Carbohydrate–Lectin Interaction on Graphene-Coated Surface Plasmon Resonance (SPR) Interfaces

The paper describes the detection of carbohydrate–lectin interaction on graphene-on-metal surface plasmon resonance (SPR) interfaces. Graphene-coated gold-based SPR interfaces were formed through the transfer of large-area graphene grown by chemical vapor deposition (CVD) on polycrystalline Cu foils. The method allowed successful transfer of single- and double-layered graphene sheets onto the SPR interfaces in a reproducible manner. Functionalization of the graphene interface with mannose was achieved by simple immersion into a mannose aqueous solution (100 mM), resulting in noncovalent interactions between the aromatic ring structure of graphene and mannose. The utility of the carbohydrate-modified graphene-on-gold interface for the selective and sensitive detection of carbohydrate–lectin interactions was demonstrated using model lectins from Lens culinaris (LC) and Triticum vulgaris (TV). While LC lectin binds specifically to mannopyranoside units, TV lectin has an affinity for N-acetyl glucosamine and sialic acid residues.     

Deciphering the Stepwise Binding Mode of HRG1β to HER3 by Surface Plasmon Resonance and Interaction Map

For the development of efficient anti-cancer therapeutics against the HER receptor family it is indispensable to understand the mechanistic model of the HER receptor activation upon ligand binding. Due to its high complexity the binding mode of Heregulin 1 beta (HRG1β) with its receptor HER3 is so far not understood. Analysis of the interaction of HRG1β with surface immobilized HER3 extracellular domain by time-resolved Surface Plasmon Resonance (SPR) was so far not interpretable using any regular analysis method as the interaction was highly complex. Here, we show that Interaction Map (IM) made it possible to shed light on this interaction. IM allowed deciphering the rate limiting kinetic contributions from complex SPR sensorgrams and thereby enabling the extraction of discrete kinetic rate components from the apparently heterogeneous interactions. We could resolve details from the complex avidity-driven binding mode of HRG1β with HER3 by using a combination of SPR and IM data. Our findings contribute to the general understanding that a major conformational change of HER3 during its activation is induced by a complex sequential HRG1β docking mode.     

Spectroscopic Studies of GSK3β Phosphorylation of the Neuronal Tau Protein and Its Interaction with the N-terminal Domain of Apolipoprotein E

Alzheimer disease neurons are characterized by extraneuronal plaques formed by aggregated amyloid-β peptide and by intraneuronal tangles composed of fibrillar aggregates of the microtubule-associated Tau protein. Tau is mostly found in a hyperphosphorylated form in these tangles. Glycogen synthase kinase 3β (GSK3β) is a proline-directed kinase generally considered as one of the major players that (hyper)phosphorylates Tau. The kinase phosphorylates mainly (Ser/Thr)-Pro motifs and is believed to require a priming activity by another kinase. Here, we use an in vitro phosphorylation assay and NMR spectroscopy to characterize in a qualitative and quantitative manner the phosphorylation of Tau by GSK3β. We find that three residues can be phosphorylated (Ser-396, Ser-400, and Ser-404) by GSK3β alone, without priming. Ser-404 is essential in this process, as its mutation to Ala prevents all activity of GSK3β. However, priming enhances the catalytic efficacy of the kinase, as initial phosphorylation of Ser-214 by the cAMP-dependent protein kinase (PKA) leads to the rapid modification by GSK3β of four regularly spaced additional sites. Because the regular incorporation of negative charges by GSK3β leads to a potential parallel between phospho-Tau and heparin, we investigated its interaction with the heparin/low density lipoprotein receptor binding domain of human apolipoprotein E. We indeed observed an interaction between the GSK3β-promoted regular phospho-pattern on Tau and the apolipoprotein E fragment but none in the absence of phosphorylation or the presence of an irregular phosphorylation pattern by the prolonged activity of PKA. Apolipoprotein E is therefore able to discriminate and interact with specific phosphorylation patterns of Tau.     

Molecular Analysis of the Prostacyclin Receptor’s Interaction with the PDZ1 Domain of Its Adaptor Protein PDZK1

The prostanoid prostacyclin, or prostaglandin I₂, plays an essential role in many aspects of cardiovascular disease. The actions of prostacyclin are mainly mediated through its activation of the prostacyclin receptor or, in short, the IP. In recent studies, the cytoplasmic carboxy-terminal domain of the IP was shown to bind several PDZ domains of the multi-PDZ adaptor PDZK1. The interaction between the two proteins was found to enhance cell surface expression of the IP and to be functionally important in promoting prostacyclin-induced endothelial cell migration and angiogenesis. To investigate the interaction of the IP with the first PDZ domain (PDZ1) of PDZK1, we generated a nine residue peptide (KK⁴¹¹IAACSLC⁴¹⁷) containing the seven carboxy-terminal amino acids of the IP and measured its binding affinity to a recombinant protein corresponding to PDZ1 by isothermal titration calorimetry. We determined that the IP interacts with PDZ1 with a binding affinity of 8.2 µM. Using the same technique, we also determined that the farnesylated form of carboxy-terminus of the IP does not bind to PDZ1. To understand the molecular basis of these findings, we solved the high resolution crystal structure of PDZ1 bound to a 7-residue peptide derived from the carboxy-terminus of the non-farnesylated form of IP (⁴¹¹IAACSLC⁴¹⁷). Analysis of the structure demonstrates a critical role for the three carboxy-terminal amino acids in establishing a strong interaction with PDZ1 and explains the inability of the farnesylated form of IP to interact with the PDZ1 domain of PDZK1 at least in vitro.     

Use of Surface Plasmon Resonance and Biolayer Interferometry for the Study of Protein–Protein Interactions on the Example of an Enzyme of a Glycosyl Hydrolase Subtype (EC 3.2.1) and Specific Antibodies to It

Two previously obtained, full-size, fully human antibodies that reversibly bind the active form of an enzyme belonging to the subtype EC 3.2.1, which is used for substitutive enzymatic therapy in lysosomal storage diseases, have been characterized by surface plasmon resonance and biolayer interferometry methods. It was shown under normal physiological conditions that the designed antibodies specifically bound with the antigen (KD ~ 10^–8 M) and rapidly dissociated at neutral pH in 60% ethylene glycol while leaving the enzymatic activity unchanged. Dan ue to their properties, the developed antibodies can be used in industry as affinity ligand in the isolation of therapeutic substances of the enzyme.     

Noble Metal Ion Embedded Nanocomposite Glass Materials for Optical Functionality of UV–Visible Surface Plasmon Resonance (SPR) Surface-Enhanced Raman Scattering (SERS) X-ray and Electron Microscopic Studies: An Overview

Plasmonics - Raman spectroscopy (RS) is a modern scientific analytic fingerprint technique that detects, examines, and analyzes the constituent chemical composition of various substances...     

Atomic Resolution Structure of the Orotidine 5′-Monophosphate Decarboxylase Product Complex Combined with Surface Plasmon Resonance Analysis: IMPLICATIONS FOR THE CATALYTIC MECHANISM*

Orotidine 5′-monophosphate decarboxylase (ODCase) accelerates the decarboxylation of its substrate by 17 orders of magnitude. One argument brought forward against steric/electrostatic repulsion causing substrate distortion at the carboxylate substituent as part of the catalysis has been the weak binding affinity of the decarboxylated product (UMP). The crystal structure of the UMP complex of ODCase at atomic resolution (1.03 Å) shows steric competition between the product UMP and the side chain of a catalytic lysine residue. Surface plasmon resonance analysis indicates that UMP binds 5 orders of magnitude more tightly to a mutant in which the interfering side chain has been removed than to wild-type ODCase. These results explain the low affinity of UMP and counter a seemingly very strong argument against a contribution of substrate distortion to the catalytic reaction mechanism of ODCase.     

The Interaction of Per‐O‐Acetylated Acyclic 1‐(1‐Butylindol‐3‐yl)‐1‐deoxy‐ketoses with Silylated Uracil

The per‐O‐acetylated open chain derivatives of 1‐(1‐butylindol‐3‐yl)‐1‐deoxy‐1‐L‐sorbose and 1‐(1‐butylindol‐3‐yl)‐1‐deoxy‐L‐tagatose, which are readily available by alkaline degradation of 1‐butylascorbigen followed by acetylation, were used in a nucleoside‐type synthesis. The interaction of these ketoses derivatives with bis‐(trimethylsilyl)‐uracil yielded in each case a mixture of (E)‐2,4,5,6‐tetra‐O‐acetyl‐1‐(1‐butylindol‐3‐yl)‐1,3‐dideoxy‐3‐(uracil‐1‐yl)‐L‐xylo‐hexa‐1‐enitol and (E)‐2,4,5,6‐tetra‐O‐acetyl‐1‐(1‐butylindol‐3‐yl)‐1,3‐dideoxy‐3‐(uracil‐1‐yl)‐L‐lyxo‐hexa‐1‐enitol, which were separated by preparative HPLC. The deacetylation of each of these compounds by MeONa in MeOH produced a mixture of 1‐(1‐butylindol‐3‐yl)‐1,3‐dideoxy‐4‐O‐methyl‐3‐(uracil‐1‐yl)‐α‐L‐sorbopyranose and 1‐(1‐butylindol‐3‐yl)‐1,3‐dideoxy‐4‐O‐methyl‐3‐(uracil‐1‐yl)‐β‐D‐fructopyranose, which were also separated by HPLC, the structures were confirmed by NMR.     

Probing the Solution Structure of IκB Kinase (IKK) Subunit γ and Its Interaction with Kaposi Sarcoma-associated Herpes Virus Flice-interacting Protein and IKK Subunit β by EPR Spectroscopy

Viral flice-interacting protein (vFLIP), encoded by the oncogenic Kaposi sarcoma-associated herpes virus (KSHV), constitutively activates the canonical nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) pathway. This is achieved through subversion of the IκB kinase (IKK) complex (or signalosome), which involves a physical interaction between vFLIP and the modulatory subunit IKKγ. Although this interaction has been examined both in vivo and in vitro, the mechanism by which vFLIP activates the kinase remains to be determined. Because IKKγ functions as a scaffold, recruiting both vFLIP and the IKKα/β subunits, it has been proposed that binding of vFLIP could trigger a structural rearrangement in IKKγ conducive to activation. To investigate this hypothesis we engineered a series of mutants along the length of the IKKγ molecule that could be individually modified with nitroxide spin labels. Subsequent distance measurements using electron paramagnetic resonance spectroscopy combined with molecular modeling and molecular dynamics simulations revealed that IKKγ is a parallel coiled-coil whose response to binding of vFLIP or IKKβ is localized twisting/stiffening and not large-scale rearrangements. The coiled-coil comprises N- and C-terminal regions with distinct registers accommodated by a twist: this structural motif is exploited by vFLIP, allowing it to bind and subsequently activate the NF-κB pathway. In vivo assays confirm that NF-κB activation by vFLIP only requires the N-terminal region up to the transition between the registers, which is located directly C-terminal of the vFLIP binding site.     

Interaction of (1→4)- and (1→6)-linked disaccharides with the fenton reagent under physiological conditions

Reactions of (1→4)- and (1→6)-linked disaccharides, mainly of maltose and isomaltose, with the Fenton reagent under physiological conditions were studied. Chemical characterization of oxidation products was conducted by g.l.c. and g.l.c.-m.s. of their trimethylsilyl derivatives, and the results demonstrated that (1→6)-linked disaccharides are more reactive with the hydroxyl radical (·OH) generated by the Fenton reagent than (1→4)-linked disaccharides. About 35–40% of (1→6)-and 15–20% of (1→4)-linked disaccharides were oxidatively degraded to smaller molecules after incubation for 24 h. Of the four disaccharides examined, namely, maltose, isomaltose, cellobiose, and gentiobiose, the α-(1→6)-linked disaccharide isomaltose exhibited the highest reactivity, whereas the β-(1→4)-linked disaccharide cellobiose showed the lowest. These results suggest the existence of a relationship between the configuration of the glycosidic linkage and the reactivity with ·OH in aqueous solution.     

Synthesis of Oligoribonucleotides Containing 4‐Thiouridine Using the Convertible Nucleoside Approach and the 1‐(2‐Fluorophenyl)‐4‐Methoxypiperidin‐4‐yl Group

Oligoribonucleotides containing 4‐thiouridine were prepared using the Fpmp group for protection of the 2′‐OH. Two uridine derivatives with the 1,2,4‐triazolyl and the 2‐nitrophenyl groups at position 4 were used to obtain 4‐thiouridine by postsynthetic substitution with sodium hydrogen sulfide. Both uridine derivatives allow the preparation of the desired oligonucleotides in good yields.     

The C-terminal Domain of the Long Form of Cellular FLICE-inhibitory Protein (c-FLIPL) Inhibits the Interaction of the Caspase 8 Prodomain with the Receptor-interacting Protein 1 (RIP1) Death Domain and Regulates Caspase 8-dependent Nuclear Factor κB (NF-κB) Activation

Caspase 8 plays an essential role in the regulation of apoptotic and non-apoptotic signaling pathways. The long form of cellular FLICE-inhibitory protein (c-FLIP_L) has been shown previously to regulate caspase 8-dependent nuclear factor κB (NF-κB) activation by receptor-interacting protein 1 (RIP1) and TNF receptor-associated factor 2 (TRAF2). In this study, the molecular mechanism by which c-FLIP_L regulates caspase 8-dependent NF-κB activation was further explored in the human embryonic kidney cell line HEK 293 and variant cells barely expressing caspase 8. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone greatly diminished caspase 8-dependent NF-κB activation induced by Fas ligand (FasL) when c-FLIP_L, but not its N-terminal fragment c-FLIP(p43), was expressed. The prodomain of caspase 8 was found to interact with the RIP1 death domain and to be sufficient to mediate NF-κB activation induced by FasL or c-FLIP(p43). The interaction of the RIP1 death domain with caspase 8 was inhibited by c-FLIP_L but not c-FLIP(p43). Thus, these results reveal that the C-terminal domain of c-FLIP_L specifically inhibits the interaction of the caspase 8 prodomain with the RIP1 death domain and, thereby, regulates caspase 8-dependent NF-κB activation.     

Interaction of Platelet Endothelial Cell Adhesion Molecule (PECAM) with α2,6-Sialylated Glycan Regulates Its Cell Surface Residency and Anti-apoptotic Role

The luminal sides of vascular endothelial cells are heavily covered with a so-called glycocalyx, but the precise role of the endothelial glycocalyx remains unclear. Our previous study showed that N-glycan α2,6-sialylation regulates the cell surface residency of an anti-apoptotic molecule, platelet endothelial cell adhesion molecule (PECAM), as well as the sensitivity of endothelial cells toward apoptotic stimuli. As PECAM itself was shown to be modified with biantennary N-glycans having α2,6-sialic acid, we expected that PECAM would possess lectin-like activity toward α2,6-sialic acid to ensure its homophilic interaction. To verify this, a series of oligosaccharides were initially added to observe their inhibitory effects on the homophilic PECAM interaction in vitro. We found that a longer α2,6-sialylated oligosaccharide exhibited strong inhibitory activity. Furthermore, we found that a cluster-type α2,6-sialyl N-glycan probe specifically bound to PECAM-immobilized beads. Moreover, the addition of the α2,6-sialylated oligosaccharide to endothelial cells enhanced the internalization of PECAM as well as the sensitivity to apoptotic stimuli. Collectively, these findings suggest that PECAM is a sialic acid binding lectin and that this binding property supports endothelial cell survival. Notably, our findings that α2,6-sialylated glycans influenced the susceptibility to endothelial cell apoptosis shed light on the possibility of using a glycan-based method to modulate angiogenesis.     

Escherichia coli Virulence Protein NleH1 Interaction with the v-Crk Sarcoma Virus CT10 Oncogene-like Protein (CRKL) Governs NleH1 Inhibition of the Ribosomal Protein S3 (RPS3)/Nuclear Factor κB (NF-κB) Pathway

Enterohemorrhagic Escherichia coli and other attaching/effacing bacterial pathogens cause diarrhea in humans. These pathogens use a type III secretion system to inject virulence proteins (effectors) into host cells, some of which inhibit the innate immune system. The enterohemorrhagic E. coli NleH1 effector prevents the nuclear translocation of RPS3 (ribosomal protein S3) to inhibit its participation as a nuclear “specifier” of NF-κB binding to target gene promoters. NleH1 binds to RPS3 and inhibits its phosphorylation on Ser-209 by IκB kinase-β (IKKβ). However, the precise mechanism of this inhibition is unclear. NleH1 possesses a Ser/Thr protein kinase activity that is essential both for its ability to inhibit the RPS3/NF-κB pathway and for full virulence of the attaching/effacing mouse pathogen Citrobacter rodentium. However, neither RPS3 nor IKKβ is a substrate of NleH1 kinase activity. We therefore screened ∼9,000 human proteins to identify NleH1 kinase substrates and identified CRKL (v-Crk sarcoma virus CT10 oncogene-like protein), a substrate of the BCR/ABL kinase. Knockdown of CRKL abundance prevented NleH1 from inhibiting RPS3 nuclear translocation and NF-κB activity. CRKL residues Tyr-198 and Tyr-207 were required for interaction with NleH1. Lys-159, the kinase-active site of NleH1, was necessary for its interaction with CRKL. We also identified CRKL as an IKKβ interaction partner, mediated by CRKL Tyr-198. We propose that the CRKL interaction with IKKβ recruits NleH1 to the IKKβ complex, where NleH1 then inhibits the RPS3/NF-κB pathway.     

Determination of the Oligomer Size of Amyloidogenic Protein β-Amyloid(1-40) by Single-Molecule Spectroscopy

Amyloid diseases are traditionally characterized by the appearance of inter- and intracellular fibrillar protein deposits, termed amyloid. Historically, these deposits have been thought to be the etiology of the disease. However, recent evidence suggests that small oligomers of the amyloidogenic protein/peptide are the origin of neurotoxicity. Although the importance of identifying the toxic oligomeric species is widely recognized, such identification is challenging because these oligomers are metastable, occur at low concentration, and are characterized by a high degree of heterogeneity. In this work, a fluorescently labeled β-amyloid(1-40) is used as a model amyloidogenic peptide to test the effectiveness of what we believe is a novel approach based on single-molecule spectroscopy. We find that by directly counting the photobleaching steps in the fluorescence, we can determine the number of subunits in individual β-amyloid(1-40) oligomers, which allows us to easily distinguish among different species in the mixtures. The results are further analyzed by comparison with Monte Carlo simulations to show that the variability seen in the size of photobleaching steps can be explained by assuming random dipole orientations for the chromophores in a given oligomer. In addition, by accounting for bias in the oligomer size distribution due to the need to subtract background noise, we can make the results more quantitative. Although the oligomer size determined in this work is limited to only small species, our single-molecule results are in good quantitative agreement with high-performance liquid chromatography gel filtration data and demonstrate that single-molecule spectroscopy can provide useful insights into the issues of heterogeneity and ultimately cellular toxicity in the study of amyloid diseases.     

Identification of a novel and potent inhibitor of phospholipase A(2) in a medicinal plant: crystal structure at 1.93Å and Surface Plasmon Resonance analysis of phospholipase A(2) complexed with berberine

Crystal of Russell Viper venom phospholipase A(2) complexed with an isoquinoline alkaloid, berberine from a herbaceous plant Cardiospermum halicacabum, was prepared and its structure was solved by X-ray crystallography. The crystal diffracted up to 1.93? and the structure solution clearly located the position of berberine in the active site of the enzyme. Two hydrogen bonds, one direct and the other water mediated, were formed between berberine and the enzyme. Gly 30 and His 48 made these two hydrogen bonds. Additionally, the hydrophobic surface of berberine made a number of hydrophobic contacts with side chains of neighboring amino acids. Surface Plasmon Resonance studies revealed strong binding affinity between berberine and phospholipase A(2). Enzyme inhibition studies proved that berberine is a competitive inhibitor of phospholipase A(2). It was inferred that the isoquinoline alkaloid, berberine, is a potent natural inhibitor of phospholipaseA(2).