Homo- and Heteroassociations Drive Activation of ErbB3.

Dimerization or the formation of higher-order oligomers is required for the activation of ErbB receptor tyrosine kinases. The heregulin (HRG) receptor, ErbB3, must heterodimerize with other members of the family, preferentially ErbB2, to form a functional signal transducing complex. Here, we applied single molecule imaging capable of detecting long-lived and mobile associations to measure their stoichiometry and mobility and analyzed data from experiments globally, taking the different lateral mobility of monomeric and dimeric molecular species into account. Although ErbB3 was largely monomeric in the absence of stimulation and ErbB2 co-expression, a small fraction was present as constitutive homodimers exhibiting a ∼40% lower mobility than monomers. HRG stimulation increased the homodimeric fraction of ErbB3 significantly and reduced the mobility of homodimers fourfold compared to constitutive homodimers. Expression of ErbB2 elevated the homodimeric fraction of ErbB3 even in unstimulated cells and induced a ∼2-fold reduction in the lateral mobility of ErbB3 homodimers. The mobility of ErbB2 was significantly lower than that of ErbB3, and HRG induced a less pronounced decrease in the diffusion coefficient of all ErbB2 molecules and ErbB3/ErbB2 heterodimers than in the mobility of ErbB3. The slower diffusion of ErbB2 compared to ErbB3 was abolished by depolymerizing actin filaments, whereas ErbB2 expression induced a substantial rearrangement of microfilaments, implying a bidirectional interaction between ErbB2 and actin. HRG stimulation of cells co-expressing ErbB3 and ErbB2 led to the formation of ErbB3 homodimers and ErbB3/ErbB2 heterodimers in a competitive fashion. Although pertuzumab, an antibody binding to the dimerization arm of ErbB2, completely abolished the formation of constitutive and HRG-induced ErbB3/ErbB2 heterodimers, it only slightly blocked ErbB3 homodimerization. The results imply that a dynamic equilibrium exists between constitutive and ligand-induced homo- and heterodimers capable of shaping transmembrane signaling.

[The significance of ECG recorded at hospitalization in determining the prognosis of patients treated with myocardial infarction]. / A kórházi felvétel idején rögzített EKG jelentosége a szívinfarktus miatt kezelt betegek prognózisának meghatározásában.

INTRODUCTION AND AIM: By using the database of the National Registry of Myocardial Infarction, the authors examine the prognosis of patients treated with acute myocardial infarction, in case of whom there was new or presumably new left bundle branch block (nLBBB) on the ECG recorded at hospitalization. METHOD: We recorded the details of 18 091 patients treated with acute myocardial infarction (AMI) between 1 January 2014 and 30 June 2015 in the National Registry of Myocardial Infarction. In case of 8334 patients, the clinical diagnosis was ST-elevation myocardial infarction (STEMI), whereas in 9757 cases it was non-ST elevation myocardial infarction (NSTEMI). In the STEMI population we examined the clinical characteristics and prognosis of patients with ST-elevation (n = 7937) and nLBBB (n = 397). We used the proportional hazards regression model (Cox-regression) to examine mortality. RESULTS: In the AMI patient population, we found LBBB in 1274 cases (7%). In case of STEMI clinical diagnosis, the patients belonging to the nLBBB subpopulation (n = 397) were older, and the proportion of men and the occurrence of co-morbidities was higher than in case of those who had ST-elevation on their ECG recorded at hospitalization. The mortality rate of the LBBB population was higher than that of the ST-elevation patient population in both the 30-day (25.4% versus 12.4%) and the 1-year period (47.3 versus 19.9%). Percutaneous coronary intervention (PCI) had significantly lower mortality in both populations. In the course of a multifactorial analysis we verified the independent prognostic significance of LBBB: the hazard ratio compared to ST-elevation was 1.33 (95% confidence interval: 1.10-1.62), checked for gender, age, occurrence of PCI, systolic blood pressure, cardiac frequency, serum creatinine difference, and the details of five anamneses/co-morbidities. CONCLUSION: The admission ECG has prognostic significance. Patients with LBBB have poorer prognosis compared to patients with ST-elevation on admission ECG. Orv Hetil. 2018; 159(17): 677-681.

rFRET: A comprehensive, Matlab-based program for analyzing intensity-based ratiometric microscopic FRET experiments.

Fluorescence or Förster resonance energy transfer (FRET) remains one of the most widely used methods for assessing protein clustering and conformation. Although it is a method with solid physical foundations, many applications of FRET fall short of providing quantitative results due to inappropriate calibration and controls. This shortcoming is especially valid for microscopy where currently available tools have limited or no capability at all to display parameter distributions or to perform gating. Since users of multiparameter flow cytometry usually apply these tools, the absence of these features in applications developed for microscopic FRET analysis is a significant limitation. Therefore, we developed a graphical user interface-controlled Matlab application for the evaluation of ratiometric, intensity-based microscopic FRET measurements. The program can calculate all the necessary overspill and spectroscopic correction factors and the FRET efficiency and it displays the results on histograms and dot plots. Gating on plots and mask images can be used to limit the calculation to certain parts of the image. It is an important feature of the program that the calculated parameters can be determined by regression methods, maximum likelihood estimation (MLE) and from summed intensities in addition to pixel-by-pixel evaluation. The confidence interval of calculated parameters can be estimated using parameter simulations if the approximate average number of detected photons is known. The program is not only user-friendly, but it provides rich output, it gives the user freedom to choose from different calculation modes and it gives insight into the reliability and distribution of the calculated parameters. © 2016 International Society for Advancement of Cytometry.

Maximum likelihood estimation of FRET efficiency and its implications for distortions in pixelwise calculation of FRET in microscopy.

Ratiometric determination of the efficiency of fluorescence or Förster resonance energy transfer (FRET) is one of the most widespread methods for the characterization of protein clustering and conformation. Low photon numbers, often present in pixel-by-pixel determination of FRET efficiency in digital microscopy, result in large uncertainties in the derived FRET parameter. Here, we propose a method based on maximum likelihood estimation (MLE) of FRET efficiency using photon counting detectors to overcome this limitation. Intensities measured in the donor, FRET, and acceptor channels were all assumed to follow Poisson statistics as a result of detector shot noise. The joint probability of photon numbers detected in the donor, FRET, and acceptor channels was derived using an equation describing the relationship between the three measured intensities. The FRET efficiency generating the measured photon numbers with the largest likelihood was determined iteratively providing a single FRET value for all pixels in the calculation. Since as few as 100 pixels are sufficient to provide a maximum likelihood estimate for FRET, biological variability in FRET values can be revealed by performing the analysis for regions of interests in an image. Since the algorithm provides the probability of a combination of donor, FRET, and acceptor intensities observed in each individual pixel given a certain FRET efficiency, outlier pixels with low probabilities could be excluded from the analysis. Simulations carried out with low photon numbers in the presence and absence of outlier pixels revealed that the proposed approach can reliably and reproducibly estimate FRET efficiency. In addition, systematic evaluation of the simulation results showed that the distribution of pixel-by-pixel FRET efficiencies is skewed, and the mean of these FRET values is a biased and unreliable estimate of the FRET efficiency. In the absence of outlier pixels, FRET calculated from summed donor, FRET, and acceptor intensities proved to be as reliable as MLE. We conclude that MLE of FRET outperforms calculations using summed and pixel-by-pixel intensities in biologically relevant situations involving low photon numbers and outlier pixels. © 2014 International Society for Advancement of Cytometry.

Epigallocatechin 3-O-gallate induces 67 kDa laminin receptor-mediated cell death accompanied by downregulation of ErbB proteins and altered lipid raft clustering in mammary and epidermoid carcinoma cells.

Since the administration of synthetic medicines is associated with drug resistance and undesired side effects, utilization of natural compounds could be an alternative and complementary modality to inhibit or prevent the development of tumors. Epigallocatechin 3-O-gallate (EGCG, 1), the major flavan component of green tea, and genistein (2), a soy isoflavonoid, are known to have chemopreventive and chemotherapeutic effects against cancer. This study demonstrated that both flavonoids inhibit cell proliferation, an effect enhanced under serum-free conditions. Compound 1, but not 2, induced downregulation of ErbB1 and ErbB2 in mammary and epidermoid carcinoma cells, and its inhibitory effect on cell viability was mediated by the 67 kDa laminin receptor (67LR). While 1 was superior in inducing cell death, 2 was more efficient in arresting the tumor cells in the G2/M phase. Furthermore, number and brightness analysis revealed that 1 decreased the homoclustering of a lipid raft marker, glycosylphosphatidylinositol-anchored GFP, and it also reduced the co-localization between lipid rafts and 67LR. The main conclusion made is that the primary target of 1 may be the lipid raft component of the plasma membrane followed by secondary changes in the expression of ErbB proteins. Compound 2, on the other hand, must have other unidentified targets.

Hypoxia reduces the efficiency of elisidepsin by inhibiting hydroxylation and altering the structure of lipid rafts.

The mechanism of action of elisidepsin (PM02734, Irvalec®) is assumed to involve membrane permeabilization via attacking lipid rafts and hydroxylated lipids. Here we investigate the role of hypoxia in the mechanism of action of elisidepsin. Culturing under hypoxic conditions increased the half-maximal inhibitory concentration and decreased the drug's binding to almost all cell lines which was reversed by incubation of cells with 2-hydroxy palmitic acid. The expression of fatty acid 2-hydroxylase was strongly correlated with the efficiency of the drug and inversely correlated with the effect of hypoxia. Number and brightness analysis and fluorescence anisotropy experiments showed that hypoxia decreased the clustering of lipid rafts and altered the structure of the plasma membrane. Although the binding of elisidepsin to the membrane is non-cooperative, its membrane permeabilizing effect is characterized by a Hill coefficient of ~3.3. The latter finding is in agreement with elisidepsin-induced clusters of lipid raft-anchored GFP visualized by confocal microscopy. We propose that the concentration of elisidepsin needs to reach a critical level in the membrane above which elisidepsin induces the disruption of the cell membrane. Testing for tumor hypoxia or the density of hydroxylated lipids could be an interesting strategy to increase the efficiency of elisidepsin.

Critical analysis of in vitro stability testing of spinal implants and proposal for standardization.

BACKGROUND: The American Society for Testing and Materials (ASTM), considered the gold standard worldwide, requires only testing in physiological saline solution to simulate in vivo conditions in standard testing of spinal implants. RESEARCH DESIGN AND METHODS: We conducted an in vitro study to identify an industrial lubricant with characteristics that are most similar to those of biologically lubricating fat, blood, and tissue fluids. The use of such a material could standardize the results of in vitro mechanical tests for better clinical applications. RESULTS: Our study has shown that the lubricity of physiological saline was well below that of human soft tissues and tissue fluids, and among the motor oils, Castrol GTX3 provided a testing environment similar to that of a living organism. CONCLUSIONS: With the intention of standardizing and preventing a biological hazard, we have developed a reproducible mechanical testing proposal based on our experiments, which, in addition, would allow us to avoid many misunderstandings and contingencies.

Comparative analysis of fluorescence resonance energy transfer (FRET) and proximity ligation assay (PLA).

Both fluorescence resonance energy transfer (FRET) and proximity ligation assay (PLA) are techniques used in the investigation of protein interactions but the latter has not been evaluated in a systematic way, prompting us to compare their performance quantitatively. Proteins were labeled with oligonucleotide- or fluorophore-conjugated antibodies and their proximity was analyzed by flow cytometry in order to obtain statistically robust data. Both intermolecular and intramolecular PLA signals reached saturation at high expression levels. At the same time, the FRET efficiency was independent of, while the FRET signal exhibited a strict linear correlation with the expression levels of proteins. When the density of oligonucleotide- and fluorophore-conjugated antibodies was systematically changed by competition with unlabeled antibodies the FRET signal was linearly proportional to the amount of bound fluorophore-tagged antibodies, whereas the PLA signal was again saturated. The saturation phenomenon in PLA could not be eliminated by decreasing the duration of the rolling circle amplification reaction. Our data imply that PLA is a semiquantitative measure of protein colocalizations due to non-linear effects in the reaction and that caution should be exercised when interpreting PLA data in a quantitative way.

Comprehensive Model for Epidermal Growth Factor Receptor Ligand Binding Involving Conformational States of the Extracellular and the Kinase Domains.

The epidermal growth factor (EGF) receptor (EGFR) undergoes ligand-dependent dimerization to initiate transmembrane signaling. Although crystallographic structures of the extracellular and kinase domains are available, ligand binding has not been quantitatively analyzed taking the influence of both domains into account. Here, we developed a model explicitly accounting for conformational changes of the kinase and extracellular domains, their dimerizations and ligand binding to monomeric and dimeric receptor species. The model was fitted to ligand binding data of suspended cells expressing receptors with active or inactive kinase conformations. Receptor dimers with inactive, symmetric configuration of the kinase domains exhibit positive cooperativity and very weak binding affinity for the first ligand, whereas dimers with active, asymmetric kinase dimers are characterized by negative cooperativity and subnanomolar binding affinity for the first ligand. The homodimerization propensity of EGFR monomers with active kinase domains is ∼100-times higher than that of dimers with inactive kinase domains. Despite this fact, constitutive, ligand-independent dimers are mainly generated from monomers with inactive kinase domains due to the excess of such monomers in the membrane. The experimental finding of increased positive cooperativity at high expression levels of EGFR was recapitulated by the model. Quantitative prediction of ligand binding to different receptor species revealed that EGF binds to receptor monomers and dimers in an expression-level dependent manner without significant recruitment of monomers to dimers upon EGF stimulation below the phase transition temperature of the membrane. Results of the fitting offer unique insight into the workings of the EGFR.

The Dipole Potential Modifies the Clustering and Ligand Binding Affinity of ErbB Proteins and Their Signaling Efficiency.

Although activation of the ErbB family of receptor tyrosine kinases (ErbB1-4) is driven by oligomerization mediated by intermolecular interactions between the extracellular, the kinase and the transmembrane domains, the transmembrane domain has been largely neglected in this regard. The largest contributor to the intramembrane electric field, the dipole potential, alters the conformation of transmembrane peptides, but its effect on ErbB proteins is unknown. Here, we show by Förster resonance energy transfer (FRET) and number and brightness (N&B) experiments that the epidermal growth factor (EGF)-induced increase in the homoassociation of ErbB1 and ErbB2 and their heteroassociation are augmented by increasing the dipole potential. These effects were even more pronounced for ErbB2 harboring an activating Val → Glu mutation in the transmembrane domain (NeuT). The signaling capacity of ErbB1 and ErbB2 was also correlated with the dipole potential. Since the dipole potential decreased the affinity of EGF to ErbB1, the augmented growth factor-induced effects at an elevated dipole potential were actually induced at lower receptor occupancy. We conclude that the dipole potential plays a permissive role in the clustering of ErbB receptors and that the effects of lipid rafts on ligand binding and receptor signaling can be partially attributed to the dipole potential.

Binding of trastuzumab to ErbB2 is inhibited by a high pericellular density of hyaluronan.

Although trastuzumab is an efficient drug, primary and acquired resistance is a challenging problem. The authors have previously shown in mouse xenograft experiments that masking ErbB2 by hyaluronan leads to diminished binding of the antibody and consequent resistance. In the current work, they correlated trastuzumab binding with the pericellular density of hyaluronan in ErbB2-overexpressing human breast cancer samples. A method for quantifying the relative binding of trastuzumab was developed involving constant and low-frequency background subtraction, segmenting the image to membrane and background pixels followed by evaluation of trastuzumab fluorescence, normalized with the expression level of ErbB2, only in the membrane. The normalized binding of trastuzumab showed a negative correlation with the pericellular density of hyaluronan (r = -0.52) with the effect being the most pronounced in the extreme cases (i.e., low and high hyaluronan densities predicted strong and weak binding of trastuzumab, respectively). Removal of hyaluronan by hyaluronidase digestion unmasked the trastuzumab binding epitope of ErbB2 demonstrated by a significantly increased normalized binding of the antibody. The results show that the accumulation of pericellular hyaluronan plays a crucial role in masking ErbB2.

ErbB protein modifications are secondary to severe cell membrane alterations induced by elisidepsin treatment.

Elisidepsin is a marine-derived anti-tumor agent with unique mechanism of action. It has been suggested to induce necrosis associated with severe membrane damage. Since indirect evidence points to the involvement of ErbB receptor tyrosine kinases and lipid rafts in the mechanism of action of elisidepsin, we investigated the effect of the drug on the distribution of ErbB proteins and systematically compared the elisidepsin sensitivity of cell lines overexpressing ErbB receptors. Stable expression of a single member of the ErbB family (ErbB1-3) or co-transfection of ErbB2 and ErbB3 did not modify the elisidepsin sensitivity of CHO and A431 cells. However, elisidepsin induced the redistribution of ErbB3 and two GPI-anchored proteins (transfected GPI-anchored eGFP and placental alkaline phosphatase) from the plasma membrane to intracellular vesicles without comparable effects on ErbB1 and ErbB2. Elisidepsin increased the binding of a conformational sensitive anti-ErbB3 antibody without modifying the binding of other ErbB2 or ErbB3 antibodies, and it decreased the homoassociation of both ErbB2 and ErbB3. We also found that elisidepsin decreased the fluorescence anisotropy of a membrane specific fluorescent probe and induced a blue shift in the emission spectrum of Laurdan pointing to significant changes in the order of the plasma membrane possibly associated with the formation of liquid ordered domains. Although the distribution of ErbB proteins is preferentially altered by elisidepsin, our data question their role in determining sensitivity to the drug. We assume that induction of liquid ordered domains is the primary action of elisidepsin leading to all the other observed changes.