Calcium (Ca2+) dysregulation is a hallmark of heart failure and characterized by impaired Ca2+ sequestration into the sarcoplasmic reticulum (SR) SR-Ca2+-ATPase (SERCA). We recently discovered micropeptide named DWORF (DWarf Open Reading Frame) that enhances SERCA activity displacing phospholamban (PLN), potent inhibitor. Here we show has higher apparent binding affinity for than PLN overexpression mitigates contractile dysfunction associated with overexpression, substantiating its role as...

We have used a "two-color" SERCA (sarco/endoplasmic reticulum calcium ATPase) biosensor and unique high-throughput fluorescence lifetime plate reader (FLT-PR) to develop high-precision live-cell assay designed screen for small molecules that perturb structure. A construct, in which red fluorescent protein (RFP) was fused the N terminus green (GFP) an interior loop, stably expressed HEK cell line grows monolayer or suspension. Fluorescence resonance energy transfer (FRET) from GFP RFP...

Multiple Ca2+ channel beta-subunit (Ca(v)beta) isoforms are known to differentially regulate the functional properties and membrane trafficking of high-voltage-activated channels, but precise isoform expression pattern Ca(v)beta subunits in ventricular muscle has not been fully characterized. Using sequence data from Human Genome Project define intron/exon structure four genes, we designed a systematic RT-PCR strategy screen human canine left myocardial samples for all isoforms. A total 18...

The E3 ubiquitin ligase atrophin interacting protein 4 (AIP4) mediates ubiquitination and down-regulation of the chemokine receptor CXCR4. AIP4 belongs to Nedd4-like homologous E6-AP carboxy terminus domain family ligases, which typically bind proline-rich motifs within target proteins via WW domains. intracellular domains CXCR4 lack canonical binding motifs; thus, whether is targeted directly or indirectly an adaptor remains unknown. Here, we show that can interact with a novel noncanonical...

The regulatory interaction of phospholamban (PLN) with Ca 2+ -ATPase controls the uptake calcium into sarcoplasmic reticulum, modulating heart muscle contractility. A missense mutation in PLN cytoplasmic domain (R9C) triggers dilated cardiomyopathy humans, leading to premature death. Using a combination biochemical and biophysical techniques both vitro live cells, we show that R9C increases stability pentameric assembly via disulfide bridge formation, preventing its binding as well...

To investigate the mechanism of regulation sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) by phospholamban (PLB), we expressed Cerulean-SERCA and yellow fluorescent protein (YFP)-PLB in adult rabbit ventricular myocytes using adenovirus vectors. SERCA PLB were localized sarcoplasmic mobile over multiple sarcomeres on a timescale tens seconds. We also observed robust fluorescence resonance energy transfer (FRET) from to YFP-PLB. Electrical pacing cardiac elicited cytoplasmic Ca(2+)...

The sarco-plasmic reticulum calcium pump (SERCA) plays a critical role in the contraction-relaxation cycle of muscle. In cardiac muscle, SERCA is regulated by inhibitor phospholamban. A new regulator, dwarf open reading frame (DWORF), has been reported to displace phospholamban from SERCA. Here, we show that DWORF direct activator SERCA, increasing its turnover rate absence Measurement in-cell dynamics supports this observation and demonstrates increases SERCA-dependent reuptake. These...

Phospholamban (PLB) or the sarcoplasmic reticulum Ca 2+ –ATPase (SERCA) were fused to cyan fluorescent protein (CFP) and coexpressed with PLB yellow (YFP). The expressed fluorescently tagged proteins imaged using epifluorescence total internal reflection fluorescence microscopy. YFP was selectively bleached by a focused laser beam. CFP at targeted site increased after photobleaching, indicating resonance energy transfer between CFP-SERCA/CFP-PLB YFP-PLB. donor relaxed back toward baseline as...

To investigate the effect of phosphorylation on interactions phospholamban (PLB) with itself and its regulatory target, SERCA, we measured FRET from CFP-SERCA or CFP-PLB to YFP-PLB in live AAV-293 cells. Phosphorylation PLB was mimicked by mutations S16E (PKA site) S16E/T17E (PKA+CaMKII sites). increased protein concentration up a maximum (FRET(max)) that taken represent intrinsic bound complex. The dependence yielded dissociation constants (K(D)) for PLB-PLB PLB-SERCA interactions. data...

Phospholamban (PLB) oligomerization, quaternary structure, and sarco(endo)plasmic reticulum calcium ATPase (SERCA) binding were quantified by fluorescence resonance energy transfer (FRET) in an intact cellular environment. FRET between cyan fluorescent protein-PLB yellow AAV-293 cells showed hyperbolic dependence on protein concentration, with a maximum efficiency of 45.1 +/- 1.3%. The observed corresponds to probe separation distance 58.7 0.5A(,) according computational model...

The HS-1 associated protein X-1 (HAX-1) is a ubiquitously expressed that protects cardiomyocytes from programmed cell death. Here we identify HAX-1 as regulator of contractility and calcium cycling in the heart. overexpression reduced sarcoplasmic reticulum Ca-ATPase (SERCA2) pump activity isolated vivo, leading to depressed myocyte kinetics mechanics. Conversely, downregulation enhanced contractility. inhibitory effects were abolished upon phosphorylation phospholamban, which plays...

ABSTRACT TRIM5 proteins mediate a potent block to the cross-species transmission of retroviruses, most well known being TRIM5α protein from rhesus macaques, which potently inhibits human immunodeficiency virus type 1 (HIV-1) infection. This restriction occurs at an early stage in replication cycle and is mediated by binding determinants present retroviral capsid. TRIM5α, as other TRIM family proteins, has been shown be regulated interferons (IFN). Here we show that associates with another...

The sarco(endo)plasmic reticulum calcium ATPase (SERCA) undergoes conformational changes while transporting calcium, but the details of domain motions are still unclear. objective present study was to measure distances between cytoplasmic domains SERCA2a in order reveal magnitude and direction changes. Using fluorescence microscopy live cells, we measured intramolecular resonance energy transfer (FRET) from a donor fluorescent protein fused SERCA N-terminus an acceptor either N-, P-, or...

We measured in-gel fluorescence anisotropy of phospholamban (PLB) labeled with the biarsenical fluorophore FlAsH at three different sites on cytoplasmic domain. The 6 kDa monomer bands FlAsH-tetracysPLB showed high (r = 0.29), reflecting null homotransfer and low mobility (S 0.85) nanosecond time scale lifetime. 30 (pentameric PLB) within same lanes exhibited anisotropy, suggesting intrapentameric energy between PLB subunits. labels positioned residue −6, 5, or 23 a graduated pattern...

We have detected directly the interactions of sarcolipin (SLN) and sarcoplasmic reticulum Ca-ATPase (SERCA) by measuring fluorescence resonance energy transfer (FRET) between fusion proteins labeled with cyan fluorescent protein (donor) yellow (acceptor). SLN is a membrane that helps control contractility regulating SERCA activity in fast-twitch atrial muscle. Here we used FRET microscopy spectroscopy baculovirus expression insect cells to provide direct evidence for: 1) oligomerization 2)...

Abstract Background The TRIM5 proteins are cellular restriction factors that prevent retroviral infection in a species-specific manner. Multiple experiments indicate activity requires accessory host factors, including E2-enzymes. To better understand the mechanism of restriction, we conducted yeast-two hybrid screens to identify bind two orthologues. Results only cDNAs scored on repeat testing with both orthologues were proteasome subunit PSMC2 and ubiquitin. Using co-immunoprecipitation...