Toxoplasma ceramide synthases: Gene duplication, functional divergence, and roles in parasite fitness.

Toxoplasma gondii is an obligate, intracellular apicomplexan protozoan parasite of both humans and animals that can cause fetal damage and abortion and severe disease in the immunosuppressed. Sphingolipids have indispensable functions as signaling molecules and are essential and ubiquitous components of eukaryotic membranes that are both synthesized and scavenged by the Apicomplexa. Ceramide is the precursor for all sphingolipids, and here we report the identification, localization and analyses of the Toxoplasma ceramide synthases TgCerS1 and TgCerS2. Interestingly, we observed that while TgCerS1 was a fully functional orthologue of the yeast ceramide synthase (Lag1p) capable of catalyzing the conversion of sphinganine to ceramide, in contrast TgCerS2 was catalytically inactive. Furthermore, genomic deletion of TgCerS1 using CRISPR/Cas-9 led to viable but slow-growing parasites indicating its importance but not indispensability. In contrast, genomic knock out of TgCerS2 was only accessible utilizing the rapamycin-inducible Cre recombinase system. Surprisingly, the results demonstrated that this "pseudo" ceramide synthase, TgCerS2, has a considerably greater role in parasite fitness than its catalytically active orthologue (TgCerS1). Phylogenetic analyses indicated that, as in humans and plants, the ceramide synthase isoforms found in Toxoplasma and other Apicomplexa may have arisen through gene duplication. However, in the Apicomplexa the duplicated copy is hypothesized to have subsequently evolved into a non-functional "pseudo" ceramide synthase. This arrangement is unique to the Apicomplexa and further illustrates the unusual biology that characterize these protozoan parasites.

Independent Membrane Binding Properties of the Caspase Generated Fragments of the Beaded Filament Structural Protein 1 (BFSP1) Involves an Amphipathic Helix.

BACKGROUND: BFSP1 (beaded filament structural protein 1) is a plasma membrane, Aquaporin 0 (AQP0/MIP)-associated intermediate filament protein expressed in the eye lens. BFSP1 is myristoylated, a post-translation modification that requires caspase cleavage at D433. Bioinformatic analyses suggested that the sequences 434-452 were α-helical and amphipathic. METHODS AND RESULTS: By CD spectroscopy, we show that the addition of trifluoroethanol induced a switch from an intrinsically disordered to a more α-helical conformation for the residues 434-467. Recombinantly produced BFSP1 fragments containing this amphipathic helix bind to lens lipid bilayers as determined by surface plasmon resonance (SPR). Lastly, we demonstrate by transient transfection of non-lens MCF7 cells that these same BFSP1 C-terminal sequences localise to plasma membranes and to cytoplasmic vesicles. These can be co-labelled with the vital dye, lysotracker, but other cell compartments, such as the nuclear and mitochondrial membranes, were negative. The N-terminal myristoylation of the amphipathic helix appeared not to change either the lipid affinity or membrane localisation of the BFSP1 polypeptides or fragments we assessed by SPR and transient transfection, but it did appear to enhance its helical content. CONCLUSIONS: These data support the conclusion that C-terminal sequences of human BFSP1 distal to the caspase site at G433 have independent membrane binding properties via an adjacent amphipathic helix.

Cysteine synthase: multiple structures of a key enzyme in cysteine synthesis and a potential drug target for Chagas disease and leishmaniasis.

Chagas disease is a neglected tropical disease (NTD) caused by Trypanosoma cruzi, whilst leishmaniasis, which is caused by over 20 species of Leishmania, represents a group of NTDs endemic to most countries in the tropical and subtropical belt of the planet. These diseases remain a significant health problem both in endemic countries and globally. These parasites and other trypanosomatids, including T. theileri, a bovine pathogen, rely on cysteine biosynthesis for the production of trypanothione, which is essential for parasite survival in hosts. The de novo pathway of cysteine biosynthesis requires the conversion of O-acetyl-L-serine into L-cysteine, which is catalysed by cysteine synthase (CS). These enzymes present potential for drug development against T. cruzi, Leishmania spp. and T. theileri. To enable these possibilities, biochemical and crystallographic studies of CS from T. cruzi (TcCS), L. infantum (LiCS) and T. theileri (TthCS) were conducted. Crystal structures of the three enzymes were determined at resolutions of 1.80 Šfor TcCS, 1.75 Šfor LiCS and 2.75 Šfor TthCS. These three homodimeric structures show the same overall fold and demonstrate that the active-site geometry is conserved, supporting a common reaction mechanism. Detailed structural analysis revealed reaction intermediates of the de novo pathway ranging from an apo structure of LiCS and holo structures of both TcCS and TthCS to the substrate-bound structure of TcCS. These structures will allow exploration of the active site for the design of novel inhibitors. Additionally, unexpected binding sites discovered at the dimer interface represent new potential for the development of protein-protein inhibitors.

AmiP from hyperthermophilic Thermus parvatiensis prophage is a thermoactive and ultrathermostable peptidoglycan lytic amidase.

Bacteriophages encode a wide variety of cell wall disrupting enzymes that aid the viral escape in the final stages of infection. These lytic enzymes have accumulated notable interest due to their potential as novel antibacterials for infection treatment caused by multiple-drug resistant bacteria. Here, the detailed functional and structural characterization of Thermus parvatiensis prophage peptidoglycan lytic amidase AmiP, a globular Amidase_3 type lytic enzyme adapted to high temperatures is presented. The sequence and structure comparison with homologous lytic amidases reveals the key adaptation traits that ensure the activity and stability of AmiP at high temperatures. The crystal structure determined at a resolution of 1.8 Å displays a compact α/ß-fold with multiple secondary structure elements omitted or shortened compared with protein structures of similar proteins. The functional characterization of AmiP demonstrates high efficiency of catalytic activity and broad substrate specificity toward thermophilic and mesophilic bacteria strains containing Orn-type or DAP-type peptidoglycan. The here presented AmiP constitutes the most thermoactive and ultrathermostable Amidase_3 type lytic enzyme biochemically characterized with a temperature optimum at 85°C. The extraordinary high melting temperature Tm 102.6°C confirms fold stability up to approximately 100°C. Furthermore, AmiP is shown to be more active over the alkaline pH range with pH optimum at pH 8.5 and tolerates NaCl up to 300 mM with the activity optimum at 25 mM NaCl. This set of beneficial characteristics suggests that AmiP can be further exploited in biotechnology.

CPR-C4 is a highly conserved novel protease from the Candidate Phyla Radiation with remote structural homology to human vasohibins.

The Candidate Phyla Radiation is a recently uncovered and vast expansion of the bacterial domain of life, made up of largely uncharacterized phyla that lack isolated representatives. This unexplored territory of genetic diversity presents an abundance of novel proteins with potential applications in the life-science sectors. Here, we present the structural and functional elucidation of CPR-C4, a hypothetical protein from the genome of a thermophilic Candidate Phyla Radiation organism, identified through metagenomic sequencing. Our analyses revealed that CPR-C4 is a member of a family of highly conserved proteins within the Candidate Phyla Radiation. The function of CPR-C4 as a cysteine protease was predicted through remote structural similarity to the Homo sapiens vasohibins and subsequently confirmed experimentally with fluorescence-based activity assays. Furthermore, detailed structural and sequence alignment analysis enabled identification of a noncanonical cysteine-histidine-leucine(carbonyl) catalytic triad. The unexpected structural and functional similarities between CPR-C4 and the human vasohibins suggest an evolutionary relationship undetectable at the sequence level alone.

Chalcones identify cTXNPx as a potential antileishmanial drug target.

With current drug treatments failing due to toxicity, low efficacy and resistance; leishmaniasis is a major global health challenge that desperately needs new validated drug targets. Inspired by activity of the natural chalcone 2',6'-dihydroxy-4'-methoxychalcone (DMC), the nitro-analogue, 3-nitro-2',4',6'- trimethoxychalcone (NAT22, 1c) was identified as potent broad spectrum antileishmanial drug lead. Structural modification provided an alkyne containing chemical probe that labelled a protein within the parasite that was confirmed as cytosolic tryparedoxin peroxidase (cTXNPx). Crucially, labelling is observed in both promastigote and intramacrophage amastigote life forms, with no evidence of host macrophage toxicity. Incubation of the chalcone in the parasite leads to ROS accumulation and parasite death. Deletion of cTXNPx, by CRISPR-Cas9, dramatically impacts upon the parasite phenotype and reduces the antileishmanial activity of the chalcone analogue. Molecular docking studies with a homology model of in-silico cTXNPx suggest that the chalcone is able to bind in the putative active site hindering access to the crucial cysteine residue. Collectively, this work identifies cTXNPx as an important target for antileishmanial chalcones.

Flavonoid-based inhibitors of the Phi-class glutathione transferase from black-grass to combat multiple herbicide resistance.

The evolution and growth of multiple-herbicide resistance (MHR) in grass weeds continues to threaten global cereal production. While various processes can contribute to resistance, earlier work has identified the phi class glutathione-S-transferase (AmGSTF1) as a functional biomarker of MHR in black-grass (Alopecurus myosuroides). This study provides further insights into the role of AmGSTF1 in MHR using a combination of chemical and structural biology. Crystal structures of wild-type AmGSTF1, together with two specifically designed variants that allowed the co-crystal structure determination with glutathione and a glutathione adduct of the AmGSTF1 inhibitor 4-chloro-7-nitro-benzofurazan (NBD-Cl) were obtained. These studies demonstrated that the inhibitory activity of NBD-Cl was associated with the occlusion of the active site and the impediment of substrate binding. A search for other selective inhibitors of AmGSTF1, using ligand-fishing experiments, identified a number of flavonoids as potential ligands. Subsequent experiments using black-grass extracts discovered a specific flavonoid as a natural ligand of the recombinant enzyme. A series of related synthetic flavonoids was prepared and their binding to AmGSTF1 was investigated showing a high affinity for derivatives bearing a O-5-decyl-α-carboxylate. Molecular modelling based on high-resolution crystal structures allowed a binding pose to be defined which explained flavonoid binding specificity. Crucially, high binding affinity was linked to a reversal of the herbicide resistance phenotype in MHR black-grass. Collectively, these results present a nature-inspired new lead for the development of herbicide synergists to counteract MHR in weeds.

Multiple Occurrences of a 168-Nucleotide Deletion in SARS-CoV-2 ORF8, Unnoticed by Standard Amplicon Sequencing and Variant Calling Pipelines.

Genomic surveillance of the SARS-CoV-2 pandemic is crucial and mainly achieved by amplicon sequencing protocols. Overlapping tiled-amplicons are generated to establish contiguous SARS-CoV-2 genome sequences, which enable the precise resolution of infection chains and outbreaks. We investigated a SARS-CoV-2 outbreak in a local hospital and used nanopore sequencing with a modified ARTIC protocol employing 1200 bp long amplicons. We detected a long deletion of 168 nucleotides in the ORF8 gene in 76 samples from the hospital outbreak. This deletion is difficult to identify with the classical amplicon sequencing procedures since it removes two amplicon primer-binding sites. We analyzed public SARS-CoV-2 sequences and sequencing read data from ENA and identified the same deletion in over 100 genomes belonging to different lineages of SARS-CoV-2, pointing to a mutation hotspot or to positive selection. In almost all cases, the deletion was not represented in the virus genome sequence after consensus building. Additionally, further database searches point to other deletions in the ORF8 coding region that have never been reported by the standard data analysis pipelines. These findings and the fact that ORF8 is especially prone to deletions, make a clear case for the urgent necessity of public availability of the raw data for this and other large deletions that might change the physiology of the virus towards endemism.

Structure-functional relationship of cellular retinoic acid-binding proteins I and II interacting with natural and synthetic ligands.

A detailed understanding of the interactions between small-molecule ligands and their proposed binding targets is of the utmost importance for modern drug-development programs. Cellular retinoic acid-binding proteins I and II (CRABPI and CRABPII) facilitate a number of vital retinoid signalling pathways in mammalian cells and offer a gateway to manipulation of signalling that could potentially reduce phenotypes in serious diseases, including cancer and neurodegeneration. Although structurally very similar, the two proteins possess distinctly different biological functions, with their signalling influence being exerted through both genomic and nongenomic pathways. In this article, crystal structures are presented of the L29C mutant of Homo sapiens CRABPI in complex with naturally occurring fatty acids (1.64 Šresolution) and with the synthetic retinoid DC645 (2.41 Šresolution), and of CRABPII in complex with the ligands DC479 (1.80 Šresolution) and DC645 (1.71 Šresolution). DC645 and DC479 are two potential drug compounds identified in a recent synthetic retinoid development program. In particular, DC645 has recently been shown to have disease-modifying capabilities in neurodegenerative disease models by activating both genomic and nongenomic signalling pathways. These co-crystal structures demonstrate a canonical binding behaviour akin to that exhibited with all-trans-retinoic acid and help to explain how the compounds are able to exert an influence on part of the retinoid signalling cascade.

Crystal structure of the GDP-bound GTPase domain of Rab5a from Leishmania donovani.

Eukaryotic Rab5s are highly conserved small GTPase-family proteins that are involved in the regulation of early endocytosis. Leishmania donovani Rab5a regulates the sorting of early endosomes that are involved in the uptake of essential nutrients through fluid-phase endocytosis. Here, the 1.80 Šresolution crystal structure of the N-terminal GTPase domain of L. donovani Rab5a in complex with GDP is presented. The crystal structure determination was enabled by the design of specific single-site mutations and two deletions that were made to stabilize the protein for previous NMR studies. The structure of LdRab5a shows the canonical GTPase fold, with a six-stranded central mixed ß-sheet surrounded by five α-helices. The positions of the Switch I and Switch II loops confirm an open conformation, as expected in the absence of the γ-phosphate. However, in comparison to other GTP-bound and GDP-bound homologous proteins, the Switch I region traces a unique disposition in LdRab5a. One magnesium ion is bound to the protein at the GTP-binding site. Molecular-dynamics simulations indicate that the GDP-bound structure exhibits higher stability than the apo structure. The GDP-bound LdRab5a structure presented here will aid in efforts to unravel its interactions with its regulators, including the guanine nucleotide-exchange factor, and will lay the foundation for a structure-based search for specific inhibitors.

Classical pathways of gene regulation by retinoids.

Retinoic acid receptors were discovered during early studies of the actions and mechanisms of essential vitamins. Vitamin A is metabolized in the body to retinoic acid (RA) which is a key compound in the control of many developmental processes in chordates. These functions are mediated by a subfamily of nuclear receptors, divided into two classes, the retinoic acid receptors (RAR) and the retinoid X receptors (RXR). Each class is encoded by three closely related genes that are located on different chromosomes. The three proteins in each class are designated α, ß and γ, respectively. A wealth of structural studies have shown that they all share the same architecture including a DNA-binding domain connected by a flexible linker to the ligand and co-activator binding domain. Retinoic acid incorporation into the ligand-binding domain leads to a conformational change enabling the formation of RAR homodimers or RAR/RXR heterodimers that in turn bind specifically to target DNA sequences. The consensus sequences located on the promotors of regulated genes are known as retinoic acid response elements (RARE). The activated RAR/RXR homodimers recruit co-activators with histone acetylase activity leading to an opening of the chromatin structure and enabling downstream transcription of regulated genes. These canonical pathways describe the control mechanism for the majority of developmental processes mediated by retinoic acid and its derivatives.

GSP4PDB: a web tool to visualize, search and explore protein-ligand structural patterns.

BACKGROUND: In the field of protein engineering and biotechnology, the discovery and characterization of structural patterns is highly relevant as these patterns can give fundamental insights into protein-ligand interaction and protein function. This paper presents GSP4PDB, a bioinformatics web tool that enables the user to visualize, search and explore protein-ligand structural patterns within the entire Protein Data Bank. RESULTS: We introduce the notion of graph-based structural pattern (GSP) as an abstract model for representing protein-ligand interactions. A GSP is a graph where the nodes represent entities of the protein-ligand complex (amino acids and ligands) and the edges represent structural relationships (e.g. distances ligand - amino acid). The novel feature of GSP4PDB is a simple and intuitive graphical interface where the user can "draw" a GSP and execute its search in a relational database containing the structural data of each PDB entry. The results of the search are displayed using the same graph-based representation of the pattern. The user can further explore and analyse the results using a wide range of filters, or download their related information for external post-processing and analysis. CONCLUSIONS: GSP4PDB is a user-friendly and efficient application to search and discover new patterns of protein-ligand interaction.

Obtaining Tertiary Protein Structures by the ab Initio Interpretation of Small Angle X-ray Scattering Data.

Small angle X-ray scattering (SAXS) is an important tool for investigating the structure of proteins in solution. We present a novel ab initio method representing polypeptide chains as discrete curves used to derive a meaningful three-dimensional model from only the primary sequence and SAXS data. High resolution structures were used to generate probability density functions for each common secondary structural element found in proteins, which are used to place realistic restraints on the model curve's geometry. This is coupled with a novel explicit hydration shell model in order to derive physically meaningful three-dimensional models by optimizing against experimental SAXS data. The efficacy of this model is verified on an established benchmark protein set, and then it is used to predict the lysozyme structure using only its primary sequence and SAXS data. The method is used to generate a biologically plausible model of the coiled-coil component of the human synaptonemal complex central element protein.

Crystal structures of the Bacillus subtilis prophage lytic cassette proteins XepA and YomS.

As part of the Virus-X Consortium that aims to identify and characterize novel proteins and enzymes from bacteriophages and archaeal viruses, the genes of the putative lytic proteins XepA from Bacillus subtilis prophage PBSX and YomS from prophage SPß were cloned and the proteins were subsequently produced and functionally characterized. In order to elucidate the role and the molecular mechanism of XepA and YomS, the crystal structures of these proteins were solved at resolutions of 1.9 and 1.3 Å, respectively. XepA consists of two antiparallel ß-sandwich domains connected by a 30-amino-acid linker region. A pentamer of this protein adopts a unique dumbbell-shaped architecture consisting of two discs and a central tunnel. YomS (12.9 kDa per monomer), which is less than half the size of XepA (30.3 kDa), shows homology to the C-terminal part of XepA and exhibits a similar pentameric disc arrangement. Each ß-sandwich entity resembles the fold of typical cytoplasmic membrane-binding C2 domains. Only XepA exhibits distinct cytotoxic activity in vivo, suggesting that the N-terminal pentameric domain is essential for this biological activity. The biological and structural data presented here suggest that XepA disrupts the proton motive force of the cytoplasmatic membrane, thus supporting cell lysis.

A C-terminal CXCL8 peptide based on chemokine-glycosaminoglycan interactions reduces neutrophil adhesion and migration during inflammation.

Leucocyte recruitment is critical during many acute and chronic inflammatory diseases. Chemokines are key mediators of leucocyte recruitment during the inflammatory response, by signalling through specific chemokine G-protein-coupled receptors (GPCRs). In addition, chemokines interact with cell-surface glycosaminoglycans (GAGs) to generate a chemotactic gradient. The chemokine interleukin-8/CXCL8, a prototypical neutrophil chemoattractant, is characterized by a long, highly positively charged GAG-binding C-terminal region, absent in most other chemokines. To examine whether the CXCL8 C-terminal peptide has a modulatory role in GAG binding during neutrophil recruitment, we synthesized the wild-type CXCL8 C-terminal [CXCL8 (54-72)] (Peptide 1), a peptide with a substitution of glutamic acid (E) 70 with lysine (K) (Peptide 2) to increase positive charge; and also, a scrambled sequence peptide (Peptide 3). Surface plasmon resonance showed that Peptide 1, corresponding to the core CXCL8 GAG-binding region, binds to GAG but Peptide 2 binding was detected at lower concentrations. In the absence of cellular GAG, the peptides did not affect CXCL8-induced calcium signalling or neutrophil chemotaxis along a diffusion gradient, suggesting no effect on GPCR binding. All peptides equally inhibited neutrophil adhesion to endothelial cells under physiological flow conditions. Peptide 2, with its greater positive charge and binding to polyanionic GAG, inhibited CXCL8-induced neutrophil transendothelial migration. Our studies suggest that the E70K CXCL8 peptide, may serve as a lead molecule for further development of therapeutic inhibitors of neutrophil-mediated inflammation based on modulation of chemokine-GAG binding.

Relative Binding Energies Predict Crystallographic Binding Modes of Ethionamide Booster Lead Compounds.

Transcriptional repressor EthR from Mycobacterium tuberculosis is a valuable target for antibiotic booster drugs. We previously reported a virtual screening campaign to identify EthR inhibitors for development. Two ligand binding orientations were often proposed, though only the top scoring pose was utilized for filtering of the large data set. We obtained biophysically validated hits, some of which yielded complex crystal structures. In some cases, the crystallized binding mode and top scoring mode agree, while for others an alternate ligand binding orientation was found. In this contribution, we combine rigid docking, molecular dynamics simulations, and the linear interaction energy method to calculate binding free energies and derive relative binding energies for a number of EthR inhibitors in both modes. This strategy allowed us to correctly predict the most favorable orientation. Therefore, this widely applicable approach will be suitable to triage multiple binding modes within EthR and other potential drug targets with similar characteristics.

A unique dynamin-related protein is essential for mitochondrial fission in Toxoplasma gondii.

The single mitochondrion of apicomplexan protozoa is thought to be critical for all stages of the life cycle, and is a validated drug target against these important human and veterinary parasites. In contrast to other eukaryotes, replication of the mitochondrion is tightly linked to the cell cycle. A key step in mitochondrial segregation is the fission event, which in many eukaryotes occurs by the action of dynamins constricting the outer membrane of the mitochondria from the cytosolic face. To date, none of the components of the apicomplexan fission machinery have been identified and validated. We identify here a highly divergent, dynamin-related protein (TgDrpC), conserved in apicomplexans as essential for mitochondrial biogenesis and potentially for fission in Toxoplasma gondii. We show that TgDrpC is found adjacent to the mitochondrion, and is localised both at its periphery and at its basal part, where fission is expected to occur. We demonstrate that depletion or dominant negative expression of TgDrpC results in interconnected mitochondria and ultimately in drastic changes in mitochondrial morphology, as well as in parasite death. Intriguingly, we find that the canonical adaptor TgFis1 is not required for mitochondrial fission. The identification of an Apicomplexa-specific enzyme required for mitochondrial biogenesis and essential for parasite growth highlights parasite adaptation. This work paves the way for future drug development targeting TgDrpC, and for the analysis of additional partners involved in this crucial step of apicomplexan multiplication.

How to Stabilize Protein: Stability Screens for Thermal Shift Assays and Nano Differential Scanning Fluorimetry in the Virus-X Project.

The Horizon2020 Virus-X project was established in 2015 to explore the virosphere of selected extreme biotopes and discover novel viral proteins. To evaluate the potential biotechnical value of these proteins, the analysis of protein structures and functions is a central challenge in this program. The stability of protein sample is essential to provide meaningful assay results and increase the crystallizability of the targets. The thermal shift assay (TSA), a fluorescence-based technique, is established as a popular method for optimizing the conditions for protein stability in high-throughput. In TSAs, the employed fluorophores are extrinsic, environmentally-sensitive dyes. An alternative, similar technique is nano differential scanning fluorimetry (nanoDSF), which relies on protein native fluorescence. We present here a novel osmolyte screen, a 96-condition screen of organic additives designed to guide crystallization trials through preliminary TSA experiments. Together with previously-developed pH and salt screens, the set of three screens provides a comprehensive analysis of protein stability in a wide range of buffer systems and additives. The utility of the screens is demonstrated in the TSA and nanoDSF analysis of lysozyme and Protein X, a target protein of the Virus-X project.

Fluorescent Retinoic Acid Analogues as Probes for Biochemical and Intracellular Characterization of Retinoid Signaling Pathways.

Retinoids, such as all- trans-retinoic acid (ATRA), are endogenous signaling molecules derived from vitamin A that influence a variety of cellular processes through mediation of transcription events in the cell nucleus. Because of these wide-ranging and powerful biological activities, retinoids have emerged as therapeutic candidates of enormous potential. However, their use has been limited, to date, due to a lack of understanding of the complex and intricate signaling pathways that they control. We have designed and synthesized a family of synthetic retinoids that exhibit strong, intrinsic, solvatochromatic fluorescence as multifunctional tools to interrogate these important biological activities. We utilized the unique photophysical characteristics of these fluorescent retinoids to develop a novel in vitro fluorometric binding assay to characterize and quantify their binding to their cellular targets, including cellular retinoid binding protein II (CRABPII). The dihydroquinoline retinoid, DC360, exhibited particularly strong binding ( Kd = 34.0 ± 2.5 nM), and we further used X-ray crystallography to determine the structure of the DC360-CRABPII complex to 1.8 Å, which showed that DC360 occupies the known hydrophobic retinoid binding pocket. Finally, we used confocal fluorescence microscopy to image the cellular behavior of the compounds in cultured human epithelial cells, highlighting a fascinating nuclear localization, and used RNA sequencing to confirm that the compounds regulate cellular processes similar to those of ATRA. We anticipate that the unique properties of these fluorescent retinoids can now be used to cast new light on the vital and highly complex retinoid signaling pathway.