Improved production of ß-carotene in light-powered Escherichia coli by co-expression of Gloeobacter rhodopsin expression.

BACKGROUND: Providing sufficient and usable energy for the cell factory has long been a heated issue in biosynthesis as solar energy has never been rooted out from the strategy for improvement, and turning Escherichia coli (E. coli) into a phototrophic host has multiple captivating qualities for biosynthesis. In this study, ß-carotene was a stable compound for production in E. coli with the expression of four enzymes (CrtE, CrtB, CrtI, CrtY) for production due to its light-harvesting feature as an antenna pigment and as an antioxidant and important precursor for human health. The expression of Gloeobacter rhodopsin (GR) in microbial organisms was proved to have potential for application. RESULTS: The expression of fusion protein, GR-GFP, in E. coli showed visible GFP signal under fluorescent microscopy, and its in vivo proton pumping activity signal can be detected in induced photocurrent by electrodes on the chip under intervals of illumination. To assess the phototrophic synthesis ability of the host strain compared to wild-type and vector control strain in chemostat batch with illumination, the expression of red fluorescent protein (RFP) as a target protein showed its yield improvement in protein assay and also reflected its early dominance in RFP fluorescence signal during the incubation, whereas the synthesis of ß-carotene also showed yield increase by 1.36-fold and 2.32-fold compared with its wildtype and vector control strain. To investigate the effect of GR-GFP on E. coli, the growth of the host showed early rise into the exponential phase compared to the vector control strain and glucose turnover rate was elevated in increased glucose intake rate and upregulation of ATP-related genes in glycolysis (PtsG, Pgk, Pyk). CONCLUSION: We reported the first-time potential application of GR in the form of fusion protein GR-GFP. Expression of GR-GFP in E. coli improved the production of ß-carotene and RFP. Our work provides a strain of E. coli harboring phototrophic metabolism, thus paving path to a more sustainable and scalable production of biosynthesis.

Insm1 promotes differentiation of retinal progenitor cells toward photoreceptor cells in the developing retina through up-regulation of SHH.

This article investigated the effect of Insm1 on RPC differentiation in mice and the underlying mechanism. The retinal tissues of mouse embryo at 12.5 days (E12.5) and postnatal 14 days (P14) were collected, following by the detection of Insm1 and corresponding markers by immunofluorescent staining. RPCs isolated from retinal tissues at P1 were cultured in culture medium for 7 days. The differentiation of photoreceptor and glial cells was assessed after RPCs transferred to the differentiation medium for 20 days. Next, the effect of Insm1 overexpression on the differentiation of RPCs toward rod photoreceptor and glial cells were assessed. Insm1 was highly expressed in RPCs of retinal tissues and decline in photoreceptor cells, while hardly expressed in glial cells. Based on the results of Pax-6 positive immunofluorescent staining and flow cytometry detection, RPCs were successfully isolated from retinal tissues. After the culture in differentiation medium, RPCs showed positive staining of Rhodopsin and glial fibrillary acidic protein (GFAP). Further results showed that overexpression of Insm1 significantly increased the percentage of Rhodopsin positive cells, and up-regulated Sonic Hedgehog (SHH), hairy and enhancer of split homolog-1(Hes1), S-opsin and Rhodopsin levels, while decreased the percentage of Glutamine synthetase positive cells, and reduced Glutamine synthetase and GFAP levels. Whereas, the effect of Insm1 overexpression on these protein levels were partly abolished by the knockdown of SHH or Hes1. We conclude that Insm1 promotes the differentiation of RPCs into photoreceptor cells in the developing retina through up-regulation of SHH.

High dose expression of heme oxigenase-1 induces retinal degeneration through ER stress-related DDIT3.

BACKGROUND: Oxidative stress is a common cause of neurodegeneration and plays a central role in retinal degenerative diseases. Heme oxygenase-1 (HMOX1) is a redox-regulated enzyme that is induced in neurodegenerative diseases and acts against oxidative stress but can also promote cell death, a phenomenon that is still unexplained in molecular terms. Here, we test whether HMOX1 has opposing effects during retinal degeneration and investigate the molecular mechanisms behind its pro-apoptotic role. METHODS: Basal and induced levels of HMOX1 in retinas are examined during light-induced retinal degeneration in mice. Light damage-independent HMOX1 induction at two different expression levels is achieved by intraocular injection of different doses of an adeno-associated virus vector expressing HMOX1. Activation of Müller glial cells, retinal morphology and photoreceptor cell death are examined using hematoxylin-eosin staining, TUNEL assays, immunostaining and retinal function are evaluated with electroretinograms. Downstream gene expression of HMOX1 is analyzed by RNA-seq, qPCR examination and western blotting. The role of one of these genes, the pro-apoptotic DNA damage inducible transcript 3 (Ddit3), is analyzed in a line of knockout mice. RESULTS: Light-induced retinal degeneration leads to photoreceptor degeneration and concomitant HMOX1 induction. HMOX1 expression at low levels before light exposure prevents photoreceptor degeneration but expression at high levels directly induces photoreceptor degeneration even without light stress. Photoreceptor degeneration following high level expression of HMOX1 is associated with a mislocalization of rhodopsin in photoreceptors and an increase in the expression of DDIT3. Genetic deletion of Ddit3 in knockout mice prevents photoreceptor cell degeneration normally resulting from high level HMOX1 expression. CONCLUSION: The results reveal that the expression levels determine whether HMOX1 is protective or deleterious in the retina. Furthermore, in contrast to the protective low dose of HMOX1, the deleterious high dose is associated with induction of DDIT3 and endoplasmic reticulum stress as manifested, for instance, in rhodopsin mislocalization. Hence, future applications of HMOX1 or its regulated targets in gene therapy approaches should carefully consider expression levels in order to avoid potentially devastating effects.

Potassium channel-based optogenetic silencing.

Optogenetics enables manipulation of biological processes with light at high spatio-temporal resolution to control the behavior of cells, networks, or even whole animals. In contrast to the performance of excitatory rhodopsins, the effectiveness of inhibitory optogenetic tools is still insufficient. Here we report a two-component optical silencer system comprising photoactivated adenylyl cyclases (PACs) and the small cyclic nucleotide-gated potassium channel SthK. Activation of this 'PAC-K' silencer by brief pulses of low-intensity blue light causes robust and reversible silencing of cardiomyocyte excitation and neuronal firing. In vivo expression of PAC-K in mouse and zebrafish neurons is well tolerated, where blue light inhibits neuronal activity and blocks motor responses. In combination with red-light absorbing channelrhodopsins, the distinct action spectra of PACs allow independent bimodal control of neuronal activity. PAC-K represents a reliable optogenetic silencer with intrinsic amplification for sustained potassium-mediated hyperpolarization, conferring high operational light sensitivity to the cells of interest.

Optogenetic inhibition of behavior with anion channelrhodopsins.

Optogenetics uses light exposure to manipulate physiology in genetically modified organisms. Abundant tools for optogenetic excitation are available, but the limitations of current optogenetic inhibitors present an obstacle to demonstrating the necessity of neuronal circuits. Here we show that anion channelrhodopsins can be used to specifically and rapidly inhibit neural systems involved in Drosophila locomotion, wing expansion, memory retrieval and gustation, thus demonstrating their broad utility in the circuit analysis of behavior.

Optogenetic analysis of synaptic transmission in the central nervous system of the nematode Caenorhabditis elegans.

A reliable method for recording evoked synaptic events in identified neurons in Caenorhabditis elegans would greatly accelerate our understanding of its nervous system at the molecular, cellular and network levels. Here we describe a method for recording synaptic currents and potentials from identified neurons in nearly intact worms. Dissection and exposure of postsynaptic neurons is facilitated by microfabricated agar substrates, and ChannelRhodopsin-2 is used to stimulate presynaptic neurons. We used the method to analyse functional connectivity between a polymodal nociceptor and a command neuron that initiates a stochastic escape behaviour. We find that escape probability mirrors the time course of synaptic current in the command neuron. Moreover, synaptic input increases smoothly as stimulus strength is increased, suggesting that the overall input-output function of the connection is graded. We propose a model in which the energetic cost of escape behaviours in C. elegans is tuned to the intensity of the threat.

AAV delivery of wild-type rhodopsin preserves retinal function in a mouse model of autosomal dominant retinitis pigmentosa.

Autosomal dominant retinitis pigmentosa (ADRP) is frequently caused by mutations in RHO, the gene for rod photoreceptor opsin. Earlier, a study on mice carrying mutated rhodopsin transgenes on either RHO + / + or RHO + /- backgrounds suggested that the amount of wild-type rhodopsin affected survival of photoreceptors. Therefore, we treated P23H RHO transgenic mice with adeno-associated virus serotype 5 (AAV5) expressing a cDNA clone of the rhodopsin gene (RHO301) that expressed normal opsin from the mouse opsin promoter. Analysis of the electroretinogram (ERG) demonstrated that increased expression of RHO301 slowed the rate of retinal degeneration in P23H mice: at 6 months, a-wave amplitudes were increased by 100% and b-wave amplitudes by 79%. In contrast, nontransgenic mice injected with AAV5 RHO301 demonstrated a decrease in the ERG, confirming the damaging effect of rhodopsin overproduction in normal photoreceptors. In P23H mice, the increase in the ERG amplitudes was correlated with improvement of retinal structure: the thickness of the outer nuclear layer in RHO301-treated eyes was increased by 80% compared with control eyes. These findings suggest that the wild-type RHO gene can be delivered to rescue retinal degeneration in mice carrying a RHO mutation and that increased production of normal rhodopsin can suppress the effect of the mutated protein. These findings make it possible to treat ADRP caused by different mutations of RHO with the expression of wild-type RHO.

Early receptor current of wild-type and transducin knockout mice: photosensitivity and light-induced Ca2+ release.

We have used suction-electrode recording to measure the early receptor current (ERC) from single, isolated mammalian photoreceptors. When a wild-type mouse rod was illuminated with light sufficient to close all the cGMP-gated channels, a succeeding bright laser flash bleaching a large proportion of the visual pigment produced an ERC, which at 37 degrees C consisted primarily of a single component of transient positive current. The amplitude of total charge movement of this component declined exponentially with successive flashes, consistent with the direct proportionality of the ERC to the quantity of pigment bleached. From the constant of exponential decline, it was possible to estimate the in vivo photosensitivity of mouse rhodopsin to be about 6 x 10(-9)microm(2) per molecule. We have also measured the ERC from rods of transducin-knockout mice, for which previous illumination to close the cGMP-gated channels was not required. The ERC of these rods was similar to that of wild-type rods but was followed by a slow component of outward current whose maximum amplitude in some cells approached that of the normal light response. This slow current was blocked by l-cis diltiazem, indicating that it was produced by ion flux through the cyclic nucleotide-gated channels of the outer segment; however, it could not have been produced by the normal transduction cascade, since it was recorded from rods lacking transducin. Since it was depressed by prior incorporation of the Ca(2+) buffer BAPTA, it was probably generated by light-activated Ca(2+) release earlier demonstrated in salamander and zebrafish. Recordings of the ERC from normal and mutant mice may provide a useful tool for the analysis of models of retinal disease, as well as exploration of the molecular origin of light-activated Ca(2+) release.

Ocular disposition and tolerance of ganciclovir-loaded albumin nanoparticles after intravitreal injection in rats.

Cytomegalovirus (CMV) infection mainly affects endothelial cells of ocular vessels, optic nerve and the retina, resulting in direct or autoimmune damages, uveoretinitis and disturbed vision. The use of colloidal carriers for the intravitreal delivery of ganciclovir may prolong its residence in the eye, minimizing the opacification observed for macroscopic implants. The aim of this work was to evaluate the ocular toxicity induced by the prolonged presence of ganciclovir-loaded bovine serum albumin nanoparticles after their intravitreal injection. The intraocular disposition of these carriers was also studied by immunochemistry. Two weeks post-injection, a significant amount of nanoparticles remained in the vitreous cavity, mainly in a thin layer overlying the retina and in the area close to the blood aqueoUs barrier. Their prolonged residence in the eve seemed to be well tolerated and the histological evaluation of the retina, mainly the photoreceptor layer, and adjacent tissues revealed the absence of inflammatory reactions or alterations in the tissue architecture (i.e. cellular infiltrations or vascular inflammation). In addition, nanoparticles neither alter the expression and distribution of arrestin and rhodopsin autoantigens nor the mineralocorticoid receptor. In summary, the vision was not affected by autoimmune phenomena or alterations in the behavior of ophthalmic cells due to the intravitreal injection of these nanoparticles.

Chemical modification of transducin with iodoacetic acid: transducin-alpha carboxymethylated at Cys(347) allows transducin binding to Light-activated rhodopsin but prevents its release in the presence of GTP.

Modification of transducin (T) with iodoacetic acid (IAA) inhibited its light-dependent guanine nucleotide-binding activity. Approximately 1 mol of [(3)H]IAA was incorporated per mole of T. Cys(347), located on the alpha-subunit of T (T(alpha)), was identified as the major labeled residue in the [(3)H]IAA-modified holoenzyme. In contrast, Cys(135) and Cys(347) were modified with [(3)H]IAA in the isolated T(alpha). IAA-modified T was able to bind tightly to photoexcited rhodopsin (R*), but GTP did not promote the dissociation of the complex between alkylated T and R*. In addition, R* protected against the inhibition of T by IAA. A comparable inactivation of T and analogous interactions between T and R* were observed when 2-nitro 5-thiocyanobenzoic acid (NTCBA) was used as the modifying reagent (J. O. Ortiz and J. Bubis, 2001, Effects of differential sulfhydryl group-specific labeling on the rhodopsin and guanine nucleotide binding activities of transducin, Arch. Biochem. Biophys. 387, 233-242). However, while carboxymethylated T was capable of liberating GDP in the presence of R*, NTCBA-modified T was unable to release the guanine nucleotide diphosphate upon incubation with the photoactivated receptor. Thus, IAA-labeling stabilized a T:R* complex intermediate carrying the empty nucleotide pocket conformation of T. On the other hand, NTCBA-modified T seemed to be "locked" in the GDP-bound state of T, even in the presence of R*.

Effects of differential sulfhydryl group-specific labeling on the rhodopsin and guanine nucleotide binding activities of transducin.

The role of transducin sulfhydryl groups was examined by chemical modification with four different reagents: 4-acetamido-4'-maleimidyl-stilbene-2, 2' disulfonic acid (AMDA); 4-vinyl pyridine (VP); 2-nitro-5-thiocyano benzoic acid (NTCBA); and 2, 5-dimethoxystilbene-4'-maleimide (DM). All these compounds rapidly inhibited the [3H]GMPpNp-binding activity of transducin stimulated by photoexcited rhodopsin (R*). Sedimentation experiments showed that the labeling of transducin with AMDA or VP hindered its binding to R* while NTCBA-modified transducin was capable of interacting with the photoreceptor protein. In contrast, DM-labeled transducin precipitated even in the absence of R*. Photoactivated rhodopsin was capable of protecting against the observed AMDA and NTCBA inhibition in transducin function, but not against the inactivation caused by VP or DM. These results suggest the existence of different functional cysteines on transducin that are located in the proximity of the interaction site with the photoreceptor protein, near the guanine nucleotide binding site, or in regions involved in the structural changes taking place upon protein activation. With the use of these reagents, transducin appears to be "frozen" in various conformational stages of its cycle, providing conditions for studying two of the initial steps of the visual process: the light-dependent binding of transducin to rhodopsin and the transducin guanine nucleotide exchange reaction.

Light adaptation and dark adaptation of human rod photoreceptors measured from the a-wave of the electroretinogram.

1. We recorded the a-wave of the human electroretinogram from subjects with normal vision, using a corneal electrode and ganzfeld (full-field) light stimulation. From analysis of the rising phase of rod-isolated flash responses we determined the maximum size (amax) of the a-wave, a measure of the massed circulating current of the rods, and the amplification constant (A) of transduction within the rod photoreceptors. 2. During light adaptation by steady backgrounds the maximal response was reduced, as reported previously. amax declined approximately as I0/(I0 + IB), where IB is retinal illuminance and I0 is a constant. In different subjects I0 ranged from 40 to 100 trolands, with a mean of 70 trolands, corresponding to about 600 photoisomerizations s-1 per rod. (1 troland is the retinal illuminance that results when a surface luminance of 1 cd m-2 is viewed through a pupil area of 1 mm2.) The amplification constant A decreased only slightly in the presence of steady backgrounds. 3. Following a full bleach amax recovered along an S-shaped curve over a period of 30 min. There was no detectable response for the first 5 min, and half-maximal recovery took 13-17 min. 4. The apparent amplification constant decreased at early times after large bleaches. However, upon correction for reduced light absorption due to loss of pigment, with regeneration of rhodopsin occurring with a time constant of 9-15 min in different subjects, it appeared that the true value of A was probably unchanged by bleaching. 5. The recovery of amax following a bleach could be converted into recovery of equivalent background intensity, using a 'Crawford transformation' derived from the light adaptation results. Following bleaches ranging from 10 to > 99 %, the equivalent background intensity decayed approximately exponentially, with a time constant of about 3 min. 6. The time taken for amax to recover to a fixed proportion of its original level increased approximately linearly (rather than logarithmically) with fractional bleach, with a slope of about 12 min per 100 % bleach. Similar behaviour has previously been seen in psychophysical dark adaptation experiments, for the dependence of the 'second component' of recovery on the level of bleaching.

Effect of rhodopsin C-terminal peptide on photoresponses in functionally intact rod outer segments.

The protein-protein interactions that underlie shut-off of the light-activated rhodopsin were studied using synthetic peptides derived from C-terminal region of the rhodopsin. The photoresponses were recorded in whole-cell voltage clamp from rod outer segments (ROS) that were internally dialyzed with an intracellular solution containing the synthetic peptides. This was the first time that synthetic peptides have been used in functionally intact ROS. None of the tested peptides promoted the shut-off of the photolyzed rhodopsin (R) by stimulating the binding of an activated arrestin to non-phosphorylated R, contrary to what was expected from in vitro experiments (Puig et al. FEBS Lett. 362: 185-188, 1995).

Molecular basis for interactions of G protein betagamma subunits with effectors.

Both the alpha and betagamma subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins) communicate signals from receptors to effectors. Gbetagamma subunits can regulate a diverse array of effectors, including ion channels and enzymes. Galpha subunits bound to guanine diphosphate (Galpha-GDP) inhibit signal transduction through Gbetagamma subunits, suggesting a common interface on Gbetagamma subunits for Galpha binding and effector interaction. The molecular basis for interaction of Gbetagamma with effectors was characterized by mutational analysis of Gbeta residues that make contact with Galpha-GDP. Analysis of the ability of these mutants to regulate the activity of calcium and potassium channels, adenylyl cyclase 2, phospholipase C-beta2, and beta-adrenergic receptor kinase revealed the Gbeta residues required for activation of each effector and provides evidence for partially overlapping domains on Gbeta for regulation of these effectors. This organization of interaction regions on Gbeta for different effectors and Galpha explains why subunit dissociation is crucial for signal transmission through Gbetagamma subunits.

Effect of detergents and lipids on transducin photoactivation by rhodopsin.

Rhodopsin samples, isolated using four different extraction procedures, were used to investigate the photodependent activation of the GTPase activity of transducin. A complete inhibition of transducin light-dependent GTP hydrolytic activity was observed when rhodopsin purified in the presence of 1% digitonin, following rod outer segment (ROS) solubilization with 1% 3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate (CHAPS), was used for its activation [0 pmol of inorganic phosphate (Pi) released/min/pmol of rhodopsin]. Rhodopsin, isolated in the presence of 1% digitonin following ROS solubilization with 1% digitonin, was capable of stimulating slightly transducin GTPase activity, with an initial rate of 1 pmol of GTP hydrolyzed/min/pmol of rhodopsin. However, rhodopsin purified in the presence of 0.2% n-dodecyl-beta-D-maltoside (DM), following ROS solubilization with either 1% CHAPS or 1% DM, stimulated the enzymatic activity of transducin in a light-dependent manner, with an initial rate of 5 pmol of Pi released/min/pmol of rhodopsin. Addition of 0.075% egg phosphatidylcholine (PC) to the four different solubilized rhodopsin samples significantly enhanced light-stimulated GTP hydrolysis by transducin, with initial rates increasing from 0 to 1, 1 to 2, and 5 to 30 pmol of Pi released/min/pmol of rhodopsin, respectively. Furthermore, DM-solubilized rhodopsin induced the hydrolysis of the maximum amount of GTP by transducin at 0.0075% PC, while digitonin-solubilized rhodopsin only stimulated the GTPase activity of transducin to a similar value, when the amount of the photoreceptor protein was increased 4-fold and 0.15% PC was added to the assay mixture. These results suggest that the effective photoactivation of transducin by rhodopsin requires phospholipids, which seem to be differentially eliminated with the detergent extraction procedure utilized during ROS membranes solubilization and photopigment isolation.

Receptor and betagamma binding sites in the alpha subunit of the retinal G protein transducin.

Transmembrane receptors for hormones, neurotransmitters, light, and odorants mediate their cellular effects by activating heterotrimeric guanine nucleotide-binding proteins (G proteins). Crystal structures have revealed contact surfaces between G protein subunits, but not the surfaces or molecular mechanism through which Galphabetagamma responds to activation by transmembrane receptors. Such a surface was identified from the results of testing 100 mutant alpha subunits of the retinal G protein transducin for their ability to interact with rhodopsin. Sites at which alanine substitutions impaired this interaction mapped to two distinct Galpha surfaces: a betagamma-binding surface and a putative receptor-interacting surface. On the basis of these results a mechanism for receptor-catalyzed exchange of guanosine diphosphate for guanosine triphosphate is proposed.

A general method for mapping tertiary contacts between amino acid residues in membrane-embedded proteins.

A general method for mapping tertiary interactions in membrane proteins using the visual pigment rhodopsin as a model is presented. In this approach, the protein is first assembled from two separately expressed gene fragments encoding nonoverlapping segments of the full-length polypeptide. Cys residues are then introduced into each of the two fragments such that juxtaposed residues are able to form disulfide cross-links in the protein either spontaneously or with the assistance of a Cu(2+)-(phenanthroline)3 oxidant. The cross-linked polypeptides are identified from a characteristic mobility shift on sodium dodecyl sulfate (SDS) gels as detected by Western blot analysis where the covalently bound heterodimer migrates with a mobility essentially identical to that of the native, full-length protein. Three different split rhodopsin mutants were prepared: one with a split in the loop connecting helices 3 and 4 (the 3/4 loop), one with a split in the 4/5 loop, and one with a split in the 5/6 loop. Each of these proteins when purified from transfected COS cells bound 11-cis-retinal, had a native absorption maximum at 500 nm, and activated transducin in a light-dependent manner. The cross-linking assay was tested with the rhodopsin mutant split in the 5/6 loop using the rho-1D4 antibody (which recognizes the carboxy terminal eight amino acids of rhodopsin) to detect the proteins on Western blots of SDS gels. Cys residues were substituted for Val-204 in the amino terminal fragment and Phe-276 in the carboxy terminal fragment of the rhodopsin mutant because Schwartz and co-workers [Elling et al.(ABSTRACT TRUNCATED AT 250 WORDS)