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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

Effects of ssDNA on the conformational equilibrium of the SERCA/PLN complex (a) Portions of the [13C, 13C]-DARR spectra indicating the conformational equilibrium between the different states of PLN as probed by the alanine residues in the cytoplasmic domain. A small population of AFA PLN exists in the B (bound) state, upon phosphorylation, a higher population transitions to the B state (with a slight shift upon the phosphorylation), a further shift occurs upon addition of ssDNA to the D (DNA) bound state. (b) Proposed regulatory model of SERCA by PLN and effects of ssDNA. ssDNA shifts the monomer to pentamer equilibrium toward the oligomeric state. Phosphorylation of Ser16 shifts the equilibrium toward the non-inhibitory bound state (B-state). ssDNA is proposed to bind phospholamban and mimic the phosphorylated state, shifting the equilibrium toward a non-inhibitory D-state.
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f6: Effects of ssDNA on the conformational equilibrium of the SERCA/PLN complex (a) Portions of the [13C, 13C]-DARR spectra indicating the conformational equilibrium between the different states of PLN as probed by the alanine residues in the cytoplasmic domain. A small population of AFA PLN exists in the B (bound) state, upon phosphorylation, a higher population transitions to the B state (with a slight shift upon the phosphorylation), a further shift occurs upon addition of ssDNA to the D (DNA) bound state. (b) Proposed regulatory model of SERCA by PLN and effects of ssDNA. ssDNA shifts the monomer to pentamer equilibrium toward the oligomeric state. Phosphorylation of Ser16 shifts the equilibrium toward the non-inhibitory bound state (B-state). ssDNA is proposed to bind phospholamban and mimic the phosphorylated state, shifting the equilibrium toward a non-inhibitory D-state.

Mentions: Previously, we found that PLN bound to SERCA undergoes conformational transitions between three major states (Fig. 6b): an inhibitory T state, with the transmembrane (TM) domain bound to SERCA and the cytoplasmic domain associated to the membrane; an inhibitory R state, with the TM domain bound to SERCA and the cytoplasmic domain unfolded and dissociated from the membrane; and a sparsely populated, non-inhibitory B or bound state, with both the TM and cytoplasmic domains interacting with SERCA28. PLN phosphorylation at Ser16 shifts the equilibrium toward the B state, undergoing a local structural rearrangement and effectively reversing PLN’s inhibition of SERCA. To assess the effect of ssDNA on the PLN conformational equilibrium in the presence of SERCA, we utilized PLNAFA, which simplifies the interpretation of the ssDNA binding on the SERCA/PLN complex by eliminating the monomer/pentamer equilibrium. To resolve the resonances in the aliphatic region of the spectrum, the cytosolic domain Ia of PLNAFA was selectively labeled at 6 different sites (Val4, Leu7, Ala11, Ala15, Ile12, Ile18). These cytosolic residues (in the absence of SERCA) display chemical shift perturbations upon the addition of ssDNA, indicating binding between ssDNA and PLNAFA (Fig. S6). Dipolar assisted rotational resonance (DARR) MAS [13C, 13C] experiments of U-13C/15N labeled PLN in complex with unlabeled SERCA were performed in the absence and presence of ssDNA (Fig. 6). Specifically, the DARR peaks corresponding to the Cα/Cβ correlations of the two Ala residues are sensitive to the conformational equilibrium between the different states and can be used as reporter residues (Fig. 6). In the absence of SERCA at 20 °C, these two residues populate a major conformational state (T state) and a sparsely populated R state. Upon addition of SERCA, there is a population shift toward the bound state that is augmented upon phosphorylation of PLN. Upon binding ssDNA, we observe a progressive shift of the two Ala residues of PLNAFA toward lower fields, similar to what is observed with the non-inhibitory phosphorylated form of PLN. These results strongly support the fluorescence studies and show that ssDNA does not detach PLN from SERCA; rather it shifts the conformational equilibrium toward a non-inhibitory or D (DNA bound) state in a manner similar to the phosphorylated state. A hypothetical model of the conformational equilibrium of the SERCA/PLN complex and the effects of ssDNA is reported in Fig. 6B.


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)

Effects of ssDNA on the conformational equilibrium of the SERCA/PLN complex (a) Portions of the [13C, 13C]-DARR spectra indicating the conformational equilibrium between the different states of PLN as probed by the alanine residues in the cytoplasmic domain. A small population of AFA PLN exists in the B (bound) state, upon phosphorylation, a higher population transitions to the B state (with a slight shift upon the phosphorylation), a further shift occurs upon addition of ssDNA to the D (DNA) bound state. (b) Proposed regulatory model of SERCA by PLN and effects of ssDNA. ssDNA shifts the monomer to pentamer equilibrium toward the oligomeric state. Phosphorylation of Ser16 shifts the equilibrium toward the non-inhibitory bound state (B-state). ssDNA is proposed to bind phospholamban and mimic the phosphorylated state, shifting the equilibrium toward a non-inhibitory D-state.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: Effects of ssDNA on the conformational equilibrium of the SERCA/PLN complex (a) Portions of the [13C, 13C]-DARR spectra indicating the conformational equilibrium between the different states of PLN as probed by the alanine residues in the cytoplasmic domain. A small population of AFA PLN exists in the B (bound) state, upon phosphorylation, a higher population transitions to the B state (with a slight shift upon the phosphorylation), a further shift occurs upon addition of ssDNA to the D (DNA) bound state. (b) Proposed regulatory model of SERCA by PLN and effects of ssDNA. ssDNA shifts the monomer to pentamer equilibrium toward the oligomeric state. Phosphorylation of Ser16 shifts the equilibrium toward the non-inhibitory bound state (B-state). ssDNA is proposed to bind phospholamban and mimic the phosphorylated state, shifting the equilibrium toward a non-inhibitory D-state.
Mentions: Previously, we found that PLN bound to SERCA undergoes conformational transitions between three major states (Fig. 6b): an inhibitory T state, with the transmembrane (TM) domain bound to SERCA and the cytoplasmic domain associated to the membrane; an inhibitory R state, with the TM domain bound to SERCA and the cytoplasmic domain unfolded and dissociated from the membrane; and a sparsely populated, non-inhibitory B or bound state, with both the TM and cytoplasmic domains interacting with SERCA28. PLN phosphorylation at Ser16 shifts the equilibrium toward the B state, undergoing a local structural rearrangement and effectively reversing PLN’s inhibition of SERCA. To assess the effect of ssDNA on the PLN conformational equilibrium in the presence of SERCA, we utilized PLNAFA, which simplifies the interpretation of the ssDNA binding on the SERCA/PLN complex by eliminating the monomer/pentamer equilibrium. To resolve the resonances in the aliphatic region of the spectrum, the cytosolic domain Ia of PLNAFA was selectively labeled at 6 different sites (Val4, Leu7, Ala11, Ala15, Ile12, Ile18). These cytosolic residues (in the absence of SERCA) display chemical shift perturbations upon the addition of ssDNA, indicating binding between ssDNA and PLNAFA (Fig. S6). Dipolar assisted rotational resonance (DARR) MAS [13C, 13C] experiments of U-13C/15N labeled PLN in complex with unlabeled SERCA were performed in the absence and presence of ssDNA (Fig. 6). Specifically, the DARR peaks corresponding to the Cα/Cβ correlations of the two Ala residues are sensitive to the conformational equilibrium between the different states and can be used as reporter residues (Fig. 6). In the absence of SERCA at 20 °C, these two residues populate a major conformational state (T state) and a sparsely populated R state. Upon addition of SERCA, there is a population shift toward the bound state that is augmented upon phosphorylation of PLN. Upon binding ssDNA, we observe a progressive shift of the two Ala residues of PLNAFA toward lower fields, similar to what is observed with the non-inhibitory phosphorylated form of PLN. These results strongly support the fluorescence studies and show that ssDNA does not detach PLN from SERCA; rather it shifts the conformational equilibrium toward a non-inhibitory or D (DNA bound) state in a manner similar to the phosphorylated state. A hypothetical model of the conformational equilibrium of the SERCA/PLN complex and the effects of ssDNA is reported in Fig. 6B.

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