A Thia-Analogous Indirubin N-Glycoside Disrupts Mitochondrial Function and Causes the Death of Human Melanoma and Cutaneous Squamous Cell Carcinoma Cells.

Skin cancer is the most common malignant disease worldwide and, therefore, also poses a challenge from a pharmacotherapeutic perspective. Derivatives of indirubin are an interesting option in this context. In the present study, the effects of 3-[3'-oxo-benzo[b]thiophen-2'-(Z)-ylidene]-1-(ß-d-glucopyranosyl)-oxindole (KD87), a thia-analogous indirubin N-glycoside, on the viability and mitochondrial properties of melanoma (A375) and squamous cell carcinoma cells (A431) of the skin were investigated. In both cell lines, KD87 caused decreased viability, the activation of caspases-3 and -7, and the inhibition of colony formation. At the mitochondrial level, a concentration-dependent decrease in both the basal and ATP-linked oxygen consumption rate and in the reserve capacity of oxidative respiration were registered in the presence of KD87. These changes were accompanied by morphological alterations in the mitochondria, a release of mitochondrial cytochrome c into the cytosol and significant reductions in succinate dehydrogenase complex subunit B (SDHB, subunit of complex II) in A375 and A431 cells and NADH:ubiquinone oxidoreductase subunit B8 (NDUFB8, subunit of complex I) in A375 cells. The effect of KD87 was accompanied by a significant upregulation of the enzyme heme oxygenase-1, whose inhibition led to a partial but significant reduction in the metabolic-activity-reducing effect of KD87. In summary, our data show a mitochondria-targeting effect of KD87 as part of the cytotoxic effect of this compound on skin cancer cells, which should be considered in future studies with this class of compounds.

The Combination of Δ9-Tetrahydrocannabinol and Cannabidiol Suppresses Mitochondrial Respiration of Human Glioblastoma Cells via Downregulation of Specific Respiratory Chain Proteins.

Phytocannabinoids represent a promising approach in glioblastoma therapy. Previous work has shown that a combined treatment of glioblastoma cells with submaximal effective concentrations of psychoactive Δ9-tetrahydrocannabinol (THC) and non-psychoactive cannabidiol (CBD) greatly increases cell death. In the present work, the glioblastoma cell lines U251MG and U138MG were used to investigate whether the combination of THC and CBD in a 1:1 ratio is associated with a disruption of cellular energy metabolism, and whether this is caused by affecting mitochondrial respiration. Here, the combined administration of THC and CBD (2.5 µM each) led to an inhibition of oxygen consumption rate and energy metabolism. These effects were accompanied by morphological changes to the mitochondria, a release of mitochondrial cytochrome c into the cytosol and a marked reduction in subunits of electron transport chain complexes I (NDUFA9, NDUFB8) and IV (COX2, COX4). Experiments with receptor antagonists and inhibitors showed that the degradation of NDUFA9 occurred independently of the activation of the cannabinoid receptors CB1, CB2 and TRPV1 and of usual degradation processes mediated via autophagy or the proteasomal system. In summary, the results describe a previously unknown mitochondria-targeting mechanism behind the toxic effect of THC and CBD on glioblastoma cells that should be considered in future cancer therapy, especially in combination strategies with other chemotherapeutics.

Implementation of a combined CDK inhibition and arginine-deprivation approach to target arginine-auxotrophic glioblastoma multiforme cells.

Constitutive activation of cyclin-dependent kinases (CDKs) or arginine auxotrophy are hallmarks of Glioblastoma multiforme (GBM). The latter metabolic defect renders tumor cells vulnerable to arginine-depleting substances, such as arginine deiminase from Streptococcus pyogenes (SpyADI). Previously, we confirmed the susceptibility of patient-derived GBM cells towards SpyADI as well as CDK inhibitors (CDKis). To improve therapeutic effects, we here applied a combined approach based on SpyADI and CDKis (dinaciclib, abemaciclib). Three arginine-auxotrophic patient-derived GBM lines with different molecular characteristics were cultured in 2D and 3D and effects of this combined SpyADI/CDKi approach were analyzed in-depth. All CDKi/SpyADI combinations yielded synergistic antitumoral effects, especially when given sequentially (SEQ), i.e., CDKi in first-line and most pronounced in the 3D models. SEQ application demonstrated impaired cell proliferation, invasiveness, and viability. Mitochondrial impairment was demonstrated by increasing mitochondrial membrane potential and decreasing oxygen consumption rate and extracellular acidification rate after SpyADI/abemaciclib monotherapy or its combination regimens. The combined treatment even induced autophagy in target cells (abemaciclib/SpyADI > dinaciclib/SpyADI). By contrast, the unfolded protein response and p53/p21 induced senescence played a minor role. Transmission electron microscopy confirmed damaged mitochondria and endoplasmic reticulum together with increased vacuolization under CDKi mono- and combination therapy. SEQ-abemaciclib/SpyADI treatment suppressed the DSB repair system via NHEJ and HR, whereas SEQ-dinaciclib/SpyADI treatment increased γ-H2AX accumulation and induced Rad51/Ku80. The latter combination also activated the stress sensor GADD45 and ß-catenin antagonist AXIN2 and induced expression changes of genes involved in cellular/cytoskeletal integrity. This study highlights the strong antitumoral potential of a combined arginine deprivation and CDK inhibition approach via complex effects on mitochondrial dysfunction, invasiveness as well as DNA-damage response. This provides a good starting point for further in vitro and in vivo proof-of-concept studies to move forward with this strategy.

ANT1 Activation and Inhibition Patterns Support the Fatty Acid Cycling Mechanism for Proton Transport.

Adenine nucleotide translocase (ANT) is a well-known mitochondrial exchanger of ATP against ADP. In contrast, few studies have shown that ANT also mediates proton transport across the inner mitochondrial membrane. The results of these studies are controversial and lead to different hypotheses about molecular transport mechanisms. We hypothesized that the H+-transport mediated by ANT and uncoupling proteins (UCP) has a similar regulation pattern and can be explained by the fatty acid cycling concept. The reconstitution of purified recombinant ANT1 in the planar lipid bilayers allowed us to measure the membrane current after the direct application of transmembrane potential ΔΨ, which would correspond to the mitochondrial states III and IV. Experimental results reveal that ANT1 does not contribute to a basal proton leak. Instead, it mediates H+ transport only in the presence of long-chain fatty acids (FA), as already known for UCPs. It depends on FA chain length and saturation, implying that FA's transport is confined to the lipid-protein interface. Purine nucleotides with the preference for ATP and ADP inhibited H+ transport. Specific inhibitors of ATP/ADP transport, carboxyatractyloside or bongkrekic acid, also decreased proton transport. The H+ turnover number was calculated based on ANT1 concentration determined by fluorescence correlation spectroscopy and is equal to 14.6 ± 2.5 s-1. Molecular dynamic simulations revealed a large positively charged area at the protein/lipid interface that might facilitate FA anion's transport across the membrane. ANT's dual function-ADP/ATP and H+ transport in the presence of FA-may be important for the regulation of mitochondrial membrane potential and thus for potential-dependent processes in mitochondria. Moreover, the expansion of proton-transport modulating drug targets to ANT1 may improve the therapy of obesity, cancer, steatosis, cardiovascular and neurodegenerative diseases.

STAT5 is required for lipid breakdown and beta-adrenergic responsiveness of brown adipose tissue.

OBJECTIVE: Increasing energy expenditure through activation of brown adipose tissue (BAT) thermogenesis is an attractive approach to counteract obesity. It is therefore essential to understand the molecular mechanisms that control BAT functions. Until now several members of the Janus kinase (JAK) - signal transducer and activator of transcription (STAT) pathway have been implicated as being relevant for BAT physiology. However, whether the STAT family member STAT5 is important for the thermogenic property of adipose tissues is unknown. Therefore, we have investigated the role of STAT5 in thermogenic fat in this paper. METHODS: We performed metabolic and molecular analyses using mice that harbor an adipocyte-specific deletion of Stat5a/b alleles. RESULTS: We found that STAT5 is necessary for acute cold-induced temperature maintenance and the induction of lipid mobilization in BAT following ß3-adrenergic stimulation. Moreover, mitochondrial respiration of primary differentiated brown adipocytes lacking STAT5 was diminished. Increased sensitivity to cold stress upon STAT5 deficiency was associated with reduced expression of thermogenic markers including uncoupling protein 1 (UCP1), while decreased stimulated lipolysis was linked to decreased protein kinase A (PKA) activity. Additionally, brown remodeling of white adipose tissue was diminished following chronic ß3-adrenergic stimulation, which was accompanied by a decrease in mitochondrial performance. CONCLUSION: We conclude that STAT5 is essential for the functionality and the ß-adrenergic responsiveness of thermogenic adipose tissue.

Age-related sex differences in the expression of important disease-linked mitochondrial proteins in mice.

The prevalence and progression of many illnesses, such as neurodegenerative and cardiovascular diseases, obesity, and cancer, vary between women and men, often in an age-dependent manner. A joint hallmark of these diseases is some type of mitochondrial dysfunction. While several mitochondrial proteins are known to be regulated by sex hormones, the levels of those proteins have not been systematically analyzed with regard to sex and age, and studies that consider sex and/or age differences in the protein expression are very rare. In this study, we compared the expression patterns of physiologically important mitochondrial proteins in female and male C57BL/6N mice of age cohorts frequently used in experiments. We found that sex-related differences in the expression of uncoupling proteins 1 and 3 (UCP1 and UCP3) occur in an age-dependent manner. The sex-specific expression of UCP1 and UCP3 in brown adipose tissue (BAT) was inversely correlated with differences in body weight. Expression of UCP4 in the brain, Complex I in the spleen, and Complex II in the brain and BAT was least affected by the sex of the mouse. We further demonstrated that there are serious limitations in using VDAC1 and actin as markers in western blot analyses, due to their sex- and age-specific fluctuations. Our results confirm that sex and age are important parameters and should be taken into account by researchers who examine the mechanistic aspects of diseases. HIGHLIGHTS: I.The levels of UCP1 and UCP3 protein expression differ between females and males in an age-dependent manner.II.Pre-pubertal expression of almost all proteins tested in this study does not depend on the sex of the mouse.III.Expression of VDAC1 and actin, which are often used as loading control proteins in western blot analysis, is tissue-specifically influenced by sex and age.

Molecular Mechanisms Responsible for Pharmacological Effects of Genipin on Mitochondrial Proteins.

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 µM, 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.

Important Trends in UCP3 Investigation.

Membrane uncoupling protein 3 (UCP3), a member of the mitochondrial uncoupling protein family, was discovered in 1997. UCP3's properties, such as its high homology to other mitochondrial carriers, especially to UCP2, its short lifetime and low specificity of UCP3 antibodies, have hindered progress in understanding its biological function and transport mechanism over decades. The abundance of UCP3 is highest in murine brown adipose tissue (BAT, 15.0 pmol/mg protein), compared to heart (2.7 pmol/mg protein) and the gastrocnemius muscle (1.7 pmol/mg protein), but it is still 400-fold lower than the abundance of UCP1, a biomarker for BAT. Investigation of UCP3 reconstituted in planar bilayer membranes revealed that it transports protons only when activated by fatty acids (FA). Although purine nucleotides (PN) inhibit UCP3-mediated transport, the molecular mechanism differs from that of UCP1. It remains a conundrum that two homologous proton-transporting proteins exist within the same tissue. Recently, we proposed that UCP3 abundance directly correlates with the degree of FA ß-oxidation in cell metabolism. Further development in this field implies that UCP3 may have dual function in transporting substrates, which have yet to be identified, alongside protons. Evaluation of the literature with respect to UCP3 is a complex task because (i) UCP3 features are often extrapolated from its "twin" UCP2 without additional proof, and (ii) the specificity of antibodies against UCP3 used in studies is rarely evaluated. In this review, we primarily focus on recent findings obtained for UCP3 in biological and biomimetic systems.

Glutamine regulates mitochondrial uncoupling protein 2 to promote glutaminolysis in neuroblastoma cells.

Mitochondrial uncoupling protein 2 (UCP2) is highly abundant in rapidly proliferating cells that utilize aerobic glycolysis, such as stem cells, cancer cells, and cells of the immune system. However, the function of UCP2 has been a longstanding conundrum. Considering the strict regulation and unusually short life time of the protein, we propose that UCP2 acts as a "signaling protein" under nutrient shortage in cancer cells. We reveal that glutamine shortage induces the rapid and reversible downregulation of UCP2, decrease of the metabolic activity and proliferation of neuroblastoma cells, that are regulated by glutamine per se but not by glutamine metabolism. Our findings indicate a very rapid (within 1 h) metabolic adaptation that allows the cell to survive by either shifting its metabolism to the use of the alternative fuel glutamine or going into a reversible, more quiescent state. The results imply that UCP2 facilitates glutamine utilization as an energetic fuel source, thereby providing metabolic flexibility during glucose shortage. The targeting UCP2 by drugs to intervene with cancer cell metabolism may represent a new strategy for treatment of cancers resistant to other therapies.

The Expression of Uncoupling Protein 3 Coincides With the Fatty Acid Oxidation Type of Metabolism in Adult Murine Heart.

The involvement of mitochondrial uncoupling proteins 2 and 3 in the pathogenesis of cardiovascular diseases is widely acknowledged. However, contradictory reports show that the functions of UCP2/UCP3 are still disputed. We have previously described that UCP2 is highly abundant in cells that rely on glycolysis, such as stem, cancer and activated immune cells. In contrast, high amounts of UCP3 are present in brown adipose tissue, followed by heart and skeletal muscles - all known to metabolize fatty acids (FA) to a high extent. Using two different models - mouse embryonic stem cell (mESC) differentiation to cardiomyocytes (CM) and murine heart at different developmental stages - we now tested the concept that the expression ratio between UCP2 and UCP3 indicates the metabolism type in CM. Our results revealed the tight correlation between UCP3 abundance, expression of mitochondrial fatty acid oxidation (FAO) markers and presence of multiple connections between mitochondria and lipid droplets. We further demonstrated that the time course of UCP3 expression neither coincided with the onset of the electrical activity in CM, derived from mESC, nor with the expression of respiratory chain proteins, the observation which rendered protein participation in ROS regulation unlikely. The present data imply that UCP3 may facilitate FAO by transporting FAs into mitochondria. In contrast, UCP2 was highly abundant at early stages of heart development and in mESC. Understanding, that the expression patterns of UCP3 and UCP2 in heart during development reflect the type of the cell metabolism is key to the uncovering their different functions. Their expression ratio may be an important diagnostic criterion for the degree of CM differentiation and/or severity of a heart failure.

Inhibition of mitochondrial UCP1 and UCP3 by purine nucleotides and phosphate.

Mitochondrial membrane uncoupling protein 3 (UCP3) is not only expressed in skeletal muscle and heart, but also in brown adipose tissue (BAT) alongside UCP1, which facilitates a proton leak to support non-shivering thermogenesis. In contrast to UCP1, the transport function and molecular mechanism of UCP3 regulation are poorly investigated, although it is generally agreed upon that UCP3, analogous to UCP1, transports protons, is activated by free fatty acids (FFAs) and is inhibited by purine nucleotides (PNs). Because the presence of two similar uncoupling proteins in BAT is surprising, we hypothesized that UCP1 and UCP3 are differently regulated, which may lead to differences in their functions. By combining atomic force microscopy and electrophysiological measurements of recombinant proteins reconstituted in planar bilayer membranes, we compared the level of protein activity with the bond lifetimes between UCPs and PNs. Our data revealed that, in contrast to UCP1, UCP3 can be fully inhibited by all PNs and IC50 increases with a decrease in PN-phosphorylation. Experiments with mutant proteins demonstrated that the conserved arginines in the PN-binding pocket are involved in the inhibition of UCP1 and UCP3 to different extents. Fatty acids compete with all PNs bound to UCP1, but only with ATP bound to UCP3. We identified phosphate as a novel inhibitor of UCP3 and UCP1, which acts independently of PNs. The differences in molecular mechanisms of the inhibition between the highly homologous transporters UCP1 and UCP3 indicate that UCP3 has adapted to fulfill a different role and possibly another transport function in BAT.

UCP2 up-regulation within the course of autoimmune encephalomyelitis correlates with T-lymphocyte activation.

Multiple sclerosis (MS) is an inflammatory demyelinating autoimmune disorder of the central nervous system (CNS) associated with severe neurological disability. Reactive oxygen species (ROS) and mitochondrial dysfunction play a pivotal role in the pathogenesis of this disease. Several members of the mitochondrial uncoupling protein subfamily (UCP2-UCP5) were suggested to regulate ROS by diminishing the mitochondrial membrane potential and constitute therefore a promising pharmacological target for MS. To evaluate the role of different uncoupling proteins in neuroinflammation, we have investigated their expression patterns in murine brain and spinal cord (SC) during different stages of experimental autoimmune encephalomyelitis (EAE), an animal model for MS. At mRNA and protein levels we found that only UCP2 is up-regulated in the SC, but not in brain. The increase in UCP2 expression was antigen-independent, reached its maximum between 14 and 21days in both OVA and MOG immunized animals and correlated with an augmented number of CD3+ T-lymphocytes in SC parenchyma. The decrease in abundance of UCP4 was due to neuronal injury and was only detected in CNS of MOG-induced EAE animals. The results provide evidence that the involvement of mitochondrial UCP2 in CNS inflammation during EAE may be mainly explained by the invasion of activated T-lymphocytes. This conclusion coincides with our previous observation that UCP2 is up-regulated in activated and rapidly proliferating T-cells and participates in fast metabolic re-programming of cells during proliferation.

Combined Recognition Imaging and Force Spectroscopy: A New Mode for Mapping and Studying Interaction Sites at Low Lateral Density.

We combined recognition imaging and force spectroscopy to study the interactions between receptors and ligands on the single molecule level. This method allowed the selection of a single receptor molecule reconstituted in a supported lipid membrane at low density, with the subsequent quantification of the receptor-ligand unbinding force. Based on atomic force microscopy (AFM) tapping mode, a cantilever tip carrying a ligand molecule was oscillated across a membrane. Topography and recognition images of reconstituted receptors were recorded simultaneously by analyzing the downward and upward parts of the oscillation, respectively. Functional receptor molecules were selected from the recognition image with nanometer resolution before the AFM was switched to the force spectroscopy mode, using positional feedback control. The combined mode allowed for dynamic force probing on different pre-selected molecules, resulting in higher throughput when compared with force mapping. We applied this method for a quantitative characterization of the binding mechanism between mitochondrial uncoupling protein 1 (UCP1) and its inhibitor adenosine triphosphate (ATP). Moreover the dynamics of force loading was varied to elucidate the binding dynamics and map the interaction energy landscape.

The expression of UCP3 directly correlates to UCP1 abundance in brown adipose tissue.

UCP1 and UCP3 are members of the uncoupling protein (UCP) subfamily and are localized in the inner mitochondrial membrane. Whereas UCP1's central role in non-shivering thermogenesis is acknowledged, the function and even tissue expression pattern of UCP3 are still under dispute. Because UCP3 properties regarding transport of protons are qualitatively identical to those of UCP1, its expression in brown adipose tissue (BAT) alongside UCP1 requires justification. In this work, we tested whether any correlation exists between the expression of UCP1 and UCP3 in BAT by quantification of protein amounts in mouse tissues at physiological conditions, in cold-acclimated and UCP1 knockout mice. Quantification using recombinant UCP3 revealed that the UCP3 amount in BAT (0.51ng/(µg total tissue protein)) was nearly one order of magnitude higher than that in muscles and heart. Cold-acclimated mice showed an approximate three-fold increase in UCP3 abundance in BAT in comparison to mice in thermoneutral conditions. Surprisingly, we found a significant decrease of UCP3 in BAT of UCP1 knockout mice, whereas the protein amount in skeletal and heart muscles remained constant. UCP3 abundance decreased even more in cold-acclimated UCP1 knockout mice. Protein quantification in UCP3 knockout mice revealed no compensatory increase in UCP1 or UCP2 expression. Our results do not support the participation of UCP3 in thermogenesis in the absence of UCP1 in BAT, but clearly demonstrate the correlation in abundance between both proteins. The latter is important for understanding UCP3's function in BAT.

Uncoupling protein 2 and 4 expression pattern during stem cell differentiation provides new insight into their putative function.

Apart from the first family member, uncoupling protein 1 (UCP1), the functions of other UCPs (UCP2-UCP5) are still unknown. In analyzing our own results and those previously published by others, we have assumed that UCP's cellular expression pattern coincides with a specific cell metabolism and changes if the latter is altered. To verify this hypothesis, we analyzed the expression of UCP1-5 in mouse embryonic stem cells before and after their differentiation to neurons. We have shown that only UCP2 is present in undifferentiated stem cells and it disappears simultaneously with the initiation of neuronal differentiation. In contrast, UCP4 is simultaneously up-regulated together with typical neuronal marker proteins TUJ-1 and NeuN during mESC differentiation in vitro as well as during murine brain development in vivo. Notably, several tested cell lines express UCP2, but not UCP4. In line with this finding, neuroblastoma cells that display metabolic features of tumor cells express UCP2, but not UCP4. UCP2's occurrence in cancer, immunological and stem cells indicates that UCP2 is present in cells with highly proliferative potential, which have a glycolytic type of metabolism as a common feature, whereas UCP4 is strongly associated with non-proliferative highly differentiated neuronal cells.

Fatty acids are key in 4-hydroxy-2-nonenal-mediated activation of uncoupling proteins 1 and 2.

The production of reactive oxygen species (ROS) in mitochondria is very sensitive to the proton motive force and may be decreased by mild uncoupling, mediated e.g. by mitochondrial uncoupling proteins (UCPs). UCPs were conversely hypothesized to be activated by ROS. Conclusions from experiments studying the reactive product of lipid peroxidation 4-hydroxy-2-nonenal (HNE) in isolated mitochondria and UCP knock-out mice are highly controversial. Here we investigated the molecular mechanism of HNE action by evaluating the separate contributions of lipid and protein phases of the membrane and by comparing UCP1 and UCP2, which were reconstituted in planar lipid bilayers. We demonstrated that aldehyde does not directly activate either UCP1 or UCP2. However, HNE strongly potentiated the membrane conductance increase (Gm) mediated by different long-chain fatty acids in UCP-containing and in UCP-free membranes and this suggest the involvement of both lipid-mediated and protein-mediated mechanisms with FA playing the central role. Gm increase was concentration-dependent and exhibited a typical saturation kinetic with the binding constant 0.3 mM. By using Electron Paramagnetic Resonance, membrane fluidity change could be excluded as a cause for the HNE-mediated increase in the presence of FA. The impact of the HNE binding to definite positively charged UCP amino acid residues is discussed as a possible protein-mediated mechanism of the UCP activation.

Mapping the nucleotide binding site of uncoupling protein 1 using atomic force microscopy.

A tight regulation of proton transport in the inner mitochondrial membrane is crucial for physiological processes such as ATP synthesis, heat production, or regulation of the reactive oxygen species as proposed for the uncoupling protein family members (UCP). Specific regulation of proton transport is thus becoming increasingly important in the therapy of obesity and inflammatory, neurodegenerative, and ischemic diseases. We and other research groups have shown previously that UCP1- and UCP2-mediated proton transport is inhibited by purine nucleotides. Several hypotheses have been proposed to explain the inhibitory effect of ATP, although structural details are still lacking. Moreover, the unresolved mystery is how UCP operates in vivo despite the permanent presence of high (millimolar) concentrations of ATP in mitochondria. Here we use the topographic and recognition (TREC) mode of an atomic force microscope to visualize UCP1 reconstituted into lipid bilayers and to analyze the ATP-protein interaction at a single molecule level. The comparison of recognition patterns obtained with anti-UCP1 antibody and ATP led to the conclusion that the ATP binding site can be accessed from both sides of the membrane. Using cantilever tips with different cross-linker lengths, we determined the location of the nucleotide binding site inside the membrane with 1 Å precision. Together with the recently published NMR structure of a UCP family member (Berardi et al. Nature, 2011, 476, 109-113), our data provide a valuable insight into the mechanism of the nucleotide binding and pave the way for new pharmacological approaches against the diseases mentioned above.

Quantification of uncoupling protein 2 reveals its main expression in immune cells and selective up-regulation during T-cell proliferation.

Uncoupling protein 2 (UCP2) is an inner mitochondrial membrane protein. Although the protein was discovered in 1997, its function and even its tissue distribution are still under debate. Here we present a quantitative analysis of mRNA and protein expression in various mice tissues, revealing that UCP2 is mainly expressed in organs and cells associated with the immune system. Although the UCP2 gene is present in the brain, as demonstrated using quantitative RT-PCR, the protein was not detectable in neurons under physiological conditions. Instead, we could detect UCP2 in microglia, which act in the immune defense of the central nervous system. In lymphocytes, activation led to a ten-fold increase of UCP2 protein expression simultaneously to the increase in levels of other mitochondrial proteins, whereas lymphocyte re-stimulation resulted in the selective increase of UCP2. The highest detected level of UCP2 expression in stimulated T-cells (0.54 ng/(µg total cellular protein)) was approximately 200 times lower than the level of UCP1 in brown adipose tissue from room temperature acclimated mice. Both the UCP2 expression pattern and the time course of up-regulation in stimulated T-cells imply UCP2's involvement in the immune response, probably by controlling the metabolism during cell proliferation.

Long-term Fgf23 deficiency does not influence aging, glucose homeostasis, or fat metabolism in mice with a nonfunctioning vitamin D receptor.

It is still controversial whether the bone-derived hormone fibroblast growth factor-23 (FGF23) has additional physiological functions apart from its well-known suppressive actions on renal phosphate reabsorption and vitamin D hormone synthesis. Here we analyzed premature aging, mineral homeostasis, carbohydrate metabolism, and fat metabolism in 9-month-old male wild-type (WT) mice, vitamin D receptor mutant mice (VDR(Δ/Δ)) with a nonfunctioning vitamin D receptor, and Fgf23⁻/⁻/VDR(Δ/Δ) compound mutant mice on both a standard rodent chow and a rescue diet enriched with calcium, phosphorus, and lactose. Organ atrophy, lung emphysema, and ectopic tissue or vascular calcifications were absent in compound mutants. In addition, body weight, glucose tolerance, insulin tolerance, insulin secretory capacity, pancreatic beta cell volume, and retroperitoneal and epididymal fat mass as well as serum cholesterol and triglycerides were indistinguishable between vitamin D receptor and compound mutants. In contrast to VDR(Δ/Δ) and Fgf23⁻/⁻/VDR(Δ/Δ) mice, which stayed lean, WT mice showed obesity-induced insulin resistance. To rule out alopecia and concomitantly elevated energy expenditure present in 9-month-old VDR(Δ/Δ) and Fgf23⁻/⁻/VDR(Δ/Δ) mice as a confounding factor for the lacking effect of Fgf23 deficiency on fat mass, we analyzed whole-body composition in WT, Fgf23⁻/⁻, VDR(Δ/Δ), and Fgf23⁻/⁻/VDR(Δ/Δ) mice at the age of 4 wk, when the coat in VDR(Δ/Δ) mice is still normal. Whole-body fat mass was reduced in Fgf23⁻/⁻ mice but almost identical in WT, VDR(Δ/Δ), and Fgf23⁻/⁻/VDR(Δ/Δ) mice. In conclusion, our data indicate that Fgf23 has no molecular vitamin D-independent role in aging, insulin signaling, or fat metabolism in mice.

Linking of sensor molecules with amino groups to amino-functionalized AFM tips.

The measuring tip of an atomic force microscope (AFM) can be upgraded to a specific biosensor by attaching one or a few biomolecules to the apex of the tip. The biofunctionalized tip is then used to map cognate target molecules on a sample surface or to study biophysical parameters of interaction with the target molecules. The functionality of tip-bound sensor molecules is greatly enhanced if they are linked via a thin, flexible polymer chain. In a typical scheme of tip functionalization, reactive groups are first generated on the tip surface, a bifunctional cross-linker is then attached with one of its two reactive ends, and finally the probe molecule of interest is coupled to the free end of the cross-linker. Unfortunately, the most popular functional group generated on the tip surface is the amino group, while at the same time, the only useful coupling functions of many biomolecules (such as antibodies) are also NH(2) groups. In the past, various tricks or detours were applied to minimize the undesired bivalent reaction of bifunctional linkers with adjacent NH(2) groups on the tip surface. In the present study, an uncompromising solution to this problem was found with the help of a new cross-linker ("acetal-PEG-NHS") which possesses one activated carboxyl group and one acetal-protected benzaldehyde function. The activated carboxyl ensures rapid unilateral attachment to the amino-functionalized tip, and only then is the terminal acetal group converted into the amino-reactive benzaldehyde function by mild treatment (1% citric acid, 1-10 min) which does not harm the AFM tip. As an exception, AFM tips with magnetic coating become demagnetized in 1% citric acid. This problem was solved by deprotecting the acetal group before coupling the PEG linker to the AFM tip. Bivalent binding of the corresponding linker ("aldehyde-PEG-NHS") to adjacent NH(2) groups on the tip was largely suppressed by high linker concentrations. In this way, magnetic AFM tips could be functionalized with an ethylene diamine derivative of ATP which showed specific interaction with mitochondrial uncoupling protein 1 (UCP1) that had been purified and reconstituted in a mica-supported planar lipid bilayer.