2023 GEC-CHINA
Abstract
Our project is mainly on the directed evolution of NAD+ synthases for increased intracellular NAD+ production. Recognizing the significance of NAD+ in combating aging processes, our team focuses on employing directed evolution techniques to optimize the synthesis of NAD+ enzymes in order to enhance overall cellular vitality. This paper highlights the rationale behind our approach, the methodology employed, and the potential implications for anti-aging interventions.
The Problem of Aging
With the extension of the human lifespan, the problem of global aging is becoming increasingly severe, presenting a major challenge that must be faced and resolved by all of humanity. According to statistics, in 2015, the global population over 60 years old reached 901 million and is expected to increase to 2.1 billion by 2050. Accompanying this trend are various age-related diseases , including atherosclerosis, hypertension, osteoarthritis, Alzheimer's disease, Parkinson's disease, diabetes, and cancer. The incidence of these diseases is constantly rising, posing a heavy burden on global socio-economic and healthcare systems about 15-25 trillion dollars globally. Therefore, the development of strategies to combat aging and related diseases is urgently needed.
Currently, humanity is adopting various methods to alleviate the impacts of aging and age-related diseases, including the use of nutritional supplements, various drugs, exercise, hormone therapy, and other treatment methods. Encouragingly, it is feasible to delay aging through nutritional supplements, among which Nicotinamide adenine dinucleotide (NAD+) is one of the essential molecules for slowing aging and preventing the development of age-related diseases (Partridge et al., 2020).
Background Information of NAD+
As a necessary redox carrier, the redox coenzyme NAD+ occupies a central position in energy metabolism. NAD+ receives high-energy electrons from pathways such as the citric acid cycle and fatty acid oxidation and drives the process of oxidative phosphorylation on the electron transport chain. In addition, NAD+ is indispensable in cellular life activities, being used as a cofactor or substrate by hundreds of enzymes, and participating in cell signaling transduction, DNA damage repair, epigenetic modifications, and other biological processes (Amjad et al., 2021). Maintaining cellular NAD+ levels is crucial for normal cellular life activities because NAD+ deficiency can lead to a series of diseases, including metabolic diseases, cancer, aging, and neurodegenerative diseases. Cellular NAD+ levels depend on the relative rates of NAD+ synthesis and degradation. In mammals, NAD+ synthesis mainly occurs through three pathways, including the de novo synthesis pathway initiated by tryptophan, the Preiss-Handler pathway initiated by nicotinic acid (NA), and the salvage synthesis pathway that rapidly generates NAD+ by recycling nicotinamide (NAM) and nicotinamide riboside (NR) (Verdin, 2015).
The Role of NAD+ in Cells
NAD+ is widely used as a cofactor or substrate for biochemical reactions. Thus, NAD+ is an intermediator of key cellular functions and adaptive metabolic needs. Some of these key cellular processes include metabolic pathways, redox homeostasis, sources and repair of major DNA to protect genome stability, epigenetic regulation, and more. Overall, these functions are important for maintaining system health and homeostasis. However, during aging, a decrease in NAD+ levels affect these processes.
Mechanisms of NAD+ Dependence During Aging
Major cellular processes that affect or are affected by aging include metabolic dysfunction, DNA repair failure and genomics instability, while NAD+ levels play an important role in regulating this process. NAD+ has been widely valued in the field of anti-aging and proven by a large number of scientific research results. As age increases, the levels of NAD+ continuously decline in various organisms, promoting the development of many age-related pathophysiological conditions. Therefore, restoring NAD+ levels is considered an effective intervention measure to improve or even prevent age-related functional decline (Yoshino et al., 2018).
Cellular NAD+ Metabolism
NAD+ is a key metabolite and coenzyme for multiple metabolic pathways and cellular processes. NAD+ reduction is necessary to maintain cellular energy balance and redox state. NAD+ is also continuously converted by three classes of NAD+ consuming enzymes, which utilize NAD+ as a substrate or cofactor and produce nicotinamide (NAM) as a by-product. Therefore, NAD+ mediates multiple major biological processes and is always in high demand. NAD+ is continuously synthesized, catabolized, and circulated in cells to maintain stable intracellular NAD+ levels.
Current Method to Increase NAD+
Effective methods to increase NAD+ currently include supplementing NAD+ precursors and using NAD+ synthesis enzyme agonists. However, due to issues like rapid metabolism after intake, low bioavailability, and high costs associated with NAD+ intermediates like NR and NMN, developing agonists of NAD+ synthesizing enzymes is a good way to resolve these drawbacks. However, currently developed and biologically validated NAD+ synthesizing enzyme agonists are limited and restricted to preclinical research, limiting the clinical application of NAD+-enhancing therapies.
Background Information of Directed Evolution
Directed evolution technology is an important molecular biology technique for improving protein properties. With the continuous development of technology, directed evolution has become a powerful method. By simulating the process of natural evolution, a large number of mutations are artificially created in specific proteins in the lab, resulting in a mutation library. After expressing the mutation results in cells, selections or screenings are made under specific conditions to find proteins that meet the desired characteristics (Hendel and Shoulders, 2021).
Our Approach
This project proposes to obtain a super-active mutant of NAD+ biosynthesis enzymes through directed evolution technology, thereby improving the efficiency of NAD+ biosynthesis enzymes and achieving an increase in the production of NAD+ as well as its precursors.
Reference:
- http://thesjf.com/2021/11/04/nad-metabolism-and-its-roles-in-cellular-processes-during-ageing/.
- Picture 1 from BofA Merrill Lynch Global Research.
- Picture 2-4 from @thesjf http://thesjf.com/2021/11/04/nad-metabolism-and-its-roles-in-cellular-processes-during-ageing/.
- Picture 5 from https://lewis.lab.indiana.edu/directed-evolution/.