High-Throughput Expression and Purification of Human Solute Carriers for Structural and Biochemical Studies.

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, metabolites, neurotransmitters, and pharmaceuticals. Despite having emerged as attractive therapeutic targets and markers of disease, this group of proteins is still relatively underdrugged by current pharmaceuticals. Drug discovery projects for these transporters are impeded by limited structural, functional, and physiological knowledge, ultimately due to the difficulties in the expression and purification of this class of membrane-embedded proteins. Here, we demonstrate methods to obtain high-purity, milligram quantities of human SLC transporter proteins using codon-optimized gene sequences. In conjunction with a systematic exploration of construct design and high-throughput expression, these protocols ensure the preservation of the structural integrity and biochemical activity of the target proteins. We also highlight critical steps in the eukaryotic cell expression, affinity purification, and size-exclusion chromatography of these proteins. Ultimately, this workflow yields pure, functionally active, and stable protein preparations suitable for high-resolution structure determination, transport studies, small-molecule engagement assays, and high-throughput in vitro screening.

Functional characterization of SLC39 family members ZIP5 and ZIP10 in overexpressing HEK293 cells reveals selective copper transport activity.

Zinc is the second most prevalent metal element present in living organisms, and control of its concentration is pivotal to physiology. The amount of zinc available to the cell cytoplasm is regulated by the activity of members of the SLC39 family, the ZIP proteins. Selectivity of ZIP transporters has been the focus of earlier studies which provided a biochemical and structural basis for the selectivity for zinc over other metals such as copper, iron, and manganese. However, several previous studies have shown how certain ZIP proteins exhibit higher selectivity for metal elements other than zinc. Sequence similarities suggest an evolutionary basis for the elemental selectivity within the ZIP family. Here, by engineering HEK293 cells to overexpress ZIP proteins, we have studied the selectivity of two phylogenetic clades of ZIP proteins, that is ZIP8/ZIP14 (previously known to be iron and manganese transporters) and ZIP5/ZIP10. By incubating ZIP over-expressing cells in presence of several divalent metals, we found that ZIP5 and ZIP10 are high affinity copper transporters with greater selectivity over other elements, revealing a novel substrate signature for the ZIP5/ZIP10 clade.

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.

An Overview of Cell-Based Assay Platforms for the Solute Carrier Family of Transporters.

The solute carrier (SLC) superfamily represents the biggest family of transporters with important roles in health and disease. Despite being attractive and druggable targets, the majority of SLCs remains understudied. One major hurdle in research on SLCs is the lack of tools, such as cell-based assays to investigate their biological role and for drug discovery. Another challenge is the disperse and anecdotal information on assay strategies that are suitable for SLCs. This review provides a comprehensive overview of state-of-the-art cellular assay technologies for SLC research and discusses relevant SLC characteristics enabling the choice of an optimal assay technology. The Innovative Medicines Initiative consortium RESOLUTE intends to accelerate research on SLCs by providing the scientific community with high-quality reagents, assay technologies and data sets, and to ultimately unlock SLCs for drug discovery.

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.

A widespread role for SLC transmembrane transporters in resistance to cytotoxic drugs.

Solute carriers (SLCs) are the largest family of transmembrane transporters in humans and are major determinants of cellular metabolism. Several SLCs have been shown to be required for the uptake of chemical compounds into cellular systems, but systematic surveys of transporter-drug relationships in human cells are currently lacking. We performed a series of genetic screens in a haploid human cell line against 60 cytotoxic compounds representative of the chemical space populated by approved drugs. By using an SLC-focused CRISPR-Cas9 library, we identified transporters whose absence induced resistance to the drugs tested. This included dependencies involving the transporters SLC11A2/SLC16A1 for artemisinin derivatives and SLC35A2/SLC38A5 for cisplatin. The functional dependence on SLCs observed for a significant proportion of the screened compounds suggests a widespread role for SLCs in the uptake and cellular activity of cytotoxic drugs and provides an experimentally validated set of SLC-drug associations for a number of clinically relevant compounds.

Non-conservation of folding rates in the thioredoxin family reveals degradation of ancestral unassisted-folding.

Evolution involves not only adaptation, but also the degradation of superfluous features. Many examples of degradation at the morphological level are known (vestigial organs, for instance). However, the impact of degradation on molecular evolution has been rarely addressed. Thioredoxins serve as general oxidoreductases in all cells. Here, we report extensive mutational analyses on the folding of modern and resurrected ancestral bacterial thioredoxins. Contrary to claims from recent literature, in vitro folding rates in the thioredoxin family are not evolutionarily conserved, but span at least a ∼100-fold range. Furthermore, modern thioredoxin folding is often substantially slower than ancestral thioredoxin folding. Unassisted folding, as probed in vitro, thus emerges as an ancestral vestigial feature that underwent degradation, plausibly upon the evolutionary emergence of efficient cellular folding assistance. More generally, our results provide evidence that degradation of ancestral features shapes, not only morphological evolution, but also the evolution of individual proteins.

Optogenetic Control of Nodal Signaling Reveals a Temporal Pattern of Nodal Signaling Regulating Cell Fate Specification during Gastrulation.

During metazoan development, the temporal pattern of morphogen signaling is critical for organizing cell fates in space and time. Yet, tools for temporally controlling morphogen signaling within the embryo are still scarce. Here, we developed a photoactivatable Nodal receptor to determine how the temporal pattern of Nodal signaling affects cell fate specification during zebrafish gastrulation. By using this receptor to manipulate the duration of Nodal signaling in vivo by light, we show that extended Nodal signaling within the organizer promotes prechordal plate specification and suppresses endoderm differentiation. Endoderm differentiation is suppressed by extended Nodal signaling inducing expression of the transcriptional repressor goosecoid (gsc) in prechordal plate progenitors, which in turn restrains Nodal signaling from upregulating the endoderm differentiation gene sox17 within these cells. Thus, optogenetic manipulation of Nodal signaling identifies a critical role of Nodal signaling duration for organizer cell fate specification during gastrulation.

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.

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.

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.

Mutational studies on resurrected ancestral proteins reveal conservation of site-specific amino acid preferences throughout evolutionary history.

Local protein interactions ("molecular context" effects) dictate amino acid replacements and can be described in terms of site-specific, energetic preferences for any different amino acid. It has been recently debated whether these preferences remain approximately constant during evolution or whether, due to coevolution of sites, they change strongly. Such research highlights an unresolved and fundamental issue with far-reaching implications for phylogenetic analysis and molecular evolution modeling. Here, we take advantage of the recent availability of phenotypically supported laboratory resurrections of Precambrian thioredoxins and ß-lactamases to experimentally address the change of site-specific amino acid preferences over long geological timescales. Extensive mutational analyses support the notion that evolutionary adjustment to a new amino acid may occur, but to a large extent this is insufficient to erase the primitive preference for amino acid replacements. Generally, site-specific amino acid preferences appear to remain conserved throughout evolutionary history despite local sequence divergence. We show such preference conservation to be readily understandable in molecular terms and we provide crystallographic evidence for an intriguing structural-switch mechanism: Energetic preference for an ancestral amino acid in a modern protein can be linked to reorganization upon mutation to the ancestral local structure around the mutated site. Finally, we point out that site-specific preference conservation naturally leads to one plausible evolutionary explanation for the existence of intragenic global suppressor mutations.

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.

The optogenetic promise for oncology: Episode I.

As light-based control of fundamental signaling pathways is becoming a reality, the field of optogenetics is rapidly moving beyond neuroscience. We have recently developed receptor tyrosine kinases that are activated by light and control cell proliferation, epithelial-mesenchymal transition, and angiogenic sprouting-cell behaviors central to cancer progression.

Conservation of protein structure over four billion years.

Little is known about the evolution of protein structures and the degree of protein structure conservation over planetary time scales. Here, we report the X-ray crystal structures of seven laboratory resurrections of Precambrian thioredoxins dating up to approximately four billion years ago. Despite considerable sequence differences compared with extant enzymes, the ancestral proteins display the canonical thioredoxin fold, whereas only small structural changes have occurred over four billion years. This remarkable degree of structure conservation since a time near the last common ancestor of life supports a punctuated-equilibrium model of structure evolution in which the generation of new folds occurs over comparatively short periods and is followed by long periods of structural stasis.

Protein folding drives disulfide formation.

PDI catalyzes the oxidative folding of disulfide-containing proteins. However, the sequence of reactions leading to a natively folded and oxidized protein remains unknown. Here we demonstrate a technique that enables independent measurements of disulfide formation and protein folding. We find that non-native disulfides are formed early in the folding pathway and can trigger misfolding. In contrast, a PDI domain favors native disulfides by catalyzing oxidation at a late stage of folding. We propose a model for cotranslational oxidative folding wherein PDI acts as a placeholder that is relieved by the pairing of cysteines caused by substrate folding. This general mechanism can explain how PDI catalyzes oxidative folding in a variety of structurally unrelated substrates.

Highly anomalous energetics of protein cold denaturation linked to folding-unfolding kinetics.

Despite several careful experimental analyses, it is not yet clear whether protein cold-denaturation is just a "mirror image" of heat denaturation or whether it shows unique structural and energetic features. Here we report that, for a well-characterized small protein, heat denaturation and cold denaturation show dramatically different experimental energetic patterns. Specifically, while heat denaturation is endothermic, the cold transition (studied in the folding direction) occurs with negligible heat effect, in a manner seemingly akin to a gradual, second-order-like transition. We show that this highly anomalous energetics is actually an apparent effect associated to a large folding/unfolding free energy barrier and that it ultimately reflects kinetic stability, a naturally-selected trait in many protein systems. Kinetics thus emerges as an important factor linked to differential features of cold denaturation. We speculate that kinetic stabilization against cold denaturation may play a role in cold adaptation of psychrophilic organisms. Furthermore, we suggest that folding-unfolding kinetics should be taken into account when analyzing in vitro cold-denaturation experiments, in particular those carried out in the absence of destabilizing conditions.

Single-molecule paleoenzymology probes the chemistry of resurrected enzymes.

It is possible to travel back in time at the molecular level by reconstructing proteins from extinct organisms. Here we report the reconstruction, based on sequence predicted by phylogenetic analysis, of seven Precambrian thioredoxin enzymes (Trx) dating back between ~1.4 and ~4 billion years (Gyr). The reconstructed enzymes are up to 32 °C more stable than modern enzymes, and the oldest show markedly higher activity than extant ones at pH 5. We probed the mechanisms of reduction of these enzymes using single-molecule force spectroscopy. From the force dependency of the rate of reduction of an engineered substrate, we conclude that ancient Trxs use chemical mechanisms of reduction similar to those of modern enzymes. Although Trx enzymes have maintained their reductase chemistry unchanged, they have adapted over 4 Gyr to the changes in temperature and ocean acidity that characterize the evolution of the global environment from ancient to modern Earth.