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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

ssDNA does not dissociate PLN from SERCA.(a) Fluorescence spectra of SERCA alone and upon addition of ssDNA. (b) FRET of SERCA-PLN complex (cyan) in the absence and presence of ssDNA. Upon addition of ssDNA (purple and green) the FRET signal does not change, indicating no structural changes within the range of the FRET probes. (c) SERCA-PLN FRET at a 10:1 PLN:SERCA ratio (green).
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f3: ssDNA does not dissociate PLN from SERCA.(a) Fluorescence spectra of SERCA alone and upon addition of ssDNA. (b) FRET of SERCA-PLN complex (cyan) in the absence and presence of ssDNA. Upon addition of ssDNA (purple and green) the FRET signal does not change, indicating no structural changes within the range of the FRET probes. (c) SERCA-PLN FRET at a 10:1 PLN:SERCA ratio (green).

Mentions: To probe the specific interactions between PLN and ssDNA both in the absence and presence of SERCA, we labeled SERCA with a FRET donor (AEDANS) at position Cys674 and PLN with a non-fluorescent acceptor (Dabcyl-SE) on Lys3. FRET sample preparation matched that of the coupled enzyme assay samples (see Methods). In the presence of the FRET pair, we detected a marked decrease in fluorescence signal upon formation of the complex between SERCA and PLN (Fig. 3B). Addition of an equimolar amount of 40mer ssDNA (Table 1) to the pre-formed SERCA/PLN complex under conditions similar to the activity assays did not change the FRET signal. These measurements were repeated with excess PLN, or excess ssDNA, and the results were similar to the FRET data with stoichiometrical amounts (Fig. 3B,C). The enhanced FRET and shift of the emission maximum seen with a 10-fold excess of PLN is indicative of more FRET between SERCA and PLN, resulting from saturating the PLN binding site in the ATPase (Fig. 3C). We have also recorded fluorescence of SERCAAEDANS without PLN, but in the presence of ssDNA. The fluorescence intensity was identical to that of SERCAAEDANS, illustrating that ssDNA does not interact with the ATPase alone (Fig. 3A). The FRET, ACE and fluorescence polarization data collectively demonstrate that the ssDNA interacts specifically with PLN irrespective of the presence of SERCA. Furthermore, ssDNA relieves PLN inhibition by causing a structural rearrangement of PLN without dissociating it from SERCA, in a manner resembling PLN phosphorylation at Ser16 – the endogenous mechanism for inhibition relief.


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)

ssDNA does not dissociate PLN from SERCA.(a) Fluorescence spectra of SERCA alone and upon addition of ssDNA. (b) FRET of SERCA-PLN complex (cyan) in the absence and presence of ssDNA. Upon addition of ssDNA (purple and green) the FRET signal does not change, indicating no structural changes within the range of the FRET probes. (c) SERCA-PLN FRET at a 10:1 PLN:SERCA ratio (green).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: ssDNA does not dissociate PLN from SERCA.(a) Fluorescence spectra of SERCA alone and upon addition of ssDNA. (b) FRET of SERCA-PLN complex (cyan) in the absence and presence of ssDNA. Upon addition of ssDNA (purple and green) the FRET signal does not change, indicating no structural changes within the range of the FRET probes. (c) SERCA-PLN FRET at a 10:1 PLN:SERCA ratio (green).
Mentions: To probe the specific interactions between PLN and ssDNA both in the absence and presence of SERCA, we labeled SERCA with a FRET donor (AEDANS) at position Cys674 and PLN with a non-fluorescent acceptor (Dabcyl-SE) on Lys3. FRET sample preparation matched that of the coupled enzyme assay samples (see Methods). In the presence of the FRET pair, we detected a marked decrease in fluorescence signal upon formation of the complex between SERCA and PLN (Fig. 3B). Addition of an equimolar amount of 40mer ssDNA (Table 1) to the pre-formed SERCA/PLN complex under conditions similar to the activity assays did not change the FRET signal. These measurements were repeated with excess PLN, or excess ssDNA, and the results were similar to the FRET data with stoichiometrical amounts (Fig. 3B,C). The enhanced FRET and shift of the emission maximum seen with a 10-fold excess of PLN is indicative of more FRET between SERCA and PLN, resulting from saturating the PLN binding site in the ATPase (Fig. 3C). We have also recorded fluorescence of SERCAAEDANS without PLN, but in the presence of ssDNA. The fluorescence intensity was identical to that of SERCAAEDANS, illustrating that ssDNA does not interact with the ATPase alone (Fig. 3A). The FRET, ACE and fluorescence polarization data collectively demonstrate that the ssDNA interacts specifically with PLN irrespective of the presence of SERCA. Furthermore, ssDNA relieves PLN inhibition by causing a structural rearrangement of PLN without dissociating it from SERCA, in a manner resembling PLN phosphorylation at Ser16 – the endogenous mechanism for inhibition relief.

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