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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

Functional effect of RNA and ssDNA on the SERCA/PLN complex.(a) Dissociation constants as measured by both fluorescence polarization and affinity capillary electrophoresis of both RNA and ssDNA. (b) pKCa of SERCA as measured by coupled enzyme assays. Dissociation constants versus ssDNA length obtained from fluorescence polarization (c) and affinity capillary electrophoresis (d). Sequences for (c) and (d) are found in Table 1.
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f1: Functional effect of RNA and ssDNA on the SERCA/PLN complex.(a) Dissociation constants as measured by both fluorescence polarization and affinity capillary electrophoresis of both RNA and ssDNA. (b) pKCa of SERCA as measured by coupled enzyme assays. Dissociation constants versus ssDNA length obtained from fluorescence polarization (c) and affinity capillary electrophoresis (d). Sequences for (c) and (d) are found in Table 1.

Mentions: Using affinity capillary electrophoresis (ACE), fluorescence polarization (FP), and native gel mobility shift assays, we found that short RNAs, of similar length to naturally occurring miRNA sequences, display low nanomolar dissociation constants for PLN. As an example, we report the dissociation constant of a random sequence RNA (50mer) to PLN in Fig. 1A and typical binding curves (80mer) in Fig. S1. Importantly, we found that these short RNAs not only bind, but also reverse PLN’s inhibition of SERCA. Fig. 1B shows the effect of the 50mer RNA on SERCA activity as monitored using coupled enzyme assays. The Ca2+ concentration at half maximal activity in the coupled assay curves indicates SERCA’s apparent affinity for Ca2+ ions (pKCa). When SERCA is bound to PLNWT, the ATPase activity decreases and the normalized curves show a concomitant reduction of the pKCa value. Upon binding the 50mer RNA, SERCA’s function is nearly restored (Fig. 1B), mimicking the effect of PLN phosphorylation at Ser1620. Since aptamers have been selected to target specific proteins without off-target effects on gene expression21, we tested the effects of a 50mer ssDNA random sequence. Indeed, we found that both short RNAs and ssDNAs have similar Kd values for PLN (Fig. 1A) and restoration of SERCA function (Fig. 1B).


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)

Functional effect of RNA and ssDNA on the SERCA/PLN complex.(a) Dissociation constants as measured by both fluorescence polarization and affinity capillary electrophoresis of both RNA and ssDNA. (b) pKCa of SERCA as measured by coupled enzyme assays. Dissociation constants versus ssDNA length obtained from fluorescence polarization (c) and affinity capillary electrophoresis (d). Sequences for (c) and (d) are found in Table 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Functional effect of RNA and ssDNA on the SERCA/PLN complex.(a) Dissociation constants as measured by both fluorescence polarization and affinity capillary electrophoresis of both RNA and ssDNA. (b) pKCa of SERCA as measured by coupled enzyme assays. Dissociation constants versus ssDNA length obtained from fluorescence polarization (c) and affinity capillary electrophoresis (d). Sequences for (c) and (d) are found in Table 1.
Mentions: Using affinity capillary electrophoresis (ACE), fluorescence polarization (FP), and native gel mobility shift assays, we found that short RNAs, of similar length to naturally occurring miRNA sequences, display low nanomolar dissociation constants for PLN. As an example, we report the dissociation constant of a random sequence RNA (50mer) to PLN in Fig. 1A and typical binding curves (80mer) in Fig. S1. Importantly, we found that these short RNAs not only bind, but also reverse PLN’s inhibition of SERCA. Fig. 1B shows the effect of the 50mer RNA on SERCA activity as monitored using coupled enzyme assays. The Ca2+ concentration at half maximal activity in the coupled assay curves indicates SERCA’s apparent affinity for Ca2+ ions (pKCa). When SERCA is bound to PLNWT, the ATPase activity decreases and the normalized curves show a concomitant reduction of the pKCa value. Upon binding the 50mer RNA, SERCA’s function is nearly restored (Fig. 1B), mimicking the effect of PLN phosphorylation at Ser1620. Since aptamers have been selected to target specific proteins without off-target effects on gene expression21, we tested the effects of a 50mer ssDNA random sequence. Indeed, we found that both short RNAs and ssDNAs have similar Kd values for PLN (Fig. 1A) and restoration of SERCA function (Fig. 1B).

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