Combining chemistry/biochemistry/genetics

to discover new biological pathways

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: mono(ADP-ribosyl)ation, NAD-dependent deacetylation, and poly(ADP-ribosyl)ation. These NAD-consuming reactions control many biological processes, including transcription, aging, DNA repair, mitosis, telomere maintenance, immune responses, and RNA splicing. However, for most of these NAD-consuming reactions, the biological functions are only known for a few members in the family of enzymes that catalyze the reaction. For example, mammals have 18 poly(ADP-ribose) polymerases (PARPs) that catalyze protein poly(ADP-ribosyl)ation, but the biological functions are only known for two of them, PARP-1 (required for DNA repair and transcription of certain genes) and Tankyrase-1 (promoting telomere extension and required for mitosis). Thus an important unaddressed question is what biological pathways are regulated by the other enzymes in the family that catalyze similar reactions.  Furthermore, the NAD-dependent deacetylation reactions were only discovered in 2000, and there is no sequence homology between different enzymes that catalyze different NAD-consuming reactions. Therefore it is very likely that new NAD consuming reactions are still waiting to be discovered.

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.

To discover new reactions that involve NAD and new biological processes that are regulated by known or new NAD-consuming reactions, we have synthesized various NAD analogs bearing affinity tags.  The tags enable us to trace reactions that happen in live cells. The reaction products will be purified and enriched using the affinity tag, and their structures identified by analytical methods such as LCMS. The identification of new NAD-derived small molecules will lead to the discovery of new reactions that consume NAD and the identification of modified proteins will reveal new pathways that are regulated by NAD-consuming reactions.  Genetic manipulations can be used in the process to facilitate the assignment of new reactions or pathways to particular enzymes.

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 translation elongation factor 2 (eEF-2). Diphthamide is  conserved in all eukaryotes and archeabacteria. eEF-2 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 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) required for the biosynthesis.

The proposed biosynthesis pathway is shown in Figure 1. Our goal is to use in vitro biochemistry to figure out the molecular functions of the five proteins Dph1-5 in the biosynthesis of diphthamide, and study the effects of diphthamide formation on the function of eEF-2 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). 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

 Figure 1. The proposed biosynthesis pathway for diphthamide and its ADP-ribosylation by diphtheria toxin.

Figure 3. N-, O-, and C-glycosidic linkages in carbohydrate-modified proteins.

Protein glycosylation is important for protein function and many human diseases are associated with abnormalities in protein glycosylation. Most naturally-occuring carbohydrate-protein conjugates are linked via N- or O-glycosidic bonds (Figure 1). Recently, a novel C-glycosylation has been found for many proteins, including RNase 2, IL-12, complements, erythropoietin receptor, and mucins. This C-glycosylation happens on a Trp-X-X-Trp sequence motif, with the first Trp's C2 position being modified with an a-Man.

The function of this modification is still not well understood, and the enzyme (or enzymes) responsible for this modification has yet to be identified. The wide spread occurrence of C-mannosylation suggests that this modification has important functions in normal physiology. Further studies are needed to understand this posttranslational modification. Our goal is to identify and characterize the C-mannosyltransferase, and to then use it as a tool for understanding the biological function of protein C-mannosylation.