Salvianolic acid B inhibits thrombosis and directly blocks the thrombin catalytic site.

Background: Salvianolic acid B (SAB) is a major component of Salvia miltiorrhiza root (Danshen), widely used in East/Southeast Asia for centuries to treat cardiovascular diseases. Danshen depside salt, 85% of which is made up of SAB, is approved in China to treat chronic angina. Although clinical observations suggest that Danshen extracts inhibited arterial and venous thrombosis, the exact mechanism has not been adequately elucidated. Objective: To delineate the antithrombotic mechanisms of SAB. Methods: We applied platelet aggregation and coagulation assays, perfusion chambers, and intravital microscopy models. The inhibition kinetics and binding affinity of SAB to thrombin are measured by thrombin enzymatic assays, intrinsic fluorescence spectrophotometry, and isothermal titration calorimetry. We used molecular in silico docking models to predict the interactions of SAB with thrombin. Results: SAB dose-dependently inhibited platelet activation and aggregation induced by thrombin. SAB also reduced platelet aggregation induced by adenosine diphosphate and collagen. SAB attenuated blood coagulation by modifying fibrin network structures and significantly decreased thrombus formation in mouse cremaster arterioles and perfusion chambers. The direct SAB-thrombin interaction was confirmed by enzymatic assays, intrinsic fluorescence spectrophotometry, and isothermal titration calorimetry. Interestingly, SAB shares key structural similarities with the trisubstituted benzimidazole class of thrombin inhibitors, such as dabigatran. Molecular docking models predicted the binding of SAB to the thrombin active site. Conclusion: Our data established SAB as the first herb-derived direct thrombin catalytic site inhibitor, suppressing thrombosis through both thrombin-dependent and thrombin-independent pathways. Purified SAB may be a cost-effective agent for treating arterial and deep vein thrombosis.

The NMR structure of the Orf63 lytic developmental protein from lambda bacteriophage.

The orf63 gene resides in a region of the lambda bacteriophage genome between the exo and xis genes and is among the earliest genes transcribed during infection. In lambda phage and Shiga toxin (Stx) producing phages found in enterohemorrhagic Escherichia coli (EHEC) associated with food poisoning, Orf63 expression reduces the host survival and hastens the period between infection and lysis thereby giving it pro-lytic qualities. The NMR structure of dimeric Orf63 reveals a fold consisting of two helices and one strand that all make extensive intermolecular contacts. Structure-based data mining failed to identify any Orf63 homolog beyond the family of temperate bacteriophages. A machine learning approach was used to design an amphipathic helical ligand that bound a hydrophobic cleft on Orf63 with micromolar affinity. This approach may open a new path towards designing therapeutics that antagonize the contributions of Stx phages in EHEC outbreaks.

The NMR structure of the Ea22 lysogenic developmental protein from lambda bacteriophage.

The ea22 gene resides in a relatively uncharacterized region of the lambda bacteriophage genome between the exo and xis genes and is among the earliest genes transcribed upon infection. In lambda and Shiga toxin-producing phages found in enterohemorrhagic E. coli (EHEC) associated with food poisoning, Ea22 favors a lysogenic over lytic developmental state. The Ea22 protein may be considered in terms of three domains: a short amino-terminal domain, a coiled-coiled domain, and a carboxy-terminal domain (CTD). While the full-length protein is tetrameric, the CTD is dimeric when expressed individually. Here, we report the NMR solution structure of the Ea22 CTD that is described by a mixed alpha-beta fold with a dimer interface reinforced by salt bridges. A conserved mobile loop may serve as a ligand for an unknown host protein that works with Ea22 to promote bacterial survival and the formation of new lysogens. From sequence and structural comparisons, the CTD distinguishes lambda Ea22 from homologs encoded by Shiga toxin-producing bacteriophages.

Bacteriophage-encoded 24B_1 molecule resembles herpesviral microRNAs and plays a crucial role in the development of both the virus and its host.

The 24B_1 small non-coding RNA molecule has been identified in Escherichia coli after induction of Shiga toxin-converting bacteriophage Φ24B. In this work, we focused on its direct role during phage and bacterial host development. We observed that in many aspects, this phage sRNA resembles herpesviral microRNAs. Similar to microRNAs, the mature 24B_1 is a short molecule, consisting of just 20 nucleotides. It is generated by cleaving the 80-nt long precursor transcript, and likely it undergoes a multi-step maturation process in which the Hfq protein plays an important role, as confirmed by demonstration of its binding to the 24B_1 precursor, but not to the 24B_1 mature form. Moreover, 24B_1 plays a significant role in maintaining the prophage state and reprogramming the host's energy metabolism. We proved that overproduction of this molecule causes the opposite physiological effects to the mutant devoid of the 24B_1 gene, and thus, favors the lysogenic pathway. Furthermore, the 24B_1 overrepresentation significantly increases the efficiency of expression of phage genes coding for proteins CI, CII, and CIII which are engaged in the maintenance of the prophage. It seems that through binding to mRNA of the sdhB gene, coding for the succinate dehydrogenase subunit, the 24B_1 alters the central carbon metabolism and causes a drop in the ATP intracellular level. Interestingly, a similar effect, called the Warburg switch, is caused by herpesviral microRNAs and it is observed in cancer cells. The advantage of the Warburg effect is still unclear, however, it was proposed that the metabolism of cancer cells, and all rapidly dividing cells, is adopted to convert nutrients such as glucose and glutamine faster and more efficiently into biomass. The availability of essential building blocks, such as nucleotides, amino acids, and lipids, is crucial for effective cell proliferation which in turn is essential for the prophage and its host to stay in the lysogenic state.

The prothoracicotropic hormone (PTTH) of Rhodnius prolixus (Hemiptera) is noggin-like: Molecular characterisation, functional analysis and evolutionary implications.

Prothoracicotropic hormone (PTTH) is a central regulator of insect development that regulates the production of the steroid moulting hormones (ecdysteroids) from the prothoracic glands (PGs). Rhodnius PTTH was the first brain neurohormone discovered in any animal almost 100 years ago but has eluded identification and no homologue of Bombyx mori PTTH occurs in its genome. Here, we report Rhodnius PTTH is the first noggin-like PTTH found. It differs in important respects from known PTTHs and is the first PTTH from the Hemimetabola (Exopterygota) to be fully analysed. Recorded PTTHs are widespread in Holometabola but close to absent in hemimetabolous orders. We concluded Rhodnius PTTH likely differed substantially from the known ones. We identified one Rhodnius gene that coded a noggin-like protein (as defined by Molina et al., 2009) that had extensive similarities with known PTTHs but also had two additional cysteines. Sequence and structural analysis showed known PTTHs are closely related to noggin-like proteins, as both possess a growth factor cystine knot preceded by a potential cleavage site. The gene is significantly expressed only in the brain, in a few cells of the dorsal protocerebrum. We vector-expressed the sequence from the potential cleavage site to the C-terminus. This protein was strongly steroidogenic on PGs in vitro. An antiserum to the protein removed the steroidogenic protein released by the brain. RNAi performed on brains in vitro showed profound suppression of transcription of the gene and of production and release of PTTH and thus of ecdysteroid production by PGs. In vivo, the gene is expressed throughout development, in close synchrony with PTTH release, ecdysteroid production by PGs and the ecdysteroid titre. The Rhodnius PTTH monomer is 17kDa and immunoreactive to anti-PTTH of Bombyx mori (a holometabolan). Bombyx PTTH also mildly stimulated Rhodnius PGs. The two additional cysteines form a disulfide at the tip of finger 2, causing a loop of residues to protrude from the finger. A PTTH variant without this loop failed to stimulate PGs, showing the loop is essential for PTTH activity. It is considered that PTTHs of Holometabola evolved from a noggin-like protein in the ancestor of Holometabola and Hemiptera, c.400ma, explaining the absence of holometabolous-type PTTHs from hemimetabolous orders and the differences of Rhodnius PTTH from them. Noggin-like proteins studied from Hemiptera to Arachnida were homologous with Rhodnius PTTH and may be common as PTTHs or other hormones in lower insects.

Panx1 channels promote both anti- and pro-seizure-like activities in the zebrafish via p2rx7 receptors and ATP signaling.

The molecular mechanisms of excitation/inhibition imbalances promoting seizure generation in epilepsy patients are not fully understood. Evidence suggests that Pannexin1 (Panx1), an ATP release channel, modulates the excitability of the brain. In this report, we performed electrophysiological, behavioral, and molecular phenotyping experiments on zebrafish larvae bearing genetic or pharmacological knockouts of Panx1a and Panx1b channels, each homologous to human PANX1. When Panx1a function is lost, or both channels are under pharmacological blockade, seizures with ictal-like events and seizure-like locomotion are reduced in the presence of pentylenetetrazol. Transcriptome profiling by RNA-seq demonstrates a spectrum of distinct metabolic and cell signaling states which correlate with the loss of Panx1a. Furthermore, the pro- and anticonvulsant activities of both Panx1 channels affect ATP release and involve the purinergic receptor P2rx7. Our findings suggest a subfunctionalization of Panx1 enabling dual roles in seizures, providing a unique and comprehensive perspective to understanding seizure mechanisms in the context of this channel.

Molecular Modeling the Proteins from the exo-xis Region of Lambda and Shigatoxigenic Bacteriophages.

Despite decades of intensive research on bacteriophage lambda, a relatively uncharacterized region remains between the exo and xis genes. Collectively, exo-xis region genes are expressed during the earliest stages of the lytic developmental cycle and are capable of affecting the molecular events associated with the lysogenic-lytic developmental decision. In Shiga toxin-producing E. coli (STEC) and enterohemorragic E. coli (EHEC) that are responsible for food- and water-borne outbreaks throughout the world, there are distinct differences of exo-xis region genes from their counterparts in lambda phage. Together, these differences may help EHEC-specific phage and their bacterial hosts adapt to the complex environment within the human intestine. Only one exo-xis region protein, Ea8.5, has been solved to date. Here, I have used the AlphaFold and RoseTTAFold machine learning algorithms to predict the structures of six exo-xis region proteins from lambda and STEC/EHEC phages. Together, the models suggest possible roles for exo-xis region proteins in transcription and the regulation of RNA polymerase.

HACS1 signaling adaptor protein recognizes a motif in the paired immunoglobulin receptor B cytoplasmic domain.

Hematopoietic adaptor containing SH3 and SAM domains-1 (HACS1) is a signaling protein with two juxtaposed protein-protein interaction domains and an intrinsically unstructured region that spans half the sequence. Here, we describe the interaction between the HACS1 SH3 domain and a sequence near the third immunoreceptor tyrosine-based inhibition motif (ITIM3) of the paired immunoglobulin receptor B (PIRB). From surface plasmon resonance binding assays using a mouse and human PIRB ITIM3 phosphopeptides as ligands, the HACS1 SH3 domain and SHP2 N-terminal SH2 domain demonstrated comparable affinities in the micromolar range. Since the PIRB ITIM3 sequence represents an atypical ligand for an SH3 domain, we determined the NMR structure of the HACS1 SH3 domain and performed a chemical shift mapping study. This study showed that the binding site on the HACS1 SH3 domain for PIRB shares many of the same amino acids found in a canonical binding cleft normally associated with polyproline ligands. Molecular modeling suggests that the respective binding sites in PIRB ITIM3 for the HACS1 SH3 domain and the SHP2 SH2 domain are too close to permit simultaneous binding. As a result, the HACS1-PIRB partnership has the potential to amalgamate signaling pathways that influence both immune and neuronal cell fate.

Ea22 Proteins from Lambda and Shiga Toxin-Producing Bacteriophages Balance Structural Diversity with Functional Similarity.

Enterohemorrhagic Escherichia coli (EHEC) outbreaks are commonly associated with contaminated food sources. Unlike normal intestinal bacteria, EHEC are lysogens of lambdoid bacteriophages that also carry a gene for Shiga toxin. Oxidative attack by the immune system or other stressors on the bacterial host can activate the lytic pathway of the latent phage genome to produce phage progeny and the release of Shiga toxin into the surrounding tissues. Within the genomes of bacteriophage λ and Shiga toxin-expressing (Stx+) phages such as φ24B and φP27, there is a conserved set of open reading frames that is located between the exo and xis genes that influences the lysogenic-lytic decision. In this report, we have focused on the largest exo-xis region open reading frame termed ea22 that has been shown previously to have prolysogenic properties. Using a variety of biophysical and bioinformatic methods, we demonstrate that λ and φP27 Ea22 proteins are tetrameric in solution and can be considered in terms of an amino-terminal region, a central coiled-coil region, and a carboxy-terminal region. The carboxy-terminal regions of λ and φ24B Ea22, expressed on their own, form dimers with exceptional thermostability. Limited proteolysis of φP27 Ea22 also identified a C-terminal region along the predicted boundaries. While the three Ea22 proteins all appear to have the hallmarks of a domain in their respective C-terminal regions, each sequence is remarkably dissimilar. To reconcile this difference among Ea22 proteins from λ and Stx+ phages alike, we speculate that each Ea22 may achieve the same function by targeting different components of the same regulatory process in the host.

The ea22 gene of lambdoid phages: preserved prolysogenic function despite of high sequence diversity.

The exo-xis region of lambdoid phages contains open reading frames and genes that appear to be evolutionarily important. However, this region has received little attention up to now. In this study, we provided evidence that ea22, the largest gene of this region, favors the lysogenic pathway over the lytic pathway in contrast to other characterized exo-xis region genes including ea8.5, orf61, orf60a, and orf63. Our assays also suggest some functional analogies between Ea22 and the phage integrase protein (Int). While it is unsurprising that Ea22 operates similarly in both λ and Stx phages, we have observed some distinctions that may arise from considerable sequence dissimilarity at the carboxy termini of each protein.

Tubulin-Dependent Transport of Connexin-36 Potentiates the Size and Strength of Electrical Synapses.

Connexin-36 (Cx36) electrical synapses strengthen transmission in a calcium/calmodulin (CaM)/calmodulin-dependent kinase II (CaMKII)-dependent manner similar to a mechanism whereby the N-methyl-D-aspartate (NMDA) receptor subunit NR2B facilitates chemical transmission. Since NR2B-microtubule interactions recruit receptors to the cell membrane during plasticity, we hypothesized an analogous modality for Cx36. We determined that Cx36 binding to tubulin at the carboxy-terminal domain was distinct from Cx43 and NR2B by binding a motif overlapping with the CaM and CaMKII binding motifs. Dual patch-clamp recordings demonstrated that pharmacological interference of the cytoskeleton and deleting the binding motif at the Cx36 carboxyl-terminal (CT) reversibly abolished Cx36 plasticity. Mechanistic details of trafficking to the gap-junction plaque (GJP) were probed pharmacologically and through mutational analysis, all of which affected GJP size and formation between cell pairs. Lys279, Ile280, and Lys281 positions were particularly critical. This study demonstrates that tubulin-dependent transport of Cx36 potentiates synaptic strength by delivering channels to GJPs, reinforcing the role of protein transport at chemical and electrical synapses to fine-tune communication between neurons.

Roles of orf60a and orf61 in Development of Bacteriophages λ and Φ24B.

The exo-xis region of lambdoid bacteriophage genomes contains several established and potential genes that are evolutionarily conserved, but not essential for phage propagation under laboratory conditions. Nevertheless, deletion or overexpression of either the whole exo-xis region and important regulatory elements can significantly influence the regulation of phage development. This report defines specific roles for orf60a and orf61 in bacteriophage λ and Φ24B, a specific Shiga toxin-converting phage with clinical relevance. We observed that mutant phages bearing deletions of orf60a and orf61 impaired two central aspects of phage development: the lysis-versus-lysogenization decision and prophage induction. These effects were more pronounced for phage Φ24B than for λ. Surprisingly, adsorption of phage Φ24B on Escherichia coli host cells was less efficient in the absence of either orf60a or orf61. We conclude that these open reading frames (ORFs) play important, but not essential, roles in the regulation of lambdoid phage development. Although phages can propagate without these ORFs in nutrient media, we suggest that they may be involved in the regulatory network, ensuring optimization of phage development under various environmental conditions.

Bad Phages in Good Bacteria: Role of the Mysterious orf63 of λ and Shiga Toxin-Converting Φ24B Bacteriophages.

Lambdoid bacteriophages form a group of viruses that shares a common schema of genome organization and lifecycle. Some of them can play crucial roles in creating the pathogenic profiles of Escherichia coli strains. For example, Shiga toxin-producing E. coli (STEC) acquired stx genes, encoding Shiga toxins, via lambdoid prophages (Stx phages). The results obtained so far present the evidence for the relation between the exo-xis region of the phage genome and lambdoid phage development, however molecular mechanisms of activities of the exo-xis genes' products are still unknown. In view of this, we decided to determine the influence of the uncharacterized open reading frame orf63 of the exo-xis region on lambdoid phages development using recombinant prophages, λ and Stx phage Φ24B. We have demonstrated that orf63 codes for a folded protein, thus, it is a functional gene. NMR spectroscopy and analytical gel filtration were used to extend this observation further. From backbone chemical shifts, Orf63 is oligomeric in solution, likely a trimer and consistent with its small size (63 aa.), is comprised of two helices, likely intertwined to form the oligomer. We observed that the deletion of phage orf63 does not impair the intracellular lambdoid phage lytic development, however delays the time and decreases the efficiency of prophage induction and in consequence results in increased survival of E. coli during phage lytic development. Additionally, the deletion of phage orf63 negatively influences expression of the major phage genes and open reading frames from the exo-xis region during prophage induction with hydrogen peroxide. We conclude, that lambdoid phage orf63 may have specific functions in the regulation of lambdoid phages development, especially at the stage of the lysis vs. lysogenization decision. Besides, orf63 probably participates in the regulation of the level of expression of essential phage genes and open reading frames from the exo-xis region during prophage induction.

Tombusvirus polymerase: Structure and function.

Tombusviruses are small icosahedral viruses that possess plus-sense RNA genomes ∼4.8kb in length. The type member of the genus, tomato bushy stunt virus (TBSV), encodes a 92kDa (p92) RNA-dependent RNA polymerase (RdRp) that is responsible for viral genome replication and subgenomic (sg) mRNA transcription. Several functionally relevant regions in p92 have been identified and characterized, including transmembrane domains, RNA-binding segments, membrane targeting signals, and oligomerization domains. Moreover, conserved tombusvirus-specific motifs in the C-proximal region of the RdRp have been shown to modulate viral genome replication, sg mRNA transcription, and trans-replication of subviral replicons. Interestingly, p92 is initially non-functional, and requires an accessory viral protein, p33, as well as viral RNA, host proteins, and intracellular membranes to become active. These and other host factors, through a well-orchestrated process guided by the viral replication proteins, mediate the assembly of membrane-associated virus replicase complexes (VRCs). Here, we describe what is currently known about the structure and function of the tombusvirus RdRp and how it utilizes host components to build VRCs that synthesize viral RNAs.

Structural and Functional Consequences of Connexin 36 (Cx36) Interaction with Calmodulin.

Functional plasticity of neuronal gap junctions involves the interaction of the neuronal connexin36 with calcium/calmodulin-dependent kinase II (CaMKII). The important relationship between Cx36 and CaMKII must also be considered in the context of another protein partner, Ca2+ loaded calmodulin, binding an overlapping site in the carboxy-terminus of Cx36. We demonstrate that CaM and CaMKII binding to Cx36 is calcium-dependent, with Cx36 able to engage with CaM outside of the gap junction plaque. Furthermore, Ca2+ loaded calmodulin activates Cx36 channels, which is different to other connexins. The NMR solution structure demonstrates that CaM binds Cx36 in its characteristic compact state with major hydrophobic contributions arising from W277 at anchor position 1 and V284 at position 8 of Cx36. Our results establish Cx36 as a hub binding Ca2+ loaded CaM and they identify this interaction as a critical step with implications for functions preceding the initiation of CaMKII mediated plasticity at electrical synapses.

A lack of peptide binding and decreased thermostability suggests that the CASKIN2 scaffolding protein SH3 domain may be vestigial.

BACKGROUND: CASKIN2 is a neuronal signaling scaffolding protein comprised of multiple ankyrin repeats, two SAM domains, and one SH3 domain. The CASKIN2 SH3 domain for an NMR structural determination because its peptide-binding cleft appeared to deviate from the repertoire of aromatic enriched amino acids that typically bind polyproline-rich sequences. RESULTS: The structure demonstrated that two non-canonical basic amino acids (K290/R319) in the binding cleft were accommodated well in the SH3 fold. An K290Y/R319W double mutant restoring the typical aromatic amino acids found in the binding cleft resulted in a 20 °C relative increase in the thermal stability. Considering the reduced stability, we speculated that the CASKIN2 SH3 could be a nonfunctional remnant in this scaffolding protein. CONCLUSIONS: While the NMR structure demonstrates that the CASKIN2 SH3 domain is folded, its cleft has suffered two substitutions that prevent it from binding typical polyproline ligands. This observation led us to additionally survey and describe other SH3 domains in the Protein Data Bank that may have similarly lost their ability to promote protein-protein interactions.

A new mode of SAM domain mediated oligomerization observed in the CASKIN2 neuronal scaffolding protein.

BACKGROUND: CASKIN2 is a homolog of CASKIN1, a scaffolding protein that participates in a signaling network with CASK (calcium/calmodulin-dependent serine kinase). Despite a high level of homology between CASKIN2 and CASKIN1, CASKIN2 cannot bind CASK due to the absence of a CASK Interaction Domain and consequently, may have evolved undiscovered structural and functional distinctions. RESULTS: We demonstrate that the crystal structure of the Sterile Alpha Motif (SAM) domain tandem (SAM1-SAM2) oligomer from CASKIN2 is different than CASKIN1, with the minimal repeating unit being a dimer, rather than a monomer. Analytical ultracentrifugation sedimentation velocity methods revealed differences in monomer/dimer equilibria across a range of concentrations and ionic strengths for the wild type CASKIN2 SAM tandem and a structure-directed double mutant that could not oligomerize. Further distinguishing CASKIN2 from CASKIN1, EGFP-tagged SAM tandem proteins expressed in Neuro2a cells produced punctae that were distinct both in shape and size. CONCLUSIONS: This study illustrates a new way in which neuronal SAM domains can assemble into large macromolecular assemblies that might concentrate and amplify synaptic responses.

A Model for Dimerization of the SOX Group E Transcription Factor Family.

Group E members of the SOX transcription factor family include SOX8, SOX9, and SOX10. Preceding the high mobility group (HMG) domain in each of these proteins is a thirty-eight amino acid region that supports the formation of dimers on promoters containing tandemly inverted sites. The purpose of this study was to obtain new structural insights into how the dimerization region functions with the HMG domain. From a mutagenic scan of the dimerization region, the most essential amino acids of the dimerization region were clustered on the hydrophobic face of a single, predicted amphipathic helix. Consistent with our hypothesis that the dimerization region directly contacts the HMG domain, a peptide corresponding to the dimerization region bound a preassembled HMG-DNA complex. Sequence conservation among Group E members served as a basis to identify two surface exposed amino acids in the HMG domain of SOX9 that were necessary for dimerization. These data were combined to make a molecular model that places the dimerization region of one SOX9 protein onto the HMG domain of another SOX9 protein situated at the opposing site of a tandem promoter. The model provides a detailed foundation for assessing the impact of mutations on SOX Group E transcription factors.

Solution structure and peptide binding of the PTB domain from the AIDA1 postsynaptic signaling scaffolding protein.

AIDA1 links persistent chemical signaling events occurring at the neuronal synapse with global changes in gene expression. Consistent with its role as a scaffolding protein, AIDA1 is composed of several protein-protein interaction domains. Here we report the NMR structure of the carboxy terminally located phosphotyrosine binding domain (PTB) that is common to all AIDA1 splice variants. A comprehensive survey of peptides identified a consensus sequence around an NxxY motif that is shared by a number of related neuronal signaling proteins. Using peptide arrays and fluorescence based assays, we determined that the AIDA1 PTB domain binds amyloid protein precursor (APP) in a similar manner to the X11/Mint PTB domain, albeit at reduced affinity (∼10 µM) that may allow AIDA1 to effectively sample APP, as well as other protein partners in a variety of cellular contexts.

The solution structures of two prophage homologues of the bacteriophage λ Ea8.5 protein reveal a newly discovered hybrid homeodomain/zinc-finger fold.

A cluster of genes in the exoxis region of bacteriophage λ are capable of inhibiting the initiation of DNA synthesis in Escherichia coli. The most indispensible gene in this region is ea8.5. Here, we report the nuclear magnetic resonance structures of two ea8.5 orthologs from enteropathogenic E. coli and Pseudomonas putida prophages. Both proteins are characterized by a fused homeodomain/zinc-finger fold that escaped detection by primary sequence search methods. While these folds are both associated with a nucleic acid binding function, the amino acid composition suggests otherwise, leading to the possibility that Ea8.5 associates with other viral and host proteins.