However, repeated episodes of brief periods of ischemia followed by reperfusion or ischemic preconditioning (IP) have been shown to improve cardiac function and exert cardioprotective actions against the adverse effects of prolonged I/R injury. This protective action of IP in attenuating myocardial damage and subcellular remodeling is likely to be due to marked reductions in the occurrence of oxidative stress and intracellular Ca2+-overload in cardiomyocytes. In addition, the beneficial actions of IP have been attributed to the depression of proteolytic activities and inflammatory levels of cytokines as well as the activation of the nuclear factor erythroid factor 2-mediated signal transduction pathway. Accordingly, this review is intended to describe some of the changes in subcellular organelles, which are induced in cardiomyocytes by I/R for the occurrence of oxidative stress and intracellular Ca2+-overload and highlight some of the mechanisms for explaining the cardioprotective effects of IP.It is now well established that ischemia/reperfusion (I/R) injury is associated with the compromised recovery of cardiac contractile function. Such an adverse effect of I/R injury in the heart is attributed to the development of oxidative stress and intracellular Ca In this regard, the beneficial effects of Ca2+-antagonists [+-H+ exchange inhibitors [2+-overload [2+-overload and the occurrence of oxidative stress [2+-overload with respect to the modification of subcellular defects due to I/R injury [2+-handling by subcellular organelles will be described in hearts subjected to I/R injury. Furthermore, the effects of IP on some factors, such as inflammatory cytokines and proteolytic activities, as well as the nuclear factor erythroid factor 2 (Nrf2) signal transduction pathway, which are known to modulate subcellular defects due to I/R injury, will be outlined. The generation of reactive oxygen species (ROS) and subsequent oxidative stress is known to increase apoptosis [Although myocardial ischemia due to a partial or complete blockage of the coronary artery is known to induce marked changes in cardiac function and metabolism, as well as the ultrastructural integrity of cardiomyocytes ,2, the pagonists ,10 and Nhibitors ,12,13,14overload ,19,20,21overload  suggest e stress . It is ae stress ,23. ThisR injury . In partpoptosis , necrosipoptosis ,25, inflpoptosis  and impapoptosis ,27. In fpoptosis ,29,30,312+-homeostasis in cardiomyocytes, it is necessary to describe the impact of I/R on the Ca2+-handling properties of the two major subcellular organelles, namely SL and SR. It has become evident that ROS and other oxidant molecules are involved in the genesis of intracellular Ca2+-overload, myocardial cell damage and subsequent contractile dysfunction due to I/R injury [2+-movements in response to excessive ROS generation and the inability of cardiomyocytes to scavenge ROS by endogenous antioxidant systems (a state of oxidative stress) results in the development of intracellular Ca2+-overload subsequent to I/R injury [2+-channel density has been reported to decrease due to hypoxia/regeneration and myocardial ischemia [2+-overload, as it would decrease Ca2+-influx. On the other hand, the activities of both SL Na+-Ca2+ exchange and the SL Ca2+-pump have been observed to be markedly reduced subsequent to hypoxia or I/R injury [2+-efflux and have been suggested to produce intracellular Ca2+-overload. Other SL activities, such as Na+-K+-ATPase, Ca2+/Mg2+ ecto-ATPase and superficial store of Ca2+ [2+-influx in the cell indirectly. It should be noted that the actions of ROS as well as I/R injury on the SL Na+-Ca2+ exchange, Na+-K+-ATPase and Ca2+/Mg2+ ecto-ATPase seem to depend on the type of oxyradical generating system and the time of exposure to these interventions [2+ transporters [2+-fluxes, but on balance would result in the development of intracellular Ca2+-overload in cardiomyocytes. It should also be mentioned that oxidative stress generated due to I/R injury has been shown to cause alterations in excitation–contraction coupling as well as induce cardiac arrhythmias, heart dysfunction and myocardial cell damage [In order to understand the importance of the beneficial actions of IP with respect to CaR injury ,4,5,32. R injury ,34,35. DR injury ,37,38,39ischemia ,41, or wischemia , this meR injury ,44,45, aR injury ,47,48,49 of Ca2+ ,57,58,59ventions ,61,62. Isporters ,64,65,66sporters ,78,79,80sporters ,82. Froml damage ,87,88,892+- transporting activities are also known to occur under conditions of I/R as well as upon exposure of the heart to different oxidant molecules. In this regard, a reduced ability of cardiac SR Ca2+-uptake due to oxyradicals has been attributed to attenuation of Ca2+-stimulated ATPase activities [2+-release channels has also been observed following exposure of the SR to ROS [2+-release from SR under the in vivo situations for oxidative stress. The rotenone-insensitive NADH cytochrome C reductase activity of the cardiac SR has also been reported to be reduced in response to oxyradicals and has been linked to SR lipid peroxidation [2+-uptake and Ca2+-stimulated ATPase activities of skeletal muscle SR are depressed by ROS, this effect was suggested to be due to the inhibition of the sulfhydryl groups of the Ca2+-pump ATPase rather than membrane lipid peroxidation [2+-pump activities due to the generation of oxidants [2+-overload in cardiomyocytes. It is therefore clear that several subcellular Ca2+-handling proteins can be targeted as mechanisms for defects in SL and SR Ca2+-handling activities and inducing intracellular Ca2+-overload due to I/R injury. Furthermore, it should also be pointed out that cardiac dysfunction due to I/R injury is not limited to defects in SL and SR membranes but to other subcellular organelles such as the mitochondria, myofibrils and extracellular matrix, which are also affected in inducing myocardial abnormalities [Changes in SR Cativities ,91,92. AR to ROS , but thixidation . Althougxidation ,95. Intexidation , are conoxidants . Overallmalities ,22,23,32It has been demonstrated that IP limits the infarct size, and attenuates necrosis, apoptosis as well as cardiac dysfunction due to I/R injury ,102,103.The glycocalyx is particularly susceptible to I/R injury, as it is the first to be exposed to ROS, but IP has been shown to provide partial protection . Further1- adrenoceptor [+ due to the exchange with protons via the activation of the SL Na+-H+ exchanger (NHE), which causes the activation of the SL Na+-Ca2+ exchanger in reverse mode, resulting in an increase in the intracellular Ca2+-concentration [2+-overload [There are a variety of receptors and ion channels/pumps located in the SL membrane that are considered to influence cardiomyocyte function through the activation of downstream signal transduction processes and are affected by I/R injury, and thus may serve as targets for providing cardioprotection in IP. For example, the activation of several different receptor types, including adenosine receptors , angiotenoceptor ,121 and noceptor ,123, havntration ; thus, toverload . The ben+-K+-ATPase (sodium pump) is known to play an important role in the maintenance of the membrane potential and cation transport across the SL membrane [+-K+-ATPase activity directly maintains intracellular Na+ and K+ concentrations as well as indirectly maintains intracellular Ca2+-concentration [+-K+-ATPase is the catalytic domain and occurs in three isoforms: α1 is the most abundant but with a lower affinity for cardiac glycosides, as compared to the α2 and α3 subunits [+-K+-ATPase activity [+-K+-ATPase can be considered as a viable mechanism for cardioprotection due to IP. In this regard, a reduction of the intracellular Ca2+-concentration has been reported to be due to the IP-induced preservation of Na+-K+-ATPase activity in the I/R hearts [+-K+-ATPase is considered to play a signaling function [+-K+-ATPase/cSrc receptor complex and the subsequent stimulation of the key signaling mediators [+-K+-ATPase-mediated opening of the cardiac SL K+-ATP channels [2+-concentration, although these effects have been suggested to involve the mitochondrial K+-ATP channels [The Namembrane ; specifintration . The α ssubunits , while tactivity  and contR hearts . It shoufunction ,133,134 ediators ,136. Thechannels  and the channels .+-ATP channels and the mitochondrial K+-ATP channels have been extensively studied in the cardioprotective effects of IP. Although it is now generally believed that the mitochondrial K+-ATP channels play a more significant role in the beneficial effects of IP, the SL K+-ATP channels have increased in importance for inducing the harmful effects of oxidative stress [+-ATP channels because they are in an open state when exposed to ROS and this correlated well with the cytoprotective properties [+-ATP channels appear to act as an effector of IP and facilitated in improving functional recovery. This proposition was evident when the cardioprotective effects induced by the activation of the SL K+-ATP were observed in the stress period, but not during IP. On the other hand, by using isolated adult rat cardiomyocytes and an isoflurane-induced protection technique, both SL and mitochondrial K+-ATP channels were observed to participate in the IP-induced cardioprotection [It is pointed out that cardiac SL Ke stress . Earlieroperties ,142,143.otection .+-ATP channels) knockout mouse model [+-ATP channels are important in preventing cardiomyocyte apoptosis and mitochondrial damage during oxidative stress, as inhibition of SL K+-ATP channels promoted oxidative stress-induced apoptosis. Mitochondrial Ca2+-loading has been reported to be markedly increased upon the inhibition of SL K+-ATP channel in cultured HL-1 and neonatal cardiomyocytes [It should also be mentioned that the beneficial actions of IP have been reported to be lost in a cardiac specific Kir 6.2  by decreasing its activity and causing an increase in the SL Na+-Ca2+ exchanger (NCX) activity. These observations indicate that redox-dependent SR Ca2+ depletion may be partly affected by a reciprocal regulation of SERCA 2 and NCX [2+-stimulated ATPase activity [2+-overload [Under conditions of I/R and/or oxidative stress marked changes in SR Cathe cell ,149,150.function ,152. It  and NCX . Furtheractivity . On the overload .+-ATP channel acts not only as an effecter of IP, but also as a trigger, suggesting that its activation is required for both IP as well as during exposure to oxidative stress [+-ATP channel, the mitochondrial K+-ATP channel has been reported to primarily have an effect on the infarct size [+-ATP channel, which protects against ischemia-induced ventricular arrhythmias [+-ATP channels, which is protective [+-ATP channels play an important role in IP-induced cardioprotection against I/R injury. On the other hand, donors of nitroxyl (the one-electron reduced form of nitric oxide) have been shown to provide protection against I/R injury in a manner similar to IP [+-ATP channels [There is a wealth of information available in the literature regarding the role of mitochondrial alterations in IP-induced cardioprotection in I/R hearts. ,157,158.e stress . Of noterct size . Pharmachythmias . ROS relotective . It is tar to IP , with thchannels .2+ overload [During IP, the mitochondria also undergo other modifications. In this regard, IP increases the expression level of manganese superoxide dismutase (MnSOD) via the formation of NFκB and activator protein-1 (AP-1) . The incoverload .+-ATP channel and result in cardioprotection [c release and ROS production [2+-overload and the impairing of their function. These mitochondrial defects are attenuated by IP through the participation of diverse mechanisms.It needs to be emphasized that complex I (NADH ubiquinone oxidoreductase) is the point of entry of the electron into the mitochondria and is a major site of ROS generation ,169. An otection . Glyceraotection ,173. Thiotection . Furtherotection . These foduction ,176. Ove+-K+-ATPase mRNA levels, protein contents and activity have been reported. Based on the analysis of information in our paper [+-K+-ATPase mRNA and protein levels as well as the activity, IP reduced the I/R-induced changes, demonstrating that cardioprotection due to IP involves maintenance of Na+-K+-ATPase. +-K+-ATPase activity and Na+-K+-ATPase isoform  protein contents and mRNA levels in the I/R hearts [2+-handling properties of hearts subjected to a prolonged period of I/R. From the data in 2+-release and Ca2+-uptake activities, as well as SR Ca2+-stimulated ATPase activity in preischemic, ischemic and ischemic–reperfused control and preconditioned hearts, it has been suggested that IP prevents cardiac contractile dysfunction by protecting SR Ca2+ movements in cardiomyocytes [2+ movements by reducing the development of I/R-induced oxidative stress in cardiomyocytes. These earlier findings provide evidence that an attenuation of the I/R-induced defects in both the SL and the SR can be considered as important mechanisms for the beneficial actions of IP-induced cardioprotection.While the aforementioned has described the IP-induced cardioprotection of subcellular organelle defects due to I/R, we have earlier examined some of the SL and SR mechanisms for cardioprotection induced by IP. Specifically, the effects of IP on Naur paper , while IR hearts . In addimyocytes . In addimyocytes . Based omyocytes ,152, it It should be pointed out that the expression and activities of different SL and SR proteins are altered in I/R, which are attenuated by IP ,108,177,2+-concentration and subsequent I/R injury. Some of these factors include the production of inflammatory cytokines, the activation of proteases as well as Nrf2 signal transduction. By virtue of their ability to influence intracellular Ca2+-concentration, it is possible that an attenuation of the I/R-induced increases in inflammatory cytokines and proteolytic activity, as well as the regulation of Nrf2 signaling processes, may serve as important mechanisms in IP-induced cardioprotection. These aspects are briefly described in the following sections.Reperfusion of the ischemic heart is vital to restore cardiac function, reduce infarct size and salvage the viable myocardium . From th2+-handling and subsequent cardiac dysfunction in I/R injury [The inflammatory cytokines, TNF-α, IL-1 and IL-6, are known to influence cardiomyocyte CaR injury ,180,181,R injury . It is nR injury ; indeed,R injury . Late IPR injury . InteresR injury . IP has R injury . The infR injury .It is pointed out that while the adverse effects of IL-1 in I–R are known to be due to the activation of superoxide dismutase and cardiac remodeling, IL-1 has been shown to be cardioprotective in IP . On the 2+-overload has also been reported to diminish the I/R-induced activation of calpain and MMP [2+-overload, may both play a key role in the activation of proteolytic activities subsequent to I/R [Proteases play an important role in the degradation of misfolded or malfunctional proteins, as well as in the routine turnover of the extracellular matrix and other subcellular organelles, and thus are essential in the maintenance of cell homeostasis ,195,196. and MMP . Thus, it to I/R . It shout to I/R . Interest to I/R .2+ levels [Nrf2 is a transcription factor that controls cellular defense responses , particu+ levels , and thu+ levels . In addi+ levels ,217. Ind+ levels . In Nrf2+ levels . The act+ levels . In this+ levels . It was + levels .2+-overload in the I/R heart. A schematic representation of some critical events in eliciting improved cardiac function due to IP in the I/R hearts is given in 2+-overload, oxidative stress can directly cause subcellular and molecular abnormalities. While the initial functional changes occur rapidly under conditions of oxidative stress, the delay in functional recovery of the ischemic heart upon reperfusion may be due to alterations in the gene expression levels of SL and SR proteins, including those for other subcellular organelles such as the mitochondria, myofibrils and extracellular matrix. On the other hand, IP attenuates the occurrence of intracellular Ca2+-overload and oxidative stress in the I/R hearts, and thus reduces the I/R-induced subcellular remodeling and cardiac dysfunction. In addition, attenuation of proteolysis, but activation of Nrf2-mediated signal transduction, can also be considered as one of the important mechanisms of beneficial action of IP-induced cardioprotection. However, the translation of experimental interventions to clinical therapies remains to be a challenge.From the foregoing discussion, it is evident that IP-induced cardioprotection is associated with the reduction of I/R-induced oxidative stress and the subsequent depression in the development of intracellular Ca


Patients with myelodysplastic syndrome (MDS) require chronic red blood cell (RBC) transfusion due to anemia. Multiple RBC transfusions cause secondary iron overload and subsequent excessive generation of reactive oxygen species (ROS), which leads to mutations, cell death, organ failure, and inferior disease outcomes. We hypothesize that iron loading promotes AML development by increasing oxidative stress and disrupting important signaling pathways in the bone marrow cells (BMCs). Conversely, iron chelation therapy (ICT) may reduce AML risk by lowering iron burden in the iron-loaded animals.We utilized a radiation-induced acute myeloid leukemia (RI-AML) animal model. Iron overload was introduced via intraperitoneal injection of iron dextran, and iron chelation via oral gavage of deferasirox. A total of 86 irradiated B6D2F1 mice with various levels of iron burden were monitored for leukemia development over a period of 70 weeks. The Kaplan-Meier estimator was utilized to assess AML free survival. In addition, a second cohort of 30 mice was assigned for early analysis at 5 and 7 months post-irradiation. The BMCs of the early cohort were assessed for alterations of signaling pathways, DNA damage response and gene expression. Statistical significance was established using Student’s t-test or ANOVA.Iron loading in irradiated B6D2F1 mice accelerated RI-AML development. However, there was a progressive decrease in AML risk for irradiated mice with increase in iron burden from 7.5 to 15 to 30 mg. In addition, ICT decreased AML incidence in the 7.5 mg iron-loaded irradiated mice, while AML onset was earlier for the 30 mg iron-loaded irradiated mice that received ICT. Furthermore, analysis of BMCs from irradiated mice at earlier intervals revealed accelerated dysregulation of signaling pathways upon iron loading, while ICT partially mitigated the effects.We concluded that iron is a promoter of leukemogenesis in vivo up to a peak iron dose, but further iron loading decreases AML risk by increasing cell death. ICT can partially mitigate the adverse effects of iron overload, and to maximize its benefit this intervention should be undertaken prior to the development of extreme iron overload.The online version contains supplementary material available at 10.1186/s12885-021-08259-9. Myelodysplastic syndrome (MDS) patients who are chronically anemic require sustained red blood cell (RBC) transfusion, which inevitably leads to secondary hemochromatosis with significant pathophysiological consequences. To mitigate the risk of iron-related morbidity and premature mortality, iron chelation therapy (ICT) is recommended in transfusion dependent iron-overloaded patients with lower risk MDS, even though the evidence that ICT is effective in influencing these outcomes is less extensive than it is for patients with thalassemia major .Retrospective reviews of registry data suggested that iron overload in MDS may promote the development of acute myeloid leukemia (AML) . AlthougProof that iron overload promotes the progression of MDS to AML would have a profound effect on the aims and breadth of ICT in this disease, and could have a positive impact in both low and high risk MDS. We utilized a radiation-induced AML (RI-AML) B6D2F1 mouse model to investigate the hypothesis that extrinsic iron overload can promote AML development.B6D2F1 mice were obtained from and Charles River Canada . Upon arrival, all mice were randomized and housed in unisex groups of 5 or less per cage in a temperature and humidity-controlled room maintained on a 12:12 h light/dark cycle in a pathogen-free facility at the Sunnybrook Research Institute.Leuconostoc spp., Mr ~ 6000) was delivered by intraperitoneal injection for 5 days per week until the desired iron burden was reached. Iron burden is defined as the total amount of excess iron in the body after iron loading and/or ICT. ICT was initiated after the end of iron loading. Deferasirox  was suspended in 0.5% hydroxypropylcellulose  and administered by oral gavage at 10 or 40 mg/kg/day for 7 days a week over 4 or 8 weeks.All chemicals were purchased from Sigma-Aldrich  unless stated otherwise. For radiation treatment, nine-week-old male B6D2F1 mice were subjected to non-lethal total-body irradiation at 300 cGy delivered in a Cs-137 small animal irradiator. Three hours after irradiation, the mice were inoculated with 0.5 mg dexamethasone sodium phosphate  by subcutaneous injection. The use of dexamethasone has been reported to increase leukemia incidence by up to 50% in irradiated SJL/J mice . Mice beN = 13), 7.5/0 (N = 13), 15/0 (N = 10), 30/0 (N = 10), 7.5/10 (N = 10), 7.5/40 (N = 10), 30/10 (N = 10), and 30/40 (N = 10). The individual mouse was considered to be an experimental unit. The sample size calculation was based on 30% RI-AML incident rate [N = 5 per group); 0/0, 7.5/0, 7.5/40 for 7 months (N = 5 per group).The main cohort Fig.  consisteThe body weight of all irradiated B6D2F1 mice was measured weekly for the 70 weeks post-irradiation observation period. Overt leukemia was suspected when the subject lost 20% of its body weight, showed signs of illness, and presented leukemic blasts in its tail vein peripheral blood (PB) smear. Diagnosis of AML was made based on the Bethesda proposals for classification of nonlymphoid hematopoietic neoplasms in mice . The micBMCs for each mouse were obtained from the remaining hind limb bones by flushing the medullary cavity with PBS containing 2% fetal bovine serum . Mature RBC from the BMCs were lysed using ACK lysing buffer (Life Technologies). The BMCs were then washed and resuspended in PBS with 2% FBS for subsequent flow cytometry and DNA/RNA/protein analysis  between different treatment groups was plotted on Kaplan-Meier (KM) survival curves and analyzed by the Mantel-Cox test or log-rank test for trend. All statistical tests were two sided. Clustergram for RT2 Profiler PCR Arrays was created by the web-based algorithm provided by QIAGEN.All figures and statistical analysis were prepared by GraphPad Prism 5  or Microsoft Excel. Data were presented as mean ± SD. Statistical significance was determined by Student’s t-test or analysis of variance (ANOVA) and set at + population, was also expanded in the PB in comparison to B220+CD3e+ lymphoid population  hematopoietic populations  of the 86 mice were found dead or needed to be sacrificed due to illness Table . Among tion Fig.  d, e. Thion Fig. , homogenion Fig. , expansions Fig. , and theMCs Fig. .Table 1P = 0.06 and P = 0.12, respectively) or with each other .Based on the Bethesda proposals for classification of nonlymphoid hematopoietic neoplasms , 7 mice P < 0.01). The mice that receive high-dose deferasirox at 40 mg/kg/day had inferior AFS than their low-dose counterpart . Low-dose ICT had the opposite effect on the 30 mg iron-loaded group, in which a more rapid AML onset with inferior AFS was observed after deferasirox treatment  from a separate cohort of mice at earlier intervals of 5 and 7 months after irradiation Fig. . We injeAssessments of the iBMCs from the earlier cohort alongside the AML group suggested a pattern of progressive molecular changes including alteration of signaling pathways, DNA damage response, and gene expression pattern see . We charP < 0.01). Low-dose ICT with the 30 mg iron-loaded mice might have decreased the pro-death effect of iron, but ironically enabled the survival of AML-initiating cells, thereby increased the RI-AML rate .We proposed a model to describe the observed RI-AML rate in relation to iron burden Fig. . In thisAnemic MDS patients acquire iron overload as a consequence of chronic transfusion . HoweverAt 7.5 mg, iron was a potent promoter of RI-AML with shortened latency at 6 months and 38% incidence rate. Analysis of the early cohort suggested further gene expression dysregulations in the iiBMCs when compared to iBMCs, which is consistent with the notion of a more advanced pre-leukemic stage for the iiBMCs. The amount of iron burden in this study is generally consistent with the literature and reasonably translatable to the clinical setting. First, a large portion of the reported studies on iron-dependent carcinogenesis were conducted using iron dextran at approximately 21 g . NeverthWe observed a biphasic relationship between iron burden and RI-AML rate, possibly as the combined pro-mutagenic and pro-death effects of iron. A similar biphasic model has been proposed for radiation oncogenesis  and demoRUNX1-S291fs mutation [There were several limitations in our study. We screened a wide range of iron burden by means of iron loading and chelation dosage, which enable the establishment of the relationship between secondary iron overload and RI-AML. Conversely, the statistical power of our findings is limited by the relatively small sample size per treatment group. Jin et al. reported impaired hematopoietic progenitors and shortened overall survival upon iron loading in a MDS mouse model that utilize the mutation . Howevermutation , 19. In In this proof-of-concept study, we have demonstrated the ability of iron to promote RI-AML. We also observed a biphasic relationship between iron burden and the rate of RI-AML. ICT reduced the risk of RI-AML at low iron burden, but the effect was reversed at high iron burden. Our findings may have clinical implications to MDS patients who have secondary iron overload due to chronic RBC transfusion.Additional file 1.


DNA double strand breaks (DSBs) are repaired in eukaryotes by one of several cellular mechanisms. The decision-making process controlling DSB repair takes place at the step of DNA end resection, the nucleolytic processing of DNA ends, which generates single-stranded DNA overhangs. Dependent on the length of the overhang, a corresponding DSB repair mechanism is engaged. Interestingly, nucleosomes—the fundamental unit of chromatin—influence the activity of resection nucleases and nucleosome remodelers have emerged as key regulators of DSB repair. Nucleosome remodelers share a common enzymatic mechanism, but for global genome organization specific remodelers have been shown to exert distinct activities. Specifically, different remodelers have been found to slide and evict, position or edit nucleosomes. It is an open question whether the same remodelers exert the same function also in the context of DSBs. Here, we will review recent advances in our understanding of nucleosome remodelers at DSBs: to what extent nucleosome sliding, eviction, positioning and editing can be observed at DSBs and how these activities affect the DSB repair decision. DNA double strand breaks are a highly toxic form of DNA damage, arising from intrinsic and extrinsic sources . EukaryoThe cellular DSB repair pathway decision is made at the step of DNA end resection, the nucleolytic processing of DSB ends . ResectiNucleosomes form obstacles to the resection nucleases . InitialNucleosome remodelers have received attention as regulators of DNA end resection and DSB repair pathway choice. These nucleosome remodelers are enzymes that in ATP-dependent fashion catalyze the breakage of histone-DNA-contacts within the nucleosome and translocate the DNA relative to histone proteins . All eukIn many eukaryotes, DNA end resection is carried out by three resection enzymes, Mre11-C, STR-Dna2 and Exo1, which act specifically at one of the two stages of the resection process initiate and thereby allow to bypass the nucleosome barrier. Currently, such an auxiliary role of Mre11-C in long-range resection lacks experimental support, but recent data suggest that short-range and long-range resection nucleases work in a coordinated fashion .Alternatively, resection enzymes will need assistance by chromatin remodelers to get past nucleosomes and it is therefore important to consider how these enzymes may be able to modify the nucleosome barrier.Eukaryotes express several nucleosome remodelers  and chroNucleosome remodelers are found to be either single protein enzymes or multi-protein complexes. Historically, four major sub-families of remodelers have been proposed , but phyIn vitro and in vivo we can discriminate at least three activities of nucleosome remodelers: 1) sliding/eviction leads to movement of nucleosomes along DNA that can even result in the removal of the entire nucleosome  posicleosome ; 3) editcleosome . Based ocleosome . In the in vivo , which cleaves preferentially non-nucleosomal DNA. Several studies that used MNase to investigate nucleosome localization around a single DSB showed eviction of DSB-proximal nucleosomes, but came to different conclusions as to whether DSB-distal nucleosomes would shift their position , ETL1 (from mouse) and SMARCAD1 (from human) are the prototypical members of a sub-family of nucleosome remodelers that is evolutionary conserved throughout eukaryotes . Historifun30 mutants scored similarly to mutants deficient in the long-range resection nucleases, when they were initially found in screens for resection-dependent repair of DSBs  in thisst cells . Rad9 orst cells , suggestst cells .In budding yeast cells lacking both Fun30 and Rad9, resection and nucleosome eviction are fully functional , suggestAlso human SMARCAD1 antagonizes 53BP1. Depletion of SMARCAD1 stabilizes 53BP1 around DSB sites . HoweverIn all, we think that previous studies collectively indicate that nucleosome remodelers may serve similar roles during DSB repair as during gene transcription with nucleosome eviction, editing and potentially even positioning taking place at DSBs. Knowledge of the specific activities of individual nucleosome remodelers and of their redundancies thereby offers the potential to get to grips with chromatin changes occurring at DSBs. Moreover, we think that studies of DSB resection and repair may be generally inspired by analogies to gene transcription. Both processes appear to be similarly affected by the presence of chromatin, with nucleosomes forming a dynamic barrier and nucleosome remodelers facilitating its bypass.Importantly, while nucleosomes clearly form a barrier to the resection nucleases, nucleosome remodelers equip cells with multiple ways to overcome this barrier. In this review, we have outlined several putative mechanisms of how bypass may occur. These include eviction, sliding and editing of nucleosomes. While we are still only beginning to understand how the nucleosome barrier is overcome, a key future question will be which bypass mechanism is chosen in which cellular scenario. Importantly, the nucleosome barrier and its dynamic nature offers additional possibilities to regulate resection and DSB repair. Moreover, critical factors of the DSB repair decision, such as 53BP1 and BRCA1, are proteins that bind and modify nucleosomes. Therefore, we propose that convergence of resection-regulatory pathways on nucleosomes is a central part of the cellular DSB repair decision.


The increase in [Ca2+]i involved Ca2+ uptake through two pathways, one blocked by oxidized ATP  and another that was blocked by the TRPV-1 antagonist A784168 (100 nM). PEI stimulation also increased IL-33 mRNA expression and protein secretion. In vivo experiments showed that acute (4.5 h) PEI exposure stimulated secretion of Th2 cytokines (IL-5 and IL-13) into bronchoalveolar lavage (BAL) fluid. Conjugation of PEI with ovalbumin also induced eosinophil recruitment and secretion of IL-5 and IL-13 into BAL fluid, which was inhibited in IL-33 receptor (ST2) deficient mice. In conclusion, PEI-induced oxidative stress stimulated type 2 immune responses by activating ATP-dependent Ca2+ uptake leading to IL-33 secretion, similar to allergens derived from Alternaria.Polyethyleneimine (PEI) induced immune responses were investigated in human bronchial epithelial (hBE) cells and mice. PEI rapidly induced ATP release from hBE cells and pretreatment with glutathione (GSH) blocked the response. PEI activated two conductive pathways, VDAC-1 and pannexin 1, which completely accounted for ATP efflux across the plasma membrane. Moreover, PEI increased intracellular Ca Removal of Ca2+ from the extracellular solution abolished the increase in [Ca2+]i induced by PEI . This result indicated that the PEI-stimulated increase in [Ca2+]i was due to an increase in Ca2+ uptake across the plasma membrane. The PEI-evoked increase in [Ca2+]i was more rapid than that produced by Alternaria, but similar to the more transient Ca2+ response induced by HDM. 10Panx (50 μM), DIDS (100 μM) and GSH (5 mM)) reduced PEI-stimulated Ca2+ uptake by amounts corresponding to their inhibitory effects on PEI-stimulated ATP release.hBE cell exposure to PEI induced a sustained increase in intracellular i . p < 0.0001, n = 5). The kinetics of PEI and ATP-induced Yo Pro-1 uptake are shown in Stimulation of hBE cells with PEI produced an increase in uptake of the fluorescent cationic dye Yo Pro-1 (mw 629). n = 55) also caused DNA fragmentation as revealed in comet assays that show significant (†p < 0.0001) tail formation following exposure for 30 min (n = 70) or with inhibitors of ATP release  or 10Panx , DNA fragmentation was significantly (* p < 0.0001) blocked. Similarly, when PEI (5 μg/mL) stimulated Ca2+ uptake is blocked by combined pretreatment with oATP (300 μM) and A784168 (100 nM), DNA damage was also inhibited .PEI . Furthermore, increased expression of IL-33 mRNA was blocked (†p < 0.0008) under conditions where oxidative stress was inhibited by pretreatment with 5 mM GSH or when the PEI-induced increase in intracellular [Ca2+] was blocked by removal of extracellular Ca2+ or when ATP release was inhibited with both DIDS (100 μM) and TVM (20 μM)  by the same pretreatment conditions that also reduced IL-33 mRNA expression ( (20 μM) A. PEI alpression C.p = 0.0113; * p = 0.0260) and IL-33 (* p = 0.0324) in BAL fluid after 4.5 h  in plasma IgE levels that were not significantly reduced in ST2 deficient mice  and cockroaches stimulate ATP release from airway epithelial cells, which functions as a critical early step in the processing, nuclear mobilization, and secretion of IL-33 ,38,39,40derstood ,43. In aderstood . Furtherderstood ,41. Fromderstood . 2+]i in response to ATP release i, providing evidence of an underlying role for purinergic signaling in regulation of the PEI response. Similarly, reducing ROS levels with GSH nearly abolished the PEI-stimulated increase in [Ca2+]i, indicating that, like Alternaria, inhibition of ATP release by reducing oxidative stress also blocks the increase in [Ca2+]i. Removal of extracellular Ca2+ completely inhibited the effect of PEI on [Ca2+]i, demonstrating that Ca2+ uptake was responsible for increasing [Ca2+]i, as previously shown for Alterneria i was produced following PEI exposure, demonstrating that PEI-stimulated Ca2+ uptake can be completely accounted for by activation of oATP and A784168 sensitive pathways. In an earlier study, the increase in [Ca2+]i evoked by Alternaria extract was completely blocked by oATP, which was shown to be the result of P2X receptor inhibition [2+]i suggesting that TRPV1 does not appear to be not involved in mediating Ca2+ uptake induced by Alternaria extract  and organic cation uptake, PEI exposure produced DNA fragmentation. Inhibiting PEI-induced oxidative stress with GSH significantly reduced fragmentation, as did inhibition of ATP release and Ca2+ uptake. Genotoxic effects of linear and branched PEI polymers were investigated previously using a human squamous carcinoma cell line (A431 cells). Branched, but not linear PEI polymers, induced some tail formation in comet assays, indicating DNA fragmentation [2+] have inhibitory effects on DNA damage that are similar to blocking oxidative stress with GSH. Given that the increase in [Ca2+]i is downstream of PEI-induced oxidative stress, it seems likely that raising intracellular [Ca2+] may stimulate DNase activity, perhaps by activating caspase 3. Further experiments will be required to determine whether increased caspase 3 activity is involved in mediating the effects of PEI on DNA fragmentation.In addition to stimulating ATP release and increasing both intracellular i or blocking ATP release reduced the response. The increase in mRNA expression was associated with a concentration-dependent increase in IL-33 secretion into the extracellular fluid, which was also inhibited by the same pretreatment conditions that reduced IL-33 mRNA expression and similar to results of previous studies showing that Alternaria stimulates IL-33 secretion  using an ATP calibration curve (range: 0.1–100 nM) and expressed as nM/cm2+ concentration ([Ca2+]i). Fluorescence was measured using a Nikon UV 20× objective at excitation wavelengths of 340 nm/380 nm and an emission wavelength of 510 nm. MetaMorph software  was used for image acquisition and data analysis. Relative changes in [Ca2+]i were determined and expressed as the fluorescence ratio when the cells were excited at 340 nm and 380 nm (F340/F380).hBE cells grown on chamber slides (48–72 h), they were washed with HBSS containing 10 mM HEPES buffer and incubated with Fura-2 AM for 1 h. The cells were washed again and replaced with HBSS buffer, then mounted onto the stage of an inverted fluorescence microscope for measurements of intracellular Cahttps://micro-manager.org/ access date: 23 October 2017). A similar protocol was used for the rhodamine B uptake experiments, where cells were incubated in HBSS containing 0.5 μM rhodamine B for 10 min prior to addition of Alternaria (100 μg/mL) for 15 min. Images of the cells were acquired using a 20× fluorescence objective (peak excitation/emission λ = 545/570 nm).hBE cells were grown on two-well chamber slides for 48 h prior to use in dye uptake experiments. Culture media was replaced with 1 mL HBSS solution containing 10 mM HEPES, pH 7.4 and 2 μM Yo Pro-1. Chamber slides were then mounted onto the stage of a fluorescence microscope and images of the cells were acquired with a Prime 95B sCMOS digital camera  using a 40× fluorescence objective (peak excitation/emission λ = 490/520 nm). Time course experiments were initiated with the addition of PEI (5 μg/mL) or Alternaria (100 μg/mL) or ATP (250 μM) and images of the cells were captured at 0.5 or 1.0 min intervals for 15 min and analyzed using Micro-Manager 1.4 software . After PEI exposure, cells were trypsinized and combined with molten LM Agarose (37 °C) at a 1:10 v/v ratio. The combined cell-LM Agarose solution was pipetted onto comet slides and placed flat at 4 °C in the dark for 10 min, then slides were immersed in 4 °C lysis solution for 30–60 min to lyse the cells. To unwind and denature the DNA, the slides were immersed in alkaline electrophoresis solution  for 20 min and then placed in an electrophoresis slide tray with a protective overlay on top. Gel electrophoresis was performed using the CometAssay® ES unit, with 4 °C alkaline electrophoresis solution at 21V for 30 min. The slides were gently immersed twice in distilled H2O, then in 70% ethanol for 5 min each. After drying the slides at 37 °C for 10 min, SYBR® Gold staining solution containing 10 mM Tris-HCl, 1 mM EDTA was placed onto the dried agarose and stained for 30 min in the dark. DNA fragmentation was visualized using an inverted fluorescence microscope with a Nikon 10x fluorescence objective at excitation/emission wavelengths of 496 nm/522 nm. Quantitative and statistical analyses were performed using CometScore 2.0 software (http://rexhoover.com/index.php?id=cometscore access date: 22 April 2021) and DNA fragmentation was expressed as percent DNA in the head and tail.The effects of PEI on DNA fragmentation were examined using a CometAssayTotal RNA was extracted from hBE cells transfected with the full-length human IL-33 gene (GET-33 cells) using RNeasy Mini Kit . One µg of total RNA quantified by Qubit Fluorometric assay  was reverse transcribed to cDNA using a High Capacity RNA to cDNA kit (ThermoFisher). One µg cDNA, TaqMan probes , and TaqMan Fast Advanced Master Mix (ThermoFisher) were used to preform qRT-PCR on a Step-One-Plus Real-time PCR machine. The expression level was normalized to the threshold cycle number (Ct) of an internal reference gene (GAPDH).−/− (Il1rl1−/−) mice on a BALB/c background were kindly provided by Dr. Andrew N. McKenzie . All animal experiments were performed with the approval of and following the regulatory guidelines and standards set by the Institutional Animal Care and Use Committee of Mayo Clinic . Female mice ages 6–13 weeks old were held under specific pathogen-free conditions prior to use in experiments. Wild-type (WT) BALB/c mice were purchased from Jackson Laboratories. ST2−/− mice were intranasally challenged with 10 µg ovalbumin (OVA) in the absence of an adjuvant with or without 25 µg PEI following the treatment scheme below. After the final administration bronchoalveolar lavage (BAL) fluid was collected through a tracheostomy tube at 1 h after euthanasia with pentobarbital. Cell numbers in BAL fluid were counted using a hemocytometer. Eosinophils, neutrophils, lymphocytes, or macrophages were identified using standard morphologic criteria under light microscopy and percentages of these cells were determined.For acute PEI exposure, WT BALB/c mice were administered a single intranasal (i.n.) dose of phosphate buffered saline solution (PBS) with 12.5 µg PEI under isoflurane anesthesia. In chronic exposure experiments, WT BALB/c or ST2Measurement of IL-5, IL-13, and IL-33 concentrations in BAL fluid and GET-33 cell culture media or Hank’s buffered saline solution (HBSS) were performed using Quantikine or Duoset ELISA kits , following the manufacturer’s instructions. Note that sensitivity for IL-5 was 8 pg/mL, for IL-13, 4 pg/mL and for IL-33, 14 pg/mL. Protein concentrations in the BAL fluid were quantitated using a BCA Protein Assay Kit .IgE measurements were performed following the protocol by Kobayashi et al., 2009 . BrieflyData are presented as means ± standard error. Statistical comparisons between means from multiple treatment conditions were determined using a Brown-Forsythe and Welch one-way ANOVA followed by Dunnett’s T3 posttest or by a standard one-way ANOVA followed by Tukey’s multiple comparisons test. A two-tailed, unpaired t test was used for statistical comparisons between two means. Graphics and statistical analyses were performed using GraphPad PRISM 8.0 .2+]. Most of the ATP release occurred in response to oxidative stress and Ca2+ uptake was required for increasing IL-33 mRNA transcription and secretion into the extracellular media. These findings indicate that mucosal adjuvanticity associated with PEI alone or with PEI polyplexes requires early induction of ATP release as a means of promoting IL-33 mobilization and secretion from the airway epithelium. Subsequent interaction of IL-33 with ST2 receptors associated with ILC2 cells, Th2-type CD4+ T cells, and perhaps other inflammatory cells facilitates the production and release of Th2 cytokines like IL-5 and IL-13, which ultimately leads to development of an allergic inflammatory response.The findings of the present study provide new insights into the mechanisms by which PEI induces type 2 immunity. The airway epithelial response to PEI exposure was similar to that of certain allergens that stimulate ATP release and subsequently increase intracellular [Ca


Histone methylation tightly regulates chromatin accessibility, transcription, proliferation, and cell differentiation, and its perturbation contributes to oncogenic reprogramming of cells. In particular, many myeloid malignancies show evidence of epigenetic dysregulation. Jumonji C (JmjC) domain-containing proteins comprise a large and diverse group of histone demethylases (KDMs), which remove methyl groups from lysines in histone tails and other proteins. Cumulating evidence suggests an emerging role for these demethylases in myeloid malignancies, rendering them attractive targets for drug interventions. In this review, we summarize the known functions of Jumonji C (JmjC) domain-containing proteins in myeloid malignancies. We highlight challenges in understanding the context-dependent mechanisms of these proteins and explore potential future pharmacological targeting. Non-genetic chromatin alterations play a significant role in disease development, maintenance, and relapse of myeloid malignancies. Among epigenetic modifying enzymes, histone demethylases have gained special attention in recent years for their involvement in myeloid malignancies. Histone lysine demethylases (KDMs) are subdivided into two subclasses, FAD-dependent and Jumonji C (JmjC) domain-containing (JMJD) demethylases . AlthougFor all abbreviations of important proteins used in the context of normal hematopoiesis and myeloid malignancies, please refer to 2 .,12.11,12ML cells . These mML cells . As a meML cells . These dG12D-driven AML mouse model. Mice displayed shortened survival upon loss of Kdm2b, while overexpression significantly extended survival of KrasG12D-induced AML . Me. Me31]. 1/2 at the NFE2 promoter region, resulting in a positive feedback loop . Lo. Lo35].  complex ,90,91. P complex . Thus, K+ cells, indicating an involvement of KDM4B in AML leukemogenesis  demethylase ALKBH5 through removal of repressive H3K9me3 at its promoter . Kn. Kn+ cel+ HSCs. This is crucial as KDM5 expression is required for normal stem cell function and therapeutic applicability might thus be hampered by toxicity.In AML, Wong et al. demonstrated that KDM5B negatively regulates leukemogenesis in both mouse and human MLL-rearranged AML cells  through 3 demethylation) . KD. KD+ BM pression . Fittingpression . Likewispression . Togethe3 and increases H3K4me3 levels through its demethylase activity and association with the H3K4 methyltransferase complex, respectively [Although the abovementioned studies focus on KMD6B as a potential oncogene in disease initiation and progression of myeloid malignancy, Yu et al. found a significant role of KDM6B in the differentiation of AML FAB subtypes M2 and M3. Combining ChIP and RNA-sequencing the authors show that KDM6B enables the expression of myeloid differentiation genes. Mechanistically KDM6B reduces H4K27meectively . CEBPB iectively . Knockdoectively . As showThe KDM7 subfamily of the Jumonji domain-containing histone demethylases encompasses three members, KDM7A, KDM7B, and KDM7C. As reviewed by Markolovic et al., all enzymes possess catalytic activity towards mono-, di-, and tri-methylated lysine residues . H3K9, HKdm5b knockout PB cells in a BM transplantation [KDM5B is constitutively knocked out, it is dispensable for HSC function. This raises the question whether other KDMs compensate for embryogenic loss of this enzyme. Another obstacle in defining the role of individual KDMs derives from contradictory, context-dependent findings. For example, KDM2B has been described as oncogenic by repressing the tumor suppressor p15 in AML [Recent research on JmjC domain-containing histone demethylases has revealed clear contributions to disease initiation and progression in myeloid malignancies. However, this emerging field faces several challenges including heterogeneity, potential functional redundancy, and substantial context-sensitivity of function both between KDM families and among members of individual families. For example, KDM5B is required for normal HSC function as witnessed by compromised reconstitution of antation . However5 in AML . However5 in AML ,70,71. L5 in AML .Evidence suggesting oncogenic potential has led to the development of pharmacological KDM inhibitors. Future studies will determine if inhibition of a single KDM is sufficient to reduce leukemic growth or whether other KDMs compensate, abrogating any effect. Moreover, is there a therapeutic window, can healthy cells be spared? Given the high degree of similarity between individual KDMs, it seems likely that inhibitors will affect multiple members of the same family. Nonetheless, with careful drug design and optimization, KDM inhibition might serve to overcome non-genetic drug resistance in myeloid malignancies ,138,139.


However, the mechanisms by which extracellular Ca2+ (Ca2+ex) and the CaSR mediate networks of intracellular Ca2+-signaling and players involved throughout the life cycle of CaSR are largely unknown. Here we report the first CaSR protein–protein interactome with 94 novel putative and 8 previously published interactors using proteomics. Ca2+ex promotes enrichment of 66% of the identified CaSR interactors, pertaining to Ca2+ dynamics, endocytosis, degradation, trafficking, and primarily to protein processing in the endoplasmic reticulum (ER). These enhanced ER-related processes are governed by Ca2+ex-activated CaSR which directly modulates ER-Ca2+ (Ca2+ER), as monitored by a novel ER targeted Ca2+-sensor. Moreover, we validated the Ca2+ex dependent colocalizations and interactions of CaSR with ER-protein processing chaperone, 78-kDa glucose regulated protein (GRP78), and with trafficking-related protein. Live cell imaging results indicated that CaSR and vesicle-associated membrane protein-associated A (VAPA) are inter-dependent during Ca2+ex induced enhancement of near-cell membrane expression. This study significantly extends the repertoire of the CaSR interactome and reveals likely novel players and pathways of CaSR participating in Ca2+ER dynamics, agonist mediated ER-protein processing and surface expression.G-protein-coupled receptors (GPCRs) are a target for over 34% of current drugs. The calcium-sensing receptor (CaSR), a family C GPCR, regulates systemic calcium (Ca CaSR helps maintains a tight systemic Ca2+ homeostasis between the extracellular space (10−3 M) and cytosol (10−7 to 10−8 M) to control numerous processes including cellular communication, secretion, apoptosis, chemotactic responses, cell proliferation, cytoskeletal rearrangements, ion channel activity, the control of gene expression, and cell differentiation6. Thus, homeostasis is critical for many (patho)physiological processes in multiple organs, including parathyroid, kidney, heart, bone, brain, and skin7. CaSR activated by extracellular Ca2+ (Ca2+ex) triggers multiple intracellular signaling pathways transduced through heterotrimeric G proteins; Gq/11, Gi/o, G12/13, and Gs11 , including, Ca2+cyt, Ca2+ER and mitochondrial Ca2+ (Ca2+mito)14. One of the major CaSR mediated Gq/11 transduced pathways involved the activation of phospholipase C (PLC), which in turn raises the cytosolic Ca2+ (Ca2+cyt) and results in Ca2+cyt oscillation through inositol triphosphate (IP3) induced endoplasmic reticulum Ca2+ (Ca2+ER) release12.Calcium-sensing receptor (CaSR) belongs to class-C of the largest cell surface receptor family, the G-protein-coupled-receptors (GPCR’s), which are targets to over 34% of the Food and Drug Administration (FDA) approved drugs in the United Statess11 Fig. . Thereof2+ signaling is integral to protein biosynthesis and trafficking, including regulation of CaSR abundance, as well as inducing its active conformation and dictating its dynamic life cycle15. Prolonged exposure to Ca2+ex is known to mobilize the intracellular pool of nascent CaSR from the ER16, Golgi, and ER-Golgi intermediate compartments (ERGIC) to the plasma membrane through anterograde transport15. CaSR is an exceptional receptor with minimal functional desensitization in the chronic presence of agonist17. Subsequently, CaSR undergoes retrograde transport to lysosome or proteasome for degradation15. Perturbation in protein biosynthesis and trafficking are observed in physiological disorders, including diabetes mellitus and vascular and neurological diseases that alter Ca2+ mediated signaling, such as in the ER15. Additionally, mutations in CaSR and its binding partners and subsequent dysfunctions in CaSR mediated Ca2+ signaling are closely associated with calciotropic , neonatal severe hyperparathyroidism (NSHPT), autosomal dominant hypocalcemia (ADH), and secondary hyperparathyroidism) and noncalciotropic disorders 20.This CaSR mediated crosstalk between the extra- and intra- cellular Ca31. Proteins involved in anterograde trafficking of CaSR are small GTP binding proteins , cargo/chaperones  and interacting proteins . Further, CaSR endocytosis is facilitated by G protein receptor kinases (GRKs)29, protein kinase C30 and β-arrestins31. A recent study has shown an interaction between CaSR and AP2S1, and AP2S1 has been shown to facilitate CaSR endocytosis33. Finally, CaSR is degraded in the proteasome or lysosome following ubiquitination by E3 ubiquitin ligase, dorfin34. While the physiological significance of CaSR-mediated Ca2+ signaling and information on sparse CaSR-binding partners has been established, knowledge related to how Ca2+ex and CaSR orchestrate Ca2+ dynamics and signaling as well how they harmonize key regulators for critical cellular processes is incomplete. This is due in part to limitations involved in studying membrane proteins, and challenges in capturing transient interactions32. In our study we have aimed to capture proteins that interact with CaSR throughout its life cycle from signaling, internalization, endocytosis and synthesis to insertion through agonist-derived insertional signaling (ADIS)15.To date, only a handful of CaSR interactors have been identified2+ex in HEK293 cells. Many previous CaSR related studies have been carried out in HEK293 cells35. We mapped 94 novel putative and 8 previously published CaSR interactors. Further, we revealed a distinct Ca2+ex dependent enrichment in gene ontology annotations related to the ER. Moreover, we characterized Ca2+ex mediated interactions of CaSR with two major regulatory proteins, vesicle-associated membrane protein-associated A (VAPA) involved in anterograde trafficking37 from the ER to the Golgi and 78-kDa glucose regulated protein (GRP78/Bip/HSPA5) involved in protein processing in the ER38. We have shown that CaSR and VAPA are inter-dependent of each other for the Ca2+ex enhanced near-cell membrane expression in Cos7 cells. Thus, combining our study with known functions, we provide a larger repertoire of the CaSR interactome in HEK293 cells, and reveal likely novel players and pathways of CaSR participating in: Ca2+ER dynamics; agonist mediated ER-protein processing, such as by GRP78; and surface expression via key regulators such as VAPA in different tissues. The established CaSR-PPI offers a novel paradigm for understanding the molecular bases of CaSR associated diseases and facilitating drug development.To address the above long-standing question, we have characterized the first CaSR-protein–protein interaction (CaSR-PPI) network using quantitative proteomics with tandem mass spectrometry (LC–MS/MS) coupled with Co-IP, with and without Ca41. Further, to establish the role of Ca2+ex in interactions, the serum-starved cells were treated with either 4 mM Ca2+ex 43 or 2 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetra acetic acid (EGTA) (to chelate Ca2+ex), followed by subjection to immunoprecipitation with anti-FLAG antibody, LC–MS/MS (90% sample) and western-blot (10% sample)  or empty pcDNA3.1 (negative control)le) Fig. . Serum s2+ex and EGTA treatment conditions  treatments and IP for the positive and negative controls were performed in triplicates; (ii) identified proteins were at least twofold enriched in the CaSR transfected samples over the negative controls, i.e., log2 (HEK293 + CaSR-FLAG pcDNA/Hek293 + pcDNA3.1) ≥ 1.00; (iii) P-values were calculated using a two-sided Student’s t-test, with a null hypothesis that there is no difference in protein relative abundance between the two groups, and a two tailed t-test with P ≤ 0.05 between the groups being statistically significant; (iv) proteins had a minimum peptide spectrum match (PSM) of two for at least two replicates, and; (v) proteins were identified with at least one unique peptide. To compliment the study, LC–MS/MS was carried out on the total cell lysates to discern if the changes in enrichment after IP were due to the change in the total cellular expressions of these protein after Ca2+ex or EGTA treatments. In all, 88% of the 106 proteins of interest displayed no significant change in the total protein expression  ≤ 0.58 and >  − 0.58])  Fig. , implyini)45, GNB2 (Gβ2)45, YWHAQ (14–3-3 θ)28, YWHAZ (14-3-3 ζ)28, YWHAE (14-3-3 ε)28, YWHAG (14-3-3 γ)28, TMED9 (p24)23 and CANX 15. Additionally, 44 were exclusively identified in the presence of Ca2+ex, 13 exclusively in EGTA, and 45 were found common in both conditions  ≥ 1, -log10 (P) ≥ 1.3] potential CaSR interactors in the presence of Ca2+ex  ≥ 1.58]. CaSR had the highest log2 intensity (fold change) in both samples treated with either Ca2+ex at 11.61 ± 1.07 [and −log10 (P) = 3.05] or EGTA at 14.32 ± 0.76 [and −log10(P) = 3.85].Out of 102 CaSR interacting partners, eight previously known CaSR interactors involved in signaling and trafficking were re-validated: GNAI   = 1.00–5.42)  =  ≤ − 0.58]. These proteins included 14-3-3-θ (YWHAQ), 14-3-3-ζ (YWHAZ), 14-3-3-ε (YWHAE), Tubulin α-1C (TUBA1C) and THO complex subunit 4 (ALYREF)  ≤ 0.58 and >  − 0.58]  Fig. . Remarka2+ex enriched CaSR interactors, consisting of cellular compartment, molecular function, and biological process using Database for Annotation, Visualization, and Integrated Discovery (DAVID), version 6.848 52. The CaSR-PPI scored a local clustering coefficient of 0.492 with 1062 edges and a PPI enrichment of P < 1.00E−16 with high confidence 45 and GNB2 (Gβ2)45 57 57  63   designed with Ca2+ binding site on the surface of the beta barrel in a chromophore sensitive location of enhanced green fluorescent protein (EGFP)70. The inositol 1,4,5-triphosphate receptor (IP3R) is a Ca2+ release receptor, and sarco-endoplasmic reticulum calcium ATPase (SERCA) is a Ca2+ uptake receptor located on the membranes of the ER that regulate Ca2+ER  as compared to that in mono-expressed cells . This not only reiterated that Ca2+ex drives CaSR cell membrane expression, but also confirmed that overexpression of VAPA amplified this process. This implied that VAPA could play a prominent role in Ca2+ex-sensing of CaSR by controlling ER-protein processing and near surface expression.To explore the role of VAPA in CaSR expression and function, live cell imaging of Cos7 cells either expressing both EGFP-CaSR and mCherry-VAPA   Fig. B38. Our 457 Fig. . We furtlot Fig. . Furtheral Figs.  and 4 mMns Figs.  using hills Fig. . Furtherlls Fig. B.2+ex in signaling, systemic Ca2+ homeostasis and consequently, in many calciotropic and non-calciotropic (patho)physiological conditions19. Numerous studies have reported the role of Ca2+ER in protein processing, trafficking, and synthesis. However, both the players and the mechanism regarding how the Ca2+ex and the CaSR mediate networks of intracellular Ca2+-signaling and related processes remain largely unknown. In our study, we report the first richly annotated CaSR-PPI with 102 interactors, of which 94 are novel. Our comparative proteomics study with low and high Ca2+ex, along with functional studies and imaging, reveal a Ca2+ex dependent alteration of CaSR signaling networks, with two-third of the 102-PPI preferably occurring in high Ca2+ex. However, the effects of Ca2+ex resonates through proteins involved in downstream processes including major Ca2+ dependent organellar processes, such as the GPCR signaling, ER related protein processing and trafficking, quality control, endocytosis, and mitochondria transportation  that act as chaperones, buffers, and channels involved in maintaining [Ca2+] in intracellular organelles74, such as SERCA2b53, calnexin (CANX), GRP78, GRP94, PDIA375 and TMCO154, are visualized to be enriched in our proteomics data. Studies have identified a direct link of Ca2+ binding SERCA-2b with CaSR76 and glucagon receptor 39. CaSR calcilytic has been shown to attenuate CaSR-induced sarcoplasmic reticulum (SR)-mitochondria crosstalk in a rat cardiomyocyte model14. The presence of mitochondrial complexes involved in Ca2+i handling and in oxidative stress/early events of apoptosis56, including, VDAC2, SLC25A, TIMM50, ATP5A1, HSPD1 and NDUFA4, and as well as PRDX1in the cytosol, may indicate indirect binding with CaSR, the presence of mitochondrial CaSR77, or a role of CaSR in Ca2+ mediated oxidative stress78. Meanwhile, during trafficking, Ca2+ex is known to mobilize the intracellular ER pool of CaSR to the plasma membrane79. ER chaperones and regulators are dependent on optimal [Ca2+ER] for proper post-translational processing, folding, and export of proteins81. Our proteomic and gene ontology results delineate a direct link of the Ca2+ex/CaSR mediated Ca2+ER signaling to protein biosynthesis and quality control in the ER based on the enrichment of a significant number of proteins pertaining sequentially to protein-folding, glycosylation and unfolded protein responses in the ER82. Identification of interactors such as CANX further validates the biological significance of Ca2+ex dependent CaSR-PPI, as CANX is a known CaSR interactor with high Ca2+ binding affinity and is known to interact transiently with various soluble non-native conformers of glycoproteins83.Interestingly, Ca2+ex manifested CaSR trafficking associated interactors, such as VAP proteins in the ERGICs is another major implication of our study. VAPA is known to form contact sites between ER and various organelles for Ca2+ and lipid regulation71 as well as to regulate the anterograde trafficking of properly-folded androgen receptor (a GPCR) and oxysterol-binding protein related protein 3 from the ER to Golgi61. VAPB is an interactor of GPCRs, such as glucagon39 and melatonin receptor type 1A44. The utility of the CaSR interactome was ascertained by reconfirming the MS data of Ca2+ex dependent interactions between GRP78 and VAPA, potentially regulating protein processing in the ER and trafficking, respectively, using orthogonal analysis of Co-IP and western blot. Additionally, the CaSR mediated Ca2+ex enhanced VAPA near cell membrane expressions as well as VAPA dependent Ca2+ex enhanced CaSR surface expressions were established. This supports the prior knowledge that Ca2+ex drives CaSR cell membrane expression. Our work further showed that overexpression of VAPA amplified this process. This implied that VAPA could play a prominent role in Ca2+ex-sensing of CaSR by controlling ER-protein processing and near surface expression. Similarly, CaSR mediated Ca2+ signaling regulated VAPA expression near the cell membrane, hence potentially increasing the ER-plasma membrane contact site establishments. This could explain the agonist driven rapid mobilization of CaSR to cell membranes that has been observed in previous studies15.Identification of Ca23, although a single peptide was identified. Additionally, ubiquitously expressed GTPases, Rabs  have been previously identified as CaSR interactors84. Our study complements this list with Rab659. Another protein involved with CaSR trafficking through ERGIC, 14-3-3 ζ, has a distinct function in lowering CaSR membrane expression32. Interestingly, our data shows that 14-3-3  binds CaSR at lower [Ca2+ex] and may retain CaSR to ER in the absence of Ca2+ by disruption of the contact. Degradation and endocytosis are major checkpoints for proper functioning of CaSR and the interactome elucidated in this study identifies additional players in these pathways. GPCRs including, adenosine, dopamine-, P2Y, PAR1, glucagon, GABA, mGluRs and CaSR receptor are known to undergo agonist-induced ubiquitination leading to internalization and lysosomal degradation65. Ca2+ dependence and the role of Ca2+ binding protein, such as CaM, in the regulation of ubiquitin is known85. Our result showing Ca2+ex dependent enrichment of E3 ubiquitin ligases, STUB1 and RanBP2 complements their study. The desensitization of CaSR occurs through endocytosis from the plasma membrane by Ras-related proteins and CLTC. In this study, we report the enrichment of Ca2+ex dependent regulators of endocytosis: Rab564, Rab1862 and CLTC.Our work has independently verified VAPA and GRP78 as CaSR interactors using Co-IP, western blot and imaging analysis. The remaining interactors discussed further in this study remain to be validated with additional direct biochemical studies, such as BRET and FRET. Our result identified p24, which is known to interact with CaSR early in the secretory pathway and assist in transportation to and from the ERGIC2+cyt oscillations which code for biological processes. Likewise, we propose that the Ca2+ER perturbation dictates protein maturation through the co-translational translocation and calnexin cycle of nascent CaSR polypeptides in the ER. Our data indicate detection of G-proteins, Gαi and Gβ2 exclusively in the presence of Ca2+ex. Heterotrimeric G-proteins are known GPCR interactors45 and Ca2+ex activated CaSR transduces diverse downstream signaling through GTP-bound Gα and Gβγ dissociation. Gαi mediates inhibition of the cAMP dependent pathway through inhibition of adenylate cyclase86. On the other hand, Gβ2 may modulate ion channels87, anterograde trafficking15, microtubule assembly88 and ubiquitination of GPCR89. Upregulation of the signal transducing G-proteins reaffirms the direct correlation of Ca2+ex and CaSR downstream signaling.CaSR signaling through G-proteins relays Ca2+ for 2 h Ca2+ treatment may possibly depict physiological interactions in tissues maintained at higher [Ca2+], such as bone environment (reported as high as 10 mM)90. Our interactome may also depict the severe physiological implication of high Ca2+ conditions, such as during severe hypercalcemia where the serum calcium levels are above 2.65 mM91. In our study we have aimed to capture proteins that interact with CaSR throughout its life cycle from signaling, internalization, endocytosis, synthesis to insertion through agonist-derived insertional signaling (ADIS)15. Our previous study reported that two-thirds of the CaSR remains on the cell surface and only 30% is internalized with 4 mM Ca2+ exposure for 2 h35. In order to understand the physiological relevance, we: (i) used live cell imaging  captured the temporal effect of CaSR activation in ER-Ca2+ change within few seconds  signal transduction, (ii) maturation of nascent polypeptides, (iii) trafficking, (iv) quality control through degradation, (v) desensitization and (vi) Ca2+i handling. This study expands the repertoire of the CaSR interactome through the identification of 94 putative novel CaSR interactors. We were also able to recapitulate eight previously identified interactors of CaSR and several interactors previously known for other members of GPCR family, indicating overlapping signaling cascade and intracellular processes. Additionally, our study provides further evidence of Ca2+ex dependent association of CaSR with important trafficking and protein processing proteins, VAPA and GRP78. Taken together, our work provides a powerful resource for research in Ca2+ex/CaSR signaling and putative targets for CaSR-based therapeutics.However, our work provides a powerful resource for elucidating Ca371 and Thr372) in pcDNA3.1 and EGFP-CaSR  were used for transfection for negative and positive controls, respectively. pEGFPC1-hVAPA and pcDNA3.1 GRP78 were obtained from Addgene. mCherry-VAPA was constructed from pEGFPC1-hVAPA (Addgen) and mCherry in pcDNA3.1. The EGFP was removed from pEGFPC1-hVAPA between restriction sites Nhel (895) and Xhol (985), and mCherry with the paired sticky ends was fused to the N terminus of VAPA. For TIRF imaging, mCherry-VAPA and EGFP-CaSR were used for the transfection. The purified plasmids were prepared using a Mini Prep Kit .Empty pcDNA3.1, human CaSR with FLAG-tag (FLAG-hCaSR) . At 48 h post-transfection, cells were washed with Hank’s Balanced Salt Solution (HBSS)  at 37 °C, incubated in starving medium low glucose DMEM, 0 mM Ca2+ with 0.1% BSA for 30 min and finally treated with various [Ca2+] or 2 mM EGTA at various time points. For TIRF imaging, Cos-7 cells were cultured on 22 mm × 40 mm coverslips pre-coated with Poly-l-lysine solution. Transient transfection with 1 µg of mCherry-VAPA or EGFP-CaSR was performed for the mono-transfection, and 1 µg of mCherry-VAPA plus 1 µg of EGFP-CaSR was performed for the co-transfection in each coverslip using lipofectamine 3000 in the same medium following the manufacturer’s protocol . Cell images were collected at 48 h post-transfection.Monolayer culture of HEK293 cells and Cos-7 cells were purchased from American Type Culture Collection (ATCC CRL-1573) and cultured with high glucose (4.5 g/L) Dulbecco’s Modified Eagle Medium (DMEM)  supplemented with 10% fetal bovine serum  in a humidified environment at 37 °C with 5% COAnti-FLAG M2, mouse  was used to precipitate the CaSR/interactor complex. Anti-CaSR C0493, mouse  and Anti-GAPDH mouse  were used for western blot. Anti-VAPA  and anti-GRP78 (ab21685), rabbit  were used for western blot and immunostaining. Goat anti-rabbit IgG-AP conjugate  and goat anti-mouse IgG-AP conjugate  were used as secondary antibodies for western blot. Anti-GFP antibody mouse  was used to immunostain GFP-CaSR. Donkey anti mouse IgG (H + L) Alexa fluor 647 , goat anti-mouse IgG (H + L) Alexa Fluor 488 , goat anti-mouse IgG (H + L) Alexa Fluor 555  and goat anti-chicken IgY H&L Alexa Fluor 488  were used as secondary antibodies for immunostaining.2+ or 2 mM EGTA for 2 h. These were then washed three times with ice cold phosphate-buffered saline (PBS) with 0 mM Ca2+. Next, 600 μL of 10 mM sodium β-glycerophosphate, 50 mM Tris–Cl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 2 mM Na3VO4, 50 mM NaF, 10 mM sodium pyrophosphate supplied with proteinase inhibitor cocktail  was used to lyse cells for 30 min in ice with frequent vortex, followed by centrifugation to pellet cell debris. Cleared cell lysates were subjected to anti-FLAG immunoprecipitation prior to immunoblotting.Transfected HEK293 cells from 90 to 100% confluent 100 mm dishes were harvested after the treatment with 4 mM Ca93. Antigen was added and incubated for 10 h at 4 °C. The next day, beads were washed two times with lysis buffer and two times with PBS. The 10% beads were then suspended in 30 µL of 2× sample buffer with 5% β-mercaptoethanol and heated for 10 min at 100 °C. The rest of the 90% bead was used for on bead digestion and LC–MS/MS. For VAPA and GRP78 validations, 100% beads were used for western blot.For each condition, a total of 1.0 mg of total protein was used as measured by Bradford assay. Anti-FLAG antibody (10ug) and Protein G Dynabeads  were incubated for 30 min at room temperature in 200 µL PBS and 0.01% Tween 20, and washed once with lysis buffer. Magnetic beads were used as they give a low background of the contaminant proteinsA total input protein of 50 µg and 30 µL of the 10% bead samples were loaded in 8.5% acrylamide gels and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) to separate proteins, then transferred to nitrocellulose membranes. The membranes were blocked with 3% nonfat milk (w/v) in TBS for 2 h at room-temperature, with constant shaking. The antibodies of interest were diluted in 3% non-fat milk (w/v) and 0.2% Tween-20 in TBS (TBST). Anti-CaSR C0493, mouse was used at 1:700 dilution and HRP-conjugated mouse secondary antibody was used  at 1:1500 dilution to probe CaSR. For GAPDH, anti-GAPDH mouse was used at 1:2000 dilution and HRP-conjugated mouse secondary antibody was used  at 1:3000 dilution. For VAPA, anti-VAPA rabbit was used at 1:500 dilution, and HRP-conjugated rabbit secondary antibody was used  at 1:1500 dilution. For GRP78, anti-GRP78 rabbit was used at 1:1000 dilution, and HRP-conjugated rabbit secondary antibody was used  at 1:1500 dilution Membranes were incubated with the primary and secondary antibodies for 1 h at room-temperature with constant shaking, and finally washed with TBST. Secondary antibody was visualized using ECL detection reagents  developed on X-OMAT™ imaging film .94. The IP beads were washed three times with 1× PBS to remove detergents. To the bead, digestion buffer (50 mM NH4HCO3) was added, and the mixture was then treated with 1 mM dithiothreitol (DTT) for 30 min, followed by 5 mM iodoacetimide (IAA) for 30 min in the dark. Proteins were digested overnight with 0.5 µg of lysyl endopeptidase (Wako) and were further digested overnight with 1 µg trypsin (Promega). Resulting peptides were desalted with HLB column (Waters) and were dried under vacuum.On-bead digestion was carried out at room temperature according to the published protocol6 ions and a maximum injection time of 100 ms. The 10 most abundant ions were automatically selected for subsequent higher-energy collision dissociation (HCD) with the energy set at 28 NCE. The MS/MS settings included a resolution of 35,000, a target value of 5 × 105 ions, a maximum integration time of 108 ms, and an isolation window was set at 3.0 m/z. Ions with undetermined charge, z = 1, 8, and z > 8 were excluded.Peptides were analyzed with Nano-High Pressure Liquid Chromatography-Tandem Mass Spectrometry (nano-LC–MS/MS). Briefly, the peptides were loaded onto an in-house packed column  analytical column (Thermo Scientific) using a Dionex nanoLC system (Thermo Scientific). The column output was connected to a Q Exactive Plus mass spectrometer (Thermo Scientific) through a nanoelectrospray ion source. The mass spectrometer was controlled by Xcalibur software  and operated in the data-dependent mode in which the initial MS scan recorded the mass-to-charge ratios (m/z) of ions over the range of 350–1750 at a resolution of 70,000 with a target value of 1 × 106 ions and a maximum injection time of 120 ms. The 15 most abundant ions were automatically selected for subsequent higher-energy collision dissociation (HCD) with the energy set at 25 NCE. The MS/MS settings included a resolution of 17,500, a target value of 1 × 105 ions, a maximum integration time of 120 ms, and an isolation window set at 1.5 m/z. Ions with undetermined charge, z = 1, and z > 8 were excluded.Each sample was analyzed by nano LC–MS/MS with a Waters NanoAcquity HPLC system interfaced to a ThermoFisher Q Exactive. Peptides were loaded on a trapping column and eluted over a 75 μm analytical column at 350 nL/min; both columns were packed with Luna C18 resin (Phenomenex). The mass spectrometer was operated in the data-dependent mode in which the initial MS scan recorded the mass-to-charge ratios (m/z) of ions over the range of 300–1600 at a resolution of 70,000 with a target value of 3 × 1095. Intensities of each protein for each treatment condition were averaged from three negative control IP samples and three CaSR IP samples. The missing values were imputed as previously described using Perseus96. The following stringencies were employed to ensure robust upregulation, reproducibility, and detection: (i) treatments and IP for the positive and negative controls were performed in triplicates; (ii) identified proteins were at least two-fold enriched in the CaSR transfected samples over the negative controls, i.e., log2 (HEK293 + CaSR-FLAG pcDNA/Hek293 + pcDNA3.1) ≥ 1.00; (iii) P-values were calculated using a two-sided Student’s t-test, with a null hypothesis that there was no difference in protein relative abundance between the two groups, and a two tailed t-test with P ≤ 0.05 between the groups being statistically significant; (iv) proteins had a minimum peptide spectrum match (PSM) of two for at least two replicates, and; (v) proteins were identified with at least one unique peptide.LC–MS/MS Q-Extractive Orbitrap was used. Raw data files were analyzed with MaxQuant version 1.6.3.3 (Thermo Foundation 2.0 for RAW file reading capability) using an established Maxquant setup48, version 6.8, was used for the functional annotation and analysis of enrichment of 102 proteins identified as putative CaSR interactors. The set of total proteins identified and quantified (n = 623) was used as the background. The 102 proteins of interest and background lists were compared in each functional cluster. A two-tailed modified fisher’s exact test (EASE score of 1) with classification stringency at “medium” was employed to generate statistically significant enrichment annotations and to categorize them under annotation terms: cellular compartments, biological processes, molecular function, and KEGG pathways. Correction for multiple hypothesis testing was carried out using standard false discovery rate control methods. A Bonferroni-corrected P ≥ 0.05 and an enrichment ≥ 1.3 were used as cut-offs48. Similar analysis was performed on 102 putative CaSR interactors and 11 literature-curated CaSR interactors to represent a comprehensive CaSR PPI. Groups with annotations comprising of cellular compartments, biological processes and molecular function were presented with Cytoscape 3.7.097.The Database for Annotation, Visualization, and Integrated Discovery (DAVID)2+ and EGTA conditions) and 11 literature-curated were generated using Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) version 10.052. Interactions were identified and visualized among the 102 putative CaSR interactors (Homo sapiens). STRING used evidence from experimental and knowledge-based databases to provide confidence in functional associations or interaction through the Edge Confidence. Size of colored nodes represent evidence of known or predicted 3-dimensional protein structures.Visual representations of the PPI network for putative 102 CaSR interactors  Alexa Fluor 488 secondary antibody  for 1 h each at room temperature to stain FLAG-CaSR. Cos7 cells were incubated with anti-GFP antibody at 1:2000, anti-calreticulin antibody at 1:200, and anti-VAPA antibody at 1:125 or anti-GRP78 antibody at 1:100 in PBS with 3% BSA at room temperature for 1 h. The cos-7 cells were subsequently washed with PBS and stained with secondary antibodies goat anti-chicken IgY H&L Alexa Fluor 488 , donkey anti mouse IgG (H + L) Alexa fluor 647  and goat anti-mouse IgG (H + L) Alexa Fluor 555 , respectively for 1 h at room temperature. Nuclei were stained with 4′,6-diamidino-2-phenylindole.2+ buffer, then mounted in flow chamber with 0 mM Ca2+ buffer as the initial condition. Next, 4 mM Ca2+ buffer was added at 100 s, and then live cell images were collected for 600 s.Fluorescence images were collected using a Nikon Ti-E invert microscope equipped with Nikon 100× TIRF objective and Andor IXon Ultra 888 EMCCD camera. The cells were imaged under total internal reflection fluorescence microscopy with an imaging speed of 1 frame per second. The excitation wavelengths for EGFP-CaSR and mCherry-VAPA were 488 and 561 nm, and a Quad Band filter set  was used for filtering out the fluorescence background. A home-built dual-color imaging device with a long pass dichroitic mirror at 561 nm (Semrock) was used to split the fluorescence signals from EGFP-CaSR and mCherry-VAPA, which allowed for simultaneous collection of fluorescence images from both proteins. Another pair of fluorescent filters  were also used for dual-color imaging to minimize the color cross talk. For live cell imaging, Cos-7 cells on the coverslips were washed three times with 0 mM Ca+ and wt-CaSR were incubated with Fura-red for 30 min at 37 °C then washed with 2 mL of physiological Ringer buffer . The coverslips were mounted on a bath chamber and placed on the stage of a Leica DM6100B inverted microscope with a Hamamatsu cooled EM-CCD camera and illuminated with a Till Polychrome V Xenon lamp. Cells were illuminated at 488 nm and 550 nm, in real-time, as cells were exposed to 0.5 mM Ca2+for 200 s, followed by 4 mM Ca2+ for another 800 s and back to 0.5 mM Ca2+ for additional 200 s.HEK293 cells transfected with G-CatchERMulticolor fluorescence images of nucleus , CaSR , VAPA/GRP78 , and ER  were taken using a Zeiss LSM780 confocal microscope. To analyze the colocalization between each imaging channels, a self-written MATLAB script was used. Briefly, regions of interest (ROIs) were first identified using merged images from all imaging channels. Next, Pearson coefficients were calculated between pixel intensities at the same ROI in green, red, and purple channels. The same image analysis method was used over different cell treatment conditions.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.


JAK2-mutated myeloproliferative neoplasms. The role of YBX1 in AML, however, remained so far elusive. Here, inactivation of YBX1 confirms its role as an essential driver of leukemia development and maintenance. We identify its ability to amplify the translation of oncogenic transcripts, including MYC, by recruitment to polysomal chains. Genetic inactivation of YBX1 disrupts this regulatory circuit and displaces oncogenic drivers from polysomes, with subsequent depletion of protein levels. As a consequence, leukemia cells show reduced proliferation and are out-competed in vitro and in vivo, while normal cells remain largely unaffected. Collectively, these data establish YBX1 as a specific dependency and therapeutic target in AML that is essential for oncogenic protein expression.Persistence of malignant clones is a major determinant of adverse outcome in patients with hematologic malignancies. Despite the fact that the majority of patients with acute myeloid leukemia (AML) achieve complete remission after chemotherapy, a large proportion of them relapse as a result of residual malignant cells. These persistent clones have a competitive advantage and can re-establish disease. Therefore, targeting strategies that specifically diminish cell competition of malignant cells while leaving normal cells unaffected are clearly warranted. Recently, our group identified YBX1 as a mediator of disease persistence in  YBX1 led to a significant increase in mis-splicing of MAPK/ERK pathway members and to eradication of otherwise persistent MPN cells . Fin. Finp < ses Fig. . Here, rses Fig. . Using ises Fig. . Importaing Fig. . In conting Fig. . Consequing Fig. . Likewising Fig. . The facion Fig. .Fig. 6YBYBX1 impacts the translational output of transcripts on the protein level and thereby selectively modulates protein abundance of oncogenic drivers and influences proliferative capacity and cell competition in AML.Taken together, we propose, that YBX1 associates with target mRNAs, including MYC, and thereby modulates translational output by recruitment of relevant mRNAs to polysomal chains Fig. . ProteinYBX1 had no deleterious effects on normal hematopoiesis [Identification of therapeutic targets that are tractable vulnerabilities and selective dependencies in cancer while being dispensable for normal tissues represent the ideal prerequisite for the development of cancer therapies. Cold shock protein YBX1 has been identified as a pan-cancer dependency in publicly available CRISPR-Cas9-screens and several studies in different tumor entities , 15. Conopoiesis , making YBX1 in AML shows minor impact on mRNA abundance, while having significant effects on the cellular proteome. Moreover, no relevant increase in mis-spliced isoforms could be found in AML cells after deleting YBX1, clearly distinguishing the apparent mechanisms in AML from our previous findings in MPN, where YBX1 was acting as a relevant splicing factor [Mechanistically, we demonstrate that deletion of g factor . Using ag factor , 15. To 6A) transcripts via IGF2BP-family of proteins to facilitate RNA binding and stabilization. In line with this claim, we find that deletion of IGF2BP2 confers resistance to YBX1-inactivation in our CRISPR-Cas9 screen. Structurally, the cold-shock domain seems to be required for both IGF2BP- and mRNA-binding of YBX1. Consistent with our findings, Feng et al. report an impact of YBX1 on MYC expression and show that its expression can rescue the phenotype evoked by inactivation of YBX1.A recent report published by Feng and colleagues  complemeYBX1 can affect RNA abundance [YBX1  we found regulation of RNA-stability exclusively in RNAi- but not CRISPR-treated AML cells. In RNAi-treated samples, we observed high numbers of DEGs, including MYC, BCL2 and MCL1, consistent with findings described by Feng and colleagues (Supplementary Fig. In contrast to our findings the authors assume that regulation of RNA stability represents a major mechanism of YBX1-action in AML, similar to findings described in breast cancer . This asbundance . HoweverTaken together, our data and the findings presented by Feng et al. establish YBX1 as a selective genetic vulnerability in leukemia without major restrictions towards specific genetic subtypes.Of note, a novel small molecule, SU056, was recently reported to directly bind and inhibit YBX1 . SU056 dSupplementary Material and MethodsSupplementary data