Independent Binding Sites Research Articles

Opposed Aromatic Surfaces Behave as Independent Binding Sites for Carbohydrate Stacking: Analysis of Sandwich-like CH/π/CH Complexes

Opposed Aromatic Surfaces Behave as Independent Binding Sites for Carbohydrate Stacking: Analysis of Sandwich-like CH/π/CH Complexes

Shedding Light on Viral Shedding: Novel Insights into Nuclear Assembly, Cytoplasmic Transformation and Extracellular Vesicle Release of the BK Virus.

Despite the high prevalence of BK polyomavirus (BKPyV) and the associated risk for BKPyV-associated nephropathy (BKPyVAN) in kidney transplant (KTX) recipients, many details on viral processes such as replication, maturation, assembly and virion release from host cells have not been fully elucidated. VP1 is a polyomavirus-specific protein that is expressed in the late phase of its replicative cycle with important functions in virion assembly and infectious particle release. This study investigated the localization and time-dependent changes in the distribution of VP1-positive viral particles and their association within the spectrum of differing cell morphologies that are observed in the urine of KTX patients upon active BKPyV infection. We found highly differing recognition patterns of two anti-VP1 antibodies with respect to intracellular and extracellular VP1 localization, pointing towards independent binding sites that were seemingly associated with differing stages of virion maturation. Cells originating from single clones were stably cultured out of the urine sediment of KTX recipients with suspected BKPyVAN. The cell morphology, polyploidy, virus replication and protein production were investigated by confocal microscopy using both a monoclonal (mAb 4942) and a polyclonal rabbit anti-VP1-specific antibody (RantiVP1 Ab). Immunoblotting was performed to investigate changes in the VP1 protein. Both antibodies visualized VP1 and the mAb 4942 recognized VP1 in cytoplasmic vesicles exhibiting idiomorphic sizes when released from the cells. In contrast, the polyclonal antibody detected VP1 within the nucleus and in cytoplasm in colocalization with the endoplasmic reticulum marker CNX. At the nuclear rim, VP1 was recognized by both antibodies. Immunoblotting revealed two smaller versions of VP1 in urinary decoy cell extracts, potentially from different translation start sites as evaluated by in silico analysis. Oxford Nanopore sequencing showed integration of BKPyV DNA in chromosomes 3, 4 and 7 in one of the five tested primary cell lines which produced high viral copies throughout four passages before transcending into senescence. The different staining with two VP1-specific antibodies emphasizes the modification of VP1 during the process of virus maturation and cellular exit. The integration of BKPyV into the human genome leads to high virus production; however, this alone does not transform the cell line into a permanently cycling and indefinitely replicating one.

Multitracer Approach to Understanding the Complexity of Reactive Astrogliosis in Alzheimer's Brains.

A monoamine oxidase B (MAO-B) selective positron emission tomography (PET) tracer [11C]-deuterium-l-deprenyl holds promise for imaging reactive astrogliosis in neurodegenerative diseases, such as Alzheimer's disease (AD). Two novel PET tracers ([11C]-BU99008 and [18F]-SMBT-1) have recently been developed to assess the complexity of reactive astrogliosis in the AD continuum. We have investigated the binding properties of SMBT-1, l-deprenyl, and BU99008 in AD and cognitively normal control (CN) brains. Competition binding assays with [3H]-l-deprenyl and [3H]-BU99008 versus unlabeled SMBT-1 in postmortem AD and CN temporal and frontal cortex brains demonstrated that SMBT-1 interacted with [3H]-deprenyl at a single binding site (nM range) and with [3H]-BU99008 at multiple binding sites (from nM to μM). Autoradiography studies on large frozen postmortem AD and CN hemisphere brain sections demonstrated that 1 μM SMBT-1 almost completely displaced the [3H]-l-deprenyl binding (>90%), while SMBT-1 only partly displaced the [3H]-BU99008 binding (50-60% displacement) in cortical regions. In conclusion, SMBT-1, l-deprenyl, and BU99008 interact at the same MAO-B binding site, while BU99008 shows an additional independent binding site in AD and CN brains. The high translational power of our studies in human AD and CN brains suggests that the multitracer approach with SMBT-1, l-deprenyl, and BU99008 could be useful for imaging reactive astrogliosis.

Critical overview and comparison between models for adsorption-desorption noise in bio-chemical sensors

We critically review existing models for the adsorption-desorption noise in bio-chemical sensors, in particular the model based on simplified forward Kolmogorov equation and the models based on Langevin sources. For the latter models, we propose a generalized version to handle cases beyond the branched surface reactions (a binding site that can be alternatively occupied by different ions/molecules) and the chained reaction (a binding site that sequentially binds with ions/molecules). The models are benchmarked against kinetic Monte Carlo (kMC) simulations considering relevant case studies such as pH-sensitive ions, selective molecules binding on a functionalized surface and multi-layer adsorption on bare surfaces. It is found that although the mathematical formulation of the modeling approaches appears different, when dealing with independent binding sites, they are fully equivalent and perfectly match the kMC results. The case of competitive binding considering the correlation between the occupation of the binding sites has also been analyzed.

The effect of denaturants on protein thermal stability analyzed through a theoretical model considering multiple binding sites

A novel mathematical development applied to protein ligand binding thermodynamics is proposed, which allows the simulation, and therefore the analysis of the effects of multiple and independent binding sites to the Native and/or Unfolded protein conformations, with different binding constant values. Protein stability is affected when it binds to a small number of high affinity ligands or to a high number of low affinity ligands. Differential scanning calorimetry (DSC) measures released or absorbed energy of thermally induced structural transitions of biomolecules. This paper presents the general theoretical development for the analysis of thermograms of proteins obtained for n-ligands bound to the native protein and m-ligands bound to their unfolded form. In particular, the effect of ligands with low affinity and with a high number of binding sites (n and/or m > 50) is analyzed. If the interaction with the native form of the protein is the one that predominates, they are considered stabilizers and if the binding with the unfolded species predominates, it is expected a destabilizing effect. The formalism presented here can be adapted to fitting routines in order to simultaneously obtain the unfolding energy and ligand binding energy of the protein. The effect of guanidinium chloride on bovine serum albumin thermal stability, was successfully analyzed with the model considering low number of middle affinity binding sites to the native state and a high number of weak binding sites to the unfolded state.

Blockade of TRPV channels by intracellular spermine.

The Vanilloid thermoTRP (TRPV1-4) subfamily of TRP channels are involved in thermoregulation, osmoregulation, itch and pain perception, (neuro)inflammation and immune response, and tight control of channel activity is required for perception of noxious stimuli and pain. Here we report voltage-dependent modulation of each of human TRPV1, 3, and 4 by the endogenous intracellular polyamine spermine. As in inward rectifier K channels, currents are blocked in a strongly voltage-dependent manner, but, as in cyclic nucleotide-gated channels, the blockade is substantially reduced at more positive voltages, with maximal blockade in the vicinity of zero voltage. A kinetic model of inhibition suggests two independent spermine binding sites with different affinities as well as different degrees of polyamine permeability in TRPV1, 3, and 4. Given that block and relief occur over the physiological voltage range of action potentials, voltage-dependent polyamine block may be a potent modulator of TRPV-dependent excitability in multiple cell types.

Advanced investigation of a putative adsorption process of nine non key food odorants (non-KFOs) on the broadly tuned human olfactory receptor OR2W1: Statistical physics modeling and molecular docking study

In the present paper, statistical physics formalism was used to understand the olfactory perception via the investigation of dose-olfactory response curves of a putative adsorption process of nine non key food odorants (non-KFOs) on the broadly tuned human olfactory receptor OR2W1, in order to quantitative characterize the interactions between the nine studied non-KFOs, i. e., furfuryl sulfide, furfuryl disulfide, benzyl methyl disulfide, furfuryl methyl disulfide, benzyl methyl sulfide, 1-phenylethanethiol, benzyl mercaptan, furfuryl methyl sulfide and 3-phenylpropanol molecules and OR2W1 binding sites at a molecular level. Two advanced adsorption models have been proposed: the advanced monolayer monoenergy model (monolayer model with identical and independent olfactory receptor binding sites) (Model 1) and the advanced monolayer model with two independent types of olfactory receptor binding sites (Model 2). It was concluded that the monolayer monoenergy model was selected as the most adequate model to fit the experimental dose-olfactory response curves tabulated in literature. Actually, the numerical values of the three fitted physico-chemical parameters (RM1, n and C1) were obtained by a non-linear regression. Indeed, modeling results suggested that the number of docked non-KFOs per OR2W1 binding site n values (1.24 < n < 1.94) was always superior to 1, which indicated the non-parallel orientation of the studied odorants on the olfactory receptor and the multi-molecular adsorption mechanism. The estimated molar adsorption energy ΔEa values (ranged from 6.07 to 12.16 kJ/mol) for the nine olfactory systems confirmed the physical the exothermic characters of the adsorption process since ΔEa values were lower than 40 kJ/mol and positive. Furthermore, these estimated parameters were applied to characterize stereographically and energetically the interaction between the nine non-KFOs and OR2W1 through the determination of the human receptor binding site size distributions (RSDs) and the adsorption energy distributions (AEDs), which were spread out from 0.25 to 6.50 nm and from 0 to 22.50 kJ/mol, respectively. The docking computation between these nine non-KFOs and OR2W1 proved that the estimated binding affinities were belonged to the adsorption energies spectrum in general and the specific adsorption energy band or the molecular vibration modes limited spectrum (between 2.50 kJ/mol and 17 kJ/mol) (approximate olfactory band).

Voltage-dependent inhibition of thermo-TRPV channels by intracellular spermine.

The Vanilloid TRP (TRPV1-4) subfamily of channels is involved in thermoregulation, osmoregulation, itch and pain perception, immune response and (neuro)inflammation, and tight control of channel activity is required for perception of pain and noxious stimuli. TRPV1, 2, 4 have been shown to be overexpressed in various cancer cell types and suggested to serve as a marker of cancer progression and, in some cases, as an anti-cancer drug target. The polyamines (PAs) putrescine, spermidine and spermine, are present in sub-millimolar concentrations in the cytoplasm of virtually all cells and in the extracellular fluid. Intracellular PA levels decrease with age, while pathological alterations of PA levels affect cognitive functions and have been linked to brain ageing and degeneration, as well as to cancer. We have discovered a previously unrecognized physiological modulation of human TRPV1, 3 and 4 by the endogenous intracellular polyamine spermine. As in inward rectifier K channels, currents are blocked in a strongly voltage-dependent manner, but, as in cyclic nucleotide-gated channels, the block is substantially relieved at more positive voltages. Consistent with block relief resulting from spermine permeation, there is much weaker relief of block by the analog 1-naphthyl-acetyl spermine, with a bulky aromatic group at one end of the molecule. A kinetic model of inhibition suggests two independent spermine binding sites with different affinities as well as significantly different degrees of PA permeability in TRPV1, 3, and 4. Since block, relief, and permeation occur over the physiological voltage range of action potentials and PA concentrations, voltage-dependent PA block may be a potent modulator of TRPV-dependent excitability in multiple cell types, and at least some TRPV channels may contribute to polyamine transport. Thermo-TRPV structural insights and available literature also hint at a common mechanism of PA block of different TRP channel sub-families.

Indirect characterizations of mOR-EG: Modeling analysis of five concentration-olfactory response curves via an advanced monolayer adsorption model

The investigation of the adsorption process putatively involved in the olfactory perception of apocynin, guaiacylacetone, homovanillyl alcohol, 4-ethylguaiacol and homoguaiacol molecules on the mouse eugenol olfactory receptor mOR-EG was a very useful tool for comprising olfaction process at a molecular level. Indeed, the experimental data were correlated by using an advanced monolayer adsorption model with identical and independent binding sites. Thanks to the grand canonical ensemble in statistical physics formalism, the physico-chemical interpretations of modeling results indicated that the five odorants were adsorbed via a multi-molecular mechanism. Hence, the calculation of adsorption energies, that described the interaction between the odorant molecules and the olfactory receptor binding cavities, indicated that weak bonds were made between apocynin, guaiacylacetone, homovanillyl alcohol, 4-ethylguaiacol and homoguaiacol molecules and mOR-EG binding pockets amino acid residues. In addition, theoretical stereographic and energetic characterizations of mOR-EG were made via the determination of the olfactory receptor site size distributions (RSDs) and the adsorption energy distributions (AEDs) relative to apocynin, guaiacylacetone, homovanillyl alcohol, 4-ethylguaiacol, homoguaiacol molecules. The RSD provided the size of different binding cavities of mOR-EG. Indeed, the five RSDs spectrums situated between 0.5 and 10 nm were spread out around an average size each one. The mean values obtained from the peaks of the distributions were 2.14 nm, 2.20 nm, 2 nm, 2.10 nm and 1.83 nm for apocynin, guaiacylacetone, homovanillyl alcohol, 4-ethylguaiacol and homoguaiacol molecules, respectively. The AED gave a whole spectrum of adsorption energies that was activated by the odorant molecule. Thus, the apocynin, guaiacylacetone, homovanillyl alcohol, 4-ethylguaiacol and homoguaiacol AEDs were spread out from 5 to 27.5 kJ/mol, from 5 to 30 kJ/mol, from 5 to 35 kJ/mol, from 0 to 22.5 kJ/mol, 5 to 25 kJ/mol, respectively. The thermodynamic study, via the establishment of the adsorption entropy, indicated that the peak of the disorder was obtained when half of the binding sites were occupied. In addition, the Gibbs free enthalpy and the internal energy were determined and their negative values indicated that the adsorption phenomenon involved in the olfactory perception was spontaneous and exothermic physisorption phenomenon.

Binding stoichiometry and structural model of the HIV-1 Rev/importin β complex.

HIV-1 Rev mediates the nuclear export of intron-containing viral RNA transcripts and is essential for viral replication. Rev is imported into the nucleus by the host protein importin β (Impβ), but how Rev associates with Impβ is poorly understood. Here, we report biochemical, mutational, and biophysical studies of the Impβ/Rev complex. We show that Impβ binds two Rev monomers through independent binding sites, in contrast to the 1:1 binding stoichiometry observed for most Impβ cargos. Peptide scanning data and charge-reversal mutations identify the N-terminal tip of Rev helix α2 within Rev's arginine-rich motif (ARM) as a primary Impβ-binding epitope. Cross-linking mass spectrometry and compensatory mutagenesis data combined with molecular docking simulations suggest a structural model in which one Rev monomer binds to the C-terminal half of Impβ with Rev helix α2 roughly parallel to the HEAT-repeat superhelical axis, whereas the other monomer binds to the N-terminal half. These findings shed light on the molecular basis of Rev recognition by Impβ and highlight an atypical binding behavior that distinguishes Rev from canonical cellular Impβ cargos.

Binding of dual‐flavour compounds by soy protein isolate in aqueous model systems

SummaryBy the technique of headspace solid‐phase microextraction/gas chromatography–mass spectrometry (SPME‐GC/MS) and static headspace/gas chromatography (HS/GC), the interaction of dual‐flavours and soy protein isolates (SPI) was determinated at low flavour concentration (0.04–0.16 mm) and high flavour concentration (0.4–1.6 mm). The results show that the mixture of ethyl caprylate and undecanal that has elongated structure possesses the same binding sites on SPI and shows competitive binding; the mixture of carvone that has compact structure, at low flavour concentration, possesses different and independent binding sites on SPI, and there maybe existed noncompetitive binding. If high concentration multi‐flavour binding with SPI induces the conformational change and increased more binding sites for another single flavour, there may be existed anticompetitive binding. ‘Salt out’ effects of interaction model were observed for terpene flavours.

The TetR Family Repressor HpaR Negatively Regulates the Catabolism of 5-Hydroxypicolinic Acid in Alcaligenes faecalis JQ135 by Binding to Two Unique DNA Sequences in the Promoter of Hpa Operon.

5-Hydroxypicolinic acid (5HPA), an important natural pyridine derivative, is microbially degraded in the environment. Previously, a gene cluster, hpa, responsible for 5HPA degradation, was identified in Alcaligenes faecalis JQ135. However, the transcription regulation mechanism of the hpa cluster is still unknown. In this study, the transcription start site and promoter of the hpa operon was identified. Quantitative reverse transcription-PCR and promoter activity analysis indicated that the transcription of the hpa operon was negatively regulated by a TetR family regulator, HpaR, whereas the transcription of hpaR itself was not regulated by HpaR. Electrophoretic mobility shift assay and DNase I footprinting revealed that HpaR bound to two DNA sequences, covering the -35 region and -10 region, respectively, in the promoter region of the hpa operon. Interestingly, the two binding sequences are partially palindromic, with 3 to 4 mismatches and are complementary to each other. 5HPA acted as a ligand of HpaR, preventing HpaR from binding to promoter region and derepressing the transcription of the hpa operon. The study revealed that HpaR binds to two unique complementary sequences of the promoter of the hpa operon to negatively regulate the catabolism of 5HPA. IMPORTANCE This study revealed that the transcription of the hpa operon was negatively regulated by a TetR family regulator, HpaR. The binding of HpaR to the promoter of the hpa operon has the following unique features: (i) HpaR has two independent binding sites in the promoter of the hpa operon, covering -35 region and -10 region, respectively; (ii) the palindrome sequences of the two binding sites are complementary to each other; and (iii) both of the binding sites include a 10-nucleotide partial palindrome sequence with 3 to 4 mismatches. This study provides new insights into the binding features of the TetR family regulator with DNA sequences.

Identification, functional characterization, assembly and structure of ToxIN type III toxin-antitoxin complex from E. coli.

Toxin–antitoxin (TA) systems are proposed to play crucial roles in bacterial growth under stress conditions such as phage infection. The type III TA systems consist of a protein toxin whose activity is inhibited by a noncoding RNA antitoxin. The toxin is an endoribonuclease, while the antitoxin consists of multiple repeats of RNA. The toxin assembles with the individual antitoxin repeats into a cyclic complex in which the antitoxin forms a pseudoknot structure. While structure and functions of some type III TA systems are characterized, the complex assembly process is not well understood. Using bioinformatics analysis, we have identified type III TA systems belonging to the ToxIN family across different Escherichia coli strains and found them to be clustered into at least five distinct clusters. Furthermore, we report a 2.097 Å resolution crystal structure of the first E. coli ToxIN complex that revealed the overall assembly of the protein-RNA complex. Isothermal titration calorimetry experiments showed that toxin forms a high-affinity complex with antitoxin RNA resulting from two independent (5′ and 3′ sides of RNA) RNA binding sites on the protein. These results further our understanding of the assembly of type III TA complexes in bacteria.

Physico-chemical investigations of human olfactory receptors OR10G4 and OR2B11 activated by vanillin, ethyl vanillin, coumarin and quinoline molecules using statistical physics method

This research work is a contribution to understand the olfaction mechanism at a molecular level of vanillin, ethyl vanillin, coumarin and quinoline molecules using a modeling of a putative adsorption process by analytical model established by statistical physics formalism.A statistical physics modeling using the monolayer model with identical and independent binding sites of the responses of the two human olfactory receptors OR10G4 and OR2B11 showed that vanillin and quinoline were adsorbed with a mixed non-parallel and parallel orientation on OR10G4 and on OR2B11, respectively. However, ethyl vanillin and coumarin were anchored with a total non-parallel orientation. The adsorption energy values collected from data analysis, which were ranged from 12.51 to 20.91 kJ/mol, confirmed that the adsorption of vanillin and ethyl vanillin on OR10G4 and the adsorption of coumarin and quinoline on OR2B11were exothermic and were based on physical interactions.Furthermore, the dose-olfactory response curves of vanillin, ethyl vanillin, coumarin and quinoline provided access to OR10G4 and OR2B11 steric characterization via the calculation of the studied olfactory receptors site size distributions (RSDs). Indeed, vanillin, ethyl vanillin, coumarin and quinoline RSDs are spread from 0.3 to 12 nm, from 0.5 to 12 nm, from 0.40 to 12 nm and from 0.14 to 12 nm, respectively, with a maximum at 1.55 nm, 2.11 nm, 2.50 and 1.13 nm, respectively.Lastly, the physico-chemical model parameters can be used for the energetic characterization to confirm the physical nature of the vanillin/ethyl vanillin-OR10G4 and the coumarin/quinoline-OR2B11 interactions and to determine an olfactory band of order of 12 kJ/mol [11–23 kJ/mol], 10 kJ/mol [14–24 kJ/mol], 7 kJ/mol [9–16 kJ/mol], 15 kJ/mol [13–28 kJ/mol] for vanillin, ethyl vanillin, coumarin and quinoline, respectively, through the determination of the adsorption energy distributions (AEDs).

Molecular insights into the interaction of 5-fluorouracil and Fe3O4 nanoparticles with beta-casein: An experimental and theoretical study

We investigated the potential carrier of milk beta-casein (β-CN) and its interactions with 5-fluorouracil (5-FU) and iron oxide nanoparticles (Fe3O4 NPs). We used different spectroscopic methods of fluorescence, UV–Visble, circular dichroism (CD), synchronous fluorescence, zeta potential assay, and computational studies to clarify the protein interaction with 5-FU and Fe3O4 NPs. The fluorescence data indicated both Fe3O4 NPs and 5-FU could quench the intrinsic fluorescence of β-CN. Fluorescence measurements showed that the single interaction of β-CN with 5-FU or Fe3O4 NPs was static, while reacted β-CN with both 5-FU and Fe3O4 NPs simultaneously showed a dynamic quenching. Synchronous fluorescence data in both tests revealed that the tryptophan (Trp) residue of β-CN had a dominant role in quenching and the polarity of its microenvironment more than tyrosine (Tyr) increased in interaction with 5-FU. All the binding sites and thermodynamic parameters were obtained at 25, 37, and 42 °C. The analysis of thermodynamic parameters and Job’s plot techniques pointed to that both of these complexes with the 1:1 M ratio were exothermic (ΔH°<0) driven with the van der Waals and H-bonding interactions (in agreement with the docking results). The CD spectra in the region of far-UV and thermal denaturation study indicated minor changes in the secondary structure of β-CN in the presence of various concentrations of Fe3O4 NPs and 5-FU. Also, from the molecular dynamics (MD) analysis, as a result, the protein structure was stable during 100 ns. The outcomes highlighted that β-CN protein could form a great bind with 5-FU and Fe3O4 NPs ligands (supporting the zeta potential assay results) by independent binding sites. These results would be helpful insight to construct a potential magnetic nanocarrier β-CN base for 5-FU drug delivery.

Light- and pH-regulated Water-soluble Pseudorotaxanes Comprising a Cucurbit[7]uril and a Flavylium-based Axle.

A linear double pyridinium-terminated thread comprising a central chalcone moiety is shown to provide two independent binding sites with similar affinity for cucurbit[7]uril (CB7) macrocycles in water as judged from NMR, UV-Visible and fluorescence spectroscopies. Association results in [2] and [3]pseudorotaxanes, which are both pH and photosensitive. Switching from the neutral chalcone to the cationic flavylium form upon irradiation at 365 nm under acidic conditions provided an enhanced CB7 association (K1:1 increases from 1.2×105 M-1 to 1.5×108 M-1 ), limiting spontaneous on-thread cucurbituril shuttling. This co-conformational change in the [2]pseudorotaxane is reversible in the dark with kobs =4.1×10-4 s-1 . Threading the flavylium moiety into CB7 leads to a dramatic increase in the fluorescence quantum yield, from 0.29 in the free axle to 0.97 in the [2]pseudorotaxane and 1.0 in the [3]pseudorotaxane.

Revisiting the CooJ family, a potential chaperone for nickel delivery to [NiFe]‑carbon monoxide dehydrogenase

Nickel insertion into nickel-dependent carbon monoxide dehydrogenase (CODH) represents a key step in the enzyme activation. This is the last step of the biosynthesis of the active site, which contains an atypical heteronuclear NiFe4S4 cluster known as the C-cluster. The enzyme maturation is performed by three accessory proteins, namely CooC, CooT and CooJ. Among them, CooJ from Rhodospirillum rubrum is a histidine-rich protein containing two distinct and spatially separated Ni(II)-binding sites: a N-terminal high affinity site (HAS) and a histidine tail at the C-terminus. In 46 CooJ homologues, the HAS motif was found to be strictly conserved with a H(W/F)XXHXXXH sequence. Here, a proteome database search identified at least 150 CooJ homologues and revealed distinct motifs for HAS, featuring 2, 3 or 4 histidines. The purification and biophysical characterization of three representative members of this protein family showed that they are all homodimers able to bind Ni(II) ions via one or two independent binding sites. Initially thought to be present only in R. rubrum, this study strongly suggests that CooJ could play a significant role in CODH maturation or in nickel homeostasis.

Continuous Injection Isothermal Titration Calorimetry for In Situ Evaluation of Thermodynamic Binding Properties of Ligand-Receptor Binding Models.

We utilize a continuous injection approach (CIA) rather than the traditional incremental injection approach (IIA) to deliver ligand (or receptor) to the calorimeter cell to evaluate thermodynamic binding parameters for three common ligand-receptor binding models-single independent, competitive, and two independent binding sites-using isothermal titration calorimetry (ITC). A general mathematical expression for the binding isotherm for any binding stoichiometry under continuous delivery of ligand (or receptor) resulting in an analytical solution for the thermodynamic binding parameters is presented. The advantages of CIA include reduction in experimental time, estimation of thermodynamic binding parameter values, and automation of the experiment since thermodynamic parameters are estimated in situ. We demonstrate the inherent advantages of CIA over IIA for the three binding models. For the single independent site model, we utilized the binding of Ba2+ ions to ethylenediaminetetraacetic acid (EDTA), while competitive binding was captured by titration of Ca2+ ions into a buffered solution of Ba2+ and EDTA. We experimentally simulated a two independent binding site system by injecting Ca2+ into a solution of EDTA and 1,3-diaminopropane-N,N,N',N'-tetraacetic acid (DPTA). The results demonstrate estimation of thermodynamic parameters with greater confidence and simultaneous reduction in the experimental time of 83% and titrating reagent of 50%, as compared to IIA.

Non-invasive immunoPET imaging of PD-L1 using anti-PD-L1-B11 in breast cancer and melanoma tumor model

IntroductionImmunotherapy targeting PD-1/PD-L1 immune checkpoint inhibition (ICI) is efficacious in various solid and hematologic malignancies. However, the response rate to PD-1/PD-L1 therapy is only 15–35%. To obtain optimal clinical response to ICI therapies, a reliable assessment of tumor PD-L1 expression is needed to select appropriate patients, and a non-invasive imaging-based assessment of PD-L1 expression is critically needed. Although radiolabeled PET probes based on PD-L1 targeted therapeutic antibodies (e.g. atezolizumab) have shown encouraging results, there is concern that residual therapeutic antibody may compete for binding with the radiotracer thereby compromising imaging studies that follow treatment. Methods and resultsIn this study, we used novel anti-PD-L1-B11 clone antibody known to bind to a different epitope of PD-L1 than the therapeutic antibodies to avoid potential saturation effects. The anti-PD-L1-B11 clone was radiolabeled with zirconium-89 and evaluated to detect PD-L1 expression in various in vitro and in vivo cancer model systems in comparison with [89Zr]Zr-DFO-NCS-atezolizumab. In vitro binding parameters of anti-PD-L1-B11 were like those of atezolizumab. [89Zr]Zr-DFO-NCS-anti-PD-L1-B11 clone showed favorable properties to [89Zr]Zr-DFO-NCS-atezolizumab in an in vivo breast cancer tumor model system with higher uptake in PD-L1 expressing tumors. ConclusionOur data demonstrates that [89Zr]Zr-DFO-NCS-anti-PD-L1-B11 exhibits excellent imaging properties for the assessment PD-L1 expression. The independent binding site of anti-PD-L1-B11 relative to therapeutic anti-PD-L1 antibodies may be advantageous for anti-PD-L1 therapy monitoring.

AGO HITS-CLIP reveals distinct miRNA regulation of white and brown adipose tissue identity.

MicroRNAs (miRNAs) are short, noncoding RNAs that associate with Argonaute (AGO) to influence mRNA stability and translation, thereby regulating cellular determination and phenotype. While several individual miRNAs have been shown to control adipocyte function, including energy storage in white fat and energy dissipation in brown fat, a comprehensive analysis of miRNA activity in these tissues has not been performed. We used high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP) to comprehensively characterize the network of high-confidence, in vivo mRNA:miRNA interactions across white and brown fat, revealing >20,000 unique AGO binding sites. When coupled with miRNA and mRNA sequencing, we found an inverse correlation between depot-enriched miRNAs and their targets. To illustrate the functionality of our HITS-CLIP data set in identifying specific miRNA:mRNA interactions, we show that miR-29 is a novel regulator of leptin, an adipocyte-derived hormone that coordinates food intake and energy homeostasis. Two independent miR-29 binding sites in the leptin 3' UTR were validated using luciferase assays, and miR-29 gain and loss of function modulated leptin mRNA and protein secretion in primary adipocytes. This work represents the only experimentally generated miRNA targetome in adipose tissue and identifies multiple regulatory pathways that may specify the unique identities of white and brown fat.