ABSTRACT
BACKGROUND: The activity of von Willebrand factor (VWF) in facilitating platelet adhesion and aggregation correlates with its multimer size. Traditional ristocetin-dependent functional assays lack sensitivity to multimer sizes. Recently, nanobodies targeting the autoinhibitory module and activating VWF were identified. OBJECTIVES: To develop an assay that can differentiate the platelet-binding activity of VWF multimers. METHODS: A novel enzyme-linked immunosorbent assay (nanobody-triggered glycoprotein Ib binding assay [VWF:GPIbNab]) utilizing a VWF-activating nanobody was developed. Recombinant VWF, plasma-derived VWF (pdVWF), and selected gel-filtrated fractions of pdVWF were evaluated for VWF antigen and activity levels. A linear regression model was developed to estimate the specific activity of VWF multimers. RESULTS: Of the 3 activating nanobodies tested, 6C11 with the lowest activation effect exhibited the highest sensitivity for high-molecular-weight multimers (HMWMs) of VWF. VWF:GPIbNab utilizing 6C11 (VWF:GPIbNab6C11) produced significantly higher activity/antigen ratios for recombinant VWF (>2.0) and HMWM-enriched pdVWF fractions (>2.0) than for pdVWF (â¼1.0) or fractions enriched with shorter multimers (<1.0). The differences were much larger than those produced by VWF:GPIbNab utilizing other nanobodies, VWF:GPIbM, VWF:GPIbR, or VWF:CB assays. Linear regression analysis of 5 pdVWF fractions of various multimer sizes produced an estimated specific activity of 2.7 for HMWMs. The analysis attributed >90% of the VWF activity measured by VWF:GPIbNab6C11 to that of HMWMs, which is significantly higher than all other activity assays tested. CONCLUSION: The VWF:GPIbNab6C11 assay exhibits higher sensitivity to HMWMs than ristocetin-based and collagen-binding assays. Future studies examining the application of this assay in clinical settings and any associated therapeutic benefit of doing so are warranted.
Subject(s)
Enzyme-Linked Immunosorbent Assay , Protein Multimerization , Single-Domain Antibodies , von Willebrand Factor , von Willebrand Factor/metabolism , von Willebrand Factor/analysis , Humans , Single-Domain Antibodies/immunology , Single-Domain Antibodies/chemistry , Linear Models , Recombinant Proteins , Blood Platelets/metabolism , Platelet Aggregation/drug effects , Platelet Glycoprotein GPIb-IX Complex/metabolism , Protein Binding , Platelet Adhesiveness , Molecular WeightABSTRACT
Monofunctional Pt(II) complexes with potent efficacy to overcome the drawbacks of current platinum drugs represent a promising therapeutic approach for triple negative breast cancer (TNBC). A heterocyclic-ligated monofunctional Pt(II) complex PtL with a unique action of mode was designed and investigated. PtL induced DNA single-strand breaks and caused genomic instability in TNBC cells. Mechanism studies demonstrated that PtL disrupted lysosomal acidity and function, which in turn triggered lysosome-dependent cell death. Furthermore, PtL showed convincing suppression in the tube forming and cell migratory abilities against the metastatic potential of TNBC cells. The synthesis and investigation of PtL revealed its potential value as an anti-TNBC drug and extended the family of monofunctional Pt(II) complexes.
Subject(s)
Triple Negative Breast Neoplasms , Humans , Cell Death , Cell Line, Tumor , Lysosomes/metabolism , Platinum/pharmacology , Platinum/therapeutic use , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/metabolism , Organoplatinum Compounds/chemistry , Organoplatinum Compounds/pharmacology , Organoplatinum Compounds/therapeutic useABSTRACT
ABSTRACT: Activation of von Willebrand factor (VWF) is a tightly controlled process governed primarily by local elements around its A1 domain. Recent studies suggest that the O-glycosylated sequences flanking the A1 domain constitute a discontinuous and force-sensitive autoinhibitory module (AIM), although its extent and conformation remains controversial. Here, we used a targeted screening strategy to identify 2 groups of nanobodies. One group, represented by clone 6D12, is conformation insensitive and binds the N-terminal AIM (NAIM) sequence that is distal from A1; 6D12 activates human VWF and induces aggregation of platelet-rich plasma at submicromolar concentrations. The other group, represented by clones Nd4 and Nd6, is conformation sensitive and targets the C-terminal AIM (CAIM). Nd4 and Nd6 inhibit ristocetin-induced platelet aggregation and reduce VWF-mediated platelet adhesion under flow. A crystal structure of Nd6 in complex with AIM-A1 shows a novel conformation of both CAIM and NAIM that are primed to interact, providing a model of steric hindrance stabilized by the AIM as the mechanism for regulating GPIbα binding to VWF. Hydrogen-deuterium exchange mass spectrometry analysis shows that binding of 6D12 induces the exposure of the GPIbα-binding site in the A1 domain, but binding of inhibitory nanobodies reduces it. Overall, these results suggest that the distal portion of NAIM is involved in specific interactions with CAIM, and binding of nanobodies to the AIM could either disrupt its conformation to activate VWF or stabilize its conformation to upkeep VWF autoinhibition. These reported nanobodies could facilitate future studies of VWF functions and related pathologies.
Subject(s)
Single-Domain Antibodies , von Willebrand Factor , von Willebrand Factor/metabolism , von Willebrand Factor/chemistry , Humans , Single-Domain Antibodies/pharmacology , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/metabolism , Platelet Aggregation/drug effects , Protein Conformation , Protein Domains , Protein Binding , Platelet Adhesiveness/drug effects , Crystallography, X-Ray , Animals , Blood Platelets/metabolismABSTRACT
Studies of folded-to-misfolded transitions using model protein systems reveal a range of unfolding needed for exposure of amyloid-prone regions for subsequent fibrillization. Here, we probe the relationship between unfolding and aggregation for glaucoma-associated myocilin. Mutations within the olfactomedin domain of myocilin (OLF) cause a gain-of-function, namely cytotoxic intracellular aggregation, which hastens disease progression. Aggregation by wild-type OLF (OLFWT) competes with its chemical unfolding, but only below the threshold where OLF loses tertiary structure. Representative moderate (OLFD380A) and severe (OLFI499F) disease variants aggregate differently, with rates comparable to OLFWT in initial stages of unfolding, and variants adopt distinct partially folded structures seen along the OLFWT urea-unfolding pathway. Whether initiated with mutation or chemical perturbation, unfolding propagates outward to the propeller surface. In sum, for this large protein prone to amyloid formation, the requirement for a conformational change to promote amyloid fibrillization leads to direct competition between unfolding and aggregation.
Subject(s)
Amyloid , Glaucoma , Humans , Amyloid/metabolism , Glaucoma/genetics , Mutation , Amyloid beta-Peptides/genetics , Amyloidogenic Proteins/genetics , Protein FoldingABSTRACT
Molecular forces generated by cell receptors are infrequent and transient, and hence difficult to detect. Here we report an assay that leverages the CRISPR-associated protein 12a (Cas12a) to amplify the detection of cellular traction forces generated by as few as 50 adherent cells. The assay involves the immobilization of a DNA duplex modified with a ligand specific for a cell receptor. Traction forces of tens of piconewtons trigger the dehybridization of the duplex, exposing a cryptic Cas12-activating strand that sets off the indiscriminate Cas12-mediated cleavage of a fluorogenic reporter strand. We used the assay to perform hundreds of force measurements using human platelets from a single blood draw to extract individualized dose-response curves and half-maximal inhibitory concentrations for a panel of antiplatelet drugs. For seven patients who had undergone cardiopulmonary bypass, platelet dysfunction strongly correlated with the need for platelet transfusion to limit bleeding. The Cas12a-mediated detection of cellular traction forces may be used to assess cell state, and to screen for genes, cell-adhesion ligands, drugs or metabolites that modulate cell mechanics.
Subject(s)
CRISPR-Cas Systems , Traction , Humans , Cell Adhesion/physiology , Proteins , Carrier ProteinsABSTRACT
Flow cytometry is used routinely to measure single-cell gene expression by staining cells with fluorescent antibodies and nucleic acids. Here, we present tension-activated cell tagging (TaCT) to label cells fluorescently based on the magnitude of molecular force transmitted through cell adhesion receptors. As a proof-of-concept, we analyzed fibroblasts and mouse platelets after TaCT using conventional flow cytometry.
Subject(s)
Flow Cytometry , Animals , Mice , Cell AdhesionABSTRACT
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a clear threat to humanity. It has infected over 200 million and killed 4 million people worldwide, and infections continue with no end in sight. To control the pandemic, multiple effective vaccines have been developed, and global vaccinations are in progress. However, the virus continues to mutate. Even when full vaccine coverage is achieved, vaccine-resistant mutants will likely emerge, thus requiring new annual vaccines against drifted variants analogous to influenza. A complimentary solution to this problem could be developing antiviral drugs that inhibit SARS CoV-2 and its drifted variants. Host defense peptides represent a potential source for such an antiviral as they possess broad antimicrobial activity and significant diversity across species. We screened the cathelicidin family of peptides from 16 different species for antiviral activity and identified a wild boar peptide derivative that inhibits SARS CoV-2. This peptide, which we named Yongshi and means warrior in Mandarin, acts as a viral entry inhibitor. Following the binding of SARS-CoV-2 to its receptor, the spike protein is cleaved, and heptad repeats 1 and 2 multimerize to form the fusion complex that enables the virion to enter the cell. A deep learning-based protein sequence comparison algorithm and molecular modeling suggest that Yongshi acts as a mimetic to the heptad repeats of the virus, thereby disrupting the fusion process. Experimental data confirm the binding of Yongshi to the heptad repeat 1 with a fourfold higher affinity than heptad repeat 2 of SARS-CoV-2. Yongshi also binds to the heptad repeat 1 of SARS-CoV-1 and MERS-CoV. Interestingly, it inhibits all drifted variants of SARS CoV-2 that we tested, including the alpha, beta, gamma, delta, kappa and omicron variants.
Subject(s)
COVID-19 , Cathelicidins , Humans , SARS-CoV-2 , Antiviral AgentsABSTRACT
Phenothiazinone is a promising yet underutilized fluorophore, possibly due to the lack of a general accessibility. This study reports a robust and scalable TEMPO-mediated electrochemical method to access a variety of phenothiazinones from 2-aminothiophenols and quinones. The electrosynthesis proceeds in a simple cell architecture under mild condition, and notably carbon-halogen bond in quinones remains compared to conventional methods, enabling orthogonal downstream functionalization. Mechanistic studies corroborate that TEMPO exerts a protective effect in avoiding product decomposition at the cathode. In particular, benzophenothiazinones show intriguing luminescence in both solid and solution state, and thus their photophysical properties are scrutinized in detail. Further bio-imaging of the lipid droplets in living cells highlights the considerable promise of benzophenothiazinones as fluorescent dye in the biomedical fields.
Subject(s)
Fluorescent Dyes , Luminescence , Fluorescent Dyes/chemistry , Carbon , Electrochemical Techniques , QuinonesABSTRACT
The overall success of worldwide mass vaccination in limiting the negative effect of the COVID-19 pandemics is inevitable, however, recent SARS-CoV-2 variants of concern, especially Omicron and its sub-lineages, efficiently evade humoral immunity mounted upon vaccination or previous infection. Thus, it is an important question whether these variants, or vaccines against them, induce anti-viral cellular immunity. Here we show that the mRNA vaccine BNT162b2 induces robust protective immunity in K18-hACE2 transgenic B-cell deficient (µMT) mice. We further demonstrate that the protection is attributed to cellular immunity depending on robust IFN-γ production. Viral challenge with SARS-CoV-2 Omicron BA.1 and BA.5.2 sub-variants induce boosted cellular responses in vaccinated µMT mice, which highlights the significance of cellular immunity against the ever-emerging SARS-CoV-2 variants evading antibody-mediated immunity. Our work, by providing evidence that BNT162b2 can induce significant protective immunity in mice that are unable to produce antibodies, thus highlights the importance of cellular immunity in the protection against SARS-CoV-2.
Subject(s)
COVID-19 Vaccines , COVID-19 , Immunity, Cellular , Animals , Humans , Mice , Antibodies , Antibodies, Neutralizing , Antibodies, Viral , BNT162 Vaccine , COVID-19/prevention & control , Interferon-gamma , SARS-CoV-2 , COVID-19 Vaccines/immunologyABSTRACT
Direct access to substituted dihydrochalcones from the easily available starting materials 3-hydroxypropionitrile derivatives and arylboronic acids is described. The procedure involves a multicomponent aryl addition/hydroxyl elimination/reduction Heck approach in the presence of a Pd catalyst with excellent functional group tolerance and a wide range of substrates. In addition, mixed 1,3-diarylation of 3-hydroxypropanenitrile using two arylboronic acids with different electronic properties was also achieved.
Subject(s)
Boronic Acids , Palladium , Palladium/chemistry , Molecular Structure , Boronic Acids/chemistry , CatalysisABSTRACT
The activation of platelets and coagulation at vascular injury sites is crucial for haemostasis but can promote thrombosis and inflammation in vascular pathologies. Here, we delineate an unexpected spatio-temporal control mechanism of thrombin activity that is platelet orchestrated and locally limits excessive fibrin formation after initial haemostatic platelet deposition. During platelet activation, the abundant platelet glycoprotein (GP) V is cleaved by thrombin. We demonstrate with genetic and pharmacological approaches that thrombin-mediated shedding of GPV does not primarily regulate platelet activation in thrombus formation, but rather has a distinct function after platelet deposition and specifically limits thrombin-dependent generation of fibrin, a crucial mediator of vascular thrombo-inflammation. Genetic or pharmacologic defects in haemostatic platelet function are unexpectedly attenuated by specific blockade of GPV shedding, indicating that the spatio-temporal control of thrombin-dependent fibrin generation also represents a potential therapeutic target to improve haemostasis.
ABSTRACT
Type 2B von Willebrand disease (VWD) is an inherited bleeding disorder in which a subset of point mutations in the von Willebrand factor (VWF) A1 domain and recently identified autoinhibitory module (AIM) cause spontaneous binding to glycoprotein Ibα (GPIbα) on the platelet surface. All reported type 2B VWD mutations share this enhanced binding; however, type 2B VWD manifests as variable bleeding complications and platelet levels in patients, depending on the underlying mutation. Understanding how these mutations localizing to a similar region can result in such disparate patient outcomes is essential for detailing our understanding of VWF regulatory and activation mechanisms. In this study, we produced recombinant glycosylated AIM-A1 fragments bearing type 2B VWD mutations and examined how each mutation affects the A1 domain's thermodynamic stability, conformational dynamics, and biomechanical regulation of the AIM. We found that the A1 domain with mutations associated with severe bleeding occupy a higher affinity state correlating with enhanced flexibility in the secondary GPIbα-binding sites. Conversely, mutation P1266L, associated with normal platelet levels, has similar proportions of high-affinity molecules to wild-type (WT) but shares regions of solvent accessibility with both WT and other type 2B VWD mutations. V1316M exhibited exceptional instability and solvent exposure compared with all variants. Lastly, examination of the mechanical stability of each variant revealed variable AIM unfolding. Together, these studies illustrate that the heterogeneity among type 2B VWD mutations is evident in AIM-A1 fragments.
Subject(s)
von Willebrand Disease, Type 2 , von Willebrand Factor , Humans , Binding Sites , Blood Platelets/metabolism , Mutation , Platelet Glycoprotein GPIb-IX Complex/metabolism , von Willebrand Disease, Type 2/genetics , von Willebrand Factor/chemistry , von Willebrand Factor/genetics , von Willebrand Factor/metabolismABSTRACT
CD36 is a transmembrane glycoprotein receptor for oxidized low density lipoprotein (LDL) and other endogenous danger signals and promotes athero-thrombotic processes. CD36 has been shown to associate physically with other transmembrane proteins, including integrins, tetraspanins, and toll-like receptors, which modulate CD36-mediated cell signaling. The CD36 N-terminal transmembrane domain (nTMD) contains a GXXXG sequence motif that mediates protein-protein interactions in many membrane proteins. We thus hypothesized that the nTMD is involved in CD36 interactions with other membrane proteins. CD36 interactions with partner cell surface proteins on murine peritoneal macrophages were detected with an immunofluorescence-based proximity ligation cross linking assay (PLA) and confirmed by immunoprecipitation/immunoblot. Prior to performing these assays, cells were incubated with a synthetic 29 amino acid peptide containing the 22 amino acid of CD36 nTMD or a control peptide in which the glycine residues in GXXXG motif were replaced by valines. In functional experiments, macrophages were preincubated with peptides and then treated with oxLDL to assess LDL uptake, foam cell formation, ROS formation and cell migration. CD36 nTMD peptide treated cells compared to untreated or control peptide treated cells showed decreased CD36 surface associations with tetraspanin CD9 and ameliorated pathologically important CD36 mediated responses to oxLDL, including uptake of DiI-labeled oxLDL, foam cell formation, ROS generation, and inhibition of migration.
Subject(s)
Atherosclerosis , Macrophages , Animals , Mice , Atherosclerosis/metabolism , CD36 Antigens/metabolism , Foam Cells/metabolism , Macrophages/metabolism , Membrane Proteins , Peptides , Reactive Oxygen Species/metabolismABSTRACT
BACKGROUND: Von Willebrand factor (VWF) is a multimeric plasma protein that bridges the gap between vessel injury and platelet capture at high shear rates. Under high shear or tension, VWF can become activated upon the unfolding of its autoinhibitory module (AIM). AIM unfolding exposes the A1 domain, allowing for binding to platelet glycoprotein (GP)Ibα to initiate primary hemostasis. The characteristics of the AIM and its inhibitory properties within mouse VWF are unknown. OBJECTIVES: To determine and characterize the autoinhibitory properties of mouse VWF. METHODS: Recombinant mouse VWF A1 fragments containing or lacking the flanking regions around the A1 domain were generated. We tested the ability of these fragments to bind to human or mouse GPIbα and platelets. We compared the unfolding of mouse AIM-A1 to human AIM-A1 by single-molecule force spectroscopy. RESULTS: Recombinant mouse AIM-A1 binds with higher affinity to GPIbα than its human counterpart. Recombinant mouse proteins lacking part of the AIM show increased binding to GPIbα. Activated A1 fragments lacking the AIM can effectively agglutinate platelets across the species barrier. Using single-molecule force spectroscopy, we determined that the mouse AIM unfolds under forces similar to the human AIM. Additionally, the human AIM paired with mouse A1 largely recapitulates the behavior of human AIM-A1. CONCLUSIONS: Our results suggest that the regulation of VWF-GPIbα binding has been specifically tuned to work optimally in different rheological architectures. Differences in the AIM sequence may contribute to the difference in VWF shear response between human and mice.
Subject(s)
Platelet Glycoprotein GPIb-IX Complex , von Willebrand Factor , Humans , Mice , Animals , von Willebrand Factor/metabolism , Species Specificity , Protein Binding , Platelet Glycoprotein GPIb-IX Complex/metabolism , Blood Platelets/metabolism , Hemostasis , Recombinant Proteins/chemistryABSTRACT
Membranes with nanochannels have exhibited great potential in molecular separations, while it remains a great challenge to separate molecules with very close physical properties and kinetic diameters (e.g., ethylene/ethane) owing to the lack of size-sieving property and specific affinity. Herein, a metal confined 2D sub-nanometer channel is reported to successfully discriminate ethylene over ethane via molecular recognition and sieving. Transition metal cations are paired with polyelectrolyte anions to achieve high dissociation activity, forming reversible complexation with ethylene. Aberration-corrected transmission electron microscopy observes that the metals with size of ≈2 nm are uniformly confined in graphene oxide (GO) interlayer channels with average height of ≈0.44 nm, thereby cooperating the size-sieving effect with a molecular recognition ability toward ethylene and stimulating its selective transport over ethane. The resulting ultrathin (≈60 nm) membrane exhibits superior ethylene/ethane separation performance far beyond the polymeric upper-bound. Density functional theory (DFT) and molecular dynamic simulations reveal that the metal@2D interlayer channel provides a molecular recognition pathway for selective gas transport. The proposed metal confined in 2D channel with molecular recognition and sieving properties would have broad application in other related fields such as single-atom catalysis, sensor and energy conversion.
ABSTRACT
Microneedles can realize the intradermal and transdermal delivery of drugs. However, most conventional microneedles made of metal, polymer and ceramics are unsuitable for the delivery of mRNA drugs that are fragile and temperature-sensitive. This study explores the usage of cryomicroneedles (CryoMNs) for the intradermal delivery of mRNA molecules. Taking luciferase mRNA as an example, we first optimize the formulation of CryoMNs to maximize mRNA stability. Later, in the mouse model, we compare the delivery efficiency with the conventional subcutaneous injection for both the luciferase mRNA and COVID-19 Comirnaty mRNA vaccines, where CryoMNs delivered mRNA vaccines successfully induce specific B-cell antibody, neutralizing activity and T-cell responses. STATEMENT OF SIGNIFICANCE: mRNA vaccines are fragile and temperature-sensitive, so they are mainly delivered by intramuscular injection that often causes pain and requires clinical expertise to immunize patients. Microneedles permit convenient, fast and safe vaccination. However, existing microneedle platforms are ineffective to protect the integrity of mRNA vaccines in fabrication, storage, and administration. This work utilizes cryomicroneedles (CryoMNs) technology to intradermally deliver mRNA. In the mouse model, CryoMNs are compared with the subcutaneous injection for the delivery efficiency of both the luciferase mRNA and COVID-19 Comirnaty mRNA vaccines, where CryoMNs delivered mRNA vaccines successfully produce specific B-cell antibodies, T-cell responses, and neutralizing activity. This work is expected to provide a new delivery strategy for the emerging mRNA therapeutics.