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