The long-term goal of the Titov laboratory is to understand the molecular basis of inactivation of cellular machinery during aging. We are interested in addressing the following broad questions: What are the cellular processes whose inactivation determines the lifespan of different organisms? What are the molecular mechanisms of age-associated damage to proteins, nucleic acids and lipids? How do environmental and genetic factors modulate the rate of aging? To address these questions, we are focused on studying how changes in metabolism induced by diet and exercise modulate aging and age-associated diseases. Many studies have demonstrated that diet and exercise are associated with increase in lifespan of model organisms and a decrease in human mortality from age-associated diseases including heart disease, stroke, diabetes, cancer and neurodegenerative disorders.
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CURRENT RESEARCH IN THE LAB IS FOCUSED ON THE FOLLOWING THREE DIRECTIONS:
Genetically encoded tools for manipulation of metabolism (GEMMs)
At a cellular level, the key response to dietary manipulations and exercise involves changes in intracellular bioenergetic parameters such as ATP/ADP, NADH/NAD+, NADPH/NADP+, GSH/GSSG ratios and mitochondrial membrane potential (ΔΨm). The causal relationship between changes in these crucial parameters and downstream effects of diet and exercise is currently unknown. A key bottleneck in understanding the role of intracellular bioenergetic parameters in regulation of metabolism has been the lack of methods for direct manipulation of these parameters in vivo. To fill this methodological gap, we have introduced two genetically encoded tools – LbNOX and TPNOX – 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.
Mechanism of lifespan extension by calorie restriction
Calorie restriction (CR) has been shown to extend the lifespan of yeast, worms, flies, mice and primates, which makes it the most robust method of lifespan extension known to date. Elucidation of the mechanism of CR-mediated lifespan extension will dramatically increase our understanding of the aging process. CR induces dramatic changes in energy metabolism and gene expression but it remains unclear which specific changes in metabolism and/or gene expression are responsible for mediating the beneficial effects of calorie restriction. We’re using GEMMs to mimic the effect of CR on energy metabolism pathways of C. elegans to uncover the specific metabolic changes that are necessary and sufficient for CR-mediated extension of lifespan. To facilitate these studies, we have setup a lifespan imaging machine that allows us to automatically measure the lifespan and motility of thousands of worms.
Mathematical modeling of energy metabolism
Modulation of energy metabolism is implicated in mediating the beneficial effects of diet and exercise on aging and age-associated diseases. Regulation of energy metabolism pathways is incompletely understood because most of our knowledge about regulation of these pathways is based on experiments with purified enzymes, tissue extracts and isolated mitochondria that do not fully capture the complexities of the intracellular environment. We are using mathematical modeling in combination with experimental approaches to understand the mechanisms of regulation of energy metabolism pathways in living cells. Specifically, we want to address several unanswered questions about the regulation of energy metabolism pathways: Which regulatory mechanisms are necessary and sufficient to recapitulate in vivo regulation of metabolism? What is the mechanism and logic of Warburg, Crabtree and Pasteur effects? How can we tune allosteric regulation of metabolic pathways to mimic the beneficial effects of CR and exercise?
Titov D.V.*, Cracan V.*, Goodman R.P., Peng J., Grabarek Z., Mootha V.K. Complementation of mitochondrial electron transport chain by manipulation of NAD+/NADH ratio. Science. 2016 Apr 8; 352(6282):231-5.
Cracan V.*, Titov D.V.*, Sheng H., Grabarek Z., Mootha V.K. Genetically encoded tool for manipulation of NADP+/NADPH ratio. Nature Chemical Biology. 2017 Oct; 13(10):1088-1095.
Titov D.V., Gilman B., He Q.-L., Bhat S., Low W.-K., Dang Y., Smeaton M., Demain A.L., Miller P.S., Kugel J.F., Goodrich J.A., Liu J.O. XPB, a Subunit of TFIIH, is a Target of the Natural Product Triptolide. Nature Chemical Biology. 2011 Mar;7(3):182-8.
* – these authors contributed equally to this work.
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