Introduction: Sirtuin 1 (SIRT1) is a crucial enzyme in the human body that removes acetyl groups from lysine residues in proteins. SIRT1 influences gene expression through the deacetylation of transcription factors and histones, and it also acts upon other proteins, having the largest impact on regulation in the liver and the brain. Given SIRT1’s extensive influence on gene expression, therapies targeting SIRT1 hold promise for treating various diseases, including cancer, metabolic disorders, aging, and neurodegeneration.
However, designing effective therapies for such diseases requires a comprehensive understanding of the molecular mechanisms by which SIRT1 recognizes and deacetylates its substrates. This research aims to fill these knowledge gaps by determining the regions of the SIRT1 protein essential for substrate binding and differentiating acetylated proteins that are substrates of SIRT1 from those that are not. To achieve this, we will utilize microtubule-associated protein 1A/1B-light chain 3 (LC3) as a model substrate. LC3's known interaction with SIRT1 in autophagy pathways and its suitability for nuclear magnetic resonance (NMR) spectroscopy make it an ideal candidate for studying SIRT1's enzymatic activity.
Our current work has focused on engineering acetylated LC3 protein in quantities sufficient for studying SIRT1 and acquiring more information about this protein’s molecular structure from its NMR spectrum.
Materials and
Methods: To explore SIRT1's substrate interactions, we used LC3 as a model substrate. Recombinant LC3 protein was produced in Escherichia coli (E. coli) by incorporating a plasmid containing the LC3 gene under the control of the inducible lactose operon. Protein expression was induced with IPTG, and LC3 was purified using cation exchange and gel filtration chromatography.
Cells were grown in a carbon 13 and nitrogen 15 labeled growth medium, resulting in isotopically labeled proteins. The isotopic labeling of LC3 prepared it for two-dimensional NMR Heteronuclear Single Quantum Coherence (HSQC) spectroscopy, generating spectra with cross peaks representing each hydrogen-nitrogen bonded pair in the protein. These peaks serve as structural fingerprints, with each peak corresponding to an individual amino acid, and changes in peak positions indicating changes in their chemical environment. Additional three-dimensional NMR experiments provided data on the carbon atoms (alpha, beta, and carbonyl) of the amino acids associated with each hydrogen-nitrogen pair and neighboring amino acids, enabling precise amino acid assignments to be made to the 2D HSQC spectrum.
To produce acetylated LC3, an unnatural tRNA synthetase was used to incorporate an acetyl-lysine amino acid at a specific stop codon site in the E. coli genome. This stop codon was introduced recombinantly to replace the natural lysine codon. We are currently developing purification methods for this acetylated LC3 based on those used for the natural protein.
Results, Conclusions, and Discussions: We have currently completed over 70% of the amino acid assignments for the LC3 HSQC spectrum. Progress is ongoing, as additional high-quality 3D NMR spectra are needed to finalize these assignments. The production of acetylated LC3 was successful, although initial complications with expression necessitated the use of a different E. coli cell line lacking additional rare tRNA codons. We are currently implementing adjustments to the purification method due to a loss of positive charge on the lysine residue from acetylation, such as transitioning the LC3 from a pH 7 to a pH 5 buffer. Preliminary 2D NMR data suggests that the protein structure of LC3 remains stable at this lower pH, potentially enhancing the protein's overall positive charge and improving purification yields.
Future work will involve using different lengths of SIRT1 in combination with LC3 to identify the specific regions of SIRT1 necessary for substrate interaction. Changes in the 2D NMR spectrum will be examined to pinpoint these interaction sites. Additionally, fluorometric assays will be conducted to assess SIRT1 activity, supplementing the structural data obtained from NMR spectroscopy. By using this combined approach, we will attempt to minimize potential errors associated with studying only protein fragments and provide a more comprehensive understanding of SIRT1's enzymatic function.
The knowledge gained from this research will advance the development of therapies that modulate SIRT1 activity. By identifying the key regions of SIRT1 involved in substrate recognition and deacetylation, new therapeutic strategies can be studied to maintain SIRT1 in its active state, offering potential treatments for neurological and other disorders.
Acknowledgements (Optional): I would like to thank the members of the Angela M. Gronenborn Lab, especially Troy Krzysiak, for their continued support on this project.
