Genipin is a natural product found in the fruits of flowering plants such as Gardenia jasminoides and Gardenia americana. Interestingly, genipin is a natural cross-linking agent, meaning it can react with chemical groups on different biochemical molecules and effectively link them together (1). In addition, genipin is found in traditional Chinese medicine formulations used for a variety of diseases, ranging from reducing inflammation to cancer treatment (2). Given the extensive versatility of this compound, scientists are interested in understanding more about its mechanism of action.
In the recent article, “Molecular Mechanisms Responsible for Pharmacological Effects of Genipin on Mitochondrial Proteins,” published in Biophysical Journal, a group of researchers used extensive biochemical methods to better characterize the specific effects of genipin on mammalian cells (3). While previous work suggested genipin targets a specific protein found in the membrane of mitochondria called UCP2 (uncoupling protein 2) (4), it was unclear if other proteins could be affected by this molecule. UCP2 is part of a family of proteins in the mitochondrial membrane that dissipate the proton gradient that accumulates during oxidative phosphorylation by transporting protons into the mitochondrial inner membrane (5).
To directly probe the role of genipin on UCP1, UCP2, and UCP3, the authors of this work reconstituted each purified protein into a lipid bilayer and demonstrated that the addition of genipin caused inhibition proton transport for all three proteins. This proves genipin can act on more than just UCP2. While this experiment showed genipin could inhibit these proteins, it did not demonstrate the molecular mechanism of action, which the authors sought to determine.
Interestingly, it appeared that when reconstituted UCP1 was treated with high concentrations of genipin (1 mM), membrane conductance showed an initial decrease and then subsequent increase. The authors concluded that the UCP1-specific effects occurred at low concentration (<200 µM), followed by non-specific ion transport at higher concentrations which leads to an increase in membrane conductance. To understand this specific inhibition, the authors speculated the genipin molecules may be reacting with atoms on the protein as a result of the cross-linking behavior of this unique molecule. Using mass spectrometry, they identified two specific residues of UCP1 that become labeled by genipin, suggesting this modification could be the source of UCP inhibition via genipin.
To confirm this hypothesis, the authors performed an experiment in which they chemically blocked the residues of UCP1 labeled with genipin and investigated whether this blockage prevented inhibition. The authors expected that blocking these residues would cause UCP1 to no longer be inhibited by genipin— but to their surprise, this was not the case. In fact, blocking these residues had no impact on genipin inhibition, suggesting that this cross-linking modification by genipin was not the source of inhibition.
Previous work has shown that UCP1 is inhibited by purine nucleotides and that there are key arginine residues required for this inhibition (6). To conclude whether the mechanism of UCP1 inhibition by genipin is similar to that of purine inhibition, the authors mutated these arginine residues and in fact confirmed that genipin can no longer inhibit the protein in the absence of these critical residues. This suggests the mechanism by which purine nucleotides inhibit the proton transport functions of UCP molecules is the same for the plant-derived natural product, genipin and requires critical arginine residues situated in the UCP1 pocket.
In conclusion, this work used biochemical investigation to better understand the mechanism of action of a small-molecule drug commonly found in Chinese medicine treatments. This detailed approach allowed the authors to provide extensive evidence toward how this unique and interesting molecule impacts mammalian cells. Check out the article, available on open access at Biophysical Journal!
- Abigail Powell, Biophysical Journal Social Media Contributor
References:
(1) Manickam, B, Sreedharan, R, and Elumalai, M. (2014) ‘Genipin’ – the natural water soluble cross-linking agent and its importance in the modified drug delivery systems: an overview. Curr. Drug Deliv. https://www.ncbi.nlm.nih.gov/pubmed/24041312
(2) Koo, H, et al (2004) Antiinflammatory effects of genipin, an active principle of gardenia. Eur. J. Pharmacol. https://www.ncbi.nlm.nih.gov/pubmed/?term=15249171
(3) Kreiter, J et al (2019) Molecular mechanisms responsible for pharmacological effects of genipin on mitochondrial proteins. Biophys. J. https://www.ncbi.nlm.nih.gov/pubmed/31706565
(4) Zhang, C et al (2006) Genipin inhibits UCP2-meiated proton leak and acutely reverse obesity- and high glucose-induced beta cell dysfunction in isolated pancreatic islets. Cell Metab. https://www.ncbi.nlm.nih.gov/pubmed/16753577
(5) Palmieri, F (2013) The mitochondrial transporter family SLC25: identification, properties, and physiopathology. Mol. Aspects Med. https://www.ncbi.nlm.nih.gov/pubmed/23266187
(6) Modriansky, M et al (1997) Identification by site-directed mutagenesis of three arginines in uncoupling protein that are essential for nucleotide binding and inhibition. J. Biol. Chem. https://www.ncbi.nlm.nih.gov/pubmed/9312070