Evolution of the primate glutamate taste sensor from a nucleotide sensor

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Growth promotion of a deep-sea bacterium by sensing infrared light through a bacteriophytochrome photoreceptor

Photoreceptors are found in all kingdoms of life and bacteriophytochromes (Bphps) are the most abundant photo-sensing receptors in bacteria. Interestingly, BphPs have been linked to some bacterial physiological responses, yet most of the biological processes they regulate are still elusive, especially in non-photosynthetic bacteria. Here, we show that a bacteriophytochrome (CmoBphp) from a deep-sea bacterium Croceicoccus marinus OT19 perceives infrared light (wavelength at 940 nm) and transduces photo-sensing signals to a downstream intracellular transduction cascade for better growth. We discover that the infrared light-mediated growth promotion of C. marinus OT19 is attributed partly to the enhancement of pyruvate and propanoate metabolism. Further study suggests that CmoBphp plays a crucial role in integrating infrared light with intracellular signalling to control the bacterial growth and metabolism. This is the first report that deep-sea non-photosynthetic bacteria can sense infrared light to control growth through a bacteriophytochrome photoreceptor, thus providing new understandings towards light energy utilization by microorganisms.     

A new type of bacteriophytochrome acts in tandem with a classical bacteriophytochrome to control the antennae synthesis in Rhodopseudomonas palustris

Phytochromes are chromoproteins found in plants and bacteria that switch between two photointerconvertible forms via the photoisomerization of their chromophore. These two forms, Pr and Pfr, absorb red and far-red light, respectively. We have characterized the biophysical and biochemical properties of two bacteriophytochromes, RpBphP2 and RpBphP3, from the photosynthetic bacterium Rhodopseudomonas palustris. Their genes are contiguous and localized near the pucBAd genes encoding the polypeptides of the light harvesting complexes LH4, whose synthesis depends on the light intensity. At variance with all (bacterio)phytochromes studied so far, the light-induced isomerization of the chromophore of RpBphP3 converts the Pr form to a form absorbing at shorter wavelength around 645 nm, designated as Pnr for near red. The quantum yield for the transformation of Pr into Pnr is about 6-fold smaller than for the reverse reaction. Both RpBphP2 and RpBphP3 autophosphorylate in their dark-adapted Pr forms and transfer their phosphate to a common response regulator Rpa3017. Under semiaerobic conditions, LH4 complexes replace specifically the LH2 complexes in wild-type cells illuminated by wavelengths comprised between 680 and 730 nm. In contrast, mutants deleted in each of these two bacteriophytochromes display no variation in the composition of their light harvesting complexes whatever the light intensity. From both the peculiar properties of these bacteriophytochromes and the phenotypes of their deletion mutants, we propose that they operate in tandem to control the synthesis of LH4 complexes by measuring the relative intensities of 645 and 710 nm lights.     

N-ethylmaleimide-sensitive factor: a redox sensor in exocytosis

Vascular injury triggers endothelial exocytosis of granules, releasing pro-inflammatory and pro-thrombotic mediators into the blood. Nitric oxide (NO) and reactive oxygen species (ROS) limit vascular inflammation and thrombosis by inhibiting endothelial exocytosis. NO decreases exocytosis by regulating the activity of the N-ethylmaleimide-sensitive factor (NSF), a central component of the exocytic machinery. NO nitrosylates specific cysteine residues of NSF, thereby inhibiting NSF disassembly of the soluble NSF attachment protein receptor (SNARE). NO also modulates exocytosis of other cells; for example, NO regulates platelet activation by inhibiting alpha-granule secretion from platelets. Other radicals besides NO can regulate exocytosis as well. For example, H(2)O(2) inhibits exocytosis by oxidizing NSF. Using site-directed mutagenesis, we have defined the critical cysteine residues of NSF, and found that one particular cysteine residue, C264, renders NSF sensitive to oxidative stress. Since radicals such as NO and H(2)O(2) inhibit NSF and decrease exocytosis, NSF may act as a redox sensor, modulating exocytosis in response to changes in oxidative stress.     

A second photochromic bacteriophytochrome from Synechocystis sp. PCC 6803: spectral analysis and down-regulation by light

It now appears that photosynthetic prokaryotes and lower eukaryotes possess higher plant phytochrome-like proteins. In this work, a second phytochrome-like gene was isolated, in addition to the recently identified Cph1 phytochrome, from the Synechocystis sp. PCC 6803, and its gene product was characterized photochemically. The open reading frame sll0821 (designated cph2 in this work) has structural characteristics similar to those of the plant phytochromes and the Synechocystis Cph1 with high amino acid sequence homology in the N-terminal chromophore binding domain. The predicted Cph2 protein consists of 1276 amino acids with a calculated molecular mass of 145 kDa. Interestingly, the Cph2 protein has two putative chromophore binding domains, one around Cys-129 and the other around Cys-1022. The Cph2 was overexpressed in E. coli as an Intein/CBD (chitin binding domain) fusion and in vitro reconstituted with phycocyanobilin (PCB) or phytochromobilin (PPhiB). Both the Cph2-PCB and Cph2-PPhiB adducts showed the typical photochromic reversibility with the difference spectral maxima at 643/690 and 655/701 nm, respectively. The Cys-129 was confirmed to be the chromophore binding residue by in vitro mutagenesis and Zn(2+) fluorescence. The microenvironment of the chromophore in Cph2 seems to be similar to that in plant phytochromes. The cph2 gene expression was dark-induced and down-regulated to a basal level by light, like the cph1 gene. These observations suggest that Synechocystis species have multiple photosensory proteins, probably with distinct roles, as in higher plants.     

Skeletal muscle ryanodine receptor is a redox sensor with a well defined redox potential that is sensitive to channel modulators

Hyperreactive sulfhydryl groups associated with the Ca(2+) release protein from sarcoplasmic reticulum are shown to have a well defined reduction potential that is sensitive to the cellular environment. Ca(2+) channel activators lower the redox potential of the ryanodine receptor, which favors the oxidation of thiols and the opening of the Ca(2+) release protein. In contrast, channel inhibitors increase the redox potential, which favors the reduction of disulfides and the closure of the release protein. Modulation of redox potential of reactive thiols may be a general control mechanism by which sarcoplasmic/endoplasmic reticulum, ryanodine receptors/IP(3) receptors, control cytoplasmic Ca(2+) concentrations.     

Transmembrane redox sensor of ryanodine receptor complex

Inositol 1,4,5-trisphosphate receptors (IP(3)R) and ryanodine receptors (RyR) mediate the release of endoplasmic and sarcoplasmic reticulum (ER/SR) Ca(2+) stores and regulate Ca(2+) entry through voltage-dependent or ligand-gated channels of the plasma membrane. A prominent property of ER/SR Ca(2+) channels is exquisite sensitivity to sulfhydryl-modifying reagents. A plausible role for sulfhydryl chemistry in physiologic regulation of Ca(2+) release channels and the fidelity of Ca(2+) release from ER/SR is lacking. This study reveals the existence of a transmembrane redox sensor within the RyR1 channel complex that confers tight regulation of channel activity in response to changes in transmembrane redox potential produced by cytoplasmic and luminal glutathione. A transporter selective for glutathione is co-localized with RyR1 within the SR membrane to maintain local redox potential gradients consistent with redox regulation of ER/SR Ca(2+) release. Hyperreactive sulfhydryls previously shown to reside within the RyR1 complex (Liu, G., and Pessah, I. N. (1994) J. Biol. Chem. 269, 33028-33034) are an essential biochemical component of a transmembrane redox sensor. Transmembrane redox sensing may represent a fundamental mechanism by which ER/SR Ca(2+) channels respond to localized changes in transmembrane glutathione redox potential produced by physiologic and pathophysiologic modulators of Ca(2+) release from stores.     

Monitoring cellular redox dynamics using newly developed BRET-based redox sensor proteins

Reactive oxygen species are key factors that strongly affect the cellular redox state and regulate various physiological and cellular phenomena. To monitor changes in the redox state, we previously developed fluorescent redox sensors named Re-Q, the emissions of which are quenched under reduced conditions. However, such fluorescent probes are unsuitable for use in the cells of photosynthetic organisms because they require photoexcitation that may change intracellular conditions and induce autofluorescence, primarily in chlorophylls. In addition, the presence of various chromophore pigments may interfere with fluorescence-based measurements because of their strong absorbance. To overcome these problems, we adopted the bioluminescence resonance energy transfer (BRET) mechanism for the sensor and developed two BRET-based redox sensors by fusing cyan fluorescent protein–based or yellow fluorescent protein–based Re-Q with the luminescent protein Nluc. We named the resulting redox-sensitive BRET-based indicator probes “ROBINc” and “ROBINy.” ROBINc is pH insensitive, which is especially vital for observation in photosynthetic organisms. By using these sensors, we successfully observed dynamic redox changes caused by an anticancer agent in HeLa cells and light/dark-dependent redox changes in the cells of photosynthetic cyanobacterium Synechocystis sp. PCC 6803. Since the newly developed sensors do not require excitation light, they should be especially useful for visualizing intracellular phenomena caused by redox changes in cells containing colored pigments.     

Development of a nucleoside analog UV light sensor

Conjugation of the photosensitive nucleoside (E)-5-(2-methoxycarbonylethenyl)cytidine to biotin provided a means to attach this analogue to microparticles for dosimetry applications that require UV sensor mobility.     

A fluorescent redox sensor with tuneable oxidation potential

A fluorescent redox sensor was prepared by attachment of hydroquinones to the fluorophore rhodamine B; fluorescence is reversibly modulated by hydroquinone-centered chemical redox reactions, and oxidation potential of the sensor is tuneable by variation of hydroquinone structure.     

Aging: a shift from redox regulation to oxidative damage

Proteins, nucleic acids, and lipids can undergo various forms of oxidative modification. In numerous instances, these modifications result in irreversible loss of function. The age-dependent accumulation of oxidatively modified and dysfunctional macromolecules provides the basis for the free radical theory of aging. Pro-oxidants, however, are also capable of catalyzing fully reversible modifications to protein. It is increasingly apparent that these reactions participate in redox-dependent regulation of cell metabolism and response to stress. The adventitious use of free radical species adds complexity to the experimental and theoretical manner in which the free radical theory is to be tested and considered. Elucidation of mechanisms by which reversible oxidative processes are controlled, the components involved, and the metabolic consequences and how they are altered with age will provide new insight on the aging process and attempts to delay the inevitable.     

Structure of a bacteriophytochrome and light-stimulated protomer swapping with a gene repressor

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Highlights? Bacteriophytochrome structure containing photosensory and output transducing domain ? RpBphP1 binds to the cognate repressor RpPpsR2 on illumination with far-red light ? HOS domain is distantly related to Dhp and is essential for binding to the repressor ? Light-induced protomer swapping mechanism between dimers     

Multicolor redox sensor proteins can visualize redox changes in various compartments of the living cell

Change in the intracellular redox state is a consequence of various metabolic reactions, which simultaneously regulates various physiological phenomena in cells. Monitoring the redox state in living cells is thus very important for understanding cellular physiology. Various genetically encoded fluorescent redox sensors have therefore been developed. Recently, we developed oxidation-sensitive fluorescent proteins named Oba-Q (Sugiura, K., et al. (2015) Biochem. Biophys. Res. Commun. 457, 242–248), which exhibit dramatic quenching under oxidizing conditions. To extend the range of uses of redox sensor proteins, we refined these proteins based on the molecular architecture applied to Oba-Q, and successfully produced several redox sensor proteins based on CFP and YFP. Interestingly, some of these sensor proteins showed the reverse changes in emission compared with Oba-Q, implying remarkable fluorescence quenching under reducing conditions. We named this type of sensor protein Re-Q, reduction-sensed quenching protein. The cause of the redox-dependent fluorescence quenching could be clearly explained based on the crystal structure of Re-Q in the reduced and oxidized forms. In addition, by introducing suitable mutations into the sensors, we produced Oba-Q and Re-Q mutants exhibiting various midpoint redox potentials. This series of proteins can cover a wide range of redox potentials in the cell, so they should be applicable to various cells and even intracellular organelles. As an example, we successfully measured the redox responses in different cell compartments of cultured mammalian cells simultaneously against the anticancer reagents Kp372-1.     

Calculating the areas of polygons with a smartphone light sensor

This study explores finding the areas of polygons with a smartphone light sensor. A square and an irregular pentagon were chosen as our polygons. During the activity, the LED light was placed at the vertices of our polygons, and the illuminance values of this LED light were detected by the smartphone light sensor. The smartphone was placed on a rotating time-lapse device, and this device was placed at the intersection point of the line segments in the polygons. These line segments were also connected to the vertices of these polygons; therefore, the square was composed of four triangles and the pentagon of five triangles. While the rotating time-lapse device was rotating around itself, it captured the maximum illuminance values of the LED light. Using these maximum illuminance values, we calculated the lengths of the line segments. In addition, we knew the period of the rotating time-lapse device and the duration elapsed between two consecutive maximum illuminance values. Thus, we could calculate the angle between two consecutive line segments. With these values, we could also calculate the areas of the triangles in the polygons. Then, the areas of these triangles were summed to obtain the areas of the square and pentagon.     

Sir2 regulates skeletal muscle differentiation as a potential sensor of the redox state

Sir2 is a NAD(+)-dependent histone deacetylase that controls gene silencing, cell cycle, DNA damage repair, and life span. Prompted by the observation that the [NAD(+)]/[NADH] ratio is subjected to dynamic fluctuations in skeletal muscle, we have tested whether Sir2 regulates muscle gene expression and differentiation. Sir2 forms a complex with the acetyltransferase PCAF and MyoD and, when overexpressed, retards muscle differentiation. Conversely, cells with decreased Sir2 differentiate prematurely. To inhibit myogenesis, Sir2 requires its NAD(+)-dependent deacetylase activity. The [NAD(+)]/[NADH] ratio decreases as muscle cells differentiate, while an increased [NAD(+)]/[NADH] ratio inhibits muscle gene expression. Cells with reduced Sir2 levels are less sensitive to the inhibition imposed by an elevated [NAD(+)]/[NADH] ratio. These results indicate that Sir2 regulates muscle gene expression and differentiation by possibly functioning as a redox sensor. In response to exercise, food intake, and starvation, Sir2 may sense modifications of the redox state and promptly modulate gene expression.     

Protein sulfenation as a redox sensor: proteomics studies using a novel biotinylated dimedone analogue

Protein sulfenic acids are reactive intermediates in the catalytic cycles of many enzymes as well as the in formation of other redox states. Sulfenic acid formation is a reversible post-translational modification with potential for protein regulation. Dimedone (5,5-dimethyl-1,3-cyclohexanedione) is commonly used in vitro to study sulfenation of purified proteins, selectively "tagging" them, allowing monitoring by mass spectrometry. However dimedone is of little use in complex protein mixtures because selective monitoring of labeling is not possible. To address this issue, we synthesized a novel biotinylated derivative of dimedone, keeping the dione cassette required for sulfenate reactivity but adding the functionality of a biotin tag. Biotin-amido(5-methyl-5-carboxamidocyclohexane 1,3-dione) tetragol (biotin dimedone) was prepared in six steps, combining 3,5-dimethoxybenzoic acid (Birch reduction, ultimately leading to the dimedone unit with a carboxylate functionality), 1-amino-11-azido-3,6,9-trioxaundecane (a differentially substituted tetragol spacer), and biotin. We loaded biotin dimedone (0.1 mm, 30 min) into rat ventricular myocytes, treated them with H(2)O(2) (0.1-10,000 microm, 5 min), and monitored derivatization on Western blots using streptavidin-horseradish peroxidase. There was a dose-dependent increase in labeling of multiple proteins that was maximal at 0.1 or 1 mm H(2)O(2) and declined sharply below basal with 10 mm treatment. Cell-wide labeling was observed in fixed cells probed with avidin-FITC using a confocal fluorescence microscope. Similar H(2)O(2)-induced labeling was observed in isolated rat hearts. Hearts loaded and subjected to hypoxia showed a striking loss of labeling, which returned when oxygen was resupplied, highlighting the protein sulfenates as oxygen sensors. Cardiac proteins that were sulfenated during oxidative stress were purified with avidin-agarose and identified by separation of tryptic digests by liquid chromatography with on-line analysis by mass spectrometry.