Project Description

Research has revealed that the degradation of nicotine can effectively mitigate nicotine-induced non-alcoholic fatty liver disease (NAFLD). A study published in Nature on October 20, 2022, by a team of researchers from Peking University, the National Institutes of Health (NIH) led by Frank Gonzalez, the First Affiliated Hospital of Zhejiang University School of Medicine led by Chaohui Yu, Fudan University School of Basic Medical Sciences led by Yang Li, and the First Affiliated Hospital of Wenzhou Medical University led by Minghua Zheng, explored the process. They found that nicotine, ingested during smoking, accumulates in the gut and accelerates the progression of NAFLD. Notably, they discovered that nicotine can be efficiently degraded by the human gut commensal bacterium B. xylanisolvens. To identify the key enzyme responsible for nicotine degradation, the research team utilized chromatographic and spectroscopic techniques and identified the nicotine metabolite as 4-hydroxy-1-(3-pyridinyl)-1-butanone (HPB), which significantly differs from host nicotine metabolism products, representing a novel nicotine metabolite. Subsequently, through whole-genome sequencing and functional gene analysis, the team identified the potential nicotine degradation enzyme NicX in B. xylanisolvens, and in vitro enzymatic assays confirmed NicX's role in nicotine degradation. The researchers recruited 83 NAFLD patients, confirmed through liver biopsies, including 41 smokers and 42 non-smokers. They conducted metagenomic, targeted metabolomic analysis, and association studies on their fecal samples. The conclusion was that B. xylanisolvens-mediated nicotine degradation can protect the liver from harm in smoking populations, mitigating the progression of non-alcoholic fatty liver disease. In another study, titled "Gut bacteria alleviate smoking-related NASH by degrading out nicotine," it was also confirmed that NicX in human gut commensal bacteria possesses the ability to degrade nicotine.

In experiments conducted on mice, it has been demonstrated that the degradation of nicotine can alleviate withdrawal symptoms in these rodents. In previous research, mice were concurrently administered enzymes capable of degrading nicotine and nicotine itself. It was found that withdrawal symptoms gradually lessened in the mice several days later, and further testing two weeks later revealed that the nicotine levels in their blood had nearly reached zero, with withdrawal symptoms being almost nonexistent. This suggests that the degradation of nicotine could potentially serve as a promising smoking cessation method.

Currently, common smoking cessation methods involve smoking cessation medications, including nicotine replacement therapy and non-nicotine medications. While they help alleviate withdrawal symptoms, nicotine replacement therapy still provides nicotine, which can lead to addiction. Non-nicotine medications may not be suitable for everyone due to potential side effects. In contrast, nicotine degradation through NicX appears to offer a high efficiency without the risk of addiction. To enhance the effectiveness of nicotine degradation, we aim to improve the stability and activity of NicX through various means.

After reviewing the literature, we have decided to employ three methods to enhance the stability and activity of NicX: constructing a JI-NicX fusion protein, utilizing mutation techniques, and employing cell surface display system.

Through literature research, it was found that NicA2 is a well-studied nicotine-degrading enzyme. J1 is an albumin derived from human blood, and scientists like Song Xu and Marsida Kallupi have demonstrated the extension of NicA2's half-life by fusing NicA2 with a short peptide, J1. Therefore, we intend to construct a JI-NicX fusion protein to improve the stability of NicX. We have also constructed NicA2 and JI-NicA2 fusion proteins for comparison.

Figure 3. NicA2(left) and J1 NicA2(right)

Figure 4. NicX(left) and J1-NicX(right)

Cell surface display system involves fusing the target protein with an anchor protein to express the protein on the cell surface. In this project, we are utilizing E. coli surface display system, fusing NicX with four different anchor proteins, AIDA-I, LppOmpA, INPNC, and BrkA, to display NicX on the cell surface.

Figure 5. E.coli surface display technology

In our initial literature search, we did not find the pdb file for NicX. Therefore, we used Alphafold to predict the protein structure based on the amino acid sequence of NicX and subsequently docked nicotine molecules onto the protein structure using Autodock to identify the active site. After predicting and analyzing the structure of NicX, we introduced mutations into NicX at the active site, with the expectation that experimental screening would yield mutant variants with higher degradation efficiency.

Figure 6. Nicotine degraded by NicX(left) and NicX (right) before mutation

Figure 7. Nicotine degraded byNicX(left) and NicX (right) after mutation