Protein phosphatase 1c associated with the cardiac sodium calcium exchanger1 regulates its activity by dephosphorylating serine 68 phosphorylated phospholemman

The sodium (Na+)-calcium (Ca2+) exchanger 1 (NCX1) is an important regulator of intracellular Ca2+ homeostasis. Serine 68-phosphorylated phospholemman (pSer-68-PLM) inhibits NCX1 activity. In the context of Na+/K+-ATPase (NKA) regulation, pSer-68-PLM is dephosphorylated by protein phosphatase 1 (PP1). PP1 also associates with NCX1; however, the molecular basis of this association is unknown. In this study, we aimed to analyze the mechanisms of PP1 targeting to the NCX1-pSer-68-PLM complex and hypothesized that a direct and functional NCX1-PP1 interaction is a prerequisite for pSer-68-PLM dephosphorylation. Using a variety of molecular techniques, we show that PP1 catalytic subunit (PP1c) co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes, left ventricle lysates, and HEK293 cells. Bioinformatic analysis, immunoprecipitations, mutagenesis, pulldown experiments, and peptide arrays constrained PP1c anchoring to the K(I/V)FF motif in the first Ca2+ binding domain (CBD) 1 in NCX1. This binding site is also partially in agreement with the extended PP1-binding motif K(V/I)FF-X5–8Φ1Φ2-X8–9-R. The cytosolic loop of NCX1, containing the K(I/V)FF motif, had no effect on PP1 activity in an in vitro assay. Dephosphorylation of pSer-68-PLM in HEK293 cells was not observed when NCX1 was absent, when the K(I/V)FF motif was mutated, or when the PLM- and PP1c-binding sites were separated (mimicking calpain cleavage of NCX1). Co-expression of PLM and NCX1 inhibited NCX1 current (both modes). Moreover, co-expression of PLM with NCX1(F407P) (mutated K(I/V)FF motif) resulted in the current being completely abolished. In conclusion, NCX1 is a substrate-specifying PP1c regulator protein, indirectly regulating NCX1 activity through pSer-68-PLM dephosphorylation

Molecular Regulatory Mechanisms of the Sodium-Calcium Exchanger 1 in heart

Forekomst av hjertesvikt i befolkningen er økende. Til tross for at det de siste tiårene har skjedd en betydelig utvikling av medisinsk behandling er dette en av de vanligste dødsårsaker i den vestlige verden. Man mangler innsikt i de molekylære mekanismene som er involvert i utvikling av hjertesvikt. Identifisering av molekylære mekanismer kan bidra til å utvikle mer effektive og spesifikke terapeutiske strategier for målrettet behandling. Dette prosjektet fokuserer på Na+ /Ca2+ ionebytteren (NCX1) som er lokalisert i cellemembranen og regulerer kalsiumnivået i hjertecellen. NCX1 er en av de viktigste mekanismene som fjerner kalsium for å beholde et normalt nivå av dette ionet i cellen. Effektiv håndtering av kalsium er viktig for hjertets evne til å trekke seg sammen og slappe av igjen. Ved hjertesvikt endres kalsiumhomeostasen samt aktiviteten til NCX1. Disse endringene assosieres med økt eller/og redusert hjertekontraktilitet og kan føre til arytmier. I dette prosjektet utdyper vi forståelsen av molekylære mekanismer til tre hjerteproteiner som regulerer aktiviteten til NCX1 i det normale og det sviktende hjertet. Vi har også utviklet et peptid (pro-medikament) som muligens har potensiale til å forbedre funksjon av NCX1 ved diastolisk hjertesvikt

Analysis of a point mutation (D152V) in the transcription factor c- Myb and its effect on the interaction of c-Myb with Histone H3 and DNA

Hematopoiesis is the process by which the hematopoietic stem cells differentiate into a variety of specialized cells (mature blood cells). The development of the definitive hematopoietic cell lineages is regulated by a number of transcription factors, such as the DNA binding transcription factor, c-Myb. This factor is encoded by the c-myb proto-oncogene and loss of function of the c-myb gene results in embryonic lethality due to a failure to develop fetal liver hematopoiesis. The c-myb gene is highly expressed in immature, proliferative hematopoietic cells and its expression level declines as the immature hematopoietic cells differentiate. Ectopic expression of c-Myb inhibits differentiation of hematopoietic precursor cells. Recently, several mutants acting as knockdown alleles of c-myb was found to affect lineage commitment and differentiation by perturbing differentiation of erythoid precursors but allowing megakaryocytopoiesis. One of these mutations was a substitution of valine for aspartic acid at residue 152 in the DNA-binding domain of c-Myb. This mutant was isolated based on its property to rescue platelet defects in thrombopenic c-mpl-/- mice by producing supraphysiological expansion of megakaryocyte and platelet production. However, the molecular mechanism by which this mutation alters c-Myb function was not identified. The aim of this work was to examine the molecular mechanisms of the D152V mutation. Since the alteration lies in the DNA-binding domain (DBD) of c-Myb, it was natural to investigate changes in DNA-binding properties. In addition, the ability to associate with histone H3 was also studied. The DBD of c-Myb is composed of three tandem repeats each being similar to the chromatin interacting SANT-domain found in chromatin regulatory proteins (the Swi3, Ada2, TFIIIB, NcoR, and ISWI proteins). Together these repeats are responsible for the ability of c-Myb to bind to DNA in a sequence-specific fashion. Interestingly, the two last SANT-related repeats (R2R3) of c-Myb was recently found to interact also with the N-terminal of histone H3 (Mo et al., 2005). This was proposed to position the H3-tail for acetylation and represents a novel chromatin function of c-Myb-DBD. When the molecular mechanism altered by the D152V mutant was to be studied, we addressed both putative changes in DNA-binding properties, as well as changes in its interaction with histone H3. The latter was to see whether the D152V mutation disrupted the Myb-H3 interaction, which might be an alternative explanation of the the reduction of c-Myb activity revealed in the previous study (Carpinelli et al. 2004). GST-H3 fusion proteins were made and used to elucidate histone H3 interaction with the minimal DNA-binding domain (R2R3) of c-Myb wild type and D152V mutant. The results showed that the binding of R2R3-c-MybD152V to histone H3 was weaker than that detected in R2R3-c-Myb wild type. On the other hand, investigation of its effect on DNA-binding properties revealed unexpectedly an increase in DNA-binding activity of c-MybD152V. A possible explanation may be that the substitution of the valine for an aspartic acid causes a removal of one negatively charge on the c-Myb protein surface, resulting in an increased DNA-binding of c-MybD152V compared to wild type. In conclusion, the results presented in this thesis indicate that the interaction of c-Myb with histone H3 is reduced by the D152V mutation, which may contribute to the reduction of c-Myb activity. To pursue these findings further, endogenous gene activation assays and detection of c-Myb-histone H3 interaction at the chromosomal level would be of interest

A c-Myb mutant causes deregulated differentiation due to impaired histone binding and abrogated pioneer factor function

The transcription factor c-Myb is involved in early differentiation and proliferation of haematopoietic cells, where it operates as a regulator of self-renewal and multi-lineage differentiation. Deregulated c-Myb plays critical roles in leukaemias and other human cancers. Due to its role as a master regulator, we hypothesized it might function as a pioneer transcription factor. Our approach to test this was to analyse a mutant of c-Myb, D152V, previously reported to cause haematopoietic defects in mice by an unknown mechanism. Our transcriptome data from K562 cells indicates that this mutation specifically affects c-Myb's ability to regulate genes involved in differentiation, causing failure in c-Myb's ability to block differentiation. Furthermore, we see a major effect of this mutation in assays where chromatin opening is involved. We show that each repeat in the minimal DNA-binding domain of c-Myb binds to histones and that D152V disrupts histone binding of the third repeat. ATAC-seq data indicates this mutation impairs the ability of c-Myb to cause chromatin opening at specific sites. Taken together, our findings support that c-Myb acts as a pioneer factor and show that D152V impairs this function. The D152V mutant is the first mutant of a transcription factor specifically destroying pioneer factor function

Design of a Proteolytically Stable Sodium-Calcium Exchanger 1 Activator Peptide for In Vivo Studies

The cardiac sodium–calcium exchanger (NCX1) is important for normal Na+- and Ca2+-homeostasis and cardiomyocyte relaxation and contraction. It has been suggested that NCX1 activity is reduced by phosphorylated phospholemman (pSer68-PLM); however its direct interaction with PLM is debated. Disruption of the potentially inhibitory pSer68-PLM-NCX1 interaction might be a therapeutic strategy to increase NCX1 activity in cardiac disease. In the present study, we aimed to analyze the binding affinities and kinetics of the PLM-NCX1 and pSer68-PLM-NCX1 interactions by surface plasmon resonance (SPR) and to develop a proteolytically stable NCX1 activator peptide for future in vivo studies. The cytoplasmic parts of PLM (PLMcyt) and pSer68-PLM (pSer68-PLMcyt) were found to bind strongly to the intracellular loop of NCX1 (NCX1cyt) with similar KD values of 4.1 ± 1.0 nM and 4.3 ± 1.9 nM, but the PLMcyt-NCX1cyt interaction showed higher on/off rates. To develop a proteolytically stable NCX1 activator, we took advantage of a previously designed, high-affinity PLM binding peptide (OPT) that was derived from the PLM binding region in NCX1 and that reverses the inhibitory PLM (S68D)-NCX1 interaction in HEK293. We performed N- and C-terminal truncations of OPT and identified PYKEIEQLIELANYQV as the minimum sequence required for pSer68-PLM binding. To increase peptide stability in human serum, we replaced the proline with an N-methyl-proline (NOPT) after identification of N-terminus as substitution tolerant by two-dimensional peptide array analysis. Mass spectrometry analysis revealed that the half-life of NOPT was increased 17-fold from that of OPT. NOPT pulled down endogenous PLM from rat left ventricle lysate and exhibited direct pSer68-PLM binding in an ELISA-based assay and bound to pSer68-PLMcyt with a KD of 129 nM. Excess NOPT also reduced the PLMcyt-NCX1cyt interaction in an ELISA-based competition assay, but in line with that NCX1 and PLM form oligomers, NOPT was not able to outcompete the physical interaction between endogenous full length proteins. Importantly, cell-permeable NOPT-TAT increased NCX1 activity in cardiomyocytes isolated from both SHAM-operated and aorta banded heart failure (HF) mice, indicating that NOPT disrupted the inhibitory pSer68-PLM-NCX1 interaction. In conclusion, we have developed a proteolytically stable NCX1-derived PLM binding peptide that upregulates NCX1 activity in SHAM and HF cardiomyocytes

Design of a proteolytically stable sodium-calcium exchanger 1 activator peptide for in vivo studies

The cardiac sodium–calcium exchanger (NCX1) is important for normal Na + - and Ca 2+ -homeostasis and cardiomyocyte relaxation and contraction. It has been suggested that NCX1 activity is reduced by phosphorylated phospholemman (pSer68-PLM); however its direct interaction with PLM is debated. Disruption of the potentially inhibitory pSer68-PLM-NCX1 interaction might be a therapeutic strategy to increase NCX1 activity in cardiac disease. In the present study, we aimed to analyze the binding affinities and kinetics of the PLM-NCX1 and pSer68-PLM-NCX1 interactions by surface plasmon resonance (SPR) and to develop a proteolytically stable NCX1 activator peptide for future in vivo studies. The cytoplasmic parts of PLM (PLM cyt ) and pSer68-PLM (pSer68-PLM cyt ) were found to bind strongly to the intracellular loop of NCX1 (NCX1 cyt ) with similar K D values of 4.1 ± 1.0 nM and 4.3 ± 1.9 nM, but the PLM cyt -NCX1 cyt interaction showed higher on/off rates. To develop a proteolytically stable NCX1 activator, we took advantage of a previously designed, high-affinity PLM binding peptide (OPT) that was derived from the PLM binding region in NCX1 and that reverses the inhibitory PLM (S68D)-NCX1 interaction in HEK293. We performed N- and C-terminal truncations of OPT and identified PYKEIEQLIELANYQV as the minimum sequence required for pSer68-PLM binding. To increase peptide stability in human serum, we replaced the proline with an N-methyl-proline (NOPT) after identification of N-terminus as substitution tolerant by two-dimensional peptide array analysis. Mass spectrometry analysis revealed that the half-life of NOPT was increased 17-fold from that of OPT. NOPT pulled down endogenous PLM from rat left ventricle lysate and exhibited direct pSer68-PLM binding in an ELISA-based assay and bound to pSer68-PLM cyt with a K D of 129 nM. Excess NOPT also reduced the PLM cyt -NCX1 cyt interaction in an ELISA-based competition assay, but in line with that NCX1 and PLM form oligomers, NOPT was not able to outcompete the physical interaction between endogenous full length proteins. Importantly, cell-permeable NOPT-TAT increased NCX1 activity in cardiomyocytes isolated from both SHAM-operated and aorta banded heart failure (HF) mice, indicating that NOPT disrupted the inhibitory pSer68-PLM-NCX1 interaction. In conclusion, we have developed a proteolytically stable NCX1-derived PLM binding peptide that upregulates NCX1 activity in SHAM and HF cardiomyocytes

Full-length cardiac Na+/Ca2+ exchanger 1 protein is not phosphorylated by protein kinase A

The cardiac Na+/Ca2+ exchanger 1 (NCX1) is an important regulator of intracellular Ca2+ homeostasis and cardiac function. Several studies have indicated that NCX1 is phosphorylated by the cAMP-dependent protein kinase A (PKA) in vitro, which increases its activity. However, this finding is controversial and no phosphorylation site has so far been identified. Using bioinformatic analysis and peptide arrays, we screened NCX1 for putative PKA phosphorylation sites. Although several NCX1 synthetic peptides were phosphorylated by PKA in vitro, only one PKA site (threonine 731) was identified after mutational analysis. To further examine whether NCX1 protein could be PKA phosphorylated, wild-type and alanine-substituted NCX1-green fluorescent protein (GFP)-fusion proteins expressed in human embryonic kidney (HEK)293 cells were generated. No phosphorylation of full-length or calpain- or caspase-3 digested NCX1-GFP was observed with purified PKA-C and [γ-32P]ATP. Immunoblotting experiments with anti-PKA substrate and phosphothreonine-specific antibodies were further performed to investigate phosphorylation of endogenous NCX1. Phospho-NCX1 levels were also not increased after forskolin or isoproterenol treatment in vivo, in isolated neonatal cardiomyocytes, or in total heart homogenate. These data indicate that the novel in vitro PKA phosphorylation site is inaccessible in full-length as well as in calpain- or caspase-3 digested NCX1 protein, suggesting that NCX1 is not a direct target for PKA phosphorylation

Molecular Basis of Calpain Cleavage and Inactivation of the Sodium-Calcium Exchanger 1 in Heart Failure

Cardiac sodium (Na+)-calcium (Ca2+) exchanger 1 (NCX1) is central to the maintenance of normal Ca2+ homeostasis and contraction. Studies indicate that the Ca2+-activated protease calpain cleaves NCX1. We hypothesized that calpain is an important regulator of NCX1 in response to pressure overload and aimed to identify molecular mechanisms and functional consequences of calpain binding and cleavage of NCX1 in the heart. NCX1 full-length protein and a 75-kDa NCX1 fragment along with calpain were up-regulated in aortic stenosis patients and rats with heart failure. Patients with coronary artery disease and sham-operated rats were used as controls. Calpain co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes and left ventricle lysate. Immunoprecipitations, pull-down experiments, and extensive use of peptide arrays indicated that calpain domain III anchored to the first Ca2+ binding domain in NCX1, whereas the calpain catalytic region bound to the catenin-like domain in NCX1. The use of bioinformatics, mutational analyses, a substrate competitor peptide, and a specific NCX1-Met369 antibody identified a novel calpain cleavage site at Met369. Engineering NCX1-Met369 into a tobacco etch virus protease cleavage site revealed that specific cleavage at Met369 inhibited NCX1 activity (both forward and reverse mode). Finally, a short peptide fragment containing the NCX1-Met369 cleavage site was modeled into the narrow active cleft of human calpain. Inhibition of NCX1 activity, such as we have observed here following calpain-induced NCX1 cleavage, might be beneficial in pathophysiological conditions where increased NCX1 activity contributes to cardiac dysfunction