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Scientific result | Molecular mechanisms | Fundamental Research
By combining molecular dynamics and in vitro experiments, researchers from the Laboratory of Proteins and Membrane Systems (LPSM, I2BC/CEA-Joliot) have revealed the mode of action of sarcolipin in regulating the activity of the calcium ATPase SERCA1a, responsible for muscle relaxation.
Sarcolipin (SLN) is a 31-residue membrane peptide that
inhibits the activity of the calcium ATPase
SERCA1a, which is essential for the
function of so-called fast-twitch
muscles. In skeletal cells, SERCA1a uses ATP energy to transport calcium ions from the cytosol to the sarcoplasmic reticulum, resulting in
muscle relaxation. Sarcolipin and its regulatory action on SERCA1a has been known since the 1990s. It has been shown to slightly decrease the affinity of ATPase-Ca2+ for calcium by measuring the ATPase activity of the transporter in a very comprehensive way. Researchers from the Laboratory of Proteins and Membrane Systems (LPSM, I2BC / CEA-Joliot Institute) showed in 2014 that, at least in some species,
SLN is post-translationally modified: a
fatty acid (palmitic acid or oleic acid) is
anchored to its cysteine 9. But the mode of action of SLN is still poorly understood. In particular, the role of acylation (palmitoylation or oleoylation) remains unclear. To go further, the LPSM researchers have multiplied their approaches.
On the one hand, they carried out
in silico work to better understand the interaction of SLN with SERCA1a. They used
molecular dynamics simulation techniques and a
normal mode analysis (NMA) approach, in collaboration with
Liliane Mouawad (Institut Curie, Orsay). For the first time, this methodology used
a system that included, in addition to proteins, the membrane and water. The results of the study were published in
Frontiers in Molecular Biosciences. They allowed the authors to propose a mode of action for SLN and in particular
a "long range" effect at the phosphorylation site of the calcium pump.
In parallel, the researchers experimentally characterised the effect of SLN on the ATPase activity of SERCA1a
in vitro. For such experiments,
a source of acylated SLN is essential, but the molecule is not easily tamed. It is indeed
very difficult to purify it from cell extracts, notably because of its low abundance and hydrophobic nature. It is
also very difficult to control the level of acylation
in vivo, as little is known about these cellular mechanisms to date. Another possibility is
to synthesise it chemically, which is a
real challenge because of the hydrophobicity of the peptide, reinforced by its acylation. It was the laboratory of Prof. Ji-Shen Zheng (China) that succeeded in synthesising acylated SLN for the CEA-Joliot team. This synthesis is a first and has been published in
From there, they were able to look at the effect of SLN (palmitoylated or not) on SERCA1a by dissecting the main steps of the catalytic cycle by various enzymological approaches. In order to fully control the study system, the ATPase was produced in a recombinant system devoid of endogenous SLN (LPSM's technical platform for the expression and purification of membrane proteins, affiliated to the National Health Biology Infrastructure FRISBI https://frisbi.eu/centers/south-paris/yeast-expression/). The results were published in Scientific Reports. The team confirms the effect of Sarcolipin on calcium binding. Acylation could be involved in the oligomerisation of SLN, although further experiments are needed to clarify this property. For the first time, LPSM demonstrates the long-range effect of SLN on the ATP binding site, located about 25 Å from the membrane and SLN, as proposed in their in silico study.
The team wants to go further by comparing the in silico work with new experimental NMR studies that will provide precise structural data on the behaviour of the protein acylation in the membrane and help define its role.
Nadège Jamin (firstname.lastname@example.org) et Véronica Beswick (email@example.com)
Cédric Montigny (firstname.lastname@example.org)
Thomas Barbot, Veronica Beswick, Cédric
Montigny, Éric Quiniou, Nadège Jamin,
Liliane Mouawad. Deciphering the
Mechanism of Inhibition of SERCA1a by Sarcolipin Using Molecular Simulations.
| Frontiers in molecular biosciences,
2021 Feb 4;7:606254
Dong-Liang Huang, Cédric Montigny, Yong Zheng, Veronica Beswick, Ying Li, Xiu-Xiu Cao,
Thomas Barbot, Christine Jaxel, Jun Liang, Min Xue, Chang-Lin Tian, Nadège Jamin, and Ji- Shen Zheng. Chemical Synthesis of Native S-Palmitoylated
Membrane Proteins through Removable-Backbone-Modification-Assisted Ser/Thr
Ligation. | Angewandte Chemie, Angew
Chem Int Ed Engl. 2020 Mar 23;59(13):5178-5184
Cédric Montigny, Dong Liang Huang, Veronica Beswick, Thomas Barbot, Christine
Jaxel, Marc le Maire, Ji-Shen
Zheng, Nadège Jamin.
Sarcolipin alters SERCA1a interdomain communication by impairing binding of
both calcium and ATP | Scientific Reports,
2021 Jan 15;11(1):1641
CEA is a French government-funded technological research organisation in four main areas: low-carbon energies, defense and security, information technologies and health technologies. A prominent player in the European Research Area, it is involved in setting up collaborative projects with many partners around the world.