Fatty Acids are Key in 4-Hydroxy-2-Nonenal-Mediated Activation of Uncoupling Proteins 1 and 2

Genipin, a natural compound from Gardenia jasminoides, is a well-known compound in Chinese medicine that is used for the treatment of cancer, inflammation, and diabetes. The use of genipin in classical medicine is hindered because of its unknown molecular mechanisms of action apart from its strong cross-linking ability. Genipin is increasingly applied as a specific inhibitor of proton transport mediated by mitochondrial uncoupling protein 2 (UCP2). However, its specificity for UCP2 is questionable, and the underlying mechanism behind its action is unknown. Here, we investigated the effect of genipin in different systems, including neuroblastoma cells, isolated mitochondria, isolated mitochondrial proteins, and planar lipid bilayer membranes reconstituted with recombinant proteins. We revealed that genipin activated dicarboxylate carrier and decreased the activity of UCP1, UCP3, and complex III of the respiratory chain alongside with UCP2 inhibition. Based on competitive inhibition experiments, the use of amino acid blockers, and site-directed mutagenesis of UCP1, we propose a mechanism of genipin's action on UCPs. At low concentrations, genipin binds to arginine residues located in the UCP funnel, which leads to a decrease in UCP's proton transporting function in the presence of long chain fatty acids. At concentrations above 200 mM, the inhibitory action of genipin on UCPs is overlaid by increased nonspecific membrane conductance due to the formation of protein-genipin aggregates. Understanding the concentration-dependent mechanism of genipin action in cells will allow its targeted application as a drug in the above-mentioned diseases. SIGNIFICANCE Genipin is a well-known natural cross-linking agent for proteins, collagen, gelatin, and chitosan. However, the mechanism of its multiple effects on cells and mitochondria is under dispute. Mitochondrial UCP2 was previously revealed as an important target for genipin. We show that inhibition of UCP2 by genipin at submillimolar concentrations depends on the presence of three positively charged arginines in the funnel of the protein. This mechanism is similar to the UCP inhibition by purine nucleotides such as ATP/ADP and GTP/GDP. The effect of genipin is not specific to UCP2, which can be explained by the presence of arginines in homologous UCP1 and UCP3. This insight is crucial for the design of specific inhibitors of UCPs. FIGURE 9 Extracted ion chromatograms of genipin-modified UCP1 tryptic peptides. Signals at m/z 919.47 and 914.14 correspond to UCP1 tryptic peptide 184 NVIICTELVTYDLMKGALVNNK 206 carrying K198 (919.47; tR ¼ 21.4) and C188 (919.14; tR ¼ 24.2), respectively, modified with genipin adduct of 158 amu after incubation with 50 mM (A) and 1 mM (B) genipin. Corresponding CID spectra are provided in Fig. S1. (C) Positions of cysteine C188 and lysine K198 in UCP1, at which genipin modifications were detected. Three-dimensional structure of UCP1 was computed based on the crystallographic structure of ANT (PDB: 1OKC) using PyMol. To see this figure in color, go...

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“…10 A). This supports our previous results that modification of these amino acids in UCP1 does not affect its activity (51,55).…”

Section: The Inhibition Mechanism Of Ucps By Genipin At Low Concentrasupporting

confidence: 93%

“…Further, we used NHS, MNBS, and NEM to block the lysine, histidine, and cysteine residues of UCP1, respectively (51). As the unmodified control, UCP1 was incubated with an equal volume of water.…”

Section: Genipin At Low Concentrations Inhibits Ucp1 By Binding To Armentioning

confidence: 99%

Molecular Mechanisms Responsible for Pharmacological Effects of Genipin on Mitochondrial Proteins

Kreiter

Rupprecht

Zimmermann

et al. 2019

Biophysical Journal

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“…This synergistic effect on UCP1 activity was also found in studies of UCP1 in planar lipid bilayers. Again, 4-HNE alone had no effect on UCP1 activation but strongly potentiated membrane conductance mediated by fatty acids (57). Interestingly, these effects coincided with cysteine modification of UCP1 by 4-HNE, whereas pre-treatment of UCP1 with cysteine-alkylating agents could inhibit 4-HNEdependent potentiation (57).…”

Section: Minireview: Ros and Adipose Tissue Thermogenesis Ated Hmentioning

confidence: 91%

Mitochondrial reactive oxygen species and adipose tissue thermogenesis: Bridging physiology and mechanisms

Chouchani

Kazak

Spiegelman

2017

Journal of Biological Chemistry

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Edited by Ruma BanerjeeBrown and beige adipose tissues can catabolize stored energy to generate heat, relying on the principal effector of thermogenesis: uncoupling protein 1 (UCP1). This unique capability could be leveraged as a therapy for metabolic disease. Numerous animal and cellular models have now demonstrated that mitochondrial reactive oxygen species (ROS) signal to support adipocyte thermogenic identity and function. Herein, we contextualize these findings within the established principles of redox signaling and mechanistic studies of UCP1 function. We provide a framework for understanding the role of mitochondrial ROS signaling in thermogenesis together with testable hypotheses for understanding mechanisms and developing therapies. Brown adipose tissue (BAT)2 has long been recognized as a critical thermogenic organ, exemplified by its importance in maintenance of thermal homeostasis in cold environments (1). More recently, clusters of distinct adipocytes with thermogenic capacity have been characterized in white adipose tissue-beige adipocytes-arising in response to various physiological stimuli (2, 3). The thermogenic endowment of these tissues has prompted the reasonable hypothesis that they might be leveraged to catabolize lipid and glucose as an anti-obesity and antidiabetic therapy. As such, recent decades have seen vigorous research into the mechanisms controlling brown and beige adipose tissue identity and function. Thermogenesis in BAT and beige adipose tissue relies on the key effector protein uncoupling protein 1 (UCP1), which can dissipate the electrochemical H ϩ gradient across the inner mitochondrial membrane (4). This UCP1-mediated inducible proton "leak" uncouples protonmotive force from ATP synthesis leading to an increased rate of mitochondrial respiration and heat generation. Additionally, UCP1-independent effectors of thermogenesis have been uncovered in recent years, including utilization of creatine-dependent substrate cycling in beige adipose tissue (5). Of course, expression of UCP1 (or other effectors) per se is not sufficient to drive thermogenesis; these are gas pedals that need a foot, fuel, and an engine. Indeed, thermogenic adipocytes require exceptionally high mitochondrial content and oxidative capacity to support elevated respiration. Moreover, thermogenic respiration is regulated acutely by upstream physiological signals that are required to activate UCP1-mediated uncoupling. So, elucidating the mechanisms that activate thermogenically-poised adipocytes is critical from a therapeutic standpoint.The purpose of this review is to contextualize recent findings that provide evidence for an important signaling role by reactive oxygen species (ROS) in adipose tissue thermogenesis. Remarkably, the importance of ROS signaling in this context has been demonstrated primarily through investigation of redox metabolism using in vivo mouse and intact adipocyte models of thermogenesis. In contrast, studies of ROS and thermogenesis using in vitro and reconstituted systems have provided...

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“…In subsequent studies, a high membrane potential was suggested as a requirement for the activation of UCP-mediated uncoupling by HNE [47]. Experiments performed in a well-defined system of bilayer membranes reconstituted with recombinant UCPs [48] revealed that HNE did not directly activate either UCP1 or UCP2. However, HNE strongly potentiated the membrane proton conductance increase mediated by different long-chain FAs in UCP-containing and UCP-free membranes.…”

Section: Ros Detoxification Systemsmentioning

confidence: 99%

The Role of Phosphatidylethanolamine Adducts in Modification of the Activity of Membrane Proteins under Oxidative Stress

Pohl

Jovanović

2019

Molecules

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Reactive oxygen species (ROS) and their derivatives, reactive aldehydes (RAs), have been implicated in the pathogenesis of many diseases, including metabolic, cardiovascular, and inflammatory disease. Understanding how RAs can modify the function of membrane proteins is critical for the design of therapeutic approaches in the above-mentioned pathologies. Over the last few decades, direct interactions of RA with proteins have been extensively studied. Yet, few studies have been performed on the modifications of membrane lipids arising from the interaction of RAs with the lipid amino group that leads to the formation of adducts. It is even less well understood how various multiple adducts affect the properties of the lipid membrane and those of embedded membrane proteins. In this short review, we discuss a crucial role of phosphatidylethanolamine (PE) and PE-derived adducts as mediators of RA effects on membrane proteins. We propose potential PE-mediated mechanisms that explain the modulation of membrane properties and the functions of membrane transporters, channels, receptors, and enzymes. We aim to highlight this new area of research and to encourage a more nuanced investigation of the complex nature of the new lipid-mediated mechanism in the modification of membrane protein function under oxidative stress.

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Fatty Acids are Key in 4-Hydroxy-2-Nonenal-Mediated Activation of Uncoupling Proteins 1 and 2

Cited by 36 publications

References 48 publications

Molecular Mechanisms Responsible for Pharmacological Effects of Genipin on Mitochondrial Proteins

Molecular Mechanisms Responsible for Pharmacological Effects of Genipin on Mitochondrial Proteins

Mitochondrial reactive oxygen species and adipose tissue thermogenesis: Bridging physiology and mechanisms

The Role of Phosphatidylethanolamine Adducts in Modification of the Activity of Membrane Proteins under Oxidative Stress

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