Research Interests
Our lab is interested in the function, regulation, and targeting of enzymes that control protein post-translational modifications (PTM). After the human genome is sequenced, a major challenge to understand biology and human diseases is to understand the function of all proteins. Proteins are not always expressed, and even if expressed, their activities are not always on. Their activities are often regulated by certain stresses and signals, which is crucial for all cell signaling events. Thus, to fully understand the function of a protein, it is also crucial to understand how it is regulated. We, therefore, focus on understanding the function and regulation of PTM enzymes and use the understanding to develop small molecule inhibitors for these enzymes as potential therapeutics for treating human diseases, including cancer, autoimmune diseases, and neurodegenerative diseases.
NAD-consuming enzymes: Sirtuins and PARPs
Among the PTM enzymes that we study, we are particularly interested in NAD-consuming enzymes. These enzymes 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 enzymatic reactions, including three that modify proteins: NAD-dependent deacetylation, mono(ADP-ribosyl)ation, and poly(ADP-ribosyl)ation.
Figure 1. Known cellular enzymatic 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, certain PARPs, 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 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 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 continuing to understand the functions of sirtuins by discovering new substrates and their regulatory mechanisms. Furthermore, we are also utilizing information about the enzymatic activity of sirtuins to develop small molecule inhibitors for therapeutic applications.
Figure 2. Another class of enzymes closely related to sirtuins are the zinc-dependent histone deacetylases or HDACs. There are 11 HDACs in humans and many have very weak or no detectable deacetylase activity (HDAC4, 5, 7, 8, 9, 11). We are also interested in studying the function of these HDACs.
PARPs. PARPs catalyze either poly-ADP-ribosylation or mono-ADP-ribosylation of various substrate proteins. There are 17 PARPs in humans,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 understand the biological functions of various PARPs. Our earlier work focused on identifying the substrate proteins of PARPs. We 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.
Recently, we started to study the function of PARPs by understanding their regulation. Our work with TiPARP (PARP7) showed that it is transcriptionally up-regulated by HIF-1. The increased TiPARP expression serves to ADP-ribosylate HIF-1α and promote HIF-1α degradation, forming a negative feedback loop to turn off HIF-1 transcriptional activity (Figure 4). The most interesting part of this story is the mechanism via which TiPARP promotes the degradation of HIF-1α. We found that TiPARP, via its ADP-ribosylation activity, forms phase condensates (nuclear bodies) and recruits E3 ubiquitin ligases, thus leading to the ubiquitination and degradation of HIF-1α. We are currently investigating the detailed mechanism via which ADP-ribosylation promotes phase condensation.