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

Informazioni relative al progetto

PDPcardio

ID dell’accordo di sovvenzione: 865119

DOI

10.3030/865119

Data della firma CE 15 Gennaio 2020

Data di avvio 1 Febbraio 2020

Data di completamento 31 Gennaio 2027

Finanziato da

• EXCELLENT SCIENCE - European Research Council (ERC)

Costo totale

€ 1 998 750,00

Contributo UE

€ 1 998 750,00

Coordinato da

RHEINISCHE FRIEDRICH-WILHELMS-UNIVERSITAT BONN
Germany

Periodic Reporting for period 4 - PDPcardio (Protein phosphatase 1-disrupting peptides: Scope and mechanism of action in the treatment of heart insufficiency)

Periodo di rendicontazione: 2024-10-01 al 2026-01-31

Sintesi del contesto e degli obiettivi generali del progetto

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, and is therefore a societal burden. 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.

Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti

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. We developed this method further to compare the in vitro data with in cellulo data using the PDPs, which lead to the discovery of high confidence substrate candidates and IRS2 as new substrate of PP1 (Hoermann et al. 2024). Since PDPs release bound PP1c from its holoenzymes to modulate PP1 activity inside the cell, the data on PP1c substrate specificity is the first step toward approaching possible substrates affected by PDP treatment. In order to optimise the PDPs, we developed short peptide pharmacophores (Fontanillo et al., 2022). We also developed a synthesis procedure for heterobifunctional cyanine dyes, to allow for the monitoring of enzyme recruitment to other proteins inside cells (Maller et al., 2024). The applications of the chemical tools in cardiomyocytes is ongoing. In addition, we have published several reviews involving the subject of the project (Köhn, 2020; Kokot and Köhn, 2022; Scheinost and Köhn, 2025), and applied the PDPs in a study to establish PP1 as a phosphatase of BAG3 (Kokot et al., 2025).

References:
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.
M. Köhn "Turn and Face the Strange: A New View on Phosphatases." ACS Cent. Sci. 2020, 6, 467-477.
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.
T. Kokot, M. Köhn "Emerging insights into serine/threonine-specific phosphoprotein phosphatase function and selectivity." J. Cell. Sci. 2022, 135, jcs259618.
M. Fontanillo, M. Trebacz, C.D. Reinkemeier, D. Aviles Huerta, U. Uhrig, P. Sehr, M. Köhn "Short peptide pharmacophores developed from protein phosphatase-1 disrupting peptides (PDPs)." Bioorg. Med. Chem. 2022, 65, 116785.
B. Hoermann, E.M. Dürr, C. Ludwig, M. Ercan, M. Köhn "A strategy to disentangle direct and indirect effects on (de)phosphorylation by chemical modulators of the phosphatase PP1 in complex cellular contexts." Chem. Sci. 2024, 15, 2792-2804.
C. Maller, F. Schedel, M. Köhn "A Modular Approach for the Synthesis of Diverse Heterobifunctional Cyanine Dyes." J. Org. Chem. 2024, 89, 3844-3856.
L. Scheinost, M. Köhn "The Fascinating Intricacy of pSer/Thr-Specific Phosphatases and Their Higher-Order Complexes: Emerging Concepts." Biochemistry 2025, 64, 2506-2515.
T. Kokot, J.P. Zimmermann, Y. Chand, F. Krier, L. Reimann, L. Scheinost, N. Höfflin, A. Esch, J. Höhfeld, B. Warscheid, M. Köhn "Identification of phosphatases that dephosphorylate the co-chaperone BAG3." Life Sci. Alliance 2024, 8, e202402734.

Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora)

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 combined with in cellulo PDP treatment to study substrate specificity and gain high confidence substrates of PP1 has 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 approaches not only provides information on substrate specificity on the peptide and protein level, but also provides high confidence 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 potentially for using PP1 as tool in synthetic biology.

Collage of elements of project: Beating cardiomyocytes, PP1c-PDP structure, substrate ID approach