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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

Mapping of PLN residues involved in ssDNA binding.[1H, 13C] RINEPT spectra of PLN in DMPC lipid vesicles in the absence (black) and presence (red) of ssDNA (80mer). Blue asterisks indicate lipid signals; green asterisks indicate a change in peak shape. Dotted circles indicate missing peaks.
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f5: Mapping of PLN residues involved in ssDNA binding.[1H, 13C] RINEPT spectra of PLN in DMPC lipid vesicles in the absence (black) and presence (red) of ssDNA (80mer). Blue asterisks indicate lipid signals; green asterisks indicate a change in peak shape. Dotted circles indicate missing peaks.

Mentions: To identify the specific residues of PLN interacting with the ssDNA, we used solid-state NMR (ssNMR) spectroscopy. We reconstituted U-13C/15N labeled PLN in deuterated DMPC lipid vesicles and monitored the chemical shifts of the backbone and side chain 13C resonances in the presence and absence of ssDNA (80mer). To detect the dynamic cytoplasmic domain of PLN, we used the refocused [1H, 13C]-RINEPT experiment27, which is well suited for protein domains undergoing fast reorientation (cytoplasmic domain of PLN) and insensitive to rigid domains on the NMR time scale (PLN’s transmembrane domain)28. In the free form of PLN, the resonances corresponding to the cytoplasmic region are all detectable. Addition of ssDNA to PLN at a 1:1 molar ratio causes the intensities of several amino acids peaks in the [1H, 13C]-RINEPT spectrum to decrease, with several peaks becoming broadened beyond detection (Fig. 5). The latter indicates the rigidification of PLN’s cytoplasmic domain and an increase in rotational correlation time upon ssDNA binding. In contrast, natural abundance 13C lipid signals are not affected by ssDNA and remain essentially unchanged (Fig. 5). The most affected resonances of PLN are located at the N-terminal portion of domain Ia (i.e., Lys3, Val4, Leu7, Thr8/17, Arg9/13/14, Ala/0/11/15, Ile12/18, and Glu2/19). To confirm that the ssDNA targets the cytoplasmic domain resonances, we compared the ssNMR data with solution NMR experiments carried out on PLN reconstituted in isotropic bicelles (q = 0.33). Due to the large size of the bicelle/PLN complex, solution NMR [1H, 15N]-HSQC experiments do not detect the transmembrane residues29, but provides visualization of the cytoplasmic domain resonances. Addition of 15mer ssDNA causes extensive line broadening (residues Glu2, Thr8, Ala11, Ile12/18 Arg13/14, and Met20 - Fig. S5), which is in qualitative agreement with ssNMR experiments. Taken with the FRET data, the NMR experiments indicate that the transmembrane domain of PLN remains essentially unperturbed and attached to the ATPase, while ssDNA primarily targets the cytoplasmic domain of PLN interfering with its regulatory function of SERCA.


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)

Mapping of PLN residues involved in ssDNA binding.[1H, 13C] RINEPT spectra of PLN in DMPC lipid vesicles in the absence (black) and presence (red) of ssDNA (80mer). Blue asterisks indicate lipid signals; green asterisks indicate a change in peak shape. Dotted circles indicate missing peaks.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Mapping of PLN residues involved in ssDNA binding.[1H, 13C] RINEPT spectra of PLN in DMPC lipid vesicles in the absence (black) and presence (red) of ssDNA (80mer). Blue asterisks indicate lipid signals; green asterisks indicate a change in peak shape. Dotted circles indicate missing peaks.
Mentions: To identify the specific residues of PLN interacting with the ssDNA, we used solid-state NMR (ssNMR) spectroscopy. We reconstituted U-13C/15N labeled PLN in deuterated DMPC lipid vesicles and monitored the chemical shifts of the backbone and side chain 13C resonances in the presence and absence of ssDNA (80mer). To detect the dynamic cytoplasmic domain of PLN, we used the refocused [1H, 13C]-RINEPT experiment27, which is well suited for protein domains undergoing fast reorientation (cytoplasmic domain of PLN) and insensitive to rigid domains on the NMR time scale (PLN’s transmembrane domain)28. In the free form of PLN, the resonances corresponding to the cytoplasmic region are all detectable. Addition of ssDNA to PLN at a 1:1 molar ratio causes the intensities of several amino acids peaks in the [1H, 13C]-RINEPT spectrum to decrease, with several peaks becoming broadened beyond detection (Fig. 5). The latter indicates the rigidification of PLN’s cytoplasmic domain and an increase in rotational correlation time upon ssDNA binding. In contrast, natural abundance 13C lipid signals are not affected by ssDNA and remain essentially unchanged (Fig. 5). The most affected resonances of PLN are located at the N-terminal portion of domain Ia (i.e., Lys3, Val4, Leu7, Thr8/17, Arg9/13/14, Ala/0/11/15, Ile12/18, and Glu2/19). To confirm that the ssDNA targets the cytoplasmic domain resonances, we compared the ssNMR data with solution NMR experiments carried out on PLN reconstituted in isotropic bicelles (q = 0.33). Due to the large size of the bicelle/PLN complex, solution NMR [1H, 15N]-HSQC experiments do not detect the transmembrane residues29, but provides visualization of the cytoplasmic domain resonances. Addition of 15mer ssDNA causes extensive line broadening (residues Glu2, Thr8, Ala11, Ile12/18 Arg13/14, and Met20 - Fig. S5), which is in qualitative agreement with ssNMR experiments. Taken with the FRET data, the NMR experiments indicate that the transmembrane domain of PLN remains essentially unperturbed and attached to the ATPase, while ssDNA primarily targets the cytoplasmic domain of PLN interfering with its regulatory function of SERCA.

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