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Protein phosphatase 1-disrupting peptides: Scope and mechanism of action in the treatment of heart insufficiency

Periodic Reporting for period 2 - PDPcardio (Protein phosphatase 1-disrupting peptides: Scope and mechanism of action in the treatment of heart insufficiency)

Reporting period: 2021-08-01 to 2023-01-31

Protein phosphorylation is one of the most important means for cells to regulate processes such as muscle contraction, growth, motility, and cell death. Protein phosphatase-1 (PP1) is a ubiquitously expressed enzyme known to remove a phosphate group from a large number of the phosphorylated serines and threonines. The catalytic subunit PP1c is bound to regulatory proteins forming so-called holoenzymes. These play specific and fundamental roles in physiological processes and pathologies. One important role lies in the regulation of cardiac signaling pathways and calcium homoeostasis, which play key roles in the function of the heart. Heart insufficiency constitutes an often-occurring condition affecting all ages, leading to heart failure and consequently to the requirement of a heart transplant or death. Owing to its role in processes regulating the heartbeat, deregulation of PP1 has been implicated in cardiac dysfunctions. Powerful tools to study PP1 biology are our own developed PP1-disrupting peptides (PDPs) that selectively release PP1 activity in cells. PDP treatment counteracts the hyperactivity of kinases, which are the cellular counterplayers of phosphatases, and was shown to seal the arrhythmogenic sarcoplasmic reticulum (SR)-calcium-leak in human heart failure tissue. Mechanistic data indicated that PDP–PP1c-mediated dephosphorylation of the ryanodine receptor type 2 (RyR2) is involved in this effect. Nevertheless, given the large amount of potential PP1 substrates, so far the scope of PDP action is unknown, and therefore the mechanisms underlying this beneficial and potentially therapeutic effect of the PDPs in heart failure are unclear and currently hard to investigate. This project aims at developing a variety of chemical biology methods that will allow to determine the scope of PDP action and to elucidate PP1 biology in the healthy and diseased heart.
We developed a combined phosphopeptide library and proteomics approach to study the substrate specificity of phosphatases, in particular PP1c and PP2Ac. We found specific preferences for PP1c for sequence properties around the phosphorylated site, that depended on the folded state of the substrate (Hoermann et al, 2020; Hoermann and Köhn, 2021; Kokot et al., 2022). In addition, this method provided a variety of protein substrate candidates for PP1c. Since PDPs release bound PP1c from its holoenzymes to exert dephosphorylation activity inside the cell, the data on PP1c substrate specificity is the first step toward approaching possible substrates affected by PDP treatment. Further new chemical biology methodologies are currently being evaluated and optimized.

B. Hoermann, T. Kokot, D. Helm, S. Heinzlmeir, J.E. Chojnacki, T. Schubert, C. Ludwig, A. Berteotti, N. Kurzawa, B. Kuster, M.M. Savitski, M. Köhn “Dissecting the sequence determinants for dephosphorylation by the catalytic subunits of phosphatases PP1 and PP2A.” Nat. Commun. 2020, 11, 3583.
B. Hoermann and M. Köhn “Evolutionary crossroads of cell signaling: PP1 and PP2A substrate sites in intrinsically disordered regions.” Biochem. Soc. Trans. 2021, 49, 1065-1074.
T. Kokot, B. Hoermann, D. Helm, J.E. Chojnacki, M.M. Savitski, M. Köhn “PLDMS: Phosphopeptide Library Dephosphorylation Followed by Mass Spectrometry Analysis to Determine the Specificity of Phosphatases for Dephosphorylation Site Sequences.” Methods Mol. Biol. 2022, 2499, 43-64.
The chemical biology strategies will enable identifying the scope of PDP action in general, and in particular they will be applied here in cardiomyocytes to study the effects of PDP–bound PP1c. The new phosphopeptide library/proteomics approach to study substrate specificity of phosphatases has already been a significant breakthrough beyond the state of the art. Due to their conservation, the transient interaction with the substrate and the fast signaling inside cells, which makes it almost impossible to distinguish direct from indirect substrates, the specificity of phosphatases has always been a major bottleneck in their research. Our method not only provides information on substrate specificity on the peptide and protein level, but also provides protein substrate candidates, which is another large gain from the method. Overall, the results of this project will provide the basis to fine-tune targeting PP1 for the treatment of heart insufficiency. Furthermore, the principles and methods developed here will be applicable more generally for defining the interaction scope of target-bound ligands (drugs) as well as for using PP1 as tool in synthetic biology.