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Identification of ELC interacting proteins in zebrafish heart

ABSTRACT: The essential myosin light chain (ELC) is part of the cardiac myosin motor regulating the contractility of the heart. Recently, we have demonstrated ELC phosphorylation to be responsible not only for basal maintenance of cardiac contractility, but also for the adaptation of sarcomere function to increased demands in adult zebrafish (Scheid et al. Cardiovascular Research, 2016). However, the direct interacting kinases, phosphatases as well as the signaling pathway mediating the ELC phosphorylation are mostly unknown. Here, we aim to identify ELC interacting proteins in zebrafish heart tissue using an ELC-specific precipitation assay followed by mass spectrometry. In brief, ELC specific co-immunoprecipitation was performed by using the Dynabeads Co-immunoprecipitation Kit (Invitrogen, #14321D). Custom-specific polyclonal ELC antibodies (Eurogentec, peptide for immunization: NH2-CAPVPETPKEPEVDLK-CONH2) against zebrafish ELC were covalently coupled to DynabeadsTM M-270 Epoxy (Invitrogen, #14302D) and whole zebrafish heart protein was used as input. All steps were carried out according to the manufacturer's instructions.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Danio Rerio (zebrafish) (brachydanio Rerio)

TISSUE(S): Heart

SUBMITTER: Thomas Ruppert

LAB HEAD: Thomas Ruppert

PROVIDER: PXD016357 | Pride | 2022-07-24

REPOSITORIES: Pride

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			Action	DRS
	14-533.gif	Other
	X-14-533-01.dat	Other
	X-14-533-01.mgf	Mgf
	X-14-533-01.raw	Raw
	X-14-533-02.dat	Other

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Analysis of myosin light chain migration pattern in 2D gels of human left ventricular proteins

Project description:The myosin light chain protein family consists of two classes, the regulatory myosin light chains (RLC) and the essential myosin light chains (ELC). Functionally, the MLC proteins are directly involved in sarcomeric activity and force transmission contributing to cardiac contractility. Besides regulating contractility by protein-protein interactions alone, MLCs modulate force transmission through posttranslational phosphorylation. The MLC phosphorylation status in human cardiac disease is under investigation. In contrast to RLC, the phosphorylation pattern of ELC in human heart disease is not well understood. Here, 2-dimensional (2D) gel electrophoresis followed by ELC specific immunoblot (IB) was performed to detect the ELC phosphorylation pattern of human left ventricular tissue. Cardiac proteins were separated by isoelectric point (pI) and molecular weight (MW) (vELC: MW 21,932 kDa, pI 5.03; vRLC: MW 19 kDa, pI 4.89). Next, we performed mass-spectrometry analysis after cutting the spot-regions in question from silver-stained 2D gels of proteins from the human tissue. The aim was 1) to validate the detection of the MLC migration pattern seen in 2D IB by mass spectrometry, 2) to distinguish between atrial and ventricular MLC isoforms as these two forms might converge on 2D gel (vELC: MW 21.932 kDa, pI 5.03; aELC: 21.550 kDa, pI 4.98) and 3) to detect phosphorylated amio acid residues of ELC and RLC in human ventricular tissue.

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Analysis of myosin light chain migration pattern in 2D gels of human left ventricular proteins - proximity labeling

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Cell rearrangement during angiogenesis requires suppression of actomyosin at cell-cell contacts via PI3-kinase.

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