Employing Surface Plasmon Resonance Research Articles

A Peptide Motif Covering Splice Site B in Neuroligin-1 Binds to Aβ and Acts as a Neprilysin Inhibitor.

The most common cause of dementia among elderly people is Alzheimer's disease (AD). The typical symptom of AD is the decline of cognitive abilities, which is caused by loss of synaptic function. Amyloid-β (Aβ) oligomers play a significant role in the development of this synaptic dysfunction. Neuroligin-(NL)1 is a postsynaptic cell-adhesion molecule located in excitatory synapses and involved in the maintenance and modulation of synaptic contacts. A recent study has found that Aβ interacts with the soluble N-terminal fragment of NL1. The present study aimed to elucidate the role of NL1 in Aβ-induced neuropathology. Employing surface plasmon resonance and competitive ELISA, we confirmed the high-affinity binding of NL1 to the Aβ peptide. We also identified a sequence motif representing the NL1-binding site for the Aβ peptide and showed that a synthetic peptide modeled after this motif, termed neurolide, binds to the Aβ peptide with high affinity, comparable to the NL1-Aβ interaction. To assess the effect of neurolide in vivo, wild-type and 5XFAD mice were subcutaneously treated with this peptide for 10weeks. We observed an increase in Aβ plaque formation in the cortex of neurolide-treated 5XFAD mice. Furthermore, we showed that neurolide reduces the activity of neprilysin, the predominant Aβ-degrading enzyme in the brain. Accordingly, we suggest that neurolide is the NL1-binding site for Aβ peptide, and acts as an inhibitor of neprilysin activity. Based on these data, we confirm the involvement of NL1 in the development of AD and suggest a mechanism for NL1-induced Aβ plaque formation.

Miniature fiber optic SPR high sensitivity humidity sensor based on coated polyvinyl alcohol film

For the measurement of relative humidity, this study employed Surface Plasmon Resonance (SPR) technology, a sensor with a micro-conical fiber structure (sensing length is only 375 μm) was combined with Polyvinyl Alcohol (PVA) to create a humidity sensor that is highly sensitive, compact, dense, and exhibits good linearity. The humidity sensor is based on a gold film (thickness of about 40 nm) on the surface of the miniature fiber optic structure, and by applying PVA coating, the SPR humidity sensor shows resonance peaks in the transmission spectrum in ambient air, and as the number of layers of PVA coating (thickness) is added, the resonance peaks’ positions are red-shifted. The experimental findings demonstrated that this SPR humidity sensor’s sensitivity was 0.162 nm/%RH at an initial resonance wavelength of about 550 nm (one layer) and about 1.542 nm/%RH at an initial resonance wavelength of 656 nm (five layers) in the range of 46 %RH to 93 %RH. The wavelength and the relative humidity had a good linear connection. Temperature tests revealed the sensor has a −0.152 nm/°C temperature sensitivity between 20 °C and 50 °C and has a high linearity (0.999). Therefore, the sensor has potential applications in humidity measurement.

Abstract 6567: Affinity and cooperativity modulate ternary complex formation to drive degradation of SMARCA2

Abstract Small molecules that hijack the ubiquitin-proteasome system to degrade target proteins have gained traction as tools for chemical biology and as promising clinical candidates. Targeted degradation strategies encompass relatively large hetero-bifunctional small molecules (hSMs) that are built from target and effector ligands joined by a linker to much smaller molecular glues that have limited affinity for the target or effector proteins outside of a ternary complex. There is good reason for medicinal chemists to move from linkered hSMs toward glue-like scaffolds. Molecular glues can be considerably smaller, e.g., the IMiDs, and thus are less likely to possess atoms and functional motifs that add up to poor permeability and higher protein binding. Guided by the principle that hSMs with reduced molecular weight will have superior druglike properties, we have used SMARCA2, a potential therapeutic target in patients that have both copies of SMARCA4 inactivated, as a test of our own ability to drive rapid target degradation in relevant models. The hetero-bifunctional degraders that resulted from our work trade conventional linkers for chemical spacers that orient SMARCA2 and VHL to promote fortuitous protein-protein contacts in the ternary complex. Following in the footsteps of previously published works, we employed surface plasmon resonance (SPR), x-ray crystallography, and molecular modeling to understand the relationships between our cell-based metrics of degrader activity and the biophysical parameters that describe the ternary complex. Consequently, we found that cooperativity (α) and ternary complex affinity (KLPT) correlated well with both DC50 and initial rate of degradation. Within our series of novel SMARCA2 degraders, compound 1 exhibited exceptional cooperativity and ternary complex affinity, α = 12.8 and KLPT = 4.7 ± 1. The result of high cooperativity and enhanced affinity was rapid and near complete SMARCA2 degradation. Treatment of a cancer cell line with compound 1 resulted in 50% of SMARCA2 being degraded in under 30 minutes, and greater than 90% degradation in 2 hours. Most importantly, the near complete degradation of SMARCA2 resulted in potent effects on SWI/SNF dependent transcription in vitro and in inhibition of tumor cell growth both in vitro and in vivo. Citation Format: Ivonne Archibeque, Ken Dellamaggiore, Karen Rex, Sudipa Ghimire-Rijal, Kaylee Choi, Smither Kate, Albert Amegadzie, Ning Chen, Xiaofen Li, Ryan Wurz, Amit Vaish, Paul Hughes, Dane Mohl. Affinity and cooperativity modulate ternary complex formation to drive degradation of SMARCA2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6567.

Utilizing surface plasmon resonance as a novel method for monitoring in-vitro P-glycoprotein efflux.

P-glycoprotein (Pgp) is known for its dichotomous roles as both a safeguarding efflux transporter against xenobiotics and as a catalyst for multidrug resistance. Given the susceptibility of numerous therapeutic compounds to Pgp-mediated resistance, compliance with Food and Drug Administration (FDA) guidelines mandates an in-depth in vitro transport assay during drug development. This study introduces an innovative transport assay that aligns with these regulatory imperatives but also addresses limitations in the currently established techniques. Using Pgp-reconstituted liposomes and employing surface plasmon resonance (SPR), this study developed a distinct method of measuring the relative transport rates of Pgp substrates in a controlled microenvironment. The Pgp substrates selected for this study-quinidine, methadone, and desipramine-resulted in transport ratios that corroborate with trends previously observed. To assess the kinetics of Pgp-mediated transport, the results were analyzed by fitting the data to both currently proposed Pgp substrate translocation models-the vacuum cleaner and flippase models. While the resulting kinetic analysis in this study lends support predominantly to the vacuum cleaner model, this study most notably developed a novel method of assessing Pgp-mediated transport rates and real-time kinetics using surface plasmon resonance.

HIV-1 Gag MA domain binds to cardiolipin in a binding mode distinct from virus assemble mediator PI(4,5)P2.

The human immunodeficiency virus type 1 (HIV-1) Gag protein is responsible for facilitating HIV-1 virion assembly and budding. Our study demonstrates that cardiolipin (CL), a component found in the inner mitochondrial membrane, exhibits the highest binding affinity to the N-terminal MA domain of the HIV-1 Gag protein within the lipid group of host cells. To assess this binding interaction, we synthesized short acyl chain derivatives of CL and employed surface plasmon resonance (SPR) analysis to determine the dissociation constants (Kd) for CL and the MA domain. Simultaneously, we examined the Kd of D-myo-phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 ) derivatives, known to play a crucial role in virion formation. Among all the derivatives, Tetra-C7 -CL exhibited the lowest Kd value (Kd = 30.8 ± 6.9 μM) for MA binding on the CL analog-immobilized sensorchip, indicating a higher affinity. Similarly, the Kd value of Di-C7 -PIP2 (Kd = 36.6 ± 4.7 μM) was the lowest on the PI(4,5)P2 analog-immobilized sensorchip. Thus, Tetra-C7 -CL binds to the MA domain using a distinct binding mode while displaying a comparable binding affinity to Di-C7 -PIP2. This discovery holds significant implications for comprehending the virological importance of CL-MA domain binding, such as its subcellular distribution, including mitochondrial translocation, and involvement in viral particle formation in concert with PI(4,5)P2 . Furthermore, this study has the potential to contribute to the development of drugs in the future.

Parallel SPR and QCM-D Quantitative Analysis of CD9, CD63, and CD81 Tetraspanins: A Simple and Sensitive Way to Determine the Concentration of Extracellular Vesicles Isolated from Human Lung Cancer Cells.

Tetraspanins, including CD9, CD63, and CD81, are transmembrane biomarkers that play a crucial role in regulating cancer cell proliferation, invasion, and metastasis, as well as plasma membrane dynamics and protein trafficking. In this study, we developed simple, fast, and sensitive immunosensors to determine the concentration of extracellular vesicles (EVs) isolated from human lung cancer cells using tetraspanins as biomarkers. We employed surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation (QCM-D) as detectors. The monoclonal antibodies targeting CD9, CD63, and CD81 were oriented vertically in the receptor layer using either a protein A sensor chip (SPR) or a cysteamine layer that modified the gold crystal (QCM-D) without the use of amplifiers. The SPR studies demonstrated that the interaction of EVs with antibodies could be described by the two-state reaction model. Furthermore, the EVs' affinity to monoclonal antibodies against tetraspanins decreased in the following order: CD9, CD63, and CD81, as confirmed by the QCM-D studies. The results indicated that the developed immunosensors were characterized by high stability, a wide analytical range from 6.1 × 104 particles·mL-1 to 6.1 × 107 particles·mL-1, and a low detection limit (0.6-1.8) × 104 particles·mL-1. A very good agreement between the results obtained using the SPR and QCM-D detectors and nanoparticle tracking analysis demonstrated that the developed immunosensors could be successfully applied to clinical samples.

InP and graphene employed surface plasmon resonance sensor for measurement of sucrose concentration: a numerical approach

A surface plasmon resonance (SPR) biosensor utilizing an indium phosphide (InP) semiconductor and graphene material layer and placed on an Ag metal-based biosensor is proposed to detect the sucrose concentration in water. The SPR biosensor works on the principle of attenuated total reflection. The transverse matrix method has been utilized for the reflectance calculation. The thickness of the Ag layer, InP, and graphene are taken as 45 nm, 2 nm, and 2 nm, respectively. When three layers of InP and a single layer of graphene are taken, and the maximum sensitivity of the sensor 506 deg / RIU is obtained. The desired values of performance parameters, such as full-width half maximum and quality factor were computed and obtained as 6.94 deg and 72.86 deg RIU − 1 respectively, which recommend the significance of the proposed work. Graphene is used to enhance the bio-compatibility of the analyte with the sensing layer. The refractive index of the sensing medium is considered as 1.33. Indium phosphide is an air-stable optical material. SPR biosensors have wide applications in detecting and analyzing biomolecules and biochemicals.

Indium phosphide and black phosphorus employed surface plasmon resonance sensor for formalin detection: numerical analysis

A hybrid Kretschmann configuration-based surface plasmon resonance biosensor is investigated to detect formalin in water was proposed. The modification is done in the conventional sensor by adding the indium phosphide (InP) and black phosphorus (BP) material layer. The silver (Ag) metal thickness is 45 nm, the optimized thickness of the metal for the proposed design. The thickness of the InP and BP materials 2 and 0.34 nm are considered. For three InP layers and one BP layer, the maximum sensitivity of 250.2 deg / RIU is achieved. The BP layer is used to improve the biorecognition ability of the sensor. The performance of the sensor is analyzed using the angular interrogation method. The proposed sensor is investigated for the aqueous sensing medium. The InP is an air-stable semiconductor material and has applications in chemical, medical, and biological fields.

Conformational heterogeneity coupled with \u03b2-fibril formation of a scaffold protein involved in chronic mental illnesses

Chronic mental illnesses (CMIs) pose a significant challenge to global health due to their complex and poorly understood etiologies and hence, absence of causal therapies. Research of the past two decades has revealed dysfunction of the disrupted in schizophrenia 1 (DISC1) protein as a predisposing factor involved in several psychiatric disorders. DISC1 is a multifaceted protein that serves myriads of functions in mammalian cells, for instance, influencing neuronal development and synapse maintenance. It serves as a scaffold hub forming complexes with a variety (~300) of partners that constitute its interactome. Herein, using combinations of structural and biophysical tools, we demonstrate that the C-region of the DISC1 protein is highly polymorphic, with important consequences for its physiological role. Results from solid-state NMR spectroscopy and electron microscopy indicate that the protein not only forms symmetric oligomers but also gives rise to fibrils closely resembling those found in certain established amyloid proteinopathies. Furthermore, its aggregation as studied by isothermal titration calorimetry (ITC) is an exergonic process, involving a negative enthalpy change that drives the formation of oligomeric (presumably tetrameric) species as well as β-fibrils. We have been able to narrow down the β-core region participating in fibrillization to residues 716–761 of full-length human DISC1. This region is absent in the DISC1Δ22aa splice variant, resulting in reduced association with proteins from the dynein motor complex, viz., NDE-like 1 (NDEL1) and lissencephaly 1 (LIS1), which are crucial during mitosis. By employing surface plasmon resonance, we show that the oligomeric DISC1 C-region has an increased affinity and shows cooperativity in binding to LIS1 and NDEL1, in contrast to the noncooperative binding mode exhibited by the monomeric version. Based on the derived structural models, we propose that the association between the binding partners involves two neighboring subunits of DISC1 C-region oligomers. Altogether, our findings highlight the significance of the DISC1 C-region as a crucial factor governing the balance between its physiological role as a multifunctional scaffold protein and aggregation-related aberrations with potential significance for disease.

Peptides Derived from Vascular Endothelial Growth Factor B Show Potent Binding to Neuropilin-1.

Vascular endothelial growth factors (VEGFs) regulate significant pathways in angiogenesis, myocardial and neuronal protection, metabolism, and cancer progression. The VEGF‐B growth factor is involved in cell survival, anti‐apoptotic and antioxidant mechanisms, through binding to VEGF receptor 1 and neuropilin‐1 (NRP1). We employed surface plasmon resonance technology and X‐ray crystallography to analyse the molecular basis of the interaction between VEGF‐B and the b1 domain of NRP1, and developed VEGF‐B C‐terminus derived peptides to be used as chemical tools for studying VEGF‐B ‐ NRP1 related pathways. Peptide lipidation was used as a means to stabilise the peptides. VEGF‐B‐derived peptides containing a C‐terminal arginine show potent binding to NRP1‐b1. Peptide lipidation increased binding residence time and improved plasma stability. A crystal structure of a peptide with NRP1 demonstrated that VEGF‐B peptides bind at the canonical C‐terminal arginine binding site. VEGF‐B C‐terminus imparts higher affinity for NRP1 than the corresponding VEGF‐A165 region. This tight binding may impact on the activity and selectivity of the full‐length protein. The VEGF‐B167 derived peptides were more effective than VEGF‐A165 peptides in blocking functional phosphorylation events. Blockers of VEGF‐B function have potential applications in diabetes and non‐alcoholic fatty liver disease.

Kinetic and thermodynamic of lactoferrin – Ethoxylated-nonionic surfactants supramolecular complex formation

Understanding nonionic surfactant-protein interactions is fundamental from both technological and scientific points of view. However, there is a complete absence of kinetic data for such interactions. We employed surface plasmon resonance (SPR) to determine the kinetic and thermodynamic parameters of bovine lactoferrin-Brij58 interactions at various temperatures under physiological conditions (pH 7.4). The adsorption process was accelerated with increasing temperature, while the desorption rate decreased, resulting in a more thermodynamically stable complex. The kinetic energetic parameters obtained for the formation of the activated complex, [bLF-Brij58]‡, indicated that the potential energy barrier for [bLF-Brij58]‡ formation arises primarily from the reduction in system entropy. [bLF-Brij58]○ formation was entropically driven, indicating that hydrophobic interactions play a fundamental role in bLF interactions with Brij58.

Effective inhibition of coronavirus replication by Polygonum cuspidatum.

Background: The coronavirus disease 2019 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected more than 210 million individuals globally and resulted in over 4 million deaths since the first report in December 2019. The early use of traditional Chinese medicine (TCM) for light and ordinary patients, can rapidly improve symptoms, shorten hospitalization days and reduce severe cases transformed from light and normal. Many TCM formulas and products have a wide application in treating infectious and non-infectious diseases. Polygonum cuspidatum Sieb. et Zucc. (P. cuspidatum), is an important Traditional Chinese Medicine with actions of clearing away heat and eliminating dampness, draining the gallbladder to relieve jaundice, removing blood stasis to alleviate pain, resolving phlegm and arrest cough. In the search for anti-SARS-CoV-2, P. cuspidatum was recommended as as a therapeutic drug of COVID-19 pneumonia.In this study, we aimed to identifies P. cuspidatum is the potential broad-spectrum inhibitor for the treatment of coronaviruses infections. Methods: In the present study , we infected human malignant embryonal rhabdomyoma (RD) cells with the OC43 strain of the coronavirus, which represent an alternative model for SARS-CoV-2 and then employed the cell viability assay kit for the antiviral activity. We combined computer aided virtual screening to predicte the binding site and employed Surface plasmon resonance analysis (SPR) to comfirm the interaction between drugs and coronavirus. We employed fluorescence resonance energy transfer technology to identify drug's inhibition in the proteolytic activity of 3CLpro and Plpro. Results: Based on our results, polydatin and resveratrol derived from P. cuspidatum significantly suppressed HCoV-OC43 replication. 50% inhibitory concentration (IC50) values of polydatin inhibited SARS-CoV-2 Mpro and Plpro, MERS Mpro and Plpro were 18.66, 125, 14.6 and 25.42 μm, respectively. IC50 values of resveratrol inhibited SARS-CoV-2 Mpro and Plpro, MERS Mpro and Plpro were 29.81 ,60.86, 16.35 and19.04 μM, respectively. Finally, SPR assay confirmed that polydatin and resveratrol had high affinity to SARS-CoV-2, SARS-CoV 3Clpro, MERS-CoV 3Clpro and PLpro protein. Conclusions: we identified the antiviral activity of flavonoids polydatin and resveratrol on RD cells. Polydatin and resveratrol were found to be specific and selective inhibitors for SARS-CoV-2, 3CLpro and PLpro, viral cysteine proteases. In summary, this study identifies P. cuspidatum as the potential broad-spectrum inhibitor for the treatment of coronaviruses infections.

Anti-S100A4 Antibody Therapy Is Efficient in Treating Aggressive Prostate Cancer and Reversing Immunosuppression: Serum and Biopsy S100A4 as a Clinical Predictor.

S100A4 oncoprotein plays a critical role during prostate cancer progression and induces immunosuppression in host tissues. We hypothesized that S100A4-regulated oncogenic activity in immunosuppressed prostate tumors promotes growth of neoplastic cells, which are likely to become aggressive. In the current study, we investigated whether biopsy-S100A4 gene alteration independently predicts the outcome of disease in patients and circulatory-S100A4 is druggable target for treating immunosuppressive prostate cancer. Aided by DECIPHER-genomic test, we show biopsy-S100A4 overexpression as predictive of (i) poor ADT response and (ii) high risk of mortality in 228 radical prostatectomy-treated patients. Furthermore, analysis of tumor genome data of more than 1,000 patients with prostate cancer (PRAD/SU2C/FHCRC studies) validated the association of S100A4-alteration to poor survival and metastasis. We show that increased serum-S100A4 levels are associated to the prostate cancer progression in patients. The prerequisite for metastasis is the escape of tumor cells via vascular system. We show that extracellular-S100A4 protein as a growth factor induces vascular transmigration of prostate cancer cells and bone demineralization thus forms an ideal target for therapies for treating prostate cancer. By employing surface plasmon resonance and isothermal titration calorimetry, we show that mab6B12 antibody interacts with and neutralizes S100A4 protein. When tested for therapeutic efficacy, the mab6B12 therapy reduced the (i) osteoblastic demineralization of bone-derived MSCs, (ii) S100A4-target (NFκB/MMP9/VEGF) levels in prostate cancer cells, and (iii) tumor growth in a TRAMPC2 syngeneic mouse model. The immuno-profile analysis showed that mAb6B12-therapy (i) shifted Th1/Th2 balance (increased Stat4+/T-bet+ and decreased GATA2+/CD68+/CD45+/CD206+ cells); (ii) modulated cytokine levels in CD4+ T cells; and (iii) decreased levels of IL5/6/12/13, sTNFR1, and serum-RANTES. We suggest that S100A4-antibody therapy has clinical applicability in treating immunosuppressive prostate cancer in patients.

High-Affinity Binding of LDL Receptor-Related Protein 1 to Matrix Metalloprotease 1 Requires Protease:Inhibitor Complex Formation.

Matrix metalloprotease (MMP) activation contributes to the degradation of the extracellular matrix (ECM), resulting in a multitude of pathologies. Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifaceted endocytic and signaling receptor that is responsible for internalization and lysosomal degradation of diverse proteases, protease inhibitors, and lipoproteins along with numerous other proteins. In this study, we identified MMP-1 as a novel LRP1 ligand. Binding studies employing surface plasmon resonance revealed that both proMMP-1 and active MMP-1 bind to purified LRP1 with equilibrium dissociation constants (KD) of 19 and 25 nM, respectively. We observed that human aortic smooth muscle cells readily internalize and degrade 125I-labeled proMMP-1 in an LRP1-mediated process. Our binding data also revealed that all tissue inhibitors of metalloproteases (TIMPs) bind to LRP1 with KD values ranging from 23 to 33 nM. Interestingly, the MMP-1/TIMP-1 complex bound to LRP1 with an affinity (KD = 0.6 nM) that was 30-fold higher than that of either component alone, revealing that LRP1 prefers the protease:inhibitor complex as a ligand. Of note, modification of lysine residues on either proMMP-1 or TIMP-1 ablated the ability of the MMP-1/TIMP-1 complex to bind to LRP1. LRP1's preferential binding to enzyme:inhibitor complexes was further supported by the higher binding affinity for proMMP-9/TIMP-1 complexes than for either of these two components alone. LRP1 has four clusters of ligand-binding repeats, and MMP-1, TIMP-1, and MMP-1/TIMP-1 complexes bound to cluster III most avidly. Our results reveal an important role for LRP1 in controlling ECM homeostasis by regulating MMP-1 and MMP-9 levels.

Protective Effect of a Novel (2S, 3R, 4S)-Chromene-3-Carboxamide Derivative, Z20 Against Sepsis-Induced Organ Injury.

Sepsis, a systemic inflammatory response mediated by excessive production of diverse inflammatory cytokines, remains the vital cause of morality in the intensive care unit (ICU). TLR4-MD2 (toll-like receptor 4-myeloid differentiation factor 2) complex activated by LPS serves as an effective target to decrease the inflammation during sepsis. In this study, we evaluated the effects of a new small molecule Z20 structural based on (2S, 3R, 4S)-chromene-3-carboxamide on LPS-induced sepsis in mice. We found Z20 markedly improved the survival rate and attenuated the multiply organs injury after LPS administrationin mice. In addition, Z20 significantly alleviated organ inflammation as characterized by diminished inflammatory factors expression in vivo. Furthermore, by employing surface plasmon resonance (SPR) experiment, we identified that TLR4-MD2 complexwas the potential target for Z20. Finally, we performed the safety assessment experiment to confirm the safety of Z20 in vivo. In conclusion, Z20, as a potential TLR4-MD2 inhibitor, effectively attenuated LPS-induced organ injury and inflammation.

SPR Analysis of Protein-Protein Interactions Involving Cytochromes P450 and Cytochrome b5 Integrated into Lipid Membrane

Identification of new protein-protein interactions (PPI) and characterization of quantitative parameters of complex formation represent one of central tasks of modern protein interactomics. This study is a logical continuation of the cycle of our previous works aimed at the PPIs between components of the cytochrome P450-dependent monooxygenase system. Using an optical biosensor employing surface plasmon resonance (SPR biosensor), a comparative analysis characterizing kinetic and equilibrium parameters of complex formation between the membrane-bound hemoprotein cytochrome b5 and cytochrome P450s was performed using two different protocols for protein immobilization: (1) covalent non-oriented immobilization on the carboxymethyl dextran chip and (2) non-covalent oriented immobilization in the lipid environment. In the case of the second protocol, PPIs were characterized by 2.5-fold higher affinity due to a decrease in rate dissociation constants values of the studied complexes.

Unique surface‐exposed hydrophobic residues in the C1 domain of factor VIII contribute to cofactor function and von Willebrand factor binding

BackgroundThe identity of the amino acid regions of factor VIII (FVIII) that contribute to factor IXa (FIXa) and von Willebrand factor (VWF) binding has not been fully resolved. Previously, we observed that replacing the FVIII C1 domain for the one of factor V (FV) markedly reduces VWF binding and cofactor function. Compared to the FV C1 domain, this implies that the FVIII C1 domain comprises unique surface‐exposed elements involved in VWF and FIXa interaction. ObjectiveThe aim of this study is to identify residues in the FVIII C1 domain that contribute to VWF and FIXa binding. MethodsStructures and primary sequences of FVIII and FV were compared to identify surface‐exposed residues unique to the FVIII C1 domain. The identified residues were replaced with alanine residues to identify their role in FIXa and VWF interaction. This role was assessed employing surface plasmon resonance analysis studies and enzyme kinetic assays. ResultsFive surface‐exposed hydrophobic residues unique to the FVIII C1 domain, ie, F2035, F2068, F2127, V2130, I2139 were identified. Functional analysis indicated that residues F2068, V2130, and especially F2127 contribute to VWF and/or FIXa interaction. Substitution into alanine of the also surface‐exposed V2125, which is spatially next to F2127, affected only VWF binding. ConclusionThe surface‐exposed hydrophobic residues in C1 domain contribute to cofactor function and VWF binding. These findings provide novel information on the fundamental role of the C1 domain in FVIII life cycle.

Apolipoprotein E binds to and reduces serum levels of DNA-mimicking, pyrrolated proteins

Lysine N-pyrrolation, converting lysine residues to Nϵ-pyrrole-l-lysine, is a recently discovered post-translational modification. This naturally occurring reaction confers electrochemical properties onto proteins that potentially produce an electrical mimic to DNA and result in specificity toward DNA-binding molecules such as anti-DNA autoantibodies. The discovery of this unique covalent protein modification provides a rationale for establishing the molecular mechanism and broad functional significance of the formation and regulation of Nϵ-pyrrole-l-lysine-containing proteins. In this study, we used microbeads coupled to pyrrolated or nonpyrrolated protein to screen for binding activities of human serum-resident nonimmunoglobin proteins to the pyrrolated proteins. This screen identified apolipoprotein E (apoE) as a protein that innately binds the DNA-mimicking proteins in serum. Using an array of biochemical assays, we observed that the pyrrolated proteins bind to the N-terminal domain of apoE and that oligomeric apoE binds these proteins better than does monomeric apoE. Employing surface plasmon resonance and confocal microscopy, we further observed that apoE deficiency leads to significant accumulation of pyrrolated serum albumin and is associated with an enhanced immune response. These results, along with the observation that apoE facilitates the binding of pyrrolated proteins to cells, suggest that apoE may contribute to the clearance of pyrrolated serum proteins. Our findings uncover apoE as a binding target of pyrrolated proteins, providing a key link connecting covalent protein modification, lipoprotein metabolism, and innate immunity.

The KN-93 Molecule Inhibits Calcium/Calmodulin-Dependent Protein Kinase II (CaMKII) Activity by Binding to Ca2+/CaM

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional serine/threonine protein kinase that transmits calcium signals in various cellular processes. CaMKII is activated by calcium-bound calmodulin (Ca2+/CaM) through a direct binding mechanism involving a regulatory C-terminal α-helix in CaMKII. The Ca2+/CaM binding triggers transphosphorylation of critical threonine residues proximal to the CaM-binding site leading to the autoactivated state of CaMKII. The demonstration of its critical roles in pathophysiological processes has elevated CaMKII to a key target in the management of numerous diseases. The molecule KN-93 is the most widely used inhibitor for studying the cellular and in vivo functions of CaMKII. It is widely believed that KN-93 binds directly to CaMKII, thus preventing kinase activation by competing with Ca2+/CaM. Herein, we employed surface plasmon resonance, NMR, and isothermal titration calorimetry to characterize this presumed interaction. Our results revealed that KN-93 binds directly to Ca2+/CaM and not to CaMKII. This binding would disrupt the ability of Ca2+/CaM to interact with CaMKII, effectively inhibiting CaMKII activation. Our findings also indicated that KN-93 can specifically compete with a CaMKIIδ-derived peptide for binding to Ca2+/CaM. As indicated by the surface plasmon resonance and isothermal titration calorimetry data, apparently at least two KN-93 molecules can bind to Ca2+/CaM. Our findings provide new insight into how in vitro and in vivo data obtained with KN-93 should be interpreted. They further suggest that other Ca2+/CaM-dependent, non-CaMKII activities should be considered in KN-93–based mechanism-of-action studies and drug discovery efforts.

Au/NaGdF4: Yb3+, Er3+ hybrid fluorescent system for rapid detection of ethanol

Here, we demonstrate the well linear dependence of the fluorescent intensity of upconversion nanoparticles (UCNPs) on the concentration of ethanol at the concentration range of 25%–90% (typical ethanol concentration in Chinese liquors) by employing surface plasmon resonance (SPR) to stabilize the upconversion processes. UCNPs are successfully synthesized by a solvothermal method which emit green light centered at 540 nm under the excitation of 980 nm infrared light. We select Au nanorods with the absorption peaks centered at 525 nm and 928 nm (a wide band covered the wavelength region of 980 nm) for tuning the upconversion luminescence process of UCNPs so that the fluorescence intensity is linearly dependent on the content of ethanol from 25% to 90%. The Au/NaGdF4:Yb3+, Er3+ hybrid fluorescent system may be an ideal strategy for rapidly detecting the content of ethanol in liquor and discerning the adulterated liquor.