Simultaneous binding of Guidance Cues NET1 and RGM blocks extracellular NEO1 signaling.

During cell migration or differentiation, cell surface receptors are simultaneously exposed to different ligands. However, it is often unclear how these extracellular signals are integrated. Neogenin (NEO1) acts as an attractive guidance receptor when the Netrin-1 (NET1) ligand binds, but it mediates repulsion via repulsive guidance molecule (RGM) ligands. Here, we show that signal integration occurs through the formation of a ternary NEO1-NET1-RGM complex, which triggers reciprocal silencing of downstream signaling. Our NEO1-NET1-RGM structures reveal a "trimer-of-trimers" super-assembly, which exists in the cell membrane. Super-assembly formation results in inhibition of RGMA-NEO1-mediated growth cone collapse and RGMA- or NET1-NEO1-mediated neuron migration, by preventing formation of signaling-compatible RGM-NEO1 complexes and NET1-induced NEO1 ectodomain clustering. These results illustrate how simultaneous binding of ligands with opposing functions, to a single receptor, does not lead to competition for binding, but to formation of a super-complex that diminishes their functional outputs.

Functional landscape of SARS-CoV-2 cellular restriction.

A deficient interferon (IFN) response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been implicated as a determinant of severe coronavirus disease 2019 (COVID-19). To identify the molecular effectors that govern IFN control of SARS-CoV-2 infection, we conducted a large-scale gain-of-function analysis that evaluated the impact of human IFN-stimulated genes (ISGs) on viral replication. A limited subset of ISGs were found to control viral infection, including endosomal factors inhibiting viral entry, RNA binding proteins suppressing viral RNA synthesis, and a highly enriched cluster of endoplasmic reticulum (ER)/Golgi-resident ISGs inhibiting viral assembly/egress. These included broad-acting antiviral ISGs and eight ISGs that specifically inhibited SARS-CoV-2 and SARS-CoV-1 replication. Among the broad-acting ISGs was BST2/tetherin, which impeded viral release and is antagonized by SARS-CoV-2 Orf7a protein. Overall, these data illuminate a set of ISGs that underlie innate immune control of SARS-CoV-2/SARS-CoV-1 infection, which will facilitate the understanding of host determinants that impact disease severity and offer potential therapeutic strategies for COVID-19.

Bacterial protein domains with a novel Ig-like fold target human CEACAM receptors.

Streptococcus agalactiae, also known as group B Streptococcus (GBS), is the major cause of neonatal sepsis in humans. A critical step to infection is adhesion of bacteria to epithelial surfaces. GBS adhesins have been identified to bind extracellular matrix components and cellular receptors. However, several putative adhesins have no host binding partner characterised. We report here that surface-expressed ß protein of GBS binds to human CEACAM1 and CEACAM5 receptors. A crystal structure of the complex showed that an IgSF domain in ß represents a novel Ig-fold subtype called IgI3, in which unique features allow binding to CEACAM1. Bioinformatic assessment revealed that this newly identified IgI3 fold is not exclusively present in GBS but is predicted to be present in adhesins from other clinically important human pathogens. In agreement with this prediction, we found that CEACAM1 binds to an IgI3 domain found in an adhesin from a different streptococcal species. Overall, our results indicate that the IgI3 fold could provide a broadly applied mechanism for bacteria to target CEACAMs.

Active emulsions in living cell membranes driven by contractile stresses and transbilayer coupling.

The spatiotemporal organization of proteins and lipids on the cell surface has direct functional consequences for signaling, sorting, and endocytosis. Earlier studies have shown that multiple types of membrane proteins, including transmembrane proteins that have cytoplasmic actin binding capacity and lipid-tethered glycosylphosphatidylinositol-anchored proteins (GPI-APs), form nanoscale clusters driven by active contractile flows generated by the actin cortex. To gain insight into the role of lipids in organizing membrane domains in living cells, we study the molecular interactions that promote the actively generated nanoclusters of GPI-APs and transmembrane proteins. This motivates a theoretical description, wherein a combination of active contractile stresses and transbilayer coupling drives the creation of active emulsions, mesoscale liquid order (lo) domains of the GPI-APs and lipids, at temperatures greater than equilibrium lipid phase segregation. To test these ideas, we use spatial imaging of molecular clustering combined with local membrane order, and we demonstrate that mesoscopic domains enriched in nanoclusters of GPI-APs are maintained by cortical actin activity and transbilayer interactions and exhibit significant lipid order, consistent with predictions of the active composite model.

Preparation of recombinant neuritin protein.

Neuritin plays an important role in promoting nerve injury repair and maintaining synaptic plasticity, making it a potential therapeutic target for the treatment of nerve injury and neurodegenerative diseases. The present study aimed to obtain an active, unlabeled neuritin protein. Initially, a neuritin protein expression system with an enterokinase site was constructed in Escherichia coli. After optimizing induction conditions and screening for high expression, a neuritin recombinant protein with purity exceeding 85 % was obtained through Ni-affinity chromatography. Subsequently, unlabeled neuritin with a molecular weight of 11 kDa was obtained through the enzymatic cleavage of the His label using an enterokinase. Furthermore, a neuritin recombinant protein with purity exceeding 95 % was obtained using gel chromatography. Functional investigations revealed that neurite outgrowth of PC12 cells was stimulated by the isolated neuritin. This study establishes a method to obtain active and unlabeled neuritin protein, providing a foundation for subsequent research on its biological functions.

Automated Structure- and Sequence-Based Design of Proteins for High Bacterial Expression and Stability.

Upon heterologous overexpression, many proteins misfold or aggregate, thus resulting in low functional yields. Human acetylcholinesterase (hAChE), an enzyme mediating synaptic transmission, is a typical case of a human protein that necessitates mammalian systems to obtain functional expression. We developed a computational strategy and designed an AChE variant bearing 51 mutations that improved core packing, surface polarity, and backbone rigidity. This variant expressed at ∼2,000-fold higher levels in E. coli compared to wild-type hAChE and exhibited 20°C higher thermostability with no change in enzymatic properties or in the active-site configuration as determined by crystallography. To demonstrate broad utility, we similarly designed four other human and bacterial proteins. Testing at most three designs per protein, we obtained enhanced stability and/or higher yields of soluble and active protein in E. coli. Our algorithm requires only a 3D structure and several dozen sequences of naturally occurring homologs, and is available at http://pross.weizmann.ac.il.

Potential Involvement of the South American Lungfish Intelectin-2 in Innate-Associated Immune Modulation.

Intelectins belong to a family of lectins with specific and transitory carbohydrate interaction capabilities. These interactions are related to the activity of agglutinating pathogens, as intelectins play a significant role in immunity. Despite the prominent immune defense function of intelectins, limited information about its structural characteristics and carbohydrate interaction properties is available. This study investigated an intelectin transcript identified in RNA-seq data obtained from the South American lungfish (Lepidosiren paradoxa), namely LpITLN2-B. The structural analyses predicted LpITLN2-B to be a homo-trimeric globular protein with the fibrinogen-like functional domain (FReD), exhibiting a molecular mass of 57 kDa. The quaternary structure is subdivided into three monomers, A, B, and C, and each domain comprises 11 ß-sheets: an anti-parallel ß-sheet, a ß-hairpin, and a disordered ß-sheet structure. Molecular docking demonstrates a significant interaction with disaccharides rather than monosaccharides. The preferential interaction with disaccharides highlights the potential interaction with pathogen molecules, such as LPS and Poly(I:C). The hemagglutination assay inhibited lectins activity, especially maltose and sucrose, highlighting lectin activity in L. paradoxa samples. Overall, our results show the potential relevance of LpITLN2-B in L. paradoxa immune defense against pathogens.

ER entry pathway and glycosylation of GPI-anchored proteins are determined by N-terminal signal sequence and C-terminal GPI-attachment sequence.

Newly synthesized proteins in the secretory pathway, including glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs), need to be correctly targeted and imported into the endoplasmic reticulum (ER) lumen. GPI-APs are synthesized in the cytosol as preproproteins, which contain an N-terminal signal sequence (SS), mature protein part, and C-terminal GPI-attachment sequence (GPI-AS), and translocated into the ER lumen where SS and GPI-AS are removed, generating mature GPI-APs. However, how various GPI-APs are translocated into the ER lumen in mammalian cells is unclear. Here, we investigated the ER entry pathways of GPI-APs using a panel of KO cells defective in each signal recognition particle-independent ER entry pathway-namely, Sec62, GET, or SND pathway. We found GPI-AP CD59 largely depends on the SND pathway for ER entry, whereas prion protein (Prion) and LY6K depend on both Sec62 and GET pathways. Using chimeric Prion and LY6K constructs in which the N-terminal SS or C-terminal GPI-AS was replaced with that of CD59, we revealed that the hydrophobicity of the SSs and GPI-ASs contributes to the dependence on Sec62 and GET pathways, respectively. Moreover, the ER entry route of chimeric Prion constructs with the C-terminal GPI-ASs replaced with that of CD59 was changed to the SND pathway. Simultaneously, their GPI structures and which oligosaccharyltransferase isoforms modify the constructs were altered without any amino acid change in the mature protein part. Taking these findings together, this study revealed N- and C-terminal sequences of GPI-APs determine the selective ER entry route, which in turn regulates subsequent maturation processes of GPI-APs.

Structure of the RECK CC domain, an evolutionary anomaly.

Five small protein domains, the CC-domains, at the N terminus of the RECK protein, play essential roles in signaling by WNT7A and WNT7B in the context of central nervous system angiogenesis and blood-brain barrier formation and maintenance. We have determined the structure of CC domain 4 (CC4) at 1.65-Å resolution and find that it folds into a compact four-helix bundle with three disulfide bonds. The CC4 structure, together with homology modeling of CC1, reveals the surface locations of critical residues that were shown in previous mutagenesis studies to mediate GPR124 binding and WNT7A/WNT7B recognition and signaling. Surprisingly, sequence and structural homology searches reveal no other cell-surface or secreted domains in vertebrates that resemble the CC domain, a pattern that is in striking contrast to other ancient and similarly sized domains, such as Epidermal Growth Factor, Fibronectin Type 3, Immunoglobulin, and Thrombospondin type 1 domains, which are collectively present in hundreds of proteins.

Synthesis and some enzyme inhibition effects of isoxazoline and pyrazoline derivatives including benzonorbornene unit.

Four new and four known isoxazoline derivatives were synthesized from the reactions of benzonorbornadiene with nitrile oxides formed from the corresponding benzaldehydes. Three new and one known pyrazoline derivatives were also synthesized from the reactions of the benzonorbornadiene with nitrile imines formed from the corresponding compounds. The synthesized nitrogen-based novel heterocyclic compounds were evaluated against the human carbonic anhydrase isoenzymes I and II (hCA I and hCA II), acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) enzymes. The synthesized nitrogen-based novel heterocyclic compounds showed IC50 values in the range of 2.69-7.01 against hCA I, 2.40-4.59 against hCA II, 0.81-1.32 µM against AChE, and 20.83-1.70 µM against BChE enzymes. On the contrary, nitrogen-based novel heterocyclic compounds demonstrated Ki values between 2.93 ± 0.59-8.61 ± 1.39 against hCA I, 2.05 ± 0.62-4.97 ± 0.95 against hCA II, 0.34 ± 0.02-0.92 ± 0.17 nM against AChE, and 0.50 ± 0.04-1.20 ± 0.16 µM against BChE enzymes. The synthesized nitrogen-based novel heterocyclic compounds exhibited effective inhibition profiles against both indicated metabolic enzymes. These results may contribute to the development of new drugs particularly to treat some disorders, which are widespread in the world including glaucoma and Alzheimer's diseases.

Novel amino acid Schiff base Zn(II) complexes as new therapeutic approaches in diabetes and Alzheimer's disease: Synthesis, characterization, biological evaluation, and molecular docking studies.

Schiff bases are compounds that have gained importance in the paint industry due to their colorful nature and in the field of chemistry and biochemistry due to their biological activities. Various biological applications of Schiff bases, such as antitumor, antifungal, antibacterial, antioxidant, antituberculosis, and anthelmintic, have been widely studied. Within the scope of the study, 5-bromo-2-hydroxybenzaldehyde and amino acid methyl esters (isoleucine, phenylalanine, and methionine) and amino acid Schiff bases were synthesized first. The synthesis of the new Zn(II) complexes of these Schiff bases was carried out by the reaction of synthesized Schiff bases and Zn(OAc)2 ·2H2 O. The structures of the synthesized complexes were elucidated using elemental analysis, Fourier transform infrared, nuclear magnetic resonance, UV-visible, and thermal analysis spectroscopy techniques. These synthesized salts were found to be effective inhibitor compounds for the α-glycosidase, and acetylcholinesterase enzyme with Ki values in the range of 30.50 ± 3.82-38.17 ± 6.26 µM for α-glycosidase, 3.68 ± 0.54-10.27 ± 1.68 µM for butyrylcholinesterase, and 6.26 ± 0.83-15.73 ± 4.73 µM for acetylcholinesterase, respectively.

Structural and functional characterization of engineered bifunctional fusion proteins of CD39 and CD73 ectonucleotidases.

Extracellular diphosphate and triphosphate nucleotides are released from activated or injured cells to trigger vascular and immune P2 purinergic receptors, provoking inflammation and vascular thrombosis. These metabokines are scavenged by ectonucleoside triphosphate diphosphohydrolase-1 (E-NTPDase1 or CD39). Further degradation of the monophosphate nucleoside end products occurs by surface ecto-5'-nucleotidase (NMPase) or CD73. These ectoenzymatic processes work in tandem to promote adenosinergic responses, which are immunosuppressive and antithrombotic. These homeostatic ectoenzymatic mechanisms are lost in the setting of oxidative stress, which exacerbates inflammatory processes. We have engineered bifunctional enzymes made up from ectodomains (ECDs) of CD39 and CD73 within a single polypeptide. Human alkaline phosphatase-ectodomain (ALP-ECD) and human acid phosphatase-ectodomain (HAP-ECD) fusion proteins were also generated, characterized, and compared with these CD39-ECD, CD73-ECD, and bifunctional fusion proteins. Through the application of colorimetrical functional assays and high-performance liquid chromatography kinetic assays, we demonstrate that the bifunctional ectoenzymes express high levels of CD39-like NTPDase activity and CD73-like NMPase activity. Chimeric CD39-CD73-ECD proteins were superior in converting triphosphate and diphosphate nucleotides into nucleosides when compared with ALP-ECD and HAP-ECD. We also note a pH sensitivity difference between the bifunctional fusion proteins and parental fusions, as well as ectoenzymatic property distinctions. Intriguingly, these innovative reagents decreased platelet activation to exogenous agonists in vitro. We propose that these chimeric fusion proteins could serve as therapeutic agents in inflammatory diseases, acting to scavenge proinflammatory ATP and also generate anti-inflammatory adenosine.

A highly potent CD73 biparatopic antibody blocks organization of the enzyme active site through dual mechanisms.

The dimeric ectonucleotidase CD73 catalyzes the hydrolysis of AMP at the cell surface to form adenosine, a potent suppressor of the immune response. Blocking CD73 activity in the tumor microenvironment can have a beneficial effect on tumor eradication and is a promising approach for cancer therapy. Biparatopic antibodies binding different regions of CD73 may be a means to antagonize its enzymatic activity. A panel of biparatopic antibodies representing the pairwise combination of 11 parental monoclonal antibodies against CD73 was generated by Fab-arm exchange. Nine variants vastly exceeded the potency of their parental antibodies with ≥90% inhibition of activity and subnanomolar EC50 values. Pairing the Fabs of parents with nonoverlapping epitopes was both sufficient and necessary whereas monovalent antibodies were poor inhibitors. Some parental antibodies yielded potent biparatopics with multiple partners, one of which (TB19) producing the most potent. The structure of the TB19 Fab with CD73 reveals that it blocks alignment of the N- and C-terminal CD73 domains necessary for catalysis. A separate structure of CD73 with a Fab (TB38) which complements TB19 in a particularly potent biparatopic shows its binding to a nonoverlapping site on the CD73 N-terminal domain. Structural modeling demonstrates a TB19/TB38 biparatopic antibody would be unable to bind the CD73 dimer in a bivalent manner, implicating crosslinking of separate CD73 dimers in its mechanism of action. This ability of a biparatopic antibody to both crosslink CD73 dimers and fix them in an inactive conformation thus represents a highly effective mechanism for the inhibition of CD73 activity.

PGAP6, a GPI-specific phospholipase A2, has narrow substrate specificity against GPI-anchored proteins.

PGAP6, also known as TMEM8A, is a phospholipase A2 with specificity to glycosylphosphatidylinositol (GPI) and expressed on the surface of various cells. CRIPTO, a GPI-anchored co-receptor for a morphogenic factor Nodal, is a sensitive substrate of PGAP6. PGAP6-mediated shedding of CRIPTO plays a critical role in an early stage of embryogenesis. In contrast, CRYPTIC, a close family member of CRIPTO, is resistant to PGAP6. In this report, chimeras between CRIPTO and CRYPTIC and truncate mutants of PGAP6 were used to demonstrate that the Cripto-1/FRL1/Cryptic domain of CRIPTO is recognized by an N-terminal domain of PGAP6 for processing. We also report that among 56 human GPI-anchored proteins tested, only glypican 3, prostasin, SPACA4, and contactin-1, in addition to CRIPTO, are sensitive to PGAP6, indicating that PGAP6 has a narrow specificity toward various GPI-anchored proteins.

A multidimensional computational exploration of congenital myasthenic syndrome causing mutations in human choline acetyltransferase.

Missense mutations of human choline acetyltransferase (CHAT) are mainly associated with congenital myasthenic syndrome (CMS). To date, several pathogenic mutations have been reported, but due to the rarity and genetic complexity of CMS and difficult genotype-phenotype correlations, the CHAT mutations, and their consequences are underexplored. In this study, we systematically sift through the available genetic data in search of previously unreported pathogenic mutations and use a dynamic in silico model to provide structural explanations for the pathogenicity of the reported deleterious and undetermined variants. Through rigorous multiparameter analyses, we conclude that mutations can affect CHAT through a variety of different mechanisms: by disrupting the secondary structure, by perturbing the P-loop through long-range allosteric interactions, by disrupting the domain connecting loop, and by affecting the phosphorylation process. This study provides the first dynamic look at how mutations affect the structure and catalytic activity in CHAT and highlights the need for further genomic research to better understand the pathology of CHAT.

Biologically active phthalocyanine metal complexes: Preparation, evaluation of α-glycosidase and anticholinesterase enzyme inhibition activities, and molecular docking studies.

In this study, preparation, as well as investigation of α-glycosidase and cholinesterase (ChE) enzyme inhibition activities of furan-2-ylmethoxy-substituted compounds 1-7, are reported. Peripherally, tetra-substituted copper and manganese phthalocyanines (5 and 6) were synthesized for the first time. The substitution of furan-2-ylmethoxy groups provides remarkable solubility to the complex and redshift of the phthalocyanines Q-band. Besides, the inhibitory effects of these compounds on acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and α-glycosidase (α-Gly) enzymes have been investigated. The AChE was inhibited by these compounds (1-7) in low micromolar levels, and K i values were recorded between 11.17 ± 1.03 and 83.28 ± 11.08 µM. Against the BChE, the compounds demonstrated K i values from 7.55 ± 0.98 to 81.35 ± 12.80 µM. Also, these compounds (1-7) effectively inhibited α-glycosidase, with K i values in the range of 744.87 ± 67.33 to 1094.38 ± 88.91 µM. For α-glycosidase, the most effective K i values of phthalocyanines 3 and 6 were with K i values of 744.87 ± 67.33 and 880.36 ± 56.77 µM, respectively. Moreover, the studied metal complexes were docked with target proteins PDB ID: 4PQE, 1P0I, and 3WY1. Pharmacokinetic parameters and secondary chemical interactions that play an active role in interaction were predicted with docking simulation results. Overall, furan-2-ylmethoxy-substituted phthalocyanines can be considered as potential agents for the treatment of Alzheimer's diseases and diabetes mellitus.

Prion-like proteins as epigenetic devices of stress adaptation.

Epigenetic modifications allow cells to quickly alter their gene expression and adapt to different stresses. In addition to direct chromatin modifications, prion-like proteins have recently emerged as a system that can sense and adapt the cellular response to stressful conditions. Interestingly, such responses are maintained through prions' self-templating conformations and transmitted to the progeny of the cell that established a prion trait. Alternatively, mnemons are prion-like proteins which conformational switch encodes memories of past events and yet does not propagate to daughter cells. In this review, we explore the biology of the recently described prions found in Saccharomyces cerevisiae including [ESI+], [SMAUG+], [GAR+], [MOT3+], [MOD+], [LSB+] as well as the Whi3 mnemon. The reversibility of the phenotypes they encode allows cells to remove traits which are no longer adaptive under stress relief and chaperones play a fundamental role in all steps of prion-like proteins functions. Thus, the interplay between chaperones and prion-like proteins provides a framework to establish responses to challenging environments.

Characterization of Endogenous Human FcγRIII by Mass Spectrometry Reveals Site, Allele and Sequence Specific Glycosylation.

The importance of IgG glycosylation, Fc-gamma receptor (FcγR) single nucleotide polymorphisms and FcγR copy number variations in fine tuning the immune response has been well established. There is a growing appreciation of the importance of glycosylation of FcγRs in modulating the FcγR-IgG interaction based on the association between the glycosylation of recombinant FcγRs and the kinetics and affinity of the FcγR-IgG interaction. Although glycosylation of recombinant FcγRs has been recently characterized, limited knowledge exists on the glycosylation of endogenous human FcγRs. In order to improve the structural understanding of FcγRs expressed on human cells we characterized the site specific glycosylation of native human FcγRIII from neutrophils of 50 healthy donors and from matched plasma for 43 of these individuals. Through this analysis we have confirmed site specific glycosylation patterns previously reported for soluble FcγRIII from a single donor, identified FcγRIIIb specific Asn45 glycosylation and an allelic effect on glycosylation at Asn162 of FcγRIIIb. Identification of FcγRIIIb specific glycosylation allows for assignment of FcγRIIIb alleles and relative copy number of the two alleles where DNA/RNA is not available. Intriguingly the types of structures found to be elevated at Asn162 in the NA2 allele have been shown to destabilize the Fc:FcγRIII interaction resulting in a faster dissociation rate. These differences in glycosylation may in part explain the differential activity reported for the two alleles which have similar in vitro affinity for IgG.

Structure-based design of small bicyclic peptide inhibitors of Cripto-1 activity.

Bicyclic peptides assembled around small organic scaffolds are gaining an increasing interest as new potent, stable and highly selective therapeutics because of their uncommon ability to specifically recognize protein targets, of their small size that favor tissue penetration and of the versatility and easiness of the synthesis. We have here rationally designed bicyclic peptides assembled around a common tri-bromo-methylbenzene moiety in order to mimic the structure of the CFC domain of the oncogene Cripto-1 and, more specifically, to orient in the most fruitful way the hot spot residues H120 and W123. Through the CFC domain, Cripto-1 binds the ALK4 receptor and other protein partners supporting uncontrolled cell growth and proliferation. Soluble variants of CFC have the potential to inhibit these interactions suppressing the protein activity. A CFC analog named B3 binds ALK4 in vitro with an affinity in the nanomolar range. Structural analyses in solution via NMR and CD show that B3 has rather flexible conformations, like the parent CFC domain. The functional effects of B3 on the Cripto-1-positive NTERA cancer cell line have been evaluated showing that both CFC and B3 are cytotoxic for the cells and block the Cripto-1 intracellular signaling. Altogether, the data suggest that the administration of the soluble CFC and of the structurally related analog has the potential to inhibit tumor growth.

1,3,5-Triazine Nitrogen Mustards with Different Peptide Group as Innovative Candidates for AChE and BACE1 Inhibitors.

A series of new analogs of nitrogen mustards (4a-4h) containing the 1,3,5-triazine ring substituted with dipeptide residue were synthesized and evaluated for the inhibition of both acetylcholinesterase (AChE) and ß-secretase (BACE1) enzymes. The AChE inhibitory activity studies were carried out using Ellman's colorimetric method, and the BACE1 inhibitory activity studies were carried out using fluorescence resonance energy transfer (FRET). All compounds displayed considerable AChE and BACE1 inhibition. The most active against both AChE and BACE1 enzymes were compounds A and 4a, with an inhibitory concentration of AChE IC50 = 0.051 µM; 0.055 µM and BACE1 IC50 = 9.00 µM; 11.09 µM, respectively.