The Kelch-like ECH associated protein 1 (Keap1) is a component of a Cullin3-based Cullin-RING E3 ubiquitin ligase (CRL) multisubunit protein complex. Within the CRL, homodimeric Keap1 functions as the Cullin3 adaptor, and importantly, it is also the critical component of the E3 ligase that performs the substrate recognition. The best-characterized substrate of Keap1 is transcription factor NF-E2 p45-related factor 2 (Nrf2), which orchestrates an elaborate transcriptional program in response to environmental challenges caused by oxidants, electrophiles and pro-inflammatory agents, allowing adaptation and survival under stress conditions. Keap1 is equipped with reactive cysteine residues that act as sensors for endogenously produced and exogenously encountered small molecules (termed inducers), which have a characteristic chemical signature, reactivity with sulfhydryl groups. Inducers modify the cysteine sensors of Keap1 and impair its ability to target Nrf2 for ubiquitination and degradation. Consequently, Nrf2 accumulates, enters the nucleus and drives the transcription of its target genes, which encode a large network of cytoprotective proteins. Here we summarize the early studies leading to the prediction of the existence of Keap1, followed by the discovery of Keap1 as the main negative regulator of Nrf2. We then describe the available structural information on Keap1, its assembly with Cullin3, and its interaction with Nrf2. We also discuss the multiple cysteine sensors of Keap1 that allow for detection of a wide range of endogenous and environmental inducers, and provide fine-tuning and tight control of the Keap1/Nrf2 stress-sensing response. Deficiencies in these protective mechanisms are associated with increased disease risk and accelerated disease progression. These systems can be upregulated by various small molecules, termed inducers. Many of them are synthesized in plants, including edible plants, and are thus present in the human diet. Indeed, the majority of the health-promoting properties of diets rich in fruits and vegetables have been attributed to specific phytochemicals, which are able to induce defence systems in mammalian cells. Induction of these natural defences is protective against damage and allows adaptation and survival under conditions of stress.NFE2L2) One such defence system comprises a network of cytoprotective genes, the expression of which is regulated by transcription factor NF-E2 p45-related factor 2 , a commonly added preservative in processed food In the early 1960s, Charles Huggins found that pretreatment with small doses of 7,12-dimethylbenz[R-(methylsulfinyl)butane] H:quinone acceptor oxidoreductase 1 (NQO1) as a prototypic marker enzyme of the phase 2 response )butane]  as the pthe capacity of an extraordinary variety of seemingly unrelated anticarcinogens to induce protective enzymes can be attributed to the presence, or acquisition by metabolism, of a simple and hitherto unrecognized chemical property: that of a Michael reaction acceptor … ’, and further predicted the existence in the cell of ‘ … a protein endowed with highly reactive cysteine residue(s) that serves as the sensor for small-molecule inducers of the phase 2 response’. These were courageous statements, which were not readily embraced by the scientific community; this was the era of ‘ligand: receptor’ interactions, and chemical reactivity was yet to be recognized as a mechanism for sensing environmental signals.Initial screens using the Prochaska cell culture bioassay as well as rodent models revealed a bewildering array of structurally diverse compounds able to induce NQO1 Given these early insights, when Masayuki Yamamoto and his colleagues discovered Keap1 as the main negative regulator of Nrf2 and the expression of its downstream target genes 1.2Drosophila Kelch protein The Keap1 protein was first identified as a binding partner of Nrf2 and named Keap1 based on structural similarities with the KEAP1, but is also known as KLHL19 to conform to the naming convention of the protein family. BTB-Kelch proteins are divided into KLHL proteins and KBTB proteins. KLHL proteins typically consist of an N-terminal Broad complex, Tramtrack, and Bric à brac (BTB) domain, a BACK domain, and a C-terminal Kelch domain made up of 5–6 Kelch motif repeats. KBTB proteins consist of an N-terminal BTB domain and C-terminal Kelch domain, made up of 2–4 Kelch repeats. They occasionally also have a BACK domain.Keap1 is a member of the BTB-Kelch family of proteins. It has retained the gene name As a member of the KLHL family, Keap1 is composed of an N-terminal region (residues 1–49), a BTB domain (residues 50–179) and a C-terminal Kelch domain (residues 327–611) with an intervening BACK domain that is most commonly referred to as the intervening region  A. There Drosophila proteins Broad complex, Tramtrack, and Bric à Brac in vitroThe BTB domain is named after the The BACK domain was originally defined as a conserved region occurring in proteins with BTB domains and C-terminal Kelch repeats At the N-terminal end of the IVR is a bihelical motif known as the ‘3-box’ The Kelch domain is located at the C-terminus of Keap1. Numerous crystal structures of the Keap1 Kelch domain have now been solved, driven in part by an interest in targeting the domain with small molecule inhibitors Keap1 homodimerizes via the BTB domain, employing an interface very similar to that of the PLZF BTB domain, despite limited sequence identity between the two domains 1.3in vitro assay conducted using Keap1:Cullin3 and SPOP:Cullin3 complexes suggested that both complexes assemble with a 2:2 stoichiometry Keap1 associates with the N-terminal domain of Cullin3 via the BTB domain and 3-box. Crystal structures of BTB proteins in complex with Cullin3 have shown the interface in detail 1.4The dimeric architecture of the complex is central to its function. Two molecules of Keap1 engage a single molecule of the substrate, Nrf2. Each of the Kelch domains binds to one of two motifs in the Nrf2 protein, known as the ETGE and DLG motifs de novo synthesized Nrf2 to accumulate, translocate to the nucleus, and initiate transcription of Nrf2-dependent cytoprotective genes  or 1-biotinamido-4-butane (BMCC), led to cysteine modifications of Keap1, but in each case, the spectrum of the modified cysteines was different, as analyzed by liquid chromatography-tandem mass spectrometry. Thus, Eggler et al. A mass-spectrometry approach to detect covalent adducts in Keap1 required incubation with an inducer that binds to cysteines irreversibly, and the steroid dexamethasone 21-mesylate (Dex-mes) was initially used. This led to identification of four cysteine residues  within the IVR domain, and a fifth one, C613 in the C-terminal region of murine Keap1, all of which were modified when purified recombinant murine Keap1 was incubated with Dex-mes Unlike the stable alkylation adducts formed by Dex-mes, IAB and BMCC, binding of the dietary isothiocyanate sulforaphane to Keap1 leads to the formation of unstable thionoacyl adducts, which are labile to hydrolysis and transacylation reactions. To allow for detection of these adducts, a liquid chromatography-tandem mass spectrometry method was developed and further optimised to minimize adduct decomposition Together, the experiments with purified recombinant Keap1 established that cysteine residues in Keap1 serve as sensors for a variety of inducers. However, depending on the reaction conditions and the type of the inducer, multiple cysteine residues could be modified. It was next important to determine which of these cysteine residues function as inducer sensors in the context of the cellular environment. This question was addressed by employing both genetic and pharmacologic means. Due to its low abundance, the next set of experiments required ectopic expression of Keap1 in cells.1.6in vivo by generating transgenic mice expressing either C273A or C288A Keap1 mutants The use of ectopically-expressed Keap1 or of Keap1 carrying mutations in some of the identified inducer-modified cysteine residues revealed that substitution of C273 or C288 with either serine or alanine  rendered Keap1 unable to repress Nrf2 activity under homeostatic conditions in cells tert-butyl hydroquinone (tBHQ), but not for sensing other inducers such as the environmental toxin arsenite 2, (15-dPGJ2), which is dependent on C273 In stark contrast with the loss-of-function phenotype of the C237A/S and C288A/S mutants of Keap1, mutation of C151 to serine rendered Keap1 a constitutive repressor of Nrf2 at both homeostatic and induced states in cells 2 and prostaglandin A2 (PGA2), the electrophilic nitro-oleic acid (OA-NO2) 2+, Cd2+, Se4+, As3+2S 2+ is consistent with a previous report, which found that recombinant Keap1 binds Zn2+ stoichiometrically, with an association constant of 1011 M−12S was found to be facilitated by the formation of an intramolecular disulfide bridge between C226 and C613 It is now clear that C151 is required for sensing of nitric oxide, sulforaphane, tBHQ, diethylmaleate, dimethylfumarate, and CDDO-Im a of C151, enabling it to exist as the thiolate anion at physiological pH , the levels of Keap1 are increased. Nutrient starvation in human hepatoma (HepG2) cells caused a decrease in the levels of Keap1. Together, these results suggest that Keap1 is degraded through autophagy in a p62-dependent manner. Exposure to electrophiles, such as tBHQ, shortens the half-life of Keap1. The authors proposed that autophagy-mediated accelerated turnover of Keap1 contributes to the recovery of the Keap1 activity after modification of its sensor cysteine(s) by electrophiles.To address the fate of Keap1 after modification of its sensor cysteine(s), Hong et al. de novo synthesis, reversible modification(s) of Keap1 allow for regeneration of the inducer sensor.Some of the most potent electrophilic inducers, such as the isothiocyanates and the cyanoenones induce cytoprotective responses in the order of their electron affinity 2a protein endowed with highly reactive cysteine residue(s) that serves as the sensor for small-molecule inducers of the phase 2 response’. Several decades of research by multiple independent groups have convincingly demonstrated that this protein is Keap1. Furthermore, the extraordinary ability of Keap1 to accommodate inducers of many different shapes and sizes, and with varying degrees of reactivity, has given rise to a ‘cysteine code’, which ensures fine tuning and tight control of the Keap1/Nrf2 stress-sensing response. This response is the intended target for small molecule activators of several different chemical classes, some of which are currently in clinical trials. Further research is essential for detailed understanding of the precise consequences of targeting Keap1 for disease prevention and treatment. Acknowledgments We are extremely grateful to Cancer Research UK (C20953/A18644) and the BBSRC (BB/J007498/1) for financial support.There is no longer any scepticism regarding the existence in the cell of ‘ …


By understanding the conditions during extrusion, desirable nutritional characteristics can be incorporated while maximizing expansion to make a product with low bulk density, a fine bubble structure and acceptable organoleptic properties.Pumpkin products confer natural sweetness, desirable flavours and β-carotene, a vitamin A precursor when added as ingredients to extruded snacks. Therefore, a potential use for dried pumpkin flour is as an ingredient in ready-to-eat (RTE) snack foods. Growth in this market has driven food manufacturers to produce a variety of new high value snack foods incorporating diverse ingredients to enhance the appearance and nutritional properties of these foods. Ready-to-eat snacks were made by extruding corn grits with 5%, 10%, 15% and 20% of pumpkin flour. Snacks made from 100% corn grits were used as control products for this work. The effect of formulation and screw speeds of 250 rpm and 350 rpm on torque and specific mechanical energy , physical characteristics  and the microstructure of the snacks were studied. Increasing the screw speed resulted in a decrease of torque for all formulations. When pumpkin flour was added the specific mechanical energy (SME) decreased by approximately 45%. Increasing the percentage of pumpkin flour at the higher screw speed resulted in a harder texture for the extruded products. X-ray tomography of pumpkin flour-corn grit snacks showed that increased levels of pumpkin flour decreased both the bubble area and bubble size. However, no significant differences ( Consumer interest in ready-to-eat (RTE) snack foods is growing due to their convenience, value, attractive appearance, taste and texture . Cereal-Most snack foods contain a high proportion of corn, wheat and rice, with potato and oat and other grain products commonly added in smaller quantities. Vegetable powders are occasionally added but are not common in RTE snacks. Pumpkin is a good source of carotenes, which are responsible for the yellow colour of the flesh. It also contains appreciable levels of sugar and pectin. Sugar sweetens products that include pumpkin, while pectin is a soluble fibre that delivers many good health benefits. However, the utilization of pumpkin has been limited to the consumption of fresh products as a vegetable, or thickeners in soups and purees  and the Pumpkin flour  and Specification 220 corn grits from Corson Grain Ltd., Gisborne, New Zealand were used. The Proximate composition of corn grits and pumpkin flour, according to manufacturer data are presented in Expanded snack products were made by extruding finely milled corn endosperm (Specification 220 corn grits) as a control (100%) and corn grits mixed with pumpkin flour added at 5%, 10%, 15% and 20% on a dry weight basis of total mixture 3 kg. These ingredients were added into the extruder at a constant mass flow rate of 12.5 kg/h. Processing was carried out using a twin-screw extruder . The barrel length was 700 mm and the screw diameter was 24.7 mm. The screw configuration  was a foDuring this work the processing parameters for the extruder were recorded; these included torque, electrical energy consumption (kW) and from the energy consumption and the total mass through-put, the specific mechanical energy (SME) was calculated as kWh/kg.In this paper, the effect of adding different proportions of pumpkin flour and operating the extruder at different screw speeds on the associated torque and specific mechanical energy (SME) and the expansion ratio, bulk density, true density, hardness and bubble size distribution of extruded product is reported. The torque and specific mechanical energy (SME) response to addition of different proportions of pumpkin flour in the formulation and different screw speeds are presented in Increasing the proportion of pumpkin flour from 0% to 10% for the 350 rpm treatment decreased the expansion ratio of the extruded pellets by about 15%, but no further change occurred as the proportion of pumpkin flour was increased to 15% and 20%. At a screw speed of 250 rpm the expansion ratio did not change significantly with the proportion of pumpkin flour . As expeBulk density (BD) and true density (TD) of the expanded pellets are shown in Due to cost restraints, micro computerised axial tomography (mico-CAT) was only carried out on the extruded product made using the 250 rpm screw speed. The proportion of the cross-sectional area of the extruded pellets that was represented by void area (bubbles) for pellets containing different proportions of pumpkin flour is shown in As the proportion of pumpkin flour increased, torque and SME decreased showing that the viscosity of the melt formed within the extruder decreased during processing. This was probably due the high proportion of pectin (14%–22%) and sugar 37%) in the pumpkin flour that reduced the viscosity melt compared with a melt formed from corn grits only. This effect has been previously shown for ingredient mixes that contained high levels of sugars . Melts c% in the During this work it was possible to keep the SME near constant for all treatments except the 100% corn grits for which torque and SME were about 64% greater than for the treatments including pumpkin flour. The lubricating effect of the sugar and pectin components in the pumpkin flour were clear although it is surprising to observe the stabilizing effect with addition of 5% of pumpkin flour. This effect is evident throughout this work and the expansion ratio, hardness, densities and probably the void area of extruded product made with 10%, 15% or 20% of pumpkin flour were not significantly different. This may be because the sugar and pectin in the pumpkin flour preferentially coats the working surfaces in the extruder possibly by the formation of a depletion layer. As the proportion of pumpkin flour increased from 0% to about 10%, the extruded pellets approximately doubled in hardness but their true density and void areas did not differ significantly. This suggests that the bubble walls in the foamed pellets were stronger and less prone to fracture. Another explanation is that the bubbles increased in size while the walls became thicker; however this is not evident in the micro-CT scan data for the centre region of the pellets.This work illustrates that snack foods can be produced from combinations of corn grits and pumpkin products. Although, the texture of this product was about 40% harder than for typical corn based products the bulk density was similar up to the addition of 15% of pumpkin flour. At all levels of added pumpkin powder below 20%, the hardness of the pellets seemed to be determined by the strength of the bubble walls and not by the number and distribution of bubbles measured as the void volume in the extruded pellets or as bulk density.


Dairy desserts are complex mixtures and matrices including main components such as milk, sugar, starch, hydrocolloids, colorants and flavors, with a proteinaceous structure; they are widely consumed and present a semisolid consistency. In this work, the physicochemical and rheological properties of a dairy dessert with the addition of chickpea flour  were studied to determine the effect of the flour. The results indicated that luminosity (L*: 62.75–83.29), pH (6.35–7.11) and acidity (1.56–3.56) changed with the type of flour. The flow properties of the custards exhibited a non-Newtonian behavior that was well fitted by three flow models. The studied custard systems were stored for twelve days at 4 °C. The physicochemical and flow properties of the custards changed notably as a function of flour addition and storage time. From all samples, only four were analyzed with oscillatory tests, showing their mechanical spectra with elastic behavior. The dessert texture was also measured, founding that those formulated with Blanco Noroeste chickpea flour exhibited the highest values of hardness (0.356–0.391 N) through the twelve days. It can be concluded that those custard systems with the highest content of flour presented a very good response as a potential new dairy product. Food product development in which dairy products lead the consumer preference on one side and nutritional improvement needs on the other are important research aspects of Food Science. Dairy desserts are products highly requested and consumed in America, Europe and other countries; they are formulated with a diversity of components, such as milk, carbohydrates, colorants and flavors ,2,3,4. TSeveral studies have been focused on analyzing mixtures of polysaccharides and dairy components to improve the viscosity and consistency of semi solid dairy desserts . Since 2w/v) in yogurt, in order to evaluate its effect and determined both physical and rheological properties during 28 days of storage. They found that a higher concentration of flour caused an increase in syneresis and also the storage modulus was higher for systems with 3% of flour. Cereal-based desserts, like rice and wheat are popular in Asia, occupying an important place not only due to their taste, but also due to their nutritional quality. Jha et al. [The nutritional trend for foods enrichment has been and is very important. However, when new components are incorporated into an existing dairy formulation, the effect of such modification on food properties should be researched. Several related works have been carried out in this decade. Zare et al.  incorpora et al.  developea et al.  carried a et al.  researchThus, based on the aforementioned studies related to the incorporation of new components in foods in which there are few studies reporting incorporation of legumes in dairy products, the aim of this study was to develop a dairy dessert incorporating chickpea flour of two types, as a non-conventional flour, to improve the functional and nutritional characteristics of custards, determining their physicochemical and rheological characteristics as well.κ-carrageenan and 4.5 g of starch. For the elaboration of our custard systems, this formulation was considered and the starch was replaced by chickpea flour , at different concentrations using two types of flour from two varieties. Raw and cooked flours from Blanco Noroeste (BN) and Costa 2004 (C4) chickpea varieties, and semi-skimmed milk  were used. The base formulation (COST) for the custard consists of 88.98 mL of whole milk (3% fat), 6.5 g of sucrose, 0.02 g of κ-carrageenan) were weighted and dispersed slowly in the correspondent milk volume, for 4 min under constant stirring. Subsequently, the mixture was heated up to 90 °C on a heating plate  at level 5. When each formulation reached this temperature, it was kept for 5 min and then was allowed at cool down until room temperature. After that, the custard systems were transferred into 100 mL flasks, for refrigeration storage at 4 ± 1 °C for further analysis.The method described by Seuvré et al.  was follSixteen custard systems were prepared, following a factorial design: 2 × 2 × 4; with 2 varieties of chickpea (BN and C4), 2 types of flour (raw: R and cooked: C) and 4 concentration levels of flour . These concentrations for chickpea flour were computed to replace the starch of the original formulation (4.5 g) by considering only a 50% of starch from the chickpea, and assuming that the other part (50%) of the starch, is inter-acting with the other flour components. These substitutions, in the correspondent formulations, covered a range of 4.15 to 6.65 g of starch from the flour. Y = 86.6, x = 0.3168, y = 0.3242) placing approximately 10 g of sample into the sample plate. The reflectance mode was used for determinations of color parameters. Three replicates were completed for each sample and the experiments were carried out at 0, 4, 8 and 12 days of storage.Soluble solids were determined at room temperature with a digital refractometer , the results were given as degrees Brix at 20 °C. The pH values were determined by direct immersion, using a pH meter  at room temperature. The acidity was measured by the method 947.05 of Association of Official Analytical Chemists (AOAC) , based oγ = shear rate (1/s); ω = spindle angular velocity (rad/s); cR = container radius (m); bR = spindle radius (m); N = spindle speed ; τ = shear stress (Pa); L = spindle height (m); M = torque for this viscometer = 6.73 × 10−5 N·m. All flow determinations were performed with a digital Brookfield viscometer . The viscometer was adjusted to zero and a spindle LV (a type of viscometer with a specific torque) was set at the instrument. The measuring parameters were determined with the next relationships (1)–(3) from the manufacturer : (1)γ= 2τ = shear stress (Pa); K = consistency coefficient (Pa·sn); n = flow behavior index (dimensionless); τ0 = yield stress (Pa); and ɳp = plastic viscosity (Pa·s). The experimental flow responses or rheograms were fitted to three mathematical relationships, Power Law (PL), Herschel–Bulkley (HB) and Bingham plastic (BP) models (Equations (4)–(6), respectively):expτ = experimental shear stress (Pa); predτ = predicted shear stress ; and en = the number of experimental data .Two tests of goodness were applied to fit the obtained experimental data, the percentage of the mean error ) and the square root of the mean error ), to verify the fit of each model to the rheological behavior of each dairy dessert: Due to instrumental limitations, only a partial viscoelastic response from the studied systems was carried out, selecting four formulations of the dairy dessert as representative of all. Thus, those systems RBN4, RC44, CBN4 and CC44, including the highest starch content, were characterized by the dynamic performance. Rheological evaluations were completed using a stress-controlled rheometer  on its strain mode, with a plate-plate geometry  at constant temperature (20 ± 1 °C). In all of the experiments, 25 mL of the sample were used for the test and the samples were covered with paraffin oil to prevent water loss. The frequency sweep was conducted, with oscillation frequencies ranging from 0.1 to 100 rad/s, in which the storage modulus (G’) and loss modulus (G’’) were measured as a function of frequency. Three replicates were completed for each sample and the experiments were carried out at 1, 5 and 9 days of storage.Texture of the dairy desserts was evaluated by a texture profile analysis (TPA), performed in a Shimadzu texture meter  using a cylindrical probe of 50 mm diameter, with 10 mm of compression, 5 s of waiting time and 2 mm/s of crosshead speed. The experiments were carried out at 0, 4, 8 and 12 days after preparation.Resistant starch (RS) was measured by the method proposed by Goñi et al. . The sam® .With exception of the RS test that was completed by duplicate, the rest of the determinations were done in triplicate. All data were subjected to analysis of variance and Tukey test to determine significant differences between the dairy dessert systems, with a confidence level of 95%, using the Minitab software v.16All prepared custard systems were analyzed at days 0, 4, 8 and 12, measuring their physicochemical and rheological properties, and days 1, 5 and 9 days for dynamic response. From the obtained results for physicochemical and flow characterization, it was observed that most of the data did not exhibit important or significant changes at intermediate days; therefore, the results presented in this section correspond to 0 (fresh samples) and 12 days of storage, while, for viscoelastic and textural response, all data were included.The results of the physicochemical characterization of the studied systems are presented in Regarding the pH, systems with raw flour RC4 showed values of 6.86–7.11, higher than samples containing RBN flour (6.68–6.80) at day 0. Higher pH values that does not follow a general trend are related to the increase in the concentration and type of flour. An interaction between the flour components and milk system could be one of the possible reasons, as well as the solubilization of basic amino acids during cooking to which chickpeas was subjected . These rp < 0.05) due to the formulation, compared to the acidity decrease (1.44–1.80) showed by those systems with raw flour. On the other hand, acidity as an important determination for dairy products is related to the presence of organic acids, the acidity values are inversely related to the observed variations in pH values. Those systems with cooked flour had a small increase in acid content (2.12–2.82) and showed a significant difference . In this property, the particular response involves lower number of sites available for protein-water bind due to denaturation of proteins by the heat treatment , contribp > 0.05) on soluble solids, pH, acidity and syneresis between systems; in contrast, the type of flour generated significant differences (p < 0.05) on these parameters.Statistical analysis reveals that concentration of flour had not significant effects (p < 0.05) for custard formulations prepared with raw flour compared to day 0. The corresponding results for the same physicochemical parameters for day 12 are also included in the same The pH showed a different response, although the formulations with cooked flours showed a general decreasing trend; on the contrary, those samples with raw flour exhibited an increase in six of the eight custards.p < 0.05) with storage; for samples with raw flour five of them exhibited an augment; in contrast, seven of the systems with cooked flour decreased their water loss or syneresis. Although it is known that the presence of κ-carrageenan favors the formation of gels, it was not enough to prevent syneresis of the samples with raw flour; this phenomenon depends on additional factors affecting interactions, such as polymer–polymer, water–polymer, degree of heat treatment, type and solids concentration, pH and some salts [With regard to acidity, the studied systems showed the inverse response. Custards containing raw showed a decrease in acidity while the ones with cooked flour showed an increase from day 0 to day 12 (with only one exception). The syneresis changed significantly  with respect to the type of flour, and the concentration particularly on the b* parameter. As expected, some color changes are observed with storage time. At day 12, an important decrease was observed in the parameter L* for fourteen of the sixteen systems, with significant effect of the flour type, similarly for the a* parameter. Analysis of variance reveals that all systems showed significant effect (p < 0.05). The color changes are due to the oxidation and darkening reactions by the presence of oxygen. Clearly, a tendency of increase in values ∆E is observed with respect to flour concentration, with an exception made for CBN4.The storage time did not significantly affect the a* and b* parameters of the studied systems. Finally, the net change of color as a global color parameter showed a notable range of magnitudes in the formulations (0.63 to 10.95) during the storage period, associated with the type of flour  compared with those from raw flour systems (5–105 Pa), and a decreasing trend at day 12 in the measured shear stresses for most of the systems. The structural changes in cooked chickpea are combined with the presence of sugar that weaken the initial structure that has been seen in other dispersion systems , recordiτ0), flow behavior index (n) and consistency coefficient (K) obtained from the best fittings for the three applied models, Power Law (LP), Herschel and Bulkley (HB) and Bingham plastic (PB), based on the criterion of the root mean square error (RMSE) for the all systems, are included in The rheological parameters, such as yield stress  had a better fit to PL, four of them being cooked and the other with the lowest level of raw flour. Seven systems  showed a better fitting to HB, being four dispersions with raw and three with cooked flour, while the rest of the systems  were best fitted by BP. Therefore, most of the systems exhibited a yield stress (eleven of sixteen desserts). The systems added with cooked flour showed lower yield stress values than those systems added with raw flour. The differences are due to the type of flour and solid concentration affecting the flow response of the complex mixture of components of this particular dairy product.K), from HB and LP models in freshly prepared systems (day 0) ranged from 0.33 to 11.76 Pa sn, and a decrease at day 12 is clearly observed in most of these dessert systems (0.09–2.91 Pa sn). On the other side, those values obtained in this study for flow index (n) are in agreement with the range 0.35 to 0.60 (PL model) found by Tárrega and Costell [n-values  for desserts made with different types of inulin and milk, lower n-values indicating “a more” non-Newtonian nature.The consistency coefficient  on this hardness parameter. In contrast, storage time had no significant effect (p > 0.05) on hardness. The other TPA parameters did not show important differences between samples.Texture profile analysis (TPA) of the same four selected systems, showed clear differences among dessert hardness  as the mThe hardness values obtained in this study are similar to those reported by Szwajgier and Gustaw  for dairResistant starch is a natural component present in many foods. Some studies suggest that resistant starch have positive implications for human health, its fractions pass into the colon, which are fermented by the microorganisms producing mainly short chain fatty acids. Additionally, RS has a physiological effect similar to the dietary fiber, with functional properties and it has been observed that certain types of processing, increases the level of resistant starch ,34. w/w, dry matter; w/w, dry matter) reported by Brumovsky et al. [The amount of RS determined in the same selected four dairy desserts (added with the highest content of flour) presented a range of 0.75–1.84% (y et al.  for cassy et al.  for foury et al.  for two y et al.  for leguThe presence of resistant starch has been detected in various foods such as bread, breakfast cereals, biscuits, corn mashed, potatoes and legumes. Pereira and Leonel  reportedA dairy dessert added with raw and cooked chickpea flour was formulated, prepared and characterized. Our results demonstrated that it is possible to produce and have a good alternative for generating dairy products with good properties and potential higher nutritional value. The incorporation of chickpea flour was successful, in which the custard properties were affected by the flour chickpea variety type, the level of concentration and the storage time in a different degree. From a physical-chemical point of view, all studied formulations showed good characteristics. In the rheological characterization, it was observed that both types of flour increased the viscosity of the products, which, in turn, contributed to a viscoelastic behavior. The determination of resistant starch in this type of products indicates an added value for the application of chickpea flour into other food products, particularly in the dairy dessert of this study.


Aggregation of luciferase-containing substrates upon heat shock had a similar effect and could also expose cryptic PY elements in the substrates. We inferred that ligase activation critically requires a substantial array of clustered PY motifs and that the formation of such arrays on membranes or in polymeric aggregates may be an essential step in this mode of ligase regulation. We conclude that recruitment of α arrestins to membrane receptors and aggregation of unstable proteins after heat shock may be physiologically relevant mechanisms for triggering ubiquitination by Nedd4 family HECT domain–containing E3 ligases.The Nedd4 family of HECT domain–containing E3 ligases ubiquitinate many transcription factors and signaling proteins, and their activity is tightly regulated. Normally, intramolecular interactions curb the catalytic activity of the HECT domain, but these can be broken by the binding of PY motifs, found on substrate molecules and adaptors, to the WW domains characteristic of this E3 ligase family. This raises the prospect of substrates automatically activating the ligases, frustrating the purpose of ligase regulation. Here we show that soluble protein substrates and adaptors such as α arrestins, even with multiple PY elements, cannot activate ligase activity efficiently. However, we found that polymerization or membrane tethering of these substrates dramatically increases the ligase activity both  XY), and thus proteins bearing one or more of these motifs are candidate substrates . This shows that membrane tethering is indeed an alternative to polymerization in providing PY-dependent ligase activation by Dvl2.We co-expressed Dvl2 and WWP2 with His-UbA). They are membrane proteins with a cytoplasmic N-terminal sequence containing three PY motifs (B), which are required for activation. Fusion of this cytoplasmic domain to soluble GFP did not result in any detectable activation of co-expressed Nedd4 or any ubiquitination of the GFP fusion . Addition of the Dvl2 DIX domain to the construct did result in some activation, shown by ubiquitination of both the ligase and the GFP-Ndfip construct. This was abolished by the M4 mutation, which, like the M2 mutation, prevents DIX polymerization . Although the DIX domain is capable of self-association, it alone polymerizes inefficiently; efficient formation of large aggregates requires an additional dimerization domain, a function that can be provided by the TPR (translocated promoter region) domain from TPR-Met . Expression of an active site C/G mutant of Nedd4 did not result in ubiquitination, indicating that, under these conditions, modification was largely due to the expressed Nedd4 . Thus, polymerization can restore the ligase activation function of Ndfip2, which is otherwise dependent on membrane anchoring.If polymerization and membrane anchoring are equivalent, then a prediction is that PY motifs from a membrane protein activator would not function in soluble form but would do so when polymerized. We and others have shown previously that Ndfip1 and Ndfip2 are efficient natural activators of Nedd4 ligases , 36 FigA. They aY motifs B, which D). These are evidently stable structures rather than loose aggregates, as photobleaching of a small region was not followed by rapid recovery of fluorescence . Mutation of the Ndfip PY motifs did not affect puncta formation but abolished recruitment of Nedd4, showing that they are indeed the binding sites.Fluorescence microscopy of cells expressing these constructs clearly showed the formation of punctate cytoplasmic structures to which Nedd4 was recruited and on which ubiquitin was detected D. These Previously, we and others showed that multiple PYs were required for Ndfips to function effectively , 36. Thein vitro (E and F). In such assays, high levels of the cytoplasmic domain of Ndfip2 are sufficient to activate the ligase Itch . When fused to the DIX domain, it remained a poor substrate (as judged by its own ubiquitination) and inefficient activator (as judged by autoubiquitination of Smurf2). Only when the TPR dimerization domain was added did activation become efficient , just as for the in vivo experiments with Nedd4.To test more directly whether activation is solely due to direct interactions between the ligase and the PY-bearing construct, we performed experiments with purified proteins in vitro , E and Fase Itch . HoweverA). Like the Ndfips, they act as HECT ligase adaptors, but they are not themselves integral membrane proteins. From the results above, a prediction would be that their ability to activate Nedd4 ligases is specifically triggered by their association with membranes, thus sparing them from constitutive degradation, and that this reflects clustering of the PY motifs.The α arrestins are a group of soluble proteins typically containing a pair of PY motifs that associate with and facilitate the ubiquitination of G protein–coupled receptors  Fig. 3AAFig. 3A.B). There was also a marked reduction in the level of the membrane-targeted constructs when WWP2 was co-expressed, consistent with their ubiquitination and degradation. In this experiment, we used a version of WWP2 that lacked its potential membrane-binding C2 domain and thus should not be preferentially associated with membranes in the absence of binding sites; removal of this domain does not in itself lead to activation of WWP2 , suggesting interaction with endogenous Nedd4 family ligases. The PY mutations tested in Fig. 3D (PPXG) change the Tyr residue, which forms part of a putative YXXΦ endocytic signal. However, the effects cannot be ascribed to changes to this signal, as alternative mutations (AAXY) that do not affect it gave similar results .More generally, Fig. 3C) and activated the ligase, but only when they were polymerization-competent and had intact PY motifs (E). We conclude that the α arrestins do not activate Nedd4 ligases when they are soluble but can do so when they are membrane-associated and that this is likely due to clustering of PY motifs because polymerization has a similar effect.Finally, we fused these same C-terminal arrestin sequences to the DIX-TPR construct and tested them for activation of co-expressed Nedd4. These constructs formed punctate aggregates C and actY motifs E. We conMild heat shock results in increased ubiquitination of proteins, which is generally considered a natural response that promotes degradation of thermally denatured proteins. There is evidence that, at least in yeast, some of this is mediated by a Nedd4 family ligase . A possiA). Fluorescence microscopy revealed that the protein concentrated into visible dots following heat shock (B), which represent stable aggregates, as they did not recover after photobleaching . Furthermore, when Nedd4 was co-expressed, it was recruited to these dots (B).To test this hypothesis, we examined the fate of constructs based on firefly luciferase, a protein that is easily unfolded by heat. We first appended the Ndfip2 cytoplasmic tail to GFP-tagged luciferase and found that we could detect PY motif–dependent ubiquitination of this construct following heat shock A. Fluoreat shock B, which ese dots B.A is from endogenous ubiquitin ligases, presumably of the Nedd4 family. As further evidence for this, we co-expressed a dominant negative form of WWP2 with the active-site cysteine mutated. DFig. S1 shows that this reduced the endogenous ubiquitination of the luciferase construct relative to a control in which the WW domains were also mutated to prevent substrate interaction. This is a further indication that the heat-induced modification of these constructs is mediated by Nedd4 ligases through WW–PY interactions rather than by other types of ubiquitin ligases.The ubiquitination in Fig. 4A and B). It seems that ubiquitination promotes aggregate formation. Surprisingly, the aggregates that did form still recruited Nedd4 (B), and, indeed, ubiquitination was not completely abolished. There are several possible explanations for this. The luciferase could form co-aggregates with other proteins containing PY motifs, perhaps including chaperones, as reported in yeast . We conclude that the PY-tagged luciferase can activate Nedd4 ligases and that these can then act on other nearby substrates; in this case, co-aggregated luciferase.To see whether ubiquitination mediated by co-aggregated activators could be detected, we expressed HA-tagged luciferase together with a smaller amount of FLAG-tagged luciferase bearing the Ndfip2 cytoplasmic domain. Following heat shock, ubiquitination of the HA-luciferase could be detected, and this was noticeably enhanced when the PY-containing construct was co-expressed, relative to a PY-mutated control C. We conA significant number of proteins contain PY-like sequences, many of which are presumably within folded structures and inaccessible under normal conditions. However, a combination of unfolding and aggregation could allow them to recruit ligases following heat shock.A). Thus, weak non-canonical PY motifs may contribute to ligase activation.Luciferase itself contains the sequence APFY, potentially a weak PY motif, and so we tested the effects of mutating this to AAFY. This did both reduce and delay ubiquitination following heat shock but did not eliminate it completely A. Thus, XY motif in the HECT domain, which cannot be bound by a WW domain unless the protein is heated (XY motif prevented this (B and C). This ubiquitination was also blocked by heclin, a compound that is a general inhibitor of HECT ligases (B and C). Ubiquitination was not observed when the inactive Smurf2 HECT domain was fused to GFP , suggesting that it is not particularly aggregation-prone but must be associated with a protein that is. This example demonstrates that unmasking of cryptic PY elements, coupled with aggregation, is a plausible mechanism to account for at least some of the increased ubiquitination observed after heat shock.One well-characterized example of a cryptic PY motif is the conserved inaccessible LPs heated , 38. To ted this , B and C ligases   was monitored during a heat shock. At early times (10 min), increased ubiquitination could already be detected, and it was not inhibited by heclin; indeed, it was slightly more prominent when heclin was present. This is presumably mediated by other types of ubiquitin ligases, such as RING domain ones. At later times, however, there was a substantial further increase in ubiquitination that was abolished by heclin. Heclin inhibits essentially all HECT domain ligases, not just the Nedd4 family, so we can only draw the broad conclusion that HECT domains are involved Phaperone . Togethee.g.Fig. 4B). It is also consistent with the delayed appearance of heclin-sensitive (and thus HECT-mediated) protein ubiquitination during heat shock.An unexpected finding is that aggregation and ubiquitination appear to be mutually interdependent in that visible aggregation of luciferase-based constructs is more rapid and efficient when ubiquitination is enhanced by the provision of PY motifs. This could be due to the physical properties of long ubiquitin chains, such as a tendency to associate, but it could also be facilitated by ubiquitin-binding proteins. The predicted consequence is that the process of aggregation and ubiquitination should be autocatalytic; clustering promotes ligase activation, which promotes ubiquitination, which promotes further aggregation. Ligases such as Nedd4 contain ubiquitin binding sites on their HECT domains , 41, andOf course, there are many ubiquitin ligases in the cell, and we have shown that a substantial portion of the ubiquitination that initially follows heat shock is resistant to heclin and, thus, is presumably carried out by non-HECT ligases. How these various ubiquitination events combine to protect the cell remains to be elucidated.XG and AAXY. Plasma membrane-targeting constructs had either the N-terminal myristylation sequence MGCGCSSHPEDDGGSGGSGGS or contained residues 1–206 of human CD8A. All heat shock–sensitive constructs contained firefly luciferase with various combinations of the following: a triple HA or single FLAG tag, WT or PY motif mutant (3xPPAG) NDFIP2 (aa 1–117), an inactive C/G mutant Smurf2 HECT domain (aa 371–748) with an intact or mutated PY motif (IPPG), and/or a C-terminal EGFP. For bacterial expression, full-length Smurf2 was cloned into pGEX6P-2 . All polymerization constructs were cloned into a modified pET28a vector (Novagen) with a C-terminal triple HA tag, a PreScission cleavage site, glutathione S-transferase (GST), and a His tag. Truncations and point mutations were generated by standard procedures and verified by sequencing.The following plasmids have been described before: FLAG-Dvl2, FLAG-M2-Dvl2, FLAG-M4-Dvl2, FLAG-Dvl2-Pym, HA-tagged NEDD4 family ligases , His-ubiquitin, HA-ubiquitin, and NDFIP2 , 39, 42.6 cells were harvested in 0.8 ml of 8 m urea, 100 mm Tris (pH 7.4), 2 mmN-ethylmaleimide, and 10 mm iodoacetamide, sonicated, and incubated with 20 μl of His-Tag Dynabeads (Life Technologies) at room temperature for several hours. The beads were washed three times in 8 m urea and eluted in sample buffer. Immunoblotting was done using standard procedures, and proteins were detected using antibodies from Sigma-Aldrich , Roche (GFP), or Abcam (Smurf2 and ab53316). Heat shock experiments were carried out in a 42 °C water bath, and cells were harvested by adding 0.8 ml of 8 m urea, 100 mm Tris (pH 7.4), 2 mmN-ethylmaleimide, and 10 mm iodoacetamide. For live-cell imaging, HeLa cells were seeded into chambered coverglass (Lab-Tek) and transfected with either polyethyleneimine  or FuGENE HD transfection reagent (Promega). Confocal images were captured on a Zeiss 510 LSM using standard filter sets.HEK293T cells (acquired from the ATCC) were cultured and transfected essentially as described previously , 34, 39.Escherichia coli (Stratagene), and expression was induced with 0.5 mm isopropyl 1-thio-β-d-galactopyranoside at 18 °C overnight. Cell pellets were resuspended in PBS containing 0.5 mm tris(2-carboxyethyl)phosphine and 5% glycerol and lysed by sonication. GST fusion proteins were affinity-purified from soluble bacterial lysates by using glutathione–Sepharose  according to standard manufacturer's protocols and cleaved off GST while bound to the beads by 2-h incubation at 30 °C with PreScission protease . Soluble recombinant proteins were either concentrated with Amicon Ultra centrifugation filter devices (Millipore) and stored as 5–10 mg/ml stocks at −80 °C until required (Smurf2), or the concentration was adjusted to about 0.5–1 mg/ml (polymerization-competent proteins) and stored at −80 °C.All recombinant proteins were expressed as GST fusions. Plasmids were transformed into BL21 (DE3) CodonPlus-competent In vitro ubiquitination experiments were set up essentially as described before (d before . Assays T. M. and H. R. P. conceptualization; T. M. investigation; T. M. methodology; T. M. and H. R. P. writing-review and editing; H. R. P. supervision; H. R. P. writing-original draft.


Arabidopsis thaliana, two synergid cells situated on either side of the egg cell produce cysteine-rich chemoattractant peptide LURE that guides the pollen tube to the female gametophyte for sexual reproduction. Recently, in Arabidopsis thaliana, Pollen Receptor Kinase 3 (PRK3), along with PRK1, PRK6, and PRK8, have been predicted to be the receptors responsible for sensing LURE. These receptors belong to the Leucine Rich Repeat Receptor Like Kinases (LRR-RLKs), the largest family of receptor kinases found in Arabidopsis thaliana. How PRKs regulate the growth and development of the pollen tube remains elusive. In order to better understand the PRK-mediated signaling mechanism in pollen tube growth and guidance, we have determined the crystal structure of the extracellular domain (ecd) of PRK3 at 2.5 Å, which resembles the SERK family of plant co-receptors. The structure of ecdPRK3 is composed of a conserved surface that coincides with the conserved receptor-binding surface of the SERK family of co-receptors. Our structural analyses of PRK3 have provided a template for future functional studies of the PRK family of LRR-RLK receptors in the regulation of pollen tube development.During reproduction in flowering plants, the male gametophyte delivers an immotile male gamete to the female gametophyte in the pistil by formation of pollen tubes. In  The pollen tube is a tip growing cell that originates from the pollen grain2. The pollen tube then penetrates the surface of the stigma and the style; it reaches the ovule for fertilization and delivers immotile male gametes2. One sperm cell fertilizes the egg cell, while another sperm cell fuses with the central cell to produce the embryo and endosperm3. This highly complex process is thought be regulated by several external and internal cues4. The signaling cascade controlling the pollen tube growth ensures successful fertilization by providing directionality and guidance. It not only protects polyspermy, but also stops the arrival of an incompatible pollen tube2. Lipid-transfer protein, Transmitting Tissue-Specific protein (TTS), and Styler Pectin all have been implicated as crucial factors in this signaling process8. After the identification of the first pollen specific receptor-like kinase from Petunia inflata, more receptor like kinases such as LePRK1 and LePRK2 were isolated from Lycospersicon esculentum10. The study of LePRK1 and LePRK2 proved that these two receptor like kinases play crucial roles in the pollen tube growth, pollen-pistil interaction, and pollen tube localization10. This clearly indicated that receptor like kinases are strongly involved in the pollen-pistil interaction and plant sexual reproduction.Sexual reproduction in plants is an intricate process involving pollen-pistil interactions between male and female gametophytes. Through pollen tube formation, the male gametophyte is directed towards the female gametophyte in the pistil15. Generally, the RLK proteins have an extracellular domain, a transmembrane domain, and a kinase domain with Ser/Thr-type specificity17. Both LePRK1, LePRK2 and pollen specific receptor kinases from Petunia inflata belong to the family of Leucine-Rich-Repeat Receptor-Like Kinases (LRR-RLKs)18. Bioinformatics studies on Arabidopsis genome, conducted by McCormick et al., presented multiple pollen receptor kinase candidates that have similar amino acid sequences and topologies when compared to the LePRKs. The LRR-RLK subfamily is the largest group of RLKs in Arabidopsis, and they are involved in mediating protein-protein and protein-ligand interactions22.Plant receptor-like kinases (RLKs) play key roles in various biological processes involving plant immune defense and development. This includes defense against pathogens, stomata formation, organ and flower development, xylem development, etc.23. Various small molecules and modified peptides were thought to be involved in these cell-cell communications and signal transduction26. LAT52 and LeSTIG1 of Solanum lycopersicum as well as SCR/SP11 of Brassica sp were thought to be involved in pollen tube germination and growth32. LAT52 an essential peptide required for pollen hydration and pollen tube formation was found to be interacting with the extracellular LRR domain of LePRK229. McCormick et al. demonstrated that LAT52 interacts with LePRK2. This was a major step in understanding the role of pollen receptor kinases in pollen tube growth as well as pollen tube guidance and overall plant reproduction29. Not long ago it was discovered that two synergid cells situated on either side of the egg cell of Torenia fournieri produce cysteine rich chemoattractant peptides LUREs that guide the pollen tube to the female gametophyte for sexual reproduction35. The predicted mature peptides of LURE1 and LURE2 have six cysteine residues; the correct intramolecular disulfide bond formation is essential for their activity. There is a significant amino acid divergence between LURE1 and LURE2 except for the conserved cysteines. As LURE 1 and LURE 2 are highly diverged, it is suggested that they may bind to different receptors34. Like Torenia fournieri, defensin like LURE peptides are also found in Arabidopsis which are named AtLURE136. Interestingly, six genes including a pseudo gene encoding AtLURE1 peptides (AtLURE1.1–1.5) form a species-specific gene cluster in the genome. Four of the five AtLURE1 peptides have been demonstrated to possess pollen tube attractant activity, although AtLURE1.1 has a relatively lower activity. On the other hand, AtLURE1.5 peptide which lacks one of the six conserved cysteine residues does not have pollen tube guaidance activity36.The presence of numerous receptor kinases in the pollen tube has long puzzled biologists. It was hypothesized that a chemical or biological factor secreted from the ovule is perceived by the RLKs and that in turn regulates the pollen tube guidanceArabidopsis thaliana, AtPRK3, along with AtPRK1, AtPRK6, and AtPRK8, have been predicted as the receptors that sense the AtLURE1.2 peptide. It has been revealed that the AtPRK3 has a vital role in pollen tube growth, plant fertility, and plant reproduction37. In addition, Male Discoverer1 (MDIS1), MDIS1-Interacting Receptor-Like Kinase1 (MIK1), and MIK2, have been independently identified by another research group as potential LURE1 receptors in Arabidopsis38.In 39. Here we present the atomic structure of the extracellular domain (ecd) of the PRK3 (residues 20–237) from Arabidopsis thaliana resolved at 2.5 Å. Structural elucidation of the ecdAtPRK3 will provide insight into its function, aid in identifying ligand or receptor binding site, and will describe the role of other PRKs in the regulation of pollen tube development and function.While a lot of valuable data about peptide ligands and LRR-RKs have been obtained from genetic and biochemical experiments, visualization of ligand/LRR-RK complex structures at the atomic level is vital to understand the functions of LRR-RKs and their mediated biological processes2 with two PRK3 protein molecules in each asymmetric unit . The AtPRK3 ectodomain disulfide bonding pattern is similar to that of Somatic Embryogenesis Receptor Kinase 1 (SERK1) and SERK2. SERK1 has two disulfide bonds: one in the LRR capping domain between C58–C65 and C202–C210 located in the LRR C terminal domain. In the case of SERK2, C61–C68 and C205–C213 form disulfide bonds in the LRR capping domain and in the LRR C-terminal domain respectively. Mutations in the cysteine residues have been shown to affect the functions of FLS2, whereas the same mutations in CLV2 do not impair its function46. It has been predicted that these cysteine residues take part in protein folding, trafficking, and the binding of other proteins48.PRK3 is a type I transmembrane receptor which contains an N-terminal signal peptide domain (SP), an LRR capping domain (CD), a leucine rich repeat domain (LRR), an LRR C-terminal domain (CT), a transmembrane domain (TM), and an intracellular kinase domain (KD) Fig. . To gainit Table . The AtP49. Based on the conserved and non-conserved residues present in the LRR domain, LRRs have been classified into several families19. The AtPRK3 ectodomain belongs to the plant specific LRR family19. The AtPRK3 ectodomain contains six copies of plant specific LRR repeats ranging from 23–25 amino acid residues 52. N-glycosylation associated with the polypeptides in the endoplasmic reticulum ensures proper protein folding53. In the case of Arabidopsis, abnormal and altered N-glycosylations can affect the abiotic stress response and hamper proper plant development56. Conserved N-glycosylation patterns, especially NX(S/T) motifs, have been suggested to be important for correct ectodomain structure and function57. In the PRK3 ectodomain, the N37 glycosylation, which is present in the α-helical part of the LRR convex side, belongs to the conserved NX(S/T) motif and is believed to be essential for its correct structure and function. However, the N123 glycosylation also belongs to the conserved NX(S/T) glycosylation motif, but is present at the bottom of the concave surface. We believe that the exposed N123 glycosylation pattern on the concave side of the PRK3 ectodomain can form hydrated branches and are likely to facilitate the association with other molecules58. Further studies elucidating PRK3 glycosylation patterns are required to comprehend their actual function.Two N-glycosylation sites are identified on residues N37 and N123, with only one visible GlcNAc sugar residue conjugated on N37 and two such residues on N123 Fig. . N-linkeArabidopsis thalian to Cajanus cajan. From the previously published LRR ectodomain structures, it has been demonstrated that the concave surface residues generally interact with other proteins or ligands50. Therefore, we can safely suggest that the positively charged surface will provide a favorable interacting surface for negatively charged proteins.There is a positively charged electrostatic patch on the concave surface of the LRR domain, whereas the C terminal region is mostly negatively charged Fig. . The pos2  family of plant LRR-RLKs. The SERK family of proteins are involved in the regulation of immune responses in plants66. Arabidopsis BAK1, SERK1, and SERK2 have an extracellular domain of 213, 216 and 220 residues, respectively. The extracellular domains of BAK1, SERK1, and SERK2 also share substantial sequence similarity with the PRK3 extracellular domain. The amino acid sequence identity between ecdPRK3 and ecdBAK1, ecdSERK1, and ecdSERK2 is 32.43%, 32.32%, and 30.81%, respectively. Structural alignment between ecdPRK3 and ecdSERKs resulted in a RMSD of 1.92 Å, 1.89 Å, and 1.96 Å for BAK1, SERK1, and SERK2 respectively  motif, lies in between the LRR and transmembrane region and is a unique feature of the SERK family members19. Along with the SERK family members, this particular motif is also abundantly present in the AtPRK3 ectodomain. The LRR conserved sequence for AtPRK3 is plant specific and is as follows, LxxLxxLxLxxNxLSGxIPxx; this is different from the canonical animal conserved sequence, which is LxxLxxLxLxxNxLxxLpxxoFxx. The plant specific conserved sequence in LRR receptor ectodomains is responsible for their conformation67.The LT/SGxIP motif is very common in plant specific LRR receptors65. The crystal structures of several of the extracellular domains of SERK-receptor-ligand complexes are available68. We have examined the binding interfaces between the SERK co-receptors and their LRR receptors. All three SERK members use a conserved surface that is located on the LRR capping domain and the N-terminal portion of the concaved surface of the LRR domain to interact with their receptors to facilitate ligand binding and subsequent signaling  secreted from pollen and pistil for pollen germination, as well as growth and guidance of the pollen tube, but for A. thaliana using baculovirus-mediated insect cell expression. The PRK3 gene encoding residues 20–237 was fused to the secretion signal sequence of hemolin and then cloned into a modified pFastBac1 vector. The secreted protein was first purified by nickel-affinity chromatography using an engineered 6-histidine tag at the carboxyl terminus of the PRK3 protein, and then further purified by size-exclusion chromatography in a buffer containing 20 mM Bis-Tris, pH 6.0, and 100 mM NaCl. The purified protein was concentrated to 5 mg/ml for crystallization. The predicted molecular weight based on the amino acid sequence of the recombinant protein is 25.3 kDa. However, the apparent molecular weight of the purified recombinant protein is approximately 35 kDa presumably due to glycosylation.To elucidate the structure of AtPRK3, we expressed the extracellular domain of AtPRK3 from 2 crystal form using both hanging drop vapor diffusion and sitting drop methods at 18 °C by mixing equal volumes of the purified protein and the crystallization reservoir solution of 0.1 M Tris pH 8.5 and 18% PEG 3350(w/v). For data collection, all crystals were flash frozen in the respective crystallization conditions supplemented with 20% (v/v) glycerol. Diffraction data were collected at the 22-ID (SERCAT) beam line of the Advanced Photon Source (APS). All diffraction data were processed using the HKL200073 suite and their statistics are shown in Table The recombinant AtPRK3 protein was concentrated to 5 mg/ml. The ectodomain of AtPRK3 protein was subjected to extensive crystallization screening. The protein was crystallized in P474, and refined with REFMAC575 and PHENIX76. The crystals contain two PRK3 molecules in each asymmetric unit cell. The AtPRK3 structure model contains residues 26–233. Two asparagine residues (N37 and N123) in PRK3 are N-glycosylated. One GlcNAc sugar residue on N37 and two on N123 are visible. In addition to the observed N-glycosylation, four cysteine residues are observed to form two disulfide bonds between C53–C62 and C224–C232. The structures were analyzed using the CCP4 suite77 and the PISA server78, and the figures were made using PyMOL79.We have determined the AtPRK3 ectodomain structure by molecular replacement using the SERK1 extracellular domain structure as an initial search model (PDB ID 5IYX). The model of ecd PRK3 structure was built in COOT1 mg of purified ecdPRK3 protein was loaded onto a Superdex 200 increase 10/300 GL column  in a buffer containing 20 mM Bis-Tris (pH 6.0) and 100 mM NaCl.https://www.ebi.ac.uk/Tools/msa/clustalo/)80. Clustal Omega uses the HHalign algorithm and its default settings as its core alignment engine. The algorithm is described in Söding, J81. The default transition matrix is Gonnet, gap opening penalty is 6 bits, gap extension is 1 bit. Homology modeling analyses of the ECD structures of PRKs with PRK3 ECD structure as a template was conducted with SWISS-MODEL71. Structural superposition was rendered in PyMOL79.Amino acid sequences with single letter code were input to the Clustal Omega online server for multiple sequence alignment (The atomic coordinates and structure factors have been deposited in the Protein Data Bank under accession code 5WLS.Supplementary Information


Mapping three-dimensional (3D) surface deformation caused by an earthquake is very important for the environmental, cultural, economic and social sustainability of human beings. Synthetic aperture radar (SAR) systems made it possible to measure precise 3D deformations by combining SAR interferometry (InSAR) and multiple aperture interferometry (MAI). In this paper, we retrieve the 3D surface deformation field of the 2010 Haiti earthquake which occurred on January 12, 2010 by a magnitude 7.0 Mw by using the advanced interferometric technique that integrates InSAR and MAI data. The surface deformation has been observed by previous researchers using the InSAR and GPS method, but 3D deformation has not been measured yet due to low interferometric coherence. The combination of InSAR and MAI were applied to the ALOS PALSAR ascending and descending pairs, and were validated with the GPS in-situ measurements. The archived measurement accuracy was as little as 1.85, 5.49 and 3.08 cm in the east, north and up directions, respectively. This result indicates that the InSAR/MAI-derived 3D deformations are well matched with the GPS deformations. The 3D deformations are expected to allow us to improve estimation of the area affected by the 2010 Haiti earthquake. For advances in the geological interpretation of geohazards such as earthquakes and volcanic eruptions, three-dimensional (3D) surface deformation mapping has been becoming increasingly more essential. Synthetic aperture radar (SAR) systems enable us to measure 3D surface deformations precisely by combining SAR interferometry (InSAR) and multiple aperture interferometry (MAI) –14. The The 2010 magnitude 7.0 Mw Haiti earthquake occurred on January 12, 2010. Since its epicenter was located as close as about 25 km south west of Port-au-Prince, which is Haiti’s capital, an estimated three million people were affected and notable landmark buildings were significantly damaged by the earthquake . It was The surface deformation caused by the 2010 Haiti earthquake has been observed by InSAR and GPS methods – 28. TheAlthough it can be very important to measure 3D deformation field to improve the finite fault model accuracy, the 3D ground deformation was not retrieved from these previous studies. The reasons are as following: 1) the study area is a highly mountainous region with steep mountains and narrow valleys, 2) the data pairs used for this study have relatively long time intervals. Thus, the geometric distortion of the interferometric pairs is very high due to the SAR imaging geometry and the interferometric coherence is low as well. It all means that it is very hard to apply the InSAR and MAI techniques to the Haiti interferometric pairs. Particularly, the MAI method can have a difficulty in measuring the along-track deformation because it requires higher coherence than the InSAR method.In this paper, we three-dimensionally measure the ground surface deformation caused by the 2010 Haiti earthquake using the InSAR and MAI methods and validate the achieved accuracy of the 3D deformations from the GPS measurements. The data used for this study is the ALOS PALSAR ascending pair of FBS (fine beam single polarization) acquisitions on February 9, 2008 and February 14, 2010, and the descending pairs of FBS acquisitions on March 9, 2009 and January 25, 2010. The interferometric pairs are exactly the same to the pairs in the previous studies –29. In aOn January 12, 2010, faults associated with the Enriquillo-Plantain Garden left-lateral strike-slip fault zone (EPGFZ) in the Caribbean-North America plate boundary were struck by an Mw 7.0 earthquake . The seiThe ALOS PALSAR ascending pairs of FBS (fine beam single polarization) acquired on February 9, 2008 and February 14, 2010 and the descending pairs of FBS acquired on March 9, 2009 and January 25, 2010 were used for retrieving 3D deformations by using the InSAR and MAI combination. The interferometric pairs have relatively long time intervals, which are about 2 year for ascending pair and about 1 year for descending pairs. It seems that the long time interval of InSAR pairs and the steep topography of Haiti make the precise 3D ground deformations retrieval from the InSAR and MAI methods very difficult. The GPS measurements estimated by  were useThe InSAR measurement has been widely used for mapping ground deformations, but the MAI measurement was not always applied because of the drawbacks that requires high coherence and complicate processing procedures. Recently, the MAI performance has been notably improved by reducing the interferometric phase errors and correcting the phase distortions –19. ThusϕMAI) can be defined as follows:x is the along-track surface deformation, l is the effective antenna length (8.9m for ALOS PALSAR), and n is a normalized squint that is a fraction of the full aperture width [n = 0.5 is used for the MAI processing, l = 8.9 m for ALOS satellites and hence one fringe yields 8.9 m. When compared to the one fringe of about 11.8 cm in the InSAR processing, the measurement accuracy of MAI is much lower than that of InSAR. Suppose that a NS (North-South) strike-slip fault is formed by an earthquake, then we cannot measure the NS deformation by using the InSAR method. However, the MAI method enables us to measure the NS deformation. Therefore, the MAI measurement is very important to understand the surface deformation three-dimensionally despite the fact that the MAI measurement accuracy is much lower than the InSAR.The MAI method utilizes a sub-aperture processing technique to create two images with slightly different line-of-sight (LOS) vectors in the azimuth direction: One image is a forward-looking image and the other image is a backward-looking image. Thus, the interferometry between master and slave forward-looking images is defined as the forward-looking SAR interferometry and the interferometry between master and slave backward-looking images is denoted as the backward-looking SAR interferometry. The difference between the forward- and backward-looking InSAR is MAI, which represents the along-track deformation. The MAI phase  measured by using InSAR and MAI ascending and descending pairs can be defined by ,31:r=U⋅d can be retrieved by usingW is the weighting matrix, which can be estimated from the standard deviations in the InSAR and MAI measurements [Finally, the 3D ground surface deformation urements , 31. To To observe the surface deformation caused by the 2010 Haiti earthquake, the InSAR and MAI methods were applied to the ascending and descending ALOS PALSAR interferometric pairs. The ascending and descending InSAR interferograms have been produced by a complex multi-look operation of 8 looks in the range direction and 16 looks in the azimuth direction, which corresponds to about 60 and 50 m in range and azimuth directions, respectively. The multi-look operation has been applied by using a two-step strategy: 1) 2x4 multi-looking in the range and azimuth before the correction of the flat-Earth and topographic phase distortions and 2) 4x4 multi-looking after the correction to reduce interferometric phase noise efficiently  with a wHowever, it is certainly not easy to understand the 3D deformation field from the LOS imaging geometries. Since the 3D deformation enables us to easily understand the deformation patterns, the 3D deformation field needs to be retrieved. The along-track surface deformation presented in The spatial comparison between GPS-derived and SAR-derived 3D deformations was performed as seen in For sustainable advances in the geological interpretation of geohazards such as earthquakes and volcanic eruptions, 3D surface deformation mapping has been believed to be more and more essential. The SAR systems made possible the precise measurement of the 3D surface deformation field by using the InSAR and MAI combination. In this paper, we three-dimensionally observed the surface deformation caused by the 2010 Haiti earthquake. For the measurement, we used the ALOS PALSAR FBS ascending pair, which was acquired on February 9, 2008 and February 14, 2010, and the descending pair, which was on March 9, 2009 and January 25, 2010. Two InSAR and two MAI interferograms were generated from the ascending and descending pairs. Albeit their interferometric coherences were relatively low, the LOS and along-track deformations could be measured from the InSAR and MAI interfrograms. Most of all, it was very difficult to measure the along-track deformation, because the MAI interferograms had severe topographic distortions. However, the distortions could be successfully mitigated by the additional topographic corrections. Nevertheless, one of the two MAI interferograms had a severe ionospheric distortion, and hence we could not use the ionosphere-distorted MAI interferogram for retrieving 3D deformations.The LOS and along-track surface deformations were calculated from the InSAR and MAI interferograms. We could see clear deformations in the city of Leogane and the mountainous area between the cities of Leogane and Jacmel . When co


The calculations show that relative contribution of the catalytic bases to RNA stability observably differs from contributions of the noncatalytic bases. Distinction between the concepts of “relative stability” and “mutational stability” is suggested. As results of prediction for several models of ribozymes appear to be in agreement with the published data on the potential active site regions, the method can potentially be used for prediction of functional nucleotides from nucleic sequence.Ribozymes are functionally diverse RNA molecules with intrinsic catalytic activity. Multiple structural and biochemical studies are required to establish which nucleotide bases are involved in the catalysis. The relative energetic properties of the nucleotide bases have been analyzed in a set of the known ribozyme structures. It was found that many of the known catalytic nucleotides can be identified using only the structure without any additional biochemical data. The results of the calculations compare well with the available biochemical data on RNA stability. Extensive


Drosophila Zasp PDZ domain protein, and is required for myofibril assembly and maintenance. We show by in vitro biochemistry that the PDZ domain plus a C-terminal extension is the only area of Zasp52 involved in the interaction with α-actinin. In addition, site-directed mutagenesis of 5 amino acid residues in the N-terminal part of the PDZ domain, within the PWGFRL motif, abolish binding to α-actinin, demonstrating the importance of this motif for α-actinin binding. Rescue assays of a novel Zasp52 allele demonstrate the crucial importance of the PDZ domain for Zasp52 function. Flight assays also show that a Zasp52 mutant suppresses the α-actinin mutant phenotype, indicating that both proteins are core structural Z-disc proteins required for optimal Z-disc function.Z-discs are organizing centers that establish and maintain myofibril structure and function. Important Z-disc proteins are α-actinin, which cross-links actin thin filaments at the Z-disc and Zasp PDZ domain proteins, which directly interact with α-actinin. Here we investigate the biochemical and genetic nature of this interaction in more detail. Zasp52 is the major  α-actinin/+ heterozygous animals show irregularities in wing beat frequency, which can be suppressed by removing one copy of Zasp52. This suggests that both proteins are required at fixed levels at the Z-disc to support optimal functionality.Although Zasp PDZ domain proteins are known to bind α-actinin and play a role in muscle assembly and maintenance, the details and importance of this interaction have not been assessed. Here we demonstrate that a conserved motif in the N-terminal part of the Zasp52 PDZ domain is responsible for α-actinin binding and that a C-terminal extension of the PDZ domain is required for optimal α-actinin binding. We show using transgenic animals that in the absence of the PDZ domain no aspect of myofibril assembly can be rescued. Intriguingly,  Drosophila, Zasp52 has a PDZ, ZM (Zasp-like motif) and four LIM domains; while Zasp66 and Zasp67 only feature the N-terminal PDZ domain and a weakly conserved ZM domain. Zasp52 colocalizes with α-actinin at Z-discs and plays a role in myofibril assembly and maintenance [Drosophila, a ZASP/Cypher and ENH double knock-out in mice demonstrates partial redundancy in myofibril assembly [C. elegans ortholog ALP-1 displays defects in actin filament organization, but motility defects are much milder than in vertebrates or Drosophila [Like most animals, invertebrates have three main types of muscles, body wall, heart, and visceral muscle, but in contrast to vertebrates, all of them are striated. A particularly highly organized muscle is the indirect flight muscle (IFM) in insects, a stretch-activated fibrillar muscle . Slidingntenance , 17, 18.ntenance , 20. Furntenance . Mutationtenance , 7, 21. assembly . The sinosophila –25. Mutaosophila , 27.α-actinin heterozygous flight defect is suppressed by removal of one copy of Zasp52. Our data indicate that Zasp PDZ domain family proteins are core scaffold proteins of muscles.In this study, we explore the relationship of Zasp52 and α-actinin. We show that even though different Zasp52 deletion transgenes co-immunoprecipitate α-actinin and localize to Z-discs, only an extended PDZ domain mediates direct interaction of Zasp52 with α-actinin. Through site-directed mutagenesis we also demonstrate the importance of the PWGFRL motif in α-actinin binding. A rescue assay confirms the importance of the PDZ domain of Zasp52 for myofibril assembly. Finally, we show genetically that the Zasp52 α-actinin interaction is required for IFM function, because the We have previously shown that Zasp52 binds directly to α-actinin via an N-terminal construct containing the PDZ, ZM, and LIM1 domain . Both PDWe noticed that Mef2-Gal4-mediated expression of transgenes results in various muscle defects such as wavy myofibrils . To asseOur data show that multiple domains of Zasp52 are able to mediate Z-disc localization irrespective of expression level, and therefore raise the question if different domains of Zasp52 can bind directly to α-actinin.We have previously shown a direct interaction of Zasp52-PK containing a PDZ, ZM, and LIM1 domain with α-actinin . To deteWe previously identified 16 highly conserved amino acids in the N-terminal half of the Zasp52 PDZ domain as the defining feature of Zasp PDZ domain proteins , and proZasp52 alleles. Recently, a large number of MiMIC insertions based on the Minos transposon were created in Drosophila inserting splice acceptor sites followed by stop codons at various positions in the genome [Zasp52 locus: MI02988 after exon 2, MI07547 after exon 8, and MI00979 after exon 15 BSC427 than homozygously BSC427 adults myofibril assembly in IFM is severely disrupted. In many myofibrils, Z-discs and M-lines are no longer distinguishable (Zasp52MI00979/Df(2R)BSC427 looks similar to the previously described RNAi lines iZasp52ex20 and iZasp52ex16 (Zasp52MI07547/Df(2R)BSC427 shows no obvious defects BSC427 IFM defects by expressing our transgenes with UH3-Gal4. We started with the most common isoform, Zasp52-PR, which can indeed fully rescue all IFM defects and localizes normally to Z-discs (Zasp52MI00979/Df(2R)BSC427 mutants, which resulted in no rescue BSC427, M{3xP3-RFP.attP}ZH-86Fb, y w, and Actn8 from the Bloomington Drosophila Stock Center; UH3-Gal4 has been described previously [ΔPDZ, UAS-FLAG-Zasp52-PRΔZM, UAS-FSH-Zasp52-PK and UAS-FSH-Zasp52-LIM234 (this study). Mef2-Gal4; UAS constructs and UH3-Gal4; UAS stocks were generated by standard genetic crosses.The following fly stocks were used: Mef2-Gal4, eviously ; UAS-FLAZasp52 and α-actinin, Actn8/FM7 was crossed to y w, and Zasp52MI02988/CTG and Zasp52MI00979/CTG were crossed to Actn8/FM7 or y w and incubated at 20°C.To test for genetic interaction between y w (for RK and LIM234) and M{3xP3-RFP.attP}ZH-86Fb  embryos.Transgenic flies were generated by injecting sequence-verified plasmids into ΔPDZ (deleting amino acids 11–84 of Zasp52-PR) and pUAST-attb-Zasp52-RRΔZM (deleting amino acids 144–169 of Zasp52-PR). GST-Zasp52-RK and GST-Zasp52-LIM234 were cloned from EST LP01550 (for RK) and RH03424 (for LIM234) into pGEX-5X-1 . Zasp52-PKP18DW19F, Zasp52-PPR22T Zasp52-PPG25W, Zasp52-PPG26D, Zasp52-PDZ, Zasp52-ZM, Zasp52-LIM1, Zasp52-STOP155, Zasp52-STOP143, and Zasp52-STOP112 were synthesized or generated by site-directed mutagenesis by GenScript and cloned into pGEX-5X-1 to generate GST constructs.Flag-Zasp52-RR (FlyBase ID: FBtr0329912), FSH-Zasp52-RK (FlyBase ID: FBtr0302163) and FSH-Zasp52-LIM234 were synthesized by GenScript and cloned into pUAST-attb  and pUASt . PCR-me2 fly thoraces were homogenized in 2x SDS sample buffer. Protein samples were resolved by 8% SDS-PAGE and then detected by immunoblotting. Antibodies employed were anti-Zasp52 , anti-F2, 1 mM DTT, 5% glycerol, 0.5% Triton X-100 and complete EDTA-free protease inhibitor; Roche). Protein extracts were then incubated with prewashed anti-FLAG M2 affinity resin (Sigma-Aldrich) for 3 hours at 4°C. After incubation, the beads were washed three times with wash buffer . Bound proteins were eluted by boiling in 2x SDS sample buffer. Eluates were analyzed by SDS-PAGE and by immunoblotting. Antibodies were used at the following dilution: rat anti-α-actinin MAC276 antibody at 1:2000 (Babraham Bioscience Technologies); mouse anti-FLAG antibody at 1:5000 (Sigma-Aldrich). The immunoreaction was visualized by ECL (Millipore).70 adult fly thoraces were cut in half and were homogenized in lysis buffer  for 3 hours at 4°C. The beads retaining the GST-tagged proteins were washed three times with above buffer with 250 mM NaCl and 0.5% Triton X-100. Subsequently, rabbit skeletal muscle α-actinin (Cytoskeleton) was added and incubated for another 3 hours at 4°C. Final washes were in above buffers with 150 mM NaCl and 0.2% Triton X-100. Beads were resuspended in SDS sample buffer and analyzed by SDS-PAGE and immunoblotting.For GST pulldown assays, 2, 2 mM EGTA, 5 mM DTT, 0.5% Triton X-100, 50% glycerol) overnight at -20°C. IFMs were dissected, washed and then fixed with 4% paraformaldehyde in relaxing solution  with protease inhibitors. Incubations of primary antibodies and Alexa 594-phalloidin (ThermoFisher Scientific) were carried out overnight at 4°C, followed by secondary antibody incubation for 3 hours at room temperature. Primary antibodies used were rat anti-α-actinin MAC276 , rat anti-Kettin KIg16 MAC155 , and anti-FLAG . Fluorescently labeled secondary antibodies of the Alexa series (ThermoFisher Scientific) were used at a 1:400 dilution. Samples were mounted in ProLong Gold antifade solution (ThermoFisher Scientific).Half thoraces were glycerinated .The wing beat frequency of a fly was determined using an optical tachometer as previously described . In thisS1 FigConfocal microscopy of IFM stained with anti-Flag antibody visualizing the transgene in green, anti-α-actinin antibody to label Z-discs in purple, as well as phalloidin to visualize actin thin filaments in red. Weak UH3-Gal4-mediated expression of UAS-FSH-Zasp52-LIM234 still results in Z-disc localization and reduces overexpression defects. Scale bar, 10 μm.(TIF)Click here for additional data file.S2 FigA) Zasp52-PK, Zasp52-LIM234, Zasp52-PP, Zasp52-PDZ, Zasp52-ZM, and Zasp52-LIM1 run on a SDS-PAGE gel after purification. Asterisks indicate the fusion protein. (B) Zasp52-PP, Zasp52-STOP155, Zasp52-STOP143, Zasp52-STOP112, and Zasp52-PDZ run on a SDS-PAGE gel after purification. (C) Zasp52-PK and Zasp52-PKP18DW19F mutant variant run on a SDS-PAGE gel after purification. (D) Zasp52-PP and Zasp52-PPR22T, Zasp52-PPG25W, and Zasp52-PPG26D point mutant variants run on a SDS-PAGE gel after purification. Molecular weight marker is indicated in kD.GST and Zasp52 domain GST fusions run on a SDS-PAGE gel after purification. ((TIF)Click here for additional data file.S3 FigA) Confocal microscopy of Zasp52MI02988/Df(2R)BSC427 versus homozygous Zasp52MI02988 IFM stained with phalloidin to visualize actin thin filaments in green and anti-Kettin antibody to visualize Z-discs in red. Homozygous Zasp52MI02988 IFM show no obvious defects. (B) Quantification of IFM defects in C) Confocal microscopy of Zasp52MI00979/Df(2R)BSC427 IFM rescued with UH3-Gal4-expressed UAS-Zasp52-PR and stained with anti-Flag antibody to visualize the transgene in blue, phalloidin to visualize actin thin filaments in green and anti-Kettin antibody to visualize Z-discs in red. Zasp52-PR cannot rescue Zasp52MI00979.((TIF)Click here for additional data file.S4 FigActn8/+, Zasp52MI02988/+, and Actn8/+; Zasp52MI02988/+ exhibit no obvious defects.Confocal microscopy of IFM of heterozygotes stained with phalloidin to visualize actin thin filaments in red and anti-Kettin antibody to visualize Z-discs in green. (TIF)Click here for additional data file.


Among the 20 subfamilies of protein receptor tyrosine kinases (RTKs), Eph receptors are unique in possessing a sterile alpha motif (SAM domain) at their C-terminal ends. However, the functions of SAM domains in Eph receptors remain elusive. Here we report on a combined cell biology and quantitative fluorescence study to investigate the role of the SAM domain in EphA2 function. We observed elevated tyrosine autophosphorylation levels upon deletion of the EphA2 SAM domain (EphA2ΔS) in DU145 and PC3 prostate cancer cells and a skin tumor cell line derived from EphA1/A2 knockout mice. These results suggest that SAM domain deletion induced constitutive activation of EphA2 kinase activity. In order to explain these effects, we applied fluorescence correlation spectroscopy to investigate the lateral molecular organization of EphA2. Our results indicate that SAM domain deletion (EphA2ΔS-GFP) increases oligomerization compared to the full length receptor (EphA2FL-GFP). Stimulation with ephrinA1, a ligand for EphA2, induced further oligomerization and activation of EphA2FL-GFP. The SAM domain deletion mutant, EphA2ΔS-GFP, also underwent further oligomerization upon ephrinA1 stimulation, but the oligomers were larger than those observed for EphA2FL-GFP. Based on these results, we conclude that the EphA2 SAM domain inhibits kinase activity by reducing receptor oligomerization. Of the 58 transmembrane protein receptor tyrosine kinases (RTKs) in the human genome, 14 are Eph receptors, constituting the largest subfamily of RTKs. They are divided into EphA and EphB subclasses that bind to GPI-anchored ephrin-A and transmembrane ephrin-B ligands, respectively, with some exceptions122537Eph receptors are type-I transmembrane proteins. The extracellular domain (ECD) of Eph contains a highly conserved ligand binding domain (LBD), followed by a cysteine rich domain (CRD) and two fibronectin-type III domains (FN I & II). After the transmembrane (TM) domain, the intracellular domain (ICD) of Eph consists of a juxtamembrane segment (JMS), a kinase domain, a sterile alpha motif (SAM domain) and a PDZ binding motif11141516et al. in a reconstituted intermembrane signaling system. EphA2 was expressed by live human breast cancer cells and interacted with laterally mobile ephrinA1 on a underlying supported lipid bilayerStructural studies of the extracellular domain (ECD) of EphA2 in complex with ephrinA5 showed clusters with several binding interfaces181820et al. used a quantitative FRET method to study the dimerization of EphA3 in osmotically swelled cellsOn the intracellular side, one feature that distinguishes Eph receptors from all other members of the RTK superfamily is the presence of a sterile alpha motif (or SAM) domain at the C-terminal end. SAM domains are 60 to 90 amino acid structural modules, consisting primarily of alpha helices and are known to mediate homophilic and heterophilic protein interactions23252628The spatial arrangement of Eph receptors in the cell membrane is an important aspect of regulating the Eph signaling pathway22To interrogate the function of EphA2 SAM domain, we generated three serially truncated mutants that were tagged with enhanced green fluorescence protein (eGFP) in a retroviral expression vector . The delStimulation with recombinant ephrinA1-Fc led to strong activation and degradation of endogenous EphA2 in PC3 cells , consistin vitro, also became highly compact upon stimulation with the ephrinA1 .By measuring the diffusion of the three truncated receptor constructs  in DU145et al. reported that ligand free, inactive EphA2 forms dimers with a DK of 210 receptors/μm2. The FCS experiments reported here were performed on cells with an average receptor density of 123 receptors/μm2  values obtained from the single cell FCS data . We comptors/μm2 . This deDimerization interfaces in the ecto-domain of EphA2 have been identified by previous structural studies1819202/s) are significantly smaller than that of EphA2FL-GFP . The same retroviral vectors used for the DU145 cell experiments were used to stably transduce 728 cells, resulting in EphA1/EphA2 double knockout 728 cells with stable expression of the GFP-tagged EphA2 constructs. The morphology of 728 cells with extended lamellipodia makes them suitable for FCS measurements . FCS mea2/s (2/s), the mobility of EphA2ΔS-GFP and EphA2ΔKS-GFP is decreased, which supports increased oligomerization of these two constructs. These observations lead to the same conclusions as in DU145 cells. The similarity in the results suggests that the expression level of endogenous EphA2 in DU145 cells is much lower than that of the GFP-tagged EphA2 constructs. Together, the FCS experiments in DU145 and 728 cells confirm that deletion of SAM domain leads to oligomerization of EphA2ΔS-GFP and EphA2ΔΚS-GFP beyond that of EphA2FL-GFP.The molecular brightness of EphA2FL-GFP in 728 cells is 433 cpsm . Based oEphA2 is activated by its cognate ligands, the ephrins. Structural studies have shown that the ligand binding domain (LBD) of EphA2 and the receptor binding domain (RBD) of ephrin form a circular tetramer consisting of two LBDs and two RBDs involving three interfaces22We set out to investigate the lateral organization of ephrinA1-bound EphA2 constructs using fluorescence imaging and fluctuation spectroscopy. Ligand activation was done with ephrinA1-Fc (EA1Fc), a soluble Eph ligand resulting from the fusion of ephrinA1 to the heavy chain of human IgG1. As demonstrated in 2/s to 0.22 μm2/s , and mouse epithelial tumor cells with EphA1/EphA2 gene knockout and stable expression of GFP-tagged EphA2 mutation constructs (728). Based on the FCS measurements, we found that full length EphA2 is not significantly dimerized in DU145 cells (with endogenous EphA2) and 728 cells (without endogenous EphA2). The EphA2 constructs with deletion of both kinase and SAM domains (EphA2ΔKS) and deletion of SAM domain (EphA2ΔS) underwent increased oligomerization compared to EphA2FL-GFP in both DU145 and 728 cells. These results demonstrated that the presence of the SAM domain reduces ligand-free oligomerization. The FCS results indicate that the constitutive activation of the kinase of the SAM domain deletion construct is induced by receptor oligomerization. These results add mechanistic insight into the activity of EphA2 and the inhibitory role of the SAM domain in EphA2 oligomerization.We also performed FCS measurements on 728 cells with ephrinA1-Fc (EA1-Fc) treatment. The results indicate that EA1-Fc binding causes EphA2FL-GFP to form oligomers, while the preoligomerized EphA2ΔS-GFP and EphA2ΔKS-GFP form even larger clusters. The presence of the SAM domain in EphA2FL-GFP prevented the receptors from forming clusters as large as the EA1-induced EphA2ΔS-GFP and EphA2ΔKS-GFP clusters. This is consistent with the inhibitory tendency of SAM domain towards EphA2 oligomerization observed with ligand-free receptors.This work illustrated the unique function of SAM domain in regulating the lateral organization and activity of EphA2. Within the RTK protein family, Eph receptors are the only ones that have a C-terminal SAM domain. Apart from the known function of SAM binding with other signaling proteins that contains SH2, for example SHIP2EphA1 knockout mice were generated through the insertion of an internal ribosome entry site (IRES)-human placental alkaline phosphatase (ALPP) reporter cassette into exon II of the EphA1 gene as described previouslyEphA2 mutant mice were generated by insertion of a gene trap vector into intron 1 of the EphA2 gene, which eliminated all EphA2 gene expression−/−:EphA2−/− double knockout mice. The mice were subject to DMBA/TPA two stage carcinogenesis as described previously−9 M T3, 1% penicillin/streptomycin, 2 mM l-glutamine). After one round passage, tumor cells were maintained in keratinocyte medium on plates precoated with 25 μg/mL collagen I.All procedures involving mice were performed in accordance with guidelines set forth by the American Association for Accreditation of Laboratory Animal Care and the USPHS “Policy on Humane Care and Use of Laboratory Animals”. Studies were approved and supervised by The Case Western Reserve University Institutional Animal Care and Use Committee.Human EphA2 cDNA was obtained from Dr. Tony Hunter. Full length , SAM domain deletion , or cytoplasmic deletion including the kinase and SAM domain  were amplified with PCR with appropriate primers and cloned into pEGFP-C1 plasmid in frame with the eGFP coding sequence. The entire eGFP fusion fragments were then inserted into LZRS-Pac retrovirus vector. They were then transfected into Phoenix retroviral packaging cells to produce the retrovirus. DU145, PC3 and 728 cells were infected with retroviral-mediated gene transfer in the presence of 6 µg/ml polybrene and selected in the presence of 1 mg/ml puromycin.Subconfluent DU145, PC3 and 728 cells were treated with 3 μg/ml ephrinA1-FcCell scattering assays were carried out as described2 active area, 25 dark counts per second. Signals collected by the detector are recorded with a four-channelrouted time-correlated single photon counting (TCSPC) card  which is synchronized with the white light laser source. Data recorded by TCSPC card is input to a computer for correlation with a home-written Matlab script.Fluorescence imaging and FCS measurements were performed on a customized Nikon Eclipse Ti inverted microscope  with home-built pulsed interleaved excitation and time-correlated single-photon detection . A contiF(t)) are auto-correlated with a function asCells were cultured in 10% FBS/DMEM on collagen coated plates. For activation, cells were incubated in 1 μg/mL EA1-Fc/Opti-MEM for 20 min prior to FCS experiment. FCS measurements were performed on live cells at 37 °C. The 488 nm excitation laser was set at a power of 300 nW and only focus at flat membrane area for data collections. Each data point was the average of five 15 s measurements at the same spot. Results from 50~90 cells (data points) were shown in the box plot figure. The recorded fluorescence fluctuation signals  is the auto-correlation function and where resulted correlation function curve was fitted withN is average number of fluorescent particles, τD is the average dwell time of fluorescentwhere τT is the tripletparticles within the detection volume, F is the fraction of molecules in the triplet state, D) was calculated based on N andrelaxation time. The molecular brightness (η) and diffusion coefficient .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.