Cognate Ligand Research Articles

Discovery of CRBN-Dependent WEE1 Molecular Glue Degraders from a Multicomponent Combinatorial Library.

Small molecules promoting protein-protein interactions produce a range of therapeutic outcomes. Molecular glue degraders exemplify this concept due to their compact drug-like structures and ability to engage targets without reliance on existing cognate ligands. While cereblon molecular glue degraders containing glutarimide scaffolds have been approved for treatment of multiple myeloma and acute myeloid leukemia, the design of new therapeutically relevant monovalent degraders remains challenging. We report here an approach to glutarimide-containing molecular glue synthesis using multicomponent reactions as a central modular core-forming step. Screening the resulting library identified HRZ-1 derivatives that target casein kinase 1 α (CK1α) and Wee-like protein kinase (WEE1). Further medicinal chemistry efforts led to identification of selective monovalent WEE1 degraders that provide a potential starting point for the eventual development of a selective chemical degrader probe. The structure of the hit WEE1 degrader complex with CRBN-DDB1 and WEE1 provides a model of the protein-protein interface and ideas to rationalize the observed kinase selectivity. Our findings suggest that modular synthetic routes combined with in-depth structural characterization give access to selective molecular glue degraders and expansion of the CRBN-degradable proteome.

NusG-dependent RNA polymerase pausing is a common feature of riboswitch regulatory mechanisms.

Transcription by RNA polymerase is punctuated by transient pausing events. Pausing provides time for RNA folding and binding of regulatory factors to the paused elongation complex. We previously identified 1600 NusG-dependent pauses throughout the Bacillus subtilis genome, with ∼20% localized to 5' leader regions, suggesting a regulatory role for these pauses. We examined pauses associated with known riboswitches to determine whether pausing is a common feature of these mechanisms. NusG-dependent pauses in the fmnP, tenA, mgtE, lysP and mtnK riboswitches were in strategic positions preceding the critical decision between the formation of alternative antiterminator or terminator structures, which is a critical feature of transcription attenuation mechanisms. In vitro transcription assays demonstrated that pausing increased the frequency of termination in the presence of the cognate ligand. NusG-dependent pausing also reduced the ligand concentration required for efficient termination. In vivo expression studies with transcriptional fusions confirmed that NusG-dependent pausing is a critical component of each riboswitch mechanism. Our results indicate that pausing enables cells to sense a broader range of ligand concentrations for fine-tuning riboswitch attenuation mechanisms.

Receptor determinants for ß-arrestin functional specificity at C-X-C chemokine receptor 5 (CXCR5).

ß-arrestins are multi-faceted adaptor proteins that mediate G protein-coupled receptor (GPCR) desensitization, internalization, and signaling. It is emerging that receptor-specific determinants specify these divergent functions at GPCRs, yet this remains poorly understood. Here, we set out to identify the receptor determinants responsible for ß-arrestin-mediated regulation of the chemokine receptor CXCR5. Using bioluminescence resonance energy transfer (BRET) we show that ß-arrestin1 and ß-arrestin2 are dose-dependently recruited to CXCR5 by its cognate ligand CXCL13. The carboxy-terminal tail of CXCR5 contains several Ser/Thr residues that can be divided into 3 discrete phospho-site clusters based on their position relative to transmembrane domain 7. Mutagenesis experiments revealed that the distal and medial phospho-site clusters, but not the proximal, are required for agonist-stimulated ß-arrestin1 or ß-arrestin2 recruitment to CXCR5. Consistent with this, we provide evidence that the distal and medial, but not proximal, phospho-site clusters are required for receptor desensitization. Surprisingly, the individual phospho-site clusters are not required for agonist-stimulated internalization of CXCR5. Further, we show that CXCL13-stimulated CXCR5 internalization and ERK1/2 phosphorylation, but not desensitization, remain intact in HEK293 cells lacking ß-arrestin1 and ß-arrestin2. Our study provides evidence that b-arrestins are recruited to CXCR5 and are required for desensitization but are dispensable for internalization or signaling, suggesting that discrete receptor determinants specify the divergent functions of ß-arrestins. Significance Statement CXCL13 and CXCR5 are important in the immune system and are linked to diseases, yet regulation of CXCR5 signaling remains poorly understood. We provide evidence that a phospho-site cluster located at the extreme distal carboxyl-terminal tail of the receptor is responsible for ß-arrestin recruitment and receptor desensitization. ß-arrestins are not required for CXCL13-stimulated internalization or signaling, indicating that ß-arrestins perform only one of their functions at CXCR5 and that discrete receptor determinants specify the divergent functions of ß-arrestins.

ProCogGraph: A Graph-Based Mapping of Cognate Ligand Domain Interactions

Abstract Motivation Mappings of domain-cognate ligand interactions can enhance our understanding of the core concepts of evolution and be used to aid docking and protein design. Since the last available cognate-ligand domain database was released, the PDB has grown significantly and new tools are available for measuring similarity and determining contacts. Results We present ProCogGraph, a graph database of cognate-ligand domain mappings in PDB structures. Building upon the work of the predecessor database, PROCOGNATE, we use data-driven approaches to develop thresholds and interaction modes. We explore new aspects of domain-cognate ligand interactions, including the chemical similarity of bound cognate ligands and how domain combinations influence cognate ligand binding. Finally, we use the graph to add specificity to partial EC IDs, showing that ProCogGraph can complete partial annotations systematically through assigned cognate ligands. Availability and Implementation The ProCogGraph pipeline, database and flat files are available at https://github.com/bashton-lab/ProCogGraph and https://doi.org/10.5281/zenodo.13165851. Supplementary information Supplementary data are available at Bioinformatics Advances online.

Structure-based pose prediction: Non-cognate docking extended to macrocyclic ligands

So-called “cross-docking” is the prediction of the bound configuration of small-molecule ligands that differ from the cognate ligand of a protein co-crystal structure. This is a much more challenging problem than re-docking the cognate ligand, particularly when the new ligand is structurally dissimilar from prior known ones. We have updated the previously introduced PINC (“PINC Is Not Cognate”) benchmark which introduced the idea of temporal segregation to measure cross-docking performance. The temporal set encompasses 846 future ligands for ten targets based on information from the earliest 25% of X-ray co-crystal structures known for each target. Here, we extend the benchmark to include thirteen targets where the bound poses of 128 macrocyclic ligands are to be predicted based on knowledge from structures of bound non-macrocyclic ligands. Performance was roughly equivalent for both the temporally-split non-macrocyclic ligand set and the macrocycle prediction set. Using standard and fully automatic protocols for the Surflex-Dock and ForceGen methods, across the combined 974 non-macrocyclic and macrocyclic ligands, the top-scoring pose family was correct 68% of the time, with the top-two pose families achieving a 79% success rate. Correct poses among all those predicted were identified 92% of the time. These success rates far exceeded those observed for the alternative methods AutoDock Vina and Gnina on both sets.

Large-scale annotation of biochemically relevant pockets and tunnels in cognate enzyme–ligand complexes

Tunnels in enzymes with buried active sites are key structural features allowing the entry of substrates and the release of products, thus contributing to the catalytic efficiency. Targeting the bottlenecks of protein tunnels is also a powerful protein engineering strategy. However, the identification of functional tunnels in multiple protein structures is a non-trivial task that can only be addressed computationally. We present a pipeline integrating automated structural analysis with an in-house machine-learning predictor for the annotation of protein pockets, followed by the calculation of the energetics of ligand transport via biochemically relevant tunnels. A thorough validation using eight distinct molecular systems revealed that CaverDock analysis of ligand un/binding is on par with time-consuming molecular dynamics simulations, but much faster. The optimized and validated pipeline was applied to annotate more than 17,000 cognate enzyme–ligand complexes. Analysis of ligand un/binding energetics indicates that the top priority tunnel has the most favourable energies in 75% of cases. Moreover, energy profiles of cognate ligands revealed that a simple geometry analysis can correctly identify tunnel bottlenecks only in 50% of cases. Our study provides essential information for the interpretation of results from tunnel calculation and energy profiling in mechanistic enzymology and protein engineering. We formulated several simple rules allowing identification of biochemically relevant tunnels based on the binding pockets, tunnel geometry, and ligand transport energy profiles.Scientific contributionsThe pipeline introduced in this work allows for the detailed analysis of a large set of protein–ligand complexes, focusing on transport pathways. We are introducing a novel predictor for determining the relevance of binding pockets for tunnel calculation. For the first time in the field, we present a high-throughput energetic analysis of ligand binding and unbinding, showing that approximate methods for these simulations can identify additional mutagenesis hotspots in enzymes compared to purely geometrical methods. The predictor is included in the supplementary material and can also be accessed at https://github.com/Faranehhad/Large-Scale-Pocket-Tunnel-Annotation.git. The tunnel data calculated in this study has been made publicly available as part of the ChannelsDB 2.0 database, accessible at https://channelsdb2.biodata.ceitec.cz/.

Global analysis of neuropeptide receptor conservation across phylum Nematoda.

The phylum Nematoda is incredibly diverse and includes many parasites of humans, livestock, and plants. Peptide-activated G protein-coupled receptors (GPCRs) are central to the regulation of physiology and numerous behaviors, and they represent appealing pharmacological targets for parasite control. Efforts are ongoing to characterize the functions and define the ligands of nematode GPCRs, with already most peptide GPCRs known or predicted in Caenorhabditis elegans. However, comparative analyses of peptide GPCR conservation between C. elegans and other nematode species are limited, and many nematode GPCRs remain orphan. A phylum-wide perspective on peptide GPCR profiles will benefit functional and applied studies of nematode peptide GPCRs. We constructed a pan-phylum resource of C. elegans peptide GPCR orthologs in 125 nematode species using a semi-automated pipeline for analysis of predicted proteome datasets. The peptide GPCR profile varies between nematode species of different phylogenetic clades and multiple C. elegans peptide GPCRs have orthologs across the phylum Nematoda. We identified peptide ligands for two highly conserved orphan receptors, NPR-9 and NPR-16, that belong to the bilaterian galanin/allatostatin A (Gal/AstA) and somatostatin/allatostatin C (SST/AstC) receptor families. The AstA-like NLP-1 peptides activate NPR-9 in cultured cells and are cognate ligands of this receptor in vivo. In addition, we discovered an AstC-type peptide, NLP-99, that activates the AstC-type receptor NPR-16. In our pan-phylum resource, the phylum-wide representation of NPR-9 and NPR-16 resembles that of their cognate ligands more than those of allatostatin-like peptides that do not activate these receptors. The repertoire of C. elegans peptide GPCR orthologs varies across phylogenetic clades and several peptide GPCRs show broad conservation in the phylum Nematoda. Our work functionally characterizes the conserved receptors NPR-9 and NPR-16 as the respective GPCRs for the AstA-like NLP-1 peptides and the AstC-related peptide NLP-99. NLP-1 and NLP-99 are widely conserved in nematodes and their representation matches that of their receptor in most species. These findings demonstrate the conservation of a functional Gal/AstA and SST/AstC signaling system in nematodes. Our dataset of C. elegans peptide GPCR orthologs also lays a foundation for further functional studies of peptide GPCRs in the widely diverse nematode phylum.

Endogenous opioid signalling regulates spinal ependymal cell proliferation.

After injury, mammalian spinal cords develop scars to confine the lesion and prevent further damage. However, excessive scarring can hinder neural regeneration and functional recovery1,2. These competing actions underscore the importance of developing therapeutic strategies to dynamically modulate scar progression. Previous research on scarring has primarily focused on astrocytes, but recent evidence has suggested that ependymal cells also participate. Ependymal cells normally form the epithelial layer encasing the central canal, but they undergo massive proliferation and differentiation into astroglia following certain injuries, becoming a core scar component3-7. However, the mechanisms regulating ependymal proliferation in vivo remain unclear. Here we uncover an endogenous κ-opioid signalling pathway that controls ependymal proliferation. Specifically, we detect expression of the κ-opioid receptor, OPRK1, in a functionally under-characterized cell type known as cerebrospinal fluid-contacting neuron (CSF-cN). We also discover a neighbouring cell population that expresses the cognate ligand prodynorphin (PDYN). Whereas κ-opioids are typically considered inhibitory, they excite CSF-cNs to inhibit ependymal proliferation. Systemic administration of a κ-antagonist enhances ependymal proliferation in uninjured spinal cords in a CSF-cN-dependent manner. Moreover, a κ-agonist impairs ependymal proliferation, scar formation and motor function following injury. Together, our data suggest a paracrine signalling pathway in which PDYN+ cells tonically release κ-opioids to stimulate CSF-cNs and suppress ependymal proliferation, revealing an endogenous mechanism and potential pharmacological strategy for modulating scarring after spinal cord injury.

Mechanistic analysis of Riboswitch Ligand interactions provides insights into pharmacological control over gene expression

Riboswitches are structured RNA elements that regulate gene expression upon binding to small molecule ligands. Understanding the mechanisms by which small molecules impact riboswitch activity is key to developing potent, selective ligands for these and other RNA targets. We report the structure-informed design of chemically diverse synthetic ligands for PreQ1 riboswitches. Multiple X-ray co-crystal structures of synthetic ligands with the Thermoanaerobacter tengcongensis (Tte)-PreQ1 riboswitch confirm a common binding site with the cognate ligand, despite considerable chemical differences among the ligands. Structure probing assays demonstrate that one ligand causes conformational changes similar to PreQ1 in six structurally and mechanistically diverse PreQ1 riboswitch aptamers. Single-molecule force spectroscopy is used to demonstrate differential modes of riboswitch stabilization by the ligands. Binding of the natural ligand brings about the formation of a persistent, folded pseudoknot structure, whereas a synthetic ligand decreases the rate of unfolding through a kinetic mechanism. Single round transcription termination assays show the biochemical activity of the ligands, while a GFP reporter system reveals compound activity in regulating gene expression in live cells without toxicity. Taken together, this study reveals that diverse small molecules can impact gene expression in live cells by altering conformational changes in RNA structures through distinct mechanisms.

Accurate Identification of Periplasmic Urea-binding Proteins by Structure- and Genome Context-assisted Functional Analysis

ABC transporters are ancient and ubiquitous nutrient transport systems in bacteria and play a central role in defining lifestyles. Periplasmic solute-binding proteins (SBPs) are components that deliver ligands to their translocation machinery. SBPs have diversified to bind a wide range of ligands with high specificity and affinity. However, accurate assignment of cognate ligands remains a challenging problem in SBPs. Urea metabolism plays an important role in the nitrogen cycle; anthropogenic sources account for more than half of global nitrogen fertilizer. We report identification of urea-binding proteins within a large SBP sequence family that encodes diverse functions. By combining genetic linkage between SBPs, ABC transporter components, enzymes or transcription factors, we accurately identified cognate ligands, as we verified experimentally by biophysical characterization of ligand binding and crystallographic determination of the urea complex of a thermostable urea-binding homolog. Using three-dimensional structure information, these functional assignments were extrapolated to other members in the sequence family lacking genetic linkage information, which revealed that only a fraction bind urea. Using the same combined approaches, we also inferred that other family members bind various short-chain amides, aliphatic amino acids (leucine, isoleucine, valine), γ-aminobutyrate, and as yet unknown ligands. Comparative structural analysis revealed structural adaptations that encode diversification in these SBPs. Systematic assignment of ligands to SBP sequence families is key to understanding bacterial lifestyles, and also provides a rich source of biosensors for clinical and environmental analysis, such as the thermostable urea-binding protein identified here.

FGFR‑related phenotypic and functional profile of CAFs in prognostication of breast cancer (Review).

While preclinical studies consistently implicate FGFR‑signalling in breast cancer (BC) progression, clinical evidence fails to support these findings. It may be that the clinical significance of FGFR ought to be analysed in the context of the stroma, activating or repressing its function. The present review aimed to provide such a context by summarizing the existing data on the prognostic and/or predictive value of selected cancer‑associated fibroblasts (CAFs)‑related factors, that either directly or indirectly may affect FGFR‑signalling. PubMed (https://pubmed.ncbi.nlm.nih.gov/) and Medline (https://www.nlm.nih.gov/medline/medline_home.html) databases were searched for the relevant literature related to the prognostic and/or predictive significance of: CAFs phenotypic markers (αSMA, S100A4/FSP‑1, PDGFR, PDPN and FAP), CAFs‑derived cognate FGFR ligands (FGF2, FGF5 and FGF17) or inducers of CAFs' paracrine activity (TGF‑β1, HDGF, PDGF, CXCL8, CCL5, CCL2, IL‑6, HH and EGF) both expressed in the tumour and circulating in the blood. A total of 68 articles were selected and thoroughly analysed. The findings consistently identified upregulation of αSMA, S100A4/FSP‑1, PDGFR, PDPN, HDGF, PDGF, CXCL8, CCL5, CCL2, IL‑6, HH and EGF as poor prognostic markers in BC, while evaluation of the prognostic value of the remaining markers varied between the studies. The data confirm an association of CAFs‑specific features with BC prognosis, suggesting that both quantitative and qualitative profiling of the stroma might be required for an assessment of the true FGFR's clinical value.

The Impact of Nanobodies on G Protein-Coupled Receptor Structural Biology and Their Potential as Therapeutic Agents.

The family of human G protein-coupled receptors (GPCRs) comprises about 800 different members, with about 35% of current pharmaceutical drugs targeting GPCRs. However, GPCR structural biology, necessary for structure-guided drug design, has lagged behind that of other membrane proteins, and it was not until the year 2000 when the first crystal structure of a GPCR (rhodopsin) was solved. Starting in 2007, the determination of additional GPCR structures was facilitated by protein engineering, new crystallization techniques, complexation with antibody fragments, and other strategies. More recently, the use of camelid heavy-chain-only antibody fragments (nanobodies) as crystallographic chaperones has revolutionized the field of GPCR structural biology, aiding in the determination of more than 340 GPCR structures to date. In most cases, the GPCR structures solved as complexes with nanobodies (Nbs) have revealed the binding mode of cognate or non-natural ligands; in a few cases, the same Nb has acted as an orthosteric or allosteric modulator of GPCR signaling. In this review, we summarize the multiple ingenious strategies that have been conceived and implemented in the last decade to capitalize on the discovery of nanobodies to study GPCRs from a structural perspective. SIGNIFICANCE STATEMENT: G protein-coupled receptors (GPCRs) are major pharmacological targets, and the determination of their structures at high resolution has been essential for structure-guided drug design and for insights about their functions. Single-domain antibodies (nanobodies) have greatly facilitated the structural determination of GPCRs by forming complexes directly with the receptors or indirectly through protein partners.

A riboswitch-controlled TerC family transporter Alx tunes intracellular manganese concentration in Escherichia coli at alkaline pH.

Cells use transition metal ions as structural components of biomolecules and cofactors in enzymatic reactions, making transition metal ions integral cellular components. Organisms optimize metal ion concentration to meet cellular needs by regulating the expression of proteins that import and export that metal ion, often in a metal ion concentration-dependent manner. One such regulation mechanism is via riboswitches, which are 5'-untranslated regions of an mRNA that undergo conformational changes to promote or inhibit the expression of the downstream gene, commonly in response to a ligand. The yybP-ykoY family of bacterial riboswitches shares a conserved aptamer domain that binds manganese ions (Mn2+). In Escherichia coli, the yybP-ykoY riboswitch precedes and regulates the expression of two different genes: mntP, which based on genetic evidence encodes an Mn2+ exporter, and alx, which encodes a putative metal ion transporter whose cognate ligand is currently in question. The expression of alx is upregulated by both elevated concentrations of Mn2+ and alkaline pH. With metal ion measurements and gene expression studies, we demonstrate that the alkalinization of media increases the cytoplasmic manganese pool, which, in turn, enhances alx expression. The Alx-mediated Mn2+ export prevents the toxic buildup of the cellular manganese, with the export activity maximal at alkaline pH. We pinpoint a set of acidic residues in the predicted transmembrane segments of Alx that play a critical role in Mn2+ export. We propose that Alx-mediated Mn2+ export serves as a primary protective mechanism that fine tunes the cytoplasmic manganese content, especially during alkaline stress.IMPORTANCEBacteria use clever ways to tune gene expression upon encountering certain environmental stresses, such as alkaline pH in parts of the human gut and high concentration of a transition metal ion manganese. One way by which bacteria regulate the expression of their genes is through the 5'-untranslated regions of messenger RNA called riboswitches that bind ligands to turn expression of genes on/off. In this work, we have investigated the roles and regulation of alx and mntP, the two genes in Escherichia coli regulated by the yybP-ykoY riboswitches, in alkaline pH and high concentration of Mn2+. This work highlights the intricate ways through which bacteria adapt to their surroundings, utilizing riboregulatory mechanisms to maintain Mn2+ levels amidst varying environmental factors.

Class B1 GPCRs: insights into multireceptor pharmacology for the treatment of metabolic disease.

The secretin-like, class B1 subfamily of seven transmembrane-spanning G protein-coupled receptors (GPCRs) consists of 15 members that coordinate important physiological processes. These receptors bind peptide ligands and use a distinct mechanism of activation that is driven by evolutionarily conserved structural features. For the class B1 receptors, the C-terminus of the cognate ligand is initially recognized by the receptor via an N-terminal extracellular domain that forms a hydrophobic ligand-binding groove. This binding enables the N-terminus of the ligand to engage deep into a large volume, open transmembrane pocket of the receptor. Importantly, the phylogenetic basis of this ligand-receptor activation mechanism has provided opportunities to engineer analogs of several class B1 ligands for therapeutic use. Among the most accepted of these are drugs targeting the glucagon-like peptide-1 (GLP-1) receptor for the treatment of type 2 diabetes and obesity. Recently, multifunctional agonists possessing activity at the GLP-1 receptor and the glucose-dependent insulinotropic polypeptide (GIP) receptor, such as tirzepatide, and others that also contain glucagon receptor activity, have been developed. In this article, we review members of the class B1 GPCR family with focus on receptors for GLP-1, GIP, and glucagon, including their signal transduction and receptor trafficking characteristics. The metabolic importance of these receptors is also highlighted, along with the benefit of polypharmacologic ligands. Furthermore, key structural features and comparative analyses of high-resolution cryogenic electron microscopy structures for these receptors in active-state complexes with either native ligands or multifunctional agonists are provided, supporting the pharmacological basis of such therapeutic agents.

Altered desensitization and internalization patterns of rodent versus human glucose-dependent insulinotropic polypeptide (GIP) receptors. An important drug discovery challenge.

The gut hormone glucose-dependent insulinotropic polypeptide (GIP) signals via the GIP receptor (GIPR), resulting in postprandial potentiation of glucose-stimulated insulin secretion. The translation of results from rodent studies to human studies has been challenged by the unexpected effects of GIPR-targeting compounds. We, therefore, investigated the variation between species, focusing on GIPR desensitization and the role of the receptor C-terminus. The GIPR from humans, mice, rats, pigs, dogs and cats was studied in vitro for cognate ligand affinity, G protein activation (cAMP accumulation), recruitment of beta-arrestin and internalization. Variants of the mouse, rat and human GIPRs with swapped C-terminal tails were studied in parallel. The human GIPR is more prone to internalization than rodent GIPRs. Despite similar agonist affinities and potencies for Gαs activation, especially, the mouse GIPR shows reduced receptor desensitization, internalization and beta-arrestin recruitment. Using an enzyme-stabilized, long-acting GIP analogue, the species differences were even more pronounced. 'Tail-swapped' human, rat and mouse GIPRs were all fully functional in their Gαs coupling, and the mouse GIPR regained internalization and beta-arrestin 2 recruitment properties with the human tail. The human GIPR lost the ability to recruit beta-arrestin 2 when its own C-terminus was replaced by the rat or mouse tail. Desensitization of the human GIPR is dependent on the C-terminal tail. The species-dependent functionality of the C-terminal tail and the different species-dependent internalization patterns, especially between human and mouse GIPRs, are important factors influencing the preclinical evaluation of GIPR-targeting therapeutic compounds.

Differential alterations of CXCR3, CXCR5 and CX3CR1 in patients with immune thrombocytopenia

As a versatile element for maintaining homeostasis, the chemokine system has been reported to be implicated in the pathogenesis of immune thrombocytopenia (ITP). However, research pertaining to chemokine receptors and related ligands in adult ITP is still limited. The states of several typical chemokine receptors and cognate ligands in the circulation were comparatively assessed through various methodologies. Multiple variable analyses of correlation matrixes were conducted to characterize the correlation signatures of various chemokine receptors or candidate ligands with platelet counts. Our data illustrated a significant decrease in relative CXCR3 expression and elevated plasma levels of CXCL4, 9–11, 13, and CCL3 chemokines in ITP patients with varied platelet counts. Flow cytometry assays revealed eminently diminished CXCR3 levels on T and B lymphocytes and increased CXCR5 on cytotoxic T cell (Tc) subsets in ITP patients with certain platelet counts. Meanwhile, circulating CX3CR1 levels were markedly higher on T cells with a concomitant increase in plasma CX3CL1 level in ITP patients, highlighting the importance of aberrant alterations of the CX3CR1-CX3CL1 axis in ITP pathogenesis. Spearman’s correlation analyses revealed a strong positive association of peripheral CXCL4 mRNA level, and negative correlations of plasma CXCL4 concentration and certain chemokine receptors with platelet counts, which might serve as a potential biomarker of platelet destruction in ITP development. Overall, these results indicate that the differential expression patterns and distinct activation states of peripheral chemokine network, and the subsequent expansion of circulating CXCR5+ Tc cells and CX3CR1+ T cells, may be a hallmark during ITP progression, which ultimately contributes to thrombocytopenia in ITP patients.

Discovery of periplasmic solute binding proteins with specificity for ketone bodies: β-hydroxybutyrate binding proteins.

Polyhydroxyalkanoates are a class of biodegradable, thermoplastic polymers which represent a major carbon source for various bacteria. Proteins which mediate the translocation of polyhydroxyalkanoate breakdown products, such as β-hydroxybutyrate (BHB)-a ketone body which in humans serves as an important biomarker, have not been well characterized. In our investigation to screen a solute-binding protein (SBP) which can act as a suitable recognition element for BHB, we uncovered insights at the intersection of bacterial metabolism and diagnostics. Herein, we identify SBPs associated with putative ATP-binding cassette transporters that specifically recognize BHB, with the potential to serve as recognition elements for continuous quantification of this analyte. Through bioinformatic analysis, we identified candidate SBPs from known metabolizers of polyhydroxybutyrate-including proteins from Cupriavidus necator, Ensifer meliloti, Paucimonas lemoignei, and Thermus thermophilus. After recombinant expression in Escherichia coli, we demonstrated with intrinsic tryptophan fluorescence spectroscopy that four candidate proteins interacted with BHB, ranging from nanomolar to micromolar affinity. Tt.2, an intrinsically thermostable protein from Thermus thermophilus, was observed to have the tightest binding and specificity for BHB, which was confirmed by isothermal calorimetry. Structural analyses facilitated by AlphaFold2, along with molecular docking and dynamics simulations, were used to hypothesize key residues in the binding pocket and to model the conformational dynamics of substrate unbinding. Overall, this study provides strong evidence identifying the cognate ligands of SBPs which we hypothesize to be involved in prokaryotic cellular translocation of polyhydroxyalkanoate breakdown products, while highlighting these proteins' promising biotechnological application.

Advances in research on spexin-mediated regulation of reproductive function in vertebrates.

Spexin (SPX, NPQ) is a 14-amino acid neuroactive peptide identified using bioinformatics. This amino acid sequence of the mature spexin peptide has been highly conserved during species evolution and is widely distributed in the central nervous system and peripheral tissues and organs. Therefore, spexin may play a role in various biological functions. Spexin, the cognate ligand for GALR2/3, acting as a neuromodulator or endocrine signaling factor, can inhibit reproductive performance. However, controversies and gaps in knowledge persist regarding spexin-mediated regulation of animal reproductive functions. This review focuses on the hypothalamic-pituitary-gonadal axis and provides a comprehensive overview of the impact of spexin on reproduction. Through this review, we aim to enhance understanding and obtain in-depth insights into the regulation of reproduction by spexin peptides, thereby providing a scientific basis for future investigations into the molecular mechanisms underlying the influence of spexin on reproductive function. Such investigations hold potential benefits for optimizing farming practices in livestock, poultry, and fish industries.

INA03: A Potent Transferrin-Competitive Antibody-Drug Conjugate against CD71 for Safer Acute Leukemia Treatment.

Innovative strategies for enhancing efficacy and overcoming drug resistance in hematologic cancers, such as using antibody-drug conjugates (ADC), have shifted the paradigm of conventional care by delivering promising outcomes in cancer therapies with a significant reduction in the risk of relapse. Transferrin receptor (TfR1), cluster of differentiation 71 (CD71), is known to be overexpressed in malignant cells and considered a potent antitumor target. Therefore, we developed an anti-CD71 ADC, INA03, a humanized antibody conjugated to monomethyl auristatin E through a 3-arylpropiolonitrile-valine-citrulline linker. In this study, we investigated the potency and safety of INA03, in competition with Transferrin (Tf), the CD71's natural ligand, as a novel strategy to specifically target highly proliferative cells. The high expression of CD71 was confirmed on different leukemic cell lines, allowing INA03 to bind efficiently. Subsequently, INA03 rapidly internalizes into lysosomal compartments, in which its cytotoxic drug is released following cathepsin B cleavage. Downregulation of CD71 expression using shRNA highlighted that INA03-induced cell death was dependent on CD71 density at the cell surface. INA03 intravenous treatment in acute leukemia mouse models significantly reduced tumor burden, increased mouse survival, and showed no residual disease compared with conventional chemotherapies. Because INA03 competes with human Tf, a double knock-in (human CD71/human Tf) competent mouse model was generated to mimic human pharmacokinetics and pharmacodynamics. INA03 administration in human CD71/hTf mice did not reveal any improper toxicities, even at high doses. Hence, these data demonstrate the promising preclinical efficacy and safety of INA03 and support its development as a novel acute leukemia treatment. Significance: The Tf receptor is believed to be undruggable because of its ubiquitous expression. By entering into competition with its cognate ligand, the Tf and INA03 ADC can safely achieve potency.

Cutting Edge: LAG3 Dimerization Is Required for TCR/CD3 Interaction and Inhibition of Antitumor Immunity.

Lymphocyte activation gene 3 (LAG3) is an inhibitory receptor that plays a critical role in controlling T cell tolerance and autoimmunity and is a major immunotherapeutic target. LAG3 is expressed on the cell surface as a homodimer but the functional relevance of this is unknown. In this study, we show that the association between the TCR/CD3 complex and a murine LAG3 mutant that cannot dimerize is perturbed in CD8+ T cells. We also show that LAG3 dimerization is required for optimal inhibitory function in a B16-gp100 tumor model. Finally, we demonstrate that a therapeutic LAG3 Ab, C9B7W, which does not block LAG3 interaction with its cognate ligand MHC class II, disrupts LAG3 dimerization and its association with the TCR/CD3 complex. These studies highlight the functional importance of LAG3 dimerization and offer additional approaches to therapeutically target LAG3.