Phosphorylation of the alpha-I motif in SYMRK drives root nodule organogenesis.

Symbiosis receptor-like kinase SYMRK is required for root nodule symbiosis between legume plants and nitrogen-fixing bacteria. To understand symbiotic signaling from SYMRK, we determined the crystal structure to 1.95 Å and mapped the phosphorylation sites onto the intracellular domain. We identified four serine residues in a conserved "alpha-I" motif, located on the border between the kinase core domain and the flexible C-terminal tail, that, when phosphorylated, drives organogenesis. Substituting the four serines with alanines abolished symbiotic signaling, while substituting them with phosphorylation-mimicking aspartates induced the formation of spontaneous nodules in the absence of bacteria. These findings show that the signaling pathway controlling root nodule organogenesis is mediated by SYMRK phosphorylation, which may help when engineering this trait into non-legume plants.

A glycan receptor kinase facilitates intracellular accommodation of arbuscular mycorrhiza and symbiotic rhizobia in the legume Lotus japonicus.

Receptors that distinguish the multitude of microbes surrounding plants in the environment enable dynamic responses to the biotic and abiotic conditions encountered. In this study, we identify and characterise a glycan receptor kinase, EPR3a, closely related to the exopolysaccharide receptor EPR3. Epr3a is up-regulated in roots colonised by arbuscular mycorrhizal (AM) fungi and is able to bind glucans with a branching pattern characteristic of surface-exposed fungal glucans. Expression studies with cellular resolution show localised activation of the Epr3a promoter in cortical root cells containing arbuscules. Fungal infection and intracellular arbuscule formation are reduced in epr3a mutants. In vitro, the EPR3a ectodomain binds cell wall glucans in affinity gel electrophoresis assays. In microscale thermophoresis (MST) assays, rhizobial exopolysaccharide binding is detected with affinities comparable to those observed for EPR3, and both EPR3a and EPR3 bind a well-defined ß-1,3/ß-1,6 decasaccharide derived from exopolysaccharides of endophytic and pathogenic fungi. Both EPR3a and EPR3 function in the intracellular accommodation of microbes. However, contrasting expression patterns and divergent ligand affinities result in distinct functions in AM colonisation and rhizobial infection in Lotus japonicus. The presence of Epr3a and Epr3 genes in both eudicot and monocot plant genomes suggest a conserved function of these receptor kinases in glycan perception.

Structure of the Wnt-Frizzled-LRP6 initiation complex reveals the basis for coreceptor discrimination.

Wnt morphogens are critical for embryonic development and tissue regeneration. Canonical Wnts form ternary receptor complexes composed of tissue-specific Frizzled (Fzd) receptors together with the shared LRP5/6 coreceptors to initiate ß-catenin signaling. The cryo-EM structure of a ternary initiation complex of an affinity-matured XWnt8-Frizzled8-LRP6 complex elucidates the basis of coreceptor discrimination by canonical Wnts by means of their N termini and linker domains that engage the LRP6 E1E2 domain funnels. Chimeric Wnts bearing modular linker "grafts" were able to transfer LRP6 domain specificity between different Wnts and enable non-canonical Wnt5a to signal through the canonical pathway. Synthetic peptides comprising the linker domain serve as Wnt-specific antagonists. The structure of the ternary complex provides a topological blueprint for the orientation and proximity of Frizzled and LRP6 within the Wnt cell surface signalosome.

Directed evolution identifies high-affinity cystine-knot peptide agonists and antagonists of Wnt/ß-catenin signaling.

Developing peptide-based tools to fine-tune growth signaling pathways, in particular molecules with exquisite selectivity and high affinities, opens up opportunities for cellular reprogramming in tissue regeneration. Here, we present a library based on cystine-knot peptides (CKPs) that incorporate multiple loops for randomization and selection via directed evolution. Resulting binders could be assembled into multimeric structures to fine-tune cellular signaling. An example is presented for the Wnt pathway, which plays a key role in the homeostasis and regeneration of tissues such as lung, skin, and intestine. We discovered picomolar affinity CKP agonists of the human LPR6 receptor by exploring the limits of the topological manipulation of LRP6 dimerization. Structural analyses revealed that the agonists bind at the first ß-propeller domain of LRP6, mimicking the natural Wnt inhibitors DKK1 and SOST. However, the CKP agonists exhibit a different mode of action as they amplify the signaling of natural Wnt ligands but do not activate the pathway by themselves. In an alveolosphere organoid model, the CKP agonists induced alveolar stem cell activity. They also stimulated growth in primary human intestinal organoids. The approach described here advances the important frontier of next-generation agonist design and could be applied to other signaling pathways to discover tunable agonist ligands.

A selective peptide inhibitor of Frizzled 7 receptors disrupts intestinal stem cells.

Regeneration of the adult intestinal epithelium is mediated by a pool of cycling stem cells, which are located at the base of the crypt, that express leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5). The Frizzled (FZD) 7 receptor (FZD7) is enriched in LGR5+ intestinal stem cells and plays a critical role in their self-renewal. Yet, drug discovery approaches and structural bases for targeting specific FZD isoforms remain poorly defined. FZD proteins interact with Wnt signaling proteins via, in part, a lipid-binding groove on the extracellular cysteine-rich domain (CRD) of the FZD receptor. Here we report the identification of a potent peptide that selectively binds to the FZD7 CRD at a previously uncharacterized site and alters the conformation of the CRD and the architecture of its lipid-binding groove. Treatment with the FZD7-binding peptide impaired Wnt signaling in cultured cells and stem cell function in intestinal organoids. Together, our data illustrate that targeting the lipid-binding groove holds promise as an approach for achieving isoform-selective FZD receptor inhibition.

Publisher Correction: A selective peptide inhibitor of Frizzled 7 receptors disrupts intestinal stem cells.

The version of this article originally published contained older versions of the Life Sciences Reporting Summary and the Supplementary Text and Figures. The error has been corrected in the HTML and PDF versions of the article.

Modulation of Exercise-Induced Hypoalgesia Following an Exercise Intervention in Healthy Subjects.

BACKGROUND: Exercise is recommended to promote and maintain health and as treatment for more than 25 diseases and pain conditions. Exercise-induced hypoalgesia (EIH), a measure of descending pain inhibitory control, has been found to be impaired in some chronic pain conditions, but it is currently unclear if EIH is modifiable. This study investigated whether a long-term exercise intervention could modulate EIH in healthy subjects. METHODS: In 38 healthy subjects, EIH was assessed as change in pressure pain threshold (PPT) after a three-minute isometric wall squat within the first week and after approximately seven weeks of military training (MT). Further, temporal summation of pain (TSP) and Knee injury and Osteoarthritis Outcome Score (KOOS) were assessed. Physical performance capacity was assessed using the Endurance 20-m shuttle run fitness test (20MSR). Hypoalgesic (EIH > 0.0 kPa) and hyperalgesic (EIH ≤ 0.0 kPa) subgroups were defined based on baseline EIH. Change in EIH following MT was used as the primary outcome. RESULTS: Increased EIH (P = 0.008), PPT (P < 0.003), and 20MSR (P < 0.001) were found following MT, with no changes in TSP and KOOS (P > 0.05). Subjects with a hyperalgesic EIH response at baseline (26% of the participants) presented significantly improved EIH following MT (P = 0.010). Finally, an association between 20MRS change and EIH change was found (r = 0.369, P = 0.023). CONCLUSIONS: MT increased EIH, especially in subjects who demonstrated a hyperalgesic response at baseline. Improvement in physical performance capacity was associated with an improvement in EIH, indicating that improvement in physical performance capacity may improve central pain mechanisms.

Curvature of designed armadillo repeat proteins allows modular peptide binding.

Designed armadillo repeat proteins (dArmRPs) were developed to create a modular peptide binding technology where each of the structural repeats binds two residues of the target peptide. An essential prerequisite for such a technology is a dArmRP geometry that matches the peptide bond length. To this end, we determined a large set (n=27) of dArmRP X-ray structures, of which 12 were previously unpublished, to calculate curvature parameters that define their geometry. Our analysis shows that consensus dArmRPs exhibit curvatures close to the optimal range for modular peptide recognition. Binding of peptide ligands can induce a curvature within the desired range, as confirmed by single-molecule FRET experiments in solution. On the other hand, computationally designed ArmRPs, where side chains have been chosen with the intention to optimally fit into a geometrically optimized backbone, turned out to be more divergent in reality, and thus not suitable for continuous peptide binding. Furthermore, we show that the formation of a crystal lattice can induce small but significant deviations from the curvature adopted in solution, which can interfere with the evaluation of repeat protein scaffolds when high accuracy is required. This study corroborates the suitability of consensus dArmRPs as a scaffold for the development of modular peptide binders.

Structure and Energetic Contributions of a Designed Modular Peptide-Binding Protein with Picomolar Affinity.

Natural armadillo repeat proteins (nArmRP) like importin-α or ß-catenin bind their target peptides such that each repeat interacts with a dipeptide unit within the stretched target peptide. However, this modularity is imperfect and also restricted to short peptide stretches of usually four to six consecutive amino acids. Here we report the development and characterization of a regularized and truly modular peptide-specific binding protein, based on designed armadillo repeat proteins (dArmRP), binding to peptides of alternating lysine and arginine residues (KR)n. dArmRP were obtained from nArmRP through cycles of extensive protein engineering, which rendered them more uniform. This regularity is reflected in the consistent binding of dArmRP to (KR)-peptides, where affinities depend on the lengths of target peptides and the number of internal repeats in a very systematic manner, thus confirming the modularity of the interaction. This exponential dependency between affinity and recognition length suggests that each module adds a constant increment of binding energy to sequence-specific recognition. This relationship was confirmed by comprehensive mutagenesis studies that also reveal the importance of individual peptide side chains. The 1.83 Å resolution crystal structure of a dArmRP with five identical internal repeats in complex with the cognate (KR)5 peptide proves a modular binding mode, where each dipeptide is recognized by one internal repeat. The confirmation of this true modularity over longer peptide stretches lays the ground for the design of binders with different specificities and tailored affinities by the assembly of dipeptide-specific modules based on armadillo repeats.

G-quadruplexes are specifically recognized and distinguished by selected designed ankyrin repeat proteins.

We introduce designed ankyrin repeat binding proteins (DARPins) as a novel class of highly specific and structure-selective DNA-binding proteins, which can be functionally expressed within all cells. Human telomere quadruplex was used as target to select specific binders with ribosome display. The selected DARPins discriminate the human telomere quadruplex against the telomeric duplex and other quadruplexes. Affinities of the selected binders range from 3 to 100 nM. CD studies confirm that the quadruplex fold is maintained upon binding. The DARPins show different specificity profiles: some discriminate human telomere quadruplexes from other quadruplex-forming sequences like ILPR, c-MYC and c-KIT, while others recognize two of the sequences tested or even all quadruplexes. None of them recognizes dsDNA. Quadruplex-binding DARPins constitute valuable tools for specific detection at very small scales and for the in vivo investigation of quadruplex DNA.

Modular peptide binding: from a comparison of natural binders to designed armadillo repeat proteins.

Several binding scaffolds that are not based on immunoglobulins have been designed as alternatives to traditional monoclonal antibodies. Many of them have been developed to bind to folded proteins, yet cellular networks for signaling and protein trafficking often depend on binding to unfolded regions of proteins. This type of binding can thus be well described as a peptide-protein interaction. In this review, we compare different peptide-binding scaffolds, highlighting that armadillo repeat proteins (ArmRP) offer an attractive modular system, as they bind a stretch of extended peptide in a repeat-wise manner. Instead of generating each new binding molecule by an independent selection, preselected repeats - each complementary to a piece of the target peptide - could be designed and assembled on demand into a new protein, which then binds the prescribed complete peptide. Stacked armadillo repeats (ArmR), each typically consisting of 42 amino acids arranged in three α-helices, build an elongated superhelical structure which enables binding of peptides in extended conformation. A consensus-based design approach, complemented with molecular dynamics simulations and rational engineering, resulted in well-expressed monomeric proteins with high stability. Peptide binders were selected and several structures were determined, forming the basis for the future development of modular peptide-binding scaffolds.

Synthetic Multivalent Disulfide-Constrained Peptide Agonists Potentiate Wnt1/ß-Catenin Signaling via LRP6 Coreceptor Clustering.

Wnt ligands are critical for tissue homeostasis and form a complex with LRP6 and frizzled coreceptors to initiate Wnt/ß-catenin signaling. Yet, how different Wnts achieve various levels of signaling activation through distinct domains on LRP6 remains elusive. Developing tool ligands that target individual LRP6 domains could help elucidate the mechanism of Wnt signaling regulation and uncover pharmacological approaches for pathway modulation. We employed directed evolution of a disulfide constrained peptide (DCP) to identify molecules that bind to the third ß-propeller domain of LRP6. The DCPs antagonize Wnt3a while sparing Wnt1 signaling. Using PEG linkers with different geometries, we converted the Wnt3a antagonist DCPs to multivalent molecules that potentiated Wnt1 signaling by clustering the LRP6 coreceptor. The mechanism of potentiation is unique as it occurred only in the presence of extracellular secreted Wnt1 ligand. While all DCPs recognized a similar binding interface on LRP6, they displayed different spatial orientations that influenced their cellular activities. Moreover, structural analyses revealed that the DCPs exhibited new folds that were distinct from the parent DCP framework they were evolved from. The multivalent ligand design principles highlighted in this study provide a path for developing peptide agonists that modulate different branches of cellular Wnt signaling.

Acute exercise of painful muscles does not reduce the hypoalgesic response in young healthy women - a randomized crossover study.

OBJECTIVES: Exercise-induced hypoalgesia (EIH) is characterized by an increase in pain threshold following acute exercise. EIH is reduced in some individuals with chronic musculoskeletal pain, although the mechanisms are unknown. It has been hypothesized that this may relate to whether exercises are performed in painful or non-painful body regions. The primary aim of this randomized experimental crossover study was to investigate whether the presence of pain per se in the exercising muscles reduced the local EIH response. The secondary aim was to investigate if EIH responses were also reduced in non-exercising remote muscles. METHODS: Pain-free women (n=34) participated in three separate sessions. In session 1, the maximal voluntary contraction (MVC) for a single legged isometric knee extension exercise was determined. In sessions 2 and 3, pressure pain thresholds (PPT) were assessed at the thigh and shoulder muscles before and after a 3-min exercise at 30 % of MVC. Exercises were performed with or without thigh muscle pain, which was induced by either a painful injection (hypertonic saline, 5.8 %) or a non-painful injection (isotonic saline, 0.9 %) into the thigh muscle. Muscle pain intensity was assessed with an 11-point numerical rating scale (NRS) at baseline, after injections, during and after exercises. RESULTS: PPTs increased at thigh and shoulder muscles after exercise with painful (14.0-24.9 %) and non-painful (14.3-19.5 %) injections and no significant between-injection EIH differences were observed (p>0.30). Muscle pain intensity was significantly higher following the painful injection compared to the non-painful injection (p<0.001). CONCLUSIONS: Exercising painful muscles did not reduce the local or remote hypoalgesic responses, suggesting that the pain-relieving effects of isometric exercises are not reduced by exercising painful body regions. ETHICAL COMMITTEE NUMBER: S-20210184. TRIAL REGISTRATION NUMBER: NCT05299268.

Nanobody-driven signaling reveals the core receptor complex in root nodule symbiosis.

Understanding the composition and activation of multicomponent receptor complexes is a challenge in biology. To address this, we developed a synthetic approach based on nanobodies to drive assembly and activation of cell surface receptors and apply the concept by manipulating receptors that govern plant symbiosis with nitrogen-fixing bacteria. We show that the Lotus japonicus Nod factor receptors NFR1 and NFR5 constitute the core receptor complex initiating the cortical root nodule organogenesis program as well as the epidermal program controlling infection. We find that organogenesis signaling is mediated by the intracellular kinase domains whereas infection requires functional ectodomains. Finally, we identify evolutionarily distant barley receptors that activate root nodule organogenesis, which could enable engineering of biological nitrogen-fixation into cereals.

Membrane fouling monitoring by 3ω sensing.

Membrane fouling significantly reduces membrane permeability, leading to higher operational expenses. In situ monitoring of membrane fouling can potentially be used to reduce operation cost by optimizing operational parameters and cleaning conditions. In this study, a platinum wire with a diameter of 20 µm was attached to the surface of a ceramic ultrafiltration membrane, and by measuring the voltage across the wire while applying an AC current, the amplitude of the third harmonic wave, the so-called 3ω signal, was obtained. Results showed increasing 3ω signals during formation of fouling layers, which correlates directly to the hydraulic resistance of the formed fouling layer in semi-dead end filtration of polymeric core shell particles and crossflow filtration of diluted milk. This is explained by the insulating effect of the fouling layers which reduces heat convection by crossflow and the different thermal conductivity in the fouling layer compared with the feed. After membrane cleaning, the permeability and the magnitude of the 3ω signal were partly restored, showing that the 3ω method can be used to monitor the effect of cleaning. The frequency of the AC current was varied so it was possible to measure the heat conductivity in the fouling layer (high frequency) and heat convection due to cross-flow (low frequency). This may potentially be used to get information of the type of fouling (heat conductivity) and thickness of the fouling layer (AC frequency where heat conductivity becomes dominating).

Senescent cells perturb intestinal stem cell differentiation through Ptk7 induced noncanonical Wnt and YAP signaling.

Cellular senescence and the senescence-associated secretory phenotype (SASP) are implicated in aging and age-related disease, and SASP-related inflammation is thought to contribute to tissue dysfunction in aging and diseased animals. However, whether and how SASP factors influence the regenerative capacity of tissues remains unclear. Here, using intestinal organoids as a model of tissue regeneration, we show that SASP factors released by senescent fibroblasts deregulate stem cell activity and differentiation and ultimately impair crypt formation. We identify the secreted N-terminal domain of Ptk7 as a key component of the SASP that activates non-canonical Wnt / Ca2+ signaling through FZD7 in intestinal stem cells (ISCs). Changes in cytosolic [Ca2+] elicited by Ptk7 promote nuclear translocation of YAP and induce expression of YAP/TEAD target genes, impairing symmetry breaking and stem cell differentiation. Our study discovers secreted Ptk7 as a factor released by senescent cells and provides insight into the mechanism by which cellular senescence contributes to tissue dysfunction in aging and disease.

Introducing Cysteines into Nanobodies for Site-Specific Labeling.

We have developed a generally applicable methodology for cysteine mutagenesis of nanobody (Nb) framework region serine residues. This strategy allows for subsequent labeling with thiol-reactive compounds without disrupting Nb antigen binding. We provide a protocol for production, labeling, and affinity determination of cysteine-engineered Nbs (cys-Nbs) with Alexa Fluor 488-maleimide and the mercury compound para-chloromercuribenzoic acid (PCMB). Alexa Fluor 488- and PCMB-labeled cys-Nbs can be used for immunofluorescence microscopy and experimental phasing in crystallography, respectively.

Hypoalgesia after exercises with painful vs. non-painful muscles in healthy subjects - a randomized cross-over study.

OBJECTIVES: Exercise-induced hypoalgesia (EIH) is a decrease in the pain sensitivity after exercise. Individuals with chronic pain show less EIH after one exercise session compared with pain-free individuals possibly due to pain in exercising muscles. The primary aim of this randomized controlled cross-over study was to compare the EIH response at the exercising thigh muscle following exercises performed with painful vs. non-painful muscles. Secondary aims were to explore if a reduced EIH response was confined to the painful muscle, and whether the muscle pain intensity and the EIH responses were negatively associated. METHODS: In two sessions, 34 pain-free participants received a painful (hypertonic saline, 5.8%) injection and a control (isotonic saline, 0.9%) injection in the right thigh muscle before performing a 3 min isometric wall squat exercise. Pressure pain thresholds (PPTs) were assessed at both thighs and the left neck/shoulder at baseline, after injections and after exercise. Pain intensities in the thighs were rated on numerical rating scales (NRS: 0-10). RESULTS: Hypertonic saline induced moderate thigh pain at rest (NRS: 4.6 ± 2.1) compared to the control injection (NRS: 0.3 ± 0.4; p<0.001). EIH at the thighs and neck/shoulder were not different between sessions (Injected thigh: 0 kPa; 95% CI: -51 to 52; Contralateral thigh: -6 kPa; 95% CI: -42 to 30; neck/shoulder: 19 kPa; 95% CI: -6 to 44). No significant associations between pain intensity ratings immediately after the Painful injection and EIH responses at any assessment sites were found (right thigh: ß=0.08, 95% CI: -12.95 to 20.64, p=0.64, left thigh: ß=-0.33, 95% CI: -27.86 to 0.44, p=0.06; neck/shoulder: ß=-0.18, 95% CI: -15.11 to 4.96, p=0.31). CONCLUSIONS: Pain in the area of an exercising muscle did not reduce local or systemic EIH responses. TRIAL REGISTRATION NUMBER: NCT04354948.

Immunohistochemical expression of fibronectin and C5b-9 in the myocardium in cases of fatal ethanol intoxication.

Data from the literature indicate that the pulmonary pressure rises in cases of ethanol intake. We have recently proposed a method for the detection of prevalent right ventricular damage in cases of fatal pulmonary thromboembolism and pulmonary fat embolism. In the present study, we compared the expression of the antibodies against fibronectin and C5b-9 in 19 cases of lethal alcohol intoxications (study group: 5 females, 14 males, mean age 46 years, mean blood ethanol concentration 3.5‰, min. 2.11‰, max. 5.31‰) to a group of 26 cases of fatal pulmonary thromboembolism (PE; group 2: 16 females, 10 males, mean age 56 years). Moreover, a group of 15 cases of hanging (group 3: 5 females, 10 males, mean age 50 years) as well as a group of 18 cases of myocardial infarction (group 4: 5 females, 13 males, mean age 61 years) were investigated as examples of typical cardiac damage due to global hypoxia during agony and ischemic damage, respectively. The results of this study show that fresh cardiac damage can be detected at both ventricles in cases of fatal ethanol intoxication with the antibody against fibronectin. The damage is prevalently localised at the right ventricle (RV), as already observed in cases of acute pulmonary hypertension determining right heart failure. The degree of damage at the RV in cases of ethanol intoxications is lower than the one observed in cases of fatal PE.

MANAGEMENT OF ENDOCRINE DISEASE: Glucocorticoid-induced adrenal insufficiency: replace while we wait for evidence?

Glucocorticoids are, besides non-steroidal anti-inflammatory drugs, the most widely used anti-inflammatory medications. Prevalence studies indicate substantial use of both systemic and locally acting agents. A recognised adverse effect of glucocorticoid treatment is adrenal insufficiency, which is highly prevalent based on biochemical testing, but its clinical implications are poorly understood. Current evidence, including randomised trials and observational studies, indicates substantial variation among patients in both risk and course of glucocorticoid-induced adrenal insufficiency, but both are currently unpredictable. Oral and intra-articular formulations, as well as long-term and high-dose treatments, carry the highest risk of glucocorticoid-induced adrenal insufficiency defined by biochemical tests. However, no route of administration, treatment duration, or dose can be considered without risk. More research is needed to estimate the risk and temporal pattern of glucocorticoid-induced adrenal insufficiency, to investigate its clinical implications, and to identify predictors of risk and prognosis. Randomized trials are required to evaluate whether hydrocortisone replacement therapy mitigates risk and symptoms of glucocorticoid-induced adrenal insufficiency in patients discontinuing glucocorticoid treatment. This review aims to provide an overview of the available evidence, pointing to knowledge gaps and unmet needs.