Targeting biased signaling by PAR1: function and molecular mechanism of parmodulins.

The G protein-coupled receptor (GPCR) protease-activated receptor 1 (PAR1) is a therapeutic target that was originally pursued with the aim of restricting platelet activation and the burden of cardiovascular diseases. In clinical studies, the use of orthosteric PAR1 inhibitors was associated with an increased risk of hemorrhage, including intracranial hemorrhage. Because (1) PAR1 is expressed by various cell types, including endothelial cells, (2) conveys in mice a physiological indispensable function for vascular development during embryogenesis, and (3) is subject to biased signaling dependent on the activating proteases, orthosteric PAR1 inhibition may be associated with unwanted side effects. Alternatively, the protease-activated protein C (aPC) and its variants can promote valuable anti-inflammatory signaling via PAR1. Most recently, small molecule allosteric modulators of PAR1 signaling, called parmodulins, have been developed. Parmodulins inhibit coagulation and platelet activation yet maintain cytoprotective effects typically provoked by PAR1 signaling upon the activation by aPC. In this study, we review the discovery of parmodulins and their preclinical data, summarize the current knowledge about their mode of action, and compare the structural interaction of parmodulin and PAR1 with that of other intracellularly binding allosteric GPCR modulators. Thus, we highlight the pharmaceutical potential and challenges associated with the future development of parmodulins.

Glomerular-tubular crosstalk via cold shock Y-box binding protein-1 in the kidney.

Glomerular-tubular crosstalk within the kidney has been proposed, but the paracrine signals enabling this remain largely unknown. The cold-shock protein Y-box binding protein 1 (YBX1) is known to regulate inflammation and kidney diseases but its role in podocytes remains undetermined. Therefore, we analyzed mice with podocyte specific Ybx1 deletion (Ybx1ΔPod). Albuminuria was increased in unchallenged Ybx1ΔPod mice, which surprisingly was associated with reduced glomerular, but enhanced tubular damage. Tubular toll-like receptor 4 (TLR4) expression, node-like receptor protein 3 (NLRP3) inflammasome activation and kidney inflammatory cell infiltrates were all increased in Ybx1ΔPod mice. In vitro, extracellular YBX1 inhibited NLRP3 inflammasome activation in tubular cells. Co-immunoprecipitation, immunohistochemical analyses, microscale cell-free thermophoresis assays, and blunting of the YBX1-mediated TLR4-inhibition by a unique YBX1-derived decapeptide suggests a direct interaction of YBX1 and TLR4. Since YBX1 can be secreted upon post-translational acetylation, we hypothesized that YBX1 secreted from podocytes can inhibit TLR4 signaling in tubular cells. Indeed, mice expressing a non-secreted YBX1 variant specifically in podocytes (Ybx1PodK2A mice) phenocopied Ybx1ΔPod mice, demonstrating a tubular-protective effect of YBX1 secreted from podocytes. Lipopolysaccharide-induced tubular injury was aggravated in Ybx1ΔPod and Ybx1PodK2A mice, indicating a pathophysiological relevance of this glomerular-tubular crosstalk. Thus, our data show that YBX1 is physiologically secreted from podocytes, thereby negatively modulating sterile inflammation in the tubular compartment, apparently by binding to and inhibiting tubular TLR4 signaling. Hence, we have uncovered an YBX1-dependent molecular mechanism of glomerular-tubular crosstalk.

Detecting Protein-Ligand Interactions with Nitroxide Based Paramagnetic Cosolutes.

NMR spectroscopy techniques can provide important information about protein-ligand interactions. Here we tested an NMR approach which relies on the measurement of paramagnetic relaxation enhancements (PREs) arising from analogous cationic, anionic or neutral soluble nitroxide molecules, which distribute around the protein-ligand complex depending on near-surface electrostatic potentials. We applied this approach to two protein-ligand systems, interleukin-8 interacting with highly charged glycosaminoglycans and the SH2 domain of Grb2 interacting with less charged phospho-tyrosine tripeptides. The electrostatic potential around interleukin-8 and its changes upon binding of glycosaminoglycans could be derived from the PRE data and confirmed by theoretical predictions from Poisson-Boltzmann calculations. The ligand influence on the PREs and NMR-derived electrostatic potentials of Grb2 SH2 was localized to a narrow protein region which allowed the localization of the peptide binding pocket. Our analysis suggests that experiments with nitroxide cosolutes can be useful for investigating protein-ligand electrostatic interactions and mapping ligand binding sites.

Sparse pseudocontact shift NMR data obtained from a non-canonical amino acid-linked lanthanide tag improves integral membrane protein structure prediction.

A single experimental method alone often fails to provide the resolution, accuracy, and coverage needed to model integral membrane proteins (IMPs). Integrating computation with experimental data is a powerful approach to supplement missing structural information with atomic detail. We combine RosettaNMR with experimentally-derived paramagnetic NMR restraints to guide membrane protein structure prediction. We demonstrate this approach using the disulfide bond formation protein B (DsbB), an α-helical IMP. Here, we attached a cyclen-based paramagnetic lanthanide tag to an engineered non-canonical amino acid (ncAA) using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry reaction. Using this tagging strategy, we collected 203 backbone HN pseudocontact shifts (PCSs) for three different labeling sites and used these as input to guide de novo membrane protein structure prediction protocols in Rosetta. We find that this sparse PCS dataset combined with 44 long-range NOEs as restraints in our calculations improves structure prediction of DsbB by enhancements in model accuracy, sampling, and scoring. The inclusion of this PCS dataset improved the Cα-RMSD transmembrane segment values of the best-scoring and best-RMSD models from 9.57 Å and 3.06 Å (no NMR data) to 5.73 Å and 2.18 Å, respectively.

[Contrast-enhanced ultrasound (CEUS) for characterisation of focal liver lesions]. / Kontrastverstärkte Sonografie (CEUS) zur Diagnostik fokaler Leberläsionen.

Due to the trend towards increased use of imaging and rising awareness among high-risk patients, gastroenterologists and hepatologists are more frequently confronted with patients with focal liver lesions. In the differentiation of these lesions, CT and MRI have increasingly found their way into primary diagnostic steps in everyday clinical practice. Contrast-enhanced sonography, on the other hand, is a very effective and cost-efficient method for assessing focal liver lesions. The success of the method is not only based on the visualisation of microvascularisation in real time. If sonography is performed by the treating physician, he can use the exact knowledge of history and clinical findings to specifically adapt the examination procedure and to interpret the sonographic findings with greater accuracy ("clinical sonography"). At the same time, the method enables the practitioner to combine diagnostics and management decisions in his or her own hands. To achieve excellent results with contrast-enhanced sonography-as with any other imaging method-it is necessary that the examiner is sufficiently qualified.This article systematically presents the sonographic characteristics of the most common liver lesions and clearly shows their contrast patterns using videos (available via QR code). The article illustrates that CEUS could-and from the authors' point of view, should-have an even greater significance in the future.

Recent Advances in Computer-Aided Structure-Based Drug Design on Ion Channels.

Ion channels play important roles in fundamental biological processes, such as electric signaling in cells, muscle contraction, hormone secretion, and regulation of the immune response. Targeting ion channels with drugs represents a treatment option for neurological and cardiovascular diseases, muscular degradation disorders, and pathologies related to disturbed pain sensation. While there are more than 300 different ion channels in the human organism, drugs have been developed only for some of them and currently available drugs lack selectivity. Computational approaches are an indispensable tool for drug discovery and can speed up, especially, the early development stages of lead identification and optimization. The number of molecular structures of ion channels has considerably increased over the last ten years, providing new opportunities for structure-based drug development. This review summarizes important knowledge about ion channel classification, structure, mechanisms, and pathology with the main focus on recent developments in the field of computer-aided, structure-based drug design on ion channels. We highlight studies that link structural data with modeling and chemoinformatic approaches for the identification and characterization of new molecules targeting ion channels. These approaches hold great potential to advance research on ion channel drugs in the future.

Improving the Modeling of Extracellular Ligand Binding Pockets in RosettaGPCR for Conformational Selection.

G protein-coupled receptors (GPCRs) are the largest class of drug targets and undergo substantial conformational changes in response to ligand binding. Despite recent progress in GPCR structure determination, static snapshots fail to reflect the conformational space of putative binding pocket geometries to which small molecule ligands can bind. In comparative modeling of GPCRs in the absence of a ligand, often a shrinking of the orthosteric binding pocket is observed. However, the exact prediction of the flexible orthosteric binding site is crucial for adequate structure-based drug discovery. In order to improve ligand docking and guide virtual screening experiments in computer-aided drug discovery, we developed RosettaGPCRPocketSize. The algorithm creates a conformational ensemble of biophysically realistic conformations of the GPCR binding pocket between the TM bundle, which is consistent with a knowledge base of expected pocket geometries. Specifically, tetrahedral volume restraints are defined based on information about critical residues in the orthosteric binding site and their experimentally observed range of Cα-Cα-distances. The output of RosettaGPCRPocketSize is an ensemble of binding pocket geometries that are filtered by energy to ensure biophysically probable arrangements, which can be used for docking simulations. In a benchmark set, pocket shrinkage observed in the default RosettaGPCR was reduced by up to 80% and the binding pocket volume range and geometric diversity were increased. Compared to models from four different GPCR homology model databases (RosettaGPCR, GPCR-Tasser, GPCR-SSFE, and GPCRdb), the here-created models showed more accurate volumes of the orthosteric pocket when evaluated with respect to the crystallographic reference structure. Furthermore, RosettaGPCRPocketSize was able to generate an improved realistic pocket distribution. However, while being superior to other homology models, the accuracy of generated model pockets was comparable to AlphaFold2 models. Furthermore, in a docking benchmark using small-molecule ligands with a higher molecular weight between 400 and 700 Da, a higher success rate in creating native-like binding poses was observed. In summary, RosettaGPCRPocketSize can generate GPCR models with realistic orthosteric pocket volumes, which are useful for structure-based drug discovery applications.

Recent Advances in NMR Protein Structure Prediction with ROSETTA.

Nuclear magnetic resonance (NMR) spectroscopy is a powerful method for studying the structure and dynamics of proteins in their native state. For high-resolution NMR structure determination, the collection of a rich restraint dataset is necessary. This can be difficult to achieve for proteins with high molecular weight or a complex architecture. Computational modeling techniques can complement sparse NMR datasets (<1 restraint per residue) with additional structural information to elucidate protein structures in these difficult cases. The Rosetta software for protein structure modeling and design is used by structural biologists for structure determination tasks in which limited experimental data is available. This review gives an overview of the computational protocols available in the Rosetta framework for modeling protein structures from NMR data. We explain the computational algorithms used for the integration of different NMR data types in Rosetta. We also highlight new developments, including modeling tools for data from paramagnetic NMR and hydrogen-deuterium exchange, as well as chemical shifts in CS-Rosetta. Furthermore, strategies are discussed to complement and improve structure predictions made by the current state-of-the-art AlphaFold2 program using NMR-guided Rosetta modeling.

Investigation of the structure of regulatory proteins interacting with glycosaminoglycans by combining NMR spectroscopy and molecular modeling - the beginning of a wonderful friendship.

The interaction of regulatory proteins with extracellular matrix or cell surface-anchored glycosaminoglycans (GAGs) plays important roles in molecular recognition, wound healing, growth, inflammation and many other processes. In spite of their high biological relevance, protein-GAG complexes are significantly underrepresented in structural databases because standard tools for structure determination experience difficulties in studying these complexes. Co-crystallization with subsequent X-ray analysis is hampered by the high flexibility of GAGs. NMR spectroscopy experiences difficulties related to the periodic nature of the GAGs and the sparse proton network between protein and GAG with distances that typically exceed the detection limit of nuclear Overhauser enhancement spectroscopy. In contrast, computer modeling tools have advanced over the last years delivering specific protein-GAG docking approaches successfully complemented with molecular dynamics (MD)-based analysis. Especially the combination of NMR spectroscopy in solution providing sparse structural constraints with molecular docking and MD simulations represents a useful synergy of forces to describe the structure of protein-GAG complexes. Here we review recent methodological progress in this field and bring up examples where the combination of new NMR methods along with cutting-edge modeling has yielded detailed structural information on complexes of highly relevant cytokines with GAGs.

Contrast-Enhanced Ultrasound Algorithms (CEUS-LIRADS/ESCULAP) for the Noninvasive Diagnosis of Hepatocellular Carcinoma - A Prospective Multicenter DEGUM Study.

BACKGROUND: This prospective multicenter study funded by the DEGUM assesses the diagnostic accuracy of standardized contrast-enhanced ultrasound (CEUS) for the noninvasive diagnosis of hepatocellular carcinoma (HCC) in high-risk patients. METHODS: Patients at high risk for HCC with a histologically proven focal liver lesion on B-mode ultrasound were recruited prospectively in a multicenter approach. Clinical and imaging data were entered via online entry forms. The diagnostic accuracies for the noninvasive diagnosis of HCC were compared for the conventional interpretation of standardized CEUS at the time of the examination (= CEUS on-site) and the two CEUS algorithms ESCULAP (Erlanger Synopsis for Contrast-enhanced Ultrasound for Liver lesion Assessment in Patients at risk) and CEUS LI-RADS (Contrast-Enhanced UltraSound Liver Imaging Reporting and Data System). RESULTS: 321 patients were recruited in 43 centers; 299 (93.1 %) had liver cirrhosis. The diagnosis according to histology was HCC in 256 cases, and intrahepatic cholangiocarcinoma (iCCA) in 23 cases. In the subgroup of cirrhotic patients (n = 299), the highest sensitivity for the diagnosis of HCC was achieved with the CEUS algorithm ESCULAP (94.2 %) and CEUS on-site (90.9 %). The lowest sensitivity was reached with the CEUS LI-RADS algorithm (64 %; p < 0.001). However, the specificity of CEUS LI-RADS (78.9 %) was superior to that of ESCULAP (50.9 %) and CEUS on-site (64.9 %; p < 0.001). At the same time, the negative predictive value (NPV) of CEUS LI-RADS was significantly inferior to that of ESCULAP (34.1 % vs. 67.4 %; p < 0.001) and CEUS on-site (62.7 %; p < 0.001). The positive predictive values of all modalities were high (around 90 %), with the best results seen for CEUS LI-RADS and CEUS on-site. CONCLUSION: This is the first multicenter, prospective comparison of standardized CEUS and the recently developed CEUS-based algorithms in histologically proven liver lesions in cirrhotic patients. Our results reaffirm the excellent diagnostic accuracy of CEUS for the noninvasive diagnosis of HCC in high-risk patients. However, on-site diagnosis by an experienced examiner achieves an almost equal diagnostic accuracy compared to CEUS-based diagnostic algorithms.

Clinical Ultrasound in Inflammatory Bowel Disease.

Ultrasound should be the first-line imaging method in the medical care of patients with inflammatory bowel disease. It can be used to differentiate between Crohn's disease and ulcerative colitis and to detect numerous complications like stenoses, fistulas, and abscesses. As the method is also highly suitable for follow-up, stressful endoscopic procedures and examinations involving radiation are less necessary. Comparison studies with MRI and CT show that ultrasound is not an inferior method and it is therefore included in all guidelines. Intensifying physician training in this method will help to ensure its comprehensive use.

Identification of the Glycosaminoglycan Binding Site of Interleukin-10 by NMR Spectroscopy.

The biological function of interleukin-10 (IL-10), a pleiotropic cytokine with an essential role in inflammatory processes, is known to be affected by glycosaminoglycans (GAGs). GAGs are highly negatively charged polysaccharides and integral components of the extracellular matrix with important functions in the biology of many growth factors and cytokines. The molecular mechanism of the IL-10/GAG interaction is unclear. In particular, experimental evidence about IL-10/GAG binding sites is lacking, despite its importance for understanding the biological role of the interaction. Here, we report the experimental determination of a GAG binding site of IL-10. Although no co-crystal structure of the IL-10·GAG complex could be obtained, its structural characterization was possible by NMR spectroscopy. Chemical shift perturbations of IL-10 induced by GAG binding were used to narrow down the location of the binding site and to assess the affinity for different GAG molecules. Subsequent observation of NMR pseudocontact shifts of IL-10 and its heparin ligand, as induced by a protein-attached lanthanide spin label, provided structural restraints for the protein·ligand complex. Using these restraints, pseudocontact shift-based rigid body docking together with molecular dynamics simulations yielded a GAG binding model. The heparin binding site is located at the C-terminal end of helix D and the adjacent DE loop and coincides with a patch of positively charged residues involving arginines 102, 104, 106, and 107 and lysines 117 and 119. This study represents the first experimental characterization of the IL-10·GAG complex structure and provides the starting point for revealing the biological significance of the interaction of IL-10 with GAGs.

Lymph Vessel Thrombus Detection in Cervical Lymphedema: A Case Series.

Although thrombosis in the terminal part of thoracic duct has been described in patients with head and neck cancer and upper vein thrombosis, normally medical imaging is not successful in depicting such lymphatic thrombosis. This case series is about three patients with acute onset of cervical lymphedema following minor trauma or exceptional physical strain. Using high-resolution ultrasound, it was possible to detect a thrombus formation in the terminal parts of the thoracic duct or the lymph duct. Within a few weeks all patients recovered without relapse for several years.

Chemoenzymatic Synthesis of Nonasulfated Tetrahyaluronan with a Paramagnetic Tag for Studying Its Complex with Interleukin-10.

Implants and artificial biomaterials containing sulfated hyaluronans have been shown to improve the healing of injured skin and bones. It is hypothesized that these effects are mediated by the binding of sulfated glycosaminoglycans (GAGs) to growth factors and cytokines, resulting in the sequestering of proteins to the wound healing site and in modulated protein activity. Given that no direct synthetic access to sulfated oligohyaluronans has been available, little is known about their protein binding and the structure of the resulting protein complexes. Here, the chemoenzymatic preparation of oligohyaluronans on the gram scale is described. Oligohyaluronans are converted into anomeric azides at the reducing end, enabling the attachment of analytical labels through an anomeric ligation reaction. A nonasulfated tetrahyaluronan-ethylenediaminetetraacetic acid derivative has been produced and used as a paramagnetic tag for the elucidation of the complex of this ligand with interleukin-10 using paramagnetic relaxation enhancement NMR analysis. The metal ion position is resolved with 1.0 Å, enabling a refined structural model of the complex.

NMR characterization of the binding properties and conformation of glycosaminoglycans interacting with interleukin-10.

The cytokine interleukin-10 (IL-10) is an important regulator of the host immune system with both pro- and anti-inflammatory functions. Glycosaminoglycans (GAGs) play a decisive role in the biology of many growth factors, e.g., for receptor binding or protection from proteolytic degradation. GAGs of the extracellular matrix inhibit IL-10 signaling, however, the molecular mechanism is so far unknown. Here, we studied the interaction between GAGs and IL-10 using a combination of nuclear magnetic resonance (NMR) spectroscopy and computer simulations. The binding region of a set of heparin and chondroitin sulfate GAG disaccharides with varying sulfation pattern were determined by saturation transfer difference (STD) NMR spectroscopy. From the initial growth rate of the STD amplification factor binding affinities were determined and KD values in the low millimolar to micromolar range were obtained. We observed the highest binding affinity to IL-10 with fully sulfated heparin; however, a hyaluronan hexasaccharide did not exhibit binding, which suggests that GAG sulfation is necessary for interaction with IL-10. For octasaccharides or longer GAGs, a cooperative binding behavior was observed, which could indicate simultaneous interaction with both dimer subunits of IL-10. Finally, structural information about the bound GAG was exemplarily obtained for a heparin tetrasaccharide fragment (ΔUA,2S-GlcNS,6S-IdoA,2S-GlcNS,6S) using transferred NOESY experiments, proton-proton scalar couplings and molecular dynamics simulations. The overall backbone conformation is only slightly changed in the presence of IL-10 and the conformational equilibrium between (1)C4 chair and (2)So skew-boat structure of the internal iduronic acid residue is preserved.

Regulation of an intracellular subtilisin protease activity by a short propeptide sequence through an original combined dual mechanism.

A distinct class of the biologically important subtilisin family of serine proteases functions exclusively within the cell and forms a major component of the bacilli degradome. However, the mode and mechanism of posttranslational regulation of intracellular protease activity are unknown. Here we describe the role played by a short N-terminal extension prosequence novel amongst the subtilisins that regulates intracellular subtilisin protease (ISP) activity through two distinct modes: active site blocking and catalytic triad rearrangement. The full-length proenzyme (proISP) is inactive until specific proteolytic processing removes the first 18 amino acids that comprise the N-terminal extension, with processing appearing to be performed by ISP itself. A synthetic peptide corresponding to the N-terminal extension behaves as a mixed noncompetitive inhibitor of active ISP with a K(i) of 1 µM. The structure of the processed form has been determined at 2.6 Å resolution and compared with that of the full-length protein, in which the N-terminal extension binds back over the active site. Unique to ISP, a conserved proline introduces a backbone kink that shifts the scissile bond beyond reach of the catalytic serine and in addition the catalytic triad is disrupted. In the processed form, access to the active site is unblocked by removal of the N-terminal extension and the catalytic triad rearranges to a functional conformation. These studies provide a new molecular insight concerning the mechanisms by which subtilisins and protease activity as a whole, especially within the confines of a cell, can be regulated.

Structural Studies on the Binding Mode of Bisphenols to PPARγ.

Bisphenol A (BPA) and bisphenol B (BPB) are widely used in the production of plastics, and their potential adverse health effects, particularly on endocrine disruption and metabolic health, have raised concern. Peroxisome proliferator-activated receptor gamma (PPARγ) plays a pivotal role in metabolic regulation and adipogenesis, making it a target of interest in understanding the development of obesity and associated health impacts. In this study, we employ X-ray crystallography and molecular dynamics (MD) simulations to study the interaction of PPARγ with BPA and BPB. Crystallographic structures reveal the binding of BPA and BPB to the ligand binding domain of PPARγ, next to C285, where binding of partial agonists as well as antagonists and inverse agonists of PPARγ signaling has been previously observed. However, no interaction of BPA and BPB with Y437 in the activation function 2 site is observed, showing that these ligands cannot stabilize the active conformation of helix 12 directly. Furthermore, free energy analyses of the MD simulations revealed that I341 has a large energetic contribution to the BPA and BPB binding modes characterized in this study.

Similar Binding Modes of cGMP Analogues Limit Selectivity in Modulating Retinal CNG Channels via the Cyclic Nucleotide-Binding Domain.

In treating retinitis pigmentosa, a genetic disorder causing progressive vision loss, selective inhibition of rod cyclic nucleotide-gated (CNG) channels holds promise. Blocking the increased Ca2+-influx in rod photoreceptors through CNG channels can potentially delay disease progression and improve the quality of life for patients. To find inhibitors for rod CNG channels, we investigated the impact of 16 cGMP analogues on both rod and cone CNG channels using the patch-clamp technique. Although modifications at the C8 position of the guanine ring did not change the ligand efficacy, modifications at the N1 and N2 positions rendered cGMP largely ineffective in activating retinal CNG channels. Notably, PET-cGMP displayed selective potential, favoring rod over cone, whereas Rp-cGMPS showed greater efficiency in activating cone over rod CNG channels. Ligand docking and molecular dynamics simulations on cyclic nucleotide-binding domains showed comparable binding energies and binding modes for cGMP and its analogues in both rod and cone CNG channels (CNGA1 vs CNGA3 subunits). Computational experiments on CNGB1a vs CNGB3 subunits showed similar binding modes albeit with fewer amino acid interactions with cGMP due to an inactivated conformation of their C-helix. In addition, no clear correlation could be observed between the computational scores and the CNG channel efficacy values, suggesting additional factors beyond binding strength determining ligand selectivity and potency. This study highlights the importance of looking beyond the cyclic nucleotide-binding domain and toward the gating mechanism when searching for selective modulators. Future efforts in developing selective modulators for CNG channels should prioritize targeting alternative channel domains.

Ferric reduction by a CYBDOM protein counteracts increased iron availability in root meristems induced by phosphorus deficiency.

To mobilize sparingly available phosphorus (P) in the rhizosphere, many plant species secrete malate to release P sorbed onto (hydr)oxides of aluminum and iron (Fe). In the presence of Fe, malate can provoke Fe over-accumulation in the root apoplast, triggering a series of events that inhibit root growth. Here, we identified HYPERSENSITIVE TO LOW P1 (HYP1), a CYBDOM protein constituted of a DOMON and a cytochrome b561 domain, as critical to maintain cell elongation and meristem integrity under low P. We demonstrate that HYP1 mediates ascorbate-dependent trans-plasma membrane electron transport and can reduce ferric and cupric substrates in Xenopus laevis oocytes and in planta. HYP1 expression is up-regulated in response to P deficiency in the proximal zone of the root apical meristem. Disruption of HYP1 leads to increased Fe and callose accumulation in the root meristem and causes significant transcriptional changes in roots. We further demonstrate that HYP1 activity overcomes malate-induced Fe accumulation, thereby preventing Fe-dependent root growth arrest in response to low P. Collectively, our results uncover an ascorbate-dependent metalloreductase that is critical to protect root meristems of P-deficient plants from increased Fe availability and provide insights into the physiological function of the yet poorly characterized but ubiquitous CYBDOM proteins.