Uncoupling Protein 2 and 4 Expression Pattern during Stem Cell Differentiation Provides New Insight into Their Putative Function

Rupprecht, Anne; Sittner, D.; Smorodchenko, Alina; Hilse, Karolina E.; Goyn, Justus; Moldzio, Rudolf; Seiler, A.; Bräuer, Anja U.; Pohl, Elena E.

doi:10.1371/journal.pone.0088474

Cited by 35 publications

(47 citation statements)

References 56 publications

(75 reference statements)

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“…UCP4 may be predominantly located in the neuronal cell body (8), where it would dissipate the excessive proton gradient. This location would also better fit UCP4's function in neuronal metabolism (7,27). Indeed, performing two-color dSTORM experiments in the neuronal cell body, we found nearly all mitochondria to be double positive for UCP4/ATP synthase (92.1%) (Fig.…”

Section: Resultsmentioning

confidence: 75%

Superresolution microscopy reveals spatial separation of UCP4 and F ₀ F ₁ -ATP synthase in neuronal mitochondria

Klotzsch

Smorodchenko²,

Löfler

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Because different proteins compete for the proton gradient across the inner mitochondrial membrane, an efficient mechanism is required for allocation of associated chemical potential to the distinct demands, such as ATP production, thermogenesis, regulation of reactive oxygen species (ROS), etc. Here, we used the superresolution technique dSTORM (direct stochastic optical reconstruction microscopy) to visualize several mitochondrial proteins in primary mouse neurons and test the hypothesis that uncoupling protein 4 (UCP4) and F 0 F 1 -ATP synthase are spatially separated to eliminate competition for the proton motive force. We found that UCP4, F 0 F 1 -ATP synthase, and the mitochondrial marker voltage-dependent anion channel (VDAC) have various expression levels in different mitochondria, supporting the hypothesis of mitochondrial heterogeneity. Our experimental results further revealed that UCP4 is preferentially localized in close vicinity to VDAC, presumably at the inner boundary membrane, whereas F 0 F 1 -ATP synthase is more centrally located at the cristae membrane. The data suggest that UCP4 cannot compete for protons because of its spatial separation from both the proton pumps and the ATP synthase. Thus, mitochondrial morphology precludes UCP4 from acting as an uncoupler of oxidative phosphorylation but is consistent with the view that UCP4 may dissipate the excessive proton gradient, which is usually associated with ROS production. mitochondrial membrane proteins | proton diffusion | direct stochastic optical reconstruction microscopy | uncoupling | reactive oxygen species M itochondria are involved in a wide range of cell functions, including fatty acid oxidation, calcium homeostasis, apoptosis, reactive oxygen species (ROS) signaling, and above all, production of ATP (1, 2). In neurons, these organelles are transported along neuronal processes to provide energy for areas of high energy demand, such as synapses (3). To support their functions, mitochondria exhibit a complex morphology consisting of separate and functionally distinct outer mitochondrial membrane (OMM) and inner mitochondrial membrane (IMM). The latter is structurally organized into two domains: an inner boundary membrane (IBM) and a cristae membrane (CM) (4). The current hypotheses imply that the morphology/topology of the IMM is tightly related to biochemical function, the energy state, and the pathophysiological state of mitochondria (5). Whereas the OMM contains porins [e.g., voltage-dependent anion channel (VDAC)], which mediate its permeability to molecules up to 10 kDa, the IMM topology is highly complex. It is comprised of different transport proteins, the ATP synthase (complex V), and complexes I, III, and IV of the electron transport chain, which are responsible for generating the proton motive force (pmf); pmf represents the driving force for not only ATP synthesis, but also other protein-mediated transport activities (for example, phosphate, pyruvate, and glutamate transport). Uncoupling protein 1 (UCP1; thermogenin), a memb...

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Section: Resultsmentioning

confidence: 75%

Superresolution microscopy reveals spatial separation of UCP4 and F ₀ F ₁ -ATP synthase in neuronal mitochondria

Klotzsch

Smorodchenko²,

Löfler

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…A recent comparison of wild type and UCP2 -/-or UCP3 -/-knockout mice [21] implied that both proteins are not involved in H + transport. Unfortunately, the tissue(s) chosen in this study were previously shown to have no presence or very low abundance of UCP2/UCP3 under physiological conditions [38,39].…”

Section: Reactive Oxygen Species and Their Derivatives Reactive Aldementioning

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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“…It was previously shown that neuroblastoma cell line N18TG2 expresses UCP2 (22). To determine whether genipin ( Fig.…”

Section: Genipin Does Not Influence Ucp2 Expression But Decreases Thementioning

confidence: 99%

“…One of the possible reasons can be that in many studies, UCP2 is artificially expressed in cells in which it is normally absent. Our group and others have recently shown that UCP2 is confined to rapidly proliferating cells that rely on glycolysis, such as stem cells, activated immunological cells, and cancer cells (22,28,53). The expression of UCP2 in cells that have other metabolic pathways can seriously affect the physiological function of UCP2.…”

Section: Genipin Influenced the Activity Of Membrane Proteins Differementioning

confidence: 99%

“…UCP2 is a member of the mitochondrial anion transporter superfamily (19) and is highly homologous to other members of the UCP subfamily, such as UCP3 and UCP1 (20). It has been identified in rapidly proliferating cells that rely on glycolysis, such as stem cells (21,22), activated immunological cells (23), cancer cells, and immortalized cell lines (22,24,25). UCP2 mediates a regulated proton leak through the inner mitochondrial membrane and was recently suggested to transport small C4 metabolites (26).…”

Section: Introductionmentioning

confidence: 99%

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Molecular Mechanisms Responsible for Pharmacological Effects of Genipin on Mitochondrial Proteins

Kreiter

Rupprecht

Zimmermann

et al. 2019

Biophysical Journal

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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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Uncoupling Protein 2 and 4 Expression Pattern during Stem Cell Differentiation Provides New Insight into Their Putative Function

Cited by 35 publications

References 56 publications

Superresolution microscopy reveals spatial separation of UCP4 and F ₀ F ₁ -ATP synthase in neuronal mitochondria

Superresolution microscopy reveals spatial separation of UCP4 and F ₀ F ₁ -ATP synthase in neuronal mitochondria

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

Molecular Mechanisms Responsible for Pharmacological Effects of Genipin on Mitochondrial Proteins

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Uncoupling Protein 2 and 4 Expression Pattern during Stem Cell Differentiation Provides New Insight into Their Putative Function

Cited by 35 publications

References 56 publications

Superresolution microscopy reveals spatial separation of UCP4 and F 0 F 1 -ATP synthase in neuronal mitochondria

Superresolution microscopy reveals spatial separation of UCP4 and F 0 F 1 -ATP synthase in neuronal mitochondria

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

Molecular Mechanisms Responsible for Pharmacological Effects of Genipin on Mitochondrial Proteins

Contact Info

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Superresolution microscopy reveals spatial separation of UCP4 and F ₀ F ₁ -ATP synthase in neuronal mitochondria

Superresolution microscopy reveals spatial separation of UCP4 and F ₀ F ₁ -ATP synthase in neuronal mitochondria