Psammomys obesus, the rodent desert gerbil which is a unique polygenic natural animal model of obesity. Our results show that obese animals exhibit a strong preference for lipid solutions in a two-bottle test. Interestingly, the expression of CD36, a lipido-receptor, in taste buds cells (TBC), isolated from circumvallate papillae, was decreased at mRNA level, but remained unaltered at protein level, in obese animals. We further studied the effects of linoleic acid (LA), a long-chain fatty acid, on the increases in free intracellular calcium (Ca2+) concentrations, [Ca2+]i, in the TBC of P. obesus. LA induced increases in [Ca2+]i, largely via CD36, from intracellular pool, followed by the opening of store-operated Ca2+ (SOC) channels in the TBC of these animals. The action of this fatty acid on the increases in [Ca2+]i was higher in obese animals than that in controls. However, the release of Ca2+ from intracellular stores, studied also by employing thapsigargin, was lower in TBC of obese animals than control rodents. In this study, we show, for the first time, that increased lipid intake and altered Ca2+ signaling in TBC are associated with obesity in Psammomys obesus.Since the increasing prevalence of obesity is one of the major health problems of the modern era, understanding the mechanisms of oro-gustatory detection of dietary fat is critical for the prevention and treatment of obesity. We have conducted the present study on  According to the World Health Organization (WHO) et al.et al.It is generally assumed that excessive intake of dietary fat contributes to weight gain and obesity. There are several factors which influence fat consumption, including energy density and palatability 2+ signaling in CD36-positive taste bud cells, regulated by stromal interaction molecule 1 (STIM1) via CD36, is implicated in spontaneous preference for fat et al.The lipid-binding glycoprotein CD36, expressed on circumvallate papillae (CVP) of mouse and rat tongue, has been implicated in oro-gustatory perception of dietary lipids Psammomys obesus, a desert gerbil, is a unique polygenic animal model for obesity and type 2 diabetes P. obesus relative to their lean littermates P. obesus, accompanied by low liver glucose-6-phosphate hydrolysis, contributes to insulin resistant state, with a high lipogenic activity P. obesus showed a hepatic deterioration which was accompanied by enhanced oxidative stress, further contributing to deleterious outcomes of insulin resistance P. obesus are not yet known and deserve deep investigation.Several rodent models have been used to investigate the pathogenesis of obesity but they do not reflect the human disease sufficiently. P. obesus.Keeping in view the afore-mentioned arguments, it was thought worthwhile to assess the impact of obesity on lipid taste perception and calcium signaling in CD36-positive taste bud cells (TBC) isolated from circumvallate papillae of The culture medium RPMI 1640 and L-glutamine were purchased from Lonza Verviers SPRL . Fura-2/AM was procured from Invitrogen . Elastase and dispase were purchased from Serlabo  and Roche Diagnostics , respectively. Anti-CD36 antibody coupled to phycoerythrin and anti-α-gustducin antibody were procured from Santa Cruz Biotechnology Inc. (USA). Anti-CD36 antibody, used for western blots, was procured from R & D (UK). Sulfo-N-succinimidyl-oleate (SSO) was a generous gift from JF Glatz . All other chemicals including linoleic acid (18∶2 n-6), collagenase type-I, and trypsin inhibitor were obtained from Sigma Chemicals .Psammomys obesus) were trapped in the area of Beni-Abbes (30°7′ North latitude and 2°10′ West longitude) in Algerian West Sahara and transported to Algiers. When the sand rats were captured, they were subjected to acclimatization in the animal house from 15 to 30 days. The animals were maintained in suitable cages under controlled temperature and light conditions. The animals were identified for the sex. The age of male gerbils, used in our study, was approximatively from 2 to 3 months, based on the body weight. The weighing is the main selection criteria in most of the studies on these animals. The animals were weighed and, at the beginning of the experiments, their body weights were 72±5 g. Each group consisted of 10 animals. The gerbils of control group were maintained on Salsola foetida throughout the experimentation. The animals of obese group were progressively given the laboratory diet and, after a period of 4 weeks, they were completely maintained on it until the duration of the experimentation, i.e., 18 weeks.The rodents  4%, minerals 7.1%. The Salsola foetida contained the following: proteins 3.53%, lipids 0.4%, carbohydrates: 8.42%, humidity/water 80.79%, fibers (cellulose) 5.97%, minerals 6.86% ad libitum except for the taste preference tests (see here-after).For control animals, we chose the desert plant All experimental procedures were approved by the Algerian Institutional Animal Care Committee which belongs National Administration of Algerian Higher Education and Scientific Research (Algiers). The study was a part of a bilateral Franco-Algerian collaborative project “Tassili” (grant number:12MDU855). The authorization to capture the animals in desert region was given by the Ministry of Higher Education, Algeria. The animals were sacrificed by cervical dislocation.Each animal was monitored for body weight, blood glucose and insulin. For biochemical analysis, fasted animals were killed by cervical decapitation at the end of treatments, without anesthesia to avoid any further stress, and blood samples were collected in tubes containing heparin. Plasma glucose and lipids fractions were measured by a spectrophotometric method adapted on a Cobas Mira automatic analyser. Plasma immunoreactive insulin was estimated by the Phadebas insulin test. Rat insulin (Novo) was used as standard.et al.Extraction of hepatic lipids  was carried out according to Folch The experiments on the spontaneous preference for lipid-enriched solutions were performed by means of two-bottle preference test. The animals first accustomed to water drinking in two bottles for a period of 24 hrs. The next day, the animals were subjected, during the diurnal period, to two bottles: one control and one test bottle, both containing 0.3% xanthan gum (Sigma Aldrich) homogenized in water. In the test bottle, 1% of colza oil was added. The xanthan gum was used to emulsify the oil and to minimize textural cues between the two solutions. To avoid the development of side preferences, positions of the bottles were changed during each experiment. After 12 hours, consumption of each solution was analyzed by weighing the bottles and preference for the experimental solution was estimated by calculating the ratio between the consumption of the experimental solution and the total consumption (in grams). We also assessed whether obese gerbils exhibit an altered sweet preference by subjecting them, similarly, to two bottle-test: one with water and another with 4% sucrose (w/v).Psammomys obesus were isolated according to our previously published procedure 2; 1 mM MgCl2; 10 mM glucose; 10 mM Na+ pyruvate, pH 7.4). CD36-positive cells were isolated by incubating lingual epithelium in RPMI 1640 medium containing 2 mM EDTA, 1.2 mg/ml elastase, 0.6 mg/ml collagenase (type I), and 0.6 mg/ml trypsin inhibitor at 37°C for 10 minutes, followed by centrifugation . The mixture of different cell populations was incubated with anti-CD36 antibody coupled to phycoerythrin for 2 hours, followed by a wash with PBS, pH 7.4 , and resuspended in a solution containing microbeads coupled to anti-phycoerythrin IgG. The CD36-positive TBC were isolated by passing through the MACS columns of the Miltenyi magnet system. Both the cell populations, after separation, were suspended in fresh RPMI 1640 medium containing 10% fetal calf serum, 200 U/ml penicillin, and 0.2 mg/ml streptomycin, seeded onto a BD BioCoat Poly-D-Lysine-coated dishes, and cultured for 24 hours. At the end of this period, the cells were used for the experiments or stained with trypan blue to assess their viability.The CD36-positive cells from lingual CVP of http://www.ncbi.nlm.nih.gov/blast.cgi) was used for the verification of primers. Because the genes studied have not yet been sequenced for P. obesus, we designed primers based on the conserved regions in mRNA of the three well-characterized species: Homosapiens, Rattus norvegicus, and Mus musculus5′-AGAGGGAAATCGTGCGTGAC-3′, Reverse: 5′-CAATAGTGATGACCTGGCCGT-3′); CD36 ; α-gustducin . The amplification was carried out in a total volume of 25 μl, which contained 12.5 μl SYBR Green Supermix buffer  and 12.5 μl (0.3 μM) of each primer and diluted cDNA. Results were evaluated by iCycler iQ software and relative quantification of mRNA in different groups was determined.Total RNA was extracted from CD36-positive TBC by using TRIzol (Invitrogen) and subjected to DNase treatment using the RNAse-free DNAse Set (QIAGEN). One microgram of total RNA was reverse transcribed with SuperScript II RNAse H reverse transcriptase using oligo(dT) according to the manufacturer's instructions (Invitrogen). RT-PCR was performed on the iCycler iQ real-time detection system, and amplification was undertaken by using SYBR Green I detection. Primers against the genes of interest were designed using the available mRNA sequence information in NCBI Genbank. Basic local alignment search tool , cells lysates were used immediately or stored at −20°C. The protein content was determined by bicinchoninic acid . Denatured proteins (30 µg) were separated by SDS-PAGE (10%) and transferred to polyvinylidine difluoride (PVDF) membranes, and immunodetection was performed by using anti-CD36 antibodies (1∶1000 dilution). After treating the membranes with peroxidase-conjugated anti-goat secondary antibodies, peroxidase activity was detected with ECL reagents .The CD36-positive TBC, isolated from lingual CVP of control and obese animals, were lysed with 50 µl of buffer containing the following: 20 mM, HEPES pH 7.3; 1 mM, EDTA; 1 mM, EGTA; 0.15 mM, NaCl; 1%, Triton X-100; 10% glycerol; 1 mM, phenylmethylsulfonyl fluoride (PMSF); 2 mM, sodium orthovanadate and anti-protease cocktail (2 µl in 1 ml of buffer). After centrifugation (13 000 6/ml) were cultured on WillCodish wells with a glass bottom and loaded with Fura-2/AM (1 µM) for 60 min at 37°C in loading buffer which contained the following: 110 mM NaCl; 5.4 mM KCl; 25 mM NaHCO3; 0.8 mM MgCl2; 0.4 mM KH2PO4; 20 mM Hepes-Na; 0.33 mM Na2HPO4; 1.2 mM CaCl2, pH 7.4. After loading, the cells were washed three times and remained suspended in the identical buffer. The changes in intracellular Ca2+ (F340/F380) were monitored under the Nikon microscope (TiU) by using S-fluor 40× oil immersion objective. The planes were taken at Z intervals of 0.3 μm, and NIS-Elements software was used to deconvolve the images. The microscope was equipped with EM-CCD (Lucas) camera for real time recording of 16-bit digital images. The dual excitation fluorescence imaging system was used for studies of individual cells. The changes in [Ca2+]i were expressed as ΔRatio, which was calculated as the difference between the peak F340/F380 ratios. The data were summarized from the large number of individual cells . For experiments in Ca2+-free medium, CaCl2 was replaced by EGTA (2 mM).The CD36-positive TBC . Data are presented as means ± SEM. The significance of the differences between mean values was determined by analysis of variance one way, followed by a least-significant-difference (LSD) test. For all the tests, the significance level chosen was p<0.05.The gerbils fed a high caloric chow developed metabolic syndrome like features, with an increase in their body weight . The levPsammomys obesus. This increase in hepatic mass is positively correlated with hyperinsulinemia, insulin resistance, hypertriglyceridemia and hepatic triglycerides. In this investigation, we also measured the hepatic mass-body weight ratio which was increased in obese animals , the CD36 inhibitor 2+ signaling in TBC of control and obese animals , induced a rapid rise in i in TBC of control and obese sand rats; however, the decrease in Ca2+ signaling was more pronounced in TBC of obese animals than that in controls. Whether LA induces the opening of store-operated Ca2+ (SOC) channels in 100% Ca2+ medium, we employed 2-aminoethoxydiphenyl borate (APB), a blocker of these channels. 2+ signaling in TBC of control and obese animals.In order to assess the implication of intracellular Ca2+-ATPase (SERCA) in mammalian cells. Over the past ten years, this guaianolide compound of plant origin has become a popular tool in most of studies directed at elucidating the implication of Ca2+ that belongs to endoplasmic reticulum, ER 2+ refilling process and, therefore, induces increases in [Ca2+]i.Thapsigargin (TG) is the most widely used inhibitor of the ubiquitous sarcoplasmic-endoplasmic Ca2+]i were lesser in obese animals than those in controls . The gerbils also exhibited a spontaneous preference for a sweet solution which was very high in obese group of animals. The molecular mechanisms of sweet preference and the implication of lingual receptors will be a subject of intensive investigation in the near future.Interestingly, increased hepatic mass was positively correlated with weight gain in obese Salsola foetida contains only 0.4% of them. It has been shown that foods high in dietary fat exert a weak effect on satiation, which leads to a form of passive overconsumption P. obesus, in a two-bottle test, clearly showed a spontaneous preference for a solution, containing colza oil.The CD36, a lipido-receptor, has been implicated in lipid taste perception in rodents 2+]i in CD36-positive TBC purified from mouse CVP 2+]i in TBC via CD36, and it was higher in obese animals than control rodents. A plausible explanation on the increase in Ca2+ signaling in obese animals is not available. It is possible that other proteins like GPR120 2+ signaling as SSO 2+]i in these cells. However, the implication of GPR120 in Ca2+ singling in these cells remains to be assessed later on. We tried to detect GPR120 in the TBC of P. obesus in western blots, by using antibodies form Abcam (ab-75313) and Santacruz (sc-48203), but without success, and it may be due to species different. In future, we will employ anti-GPR120 antibodies from other sources. Alternatively, it is also possible that altered composition of plasma membrane phospholipids of CD36-positive TBC in obese animals might be responsible for altered downstream signaling, involving the hydrolysis of phosphatidylinositols, but this hypothesis remains to be ascertained in future.We have shown that LA, by binding to CD36, triggers an increase in [Ca2+ signaling in 0% Ca2+ medium in TBC of control and obese animals show that this fatty acid evokes increases in [Ca2+]i from intracellular pool, probably from endoplasmic reticulum (ER) as demonstrated in mouse CD36-positive TBC 2+ homeostasis, the depletion of intracellular stores of Ca2+ is followed by Ca2+ influx via the opening of Ca2+ channels to refill the intracellular pools 2+ (SOC) channels, and their activity is maintained as long as the stores are not refilled 2+ from ER results in the opening of SOC channels, we employed a SOC channel blocker, which as anticipated, decreased significantly the LA-induced calcium response in 100% Ca2+ buffer in TBC of control and obese animals. In order to probe the implication of ER pool, we employed SERCA inhibitor and observed that TG-induced increases in [Ca2+]i in TBC of obese animals were lower than those in controls. These observations confirm the above-mentioned results on LA-induced decreases in [Ca2+]i in 0% Ca2+ buffer in TBC of obese animals, indicating a decreased recruitment of Ca2+ from the ER pool. As expected, TG also opened SOC channels both in control and obese TBC. The modification of opening of SOC channel is a specific physiological feature in these animals, as the action of ionomycin, known to open non-specifically almost all the Ca2+ channels, was not significantly different between TBC of control and obese P. obesus.However, a decrease in LA-induced Cain vivo calcium homeostasis, these animals possess regulatory adaptive mechanisms. It has been shown that plants of chenopodiacae family provide with much oxalate (300 mg/d), but little Ca2+ (30 mg/day); whereas the laboratory diet provides with low-oxalate (<100 mg/day) and high-Ca2+ (approximately 150 mg/day). P. obesus has the ability to eliminate dietary oxalate and regulate Ca2+ homeostasis via intestinal symbiotic bacteria whose number increases or decreases according to the diets As regards P. obesus is associated with increased oro-gustatory perception of dietary fat and upregulated Ca2+ signaling in circumvallate papillae, associated with altered CD36 expression. Future challenges require in-depth investigations to better understand the implication of GPR120 or the downregulation/distribution of CD36 in plasma membrane fractions of TBC during obesity since the mechanisms responsible for altered Ca2+ signaling might be involved in the regulation of feeding behavior particularly post-prandial phase of food ingestion.In conclusion, we report in this study, for the first time, that obesity in


Stents are recommended in patients with dysphagia caused by esophageal stricture, but an ideal stent does not currently exist. Thus, studies on new esophageal stents are necessary, and suitable animal models are desperately needed for these studies. The aim of this study was to establish a model of malignant esophageal stricture in rabbit for studies on stent innovation.A total of 38 New Zealand white rabbits were used in this study. Using the endoscopic submucosal injection technique, VX2 fragments were inoculated into the submucosal layer of the rabbit thoracic esophagus, and an endoscopic follow-up was subsequently performed to observe the tumor development and progression. The self-expandable metal stents were randomly deployed in rabbits with severe esophageal stricture to investigate the safety and feasibility of the animal models for stenting.P = 0.016).An endoscopic implantation procedure for VX2 tumors was completed in 34/38 rabbits, and tumor development was confirmed in 30/34 animals. The success rate of the endoscopic implantation and tumor development were 89.4%  and 88.2%  respectively. During the endoscopic follow-up period, severe esophageal stricture occurred in 22/30 rabbits with a rate of 73.3% , and 12/22 models received stent placement. During and after stent implantation, no severe stent-related complication or mortality occurred in the animal models. The rabbits that received stent placement survived longer than those without stent implantation (the mean survival time: 53.9 days versus 40.3 days, The endoscopic method is a safe and effective method for establishing a malignant esophagostenosis model in rabbits. This model can simulate the human body environment for stent deployment and is an excellent tool for the study of stent innovation for the treatment of esophageal cancer. Esophageal cancer (EC) is the sixth leading cause of cancer-related mortality and the eighth most common cancer worldwide ,2. TreatTheoretically, a good animal model of EC for stenting should be able to mimic the esophagostenosis features of the human disease and be sufficiently large to perform minimally invasive procedures designed for humans. However, there is currently a severe lack of an excellent animal model of EC to study stents -9.The VX2 tumor model, which was originally proposed by Shope and Hurst  in 1933,Here, we introduce an endoscopic method for the development of a rabbit model of malignant esophagostenosis, and further investigate the safety and feasibility of stent implantation in this model. This is the first study where rabbits with esophageal malignant tumors were used for stent development procedures.Male or female New Zealand White rabbits weighing between 2.5 and 3.0 kg were obtained from The Jiangsu Agricultural Academy of Science in China and food and water were provided ad libitum. All experimental procedures were approved by the Animal Care and Use Subcommittee at Nanjing Medical University.3) that, were previously stored in liquid nitrogen were injected deep into the hind limb of gluteal muscles of rabbits anesthetized using an intramuscular injection of 35-mg/kg pentobarbital sodium . Three weeks after implantation, the animals were sacrificed and the hind limb tumors were harvested and immediately processed. All VX2 tumors were cleaned from the surrounding tissue and gross necrotic portions of the tumors were removed.Rabbits with hind limb tumors (donor) were used to propagate and maintain VX2 tumors. Approximately 2 to 3 pieces of thawed VX2 tumor tissue fragments (approximately 0.5 mm3) and preserved in saline for fragment implantation. approximately 0.3 ml saline solution with 4 pieces of tumor fragments were placed in a 2-ml syringe on ice until they were injected into the recipient rabbit’s esophagus.The collected tumors were cut into small pieces  was inserted into the thoracic esophagus. Using a fine endoscopic needle , 0.5 ml saline was injected into the submucosal layer to elevate the mucosa layer. Next, a puncture was made using a modified endoscopic needle  across the mucosa into the submucosal layer and the success of the puncture was confirmed by further elevation of the mucosa when saline was injected. About 0.3 ml saline containing 4 tumor pieces was then injected into the submucosal layer of rabbits esophagus  was designed and manufactured , and its partial length was covered with a silicone membrane Figure . Using XThe survival time of all rabbits demonstrated severe esophageal stricture was recorded, independent of the status of stent placement. The rabbits were fed separately in cages to daily observe the quantity of food and water. Paste food or liquid food (vegetable juices) were provided for animals with dysphagia or a 50% reduction of food intake. A humane endpoint was used in this study, in which the animal models were humanely euthanized when they could not have any food or water for several days and their health was extremely weak, such that they could not stand. For the euthanasia procedure, the animal was first anesthetized via an intramuscular injection of 35-mg/kg pentobarbital sodium using the ear venous, and then with a injection of 30 ml air.P < 0.05 level. All statistical tests were performed with the SPSS 16.0 software.We calculated the success rate of tumor production, inclouding that of endoscopic implantation and tumor development. The rate of severe esophageal stricture and the rate of immediate and late complication of stents placemen were also investigated. The Kaplan-Meier method with the log rank test was used to analyze the survival of rabbits with or without stents. The statistical significance was set at the A total of 38 rabbits were used in this study. Thirty-four rabbits were successfully implanted with VX2 fragments in the esophagus and the success rate of endoscopic implantation was 89.4% . Four rabbits were removed from the study because of the esophageal perforations during the endoscopic implantation and two injured rabbits (5.3%) died after conservative treatments. After implantation, tumor development was confirmed  in the survival time between the two groups (Figure The longest survival time of rabbits with stent implantation was 74 days and the shortest survival time was 28 days with a mean survival time of 53.9 ± 13.4 days. However, the longest survival time of rabbits without stents deployed was 59 days, and the shortest survival time was 24 days with a mean survival time of 40.3 ± 11.3 days, which was significantly different P = 0.01 in the sDespite having a higher success rate of tumor growth ,16 compaEsophageal stenosis is a predominant pathological feature of EC in the clinic because it promotes rapid deterioration and death ,20. In tIn many preclinical studies, large-sized animal models, such as the dog and pig, have been generally used for stenting, but these animals were not under disease conditions, despite the fact that they could simulate the human body environment for esophageal stent deployment. Thus, they were only used to assess the safety of stenting -23. FurtThe overall complication rate and primary success rate of self-expanding esophageal stenting in clinical research were approximately 30% and 97.2%, respectively ,28. In tThe limitation of our animal model is that it is an orthotopic allograft tumor model and cannot be maintained the routine course of esophageal cancers. This study focused on whether the animal model is suitable for stent implantation and may neglect other features and utility of this animal model.In conclusion, the endoscopic method is safe and effective for the development of a rabbit model of malignant esophagostenosis to mimic the progression of this human disease. This model can simulate human the body environment for stent deployment and is an excellent tool for studying stent innovation for the treatment of esophageal cancer.The authors declare that they do not have any competing or financial interests.ZF, JH, JS, XW and GX conceived, designed and performed the experiments. YZ, XT, YS, HS and ZL analyzed the data and wrote the paper. All authors read and approved the final manuscript.Endoscopic method for implantation of VX2 tumors.Click here for file


The dynamic interplay among intracellular organelles occurs at specific membrane tethering sites, where two organellar membranes come in close apposition but do not fuse. Such membrane microdomains allow for rapid and efficient interorganelle communication that contributes to the maintenance of cell physiology. Pathological conditions that interfere with the proper composition, number, and physical vicinity of the apposing membranes initiate a cascade of events resulting in cell death. Membrane contact sites have now been identified that tether the extensive network of the endoplasmic reticulum (ER) membranes with the mitochondria, the plasma membrane (PM), the Golgi and the endosomes/lysosomes. Thus far, the most extensively studied are the MAMs, or mitochondria associated ER membranes, and the ER-PM junctions that share functional properties and crosstalk to one another. Specific molecular components that define these microdomains have been shown to promote the interaction in trans between these intracellular compartments and the transfer or exchange of Ca Although still little is known about their lipid and protein compositions and refined mode of action, the best studied membrane contact sites are those that are formed between the vast membrane network of the endoplasmic reticulum (ER) and the membranes of other cellular compartments, particularly the plasma membrane (PM) and the mitochondria. Common functions of these membrane contact sites are the regulation of Ca2+ dynamics that assures the homeostasis of intracellular Ca2+ concentration, and the synthesis/shuttling of lipids and lipid intermediates, i.e., phospholipids and (glyco)sphingolipids, such as ceramide and GM1-ganglioside (GM1). There is now evidence that these functions of membrane contact sites can be interrelated, given that changes in lipid content and concentration may alter the activity of Ca2+ binding proteins and Ca2+ channels/pumps. Cellular Ca2+ signals are initiated primarily with a rapid increase in cytosolic Ca2+ concentration that in turn controls downstream cellular processes, such as gene transcription, cell proliferation and metabolism, and neural transmission [2+ signaling, specifically at membrane contact sites, may put the cell in a state of stress and ultimately lead to cell death. A characteristic aspect of the eukaryotic cell is the complex network of intracellular compartments or organelles with distinct biological functions and delimited by a lipid bilayer that insulates their contents from the cytoplasm. Although seemingly static in appearance on electron micrographs and acting independently from one another, organelles are in a state of constant remodeling and communication, which is often achieved at tethering sites between their individual membranes, where two engaged organelles become closely juxtaposed without undergoing fusion ,2. It issmission ,8,9. Dis2+ store compartments, and on their cross talk with the PM. We will discuss how their membrane topology and composition, especially in relation to the amount of GM1-ganglioside, affects Ca2+ signaling and in turn controls cell fate.In this review, we give a condensed overview of the structural and molecular aspects of interorganellar membrane microdomains, focusing primarily on those formed between the ER and the mitochondria, as major Ca2+ flux at these two juxtaposed membrane compartments is crucial for excitation-contraction coupling. Initially thought to be present exclusively in such specialized cells like muscle cells, ER-PM junctions, also known as PAMs for PM associated ER membranes [2+ channel Orai1 or CRAC (Ca2+ release activated Ca2+ channel) in the store operated Ca2+ entry or SOCE pathway  motifs that appear to bind phosphoinositide lipids at the PM side [ et al. [In cardiac and skeletal muscle cells analogous sarcoplasmic reticulum (SR)-PM junctions have been implicated in the process that allows the transmission of signals from the PM to the SR and the local release of Ca PM side . Further PM side . Using q[ et al.  have rec[ et al.  and cont[ et al. . In addi2+ signaling, energy metabolism, cell survival and apoptosis. Structural and functional analogies between the MAMs and the ER-PM junctions suggest a strict interplay between these cellular compartments, especially as it pertains to Ca2+ dynamics and lipid shuttling.The most extensively characterized interorganellar membrane contact sites from a structural, biochemical and functional standpoint are those at the interface between the ER and the mitochondria, defined as MAMs or mitochondria associated ER membranes . These mMorphological evidence for the physical juxtaposition between ER and mitochondria emerged already in the 1960s  but it wThe MAMs are also enriched in enzymes necessary for cholesterol and steroid biosynthesis , as well2+ stores and function synergistically to maintain the cytosolic ion concentration in the nanomolar range for the proper physiology of the cell. As primary site of Ca2+ storage, the ER controls Ca2+-dependent physiological processes, such as post-translational protein folding, protein maturation and membrane stability [2+ in a process that is essential for mitochondrial bioenergetic activity [2+ from the ER to the mitochondria at the opening of the inositol 1,4,5-triphosphate (IP3)-sensitive Ca2+ channel [2+ either remains local or that Ca2+ waves dissipate from neighboring to distant mitochondria, thereby avoiding mitochondrial Ca2+ overload [ER and mitochondria are the major intracellular Catability ,26. On tactivity ,28,29,30 channel ,32 This overload ,34,35,362+ by the mitochondria occurs at the expenses of an electrical potential across mitochondrial membranes [2+ uniporter, the main transporter for mitochondrial Ca2+ uptake has now been found to consist of a complex of at least two proteins: MICU1 , which has properties of a Ca2+ uniporter or regulates its activity [2+ uptake into the mitochondria, without impairing mitochondria respiration or affecting mitochondria membrane potential. On the other hand, overexpression of MCU increases Ca2+ uptake while silencing of the protein reduces mitochondrial Ca2+ intake [The uptake of Caembranes ,37. Origactivity , and theactivity ,40. Sile+ intake ,40.2+ acts as an important cofactor that activates and regulates Ca2+-dependent dehydrogenases of the Krebs cycle for maintenance of mitochondrial respiration and ATP production [2+ receptors on the ER membranes or can be used by the ER Ca2+ ATPases to pump Ca2+ back into the ER. The discharge of Ca2+ into the cytosol, instead, follows an ion gradient and is mediated by the antiporter Letm1 (Leucine zipper-EF-hand containing transmembrane protein 1) [2+ levels are linked to different forms of cell death and mitochondria are primary effectors of the intrinsic apoptotic process, it is conceivable that disturbance of the MAMs homeostatic control of Ca2+ buffering between ER and mitochondria constitutes a potent molecular activator of apoptosis. Once in the mitochondria, Caoduction ,30. ATP otein 1) ,41. As C2+ transfer from the ER to the mitochondria [Specific ER and mitochondrial proteins co-localize at the MAMs, but the molecular mechanism(s) by which tethering between the two organellar membranes is achieved is not yet fully elucidated. In some instances, it might depend on the interaction of proteins and lipids located at the ER and the mitochondrion sides, which physically connect the membranes of the two organelles ,42. For chondria ,43. 2+ transfer from the ER to the mitochondria [2+ concentration at the MAMs, triggering an apoptotic cascade when deregulated [Additional proteins that have been found to localize at the MAMs include the chaperones Sigma-1R (receptor), calnexin, BiP and calreticulin, the trafficking molecule Rab32, the parkinson disease-related protein DJ-1 and the multifunctional sorting protein PACS-2 (phosphofurin acidic cluster sorting protein-2) ,46,47,48chondria . Intereschondria ,52,53,54egulated . Further2+ concentration at the MAMs also controls the physical distance between the ER and mitochondrial membranes [2+ from the ER that arrests the movements of neighboring mitochondria, thus easing their close opposition to the ER membrane, which favors mitochondrial Ca2+ buffering. It is noteworthy that Caembranes ,35,56. T2+ flux. The latter has been demonstrated by the use of synthetic linkers that artificially tethered the two organelles [2+ overload, whereas increasing the distance impairs mitochondrial function resulting in cell death [Going full circle, the distance between the two juxtaposed membranes at the MAMs, in turn, regulates the extent of Caganelles . Decreasll death . 2+ flux at the MAMs [2+ overload and mitochondria-mediated cell death [2+ flux [The only ER-mitochondria bona fide tether discovered so far is Mfn2 (mitofusin-2), a dynamin-related protein and potent effector of mitochondrial fusion, which is mutated in the Charcot-Marie-Tooth IIa disease . Mfn2 isthe MAMs . In agrell death . Togethe[2+ flux ,59. 2+ transfer between these organelles, underscores the cooperation of lipids and proteins in regulating physiological pathways at membrane contact sites that favor either cell survival or cell death [Although it is assumed that ER and mitochondrial membranes are linked primarily by trypsin-sensitive structures, implying an exclusively protein-based connection, there is now growing evidence for the combined role of proteins and lipids, specifically cholesterol and glycosphingolipids (GSLs), in enabling the formation of the MAMs, hence influencing their physiological function(s) ,60. Cholll death . Gangliosides comprise a class of sialic acid-containing GSLs, which occur ubiquitously in vertebrate cells. They are major constituents of the PM and are also present in the nuclear envelop ,65,66. T2+ concentration in intracellular stores [+/Ca2+ exchanger (NCX) isoform present in the inner nuclear membrane by tightly associating with it [2+ from the nucleoplasm to the nuclear envelope lumen and in turn to the ER, which constitutes an alternative route to that controlled by the ER Ca2+ pump SERCA for maintaining Ca2+ homeostasis [2+ transfer from the nucleoplasm to the ER. In addition, cerebellar granule neurons isolated from mice deficient for the GM2/GD2 synthase  that lack complex gangliosides, including GM1, showed elevation of intracellular Ca2+ under depolarizing levels of potassium, which is associated with extensive apoptosis [2+ concentration [Among the GSLs, GM1 is the only ganglioside that has been experimentally shown to modulate Car stores ,72,73,74r stores ,76,77. S with it ,79. Thes with it . This toeostasis ,81. In fpoptosis . Remarkantration .2+ signaling. These, and our own, studies have now established the importance of maintaining physiological levels of GM1 in cell membranes, particularly neuronal membranes, to avoid the initiation of an apoptotic cascade downstream of deregulated Ca2+ homeostasis [β-gal  [−/−β-gal mice have demonstrated that impaired lysosomal degradation of GM1 in neurons is accompanied by the abnormal redistribution of the ganglioside from the PM to ER membranes. Accumulation of GM1 at this site causes depletion of the ER Ca2+ store, which effectively activates the UPR, initially as survival mechanism. However, sustained activation of the UPR molecular effectors due to continuous GM1 buildup, and hence release of ER Ca2+, ultimately triggers a UPR-mediated apoptotic process [Increased levels of GM1 above its physiological threshold are as deleterious for neurons as the complete lack of the ganglioside, and activate a cell death pathway dependent on loss of Caeostasis ,74. Pathosidase) . The disosidase) ,86. Init process ,74.−/−β-gal mouse brains revealed the presence of an increased number of ER vesicles tethered to mitochondria, compared to the number of this vesicles found associated to wild-type mitochondria  in the −/−β-gal GEMs, and the co-precipitation of P-IP3R1 with GM1 suggest a physical and functional interaction between these two molecules. Concomitantly, the levels of VDAC-1 and GRP75 were also higher in the −/−β-gal GEMs than in the wild-type samples, supporting the notion that increased levels of GM1 promote the tethering of the ER-mitochondrial membranes at the GEMs. In these lipid-rich microdomains, high concentration of GM1 likely alters the dynamics of the lipid microenvironment, favoring the segregation or the clustering of an active IP3R-1 along with VDAC1 and GRP75, and promoting the formation of a Ca2+ mega-pore. This allows for a more sustained diffusion of Ca2+ into the mitochondrial matrix, which disrupts the bioenergetic activities of the organelle, and activates the mitochondrial leg of the intrinsic apoptotic pathway [2+ overload into the mitochondria in −/−β-gal cells and brain tissue [c), and formation of the apoptosome, which signs the onset of programmed cell death. The fact that GM1-mediated PTP opening and MMP are antagonized by PTP inhibitors and Ca2+ chelators, and rescued in cells double deficient for −/−β-gal and cyclophilin D, a structural component of the PTP, suggest that GM1 acts directly at the pore level in a Ca2+-dependent manner [GEMs are dynamic microdomains present both in the PM and intracellular membranes. They share several protein components with the caveolae and the lipid rafts, and have been proposed to function as signaling platforms by facilitating the clustering of proteins as well as protein–protein interactions ,89. This pathway . Mitocho pathway ,92, haven tissue . Openingt manner . −/−β-gal cells with MBCD (methyl β-cyclodextrin), which efficiently extracts GM1 from the MAMs, rescues opening of the PTP, dissipation of the potential, and apoptosis [2+ flux at the MAMs/GEMs and in Ca2+-dependent apoptosis.The mitochondrial damage that ensues upon abnormal GM1 accumulation in the GEMs also underscores the importance of these microdomains in maintaining a normal electrochemical gradient across mitochondrial membranes. In fact, treatment of poptosis . A simil2+ regulation in excess of GM1, it is tempting to speculate that an abnormal concentration of GM1 at the ER-PM junctions may also impact on the structure and function of Ca2+ channels at these Ca2+ microdomains. This could affect homeostatic Ca2+ flux between the PM and the cytosol, or the PM and the ER, and hence the physiological refill of ER Ca2+ stores.It is conceivable that the sequence of events that leads to GM1 buildup at the MAMs/GEMs and ER-mitochondrial apoptosis begins with abnormal levels of GM1 at PM, the site where the ganglioside is primarily localized under physiological conditions. In fact, a question that still remains unanswered is how GM1 redistributes to the ER membranes and hence to the MAMs/GEMs. One hypothesis is that it is transported from the PM to intracellular membranes via caveolae or lipid rafts. Supporting evidence for this is the fact that GM1, as receptor for cholera toxin at the PM, is internalized via caveolar-/non-caveolar- or clathrin-mediated endocytosis and transported to the Golgi compartment and to the ER membrane . A seconi.e., Mfn2) that specify membrane contact sites may regulate both processes in response to certain physiological or pathological cues. Future studies in this area of research promise to uncover new aspects of cell biology that are orchestrated by interorganellar membrane microdomains. It is now widely accepted that intracellular compartments are in constant interaction and communication with one another. This interplay between organelles occurs via membrane tethering sites that come in close vicinity in trans and facilitate the exchange or transfer of molecules and ions to preserve the physiological status of a cell. Membrane microdomains need to acquire or maintain a specific topology and lipid/protein composition in order to be fully active. In contrast, pathological conditions that interfere with their structure and/or function result in cell death. Intriguingly, different organellar membranes may come in close apposition to tether or fuse. We are now beginning to understand that some of the molecular players (


Dephosphorylated PLN inhibits SERCA2a and PLN phosphorylation, at either Ser16 by PKA or Thr17 by Ca2+-calmodulin-dependent protein kinase (CaMKII), reverses this inhibition. Through this mechanism, PLN is a key modulator of SR Ca2+ uptake, Ca2+ load, contractility, and relaxation. PLN phosphorylation is also the main determinant of β1-adrenergic responses in the heart. Although phosphorylation of Thr17 by CaMKII contributes to this effect, its role is subordinate to the PKA-dependent increase in cytosolic Ca2+, necessary to activate CaMKII. Furthermore, the effects of PLN and its phosphorylation on cardiac function are subject to additional regulation by its interacting partners, the anti-apoptotic HAX-1 protein and Gm or the anchoring unit of protein phosphatase 1. Regulation of PLN activity by this multimeric complex becomes even more important in pathological conditions, characterized by aberrant Ca2+-cycling. In this scenario, CaMKII-dependent PLN phosphorylation has been associated with protective effects in both acidosis and ischemia/reperfusion. However, the beneficial effects of increasing SR Ca2+ uptake through PLN phosphorylation may be lost or even become deleterious, when these occur in association with alterations in SR Ca2+ leak. Moreover, a major characteristic in human and experimental heart failure (HF) is depressed SR Ca2+ uptake, associated with decreased SERCA2a levels and dephosphorylation of PLN, leading to decreased SR Ca2+ load and impaired contractility. Thus, the strategy of altering SERCA2a and/or PLN levels or activity to restore perturbed SR Ca2+ uptake is a potential therapeutic tool for HF treatment. We will review here the role of CaMKII-dependent phosphorylation of PLN at Thr17 on cardiac function under physiological and pathological conditions.Phospholamban (PLN) is a phosphoprotein in cardiac sarcoplasmic reticulum (SR) that is a reversible regulator of the Ca During a normal excitation-contraction-coupling cycle (ECC), Ca2+ enters the cell through the L-type Ca2+ channels leading to activation of the ryanodine receptors (RyR2) in the SR and release of Ca2+ from this membrane system. This Ca2+-induced-Ca2+-release mechanism  and to a lesser extent by the Na+/Ca2+ exchanger (NCX). Thus, the SR is the major regulator of Ca2+-handling during the cardiac excitation-contraction-relaxation cycle  is altered Caechanism  underlieon cycle .2+-affinity of SERCA2a (16 by cyclic AMP (cAMP)- and cGMP-dependent protein kinases, Thr17 by Ca2+-calmodulin-dependent protein kinase II (CaMKII), and Ser16 by protein kinase C , an SR associated protein . PLN is  SERCA2a . PLN cankinase C . Phosphot hearts , cGMP pht hearts . Howevermulation . Dephospmulation , reverse16 and phosphorylation enhanced SERCA2a activity and Ca2+-uptake (2+-CaM-kinase (CaMKII) at Thr17 and this phosphorylation occurred independently of its PKA-phosphorylation (2+-transport through an increase in the apparent affinity of the SERCA2a for Ca2+ (KCa). Thus, it was initially proposed that phosphorylated PLN acts as a stimulator of cardiac SERCA2a activity. However, in the late 1980s and early 1990s, there were two significant breakthroughs: (a) in vitro studies of reconstituted SR membrane systems n of PLN , which dn of PLN , has led16 and Thr17 phosphorylation sites. Interestingly, phosphorylation of PLN diminishes its binding to HAX-1, indicating that this interaction may be physiologically relevant in the heart , which is ubiquitously expressed in mitochondria and SR. HAX-1 physically interacts with PLN and the binding region of PLN includes amino acids 16–22 with both Serhe heart . Indeed,hibition .2+-cycling and cardiac function. PP1 is a negative regulator of PLN activity through its dephosphorylation and increased inhibition of SERCA2a. Interestingly, the type 1 enzyme is modulated by its endogenous inhibitors, Inhibitor-1 (I-1) and Inhibitor-2 (I-2). Inhibitor-1 gets activated upon its PKA phosphorylation at Thr-35 resulting in potent inhibition of PP1 activity and amplification of the β1-adrenergic receptor stimulatory effects .Besides HAX-1, other regulatory proteins such as PKA, CAMKII and PP1 are also associated with PLN, achieving an efficient and compartmentalized complex that regulates SR Ca effects . More re effects . Thus, tFigure 2). PKA then phosphorylates several proteins in the cardiac myocytes to induce positive chronotropic, inotropic, and relaxant effects, the so-called “fight or flight response,” which is considered the most effective mechanism to acutely increase cardiac output. The underlying phosphoproteins include PLN and RyR2 at the SR level, the L-type Ca2+ channel and phospholemman, at the sarcolemma level, and troponin I (TnI), C protein, and myosin light chain, at the level of the myofibrils , Ser16 → Ala mutant PLN (PLN-S16A) or Thr17 → Ala mutant PLN (PLN-T17A) in the cardiac compartment of PLN knockout mice, indicated that the phosphorylation of Ser16 of PLN is a prerequisite for the phosphorylation of Thr17 , decreased SR Ca2+-content without changes in the myocyte response to isoproterenol . A similar conclusion should hold true for the different Ca2+ handling proteins which are phosphorylated by both kinases, like L-type Ca2+ channels or RyR2. Interestingly, sustained β1-adrenergic receptor stimulation enhanced cell contraction and Ca2+ transients by a mechanism which is largely PKA-independent but sensitive to CaMKII-inhibitors. In these studies, a shift from Ser16 to Thr17 phosphorylation pathway was observed, underscoring the role of CaMKII during prolonged β1-adrenergic stimulation  most of the isoproterenol-induced PKA-independent Ca2+ leak enhancement is mediated by Epac; and (2) the contribution of Epac to isoproterenol–induced SR Ca2+ reuptake through PLN phosphorylation, is very modest, if any, since no relaxant effects of isoproterenol could be detected after PKA inhibition.The role of CaMKII-phosphorylation of PLN was also addressed in a model with expression of a CaMKII inhibitory peptide targeted to the longitudinal SR  and phosphatase activities appears to regulate phosphorylation of Thr17 and Ser16 in PLN.As indicated above, several studies showed that CaMKII-dependent PLN phosphorylation can only occur in the intact beating heart in the presence of ß1-adrenergic stimulation, while it occurs independently of cAMP-PKA activation rylation . These f17 phosphorylation of PLN can occur in the absence of prior Ser16 phosphorylation , a fundamental physiological modulator of myocardial performance, is another example in which Thrrylation . These factivity , and ind17 phosphorylation of PLN on FDAR was also questioned on the basis of three main findings: (1) FDAR precedes the phosphorylation of Thr17 site of PLN  Mos SERCA2a , while F+ uptake ; and (3)+ uptake , althoug+ uptake .Taken together, the underlying molecular steps that encompass the FDAR process are currently unclear. Although most of the experimental evidence indicates that CaMKII is involved in FDAR, some studies have challenged this possibility and the participation of PLN in FDAR. Thus, it is likely that several rather than a single mechanism, are associated with this phenomenon.An understanding of how pH changes alter cardiac function is important for a better comprehension of some cardiac pathological situations, which are important in the clinical setting. Myocardial ischemia is particularly relevant along these lines: in human, acidosis can be detected 15 s after the occlusion of the coronary artery and is a major mechanism for the loss of contractility during ischemia . Substan2+ responsiveness , by increasing cytosolic Na+ and then Ca2+ through the NCX, was sufficient to overcome the inhibitory effect of acidosis on SERCA2a, increasing SR Ca2+ and intracellular Ca2+ transients . A major clue supporting this possibility was given by experiments showing that Ca2+ and contractile recovery during acidosis require an intact SR and CaMKII activity, suggesting that CaMKII-dependent phosphorylation at the SR level is involved in the recovery mechanism  Ca2+ and the inhibition of phosphatase induced by acidosis . Interestingly, experiments by 2+ and mechanical recovery during acidosis. However, the effects of PLN ablation mimic maximal PLN phosphorylation. Therefore the results of 2+ reuptake during acidosis is beneficial, favoring mechanical recovery as discussed above, or harmful, hindering it. An explanation to these apparent contradictory results may lie on the fact that intracellular Ca2+ and mechanical recovery during acidosis require an increase in SR Ca2+ uptake above steady state, a condition that cannot be accomplished in PLNKO mice in which basal SR Ca2+ uptake is already at maximal levels. In line with DeSantiago’s results, Acidosis produces a rapid decrease in the strength of contraction , which isiveness . This dele force . Earlieransients . Howeverrecovery , indicatechanism . Indeed,acidosis . This phacidosis . Thus, Cacidosis . These i2+ load during acidosis, responsible for the mechanical recovery, may also increase spontaneous SR Ca2+ release and produce extra-systoles . Together, these results indicate that post-acidosis CaMKII-dependent DADs are triggered by two concurrent factors: (1) acidosis-induced increase in SR Ca2+ content; and (2) relief of RyR2 and NCX, previously inhibited by acidosis.The increase in SR Casystoles . Moreoveacidosis . The retacidosis , favorin2+ and contractile recovery that occurs during stunning, a fully reversible post-ischemic dysfunction . In contrast, when SR Ca2+ reuptake is highly enhanced by ablation of PLN, post-ischemic recovery of contractile function was negligible .The role of CaMKII in I/R will be addressed in detail elsewhere in this issue. We will briefly refer here to the role of CaMKII-dependent PLN phosphorylation in this pathological situation. In the last few years, a dual effect of CaMKII-dependent protein phosphorylation  has been described in the scenario of I/R in the intact heart. The beneficial effect of CaMKII usually refers to the intracellular Cafunction . Initialerfusion . Furthered heart . These fgligible . A possiorylated . These rAs will be discussed below, this unbalance may constitute a major cause of the detrimental effect of CaMKII activation in the irreversible I/R.2+ during ischemia and reperfusion leads to mitochondrial Ca2+ accumulation, which is greatly favored by the close association between mitochondria and SR and constitutes a main event in the initiation of cell death , alleviated post-ischemic cardiac injury .Experimental evidence consistently indicates that CaMKII-inhibition is protective in the irreversible I/R injury . Althougerfusion . This fic injury , supportd in I/R , compatid in I/R , that wod in I/R . Moreovek in I/R . These aniporter . Taken t2+ handling a main cause of these two characteristic alterations. Indeed, there is evidence supporting a decrease in intracellular Ca2+-transient and diminished SR Ca2+ content, an outcome that constitutes the major origin of the altered contractility in HF  due to PLN phosphorylation, would enhance RyR2 opening due to the regulatory effect of intra-SR Ca2+. However, in the absence of additional RyR2 modifications, the increased SERCA2a activity, produced by PLN phosphorylation, may cope with the enhanced diastolic SR Ca2+ release/leak. In contrast, even moderate increases in SR Ca2+ may increase diastolic SR Ca2+ release under conditions where RyR2 activity is altered independently of intra-SR Ca2+-induced modifications, enhancing the propensity to arrhythmias and leading to mitochondrial Ca2+ overload, which favors apoptosis and necrosis. Thus, increasing SERCA2a activity by PLN phosphorylation seems to have the potential of producing salutary effects in a number of diseases, as long as these effects are achieved under conditions in which diastolic Ca2+ release is satisfactorily controlled. Future research in this area is needed to parse the contribution of different players involved in the balance/interaction between SR Ca2+ reuptake and leak, including the RyR2 regulators or the L-type Ca2+ channels. More specifically to the subject of this review, the recently described multimeric SERCA/PLN-ensemble may represent a nodal point in the interaction of several protein partners, regulating and modifying the fine-tuned control of Ca2+ cycling achieved by the duo SERCA-PLN. An intensive scrutiny of the various proteins of this new pathway will give new insights into their role in SR Ca2+ uptake control and may provide novel therapeutic avenues which can contribute to solve the abnormalities in Ca2+ handling underlying different pathological process.We have described that PLN and its CaMKII-dependent phosphorylation are part of a multimeric functionally coupled signaling complex, composed of SERCA, PLN, HAX-1, PKA, CaMKII, PP1, I-1, and Hsp20, which reversibly regulates SR CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


It provides dynamic regulation of a vast array of gene transcriptions, protein kinases, transcription factors and other complex downstream signaling cascades. For the past six decades, intracellular Ca2+ concentration has been significantly studied and still many studies are under way. Our understanding of Ca2+ signaling and the corresponding physiological phenomenon is growing exponentially. Here we focus on the improvements made in the development of probes used for Ca2+ imaging and expanding the application of Ca2+ imaging in plant science research.The calcium ion (Ca For the past six decades, calcium signaling has been a focus of study with a level of investigation higher than that of any other signaling molecule. In plants, numerous endogenous stimuli and stress signals of both biotic and abiotic nature lead to transient variation in intracellular Ca2+ concentration, which in turn activate respective downstream signaling cascades [2+ requires the ability to monitor the dynamics of its concentration in plant cells with both spatial and temporal accuracy [Knowledge of Cacascades –6. Studyaccuracy –9.2+ ([Ca2+]cyt) is maintained in the nano molar range (100–200 nM) while in many organelles it may reach micro molar to mili molar concentrations [2+ concentration [2+ dynamics at micor molar level in ER lumen and at nM level in mitochondria and peroxisomes [2+]cyt induced by various stimuli determine the final functional outcome [2+]cyt is largely dependent on the development of methodologies that can be used to accurately measure [Ca2+]cyt cyt ,22.2+ probes from the past six decades and the improvements that have been developed in this field. We also discuss the limitations of each probe and important points to consider while planning ideal Ca2+ imaging experiments in plant science.Here, we describe the development of various Ca2.in situ characterization of the 3D architecture of tissue microstructure [2+] signals [2+] in a non-invasive method and without artifacts. It is also particularly tricky to measure [Ca2+] in a physiologically relevant context, which allows the comparison of results obtained in the laboratory with the physiological status of plants in the field. CLSM makes extensive use of Ca2+ probes. However, not only CLSM can offer the possibility to couple the use of Ca2+ probes with in vivo microscopy analyses. Wide-field fluorescent microscopy has been used for both dyes and genetically encoded probes. [Ca2+]cyt was determined using the dye Fura-2 by either fluorescence ratio with a Cairn micro photometer  using absorbance and emission light [2+ imaging probes interact with Ca2+ through carboxylic acid groups. This interaction causes variation in indicator properties such as fluorescent intensity and its excitation. [Ca2+] is not measured directly; rather the indicator monitors the amount of free and complexed probe. The concentration of free Ca2+ is then measured based on effective dissociation constant (Kd) measured in vitro of the probe for Ca2+ in the specific environment.Caon light –38. Most2+]cyt is measured based on relative increase in fluorescence intensity of single wavelength. Non-ratiometric dyes such as fluo, rhod and Calcium Green-1 are used for measuring [Ca2+]cyt in plants, among them Calcium Green-11 is well-known and most commonly used in plant systems. The single excitation spectrum allows for simple instrumentation. A simple formula is used to calculate the absolute Ca2+ concentration in live plant cells. can be calculated using the following formula:The excitation spectrum of ratiometric dyes Fura-2 and Indo-1, varies according to the free CaKd is the dissociation constant, which is measured in vitro; R is the fluorescence ratio at both wavelengths Fλ2/Fλ1; Rmin is the minimum ratio value ; Rmax is the maximum ratio value; Fλ1 is the fluorescence used in the denominator at its maximum and minimum value).where 2+]cyt. Therefore using ratios avoids many of the problems related to absolute fluorescence values. On the other hand, acquisition and data manipulation is more complex due to the use of fluorescence ratios. Not all microscopes are suitable for these measurements (changing excitation/emission wavelength at suitable rates is required) and many ratiometric indicators require the use of UV excitation, which is an expensive option for confocal laser scanning microscopy.Ratiometric dyes can be corrected for unequal dye loading, photobleaching and focal plane shift, between two cells that have the same . Identification of Ca2+ sensitive AEQ from Aequorea victoria offers enormous advantage to carry out bioluminescence research. This protein was carefully extracted and purified from jelly fish to prevent the contact with Ca2+ as this would cause chemiluminescence, there by rendering the protein unsuitable for Ca2+ measurements cyt signaling in specific cell types, including those of the leaves. Therefore, AEQ can offer tissue specificity if placed under control of tissue-specific promoters.A major advantage of AEQ is that it can be selectively targeted to subcellular compartments by insertion of signal sequences unlike chemical compound dyes . Use of recombinant AEQ facilitated the understanding of Caartments –72. AEQ rocesses . Howeverrocesses  used GAL2+ concentrations by a method based on the calibration curve of Allen and coworkers [In AEQ assays the emitted light is calibrated into Caoworkers :L0 is the luminescence intensity per second and Lmax is the total amount of luminescence present in the entire sample over the course of the experiment. [Ca2+] is the calculated Ca2+ concentration, KR is the dissociation constant for the first Ca2+ ion to bind, and KTR is the binding constant of the second Ca2+ ion to bind to AEQ cyt variations are then calculated according to the equation:Leaves of plants expressing FRET-based CaR represents the FRET/CFP ratio measured during the experiment, n the Hill coefficient for YC3.6 , while Kd values are assessed for a given concentration of Ca2+ [2+]cyt variations using a cameleon probe.where  of Ca2+ . Figure 2+ indicators in plant sciences.8.2+ in living cells has seen a tremendous development in the last two decades with the evolution of genetically encoded Ca2+ indicators. Despite these improvements, we are still far from having an ideal Ca2+ probe. Here we give emphasis on future directions on the improvements of synthetic and fluorescent Ca2+ probes.Imaging CaCurrently, there are only two ratiomatric dyes fura-2 and indo-1 available since thirty years after their synthesis. Both these dyes requires UV spectrum for their excitation. As a result, there is the need for the use of expensive UV lasers for confocal microscopes, with the results of toxicity of UV illumination, lower penetration and background fluorescence. Therefore, it is necessary to develop new indicators that absorb light in the visible range.2+ indicators has reduced the demand to develop new optimized dyes. Although these protein-based probes offer many advantages over synthetic dyes they are still far from being ideal probes. Most of the GFP-based probes have CaM as Ca2+ sensing component. CaMs are also known to be important for many physiological processes in plant system. Overexpression of GFP-based probes might results in the substantial alteration of endogenous physiological processes that depend on CaM activity [Aeuuorea) would provide solutions to these problems. All these efforts will allow us to understand the complex subcellular interplay of Ca2+ signals underlying many plant physiological and developmental processes.The advent of a new generation of protein-based Caactivity ,102. Theactivity . Finding


The control of vascular smooth muscle contractility enables regulation of blood pressure, which is paramount in physiological adaptation to environmental challenges. Maintenance of stable blood pressure is crucial for health as deregulation (caused by high or low blood pressure) leads to disease progression. Vasotone is principally controlled by the cyclic nucleotide dependent protein kinases A and G, which regulate intracellular calcium and contractile protein calcium sensitivity. The classical pathways for activation of these two kinases are well established and involve the formation and activation by specific cyclic nucleotide second messengers. Recently we reported that both PKA and PKG can be regulated independently of their respective cyclic nucleotides via a mechanism whereby the kinases sense cellular oxidant production using redox active thiols. This novel redox regulation of these kinases is potentially of physiological importance, and may synergise with the classical regulatory mechanisms. Untreated, this can lead to increased risk of pathological complications including heart attacks, heart failure, peripheral artery disease, aortic aneurysms, stroke and kidney failure . To prev−) form due to their local environment. Close proximity with the basic amino acids arginine or lysine lower the pKa of cysteine thiols making them more reactive. These thiols can act as redox sensors by undergoing a multitude of different oxidative modifications as summarised in Oxidants are emerging as important physiological signalling molecules despite many years of bad press, which erroneously labeled them as being purely causative agents in disease progression. This turn around in perception is due to the failure of numerous antioxidant trials and the growing discovery of proteins and pathways that are oxidatively regulated (the “redoxome”) –8. A gre2O2) following reduction by superoxide dismutase [2O2 is able to act as a signalling molecule by fulfilling the several criteria required for the dynamic regulation of protein function. This includes regulated formation of this oxidant in response to specific stimuli, selectivity of target protein oxidation (provided by “reactive” cysteine residues only in selected targets), a change in protein function , and rapid reversibility by reducing enzymes, as well as localised changes in cellular redox due to compartmentalisation of antioxidants and oxidant-generating enzymes. This has been shown to be the case for oxidants generated transiently in response to growth factor and cytokine stimulation, where their formation is essential for the full spectrum of signalling events they can trigger [The generation of the reactive oxygen species superoxide is a continual process due to its formation as a by-product of energy metabolism crucial for cellular survival and homeostasis ,12. Furtismutase . This foismutase . H2O2 is trigger –17.2.3), a second messenger that increases Ca2+ release from the sarcoplasmic reticulum (SR) by activating IP3 receptors (IP3R). This enhances Ca2+ dependent Ca2+ entry via opening of transient receptor potential channels (TRPs) and SR store-operated Ca2+ channels [2+ interacts with calmodulin forming a complex that activates MLCK, a process which leads to enhanced vascular smooth muscle constriction [The regulation of blood pressure is maintained physiologically by changes in smooth muscle tone. Increased relaxation causes vessels to dilate, augmenting the total volume blood capacity, leading to a subsequent decrease in pressure within the blood vessel. Substantial characterisation at the molecular level has revealed that vascular smooth muscle tone is essentially controlled by the interplay between two opposing pathways, with the overall result being dependent on the rate of myosin light chain phosphorylation. The contractile response is calcium dependent and leads to increased myosin light chain kinase (MLCK) activity and inhibition of the myosin light chain phosphatase (MLCP) . This inchannels ,26. Thistriction .2+ in vascular smooth muscle cells. This decrease in Ca2+ concentration leads to inhibition of MLCK and activation of MLCP, switching the myosin light chain from the phosphorylated contractile state to the dephosphorylated relaxed state. The three known vasodilatory pathways are summarised in The contractile response of vascular smooth muscle is inhibited by three known converging pathways that prevent myofilament cross-bridge cycling to generate smooth muscle relaxation. These are the Protein kinase A (PKA), Protein kinase G (PKG) and endothelium derived hyperpolarising factor (EDHF) pathways, each of which acts as a vasodilator by decreasing intracellular Ca2) formation and diffusion from endothelial cells to smooth muscle cells increases the activity of adenylate cyclase, which converts adenosine triphosphate (ATP) to 3′–5′-cyclic adenosine monophosphate (cAMP) a direct activator of PKA [Both PKA and PKG are classically considered to be cyclic nucleotide dependent and require the formation of active vasodilators by the endothelium. In the case of PKA, increased prostacyclin  on the cell membrane, increasing its open probability [+ cation causes membrane hyperpolarisation, closing voltage-dependent Ca2+ channels, decreasing the total Ca2+ influx. Another source of intracellular Ca2+ is that extruded from the SR with the influx and efflux being regulated by several specific membrane Ca2+ handling proteins. This includes the Ca2+ importing pump SERCA, which is normally inhibited by the associated protein phospholamban. Phosphorylation of phospholamban by PKG removes the brake on SERCA activity, increasing the import of Ca2+ into the SR [Activation of PKG in smooth muscle cells leads to phosphorylation of several targets that regulate contractile protein Cabability ,33. Incro the SR ,35.q-coupled receptors and phospholipase C to increase IP3 production. This second messenger stimulates Ca2+ release from the SR, which activates MLCK. PKG is able to inhibit this pathway by phosphorylating the IP3 receptor-associated PKG-substrate (IRAG), which when modified inhibits IP3 stimulated SR Ca2+ release by the IP3R [3 by phospholipase C is suppressed by PKG-dependent phosphorylation of the regulator of G-protein signalling 2 (RGS2) [q, decreasing the GTP that is required for phospholipase C activation.Most contractile agonists work by activating smooth muscle Gthe IP3R –38. Therthe IP3R . Further2 (RGS2) ,41. WhenThe activation of MLCP is crucial for smooth muscle relaxation, which is maintained by PKG-dependent target phosphorylation. Essentially PKG is able to do this by preventing the inactivation of MLCP by Rho kinase (ROCK). The activator of ROCK, rhoA is directly phopshorylated by PKG increasing its affinity for the guanine dissociation inhibitor (GDI), preventing the translocation of RhoA to the membrane which is required for activation ,43. AnotThe enzymes PKA and PKG share much homology in terms of structure especially around the catalytic site, giving both kinases the ability to phosphorylate several common substrates. Variations in targets between the two kinases is likely due to differential localisation controlled by binding proteins, such as A-kinase anchoring proteins (AKAPs) for PKA and G-kinase anchoring proteins for PKG (GKAPs) –51. Thes2 with its exact identity remaining unknown [+, epoxyeicosatrienoic acid (EET), H2O2 and electronical communication through the gap junctions [A third biochemical process that mediates vascular dilation is the formation of EDHF, which as the name suggests is produced in endothelial cells and generates relaxation in smooth muscle cells by hyperpolarising the membrane. The synthesis of EDHF is stimulated by the neurotransmitter acetylcholine, the small peptide bradykinin, shear stress and a number of other agonists. The role of EDHF in regulating blood pressure is as prominent as the PKG and PKA pathways, with deficiency in EDHF signalling contributing to several cardiovascular pathologies including hypertension, chronic renal failure, and diabetes ,59. The  unknown . Howeverunctions –65. EvidThe classical view of blood pressure regulation involves a summation of three primary pathways that are coordinated in a complex manner as outlined above. These pathways are independently regulated but also integrate to generate a net blood pressure. Each pathway can be dynamically regulated by changes in circulating factors and in some cases shear stress with responses varying depending on the specifics of the diverse physiological/pathological conditions and between different vessel types. The inhibition of EDHF signalling or knockdown of PKG causes dysfunctional regulation of blood pressure, which highlights the importance of each pathway in controlling vasotone ,59,66. T3.2+ influx into cardiac cells stimulates the myofilaments to generate more force during contraction due to greater cross-bridge cycling. This process is carefully maintained to ensure adequate myocardial contractile performance and is primarily regulated by the autonomic nervous system, allowing the heart to adapt and function effectively during various environmental challenges [Sα), this increases cAMP production and subsequent PKA activation [As with the control of vascular smooth muscle cell contraction and relaxation, regulation of cardiac contractility is also dependent on intracellular calcium. Increased Caallenges . This inallenges . The stitivation ,69.+ inside and Na+ outside the cell. This gives the sarcolemma a negative charge on the inside with respect to the outside. During an action potential opening and then rapid closure of Na+ channels on the sarcolemma leads to depolarisation and activation of voltage-dependent Ca2+ channels, including the L-type Ca2+ channel located in the T-tubular membrane. This increases the influx of Ca2+ and the inward Ca2+ current (ICa), which contributes to the action potential plateau. The elevated Ca2+ binds to and activates the ryanodine receptors (RyRs), which are ligand-gated channels located on the SR membrane. This enhances the release of Ca2+ from the SR and is known as Ca2+-induced Ca2+ release. This amplified intracellular Ca2+ binds to cardiac troponin C (cTnC) triggering the constriction of myofilament proteins. The removal of Ca2+ from the cytosol allows dissociation of Ca2+ from troponin C and subsequent myofilament relaxation [+ channels, increasing the export of K+, closing the voltage-dependent Ca2+ channels. The intra and extracellular Na+ and K+ concentrations are returned to resting state by the sarcolemmal Na+/K+-ATPase (NKA). The extrusion of Ca2+ from the cytosol is mediated by the SR Ca2+-ATPase (SERCA), the sarcolemmal Na+/Ca2+ exchange (NCX1) and sarcolemmal Ca2+-ATPase. Increased cytosolic Ca2+ is sequestered into the SR Ca2+ store by SERCA, replacing that lost from the RyR during an action potential [2+ from the cell is mediated by NCX1, which is generally believed to import three Na+ ions for every Ca2+ ion extruded and is regulated by both transmembrane voltage and Na+ and Ca2+ concentration gradients [+ and imports Ca2+, with it flipping to the forward mode during repolarisation [2+ from the cell is the sarcolemmal Ca2+-ATPase, which hydrolyses ATP to transport Ca2+ out of the cell [The heart is a large pump that generates the force required for propelling the blood through the circulatory system by contracting and relaxing in a rhythmic fashion (sinus rhythm). The initiation of cardiac muscle contraction is controlled by the sinoatrial node (cardiac pacemaker) , which ilaxation . This ocotential . Extrusiradients . This exrisation –77. A sethe cell .2+ handling proteins increases cardiac contractility (see 2+ from the SR. One of the targets of PKA located on the SR is the Ca2+ channel the ryanodine receptor. This protein is a tetramer comprised of four type 2 RyR polypeptides (RyR2) and four FK506 binding proteins (FKBP12.6). The activity of this protein is regulated by intracellular Ca2+ with low concentrations (μM) increasing activity and high concentrations (mM) being inhibitory [2+-induced activation. With the overall effect being the augmented export of Ca2+ from the SR following an action potential [2+ handling protein that is regulated by PKA is the SERCA pump. The activity of this protein is inhibited by its interaction with the 52 amino acid membrane protein phospholamban. Phosphorylation of phospholamban at Ser-16 by PKA attenuates its ability to inhibit the SERCA pump [2+ into the SR, which enhances the rate of relaxation and also results in augmented release of Ca2+ following a second action potential from the enhanced Ca2+ SR store. The influx of extracellular Ca2+ is also enhanced by PKA through the regulation of sarcolemmal Ca2+ handling proteins. This includes the L-type Ca2+ channel that is phosphorylated by PKA at the α1C subunit at Ser1928 and the β2 subunit at Ser-478 and Ser-479, which increases the open probability of this channel [+ [2+ overload in response to β-stimulation by limiting the rise in intracellular Na+ that may help maintain the NCX1 in its forward mode. This is supported by transgenic mice that do not express PLM in which β-adrenergic stimulation does not activate NKA and that the amplitude of Ca2+ transients is significantly higher than for wild-types. The SR Ca2+ content was also increased in PLM knockout mice and was associated with an increased propensity for spontaneous Ca2+ transients and contractions in these animals [Phospho-regulation by PKA of several key Cality see . This inhibitory . PKA is otential . AnotherRCA pump . This in channel ,83. The annel [+ .This is  animals . Further2+ handling proteins, PKA also has several targets that are myofilament proteins required for cardiac constriction. Cardiac troponin I (cTnI) is a target with residues 23 and 24 being phosphorylated. This increases the rate of cardiac relaxation by enhancing the rate at which Ca2+ is dissociated from cTnC without effecting maximum actomysoin ATPase activity [As well as Caactivity –88. Anotactivity . This baactivity .2+ [2+ [2+ or extracellular influx of Ca2+. Furthermore, PKG is able to reduce the cellular intake of Ca2+ by directly inhibiting voltage activated Ca2+ channels. This is substantiated by experiments demonstrating that activation of PKG causes inhibition of L-type Ca2+ channel whole cell current after phosphorylation of the ß2a subunit at Ser496 [2+ channels compared to wild-type [Activation of PKG is also known to regulate cardiac contractility. However, its role has not been characterised to the same extent as PKA and is seen as somewhat controversial due to the biphasic effect of NO donors on the cardiac inotropy. Nevertheless a recent publication has put into perspective the likely effect of direct PKG stimulation on cardiac contractility. It is reported that NO donor concentrations that induce protein oxidation (S-nitrosylation) and not PKG activation increase cardiac contractility. In contrast, concentrations of NO donor that stimulate PKG, or the use of cGMP-analogs as direct activators, lead to decreased contractility . These f2+ ,94, and 2+ [2+ ,96. Botht Ser496 . This isild-type .4.2O2. When these samples were immunostained following Western blotting with a PKA-RI antibody most of the protein was monomeric in the controls, with a concentration-dependent increase in disulphide dimer after H2O2 treatment. The disulphide reducing agent 2-mecaptoethanol confirmed that the dimer was the result of disulphide formation as it reduced this complex back to its monomeric form.As mentioned in the introduction many proteins can act as redox sensors and signal transducers by being functionally regulated by cysteine oxidation. We undertook a project to identify such proteins with the aim of finding those that specifically formed inter-protein disulphide complexes, an anticipated oxidation product . The rat2O2 myocyte contractility was augmented, which could be inhibited by the addition of the PKA inhibitor, H89. This enhanced contractility was accompanied by an increase in phosphorylation of the PKA substrates phospholamban and troponin I, a process which was also sensitive to H89 inhibition. Furthermore, this apparent activation of PKA by oxidation was independent of cAMP. This was evident from a specific fluorescence based assays for cAMP, showing that H2O2 treatment did not augment formation of this cyclic nucleotide. Together this data suggested that oxidation can directly enhance the activity of PKAI independent of cyclic nucleotide stimulation. We hypothesised that this augmentation of PKAI substrate phosphorylation following oxidation may be mediated by the kinases ability to interact with its binding partners the AKAPs [To determine the functional significance of this oxidative modification on PKA activity the contractility of isolated adult rat ventricular myocytes was measured. In the presence of Hhe AKAPs ,49. The he AKAPs .2O2 highlighted mostly monomer in controls and a concentration dependent increase in PKG1α dimer formation which mirrored that of PKA-RI [2O2 there was a substantial time dependent decrease in perfusion pressure analogous to that of an NO donor. We speculated that this decrease in perfusion pressure by H2O2 may be due to activation of PKG1α by direct oxidation. We tested this hypothesis by measuring tension in aortic rings as readout of vascular tone. The use of the PKG inhibitor Rp-8-Br-cGMPs and the guanylate cyclase inhibitor ODQ blocked NO donor mediated vessel dilation. However, H2O2 induced vessel relaxation was only blocked by the PKG inhibitor, supporting an oxidative cyclic nucleotide independent mechanisms for PKG1α activation. Michaelis Menten kinetic analysis of recombinant PKG1α activity using a radioactivity based kinase assay confirmed that oxidation can directly activate PKG1α independent of cGMP, by increasing the enzyme affinity for its substrate by approximately 7-fold. Mutation in PKG1α of the redox sensitive cysteine 42 to a charge conserved serine residue removed the ability of this kinase to be activated and to disulphide dimerise in response to H2O2 treatment. This is compelling evidence demonstrating that oxidation of cysteine 42 is crucial for redox activation of PKG1α. The mechanism for oxidative activation of PKAI and PKG1α is summarised in The discovery that PKAI could be enzymatically regulated by oxidation independent of cyclic nucleotide stimulation lead us to speculate that PKG1α may also be redox sensitive. The rational being that both kinases share similar structural homology and also a single constitutive disulphide within the N-terminal dimerisation domain of PKG1α has been identified . Westernf PKA-RI . In hear2O2 is sometimes seen as being a non-physiological means of investigating protein oxidation. To overcome the potential skepticism associated with using exogenous oxidants we treated rat aortic smooth muscle cells with physiologically relevant concentrations of insulin, a known mediator of endogenous oxidant formation [2O2. Furthermore it was found that transnitrosylating species of NO (CysNO) are also able to mediate the formation of PKG1α and PKA-RI disulphide dimers [2O2, which has very little effect on cardiac contractility. As discussed in this article PKA and PKG have opposite effects on the cardiac ionotropy due to their ability to differentially regulate intracellular Ca2+. Based on this evidence we hypothesised that simultaneous activation of PKG may override the effect of PKA stimulation. We tested this theory using isoprenaline to activate PKA and enhance cardiac contractility. The addition of an NO donor to stimulate PKG activity reversed the ionotropic effect of isoprenaline, demonstrating that PKG can override PKA induced elevations in contractility. It is important to consider that in perfusion experiments, using an exogenous oxidant will simultaneously activate both kinases due to global cellular oxidation. During a physiological or pathological scenario change in oxidant formation are likely to occur in localised areas of the cell within close proximity to the enzymes that generate them, such as superoxide dismutase. This could allow differential changes in oxidant mediated contractility to occur due to stimulation of select pools of PKA or PKG1α.To some researchers the use of exogenous Hormation ,106. Thee dimers . A findi2O2 being at least a component of EDHF. This is supported by reports showing that the application of exogenous H2O2 can lead to relaxation in a number of blood vessels [2O2 acts as a component of EDHF is further supported by NO- and prostanoid-independent relaxation being blocked by catalase, which selectively breaks down H2O2 [2O2 has also been directly measured in porcine coronary microvessels using electron spin resonance imaging following bradykinin treatment (which is known to generate EDHF) [2O2 and vessel relaxation, both of which were inhibited by catalase. In theory oxidative activation of PKG1α should mimic the effect of EDHF by increasing the open probability of BKCa channels after phosphorylation, leading to increased hyperpolarisation and vascular relaxation [The potential physiological importance of oxidative activation of PKG1α or PKAI may tie in with EDHF signalling, with substantial evidence in favor of the oxidant H vessels –110. Theown H2O2 ,112. In own H2O2 . The prote EDHF) ,112,114.laxation ,33. Ther5.The discovery that PKAI and PKG1α are redox sensors and signal transducers that can be regulated by cellular oxidants means there needs to be an addition to the well-established models for their activation. These findings highlight the potential for oxidants to regulate both vasotone and the cardiac inotropic environment. However, further characterisation to determine the full extent of the physiological as well pathological roles of these oxidative mechanisms needs to be undertaken. It is tantalising to suggest that EDHF may be an oxidant that can induce vasodilation through direct oxidative activation of PKG1α or PKAI. However, this is yet to be determined and will likely require the development of sensitive techniques that can determine small variations in PKG1α or PKAI oxidation.


Inhibition of p53-MDM2/MDMX interaction is considered to be a promising strategy for anticancer drug design to activate wild-type p53 in tumors. We carry out molecular dynamics (MD) simulations to study the binding mechanisms of peptide and non-peptide inhibitors to MDM2/MDMX. The rank of binding free energies calculated by molecular mechanics generalized Born surface area (MM-GBSA) method agrees with one of the experimental values. The results suggest that van der Waals energy drives two kinds of inhibitors to MDM2/MDMX. We also find that the peptide inhibitors can produce more interaction contacts with MDM2/MDMX than the non-peptide inhibitors. Binding mode predictions based on the inhibitor-residue interactions show that the π–π, CH–π and CH–CH interactions dominated by shape complimentarity, govern the binding of the inhibitors in the hydrophobic cleft of MDM2/MDMX. Our studies confirm the residue Tyr99 in MDMX can generate a steric clash with the inhibitors due to energy and structure. This finding may theoretically provide help to develop potent dual-specific or MDMX inhibitors. N-terminal transactivation domain of p53 [The tumor suppressor protein p53, “the guardian of the genome,” plays a key role in maintaining the integrity of the genome by inducing cell cycle arrest and apoptosis in response to stress . It can n of p53 . In factn of p53 ,8. Almosn of p53 . Thus, tRecent studies show that MDMX is not only highly homologous (55%) to MDM2 , but alset al. shows similar affinity to MDM2/MDMX [Earlier biochemical studies using p53 peptide have proven that MDM2 binds to p53 with 10-fold higher affinity than MDMX . PreviouDM2/MDMX . This prDM2/MDMX ,26–32.et al. and able to efficiently fill the binding pockets of MDM2/MDMX, its median inhibitory concentration (IC50) values to MDM2/MDMX are 1.17 and 36 μM, respectively [et al. with IC50 values of 36 and 250 nM to MDM2/MDMX, respectively [Understanding the binding mechanisms of the peptide and non-peptide inhibitors to MDM2/MDMX at an atomic level may facilitate the development of potent dual inhibitors inhibiting the p53-MDM2/MDMX interaction and provide valuable information about the structure-affinity relationships of the p53-MDM2/MDMX complexes. A few computational studies have been performed for this purpose ,33,34. Iectively . Both ofectively . Thus itetc. [Binding free energy calculations have been proven to be powerful and valuable tools for understanding the binding mechanisms of inhibitors to proteins. To date, several effective methods have been proposed to calculate the binding free energies of protein inhibitors: free energy perturbation (FEP) , thermodetc. ,38–41. Aetc. ,42–47. Ietc. –50. ThusTo evaluate the reliable stability of MD trajectories, RMSD of backbone atoms relative to the initial minimized structure through the phase of the simulation was plotted in To acquire an atomic view of the difference between the MD-simulated structures and crystal structures, the structures of the MD-simulated complexes from the last 500 ps of MD simulations at an interval of 10 ps were superimposed with the crystal structures (plotted in Gexp) were summarized in −1, respectively. Furthermore, it is encouraging that the ranking of the experimental binding free energies are consistent with our predictions, which suggests the MD-simulation models and computational protocol tested in this study is reliable.To further evaluate the difference in the binding modes of the inhibitors to MDM2/MDMX and obtain detailed insights into the contribution of each component to the inhibitor-protein binding, the binding affinities of the inhibitors to MDM2/MDMX were estimated by performing MM-GBSA calculations based on a single-trajectory MD simulation. Because the radius parameter of chlorine atoms is missing for MM-GBSA module in Amber 10, we add the radii of 1.75 Å for chlorine to pbsa program in Amber . The calGvdw). Non-polar solvation energies (ΔGnopol), which correspond to the burial of SASA upon binding, also provide important contributions to binding. However, the contributions of the entropy changes to the free energies (−TΔS) impair the bindings of two inhibitors to MDM2/MDMX. Although the electrostatic terms (ΔGele) favored inhibitor binding, these favorable contributions were completely screened by the unfavorable stronger polar solvation energies (ΔGpol). It is noted that the electrostatic interaction of pDI6W with MDM2/MDMX is two times stronger than van der Waals energies, while the electrostatic terms of the interaction between WK23 and MDM2/MDMX is much weaker than van der Waals energy. Thus, it is concluded van der Walls energy dominates the bindings of the inhibitors in the hydrophobic cleft of two oncoproteins. This result agrees with the previous studies of several groups [As shown in l groups ,34.−1, weaker than one of pDI6W and WK23 with MDM2, respectively. Despite a high homology and similar structure of MDM2/MDMX . Thus, the residues Met53, Val92, Pro95 and Leu98 form the third hydrophobic pocket that matches the hydrophobic alkyl of Leu26′. As seen from −1 weaker than one of pDI6W with Val93, His96 and Ile99 in MDM2, respectively. This result shows that Tyr99 certainly forms a steric clash with Leu26′ and produces an important effect on the inhibitor-protein binding, which has been proven by the previous analyses of binding free energies. In addition, the strong CH–CH contacts between Lys50 and Thr27′ also provide favorable contribution to the pDI6W-MDMX binding.In the pDI6W-MDMX binding complex , the rese energy . The CH–al·mol−1 . The indal·mol−1 . In addind Leu98 . The aro−1 weaker than one of WK23 with His96 and Ile99 in MDM2, despite the binding energy of WK23 with Val92 of MDMX slightly higher than one of WK23 with Val93 of MDM2. This result shows that the orientation conflict of Tyr99 with the ring R4 of WK23 can produce important influence on the inhibitor binding. This work basically agrees with the previous studies [For the WK23-MDMX complex, the binding mode prediction of WK23 to MDMX is similar to the pDI6W-MDMX complex. It is observed that the number of the residues involving the WK23-MDMX interaction is less than the pDI6W-MDMX complex. By comparing  studies ,27,31.Based on the above analysis, three important conclusions can be obtained: (1) the peptide inhibitors can generate more interaction contacts with MDM2/MDMX than the non-peptide inhibitors; (2) The steric clash formed by Tyr99 leads to the decrease in the inhibitor-MDMX binding affinity; (3) The π–π, CH–π and CH–CH interactions dominated by the shape complementarity drive the bindings of the inhibitors in the hydrophobic cleft of MDM2/MDMX.Gpol) is closely related to the electrostatic term (ΔGele), these two components were combined into a component: polar interaction component (ΔGele + pol). Gvdw), non-polar solvation free energy (ΔGnopol), polar interaction component (ΔGele + pol) and binding free energy (ΔGgb). The alanine scanning results in different effects on the separate energy component. As seen in We performed alanine scanning on the inhibitor-MDM2/MDM2 complex to validate the contributions of free energy components to the binding. Because the polar solvation energy  for the pDI6W-MDM2 complex, four residues  for the WK23-MDM2 complex, six residues  for the pDI6W-MDM complex and four residues  for the WK23-MDMX complex. This result is due to the decrease in the number of CH–π and CH–CH contacts or the loss of π–π interaction between the inhibitor and MDM2/MDMX caused by the alanine scanning. Although the oxygen atoms of Leu54 or Met53 form a hydrogen bond with the inhibitors, this oxygen atom belongs to the protein backbone. Thus, the alanine scanning does not influence this hydrogen bond. In a word, the π–π, CH–π and CH–CH interactions of the inhibitors with the proteins govern the bindings of the inhibitors in the hydrophobic cleft of MDM2/MDMX, which agrees with the previous analysis of binding modes.To identify the binding hot spots of the protein and gain further insight into the contribution of each alanine mutation, we also calculated the change of the inhibitor-residue interaction caused by the alanine scanning  in the protein data bank (PDB) . The str−1·Å−2. Secondly, each system was minimized without restriction. And each stage was performed using the steepest descent minimization of 1000 steps followed by a conjugate gradient minimization of 2000 steps. After the minimization, the system was then heated from 0 to 300 K in 100 ps and equilibrated at 300 K for another 100 ps. Finally, we run MD simulations on each system at 1 atm and 300 K for 9 ns to make sure that a stable trajectory for each of the simulated structures was obtained. During the simulation, the SHAKE method was applied to constraint the covalent bonds involving hydrogen atoms [For each system, energy minimization and MD simulation were carried out using the sander module of the Amber 10 program. Before MD simulations, each system was subject to energy minimization in two stages to remove bad contacts between the complex and the solvent molecules. Firstly, the water molecules and counterions were minimized by freezing the solute using a harmonic constraint of a strength of 100 kcal·molen atoms . The Paren atoms ,62. The G) are approximated byFor each complex, a total number of 200 snapshots were taken from the last 2 ns of the MD trajectory with an interval 10 ps. The MM-PB/SA method and nmod module, which is implemented in Amber10, were applied to compute the binding free energies of the inhibitors to MDM2/MDMX. In this approach, the binding free energies  can further be expressed aswhere ΔEint, ΔGvdw and ΔGele represent the internal energy contribution from bonds, angles and torsions, the van der Waals and electrostatic interactions in gas phase, respectively. The solvation free energy (ΔGsol) is further divided into two components:where ΔGpol and ΔGnopol are polar and non-polar contributions to the solvation free energy, respectively. The former component was computed using the modified GB model developed by Onufriev A et al. [where ΔA et al. . The die−1·Å−2) and 0 kcal·mol−1, respectively.where SASA is the solvent-accessible surface area and was calculated with the MSMS program . In thisRij (the distance between two atoms) until the root-mean-square of the energy gradient was lower than 10−4 kcal·mol−1·Å−1. The calculation error bars are the standard errors of the mean (SE)calculated using N is the number of trajectory snapshots used in the calculation [The conformational entropies are important contributions to the inhibitor-receptor binding. Thus, normal-mode analysis was performed to compute the conformational entropy change upon the inhibitor binding. However, due to entropy calculations for large systems being extremely time consuming, we applied only 25 snapshots taken at an interval of 80 ps from the final 2 ns of the MD simulation to calculate the entropy contribution. Each snapshot was minimized with a distance-dependent dielectric function 4culation .The inhibitor-residue interaction, which is valuable to qualitatively define the binding mechanisms of the four inhibitors to MDM2, was analyzed using a per-residue-based decomposition method  and apprGvdw and ΔGele are non-bonded van der Waals interactions and electrostatic interactions, respectively, between the inhibitor and each MDM2 residue in the gas phase and ΔGgb and ΔGsurf are the polar and non-polar contributions to the inhibitor-residue interaction, respectively.where ΔCγ by replacing with a hydrogen atom [To determine the contribution of each residue in the interaction interface of the inhibitor-receptor binding and to study the detailed mechanisms at the energetic and atomic levels, computational alanine scanning was carried out on MDM2/MDMX, and the binding free energies of the inhibitors to the protein mutants were calculated by using the MM-GBSA method. The alanine mutant structures were obtained by altering the coordinates of the wild-type trajectory, which involves cutting atoms and truncating the mutated residue at gen atom . All pargen atom .In this work, MD simulation coupled with the MM-GBSA method has been carried out to probe the difference in the binding modes of the peptide and non-peptide inhibitors to MDM2/MDMX. The results confirm that the peptide inhibitors can produce more interaction contacts than the non-peptide inhibitors. The binding mode prediction of based-residue decomposition suggests that the π–π, CH–π and CH–CH interactions, dominated by the shape complementarity, govern the bindings of the inhibitors in the hydrophobic cleft of MDM2/MDMX. Lastly, we confirm that the existence of the potential steric clash formed by the residue Tyr99 due to energy and structure, make it possible to develop the potent dual inhibitors inhibiting the p53-MDM2/MDMX interaction.