To understand the mechanism by which imeglimin  decreases hepatic glucose production.We compared the effect of imeglimin and metformin on glucose production, ATP/ADP ratio, oxygen consumption rate, mitochondrial redox potential and membrane potential in primary rat hepatocytes.We found that both imeglimin and metformin dose‐dependently decreased glucose production and the ATP/ADP ratio. Moreover, they both increased mitochondrial redox potential H fluorescence) and decreased membrane potential (assessed by TMRM fluorescence). However, contrary to metformin, which inhibits mitochondrial Complex I, imeglimin did not decrease the oxygen consumption rate in intact cells. By measuring the oxygen consumption of in situ respiratory chain as a function of the concentration of NADH, we observed that imeglimin decreased the affinity of NADH for the respiratory chain but did not affect its Vmax (ie competitive inhibition) whereas metformin decreased both the Vmax and the affinity (ie uncompetitive inhibition).We conclude that imeglimin induces a kinetic constraint on the respiratory chain that does not affect its maximal activity. This kinetic constraint is offset by a decrease in the mitochondrial membrane potential, which induces a thermodynamic constraint on the ATPase responsible for a decrease in the ATP/ADP ratio. This work specifies the mitochondrial mechanism of action of Imeglimin. It is demonstrated for the first time that an inhibition of the respiratory chain can be competitive. It catalyses a sequence of redox reactions which couple an electron flux with a vectorial transfer of protons from the matrix to the intermembrane space, thereby generating the mitochondrial membrane potential. The reaction begins at the level of Complex I, a large protein complex with at least 45 subunits, when electrons pass from NADH to ubiquinone. Electrons from the ubiquinone pool then pass through Complexes III and IV to finally reduce oxygen into water. The mitochondrial membrane potential thus established is then used to synthesize ATP from ADP plus phosphate.Imeglimin is a novel oral antidiabetic agent. Its structure and proposed mechanism of action establish imeglimin as the first in a new class of tetrahydrotriazine‐containing molecules called ‘glimins’. Imeglimin's safety and efficacy as a monotherapy in Caucasian patients with type‐2 diabetesImeglimin has been shown to normalize glucose tolerance and insulin sensitivity in a murine model of type‐2 diabetes by protecting mitochondrial function from oxidative stress and favouring lipid oxidation in the liver.Metformin is the most widely prescribed drug to treat patients affected by type‐2 diabetes and is recommended as a first‐line oral therapy by both American and European guidelines. Metformin is believed to inhibit glucose production because it leads to a mild inhibition of respiratory chain Complex I,Surprisingly, both imeglimin22.1The 20 male Wistar rats  used for this study were obtained from Charles River . The study was carried out in accordance with the European Directives 86/609/EEC, 2010/63/UE on the care, welfare and treatment of animals. All the procedures were reviewed and approved by the ethics committee (Cometh Grenoble) affiliated to the animal facility #D3842110001, and authorized by the French Ministry of Research on 1 December 2017 (APAFIS#9994‐2017052214053756).2.2Imeglimin was from Poxel . Tetramethyl rhodamine methyl ester (TMRM) was purchased from Molecular Probes . Minimum essential medium, M199 medium, phosphate‐buffered saline (PBS) and antibiotics were purchased from Dutscher . NaCl, KCl, MgSO4, KH2PO4, NaHCO3 and NADH were purchased from Merck . Other reagents were obtained from Sigma .2.3Wistar rat liver hepatocytes were isolated from overnight‐starved animals by enzymatic digestion with collagenase according to the method of Berry and Friend2.42PO4, 1.2 mM MgSO4, 24 mM NaHCO3 and 1.3 mM CaCl2 (pH 7.4)] containing 2 mM sodium pyruvate and 20 mM sodium lactate, saturated with O2 : CO2 (19:1). The extracellular medium was collected after a 15, 30 then 45 minutes incubation in order to measure the glucose concentration. In parallel experiments, samples of cultured hepatocytes were withdrawn after a 30 min incubation in order to measure phosphate and adenine nucleotide contents, oxygen consumption and the affinity of NADH for the respiratory chain.After an overnight incubation, cells were quickly washed with PBS, to eliminate traces of glucose and then incubated at 37°C for 1 h in a Krebs‐Ringer‐bicarbonate‐calcium buffer free of metformin or imeglimin  metformin failed to measure metformin accumulation in mitochondria.The precise mechanism by which metformin inhibits Complex I remains a matter of debate. It has been proposed that metformin accumulates inside mitochondria, but two different laboratories using radioactive [4In this work, we have shown that both imeglimin and metformin decreased glucose production, the ATP/ADP ratio and mitochondrial membrane potential, while they both increased the mitochondrial redox potential. However, contrary to metformin, imeglimin did not decrease the oxygen consumption rate in intact cells.A decrease in the ATP/ADP ratio can be due to a decrease in ATP production or an increase in ATP consumption. A decrease in ATP production could result from either an inhibition or an uncoupling of oxidative phosphorylation, which are expected to decrease and increase the oxygen consumption rate, respectively. On the contrary, an increase in ATP consumption is expected to increase the oxygen consumption rate. Thus, the observation that imeglimin decreased the ATP/ADP ratio with no effect on the oxygen consumption rate is unusual. Moreover, the fact that imeglimin did not affect either the rotenone‐sensitive or the oligomycin‐sensitive respiration rates strongly suggests that imeglimin did not affect the ATP turnover.This conclusion raises two questions: (i) how is it possible to reduce the ATP/ADP ratio without reducing the turnover of ATP; (ii) how can a simple decrease in the ATP/ADP ratio affect hepatic glucose production?There needs to be a force to generate a net flux through an enzymatic pathway. This force, commonly named ∆G (the free energy of reaction) corresponds to the difference between the Gibbs free energy of the substrates and the Gibbs free energy of the products of the enzymatic pathway. However, despite a thermodynamically favourable ∆G (ie a negative ∆G), the net flux through an enzymatic pathway can be modulated by kinetic constraints. Importantly, a change in kinetic constraints does not systematically affect the net flux through an enzymatic pathway. This is typically what occurs during a competitive inhibition of an enzyme when the change in the ∆G (ie an increase in the force) counteracts the increase in the kinetic constraints.Considering oxidative phosphorylation as a whole, the ∆G of that reaction corresponds to the difference between the Gibbs free energy of NADH and the Gibbs free energy of ATP. By increasing the NAD(P)H fluorescence Figure  and decr+) and the membrane potential. The ∆G for the ATP synthase corresponds to the potential span between the membrane potential and the phosphate potential. A kinetic constraint at the respiratory chain level is expected to increase the redox potential and/or to decrease the membrane potential. A kinetic constraint at the ATP synthase level is expected to increase the membrane potential and/or to decrease the phosphate potential.Now considering the oxidative phosphorylation process step by step, the kinetic constraint can theoretically be located either at the respiratory chain or at the ATP synthesis level. The ∆G for the respiratory chain corresponds to the potential span between the redox potential  which does not correspond to the same degree of inhibition of hepatic glucose production  is not resolved in this work. Note, however, that we have previously reported that imeglimin inhibits ROS production induced by reverse electron flux through Complex I,sine qua non condition for preservation of a normal rate of ATP synthesis when the membrane potential decreases (Figure Because the potential span between the membrane potential and the phosphate potential is the driving force for ATP synthesis, the decrease in membrane potential induced by imeglimin is supposed to decrease ATP synthase activity. As a consequence, ATP production decreases unless phosphate potential also decreases in parallel to achieve the potential span required to sustain a similar rate of ATP synthesis as observed in control conditions. Note that imeglimin did not affect the ATP synthase activity (as assessed by the oligomycin‐sensitive oxygen consumption rate—Figure s Figure .Gluconeogenesis is an ATP‐consuming process controlled by the ATP/ADP ratio.Because gluconeogenesis from 2 lactates requires 6 ATP to produce 1 glucose, one would expect the inhibition of gluconeogenesis to decrease the ATP turnover. This may not be the case if other ATP‐consuming processes are increased. It is well known that different endergonic reactions do not require the same force to be driven.5Dissecting the mechanisms by which metformin and imeglimin inhibit gluconeogenesis requires a thermokinetic approach studying the flux‐force relationship. From a thermodynamic point of view, Figure This work was supported by Poxel SA. No other potential conflicts of interest relevant to this article were reported.GV and FL contributed to the experimental design, researched data, contributed to the discussion, and review of the manuscript. C. C.‐R. researched data. SH‐B. contributed to the experimental design, discussion and review of the manuscript. A.‐LB contributed to the discussion and review of the manuscript. É.F. contributed to the experimental design, contributed to the discussion and wrote the manuscript. É.F. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.Supplementary MaterialClick here for additional data file.


HCN4. There are very few reports about the association between HCN4 and LVNC. The aim of our study was to characterize the clinical phenotype of families with LVNC and sinus bradycardia caused by pathogenic variants of the HCN4 gene. Methods: From March 2008 to July 2021, we enrolled six patients from four families with diagnosed isolated LVNC based on the clinical presentation, family history and echocardiographic and cardiovascular magnetic resonance (CMR) evidence of LVNC. Next generation sequencing (NGS) analysis was undertaken for the evaluation of the molecular basis of the disease in each family. Results: A total of six children (median age 11 years) were recruited and followed prospectively for the median of 12 years. All six patients were diagnosed with LVNC by echocardiography, and five participants additionally by CMR. The presence of late gadolinium enhancement (LGE) was found in three children. Sinus bradycardia and dilation of the ascending aorta occurred in five studied patients. In four patients from three families, the molecular studies demonstrated the presence of rare heterozygous HCN4 variants. Conclusion: (1) The HCN4 molecular variants influence the presence of a complex LVNC phenotype, sinus bradycardia and dilation of the ascending aorta. (2) The HCN4 alteration may be associated with the early presentation of clinical symptoms and the severe course of the disease. (3) It is particularly important to assess myocardial fibrosis not only within the ventricles, but also in the atria in patients with LVNC and sinus bradycardia.Background: Left ventricular noncompaction (LVNC) is a genetically and phenotypically heterogeneous cardiomyopathy in which myocardium consists of two, distinct compacted and noncompacted layers, and prominent ventricular trabeculations and deep intertrabecular recesses are present. LVNC is associated with an increased risk of heart failure, atrial and ventricular arrhythmias and thromboembolic events. Familial forms of primary sinus bradycardia have been attributed to alterations in  Left ventricular noncompaction (LVNC) is a genetically and phenotypically heterogeneous cardiomyopathy in which myocardium consists of two distinct compacted and noncompacted layers, and prominent ventricular trabeculations and deep intertrabecular recesses are present. The frequency of LVNC has increased and it has been reported as comprising 9% of all childhood primary cardiomyopathies in recent studies ,2. This HCN4, SCN5A and ANK2. However, there are very few reports in the literature on the association between HCN4 dysfunction and LVNC [In the literature, there have been reports of children with LVNC in association with sinus bradycardia, suggesting the possibility that the two diseases may have a common cause ,10. Althand LVNC .HCN4 gene alterations.The aim of our study was to characterize the clinical phenotype of families with LVNC and sinus bradycardia caused by the From March 2008 to July 2021, we prospectively enrolled 6 patients from 4 families with diagnosed LVNC hospitalized in the Department of Cardiology of the Children’s Memorial Health Institute. The inclusion criteria were age <18 years at the time of LVNC diagnosis based on the clinical presentation  and echocardiographic evidence of isolated LVNC. The echocardiographic criteria for LVNC diagnosis were: (1) presence of a two-layer structure with a compacted (C) and noncompacted (NC) myocardial layer of trabecular meshwork with deep endomyocardial spaces; (2) maximal end-systolic ratio of NC/C layers of >2; (3) color Doppler evidence of deep perfused intertrabecular recesses . Only paThe Institutional Ethics Committee approved this study. Informed consent was obtained from all individual participants included in the study.Patients’ demographics, clinical symptoms, family history of cardiomyopathies and sudden cardiac death (SCD), as well as the results of echocardiography, 12-lead resting ECG with assessment of heart rate (HR), 24 h Holter electrocardiography (ECG) with analysis of minimal, maximal, average HR, occurrence of sinus pauses (RR pauses) >2 s and cardiovascular magnetic resonance (CMR) were collected. Information on clinical symptoms, such as chest pain, arrhythmias, episodes of syncope and past thromboembolic events, were recorded, and NYHA functional class was evaluated. Serum NT-proBNP level was analyzed in all patients. Each patient underwent genetic blood tests for evaluation of the molecular basis of the disease.Echocardiography, including two-dimensional, Doppler and M-mode imaging, was performed at rest, as previously described , using PIn all patients without contraindications, CMR studies were performed, utilizing a 1.5 T magnetic resonance scanner , as previously described . Steady-DNA samples were obtained from the peripheral blood by automatic isolation with MagCore Nucleic Acid Extractor HF16Plus . Candidate gene sequencing was performed on the HiSeq 1500 platform  using the TruSight One Sequencing Panel (Illumina) in Family 1 and the original CMHI pediatric panel of 1000 clinically relevant genes (Roche) in the remaining families. A set of 222 and 165 cardiomyopathy-associated genes were analyzed, respectively . Study phttps://gnomad.broadinstitute.org/ (accessed on 14 January 2022)), Exome Variant Server , UK10K Project (https://www.uk10k.org/ (accessed on 14 January 2022)) and in-house database comprising >5000 individuals ; (2) the pathogenicity of the variants, using in silico prediction algorithms: CADD, SIFT, MutationTaster, PolyPhen2_HDIV, PolyPhen2_HVAR, MutationAssessor, MetaSVM, MetaLR and FATHMM; (3) phenotypic descriptions in Online Mendelian Inheritance in Man ), the Human Gene Mutation Database ), ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/ (accessed on 14 January 2022)) and Pubmed. Analysis and prioritization of discovered variants considered: (1) the minor allele frequency (MAF) determined with the Genome Aggregation Database  reference sequences. Sanger sequencing was also used for parental segregation analysis.All variants were validated by Sanger sequencing and reported with the Baseline characteristics. A total of six children from four families were enrolled and followed prospectively for the median of 12 (6–13) years. The median age of patients was 11 (6–16) years. Clinical characteristics of the study patients are presented in In echocardiography, the median NC/C ratio in all patients was 2.29 2.13–2.40), which met Jenni’s criteria for the diagnosis of LVNC. Dilation of the ascending aorta exceeding the standard indexed to BSA was found in five study participants. In five of the six children, CMR imaging was performed to further assess cardiac morphology and function and to screen for features of myocardial fibrosis. In one child, CMR was not performed due to his severe clinical condition, hemodynamic instability and low body weight. Among the five patients who underwent CMR, the diagnosis of LVNC was confirmed in all according to Petersen’s criteria, with a median of NC/C ratio of 3.73 3.14–4.25). All patients studied were diagnosed with sinus bradycardia in accordance with the recommendations in the literature [–2.40, wh–4.25. AlCharacteristics of families. The pedigrees of individual families are presented in Two sisters, one 2 years old and the other 6 years old, diagnosed with LVNC, were admitted to our institute for cardiologic evaluation. Family history revealed that their grandfather (father’s father) was diagnosed with sinus bradycardia and a pacemaker was implanted. However, his echocardiographic examination is not known, and we cannot state whether there were LVNC features. The younger girl (F I-1) diagnosed with LVNC at 3 months of age and progressive heart failure was admitted to the intensive care unit with symptoms of severe heart failure (NYHA group IV) and multi-organ injury. Upon echocardiography, the NC/C ratio was 3.34 and LV size was normal, but the LV systolic function was reduced. In addition, there was a dilation of the ascending aorta. In the resting ECG recording, the heart rate was 130 beats per minute (bpm), whereas the average daily rhythm in the 24 h Holter study was 90 bpm (the child was treated with an infusion of catecholamines). In the course of the disease, the child developed ischemic cerebral stroke. The girl died due to the mechanism of sinus bradycardia. The second girl (F I-2) underwent cardiological examinations after the diagnosis of LVNC in her sister. Clinical symptoms included dyspnea and fatigue on effort (NYHA class II). The resting ECG showed sinus bradycardia (67 bpm), and, in the 24 h Holter monitoring, the average daily rhythm was 64 bpm. Echocardiography revealed LVNC features with a NC/C ratio of 2.35 and dilation of the ascending aorta. In CMR, the NC/C ratio was 4.60 and no LGE was found. In both imaging studies, the LV size and function were within the normal range. ACE inhibitors and spironolactone were included in the treatment.HCN4 molecular variant c.1444G>A p.Gly482Arg that was subsequently confirmed in her affected sister and her father. Nine of the used in silico algorithms  predicted this change as deleterious. The c.1444G>A variant was not found in the utilized frequency population databases  or in our DMG cohort. It was reported as a disease-causing variant in sinus bradycardia and myocardial noncompaction cases in HGMD (ID: CM159293), and also as pathogenic/likely pathogenic in Brugada syndrome patients in ClinVar (ID: 197253). NGS analysis performed in this family in the younger girl revealed the heterozygous known An 11-year-old girl (F II-3) was admitted for cardiac diagnostics due to observed sinus bradycardia and a positive family history of LVNC. Her mother had been diagnosed with LVNC and syncope, and a cardioverter-defibrillator (ICD) was implanted. In addition, her brother’s daughters were also diagnosed with LVNC. During hospitalization, the patient did not report any complaints and did not present any symptoms of heart failure (NYHA class I) and bradycardia. The ECG showed sinus bradycardia of 60 bpm, whereas the average 24 h heart rhythm in the Holter examination was 60 bpm. Echocardiography showed a NC/C ratio of 2.22, and the size of the ascending aorta was within the normal range. CMR examination confirmed the diagnosis of LVNC, and the NC/C ratio was 3.00, but no LGE was found. In both of the above studies, the LV size and systolic function were normal. Pharmacological treatment was not started.HCN4 molecular variant c.1454C>T p.Ala485Val was identified in the proband and further confirmed in her mother. Eight in silico algorithms  predicted this change as deleterious. The c.1454C>T variant was not found in the EVS, UK10K and DMG databases, but it was noted in gnomAD with MAF < 0.00001. This variant has no ClinVar submission but appeared in HGMD as a disease-causing variant in sinus bradycardia patients (ID: CM104901). The heterozygous known A 17-year-old patient (F III-4) was admitted to our institute due to sinus bradycardia and a family history of bradycardia and cardiomyopathy in other family members. Family history revealed that his sister was diagnosed with LVNC and sinus bradycardia, while the other sister was also treated for bradycardia. The boy’s father was diagnosed with hypertrophic cardiomyopathy and a syncope, and he had a pacemaker implanted. The patient’s clinical symptoms included only dyspnea and fatigue on effort (NYHA class II). The electrocardiogram showed a sinus rhythm of 44 bpm .The mean 24 h heart rate during the Holter test was 47 bpm. Echocardiography revealed LVNC phenotypic features with a NC/C ratio of 2.40 and dilation of the ascending aorta. In the CMR study, the NC/C ratio was 3.73 and the presence of LGE in the LV myocardial wall was found. Both echocardiography and CMR showed a normal LV size and LV systolic function. The patient was receiving treatment for heart failure  and salbutamol for sinus bradycardia.HCN4 molecular variant c.1438G>C p.Gly480Arg was found in the proband, and was also identified in his father (DNA from his sisters was not available for testing). Nine in silico algorithms  predicted this change as deleterious. The c.1438G>C variant was not present in the frequency population databases. It was reported as disease-associated polymorphism in sinus bradycardia asymptomatic association with HGMD (ID: CM073122), and was classified as pathogenic in two patients with sick sinus syndrome in ClinVar (ID: 5176).The heterozygous known The siblings, a 16-year-old girl (F IV-5) and her 11-year-old brother (F IV-6), were admitted to our Department of Cardiology due to sinus bradycardia. Family history revealed that their father had been diagnosed with sinus bradycardia and dilated cardiomyopathy (DCM); he had a pacemaker implanted. Moreover, their father’s brother also had a pacemaker implanted due to sinus bradycardia. The girl was asymptomatic, and no syncope was present. She only reported increased fatigue during exercise (NYHA class II). A nodal rhythm of approximately 39 bpm was recorded in the resting ECG. In the 24 h Holter ECG, the average daily rhythm was 55 bpm. Sick sinus syndrome (SSS) and supraventricular premature beats were diagnosed. Echocardiography revealed LVNC with a NC/C ratio of 2.06 and dilation of the ascending aorta. In the CMR examination, the NC/C ratio was 3.91, and foci of LGE were found both in the myocardial wall of the left ventricle and in the wall of the left atrium .In both echocardiography and CMR, the LV was enlarged, with a borderline systolic function. The girl received treatment for heart failure  and salbutamol due to sinus bradycardia.The second sibling, the boy, had no symptoms of sinus bradycardia or syncope. Clinical symptoms included dyspnea and fatigue on effort (NYHA class II). The heart rate on the ECG was 56 bpm, with a mean daily heart rhythm on the 24 h Holter study of 50 bpm. The patient was diagnosed with sick sinus syndrome and premature supraventricular beats, without episodes of supraventricular tachycardia. Echocardiography revealed typical LVNC features, with a NC/C ratio of 2.13 and LV enlargement with normal systolic function. There was also a dilation of the ascending aorta. CMR confirmed the diagnosis of LVNC (the NC/C ratio was 3.27), whereas the LV size and systolic function were normal. Like his sister, the boy was found to have LGE in the LV myocardial wall and in the left atrium. The patient is treated with medications such as ACE inhibitors and salbutamol. A molecular analysis performed on the girl revealed no candidate pathogenic/likely pathogenic variant in the studied cardiomyopathy panel.Left ventricular noncompaction cardiomyopathy is a genetically and phenotypically heterogeneous myocardial disease characterized by multiple prominent trabeculations and deep intertrabecular recesses. It is a relatively new, yet rare, clinical entity, but nevertheless the third most common cardiomyopathy in childhood, and is associated with congestive heart failure, arrhythmias, atrioventricular conduction disorders, sudden cardiac death and/or thromboembolic events. However, patients with isolated LVNC can also be asymptomatic and diagnosed after an abnormal ECG, echocardiogram or family screening ,25. HCN4 gene [In the literature reports, the relationship between LVNC and sinus bradycardia has been described in approximately 55% of cases, although the basis of conduction disturbances remains unclear ,26. The CN4 gene ,27,28.HCN4 are thought to predominantly underlie sinoatrial node (SAN) disorders because HCN4 expression is mostly limited to the cardiac conduction system, and especially the SAN area [HCN4 dysfunction may result in cardiac function disorders. They have been reported mainly in patients with sick sinus syndrome (SSS) and Brugada syndrome. However, in recent years, a broad spectrum of conditions, including sinus bradycardia, sinus tachycardia, atrial fibrillation, atrioventricular block, idiopathic ventricular tachycardia, left ventricular noncompaction, myocardial infraction, sudden infant death syndrome, arrhythmogenic right ventricular cardiomyopathy, dilation of the aorta and chronotropic incompetence, have been noted [Molecular variants in SAN area . HCN4 isSAN area ,29. Carden noted ,30. HCN4 pathogenic variants in LVNC patients is not fully determined, with limited data concerning, in particular, the pediatric population. This can be due to the rare occurrence of childhood LVNC, lack of widespread routine genetic testing and lack of HCN4 in the applied gene panels. Van Waning et al. [HCN4 alterations causative for the disease. HCN4 variants accounted for 10% of (likely) pathogenic variants identified in a cohort of 95 adults diagnosed with LVNC by Richard et al. [HCN4-related LVNC among 206 patients aged less than 16 years. The epidemiology of g et al.  collected et al. , as welld et al. . In a red et al.  found twHCN4 gene and emphasized the role of their familial occurrence [HCN4 gene variants, only patients who have the LVNC phenotype have been described. Moreover, the detection of pathogenic variants in patients with LVNC identifies cases (and their family members) who are at risk of developing adverse cardiac events, including arrhythmias [Milano et al., were the first to describe the coexistence of LVNC, sinus bradycardia and molecular variants in the currence . Althoughythmias .HCN4 molecular variants have been collected in the Human Gene Mutation Database Professional 2021v.3. All molecular variants identified in this study are known changes that affect highly evolutionary conserved amino acids located in close proximity with the essential pore-forming domain of the protein  pathogenic  protein . HCN4 alterations rarely manifest themselves clinically in childhood and are mainly recognized after adolescence. Only a few literature descriptions of teenage patients manifested with a complex phenotype of sinus bradycardia and LVNC can be found in literature. Ishikawa et al., described a case of a 13-year-old girl with significant sinus bradycardia requiring permanent pacing at the age of 17 years [These variants have been reported previously, mainly in adults with a complex phenotype of LVNC and sinus bradycardia and myocardial noncompaction ,33,36,3717 years  reportedHCN4 nucleotide position  leading to the same or various amino acid changes has been reported in multiple studies [HCN4 channel. The functional effect of these variants has been studied extensively, demonstrating that the p.Gly480Arg, p.Gly482Arg and p.Ala485Val variants severely abolish pacemaker currents, with a hyperpolarization shift in the voltage dependence of activation [HCN4 molecular variants has been demonstrated. The molecular etiology of heart abnormalities was confirmed in three out of four families . It should be emphasized that, in Family 4, despite the typical course of LVNC and sinus bradycardia, no molecular cause was identified. Different base substitution at the same  studies ,29,32,33tivation ,29,38,39tivation . As in ttivation ,28, in oHCN4 gene alterations with dilation of the ascending aorta. As reported by Vermeer et al., evidence was provided that dilation of the ascending aorta also constitutes a part of the clinical spectrum of HCN4 changes. Moreover, these authors demonstrated that HCN4 variant carriers have aortic dilation with age, which undoubtedly influences the prognosis and cardiac care in this group of patients [A very important aspect of the clinical evaluation of our patients is to demonstrate the presence of dilation of the ascending aorta. There are only a few studies showing an association of the patients .According to the literature, the presence of LGE in the LV myocardium is found in 40% of LVNC patients . In our The literature presents the results of a study showing that atrial fibrosis, quantified with CMR-LGE, is an important predictor of clinically significant sinus node dysfunction and sinus bradycardia requiring pacemaker implantation . It shouHCN4 molecular variants should be undertaken in patients with such a complex clinical picture. Additionally, we emphasized the role of CMR in LVNC, not only regarding diagnosis but also risk stratification and future medical treatment. This is important for everyday clinical practice and for further patient care, as it enables targeted treatment and further management.In summary, a systematic search for HCN4 molecular variant influences the presence of a complex LVNC phenotype, sinus bradycardia and dilation of the ascending aorta;The HCN4 alteration may be associated with the early presentation of clinical symptoms and severe course of the disease;It is particularly important to assess myocardial fibrosis not only within the ventricles, but also in the atria in patients with LVNC and sinus bradycardia.


The COVID-19 pandemic has caused millions of fatalities since 2019. Despite the availability of vaccines for this disease, new strains are causing rapid ailment and are a continuous threat to vaccine efficacy. Here, molecular docking and simulations identify strong inhibitors of the allosteric site of the SARS-CoV-2 virus RNA dependent RNA polymerase (RdRp). More than one hundred different flavonoids were docked with the SARS-CoV-2 RdRp allosteric site through computational screening. The three top hits were Naringoside, Myricetin and Aureusidin 4,6-diglucoside. Simulation analyses confirmed that they are in constant contact during the simulation time course and have strong association with the enzyme’s allosteric site. Absorption, distribution, metabolism, excretion and toxicity (ADMET) data provided medicinal information of these top three hits. They had good human intestinal absorption (HIA) concentrations and were non-toxic. Due to high mutation rates in the active sites of the viral enzyme, these new allosteric site inhibitors offer opportunities to drug SARS-CoV-2 RdRp. These results provide new information for the design of novel allosteric inhibitors against SARS-CoV-2 RdRp. Coronavirus disease COVID-19) appeared at the end of 2019 in Wuhan China 9 appeare,4. The aMost of the CoVs are transmitted to humans from zoonotic sources , and theSARS-CoV-2 is an RNA virus; compared to other RNA-based viruses, its genome is relatively larger in size. Among all RNA virus families, CoVs have the largest genomes (26–32 kb) . A multiThe structure of SARS-CoV-2 RdRp has been resolved through cryo-electron microscopy at a resolution of 2.9 Å in complex with its co-factors . The NSPOur current approach is based on computational studies of the allosteric site of RdRp of SARS-CoV-2. The binding of molecules to the allosteric site can cause a functional change in the overall structure of the enzyme. The allosteric site can be perturbed by non-covalent molecule binding . Covalent molecule binding can also occur at the allosteric site ,26. ThesThe current research investigates the inhibitory role of flavonoids against RdRp of SARS-CoV-2 . They arThe flavonoids with the lowest binding affinity energy (S-Score), lowest RMSD refine score and best interactions with RdRp (NSP12) of SARS-CoV-2 in complex with other co-factors will be discussed here . The topNaringoside flavonoid showed a total of nine interactions with the allosteric site residues of the target protein. There were eight hydrogen bond interactions and one interaction of the arene-cation type between the flavonoid nucleus ring-A and Arg583 of the allosteric site. The –OH group present at the ring-A made a hydrogen bond interaction with allosteric site residue Ser592. Furthermore, the two-glycoside moieties attached with the ring-A of the naringoside flavonoid formed the remaining seven H-bonds with the allosteric site amino acid residues. The –OH hydroxyl groups of the glycosides interacted with Thr604, Gly584, Thr582, Arg583, Met601, Thr582 and Ala585 of allosteric site residues. The binding affinity energy (S-Score) of naringoside with the allosteric site of the target enzyme was −19.0 kcal/mol .Aureusidin 4,6-diglucoside flavonoid belongs to the aurone class of flavonoids. It showed six interactions with the allosteric site residues of the protein. The six interactions of this flavonoid were all hydrogen bond interactions. The ketone group on ring-C of this aurone flavonoid engaged with Thr604 of the allosteric site via H-bonding. Thr604 also formed a H-bond with the ring-B of the aurone flavonoid nucleus. Arg750 and Thr586 also made two H-bonds with –OH groups of the ring-B of this flavonoid. The two-glycoside moieties attached to the ring-A of this flavonoid resulted in two interactions with allosteric site residues, Phe594 and Asn600. The individual glycosides each make single H-bonds via –OH groups. The binding affinity energy (S-Score) of this flavonoid was −19.21 kcal/mol .This flavonoid exhibited good binding energy (S-Score) of −18.17 kcal/mol. It also exhibited good interactions with the allosteric site residues; five interactions were seen and one of the interactions was an arene-cation interaction of the ring-A of the main nucleus of the flavonoid with Arg583. The ring-A also made a H-bond via its attached –OH group with Ser592. The ring-B hydroxyl group also formed a H-bond with the Thr604 allosteric amino acid, and the ring-C of this flavonoid engaged in two H-bonding interactions with Met755 and Gly584 via its two –OH groups at 4′ and 5′ positions . MyricetIn similar fashion, plant-based polyphenols and flavonoids were used against the active site of SARS-CoV-2 RdRp. These flavonoids showed lower free energy of binding as compared to our results. This difference in free energy may be due to the utilization of different software having different force field and mathematical equations for the calculation of binding free energy . Mostly,MD simulations were used to evaluate the target protein and ligand-binding stability, and their dynamic properties during the virtual simulation time. The RMSD fluctuation was computed separately for both the protein and ligand complexes in the MD simulations, and it was observed to be within 3 Å. The existence of intermolecular interactions among ligand and RdRp was monitored during the study using the simulation interaction diagram tool implemented in the Desmond MD package . The simRdRp in apo form was simulated for 100 ns of virtual time and RMSD calculated. RMSD designates how much the protein deviates from its initial resolved structure. At the start, it remained very low, between 1.6 and 2.0 Å, but at about 15 ns it fluctuated to 2.5 Å, then stabilized a bit and increased again to 3 Å at 50 ns time. After about 60 ns of the simulation, it stabilized and remained at about 2.4 Å. This fluctuation further stabilized and was found to be 2.1 Å at the end of the simulation, indicating that the protein stabilized and attained equilibrium and remained uniform in this form along the trajectory. An RMSD of 2.1 Å seems very stable for this protein .Moreover, the root mean square fluctuations (RMSF) stability studies showed that at the start and the end of the protein chain it fluctuated up to 4.8 Å, suggesting that the terminal regions of the protein are quite flexible. These sites in the protein chains have relatively high flexibility compared to the internal regions of the protein, so these kinds of fluctuations are normal in proteins. It can also be noted that the allosteric site of the protein, which lies between 500–800th residues, was stable and its RMSF values remained between 0.6 and 1.2 Å  and interacted throughout the 100 ns simulation, validating the docking results, and demonstrating that these allosteric site amino acids exhibited strong association with this ligand. As observed in the docking studies, Arg583, Ser592 and Asn600 also showed strong binding association for the duration of the 100 ns simulations studies . It can Stability studies of this complex demonstrated that during simulations, the RMSD of the protein at the starting time fluctuated and reached about 2.8 Å from 0–30 ns time of simulation. After the 30 ns mark, the protein stabilized, indicating that it reached equilibrium and had an average RMSD of about 2.4 Å at the end of the 100 ns . The lig2; Polar Surface Area (PSA) and the van der Waals surface area (MolSA) were relatively low compared to the first complex, indicating that myricetin was buried in the allosteric site pocket during the simulation  and the van der Waals surface area (MolSA) were high  in the rat model revealed that myricetin had LD50 = 3.02 mol/kg, while aureusidin 4,6-diglucoside and naringoside had LD50 of 2.65 and 2.26 mol/kg values, respectively. These have higher LD50 values, which means that these flavonoids can be acceptable and well-tolerated when consumed  (accessed on 3 December 2020) [The 2.9 Å resolution structure of the RdRp with PDB identification number 6m71 was obtained from the RSCB protein data bank . This PDer 2020) . After ter 2020) . The allThe allosteric site residues identified by Allosite Pro server were all present in the Chain-A of the RdRp and the predicted allosteric site residues identified had the following amino acid residues in the pocket. The sequence and types of the amino acid residues identified are: Thr582, Arg583, Glt584, Ala585, Thr586, Val587, Val588, Thr591, Ser592, Gly597, Asn600, Met601, Thr604, Val605, Arg750, Ser754, Met755 and Met756. This allosteric site is not yet validated through experiments.Prior to docking studies, a literature study was performed for the selection of ligands (flavonoids). We have selected most of the flavonoids based on their previous use as antiviral agents in both experimental and computational studies. This is the main reason we have tried these inhibitors in our docking and simulation study of RdRp of SARS-CoV-2. A total of one hundred flavonoids were selected for docking studies, and the structures of these selected flavonoids were prepared using ChemDraw software 10.0  for the Docking of the selected flavonoids with SARS-CoV-2 RdRp was performed through Molecular Operating Environment  using the established protocols ,50,51,52The top three lead flavonoids with the best interactions, lowest docking score (S-score) and lowest root-mean-square-deviations (RMSD) calculated via MOE and the apo-enzyme were then used for further computational studies via Schrodinger’s Desmond module MD-Simulation Software. For MD-Simulations, the best docked top three flavonoids in complex with the SARS-CoV-2 RdRp were saved in PDB format in MOE and then used for further stability studies in Schrodinger’s Desmond module . The Deshttp://lmmd.ecust.edu.cn/admetsar2) (accessed on 10 March 2021) [Computational studies involving the metabolism, absorption, toxicity and distribution of the top three selected flavonoids in a living system were performed through the admet-SAR online server (ch 2021) .We screened flavonoid phytochemicals against RdRp and utilized a novel approach of targeting the allosteric site of this enzyme. Various in silico computational tools were employed to perform these computational studies, including molecular docking, simulation and ADMET studies. Several studies have reported antiviral activities of these phytochemicals. In this computational work, it was found that they bind to the allosteric site with strong affinities, and the binding was also confirmed by molecular dynamics simulations. They make stable to semi-stable associations and their interactions were the same as seen in the molecular docking studies. The three flavonoids naringoside, myricetin and aureusidin 4,6-diglucoside showed maximum interactions with the lowest binding energies. Arg583, Thr604, Asn600 and Ser592 of the allosteric site of RdRp were confirmed via MD-simulations to interact strongly with the top three selected flavonoids through both H-bonding and hydrophobic interactions during 100 ns virtual simulation time. Moreover, ADMET prediction studies also confirmed that these flavonoids have ample bioavailability because of good HIA values; they were all also non-carcinogenic and non-toxic. The SARS-CoV-2 enzyme active site and surface proteins are mutating at high rates; therefore, exploring and targeting the allosteric sites, as performed in this research, can result in more potent novel drugs that can effectively bind and repress viral activity. In this computational research of allosteric site inhibition, the top hits identified exhibited strong binding and interactions with the allosteric site of RdRp polymerase. From this, one may assume that these identified phytochemical flavonoids can be potential drug candidates against the coronavirus.


Pseudomonassyringae are among the most active ice nucleatorsknown, enabling ice formation at temperatures close to the meltingpoint of water. The working mechanisms of INPs remain elusive, buttheir ice nucleation activity has been proposed to depend on the abilityto form large INP aggregates. Here, we provide experimental evidencethat INPs alone are not sufficient to achieve maximum freezing efficiencyand that intact membranes are critical. Ice nucleation measurementsof phospholipids and lipopolysaccharides show that these membranecomponents are not part of the active nucleation site but rather enableINP assembly. Substantially improved ice nucleation by INP assembliesis observed for deuterated water, indicating stabilization of assembliesby the stronger hydrogen bonds of D2O. Together, theseresults show that the degree of order/disorder and the assembly sizeare critically important in determining the extent to which bacterialINPs can facilitate ice nucleation.Ice-nucleating proteins(INPs) from  Certain ice nucleation active microbes enable ice formationat temperatures close to 0 °C, better than other organic or inorganicmaterial.3 Ice nucleation active bacteria cause frostdamage to plants, and in the atmosphere, they may glaciate cloudsand influence precipitation patterns.5 The best characterizedbiological ice nucleators (INs) are from the plant-associated bacteria Pseudomonas syringae.6 The abilityof P. syringae to facilitate ice nucleation is attributedto specialized ice-nucleating proteins (INPs) anchored to the outerbacterial cell membrane.8 The functional structureof the INPs has been reported to contain a hydrophobic N-terminaldomain, a hydrophilic C-terminal domain, and a large central repeatdomain presumably acting as ice nucleation sites.9 The proposed nucleation sites consist of arrays of STQTand ESSLT motifs, where threonine and serine are most conserved.10 Apart from the structural properties of theactive site, the exceptional activity of bacterial INs has been reportedto critically depend on the ability of the INPs to assemble into largeclusters.12 Based on their activity in droplet freezing experiments,the bacterial INs are oftentimes grouped into classes A–C.13 Class A INs consist of large aggregates (>30INPs) and are responsible for freezing between ∼−2 and∼−4 °C, whereas C INs consist of smaller aggregatesthat induce freezing at ∼−7.5 °C.13 Class B INs are less common and responsible for freezing between∼-5 and ∼−7 °C. The INPs are localized inthe outer bacterial membrane, as demonstrated by fractionating experimentsand the isolation of ice-nucleating vesicles shed from the membrane.14 The leaflet of the outer membrane of Gram-negative bacteria like P. syringae consists of phospholipids and complex lipopolysaccharides can havevery different effects on class A and class C INs.20 Here, we use the high-throughput twin-plate ice nucleationassay (TINA) to investigate the ice nucleation activity of the lipids1,2-dimyristoyl-3-trimethylammonium −propane (DPTAP), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (sodium counterion) (DPPG),1,2-dipalmitoyl-sn-glycero-3-phosphorylethanolamine(chloride counterion) (DPPE), phosphatidylinositol (PI), lipopolysaccharides,and the effects of deuterated water, heat, and delipidation on theice nucleation activity of P. syringae.21The formation of ice is thermodynamicallyfavored at temperatures below 0 °C, but the crystallization iskinetically hindered. As a result, pure water can be supercooled totemperatures as low as −38 °C, below which homogeneousice nucleation occurs.charides1. TurnerP. syringae and is widely used as a model for bacterial ice nucleation studies.22 The initial P. syringae solutionshad a concentration of 0.1 mg/mL and were then serially diluted 10-fold,resulting in concentrations from 1 ng/mL to 0.1 mg/mL. The cumulativeice nucleator number concentration (Nm) was calculated using Vali’s formula and represents the numberof ice nucleators per unit weight that are active above a certaintemperature.23 For the bacterial IN solutionin water, the spectrum shows two strong increases in Nm(T) around ∼−2.9 and ∼−7.5°C with plateaus between ∼−4.5 and ∼−7°C and below ∼−9.5 °C. The two rises in thespectrum reveal the presence of two classes of IN with different activationtemperatures. We attribute the observed rises at ∼−2.9and ∼−7.5 °C to class A and C IN, respectively.Class A IN supposedly consist of large INP assemblies and the lessactive class C IN consist of smaller INP assemblies. We purified theINPs of P. syringae using Folch extraction (FE) andice affinity purification (IAP). FE is based on the partitioning oflipids in a biphasic mixture of chloroform and methanol and causesa separation of lipid and protein components.24 IAP uses the ability of INPs to bind to ice, and the purificationprocess involves the incorporation of INPs into the growing ice phaseand the exclusion of impurities.25 We will referto the purified samples as “lipid” (FE extraction) and“purified INP” (FE extraction and subsequent IAP) fractions. P.syringae look different than the spectrum of P. syringae in water. For both fractions, we observe that the class A relatedincrease at ∼−2.9 °C is absent and that the totalnumber of INs is reduced. For the purified INPs, we observe an increaseat ∼−7 °C, and for the lipid fraction, an increaseat ∼−8 °C. Apparently, the removal of the lipidskeeps the class C INs largely intact, but it prevents the formationof the highly efficient class A INs. Interestingly, the lipid fractionretained significant activity. Ice nucleation activity of lipids alonecan be excluded, as demonstrated by P. syringae samples that underwent delipidationtreatment but were not separated also only showed an increase at ∼−7°C corresponding to class C INs and no increase at ∼−2.9°C .Clearly, the loss of activity of class A INs upon delipidationis irreversible, and reintroducing a lipid matrix is insufficientto restore activity of class A INs.Figure S2), and LPS. The activitiesof the lipids were determined in aqueous solutions with differentsurface coverages . We find that all ofthe lipids show negligible ice nucleation activity. The observed T50 values of ∼−25 °C aresimilar to those of pure water in our setup. T50 values are defined as the temperatures at which 50% of thedroplets are frozen. These observations are in line with previousfindings that long-chain fatty acids and surfactants are poor INs.27Pseudomonas aeruginosa, andin mixtures of the two. For P. syringae in pure D2O, the freezing spectrum looks similar to that of P. syringae in water, with a ∼4 °C shift ofthe INP-mediated freezing curve to warmer temperatures. The observedshift is consistent with the expected shift of ∼3.82 °Cbased on the higher melting point of D2O. Turner et al.previously described a third intermediate class B INs, active at around∼−5 °C, and that examining the effects of substitutingD2O for H2O allows for differentiation of thedifferent classes on the basis of their isotope-induced shifts innucleation threshold.13 As apparent from P. syringae in H2O and D2O. We explain the observed differences with higher rigidities ofINPs in D2O and fewer structural fluctuations of the INP-assembliesdue to the stronger intramolecular D-bonds.32 In fact, at a macroscopic level, there is some evidence suggestingthat D2O is a worse solvent than water and that proteinstend to reduce the surface area in contact with D2O byforming larger aggregates.34 Thus, especially smallINP aggregates, present at lower concentrations, tend to increasetheir size and thereby the ice nucleation efficiency as shown in Having established that lipids and LPSare not active componentsof the ice nucleation site, we hypothesized whether the role of themembrane might be to serve as a functional assembly matrix for thehighly efficient class A IN. 35 In these studies,the presence of borate compounds was further shown to dramaticallyreduce the ice nucleation activity of P. syringae, which was explained by the complexation of the hydroxyl groups ofthe inositol.36 We find that PI shows no icenucleation activity, and the presence of borate compounds also didnot reduce the ice nucleation activity of P. syringae . Our findings further supportthe interpreation that class C and class A INs from P. syringae do not differ in the structure of their INP building blocks butsolely in their assembly size and supramolecular ordering. After delipidationexperiments, the purified INPs displayed ice nucleation activity thatcould be assigned to class C INs, while class A activity was completelyremoved. We conclude that class C IN only consists of small INP assembliesthat do not require lipids for functionality, whereas class A aggregatesrequire an intact membrane environment for functional alignment andaggregation. Using different H2O/D2O ratios,we were able to show that the extent of the assembly of INPs and thecorresponding amount of class A and class C INs can be altered dependingon the solvent mixture. Our results unambiguously prove that maximumice nucleation activity observed in bacteria results from the associationof INPs within the membrane. Hence, studies that take only the bacterialINPs into consideration have to be taken with a caveat, as they donot resemble the bacterial system and their highly efficient INs.Our data is consistent with a mechanism, in which bacteria have toexert precise control over (1) the distance between the INP monomersat the sub-Ångstrom level, and (2) the size of the protein assembliesto achieve high ice nucleation activities.10 This mechanism explains the high sensitivity of class A INs to temperatureas well as chemicals19 that modify the properties of the cell membraneor the aggregation behavior of proteins. Given that highly activeclass A INs have also been observed in mutated E. coli cells12 suggests that INP assembly isrobust toward variations in exact membrane composition.INPs are localized in the outer bacterial membrane, suggestingthat lipids might be required for ice nucleation activity as partof the active nucleation site or by enabling precise INP assembly.The fact that we did not observe ice nucleation activity of lipidsand LPS components of the membrane implies that they are not partof the nucleation site. Interestingly, PI was previously reportedto be an important component in ice nucleation sites of bacteria andinsects.


Here we addressed the mechanisms of CREB attenuation following bursts in CREB phosphorylation. We show that phosphorylation of S133 is reversed by protein phosphatase 2A (PP2A), which is recruited to CREB through its B56 regulatory subunits. We found that a B56-binding site located at the carboxyl-terminal boundary of the KID (BS2) mediates high-affinity B56 binding, while a second binding site (BS1) located near the amino terminus of the KID mediates low affinity binding enhanced by phosphorylation of adjacent casein kinase (CK) phosphosites. Mutations that diminished B56 binding to BS2 elevated both basal and stimulus-induced phosphorylation of S133, increased CBP interaction with CREB, and potentiated the expression of CREB-dependent reporter genes. Cells from mice harboring a homozygous CrebE153D mutation that disrupts BS2 exhibited increased S133 phosphorylation stoichiometry and elevated transcriptional bursts to cAMP. These findings provide insights into substrate targeting by PP2A holoenzymes and establish a new mechanism of CREB attenuation that has implications for understanding CREB signaling in cell growth, metabolism, synaptic plasticity, and other physiologic contexts.The cAMP response element-binding protein (CREB) is an important regulator of cell growth, metabolism, and synaptic plasticity. CREB is activated through phosphorylation of an evolutionarily conserved Ser residue (S133) within its intrinsically disordered kinase-inducible domain (KID). Phosphorylation of S133 in response to cAMP, Ca The cyclic AMP response element (CRE)-binding protein (CREB) is a signal-regulated transcription factor that has served as a model for understanding phosphorylation-coupled folding, coactivator binding, and gene regulation . ConservThe canonical CREB activation pathway involves its cAMP-inducible phosphorylation on S133 by protein kinase A (PKA) , 5. S1332+, which stimulates the Ca2+-dependent phosphatase calcineurin to dephosphorylate CRTC2 —activates CREB independent of S133 phosphorylation , 13, 14.te CRTC2 . Upon ente CRTC2 , 17, 18.2+ by calmodulin kinases II and IV  and casein kinases 1 and 2  in response to genotoxic stress , 30. WitFinally, while the kinases controlling CREB phosphorylation have garnered much attention, comparatively little is known regarding the mechanisms of CREB dephosphorylation. Both PP1 and PP2A have been implicated as S133 phosphatases  while thin vivo.In this study we show that the CK cassette and S133 are coregulated during canonical CREB activation by cAMP and that recruitment of PP2A-B56 holoenzymes to a constitutive B56-docking motif enhanced by an acidic patch triggers the dephosphorylation of S133 and the CK cassette. Cells expressing CREB with disruptive mutations in this motif exhibits increased S133 phosphorylation and transcription potential. These results highlight the interdependence of CREB phosphorylation mechanisms and establish new models for probing functional impacts of CREB signaling A). To investigate the relationship between the CK cassette and canonical regulation of CREB by cAMP, we measured CREB phosphorylation in mouse embryo fibroblasts (MEFs) following a pulsatile exposure to forskolin (Fsk), which stimulates cAMP production. Phosphorylation of the CK cassette (pCK-CREB) was induced within 30 min of Fsk exposure as evidenced by a characteristic electrophoretic mobility shift that reflects CK1/2-dependent phosphorylation of S108, S111, S114, and S117  (B–D). The phosphorylation and dephosphorylation profiles of S133 were comparable between Creb+/+and CrebS111A/S111A MEFs (B and C) suggesting that phosphorylation of the CK cassette does not have a major influence on S133 kinases and phosphatases. Fsk-induced phosphorylation of the CK cassette was blocked by a combination of CK1 (D4476) and CK2 (TBB) inhibitors but not by either inhibitor alone . Consistent with previous findings, CK1/CK2 inhibition also suppressed phosphorylation of CREB in response to calicheamicin γ1 (CLM)-induced DNA damage . Finally, other agents that induce S133 phosphorylation, including PMA and serum, also increased the pCK-CREB/CREB ratio; however, CK cluster phosphorylation stoichiometry was lower than that seen in Fsk-treated cells (A and B). From the combined findings we conclude that Fsk triggers coincident phosphorylation of S133 and the CK cassette while the dephosphorylation profiles of the two motifs differ.Previous work established that, in addition to undergoing stoichiometric phosphorylation following DNA damage, the CK cassette is also phosphorylated independently of DNA damage  Fig. 1AAFig. 1A.Fig. 1B) . Washouted cells , A and BA). Of these, only BS1 is positionally conserved in the CREB paralog ATF1 . Pull-down assays revealed that a GST-CREB (99–159) fusion protein containing both BS1 and BS2 interacted with B56γ1, a member from the B56 family, but not B55α, a member from B55 family (A). In contrast to CREB (99–159), CREB (99–141) containing only BS1 exhibited weak binding to B56γ1 in GST pull-down assays . CREB (126–159) containing only BS2 bound to B56γ1 as avidly as CREB (99–159) containing both BS1 and BS2, suggesting that BS2 is the dominant B56-binding motif (B and C). Consistent with this notion, B56γ1 only weakly associated with the GST-ATF1 , and E153A, a mutation to the required residue for B56 binding within BS2, abolished CREB (99–159) binding to B56γ1 . Finally, the weak binding of B56γ1 to CREB (99–141) was potentiated by a phosphomimetic S108E mutation . This finding is consistent with studies showing that phosphorylation on second or seventh to ninth positions of B56-motifs enhanced the interactions between B56 subunits and their substrates (B), these results suggest that BS1 might be a stimulus-dependent B56 recruitment motif.Recognition of distinct short linear motifs (SLiMs) in substrates is an emerging theme for PP2A regulatory subunits  to control substrate specificities of diverse PP2A holoenzymes. B55 and B56 subunits of PP2A were recently suggested to recognize a bipartite polybasic motif  and an LFig. 25 family A. In conB and C, , B and Cng motif , B and Cbstrates , 38. Becbstrates  and cAMPbstrates B, these F). These models reveal that BS2 is structurally favorable for placing pS133 to the phosphatase active site . In contrast, the binding of BS1 requires a sharp turn in CREB for pS133 to access the phosphatase active site . The lack of residues to make sharp turns in the spacer between BS1 and pS133  makes this motif energetically ineffective to target the holoenzyme to pS133. Finally, we calculated Kcat (16.0/s) and Km (10.3 μM) of the PP2A-B56γ1 holoenzyme toward a phosphorylated pS133-CREB (129–154) peptide . Consistently, the affinity between CREB and B56γ1 was estimated to be between 7.5 and 15 μM by titration pull-down assay (D and E). Taken together, the in vitro studies implicate BS2 as functional B56-binding motif while BS1 may function as a phosphorylation-dependent B56 recruitment domain.Using crystal structures of the PP2A-B56γ1 holoenzyme (PDB code:2NPP) and B56γ1-BubR1 substrate peptide complex (PDB code:5JJA), we built structural models for the PP2A-B56γ1 holoenzyme bound to the upstream and downstream CREB motifs F. These wn assay , D and ETo investigate functional implications of B56-PP2A binding to CREB, we generated a panel of FLAG-tagged CREB mutants in which BS1, BS2, or both motifs were disrupted through substitution of the critical +5 Glu residue with either Ala or Asp. We also introduced eight Ser/Thr to Ala substitutions into the CK cassette see  to examiA and B). pS133 levels were comparable between CREBE153D and CREBE107D/E153D double mutants suggesting that BS2 is the major regulator of S133 dephosphorylation in the absence of stimulation.Whereas BS1 mutations (E107D/A) did not appreciably affect pS133 levels, BS2 (E153D/A) mutations caused a 3- to 4-fold increase in baseline S133 phosphorylation , A and BWT, CREBE107D, and CREBE153D following pulsatile exposure of HeLa cells to Fsk. CREBE153D exhibited much higher level of both basal and induced phosphorylation relative to CREBWT or CREBE107D (C and D). All three CREB proteins were dephosphorylated within 30 min after Fsk removal; however, residual levels of pS133 were higher for CREBE153D than CREBWT or CREBE107D (C and D). Similar findings were made using PMA-treated HeLa cells (E). These findings indicate that BS2 mutations increase basal and stimulus-induced CREB S133 phosphorylation; however, BS2 is not absolutely required for CREB dephosphorylation following stimulus removal.We also compared S133 phosphorylation levels between FLAG-tagged CREBREBE107D , C and DREBE107D , C and DLa cells E. These in vitro (E), we considered the possibility that an S108E mutation would accelerate S133 dephosphorylation following Fsk washout. However, the S133 dephosphorylation rate was comparable between CREBWT, CREBS108A, and CREBS108E following Fsk removal (A and B). These findings suggest that BS1 does not play a major role in the dephosphorylation of overexpressed CREB.Finally, because a phosphomimetic S108E mutation enhanced B56 binding to GST-CREB in vitro E, we con removal , A and BA). While the E107D (BS1) mutation had no discernible effect, E153D (BS2) and E107D/E153D (BS1/BS2) mutations modestly increased the proportion of CREB phosphorylated after CLM treatment . Meanwhile, the phosphomimetic S108E mutation did not significantly alter the magnitude or duration of S121 phosphorylation in CLM-treated HeLa cells. The combined findings suggest that, in addition to regulating S133 dephosphorylation, BS2 contributes to PP2A-dependent dephosphorylation of the CK cassette.We also measured impacts of BS1 and BS2 mutations on CK cassette dephosphorylation kinetics. To this end, we measured S121 phosphorylation/dephosphorylation in HeLa cells expressing FLAG-tagged wild-type and BS1/BS2-mutant CREB proteins following pulsatile exposure to the radiomimetic agent CLM. Consistent with previous work, CLM strongly induced S121 phosphorylation, and this was virtually abolished by mutation of the critical CK1/2 site at S111  Fig. 4AAFig. 4A.WT, CREBE107D (BS1), and CREBE153D (BS2) to a GST fusion with the KID-interacting (KIX) region of CBP. Consistent with its increased S133 phosphorylation (A and B), CREBE153D showed enhanced Fsk-inducible binding to the GST-KIX to relative to CREBWT while an S133A mutation abolished binding (A and C). Interestingly, disruption of CK cassette phosphorylation via the S111A mutation also significantly reduced Fsk-inducible binding of CREB to GST-KIX. This finding is consistent with the modest CREB activation defect previously seen in CREBS111A cells  or PMA (E). We conclude that inhibition of B56-PP2A binding enhances CBP binding and CREB transactivation potential.We next measured transactivation potential of wild-type and mutant Gal4-CREB fusions toward a UAS-luciferase reporter gene in AML-12 hepatocytes. While CREBe to Fsk D or PMA ) or PMA E. We conY134F mutant that exhibited more efficient phosphorylation by S133 kinases, including PKA . Although the Y134F mutation could plausibly diminish CREB recognition by pS133 antibodies, this is unlikely since the CREBY134F/E153D double mutant showed ∼3-fold upregulation of pS133 phosphorylation relative to CREBY134F (A and B). Similar findings were made using transfected HeLa cells stimulated with Fsk (C and D). On the other hand, the Y134F mutation increased Gal4-CREB transactivation potential to a greater extent than the E153D mutation and the activity of a combined Gal4-CREBY134F/E153D mutant was comparable to the Gal4-CREBY134F mutant (E). These findings suggest that the Y134F mutation activates CREB independent of increases in S133 phosphorylation stoichiometry and is dominant to the E153D mutation in transcriptional reporter assays.A previous study identified a CREBding PKA . ReasoniREBY134F , A and BREBY134F , A and Bwith Fsk , C and DF mutant E. These E153D and Gal4-CREBS133A/E153D were comparably elevated over the activity of Gal4-CREBWT (A), suggesting an S133-independent effect of BS2 on CREB transactivation potential. By contrast the S133A mutation abolished the inducible component of Gal4-CREBE153D activity in HeLa cells treated with Fsk (A) or PMA (B). As expected, the reduced transactivation potential of CREBS133A/E153D relative to CREBE153D correlated with reduced inducible binding to the CBP KIX domain (C and D). We conclude that BS2 mutations potentiate inducible CREB transactivation and CBP binding through a pS133-dependent mechanism.To test whether the transcriptional enhancement by BS2 mutations was due to increased S133 phosphorylation stoichiometry, we measured transactivation potential of Gal4-CREB fusions harboring S133A and E153D mutations. Baseline transcriptional activities of Gal4-CREB4-CREBWT A, suggeswith Fsk A or PMA ) or PMA B. As expX domain , C and DCreb locus using CRISPR/CAS9 . A full phenotypic description of CrebE153D mice will be presented elsewhere.To ascertain physiologic implications of CREB dephosphorylation by B56-PP2A, we engineered an E153D mutation into the mouse CAS9 see . Two CreCrebE153D/E153D mice relative to Creb+/+ mice . The fraction of CK1/2-phosphorylated CREB was also elevated in CrebE153D/E153D thymocytes relative to Creb+/+ thymocytes, supporting the idea that BS2 mediates dephosphorylation of the CK cassette (C). Basal and Fsk-induced phosphorylation of S133 were significantly upregulated in primary CrebE153D/E153D MEFs relative to Creb+/+ MEFs (D and E).Baseline S133 phosphorylation was elevated in the thymus, spleen, cerebrum, and cerebellum of A and B, , A–C. Thcassette C. Basal +/+ MEFs , D and ECreb+/+ and CrebE153D/E153D MEFs that had been treated by Fsk for 30 min followed by washout or treatment with PKA inhibitor, H-89. As previously observed, the S133 phosphorylation maximum was elevated in CrebE153D/E153D MEFs relative to Creb+/+ MEFs. H-89 treatment reduced pS133 levels in Creb+/+ and CrebE153D/E153D MEFs with similar kinetics; however, residual pS133 levels were higher in CrebE153D/E153D MEFs relative to Creb+/+ MEFs (F and G). Qualitatively similar results were obtained following Fsk washout (F and G). The combined findings support the role of BS2 in the phosphoregulation of endogenous CREB.To evaluate how the endogenous E153D mutation influences the rate of CREB dephosphorylation, we compared pS133 levels between +/+ MEFs , F and G washout , F and GCrebE153D mutation on the expression of a panel of CREB target genes. Fsk-inducible of the immediate-early genes Nr4a1 and Nr4a2 was significantly elevated in CrebE153D/E153D MEFs relative to Creb+/+ MEFs, with the effect being most pronounced 1 h post stimulation, when Nr4a1 and Nr4a2 levels reached their maxima (A). Maximum induced levels of Areg and Crem were also significantly elevated in CrebE153D/E153D MEFs relative to Creb+/+ MEFs following stimulation with Fsk for 3 h. By contrast, Areg and Crem expressions were not significantly different between CrebE153D/E153D and Creb+/+ MEFs at a 1 h timepoint during the inductive phase of the Fsk response. Finally, as predicted, chromatin immunoprecipitation (ChIP) experiments revealed increased pS133-CREB occupancy over a CREB-binding site within the Areg and Crem gene promoter (B). These findings suggest that increased S133 phosphorylation stoichiometry leads to upregulation of CREB target gene expression in response to Fsk.We next measured the effects of the r maxima A. Maximupromoter B. These In this study we have shown that CREB harbors functional B56-binding sites that facilitate PP2A-dependent dephosphorylation of S133, the CK cassette, and possibly other Ser/Thr residues within the CREB polypeptide. A schematic model summarizing our findings is presented in In vitro binding studies and functional experiments using overexpressed or endogenous CREBE153D proteins implicate BS2 as a major mediator of S133 and CK cassette dephosphorylation . While the effect was modest, Fsk-induced expression of a panel of CREB target genes was elevated in CrebE153D/E153D MEFs relative to Creb+/+ MEFs (A). The more limited impact of the E153D mutation on endogenous gene expression likely reflects the contributions of CRTC-dependent CREB coactivation, the influence of CREB paralogs, and other transcription factors whose activity may be induced by cAMP. It is also possible that cellular CBP/p300 levels become limiting at levels of high S133 phosphorylation stoichiometry.BS2 mutations enhanced CREB transcriptional potential ∼2 to 3 fold in reporter assays using Gal4-CREB fusions lacking C-terminal bZip and CRTC-binding domains , D and E+/+ MEFs A. The moin vitro (E). However, CREB proteins harboring disruptive E107D/A mutations or the S108E phosphomimetic mutation exhibited S133 phosphorylation/dephosphorylation rates similar to those seen for wild-type CREB when expressed in HeLa cells . This suggests that either BS1 is not functional or that B56-PP2A bound through BS1 mediates the dephosphorylation of different Ser/Thr residues on CREB or an associated factor.The functional implications of the low affinity B56-binding site, BS1, remain to be determined. Given that cAMP-induced phosphorylation of S108 within the CK cassette creates a favorable environment for B56 binding, we initially hypothesized that BS1 acts as a phosphorylation-dependent B56 recruitment domain. Consistent with this, a phosphomimetic S108E mutation stimulated the weak B56-binding activity of BS1 in vitro E. HoweveFinally, it is interesting to consider the interplay between the CK cassette, S133, and BS2 in the context of tunable CREB-dependent gene regulation. While BS2 mutations potentiated S133 phosphorylation, they also increased phosphorylation of the CK cassette, which inhibits CREB DNA binding activity at high phosphorylation stoichiometries , 32. AltCreb+/+ and CrebE153D/E153D embryos. Primary MEFs and HeLa cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Forskolin , 3-isobutyl-1-methylxanthine  or phorbol 12-myristate 13-acetate , H-89  were added to cells for indicated times after overnight serum starvation. CLM (2 nM working concentration) was a kind gift of the Pfizer Compound Transfer Program and was maintained as a 4 μM stock solution in DMSO. CK1 (D4476) and CK2 (TBB) inhibitors were added 30 min prior to Fsk treatment and were used at concentrations of 20 μM and 50 μM, respectively. Gal4-CREB luciferase assays were performed using transiently transfected AML-12 hepatocytes  essentially as described (MEFs were isolated from 14.5-day escribed .Escherichia coli strain BL21(DE3) for 16 h at 20 °C. The soluble fraction of the E. coli cell lysate was purified over GS4B resin  and further fractionated by ion exchange chromatography . His-tagged PP2A Cα subunit (1–309) was cloned into a pFastBac vector (Invitrogen) and transformed into DH10Bac competent cells (Thermo Fisher Scientific) for bacmid preparation followed by baculovirus production. Hi-5 suspension cells (Thermo Fisher Scientific) grown to a density of 1.5 × 106 cell/ml were infected with Cα baculovirus to express the protein. Cells were harvested and lysed after 48 h of infection. The soluble fraction of cell lysates was purified by Ni-NTA (QIAGEN) resin and further fractionated by Source 15Q. To assemble PP2A-B56γ1 holoenzyme, Cα subunit was first assembled with GST-Aα followed by the removal of GST- and His-tags using TEV protease. The protein mixture was further fractionated by Source 15Q to obtain pure AC dimer. Similar stochiometric amount of B56γ1 and AC dimer was then incubated in RT for 20 min and purified by gel filtration using a Superdex-200 column . WT and mutated GST-tagged CREB were cloned into pQLinkG vector (Addgene). The proteins were overexpressed 14 h at 23 °C in E. coli strain BL21 (DE3). The soluble fraction of the E. coli cell lysates was purified over GS4B resin and further fractionated by Source 15Q.All constructs were generated using a standard PCR-based cloning strategy. Cloning, expression, and purification of full-length Aα, Cα, and B56γ1 subunits of PP2A and the preparation of PP2A-B56 holoenzymes followed the procedures described in previous studies , 44. Bri2, 1 mM EGTA, 0.1% Triton X-100) supplemented with protease inhibitors and phosphatase inhibitors for 10 min on ice, and proteins were separated on an SDS–polyacrylamide gel. Antibodies used in this study include: α-pS121-CREB (8A construct was generated by replacing a BspEI-KpnI fragment of the wild-type CREB coding sequence with a synthetic Gene Block DNA fragment (IDT) encoding Ala substitutions at Thr-100, Ser-103, Thr-104, Ser-108, Ser-111, Ser-114, Ser-117, and Ser-121.Gal4-CREB  encodes the 147 aa Gal4 DNA-binding domain fused to CREB amino acids 1 to 280. Expression of untagged and FLAG-tagged CREB from pFLAG-CMV and pcDNA3.1-based vectors was carried out as described , 24, 25.121-CREB , α-pS133via the GST tag. The resin was washed with 100 μl assay buffer containing 25 mM Tris (pH 8.0), 150 mM NaCl, and 2 mM DTT for three times to remove the unbound protein. To examine the interaction between CREB and B subunits of PP2A, 100 μg of Bα, or B56γ1 was added to the immobilized GST-CREB in a 50 μl of assay buffer supplemented with 1 mg/ml of BSA to avoid nonspecific binding. After 30 min of incubation, the unbound proteins were removed, and the resin was washed three times using 100 μl assay buffer supplemented with 0.1% Triton-X 100. The proteins remaining bound to resin were examined by SDS-PAGE and visualized by Coomassie blue staining. GST without CREB was used as control. A similar procedure was used to examine the binding affinity between B56γ1 and CREB except for that the immobilized WT GST-CREB (99–159) was incubated with titrated concentrations of B56γ1 for the pull-down assay. GST-KIX pull-down assays of transfected CREB proteins were performed as previously described  was bound to 5 μl of glutathione resin escribed .2 were dispensed in a 384-well, low-volume, round-bottom plates (Corning P/N 3677). Ten microliter of titrated concentrations (twofold dilutions from 250 μM to 3.9 μM) of the substrate prepared in assay buffer and a buffer control were then added to each well. Immediately, the microplate with samples was read by a plate reader at excitation 485 nm and emission 530 nm in the kinetic mode to collect data every 10 s for a total of 10 min. The steady-state kinetics of the PP2A-B56γ1 holoenzymes were determined by fitting the Michaelis–Menten equation  in 10 μl assay buffer containing 25 mM Tris HCl (pH 8.0), 150 mM NaCl, and 50 μM MnClIn Equation Creb1 gene using the MIT CRISPR Design Tool. The aggregate gRNA score of 58 was considered suitable for gene targeting. Four possible off-target cleavage sites with a cutting frequency determination (CFD) score of >0.543 were identified using GUIDE-Seq ; Gm13518-mmu-mir-195b). Three of these are intergenic and none are in linkage disequilibrium with the Creb1 locus. Briefly, two independent CrebE153D founder lines were obtained and yielded identical results. Tail samples were taken at weaning, and the targeted region was characterized using targeted ultradeep sequencing. The targeted region was PCR amplified  samples were indexed and pooled, and the pool was sequenced on a MiSeq 2 × 250 Nano. Resultant sequences were quality filtered, trimmed, and analyzed with CRISPResso (https://www.nature.com/articles/nbt.3583). Founders were backcrossed to B6J, unedited mice, and F1s were characterized similarly.We employed CRISPR/CAS9 to introduce an E153D (BS2) mutation into the C57BL/6 mouse germline. A preferred gRNA target (GACTTTTCTTCTTCAATCCTTGG) was identified 9 bp upstream of codon 153 in the mouse Creb+/+ and CrebE153D/E153D mice from the same litters were used for all subsequent experiments.Heterozygous parents were used to give rise to experimental mice, allowing litters to contain a mix of genotypes. Thus, The study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal protocol was approved by the Campus Animal Care and Use Committee of the University of Wisconsin, Madison.Crem , Areg , Nr4a1 , Nr4a2 , and Gapdh .RNA analysis was carried out as described . The genCrem , ChIP-mAreg . The results were expressed as the relative fold occupancy of the target precipitation as compared with the input.Primary MEFs were cross-linked using 1% formaldehyde (Sigma) for 15 min, lysed and sonicated, and chromatin was immunoprecipitated with α-CREB  and α-pS133 CREB . After washing, the immunoprecipitated chromatin was reverse cross-linked and the liberated DNA fragments were purified using Qiagen spin columns. ChIP samples were measured by quantitative real-time PCR using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad) and SYBR Green. Primer sequences for the promoters: ChIP-mt test contained in Prism8 software. Data are presented as the mean ± standard error (SEM) from minimum three independent experiments. Differences were considered significant with ns: p > 0.05, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.Statistical analysis was performed using the unpaired, two-tailed Student’s All data have been included within the manuscript.This article contains The authors declare that they have no conflicts of interest with the contents of this article.