Broadening the Utility of Farnesyltransferase-Catalyzed Protein Labeling Using Norbornene-Tetrazine Click Chemistry.

Bioorthogonal chemistry has gained widespread use in the study of many biological systems of interest, including protein prenylation. Prenylation is a post-translational modification, in which one or two 15- or 20-carbon isoprenoid chains are transferred onto cysteine residues near the C-terminus of a target protein. The three main enzymes─protein farnesyltransferase (FTase), geranylgeranyl transferase I (GGTase I), and geranylgeranyl transferase II (GGTase II)─that catalyze this process have been shown to tolerate numerous structural modifications in the isoprenoid substrate. This feature has previously been exploited to transfer an array of farnesyl diphosphate analogues with a range of functionalities, including an alkyne-containing analogue for copper-catalyzed bioconjugation reactions. Reported here is the synthesis of an analogue of the isoprenoid substrate embedded with norbornene functionality (C10NorOPP) that can be used for an array of applications, ranging from metabolic labeling to selective protein modification. The probe was synthesized in seven steps with an overall yield of 7% and underwent an inverse electron demand Diels-Alder (IEDDA) reaction with tetrazine-containing tags, allowing for copper-free labeling of proteins. The use of C10NorOPP for the study of prenylation was explored in the metabolic labeling of prenylated proteins in HeLa, COS-7, and astrocyte cells. Furthermore, in HeLa cells, these modified prenylated proteins were identified and quantified using label-free quantification (LFQ) proteomics with 25 enriched prenylated proteins. Additionally, the unique chemistry of C10NorOPP was utilized for the construction of a multiprotein-polymer conjugate for the targeted labeling of cancer cells. That construct was prepared using a combination of norbornene-tetrazine conjugation and azide-alkyne cycloaddition, highlighting the utility of the additional degree of orthogonality for the facile assembly of new protein conjugates with novel structures and functions.

Designed Ankyrin Repeat Proteins as Actin Labels of Distinct Cytoskeletal Structures in Living Cells.

The orchestrated assembly of actin and actin-binding proteins into cytoskeletal structures coordinates cell morphology changes during migration, cytokinesis, and adaptation to external stimuli. The accurate and unbiased visualization of the diverse actin assemblies within cells is an ongoing challenge. We describe here the identification and use of designed ankyrin repeat proteins (DARPins) as synthetic actin binders. Actin-binding DARPins were identified through ribosome display and validated biochemically. When introduced or expressed inside living cells, fluorescently labeled DARPins accumulated at actin filaments, validated through phalloidin colocalization on fixed cells. Nevertheless, different DARPins displayed different actin labeling patterns: some DARPins labeled efficiently dynamic structures, such as filopodia, lamellipodia, and blebs, while others accumulated primarily in stress fibers. This differential intracellular distribution correlated with DARPin-actin binding kinetics, as measured by fluorescence recovery after photobleaching experiments. Moreover, the rapid arrest of actin dynamics induced by pharmacological treatment led to the fast relocalization of DARPins. Our data support the hypothesis that the localization of actin probes depends on the inherent dynamic movement of the actin cytoskeleton. Compared to the widely used LifeAct probe, one DARPin exhibited enhanced signal-to-background ratio while retaining a similar ability to label stress fibers. In summary, we propose DARPins as promising actin-binding proteins for labeling or manipulation in living cells.

A DARPin promotes faster onset of botulinum neurotoxin A1 action.

In this study, we characterize Designed Ankyrin Repeat Proteins (DARPins) as investigative tools to probe botulinum neurotoxin A1 (BoNT/A1) structure and function. We identify DARPin-F5 that completely blocks SNAP25 substrate cleavage by BoNT/A1 in vitro. X-ray crystallography reveals that DARPin-F5 inhibits BoNT/A1 activity by interacting with a substrate-binding region between the α- and ß-exosite. This DARPin does not block substrate cleavage of BoNT/A3, indicating that DARPin-F5 is a subtype-specific inhibitor. BoNT/A1 Glu-171 plays a critical role in the interaction with DARPin-F5 and its mutation to Asp, the residue found in BoNT/A3, results in a loss of inhibition of substrate cleavage. In contrast to the in vitro results, DARPin-F5 promotes faster substrate cleavage of BoNT/A1 in primary neurons and muscle tissue by increasing toxin translocation. Our findings could have important implications for the application of BoNT/A1 in therapeutic areas requiring faster onset of toxin action combined with long persistence.

DARPins detect the formation of hetero-tetramers of p63 and p73 in epithelial tissues and in squamous cell carcinoma.

The two p53 homologues p63 and p73 regulate transcriptional programs in epithelial tissues and several cell types in these tissues express both proteins. All members of the p53 family form tetramers in their active state through a dedicated oligomerization domain that structurally assembles as a dimer of dimers. The oligomerization domain of p63 and p73 share a high sequence identity, but the p53 oligomerization domain is more divergent and it lacks a functionally important C-terminal helix present in the other two family members. Based on these structural differences, p53 does not hetero-oligomerize with p63 or p73. In contrast, p63 and p73 form hetero-oligomers of all possible stoichiometries, with the hetero-tetramer built from a p63 dimer and a p73 dimer being thermodynamically more stable than the two homo-tetramers. This predicts that in cells expressing both proteins a p632/p732 hetero-tetramer is formed. So far, the tools to investigate the biological function of this hetero-tetramer have been missing. Here we report the generation and characterization of Designed Ankyrin Repeat Proteins (DARPins) that bind with high affinity and selectivity to the p632/p732 hetero-tetramer. Using these DARPins we were able to confirm experimentally the existence of this hetero-tetramer in epithelial mouse and human tissues and show that its level increases in squamous cell carcinoma.

Diversity-oriented synthesis encoded by deoxyoligonucleotides.

Diversity-oriented synthesis (DOS) is a powerful strategy to prepare molecules with underrepresented features in commercial screening collections, resulting in the elucidation of novel biological mechanisms. In parallel to the development of DOS, DNA-encoded libraries (DELs) have emerged as an effective, efficient screening strategy to identify protein binders. Despite recent advancements in this field, most DEL syntheses are limited by the presence of sensitive DNA-based constructs. Here, we describe the design, synthesis, and validation experiments performed for a 3.7 million-member DEL, generated using diverse skeleton architectures with varying exit vectors and derived from DOS, to achieve structural diversity beyond what is possible by varying appendages alone. We also show screening results for three diverse protein targets. We will make this DEL available to the academic scientific community to increase access to novel structural features and accelerate early-phase drug discovery.

Nuclear myosin VI maintains replication fork stability.

The actin cytoskeleton is of fundamental importance for cellular structure and plasticity. However, abundance and function of filamentous actin in the nucleus are still controversial. Here we show that the actin-based molecular motor myosin VI contributes to the stabilization of stalled or reversed replication forks. In response to DNA replication stress, myosin VI associates with stalled replication intermediates and cooperates with the AAA ATPase Werner helicase interacting protein 1 (WRNIP1) in protecting these structures from DNA2-mediated nucleolytic attack. Using functionalized affinity probes to manipulate myosin VI levels in a compartment-specific manner, we provide evidence for the direct involvement of myosin VI in the nucleus and against a contribution of the abundant cytoplasmic pool during the replication stress response.

Homeostasis at different backgrounds: The roles of overlayed feedback structures in vertebrate photoadaptation.

We have studied the resetting behavior of eight basic integral controller motifs with respect to different but constant backgrounds. We found that the controllers split symmetrically into two classes: one class, based on derepression of the compensatory flux, leads to more rapid resetting kinetics as backgrounds increase. The other class, which directly activates the compensatory flux, shows a slowing down in the resetting at increased backgrounds. We found a striking analogy between the resetting kinetics of vertebrate photoreceptors and controllers based on derepression, i.e. vertebrate rod or cone cells show decreased sensitivities and accelerated response kinetics as background illuminations increase. The central molecular model of vertebrate photoadaptation consists of an overlay of three negative feedback loops with cytosolic calcium ([Formula: see text]), cyclic guanosine monophosphate (cGMP) and cyclic nucleotide-gated (CNG) channels as components. While in one of the feedback loops the extrusion of [Formula: see text] by potassium-dependent sodium-calcium exchangers (NCKX) can lead to integral control with cGMP as the controlled variable, the expected robust perfect adaptation of cGMP is lost, because of the two other feedback loops. They avoid that [Formula: see text] levels become too high and toxic. Looking at psychophysical laws, we found that in all of the above mentioned basic controllers Weber's law is followed when a "just noticeable difference" (threshold) of 1% of the controlled variable's set-point was considered. Applying comparable threshold pulses or steps to the photoadaptation model we find, in agreement with experimental results, that Weber's law is followed for relatively high backgrounds, while Stephens' power law gives a better description when backgrounds are low. Limitations of our photoadaption model, in particular with respect to potassium/sodium homeostasis, are discussed. Finally, we discuss possible implication of background perturbations in biological controllers when compensatory fluxes are based on activation.

Enzymatic Construction of DARPin-Based Targeted Delivery Systems Using Protein Farnesyltransferase and a Capture and Release Strategy.

Protein-based conjugates have been extensively utilized in various biotechnological and therapeutic applications. In order to prepare homogeneous conjugates, site-specific modification methods and efficient purification strategies are both critical factors to be considered. The development of general and facile conjugation and purification strategies is therefore highly desirable. Here, we apply a capture and release strategy to create protein conjugates based on Designed Ankyrin Repeat Proteins (DARPins), which are engineered antigen-binding proteins with prominent affinity and selectivity. In this case, DARPins that target the epithelial cell adhesion molecule (EpCAM), a diagnostic cell surface marker for many types of cancer, were employed. The DARPins were first genetically modified with a C-terminal CVIA sequence to install an enzyme recognition site and then labeled with an aldehyde functional group employing protein farnesyltransferase. Using a capture and release strategy, conjugation of the labeled DARPins to a TAMRA fluorophore was achieved with either purified proteins or directly from crude E. coli lysate and used in subsequent flow cytometry and confocal imaging analysis. DARPin-MMAE conjugates were also prepared yielding a construct manifesting an IC50 of 1.3 nM for cell killing of EpCAM positive MCF-7 cells. The method described here is broadly applicable to enable the streamlined one-step preparation of protein-based conjugates.

Programmable DARPin-based receptors for the detection of thrombotic markers.

Cellular therapies remain constrained by the limited availability of sensors for disease markers. Here we present an integrated target-to-receptor pipeline for constructing a customizable advanced modular bispecific extracellular receptor (AMBER) that combines our generalized extracellular molecule sensor (GEMS) system with a high-throughput platform for generating designed ankyrin repeat proteins (DARPins). For proof of concept, we chose human fibrin degradation products (FDPs) as markers with high clinical relevance and screened a DARPin library for FDP binders. We built AMBERs equipped with 19 different DARPins selected from 160 hits, and found 4 of them to be functional as heterodimers with a known single-chain variable fragments binder. Tandem receptors consisting of combinations of the validated DARPins are also functional. We demonstrate applications of these AMBER receptors in vitro and in vivo by constructing designer cell lines that detect pathological concentrations of FDPs and respond with the production of a reporter and a therapeutic anti-thrombotic protein.

Disrupting the HDAC6-ubiquitin interaction impairs infection by influenza and Zika virus and cellular stress pathways.

The deacetylase HDAC6 has tandem catalytic domains and a zinc finger domain (ZnF) binding ubiquitin (Ub). While the catalytic domain has an antiviral effect, the ZnF facilitates influenza A virus (IAV) infection and cellular stress responses. By recruiting Ub via the ZnF, HDAC6 promotes the formation of aggresomes and stress granules (SGs), dynamic structures associated with pathologies such as neurodegeneration. IAV subverts the aggresome/HDAC6 pathway to facilitate capsid uncoating during early infection. To target this pathway, we generate designed ankyrin repeat proteins (DARPins) binding the ZnF; one of these prevents interaction with Ub in vitro and in cells. Crystallographic analysis shows that it blocks the ZnF pocket where Ub engages. Conditional expression of this DARPin reversibly impairs infection by IAV and Zika virus; moreover, SGs and aggresomes are downregulated. These results validate the HDAC6 ZnF as an attractive target for drug discovery.

Sortase-Mediated Site-Specific Conjugation and 89Zr-Radiolabeling of Designed Ankyrin Repeat Proteins for PET.

Designed ankyrin repeat proteins (DARPins) are genetically engineered proteins that exhibit high specificity and affinity toward specific targets. Here, the G3-DARPin, which binds the HER2/neu receptor, was site-specifically modified with enzymatic methods and 89Zr-radiolabeled for applications in positron emission tomography (PET). Sortase A transpeptidation was used to install a desferrioxamine B (DFO) chelate bearing a reactive triglycine group to the C-terminal sortase tag of the G3-DARPin, and 89Zr-radiolabeling produced a novel 89ZrDFO-G3-DARPin radiotracer that can detect HER2/neu-positive tumors. The triglycine probe, DFO-Gly3 (1), was synthesized in 29% overall yield. After sortase A transpeptidation and purification from the nonfunctionalized protein component, the DFO-G3-DARPin product was radiolabeled to give 89ZrDFO-G3-DARPin. Binding specificity was assessed in HER2/neu-expressing BT-474 and SK-OV-3 cellular assays. The pharmacokinetics, tumor uptake, and specificity of 89ZrDFO-G3-DARPin were measured in vivo by PET imaging and confirmed by final time point (24 h) biodistribution experiments in female athymic nude mice bearing BT-474 xenografts. Sortase A transpeptidation afforded the site-specific and stoichiometrically precise functionalization of DFO-G3-DARPin with one chelate per protein. The modified DFO-G3-DARPin was purified from the nonfunctionalized DARPin by using Ni-NTA affinity chromatography. 89ZrDFO-G3-DARPin was obtained with a radiochemical purity of >95% measured by radio-size-exclusion chromatography. BT-474 tumor uptake at 24 h postadministration reached 4.41 ± 0.67 %ID/g (n = 3) with an approximate ∼70% reduction in tumor-associated activity in the blocking group (1.26 ± 0.29 %ID/g; 24 h postadministration, n = 5, P-value of <0.001). Overall, the site-specific, enzyme-mediated functionalization and characterization of 89ZrDFO-G3-DARPin in HER2/neu positive BT-474 xenografts demonstrate that DARPins are an attractive platform for generating a new class of protein-based radiotracers for PET. The specific uptake and retention of 89ZrDFO-G3-DARPin in tumors and clearance from most background tissues produced PET images with high tumor-to-background contrast.

Climate shapes the seed germination niche of temperate flowering plants: a meta-analysis of European seed conservation data.

BACKGROUND AND AIMS: Interactions between ecological factors and seed physiological responses during the establishment phase shape the distribution of plants. Yet, our understanding of the functions and evolution of early-life traits has been limited by the scarcity of large-scale datasets. Here, we tested the hypothesis that the germination niche of temperate plants is shaped by their climatic requirements and phylogenetic relatedness, using germination data sourced from a comprehensive seed conservation database of the European flora (ENSCOBASE). METHODS: We performed a phylogenetically informed Bayesian meta-analysis of primary data, considering 18 762 germination tests of 2418 species from laboratory experiments conducted across all European geographical regions. We tested for the interaction between species' climatic requirements and germination responses to experimental conditions including temperature, alternating temperature, light and dormancy-breaking treatments, while accounting for between-study variation related to seed sources and seed lot physiological status. KEY RESULTS: Climate was a strong predictor of germination responses. In warm and seasonally dry climates the seed germination niche includes a cold-cued germination response and an inhibition determined by alternating temperature regimes and cold stratification, while in climates with high temperature seasonality opposite responses can be observed. Germination responses to scarification and light were related to seed mass but not to climate. We also found a significant phylogenetic signal in the response of seeds to experimental conditions, providing evidence that the germination niche is phylogenetically constrained. Nevertheless, phylogenetically distant lineages exhibited common germination responses under similar climates. CONCLUSION: This is the first quantitative meta-analysis of the germination niche at a continental scale. Our findings showed that the germination niches of European plants exhibit evolutionary convergence mediated by strong pressures at the macroclimatic level. In addition, our methodological approach highlighted how large datasets generated by conservation seed banking can be valuable sources to address questions in plant macroecology and evolution.

Structural basis of adenylyl cyclase 9 activation.

Adenylyl cyclase 9 (AC9) is a membrane-bound enzyme that converts ATP into cAMP. The enzyme is weakly activated by forskolin, fully activated by the G protein Gαs subunit and is autoinhibited by the AC9 C-terminus. Although our recent structural studies of the AC9-Gαs complex provided the framework for understanding AC9 autoinhibition, the conformational changes that AC9 undergoes in response to activator binding remains poorly understood. Here, we present the cryo-EM structures of AC9 in several distinct states: (i) AC9 bound to a nucleotide inhibitor MANT-GTP, (ii) bound to an artificial activator (DARPin C4) and MANT-GTP, (iii) bound to DARPin C4 and a nucleotide analogue ATPαS, (iv) bound to Gαs and MANT-GTP. The artificial activator DARPin C4 partially activates AC9 by binding at a site that overlaps with the Gαs binding site. Together with the previously observed occluded and forskolin-bound conformations, structural comparisons of AC9 in the four conformations described here show that secondary structure rearrangements in the region surrounding the forskolin binding site are essential for AC9 activation.

Asymmetric requirement of Dpp/BMP morphogen dispersal in the Drosophila wing disc.

How morphogen gradients control patterning and growth in developing tissues remains largely unknown due to lack of tools manipulating morphogen gradients. Here, we generate two membrane-tethered protein binders that manipulate different aspects of Decapentaplegic (Dpp), a morphogen required for overall patterning and growth of the Drosophila wing. One is "HA trap" based on a single-chain variable fragment (scFv) against the HA tag that traps HA-Dpp to mainly block its dispersal, the other is "Dpp trap" based on a Designed Ankyrin Repeat Protein (DARPin) against Dpp that traps Dpp to block both its dispersal and signaling. Using these tools, we found that, while posterior patterning and growth require Dpp dispersal, anterior patterning and growth largely proceed without Dpp dispersal. We show that dpp transcriptional refinement from an initially uniform to a localized expression and persistent signaling in transient dpp source cells render the anterior compartment robust against the absence of Dpp dispersal. Furthermore, despite a critical requirement of dpp for the overall wing growth, neither Dpp dispersal nor direct signaling is critical for lateral wing growth after wing pouch specification. These results challenge the long-standing dogma that Dpp dispersal is strictly required to control and coordinate overall wing patterning and growth.

Distinct conformations of the HIV-1 V3 loop crown are targetable for broad neutralization.

The V3 loop of the HIV-1 envelope (Env) protein elicits a vigorous, but largely non-neutralizing antibody response directed to the V3-crown, whereas rare broadly neutralizing antibodies (bnAbs) target the V3-base. Challenging this view, we present V3-crown directed broadly neutralizing Designed Ankyrin Repeat Proteins (bnDs) matching the breadth of V3-base bnAbs. While most bnAbs target prefusion Env, V3-crown bnDs bind open Env conformations triggered by CD4 engagement. BnDs achieve breadth by focusing on highly conserved residues that are accessible in two distinct V3 conformations, one of which resembles CCR5-bound V3. We further show that these V3-crown conformations can, in principle, be attacked by antibodies. Supporting this conclusion, analysis of antibody binding activity in the Swiss 4.5 K HIV-1 cohort (n = 4,281) revealed a co-evolution of V3-crown reactivities and neutralization breadth. Our results indicate a role of V3-crown responses and its conformational preferences in bnAb development to be considered in preventive and therapeutic approaches.

Half-life extension of efficiently produced DARPin serum albumin fusions as a function of FcRn affinity and recycling.

Serum albumin shows slow clearance from circulation due to neonatal Fc receptor (FcRn)-mediated recycling and has been used for half-life extension. We report here fusions to a high-affinity DARPin, binding to Epithelial Cell Adhesion Molecule (EpCAM). We developed a novel, efficient expression system for such fusion proteins in Pichia pastoris with titers above 300 mg/L of lab-scale shake-flask culture. Since human serum albumin (HSA) does not bind to the murine FcRn, half-lives of therapeutic candidates are frequently measured in human FcRn transgenic mice, limiting useable tumor models. Additionally, serum albumins with extended half-life have been designed. We tested HSA7, motivated by its previously claimed extraordinarily long half-life in mice, which we could not confirm. Instead, we determined a half-life of only 29 h for HSA7, comparable to MSA. The fusion of HSA7 to a DARPin showed a similar half-life. To rationalize these findings, we measured binding kinetics and affinities to murine and human FcRn. Briefly, HSA7 showed affinity to murine FcRn only in the micromolar range, comparable to MSA to its cognate murine FcRn, and an affinity in the nanomolar range only to the human FcRn. This explains the comparable half-life of MSA and HSA7 in mice, while wild-type-HSA has a half-life of only 21 h, as it does not bind the murine FcRn and is not recycled. Thus, HSA-fusions with improved FcRn-affinity, such as HSA7, can be used for preclinical experiments in mice when FcRn transgenes cannot be used, as they reflect better the complex FcRn-mediated recycling and distribution mechanisms.

Design Guidelines for Database-Driven Internet of Things-Enabled Dynamic Spectrum Access.

The radio-frequency spectrum shortage, which is primarily caused by the fixed allocation policy, is one of the main bottlenecks to the deployment of existing wireless communication networks, and to the development of new ones. The dynamic spectrum access policy is foreseen as the solution to this problem, since it allows shared spectrum usage by primary licensed and secondary unlicensed networks. In order to turn this policy into reality, the secondary network must be capable of acquiring reliable, real-time information on available bands within the service area, which can be achieved by means of spectrum sensing, spectrum occupancy databases, or a combination of them. This Review presents guidelines related to the design of a framework that can be adopted to foster dynamic spectrum access policies. The framework applies special-purpose Internet of Things (IoT) devices that perform spectrum sensing, subsequently feeding a spectrum occupancy database, which in turn will be used by the secondary network to gather information on location-dependent spectrum availability. The guidelines address technological enablers capable of making the framework feasible, reliable and secure.

Designed Ankyrin Repeat Proteins as Novel Binders for Ultrasound Molecular Imaging.

Clinical translation of ultrasound molecular imaging will depend on the development of binders that can easily be generated, manufactured and coupled, and that are compatible with in vivo use. We describe targeted microbubbles (MBs) using designed ankyrin repeat proteins (DARPins) as a novel class of such translatable binders. Candidate DARPin binders for vascular cell adhesion molecule 1, an endothelial cell adhesion molecule involved in inflammatory processes, were selected using ribosome display and coupled to MBs. Flow-chamber assays of five MBs carrying high-affinity binders showed selective retention on endothelial cells activated by tumor necrosis factor-α for two binders compared with a MB carrying a control DARPin. In vivo ultrasound molecular imaging in a murine hind-limb inflammation model demonstrated up to a fourfold signal enhancement for three of the five MBs versus control. However, there was no correlation between results from flow-chamber assays and in vivo imaging. Thus, we conclude that ultrasound molecular imaging of inflammation using DARPin binders is feasible per se, but that screening of candidates cannot be accomplished with flow-chamber assays as used in our study.