Possible Involvement of Protein Binding Inhibition in Changes in Dexmedetomidine Concentration in Extracorporeal Circuits during Midazolam Use.

It was recently reported that the dexmedetomidine concentration within the extracorporeal circuit decreases with co-administration of midazolam. In this study, we investigated whether displacement of dexmedetomidine by midazolam from the binding site of major plasma proteins, human serum albumin (HSA) and α1-acid glycoprotein (AAG), would increase levels of free dexmedetomidine that could be adsorbed to the circuit. Equilibrium dialysis experiments indicated that dexmedetomidine binds to a single site on both HSA and AAG with four times greater affinity than midazolam. Midazolam-mediated inhibition of the binding of dexmedetomidine to HSA and AAG was also examined. The binding of dexmedetomidine to these proteins decreased in the presence of midazolam. Competitive binding experiments suggested that the inhibition of binding by midazolam was due to competitive displacement at site II of HSA and due to non-competitive displacement at the site of AAG. Thus, our present data indicate that free dexmedetomidine displaced by midazolam from site II of HSA or from AAG is adsorbed onto extracorporeal circuits, resulting in a change in the dexmedetomidine concentration within the circuit.

N-Terminal Fragment of Cardiac Myosin Binding Protein-C Increases the Duration of Actin-Myosin Interaction.

Cardiac myosin binding protein-C (cMyBP-C) located in the C-zone of myocyte sarcomere is involved in the regulation of myocardial contraction. Its N-terminal domains C0, C1, C2, and the m-motif between C1 and C2 can bind to the myosin head and actin of the thin filament and affect the characteristics of their interaction. Measurements using an optical trap showed that the C0-C2 fragment of cMyBP-C increases the interaction time of cardiac myosin with the actin filament, while in an in vitro motility assay, it dose-dependently reduces the sliding velocity of actin filaments. Thus, it was found that the N-terminal part of cMyBP-C affects the kinetics of the myosin cross-bridge.

Unraveling blunt-end RNA binding and ATPase-driven translocation activities of the RIG-I family helicase LGP2.

The RIG-I family helicases, comprising RIG-I, MDA5 and LGP2, are cytoplasmic RNA sensors that trigger an antiviral immune response by specifically recognizing foreign RNAs. While LGP2 lacks the signaling domain necessary for immune activation, it plays a vital role in regulating the RIG-I/MDA5 signaling pathway. In this study, we investigate the mechanisms underlying this regulation by examining the oligomeric state, RNA binding specificity, and translocation activity of human LGP2 and the impact of ATPase activity. We show that LGP2, like RIG-I, prefers binding blunt-ended double-stranded (ds) RNAs over internal dsRNA regions or RNA overhangs and associates with blunt-ends faster than with overhangs. Unlike RIG-I, a 5'-triphosphate (5'ppp), Cap0, or Cap1 RNA-end does not influence LGP2's RNA binding affinity. LGP2 hydrolyzes ATP in the presence of RNA but at a 5-10 fold slower rate than RIG-I. Nevertheless, LGP2 uses its ATPase activity to translocate and displace biotin-streptavidin interactions. This activity is significantly hindered by a methylated RNA patch, particularly on the 3'-strand, suggesting a 3'-strand tracking mechanism like RIG-I. The preference of LGP2 for blunt-end RNA binding, its insensitivity to Cap0/Cap1 modification, and its translocation/protein displacement ability have substantial implications for how LGP2 regulates the RNA sensing process by MDA5/RIG-I.

Glutamine-rich regions of the disordered CREB transactivation domain mediate dynamic intra- and intermolecular interactions.

The cyclic AMP response element (CRE) binding protein (CREB) is a transcription factor that contains a 280-residue N-terminal transactivation domain and a basic leucine zipper that mediates interaction with DNA. The transactivation domain comprises three subdomains, the glutamine-rich domains Q1 and Q2 and the kinase inducible activation domain (KID). NMR chemical shifts show that the isolated subdomains are intrinsically disordered but have a propensity to populate local elements of secondary structure. The Q1 and Q2 domains exhibit a propensity for formation of short ß-hairpin motifs that function as binding sites for glutamine-rich sequences. These motifs mediate intramolecular interactions between the CREB Q1 and Q2 domains as well as intermolecular interactions with the glutamine-rich Q1 domain of the TATA-box binding protein associated factor 4 (TAF4) subunit of transcription factor IID (TFIID). Using small-angle X-ray scattering, NMR, and single-molecule Förster resonance energy transfer, we show that the Q1, Q2, and KID regions remain dynamically disordered in a full-length CREB transactivation domain (CREBTAD) construct. The CREBTAD polypeptide chain is largely extended although some compaction is evident in the KID and Q2 domains. Paramagnetic relaxation enhancement reveals transient long-range contacts both within and between the Q1 and Q2 domains while the intervening KID domain is largely devoid of intramolecular interactions. Phosphorylation results in expansion of the KID domain, presumably making it more accessible for binding the CBP/p300 transcriptional coactivators. Our study reveals the complex nature of the interactions within the intrinsically disordered transactivation domain of CREB and provides molecular-level insights into dynamic and transient interactions mediated by the glutamine-rich domains.

The Axin scaffold protects the kinase GSK3ß from cross-pathway inhibition.

Multiple signaling pathways regulate the kinase GSK3ß by inhibitory phosphorylation at Ser9, which then occupies the GSK3ß priming pocket and blocks substrate binding. Since this mechanism should affect GSK3ß activity toward all primed substrates, it is unclear why Ser9 phosphorylation does not affect other GSK3ß-dependent pathways, such as Wnt signaling. We used biochemical reconstitution and cell culture assays to evaluate how Wnt-associated GSK3ß is insulated from cross-activation by other signals. We found that the Wnt-specific scaffold protein Axin allosterically protects GSK3ß from phosphorylation at Ser9 by upstream kinases, which prevents accumulation of pS9-GSK3ß in the Axin•GSK3ß complex. Scaffold proteins that protect bound proteins from alternative pathway reactions could provide a general mechanism to insulate signaling pathways from improper crosstalk.

Chain-specificity of laminin α1-5 LG45 modules in the recognition of carbohydrate-linked receptors and intramolecular binding.

Laminins are a family of heterotrimers composed of α-, ß-, and γ-chains in the basement membrane. Five α chains contain laminin globular (LG) domain consisting of five tandem modules (LG1-5 modules) at their C-terminus. Each LG45 modules is connected to a compact cloverleaf-shaped structure of LG1-3 through a flexible linker. Although the accumulated studies of the LG45 modules have suggested differences in each α chain regarding the binding of carbohydrate chain and intramolecular interaction, this remains unclear. In this study, to characterize their functions comparatively, we produced recombinant proteins of LG45 modules of human laminin α1-5 chains. Dystroglycan (DG) modified with matriglycan readily bound to the LG45 modules of α1 and α2 chains but not to the other α chains. In contrast, heparin bound to the LG45 modules of the α chains, except for α2. The binding of heparan sulfate/heparin-linked syndecans (SDCs) to LG45 modules was influenced by their core proteins. Furthermore, the α1 and α4LG45 modules bound to SDCs in a pH-dependent manner. A cell adhesion assay showed that HEK293 cells could readily adhere to the LG45 modules of α3-5 chains through a combination of SDCs and integrins. Moreover, α5LG45 modules bound to the E8 fragment, which includes the C-terminus of the laminin coiled-coil (LCC) domain and LG1-3 modules, but α2LG45 modules did not. The results suggested that although α5LG45 modules was fixed within the LG domain, α2LG45 modules was freely placed in the vicinity of LG1-3. Our findings provide information for investigation of the structural and functional diversity of basement membranes.

GPCR Binding and JNK3 Activation by Arrestin-3 Have Different Structural Requirements.

Arrestins bind active phosphorylated G protein-coupled receptors (GPCRs). Among the four mammalian subtypes, only arrestin-3 facilitates the activation of JNK3 in cells. In available structures, Lys-295 in the lariat loop of arrestin-3 and its homologue Lys-294 in arrestin-2 directly interact with the activator-attached phosphates. We compared the roles of arrestin-3 conformational equilibrium and Lys-295 in GPCR binding and JNK3 activation. Several mutants with enhanced ability to bind GPCRs showed much lower activity towards JNK3, whereas a mutant that does not bind GPCRs was more active. The subcellular distribution of mutants did not correlate with GPCR recruitment or JNK3 activation. Charge neutralization and reversal mutations of Lys-295 differentially affected receptor binding on different backgrounds but had virtually no effect on JNK3 activation. Thus, GPCR binding and arrestin-3-assisted JNK3 activation have distinct structural requirements, suggesting that facilitation of JNK3 activation is the function of arrestin-3 that is not bound to a GPCR.

Crosstalking interactions between P2X4 and 5-HT3A receptors.

Ionotropic receptors are ligand-gated ion channels triggering fast neurotransmitter responses. Among them, P2X and 5-HT3 receptors have been shown to physically interact each other and functionally inducing cross inhibitory responses. Nevertheless, despite the importance of P2X4 and 5-HT3A receptors that mediate for example neuropathic pain and psychosis respectively, complementary evidence has recently started to move forward in the understanding of this interaction. In this review, we discuss current evidence supporting the mechanism of crosstalking between both receptors, from the structural to the transduction pathway level. We expect this work may guide the design of further experiments to obtain a comprehensive view for the neuropharmacological role of these interacting receptors. This article is part of the Special Issue on "The receptor-receptor interaction as a new target for therapy".

AgTx2-GFP, Fluorescent Blocker Targeting Pharmacologically Important Kv1.x (x = 1, 3, 6) Channels.

The growing interest in potassium channels as pharmacological targets has stimulated the development of their fluorescent ligands (including genetically encoded peptide toxins fused with fluorescent proteins) for analytical and imaging applications. We report on the properties of agitoxin 2 C-terminally fused with enhanced GFP (AgTx2-GFP) as one of the most active genetically encoded fluorescent ligands of potassium voltage-gated Kv1.x (x = 1, 3, 6) channels. AgTx2-GFP possesses subnanomolar affinities for hybrid KcsA-Kv1.x (x = 3, 6) channels and a low nanomolar affinity to KcsA-Kv1.1 with moderate dependence on pH in the 7.0-8.0 range. Electrophysiological studies on oocytes showed a pore-blocking activity of AgTx2-GFP at low nanomolar concentrations for Kv1.x (x = 1, 3, 6) channels and at micromolar concentrations for Kv1.2. AgTx2-GFP bound to Kv1.3 at the membranes of mammalian cells with a dissociation constant of 3.4 ± 0.8 nM, providing fluorescent imaging of the channel membranous distribution, and this binding depended weakly on the channel state (open or closed). AgTx2-GFP can be used in combination with hybrid KcsA-Kv1.x (x = 1, 3, 6) channels on the membranes of E. coli spheroplasts or with Kv1.3 channels on the membranes of mammalian cells for the search and study of nonlabeled peptide pore blockers, including measurement of their affinity.

Characterization of the TBR1 interactome: variants associated with neurodevelopmental disorders disrupt novel protein interactions.

TBR1 is a neuron-specific transcription factor involved in brain development and implicated in a neurodevelopmental disorder (NDD) combining features of autism spectrum disorder (ASD), intellectual disability (ID) and speech delay. TBR1 has been previously shown to interact with a small number of transcription factors and co-factors also involved in NDDs (including CASK, FOXP1/2/4 and BCL11A), suggesting that the wider TBR1 interactome may have a significant bearing on normal and abnormal brain development. Here, we have identified approximately 250 putative TBR1-interaction partners by affinity purification coupled to mass spectrometry. As well as known TBR1-interactors such as CASK, the identified partners include transcription factors and chromatin modifiers, along with ASD- and ID-related proteins. Five interaction candidates were independently validated using bioluminescence resonance energy transfer assays. We went on to test the interaction of these candidates with TBR1 protein variants implicated in cases of NDD. The assays uncovered disturbed interactions for NDD-associated variants and identified two distinct protein-binding domains of TBR1 that have essential roles in protein-protein interaction.

The influence of several nutritional supplements on the rational use of cabozantinib.

To promote the rational use of cabozantinib (CBZ), this paper studied the influence of several nutritional supplements on the interaction between CBZ and bovine serum albumin (BSA), an appropriate alternative model for human serum albumin (HSA) that is one of the important transporter proteins in plasma, by fluorescence spectroscopy and UV-vis spectroscopy. The results showed that CBZ could quench the fluorescence of BSA via a dynamic-static quenching process, and the six nutritional supplements did not change the quenching mode of BSA by CBZ. However, all of them could reduce the binding constant of the CBZ-BSA system at 293 K and increase the polarity around tryptophan residues. Among them, nicotinamide and vitamin B12 (VB12 ) had a greater effect on the binding constants of the CBZ-BSA system. In the meantime, the thermodynamic parameters of the CBZ-BSA system were examined, indicating that the interaction of CBZ with BSA was spontaneous and dominated by hydrophobic forces. Further research discovered that the combining of CBZ with BSA was primarily located within Site I of BSA, and the binding distance r was 2.48 nm. Consequently, while taking CBZ, patients should use VB12 and nicotinamide carefully, which may interfere with the transport of drugs.

Thyroid hormone activities of neutral and anionic hydroxylated polybrominated diphenyl ethers to thyroid receptor ß: A molecular dynamics study.

Hydroxylated polybrominated diphenyl ethers (OH-PBDEs) have been identified as the strong endocrine disrupting chemicals to humans, which show structural similarity with endogenous thyroid hormones (THs) and thus disrupt the functioning of THs through competitive binding with TH receptors (TRs). Although previous studies have reported the hormone activities of some OH-PBDEs on TH receptor ß (TRß), the interaction mechanism remains unclear. Furthermore, hydroxyl dissociation of OH-PBDEs may alter their TR disrupting activities, which has not yet been investigated in depth. In this work, we selected 18 OH-PBDEs with neutral and anionic forms and performed molecular dynamics (MD) simulations to estimate their binding interactions with the ligand binding domain (LBD) of TRß. The results demonstrate that most of OH-PBDEs have stronger binding affinities to TRß-LBD than their anionic counterparts, and the hydroxyl dissociation of ligands differentiate the major driving force for their binding. More Br atoms in OH-PBDEs can result in stronger binding potential with TRß-LBD. Moreover, 5 hydrophobic residues, including Met313, Leu330, Ile276, Leu346, and Phe272, are identified to have important contributions to bind OH-PBDEs. These results clarify the binding mechanism of OH(O-)-PBDEs to TRß-LBD at the molecular level, which can provide a solid theoretical basis for accurate assessment of TH disrupting effects of these chemicals.

Mechanochemical tuning of a kinesin motor essential for malaria parasite transmission.

Plasmodium species cause malaria and kill hundreds of thousands annually. The microtubule-based motor kinesin-8B is required for development of the flagellated Plasmodium male gamete, and its absence completely blocks parasite transmission. To understand the molecular basis of kinesin-8B's essential role, we characterised the in vitro properties of kinesin-8B motor domains from P. berghei and P. falciparum. Both motors drive ATP-dependent microtubule gliding, but also catalyse ATP-dependent microtubule depolymerisation. We determined these motors' microtubule-bound structures using cryo-electron microscopy, which showed very similar modes of microtubule interaction in which Plasmodium-distinct sequences at the microtubule-kinesin interface influence motor function. Intriguingly however, P. berghei kinesin-8B exhibits a non-canonical structural response to ATP analogue binding such that neck linker docking is not induced. Nevertheless, the neck linker region is required for motility and depolymerisation activities of these motors. These data suggest that the mechanochemistry of Plasmodium kinesin-8Bs is functionally tuned to support flagella formation.

Identification of orphan ligand-receptor relationships using a cell-based CRISPRa enrichment screening platform.

Secreted proteins, which include cytokines, hormones, and growth factors, are extracellular ligands that control key signaling pathways mediating cell-cell communication within and between tissues and organs. Many drugs target secreted ligands and their cell surface receptors. Still, there are hundreds of secreted human proteins that either have no identified receptors ('orphans') or are likely to act through cell surface receptors that have not yet been characterized. Discovery of secreted ligand-receptor interactions by high-throughput screening has been problematic, because the most commonly used high-throughput methods for protein-protein interaction (PPI) screening are not optimized for extracellular interactions. Cell-based screening is a promising technology for the deorphanization of ligand-receptor interactions, because multimerized ligands can enrich for cells expressing low affinity cell surface receptors, and such methods do not require purification of receptor extracellular domains. Here, we present a proteo-genomic cell-based CRISPR activation (CRISPRa) enrichment screening platform employing customized pooled cell surface receptor sgRNA libraries in combination with a magnetic bead selection-based enrichment workflow for rapid, parallel ligand-receptor deorphanization. We curated 80 potentially high-value orphan secreted proteins and ultimately screened 20 secreted ligands against two cell sgRNA libraries with targeted expression of all single-pass (TM1) or multi-pass transmembrane (TM2+) receptors by CRISPRa. We identified previously unknown interactions in 12 of these screens, and validated several of them using surface plasmon resonance and/or cell binding assays. The newly deorphanized ligands include three receptor protein tyrosine phosphatase (RPTP) ligands and a chemokine-like protein that binds to killer immunoglobulin-like receptors (KIRs). These new interactions provide a resource for future investigations of interactions between the human-secreted and membrane proteomes.

Short Arrestin-3-Derived Peptides Activate JNK3 in Cells.

Arrestins were first discovered as suppressors of G protein-mediated signaling by G protein-coupled receptors. It was later demonstrated that arrestins also initiate several signaling branches, including mitogen-activated protein kinase cascades. Arrestin-3-dependent activation of the JNK family can be recapitulated with peptide fragments, which are monofunctional elements distilled from this multi-functional arrestin protein. Here, we use maltose-binding protein fusions of arrestin-3-derived peptides to identify arrestin elements that bind kinases of the ASK1-MKK4/7-JNK3 cascade and the shortest peptide facilitating JNK signaling. We identified a 16-residue arrestin-3-derived peptide expressed as a Venus fusion that leads to activation of JNK3α2 in cells. The strength of the binding to the kinases does not correlate with peptide activity. The ASK1-MKK4/7-JNK3 cascade has been implicated in neuronal apoptosis. While inhibitors of MAP kinases exist, short peptides are the first small molecule tools that can activate MAP kinases.

Antibody for Serine 65 Phosphorylated Ubiquitin Identifies PLK1-Mediated Phosphorylation of Mitotic Proteins and APC1.

Deciphering the protein posttranslational modification (PTM) code is one of the greatest biochemical challenges of our time. Phosphorylation and ubiquitylation are key PTMs that dictate protein function, recognition, sub-cellular localization, stability, turnover and fate. Hence, failures in their regulation leads to various disease. Chemical protein synthesis allows preparation of ubiquitinated and phosphorylated proteins to study their biochemical properties in great detail. However, monitoring these modifications in intact cells or in cell extracts mostly depends on antibodies, which often have off-target binding. Here, we report that the most widely used antibody for ubiquitin (Ub) phosphorylated at serine 65 (pUb) has significant off-targets that appear during mitosis. These off-targets are connected to polo-like kinase 1 (PLK1) mediated phosphorylation of cell cycle-related proteins and the anaphase promoting complex subunit 1 (APC1).

Mechanism of the cadherin-catenin F-actin catch bond interaction.

Mechanotransduction at cell-cell adhesions is crucial for the structural integrity, organization, and morphogenesis of epithelia. At cell-cell junctions, ternary E-cadherin/ß-catenin/αE-catenin complexes sense and transmit mechanical load by binding to F-actin. The interaction with F-actin, described as a two-state catch bond, is weak in solution but is strengthened by applied force due to force-dependent transitions between weak and strong actin-binding states. Here, we provide direct evidence from optical trapping experiments that the catch bond property principally resides in the αE-catenin actin-binding domain (ABD). Consistent with our previously proposed model, the deletion of the first helix of the five-helix ABD bundle enables stable interactions with F-actin under minimal load that are well described by a single-state slip bond, even when αE-catenin is complexed with ß-catenin and E-cadherin. Our data argue for a conserved catch bond mechanism for adhesion proteins with structurally similar ABDs. We also demonstrate that a stably bound ABD strengthens load-dependent binding interactions between a neighboring complex and F-actin, but the presence of the other αE-catenin domains weakens this effect. These results provide mechanistic insight to the cooperative binding of the cadherin-catenin complex to F-actin, which regulate dynamic cytoskeletal linkages in epithelial tissues.

Zinc Modulation of Neuronal Calcium Sensor Proteins: Three Modes of Interaction with Different Structural Outcomes.

Neuronal calcium sensors (NCSs) are the family of EF-hand proteins mediating Ca2+-dependent signaling pathways in healthy neurons and neurodegenerative diseases. It was hypothesized that the calcium sensor activity of NCSs can be complemented by sensing fluctuation of intracellular zinc, which could further diversify their function. Here, using a set of biophysical techniques, we analyzed the Zn2+-binding properties of five proteins belonging to three different subgroups of the NCS family, namely, VILIP1 and neurocalcin-δ/NCLD (subgroup B), recoverin (subgroup C), as well as GCAP1 and GCAP2 (subgroup D). We demonstrate that each of these proteins is capable of coordinating Zn2+ with a different affinity, stoichiometry, and structural outcome. In the absence of calcium, recoverin and VILIP1 bind two zinc ions with submicromolar affinity, and the binding induces pronounced conformational changes and regulates the dimeric state of these proteins without significant destabilization of their structure. In the presence of calcium, recoverin binds zinc with slightly decreased affinity and moderate conformational outcome, whereas VILIP1 becomes insensitive to Zn2+. NCALD binds Zn2+ with micromolar affinity, but the binding induces dramatic destabilization and aggregation of the protein. In contrast, both GCAPs demonstrate low-affinity binding of zinc independent of calcium, remaining relatively stable even at submillimolar Zn2+ concentrations. Based on these data, and the results of structural bioinformatics analysis, NCSs can be divided into three categories: (1) physiological Ca2+/Zn2+ sensor proteins capable of binding exchangeable (signaling) zinc (recoverin and VILIP1), (2) pathological Ca2+/Zn2+ sensors responding only to aberrantly high free zinc concentrations by denaturation and aggregation (NCALD), and (3) Zn2+-resistant, Ca2+ sensor proteins (GCAP1, GCAP2). We suggest that NCS proteins may therefore govern the interconnection between Ca2+-dependent and Zn2+-dependent signaling pathways in healthy neurons and zinc cytotoxicity-related neurodegenerative diseases, such as Alzheimer's disease and glaucoma.

Ultrasensitive Label-Free Detection of Protein-Membrane Interaction Exemplified by Toxin-Liposome Insertion.

Measuring the high-affinity binding of proteins to liposome membranes remains a challenge. Here, we show an ultrasensitive and direct detection of protein binding to liposome membranes using high throughput second harmonic scattering (SHS). Perfringolysin O (PFO), a pore-forming toxin, with a highly membrane selective insertion into cholesterol-rich membranes is used. PFO inserts only into liposomes with a cholesterol concentration >30%. Twenty mole-percent cholesterol results in neither SHS-signal deviation nor pore formation as seen by cryo-electron microscopy of PFO and liposomes. PFO inserts into cholesterol-rich membranes of large unilamellar vesicles in an aqueous solution with Kd = (1.5 ± 0.2) × 10-12 M. Our results demonstrate a promising approach to probe protein-membrane interactions below sub-picomolar concentrations in a label-free and noninvasive manner on 3D systems. More importantly, the volume of protein sample is ultrasmall (<10 µL). These findings enable the detection of low-abundance proteins and their interaction with membranes.

There is nothing exempt from the peril of mutation - The Omicron spike.

The 2019 corona virus disease (COVID-19) has caused a global chaos, where a novel Omicron variant has challenged the healthcare system, followed by which it has been referred to as a variant of concern (VOC) by the World Health Organization (WHO), owing to its alarming transmission and infectivity rate. The large number of mutations in the receptor binding domain (RBD) of the spike protein is responsible for strengthening of the spike-angiotensin-converting enzyme 2 (ACE2) interaction, thereby explaining the elevated threat. This is supplemented by enhanced resistance of the variant towards pre-existing antibodies approved for the COVID-19 therapy. The manuscript brings into light failure of existing therapies to provide the desired effect, however simultaneously discussing the novel possibilities on the verge of establishing suitable treatment portfolio. The authors entail the risks associated with omicron resistance against antibodies and vaccine ineffectiveness on one side, and novel approaches and targets - kinase inhibitors, viral protease inhibitors, phytoconstituents, entry pathways - on the other. The manuscript aims to provide a holistic picture about the Omicron variant, by providing comprehensive discussions related to multiple aspects of the mutated spike variant, which might aid the global researchers and healthcare experts in finding an optimised solution to this pandemic.