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Rheostatic Regulation of the SERCA/Phospholamban Membrane Protein Complex Using Non-Coding RNA and Single-Stranded DNA oligonucleotides.

Soller KJ, Verardi R, Jing M, Abrol N, Yang J, Walsh N, Vostrikov VV, Robia SL, Bowser MT, Veglia G - Sci Rep (2015)

Bottom Line: Both in HEK cells expressing the SERCA/PLN complex, as well as in cardiac sarcoplasmic reticulum preparations, these short oligonucleotides bind and reverse PLN's inhibitory effects on SERCA, increasing the ATPase's apparent Ca(2+) affinity.Solid-state NMR experiments revealed that ssDNA interacts with PLN specifically, shifting the conformational equilibrium of the SERCA/PLN complex from an inhibitory to a non-inhibitory state.Importantly, we achieved rheostatic control of SERCA function by modulating the length of ssDNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455.

ABSTRACT
The membrane protein complex between sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) and phospholamban (PLN) is a prime therapeutic target for reversing cardiac contractile dysfunctions caused by calcium mishandling. So far, however, efforts to develop drugs specific for this protein complex have failed. Here, we show that non-coding RNAs and single-stranded DNAs (ssDNAs) interact with and regulate the function of the SERCA/PLN complex in a tunable manner. Both in HEK cells expressing the SERCA/PLN complex, as well as in cardiac sarcoplasmic reticulum preparations, these short oligonucleotides bind and reverse PLN's inhibitory effects on SERCA, increasing the ATPase's apparent Ca(2+) affinity. Solid-state NMR experiments revealed that ssDNA interacts with PLN specifically, shifting the conformational equilibrium of the SERCA/PLN complex from an inhibitory to a non-inhibitory state. Importantly, we achieved rheostatic control of SERCA function by modulating the length of ssDNAs. Since restoration of Ca(2+) flux to physiological levels represents a viable therapeutic avenue for cardiomyopathies, our results suggest that oligonucleotide-based drugs could be used to fine-tune SERCA function to counterbalance the extent of the pathological insults.

No MeSH data available.


Related in: MedlinePlus

FRET experiments in living cells.ssDNA increased the affinity of the SERCA/PLN interaction, and decreased FRETmax. ssDNA induces a conformational change of the complex, increasing fluorescent probe separation distance.
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f4: FRET experiments in living cells.ssDNA increased the affinity of the SERCA/PLN interaction, and decreased FRETmax. ssDNA induces a conformational change of the complex, increasing fluorescent probe separation distance.

Mentions: The SERCA/PLN interaction studies were also carried out in HEK cells. Specifically, FRET in-cell assays were used to investigate the interactions of ssDNA with free PLN and in complex with SERCA2324. We utilized PLNAFA, a pentamer-destabilizing mutant with full inhibitory activity to minimize PLN oligomerization and detect small changes in binding affinity. Although it runs as a monomer on SDS-PAGE, PLNAFA has a slight tendency to form pentamers in membranes and exhibits a significant intra-pentameric FRET when overexpressed in living cells. A cell-by-cell comparison of FRET with PLN expression level revealed a hyperbolic dependence of FRET efficiency on protein concentration for a population of HEK cells expressing the fluorescent proteins Cer-PLNAFA and YFP-PLNAFA, fused at the N-terminus of PLN (Fig. S4). From this curve, we calculated the apparent dissociation constant for oligomerization (KD1) as well as the intrinsic FRET efficiency of the PLN oligomer (FRETmax), reporting on the inter-protomer binding affinity and quaternary structure changes, respectively. In the absence of ssDNA, we found that PLNAFA is able to form pentamers, though with reduced propensity compared to PLNWT. In contrast, addition of the 50-mer ssDNA to the cells containing plasmids encoding for Cer-PLNAFA and YFP-PLNAFA induced a notable increase in the apparent PLN-PLN affinity, representing an approximately 20% increase in PLN oligomerization (Fig. S4). A significant decrease in FRETmax was observed, which corresponds to an increase of the donor-acceptor distance (i.e., increased interprotomer affinity) as previously observed for PLNR9C and its phosphomimetic mutants2526. For the SERCA/PLN complex, cell transfection with 50-mer ssDNA had striking effects both on the binding affinity and the structure of the complex. Unlike the in vitro fluorescence experiments in which the AEDANS donor probe was attached at Cys674 in the SERCA’s P-domain, the Cer probe placed on the A-domain of SERCA is able to detect a four-fold increase of PLN affinity for SERCA upon oligonucleotide addition, with a concomitant decrease of SERCA/PLN FRETmax (Fig. 4). Taken with the in vitro ATPase activity assays, these data demonstrate that ssDNA binding to PLN mimics both the structural and functional effects of phosphorylation on the SERCA/PLN regulatory complex. The decrease of FRETmax upon complex formation is suggestive of a structural rearrangement of PLN within the complex; rather than the dissociation of PLN from the ATPase.


Rheostatic Regulation of the SERCA/Phospholamban Membrane Protein Complex Using Non-Coding RNA and Single-Stranded DNA oligonucleotides.

Soller KJ, Verardi R, Jing M, Abrol N, Yang J, Walsh N, Vostrikov VV, Robia SL, Bowser MT, Veglia G - Sci Rep (2015)

FRET experiments in living cells.ssDNA increased the affinity of the SERCA/PLN interaction, and decreased FRETmax. ssDNA induces a conformational change of the complex, increasing fluorescent probe separation distance.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4543939&req=5

f4: FRET experiments in living cells.ssDNA increased the affinity of the SERCA/PLN interaction, and decreased FRETmax. ssDNA induces a conformational change of the complex, increasing fluorescent probe separation distance.
Mentions: The SERCA/PLN interaction studies were also carried out in HEK cells. Specifically, FRET in-cell assays were used to investigate the interactions of ssDNA with free PLN and in complex with SERCA2324. We utilized PLNAFA, a pentamer-destabilizing mutant with full inhibitory activity to minimize PLN oligomerization and detect small changes in binding affinity. Although it runs as a monomer on SDS-PAGE, PLNAFA has a slight tendency to form pentamers in membranes and exhibits a significant intra-pentameric FRET when overexpressed in living cells. A cell-by-cell comparison of FRET with PLN expression level revealed a hyperbolic dependence of FRET efficiency on protein concentration for a population of HEK cells expressing the fluorescent proteins Cer-PLNAFA and YFP-PLNAFA, fused at the N-terminus of PLN (Fig. S4). From this curve, we calculated the apparent dissociation constant for oligomerization (KD1) as well as the intrinsic FRET efficiency of the PLN oligomer (FRETmax), reporting on the inter-protomer binding affinity and quaternary structure changes, respectively. In the absence of ssDNA, we found that PLNAFA is able to form pentamers, though with reduced propensity compared to PLNWT. In contrast, addition of the 50-mer ssDNA to the cells containing plasmids encoding for Cer-PLNAFA and YFP-PLNAFA induced a notable increase in the apparent PLN-PLN affinity, representing an approximately 20% increase in PLN oligomerization (Fig. S4). A significant decrease in FRETmax was observed, which corresponds to an increase of the donor-acceptor distance (i.e., increased interprotomer affinity) as previously observed for PLNR9C and its phosphomimetic mutants2526. For the SERCA/PLN complex, cell transfection with 50-mer ssDNA had striking effects both on the binding affinity and the structure of the complex. Unlike the in vitro fluorescence experiments in which the AEDANS donor probe was attached at Cys674 in the SERCA’s P-domain, the Cer probe placed on the A-domain of SERCA is able to detect a four-fold increase of PLN affinity for SERCA upon oligonucleotide addition, with a concomitant decrease of SERCA/PLN FRETmax (Fig. 4). Taken with the in vitro ATPase activity assays, these data demonstrate that ssDNA binding to PLN mimics both the structural and functional effects of phosphorylation on the SERCA/PLN regulatory complex. The decrease of FRETmax upon complex formation is suggestive of a structural rearrangement of PLN within the complex; rather than the dissociation of PLN from the ATPase.

Bottom Line: Both in HEK cells expressing the SERCA/PLN complex, as well as in cardiac sarcoplasmic reticulum preparations, these short oligonucleotides bind and reverse PLN's inhibitory effects on SERCA, increasing the ATPase's apparent Ca(2+) affinity.Solid-state NMR experiments revealed that ssDNA interacts with PLN specifically, shifting the conformational equilibrium of the SERCA/PLN complex from an inhibitory to a non-inhibitory state.Importantly, we achieved rheostatic control of SERCA function by modulating the length of ssDNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455.

ABSTRACT
The membrane protein complex between sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) and phospholamban (PLN) is a prime therapeutic target for reversing cardiac contractile dysfunctions caused by calcium mishandling. So far, however, efforts to develop drugs specific for this protein complex have failed. Here, we show that non-coding RNAs and single-stranded DNAs (ssDNAs) interact with and regulate the function of the SERCA/PLN complex in a tunable manner. Both in HEK cells expressing the SERCA/PLN complex, as well as in cardiac sarcoplasmic reticulum preparations, these short oligonucleotides bind and reverse PLN's inhibitory effects on SERCA, increasing the ATPase's apparent Ca(2+) affinity. Solid-state NMR experiments revealed that ssDNA interacts with PLN specifically, shifting the conformational equilibrium of the SERCA/PLN complex from an inhibitory to a non-inhibitory state. Importantly, we achieved rheostatic control of SERCA function by modulating the length of ssDNAs. Since restoration of Ca(2+) flux to physiological levels represents a viable therapeutic avenue for cardiomyopathies, our results suggest that oligonucleotide-based drugs could be used to fine-tune SERCA function to counterbalance the extent of the pathological insults.

No MeSH data available.


Related in: MedlinePlus