CDK6 activity in a recurring convergent kinase network motif.

In humans, more than 500 kinases phosphorylate ~15% of all proteins in an emerging phosphorylation network. Convergent local interaction motifs, in which ≥two kinases phosphorylate the same substrate, underlie feedback loops and signal amplification events but have not been systematically analyzed. Here, we first report a network-wide computational analysis of convergent kinase-substrate relationships (cKSRs). In experimentally validated phosphorylation sites, we find that cKSRs are common and involve >80% of all human kinases and >24% of all substrates. We show that cKSRs occur over a wide range of stoichiometries, in many instances harnessing co-expressed kinases from family subgroups. We then experimentally demonstrate for the prototypical convergent CDK4/6 kinase pair how multiple inputs phosphorylate the tumor suppressor retinoblastoma protein (RB) and thereby hamper in situ analysis of the individual kinases. We hypothesize that overexpression of one kinase combined with a CDK4/6 inhibitor can dissect convergence. In breast cancer cells expressing high levels of CDK4, we confirm this hypothesis and develop a high-throughput compatible assay that quantifies genetically modified CDK6 variants and inhibitors. Collectively, our work reveals the occurrence, topology, and experimental dissection of convergent interactions toward a deeper understanding of kinase networks and functions.

Optogenetic delivery of trophic signals in a genetic model of Parkinson's disease.

Optogenetics has been harnessed to shed new mechanistic light on current and future therapeutic strategies. This has been to date achieved by the regulation of ion flow and electrical signals in neuronal cells and neural circuits that are known to be affected by disease. In contrast, the optogenetic delivery of trophic biochemical signals, which support cell survival and are implicated in degenerative disorders, has never been demonstrated in an animal model of disease. Here, we reengineered the human and Drosophila melanogaster REarranged during Transfection (hRET and dRET) receptors to be activated by light, creating one-component optogenetic tools termed Opto-hRET and Opto-dRET. Upon blue light stimulation, these receptors robustly induced the MAPK/ERK proliferative signaling pathway in cultured cells. In PINK1B9 flies that exhibit loss of PTEN-induced putative kinase 1 (PINK1), a kinase associated with familial Parkinson's disease (PD), light activation of Opto-dRET suppressed mitochondrial defects, tissue degeneration and behavioral deficits. In human cells with PINK1 loss-of-function, mitochondrial fragmentation was rescued using Opto-dRET via the PI3K/NF-кB pathway. Our results demonstrate that a light-activated receptor can ameliorate disease hallmarks in a genetic model of PD. The optogenetic delivery of trophic signals is cell type-specific and reversible and thus has the potential to inspire novel strategies towards a spatio-temporal regulation of tissue repair.

Microbial methionine transporters and biotechnological applications.

Methionine (Met) is an essential amino acid with commercial value in animal feed, human nutrition, and as a chemical precursor. Microbial production of Met has seen intensive investigation towards a more sustainable alternative to the chemical synthesis that currently meets the global Met demand. Indeed, efficient Met biosynthesis has been achieved in genetically modified bacteria that harbor engineered enzymes and streamlined metabolic pathways. Very recently, the export of Met as the final step during its fermentative production has been studied and optimized, primarily through identification and expression of microbial Met efflux transporters. In this mini-review, we summarize the current knowledge on four families of Met export and import transporters that have been harnessed for the production of Met and other valuable biomolecules. These families are discussed with respect to their function, gene regulation, and biotechnological applications. We cover methods for identification and characterization of Met transporters as the basis for the further engineering of these proteins and for exploration of other solute carrier families. The available arsenal of Met transporters from different species and protein families provides blueprints not only for fermentative production but also synthetic biology systems, such as molecular sensors and cell-cell communication systems. KEY POINTS: • Sustainable production of methionine (Met) using microbes is actively explored. • Met transporters of four families increase production yield and specificity. • Further applications include other biosynthetic pathways and synthetic biology.

Monitoring hippocampal glycine with the computationally designed optical sensor GlyFS.

Fluorescent sensors are an essential part of the experimental toolbox of the life sciences, where they are used ubiquitously to visualize intra- and extracellular signaling. In the brain, optical neurotransmitter sensors can shed light on temporal and spatial aspects of signal transmission by directly observing, for instance, neurotransmitter release and spread. Here we report the development and application of the first optical sensor for the amino acid glycine, which is both an inhibitory neurotransmitter and a co-agonist of the N-methyl-D-aspartate receptors (NMDARs) involved in synaptic plasticity. Computational design of a glycine-specific binding protein allowed us to produce the optical glycine FRET sensor (GlyFS), which can be used with single and two-photon excitation fluorescence microscopy. We took advantage of this newly developed sensor to test predictions about the uneven spatial distribution of glycine in extracellular space and to demonstrate that extracellular glycine levels are controlled by plasticity-inducing stimuli.

Spatio-temporally precise activation of engineered receptor tyrosine kinases by light.

Receptor tyrosine kinases (RTKs) are a large family of cell surface receptors that sense growth factors and hormones and regulate a variety of cell behaviours in health and disease. Contactless activation of RTKs with spatial and temporal precision is currently not feasible. Here, we generated RTKs that are insensitive to endogenous ligands but can be selectively activated by low-intensity blue light. We screened light-oxygen-voltage (LOV)-sensing domains for their ability to activate RTKs by light-activated dimerization. Incorporation of LOV domains found in aureochrome photoreceptors of stramenopiles resulted in robust activation of the fibroblast growth factor receptor 1 (FGFR1), epidermal growth factor receptor (EGFR) and rearranged during transfection (RET). In human cancer and endothelial cells, light induced cellular signalling with spatial and temporal precision. Furthermore, light faithfully mimicked complex mitogenic and morphogenic cell behaviour induced by growth factors. RTKs under optical control (Opto-RTKs) provide a powerful optogenetic approach to actuate cellular signals and manipulate cell behaviour.

Light-assisted small-molecule screening against protein kinases.

High-throughput live-cell screens are intricate elements of systems biology studies and drug discovery pipelines. Here, we demonstrate an optogenetics-assisted method that avoids the need for chemical activators and reporters, reduces the number of operational steps and increases information content in a cell-based small-molecule screen against human protein kinases, including an orphan receptor tyrosine kinase. This blueprint for all-optical screening can be adapted to many drug targets and cellular processes.

Green-Light-Induced Inactivation of Receptor Signaling Using Cobalamin-Binding Domains.

Optogenetics and photopharmacology provide spatiotemporally precise control over protein interactions and protein function in cells and animals. Optogenetic methods that are sensitive to green light and can be used to break protein complexes are not broadly available but would enable multichromatic experiments with previously inaccessible biological targets. Herein, we repurposed cobalamin (vitamin B12) binding domains of bacterial CarH transcription factors for green-light-induced receptor dissociation. In cultured cells, we observed oligomerization-induced cell signaling for the fibroblast growth factor receptor 1 fused to cobalamin-binding domains in the dark that was rapidly eliminated upon illumination. In zebrafish embryos expressing fusion receptors, green light endowed control over aberrant fibroblast growth factor signaling during development. Green-light-induced domain dissociation and light-inactivated receptors will critically expand the optogenetic toolbox for control of biological processes.

A Phytochrome Sensory Domain Permits Receptor Activation by Red Light.

Optogenetics and photopharmacology enable the spatio-temporal control of cell and animal behavior by light. Although red light offers deep-tissue penetration and minimal phototoxicity, very few red-light-sensitive optogenetic methods are currently available. We have now developed a red-light-induced homodimerization domain. We first showed that an optimized sensory domain of the cyanobacterial phytochrome 1 can be expressed robustly and without cytotoxicity in human cells. We then applied this domain to induce the dimerization of two receptor tyrosine kinases-the fibroblast growth factor receptor 1 and the neurotrophin receptor trkB. This new optogenetic method was then used to activate the MAPK/ERK pathway non-invasively in mammalian tissue and in multicolor cell-signaling experiments. The light-controlled dimerizer and red-light-activated receptor tyrosine kinases will prove useful to regulate a variety of cellular processes with light.

Quantification of riboflavin, flavin mononucleotide, and flavin adenine dinucleotide in mammalian model cells by CE with LED-induced fluorescence detection.

Cultured mammalian cells essential are model systems in basic biology research, production platforms of proteins for medical use, and testbeds in synthetic biology. Flavin cofactors, in particular flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are critical for cellular redox reactions and sense light in naturally occurring photoreceptors and optogenetic tools. Here, we quantified flavin contents of commonly used mammalian cell lines. We first compared three procedures for extraction of free and noncovalently protein-bound flavins and verified extraction using fluorescence spectroscopy. For separation, two CE methods with different BGEs were established, and detection was performed by LED-induced fluorescence with limit of detections (LODs 0.5-3.8 nM). We found that riboflavin (RF), FMN, and FAD contents varied significantly between cell lines. RF (3.1-14 amol/cell) and FAD (2.2-17.0 amol/cell) were the predominant flavins, while FMN (0.46-3.4 amol/cell) was found at markedly lower levels. Observed flavin contents agree with those previously extracted from mammalian tissues, yet reduced forms of RF were detected that were not described previously. Quantification of flavins in mammalian cell lines will allow a better understanding of cellular redox reactions and optogenetic tools.

Flipping the Photoswitch: Ion Channels Under Light Control.

Nature has incorporated small photochromic molecules, colloquially termed 'photoswitches', in photoreceptor proteins to sense optical cues in phototaxis and vision. While Nature's ability to employ light-responsive functionalities has long been recognized, it was not until recently that scientists designed, synthesized and applied synthetic photochromes to manipulate many of which open rapidly and locally in their native cell types, biological processes with the temporal and spatial resolution of light. Ion channels in particular have come to the forefront of proteins that can be put under the designer control of synthetic photochromes. Photochromic ion channel controllers are comprised of three classes, photochromic soluble ligands (PCLs), photochromic tethered ligands (PTLs) and photochromic crosslinkers (PXs), and in each class ion channel functionality is controlled through reversible changes in photochrome structure. By acting as light-dependent ion channel agonists, antagonist or modulators, photochromic controllers effectively converted a wide range of ion channels, including voltage-gated ion channels, 'leak channels', tri-, tetra- and pentameric ligand-gated ion channels, and temperature-sensitive ion channels, into man-made photoreceptors. Control by photochromes can be reversible, unlike in the case of 'caged' compounds, and non-invasive with high spatial precision, unlike pharmacology and electrical manipulation. Here, we introduce design principles of emerging photochromic molecules that act on ion channels and discuss the impact that these molecules are beginning to have on ion channel biophysics and neuronal physiology.

Optimized design and in vivo application of optogenetically functionalized Drosophila dopamine receptors.

Neuromodulatory signaling via G protein-coupled receptors (GPCRs) plays a pivotal role in regulating neural network function and animal behavior. The recent development of optogenetic tools to induce G protein-mediated signaling provides the promise of acute and cell type-specific manipulation of neuromodulatory signals. However, designing and deploying optogenetically functionalized GPCRs (optoXRs) with accurate specificity and activity to mimic endogenous signaling in vivo remains challenging. Here we optimize the design of optoXRs by considering evolutionary conserved GPCR-G protein interactions and demonstrate the feasibility of this approach using two Drosophila Dopamine receptors (optoDopRs). These optoDopRs exhibit high signaling specificity and light sensitivity in vitro. In vivo, we show receptor and cell type-specific effects of dopaminergic signaling in various behaviors, including the ability of optoDopRs to rescue the loss of the endogenous receptors. This work demonstrates that optoXRs can enable optical control of neuromodulatory receptor-specific signaling in functional and behavioral studies.

Light-activated receptor tyrosine kinases: Designs and applications.

Receptor tyrosine kinases (RTKs) are a large and essential membrane receptor family. The molecular mechanisms and physiological consequences of RTK activation depend on, for example, ligand identity, subcellular localization, and developmental or disease stage. In the past few years, genetically-encoded light-activated RTKs (Opto-RTKs) have been developed to dissect these complexities by providing reversible and spatio-temporal control over cell signaling. These methods have very recently matured to include highly-sensitive multi-color actuators. The new ability to regulate RTK activity with high precision has been recently harnessed to gain mechanistic insights in subcellular, tissue, and animal models. Because of their sophisticated engineering, Opto-RTKs may only mirror some aspects of natural activation mechanisms but nevertheless offer unique opportunities to study RTK signaling and physiology.

Structure-guided optimization of light-activated chimeric G-protein-coupled receptors.

G-protein-coupled receptors (GPCRs) are the largest human receptor family and involved in virtually every physiological process. One hallmark of their function is specific coupling to selected signaling pathways. The ability to tune this coupling would make development of receptors with new capabilities possible. Complexes of GPCRs and G-proteins have recently been resolved at high resolution, but this information was in only few cases harnessed for rational receptor engineering. Here, we demonstrate structure-guided optimization of light-activated OptoXRs. Our hypothesis was that incorporation of GPCR-Gα contacts would lead to improved coupling. We first evaluated structure-based alignments for chimeric receptor fusion. We then show in a light-activated ß2AR that including Gα contacts increased signaling 7- to 20-fold compared with other designs. In turn, contact elimination diminished function. Finally, this platform allowed optimization of a further OptoXR and spectral tuning. Our work exemplifies structure-based OptoXR development for targeted cell and network manipulation.

Formation of Kiss1R/GPER Heterocomplexes Negatively Regulates Kiss1R-mediated Signalling through Limiting Receptor Cell Surface Expression.

Kisspeptin receptor (Kiss1R) is an important receptor that plays central regulatory roles in reproduction by regulating hormone release in the hypothalamus. We hypothesize that the formation of heterocomplexes between Kiss1R and other hypothalamus G protein-coupled receptors (GPCRs) affects their cellular signaling. Through screening of potential interactions between Kiss1R and hypothalamus GPCRs, we identified G protein-coupled estrogen receptor (GPER) as one interaction partner of Kiss1R. Based on the recognised function of kisspeptin and estrogen in regulating the reproductive system, we investigated the Kiss1R/GPER heterocomplex in more detail and revealed that complex formation significantly reduced Kiss1R-mediated signaling. GPER did not directly antagonize Kiss1R conformational changes upon ligand binding, but it rather reduced the cell surface expression of Kiss1R. These results therefore demonstrate a regulatory mechanism of hypothalamic hormone receptors via receptor cooperation in the reproductive system and modulation of receptor sensitivity.

A Light-Oxygen-Voltage Receptor Integrates Light and Temperature.

Sensory photoreceptors enable organisms to adjust their physiology, behavior, and development in response to light, generally with spatiotemporal acuity and reversibility. These traits underlie the use of photoreceptors as genetically encoded actuators to alter by light the state and properties of heterologous organisms. Subsumed as optogenetics, pertinent approaches enable regulating diverse cellular processes, not least gene expression. Here, we controlled the widely used Tet repressor by coupling to light-oxygen-voltage (LOV) modules that either homodimerize or dissociate under blue light. Repression could thus be elevated or relieved, and consequently protein expression was modulated by light. Strikingly, the homodimeric RsLOV module from Rhodobacter sphaeroides not only dissociated under light but intrinsically reacted to temperature. The limited light responses of wild-type RsLOV at 37 °C were enhanced in two variants that exhibited closely similar photochemistry and structure. One variant improved the weak homodimerization affinity of 40 µM by two-fold and thus also bestowed light sensitivity on a receptor tyrosine kinase. Certain photoreceptors, exemplified by RsLOV, can evidently moonlight as temperature sensors which immediately bears on their application in optogenetics and biotechnology. Properly accounted for, the temperature sensitivity can be leveraged for the construction of signal-responsive cellular circuits.

Acute and chronic effects of a light-activated FGF receptor in keratinocytes in vitro and in mice.

FGFs and their high-affinity receptors (FGFRs) play key roles in development, tissue repair, and disease. Because FGFRs bind overlapping sets of ligands, their individual functions cannot be determined using ligand stimulation. Here, we generated a light-activated FGFR2 variant (OptoR2) to selectively activate signaling by the major FGFR in keratinocytes. Illumination of OptoR2-expressing HEK 293T cells activated FGFR signaling with remarkable temporal precision and promoted cell migration and proliferation. In murine and human keratinocytes, OptoR2 activation rapidly induced the classical FGFR signaling pathways and expression of FGF target genes. Surprisingly, multi-level counter-regulation occurred in keratinocytes in vitro and in transgenic mice in vivo, including OptoR2 down-regulation and loss of responsiveness to light activation. These results demonstrate unexpected cell type-specific limitations of optogenetic FGFRs in long-term in vitro and in vivo settings and highlight the complex consequences of transferring optogenetic cell signaling tools into their relevant cellular contexts.

A Rationally and Computationally Designed Fluorescent Biosensor for d-Serine.

Solute-binding proteins (SBPs) have evolved to balance the demands of ligand affinity, thermostability, and conformational change to accomplish diverse functions in small molecule transport, sensing, and chemotaxis. Although the ligand-induced conformational changes that occur in SBPs make them useful components in biosensors, they are challenging targets for protein engineering and design. Here, we have engineered a d-alanine-specific SBP into a fluorescence biosensor with specificity for the signaling molecule d-serine (D-serFS). This was achieved through binding site and remote mutations that improved affinity (KD = 6.7 ± 0.5 µM), specificity (40-fold increase vs glycine), thermostability (Tm = 79 °C), and dynamic range (∼14%). This sensor allowed measurement of physiologically relevant changes in d-serine concentration using two-photon excitation fluorescence microscopy in rat brain hippocampal slices. This work illustrates the functional trade-offs between protein dynamics, ligand affinity, and thermostability and how these must be balanced to achieve desirable activities in the engineering of complex, dynamic proteins.

Pharmacology of ionotropic glutamate receptors: A structural perspective.

The impact of structural biology on the design of ligands (agonists, antagonists and modulators) for ionotropic glutamate receptors is reviewed.

Design and Application of Light-Regulated Receptor Tyrosine Kinases.

Understanding how the activity of membrane receptors and cellular signaling pathways shapes cell behavior is of fundamental interest in basic and applied research. Reengineering receptors to react to light instead of their cognate ligands allows for generating defined signaling inputs with high spatial and temporal precision and facilitates the dissection of complex signaling networks. Here, we describe fundamental considerations in the design of light-regulated receptor tyrosine kinases (Opto-RTKs) and appropriate control experiments. We also introduce methods for transient receptor expression in HEK293 cells, quantitative assessment of signaling activity in reporter gene assays, semiquantitative assessment of (in)activation time courses through Western blot (WB) analysis, and easy to implement light stimulation hardware.

All-Optical Miniaturized Co-culture Assay of Voltage-Gated Ca2+ Channels.

Light-activated proteins enable the reversible and spatiotemporal control of cellular events in optogenetics. Optogenetics is also rapidly expanding into the field of drug discovery where it provides cost-effective and noninvasive approaches for cell manipulation in high-throughput screens. Here, we present a prototypical cell-based assay that applies Channelrhodopsin2 (ChR2) to recapitulate physiological membrane potential changes and test for voltage-gated ion channel (VGIC) blockade. ChR2 and the voltage-gated Ca2+ channel 1.2 (CaV1.2) are expressed in individual HEK293 cell lines that are then co-cultured for formation of gap junctions and an electrical syncytium. This co-culture allows identification of blockers using parallel fluorescence plate readers in the 384-well plate format in an all-optical mode of operation. The assay is transferable to other VGICs by modularly combining new and existing cell lines and potentially also to other drug targets.