Muscle adaptation | Orvosi Vegytani Intézet

Smooth muscle contraction is predominantly regulated by the phosphorylation of the 20 kDa regulatory light chain  of myosin (MLC20) by the calcium/calmodulin-dependent activation of myosin light chain kinase, while relaxation is affected by the dephosphorylation of MLC20 by the myosin phosphatase (MP). MP is a heterotrimer composed of a 38 kDa type 1 protein serine/threonine phosphatase catalytic subunit (PP1c), a 130/133 kDa MYPT (myosin phosphatase targeting subunit) and 20 kDa subunits. MYPT has several important functions like targeting PP1c to the substrate and activation and regulation of MP activity. One of the potential smooth muscle effector of MP is the smoothelin-like protein 1 (SMTNL1). It contains a calponin homology domain at its C-terminus and shares sequence homology with the smoothelin family of smooth muscle-specific proteins. SMTNL1 is an early target of protein kinase G (PKG) and A (PKA) in vascular smooth muscle. SMTNL1 plays a role in cGMP/cAMP-mediated adaptation to exercise direct modulation of contractile activity in smooth muscle cells. SMTNL1 was shown to suppress MP activity toward the MLC20 in vitro. In SMTNL1 Ser-301 is phosphorylated in response to both endothelial dependent agonists as well as β-adrenergic agonists. SMTNL1 has no inhibitory effect on MLCK activity and appears not to be dephosphorylated by MP. SMTNL1 is expressed in all muscle types and in sex-related organs, too. Gender-related differences were experienced in exercise adaptation of smtnl1-/- mice. Isometric force generated in response to phenylephrine were lower in female than in male littermates. SMTNL1 expression was 30-40% lower in females relative to males, while SMTNL1 expression was reduced by exercise particularly in female mice.

In spite of recent advances in our understanding of the regulation and physiological functions of contractile proteins in vascular smooth muscle, relatively little information exists on the role of MP in the mechanism of adaptation during pregnancy in smooth muscle. Because MP is the primary effector of smooth muscle relaxation and key target of signalling pathways regulating vascular tone, we hypothesize that MYPT expression would be altered in these conditions and it might be regulated by its potential effector SMTNL1. Our goal is to carry out experiments to describe the molecular interaction between SMTNL1 and MYPT and the regulation of their protein-protein interaction through PKA/PKG signalling. We intend to determine the repressor mechanism by which SMTNL1 acts on mypt expression through steroid hormone receptors. Our aim also to investigate whether SMTNL1 is a selective cofactor of mypt or can act on other genes coding contractile and metabolic proteins or cytokines regulating smooth muscle functions.

Regulation of muscle plasticity by the smoothelin like 1 protein: The contractile properties of smooth (SM) and skeletal muscle change dramatically during pregnancy and physical training. The adaptations involve coordinated changes in the expression of specific genes encoding contractile, metabolic and signal-transduction proteins. Because myosin phosphatase (MP) is a key enzyme in regulating SM contractility, we assume that muscle adaptation is also influenced by smoothelin-like protein 1 (SMTNL1), which is a regulator of MP. Based on our previous studies of muscular changes during pregnancy, we hypothesize that SMTNL1 has a double effect on the adaptation of SM to pregnancy: it inhibits the enzyme activity of citoskeletal MP but also represses the gene expression of MYPT, the regulatory (targeting) subunit of MP by binding to the progesterone receptor (PR) after PKA/PKG phosphorylation. Our goal is to study the molecular interactions between SMTNL1 and MYPT and the effect of the PKA/PKG signalling pathway on these protein-protein interactions. We intend to determine the repressor mechanism by which SMTNL1 acts on mypt gene expression through steroid hormone receptors (PR) and we aim to identify the promoter of mypt. We will use microarray and proteomic studies to identify other SMTNL1-regulated genes/proteins that can play a role in muscle adaptations during pregnancy. By studying the molecular functions of SMTNL1, we will identify potential therapeutic targets in diseases such as muscle dysfunction, hypertension and diabetes as well as pre-eclampsia, diabetes and preterm labor in pregnancy.

Nuclear localization, function and regulation of myosin phosphatase: MP regulates contractility through the dephosphorylation of myosin light chain. Apart from the myosin, which is a classical cytoskeletal substrate of MP, other non-muscle substrates have also been identified. It draws the attention to the complex function of PP1M in different tissues and cellular processes. MYPT was found to be localized not only in the cytosol and cytoskeleton but in the nucleuses of rat aortic smooth muscle cells, primary cultures of neuronal cells as well as of human hepatocarcinoma (HepG2) cells. Our goal is to investigate the nuclear functions of PP1M by determining the subnuclear localization and the interacting proteins of MYPT. Subnuclear fractions of HepG2 cells were analysed by Western blotting and by protein phoshatase enzyme activity assays in the presence of specific PP1 inhibitors such as okadaic acid and tautomycin. The dominant nuclear protein phosphatase was found to be the PP1 in the nuclear fractions. Flag-MYPT pull down assays are carried out using nuclear fractions of HepG2 cells. The eluates are subjected to silver staining and the proteins are identified by mass spectrometry. Numerous potential nuclear MYPT1-interacting proteins were identified such as histone 1, splicing factor proteins, possible enzyme regulators of PP1M and members of the methylosome complex, f. i. the protein arginine methyltransferase 5 (PRMT5). PP1cδ was detected from the nuclear pull down eluate by Western blotting as a partner of MYPT1. We also confirmed the nuclear colocalization of MYPT1 and PP1cδ suggesting that the delta and not the alpha/ gamma isoform is the member of the holoenzyme in the nucleus. MYPT1 showed colocalization with histone 1 and presented distinct localization in the spliceosomes (nuclear splicing factor compartments of cell) by confocal microscopy suggesting that PP1M may play a role in mRNA splicing. We plan to investigate the physiological role of PP1M in the nuclear dephosphorylation processes related to the regulation of transcription, RNA splicing and the functions of the methylosome complex.