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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

Tunable control of SERCA function using ssDNA of different lengths.Coupled enzyme assays in DOPC:DOPE lipid vesicles. (a) SERCA activity normalized as a function of Ca2+ concentration. Black is SERCA alone, brown is SERCA and PLN; other colors represent addition of different lengths of ssDNA. (b) pKCa values for the different ssDNA lengths derived from the pCa values at half maximum activity of SERCA. (c) Effects of ssDNA on pig cardiac SR preparations.
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f2: Tunable control of SERCA function using ssDNA of different lengths.Coupled enzyme assays in DOPC:DOPE lipid vesicles. (a) SERCA activity normalized as a function of Ca2+ concentration. Black is SERCA alone, brown is SERCA and PLN; other colors represent addition of different lengths of ssDNA. (b) pKCa values for the different ssDNA lengths derived from the pCa values at half maximum activity of SERCA. (c) Effects of ssDNA on pig cardiac SR preparations.

Mentions: We then performed ATPase assays with varying length oligonucleotides and found they give rise to a graded effect on ATPase activity. Figure 2A depicts the normalized SERCA/PLN activity curves upon addition of ssDNA at different lengths and varying concentrations of free calcium (pCa). In the absence of ssDNA, PLN binding results in decreased Ca2+ affinity of SERCA (lowest pKCa, with the brown curve shifted furthest to the right in Fig. 2A). Addition of ssDNA to the PLN/SERCA complex shifts the activity curve toward the higher pCa, indicating that the SERCA’s apparent affinity for Ca2+ ions is increasing; ssDNA reverses the inhibitory effect of PLN (Fig. 2A,B). The functional effect trends with sequence length, but not necessarily according to the relative affinity of the oligonucleotides (Kd values). This is evidenced by the 30mer, which displays a higher Kd than expected based on length alone, but still follows the length trend seen in the activity assays (Fig. 2A,B). It should be noted that all ATPase assays were performed at saturating ssDNA concentrations. Thus, the length trend (Fig. 2) observed is structural in origin and independent of the ssDNA concentration. Remarkably, longer oligonucleotides result in complete inhibition relief, an effect that mimics phosphorylation of PLN at Ser16 by protein kinase A22. With oligonucleotide sequences longer than 50 bases, we observed no further increase of SERCA activity beyond the physiological window, indicating that the effect is mediated by the direct interaction between ssDNA and PLN.


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)

Tunable control of SERCA function using ssDNA of different lengths.Coupled enzyme assays in DOPC:DOPE lipid vesicles. (a) SERCA activity normalized as a function of Ca2+ concentration. Black is SERCA alone, brown is SERCA and PLN; other colors represent addition of different lengths of ssDNA. (b) pKCa values for the different ssDNA lengths derived from the pCa values at half maximum activity of SERCA. (c) Effects of ssDNA on pig cardiac SR preparations.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Tunable control of SERCA function using ssDNA of different lengths.Coupled enzyme assays in DOPC:DOPE lipid vesicles. (a) SERCA activity normalized as a function of Ca2+ concentration. Black is SERCA alone, brown is SERCA and PLN; other colors represent addition of different lengths of ssDNA. (b) pKCa values for the different ssDNA lengths derived from the pCa values at half maximum activity of SERCA. (c) Effects of ssDNA on pig cardiac SR preparations.
Mentions: We then performed ATPase assays with varying length oligonucleotides and found they give rise to a graded effect on ATPase activity. Figure 2A depicts the normalized SERCA/PLN activity curves upon addition of ssDNA at different lengths and varying concentrations of free calcium (pCa). In the absence of ssDNA, PLN binding results in decreased Ca2+ affinity of SERCA (lowest pKCa, with the brown curve shifted furthest to the right in Fig. 2A). Addition of ssDNA to the PLN/SERCA complex shifts the activity curve toward the higher pCa, indicating that the SERCA’s apparent affinity for Ca2+ ions is increasing; ssDNA reverses the inhibitory effect of PLN (Fig. 2A,B). The functional effect trends with sequence length, but not necessarily according to the relative affinity of the oligonucleotides (Kd values). This is evidenced by the 30mer, which displays a higher Kd than expected based on length alone, but still follows the length trend seen in the activity assays (Fig. 2A,B). It should be noted that all ATPase assays were performed at saturating ssDNA concentrations. Thus, the length trend (Fig. 2) observed is structural in origin and independent of the ssDNA concentration. Remarkably, longer oligonucleotides result in complete inhibition relief, an effect that mimics phosphorylation of PLN at Ser16 by protein kinase A22. With oligonucleotide sequences longer than 50 bases, we observed no further increase of SERCA activity beyond the physiological window, indicating that the effect is mediated by the direct interaction between ssDNA and PLN.

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