions of the proteins Dph1-7 in the biosynthesis of diphthamide, and study the effects of diphthamide formation on the function of EF2 in protein synthesis. The first step of the biosynthesis is of particular interest because of the uncommon C-C bond formation reaction, the uncommon use of SAM, and the requirement of multiple proteins (Dph1-4). Our evidences suggest that the enzyme catalyzing this step contains [4Fe-4S] cluster and uses an unusual radical reaction mechanism. We are currently try to understand how this unusual [4Fe-4S] radical enzyme works.
Figure 5. The proposed biosynthesis pathway for diphthamide and its ADP-ribosylation by diphtheria toxin.
Representative publications on Diphthamide:
- X. Su, Z. Lin, W. Chen, H. Jiang, S. Zhang, and H. Lin, “A chemogenomic approach identified yeast YLR143W as diphthamide synthetase”, Proc. Natl. Acad. Sci. USA, 109, 19983-19987 (2012).
- X. Su, W. Chen, W. Lee, H. Jiang, S. Zhang, H. Lin, “YBR246W Is Required for the Third Step of Diphthamide Biosynthesis”, J. Am. Chem. Soc.,134, 773-776 (2012)
- X. Zhu, B. Dzikovski, X. Su, A.T. Torelli, Y. Zhang, S.E. Ealick, J.H. Freed, and H. Lin. “Mechanistic understanding of Pyrococcus horikoshii Dph2, a [4Fe-4S] enzyme required for diphthamide biosynthesis”, Mol. BioSystems, 7, 74-81 (2011).
- X. Zhu, J. Kim, X. Su, and H. Lin, “Reconstitution of diphthine synthase activity in vitro”, Biochemistry, 49, 9649-9657 (2010).
- Y. Zhang, X. Zhu, A. Torelli, M. Lee, B. Dzikovski, R. M. Koralewski, E. Wang, J. Freed, C. Krebs, S. E. Ealick, H. Lin, “Diphthamide biosynthesis requires an Fe-S enzyme-generated organic radical”, Nature, 465, 891-896 (2010).