Research

My background is in chemical biology, metabolism and systems biology. I am broadly interested in developing innovative approaches to study the inner workings of biological systems. My previous research has focused on developing and applying genetically encoded tools for manipulation of redox balance and on using natural products to identify inhibitors of previously undruggable proteins. In future, I plan to build on my previous work to develop a suite of genetically encoded tools for compartment-specific manipulation of intracellular metabolic parameters and apply them to study the regulation of metabolism with the goal of gaining mechanistic insight into the beneficial effects of diet and exercise on aging and age-associated disorders.

RESEARCH ACCOMPLISHMENTS:

Genetically encoded tools for manipulation of metabolism

NADH/NAD+ and NADPH/NADP+ ratios are important metabolic parameters that are implicated in regulation of many metabolic pathways and their changes are correlated with several pathologic conditions including cancer, diabetes and the aging process itself. The causal relationship between changes in the NADH/NAD+ and NADPH/NADP+ ratios and physiological effects remains poorly understood. An important barrier to understanding the role of changes in the NADH/NAD+ and NADPH/NADP+ ratios in regulation of biological processes is the lack of methods for compartment-specific manipulation of these parameters. During my postdoctoral training, I have developed the first genetically encoded tool for compartment-specific decrease of the NADH/NAD+ ratio in mammalian cells. This tool is based on the water-forming NADH oxidase from Lactobacillus brevis (LbNOX), which catalyzes the reaction between NADH and oxygen to make NAD+ and water. In collaboration with Dr. Valentin Cracan and Dr. Zenon Grabarek, I have also developed a genetically encoded tool for decreasing intracellular NADPH/NADP+ ratio, called TPNOX, by using rational mutagenesis to switch the cofactor specificity of LbNOX to accept NADPH instead of NADH. Together, LbNOX and TPNOX represent a new genetic toolkit for manipulation of redox metabolism and can be used to study the regulation and physiological role of compartment- and tissue-specific changes of the NADH/NAD+ and NADPH/NADP+ ratios.

 

Maintenance of the NAD+/NADH ratio is an essential function of the ETC

A decline in mitochondrial electron transport chain (ETC) activity is associated with many human disorders including inborn errors of metabolism, cancer, diabetes and the aging process itself. ETC catalyzes two important reactions: i) maintenance of the NADH/NAD+ ratio by oxidation of NADH to NAD+ and ii) generation of mitochondrial membrane potential by pumping protons outside of mitochondria. It has previously been impossible to determine if a phenotype caused by ETC dysfunction is the result of the inhibition of the first or second reaction because they are coupled to each other and occur simultaneously. To uncouple these two mechanisms, I used the water-forming NADH oxidase from Lactobacillus brevis (LbNOX), which only catalyzes the first reaction, to induce a compartment-specific decrease of the NADH/NAD+ ratio in human cells. Expression of LbNOX in the cytosol or mitochondria of mammalian cell lines fully rescued the proliferative and metabolic defects caused by an impaired ETC. This result demonstrates that maintenance of the NADH/NAD+ ratio, not ATP synthesis, is an essential function of the ETC that is required for mammalian cell proliferation. In future, tissue- and compartment-specific LbNOX expression in model organisms can be used to determine whether maintenance of the NADH/NAD+ ratio or generation of mitochondrial membrane potential is responsible for different phenotypes observed due to ETC dysfunction.

 

Identification of the target of the bioactive natural product triptolide

Natural products and their derivatives have served as an invaluable source of small molecules capable of selectively targeting previously undruggable enzymes and processes (e.g. aspirin, morphine, penicillin, lovastatin, paclitaxel, rapamycin). During my PhD thesis research, I investigated the mechanism of action of several bioactive natural products with the goal of identifying novel inhibitors of previously undruggable enzymes. In particular, I focused on a natural product called triptolide that has been isolated from a traditional Chinese medicinal plant with anti-inflammatory, immunosuppressive, contraceptive and antitumor activities. I showed that triptolide works by inhibiting RNA polymerase II-dependent transcription initiation through inactivation of XPB/ERCC3, a subunit of the general transcription factor TFIIH. The mechanism of action of this natural product had remained a mystery for more than 30 years since its discovery, despite hundreds of articles published on this topic. Triptolide is the first potent and specific inhibitor of RNA polymerase II- dependent transcription initiation, which makes it a unique tool compound. Triptolide is also the first potent and specific inhibitor of a DExH-box DNA helicase (i.e. XPB/ERCC3), which opens the path for developing inhibitors for this important enzyme family. Currently, an analog of triptolide is in human clinical trials for the treatment of cancer. Identification of the target of triptolide will help facilitate its clinical development.

FUTURE DIRECTIONS:

Metabolism in multicellular organisms is regulated by two overlapping mechanisms: i) modulation of gene expression and signal transduction by hormones and ii) allosteric regulation of metabolic and signaling enzymes by intracellular metabolic parameters (e.g. NADH/NAD+, ATP/ADP and mitochondrial membrane potential). The importance of both hormonal and allosteric regulation of metabolism is well appreciated from classical biochemical studies. However, it is still poorly understood which specific changes in intracellular metabolic parameters are important for allosteric regulation of metabolism in vivo because most of our knowledge about allosteric regulation is based on studies with purified enzymes or tissue extracts, which do not fully recapitulate the intracellular environment.
A key bottleneck in understanding the role of allosteric regulation of metabolism in physiology and disease has been the lack of methods for manipulating intracellular metabolic parameters in vivo. To fill this methodological gap, we have introduced two genetically encoded tools for manipulation of NADH/NAD+ and NADPH/NADP+ ratios in living cells. We are currently working on expanding this toolkit to other metabolic parameters, which will allow us to mimic metabolic changes induced by exercise and dietary changes in cell culture and model organisms. I will apply these tools and other methods to study the regulation of metabolism with the goal of gaining mechanistic insight into the beneficial effects of diet and exercise on age-associated disorders.