EN
Regulation of the proteasome by cyclic nucleotides in cardiac proteinopathy (RP6)
Principial Investigators
Project Summary and Goals
Misfolded or mutant proteins can acquire toxic gain-of-function properties, as observed in neurodegenerative diseases, and in myofibrillar myopathy (MFM), where desmin aggregates contribute to disease pathology. Variants in desmin or associated proteins, such as the molecular chaperone αB-crystallin (CRYAB), cause MFM, a rare inherited skeletal and cardiac myopathy. Patients develop progressive degeneration of skeletal muscle, heart, and peripheral neurons, and no causal treatment is currently available. The ubiquitin–proteasome system (UPS) is a central mechanism for degrading misfolded and mutant proteins. Under cellular stress, misfolded proteins and UPS components localize to perinuclear regions, forming aggresomes that can serve as temporary degradative microdomains.
In this project, we will use our complementary expertise to determine the subcellular localization of proteasomes in CRYAB R120G and control human cardiomyocytes with fixed and live cell imaging of fluorescence-tagged proteasomal subunits. Furthermore, we will use fluorescent-based biosensors to evaluate if local cyclic nucleotide concentrations affect proteasomal activity. Finally, we will test different compounds affecting cyclic nucleotide levels on engineered heart tissues (EHTs) expressing mutant CRYAB.
Aims Addressed by the Doctoral Candidates
- Determine the subcellular localization of proteasomes and measure local cyclic nucleotide levels in CRYAB R120G and control human cardiomyocytes.
- Assess the expression levels and activity of phosphodiesterase isoforms and test the effects of selective compounds in CRYAB R120G EHTs
Key Techniques
- Human induced pluripotent stem cell culture
- Cardiomyocyte differentiation
- Förster resonance energy transfer (FRET) biosensors
- Live cell imaging
- Super-resolution microscopy
- Generation and measurement of engineered heart tissues
Publications
-
JAK1/2 inhibitor ruxolitinib reduces aggregates in cardiac proteinopathy. Alizoti E, Ewald L, Parretta S, March JJK, Meyer-Jens M, Orthey E, Conze C, Carrier L, Robbins J, Singh SR (2026). EMBO Mol Med. 18:1836-1865. -
Generation of a homozygous CRYAB p.Arg120Gly (UKEi001-A-1) from a human iPSC line. Pietsch N, Cheng J, Fazio A, Ewald L, Alizoti E, Krämer E, Orthey E, Carrier L, Singh SR (2023). Stem Cell Res. 71:103188. -
Phosphodiesterases type 2, 3 and 4 promote vascular tone in mesenteric arteries from rats with heart failure. Wang L, Hubert F, Idres S, Belacel-Ouari M, Domergue V, Domenichini S, Lefebvre F, Mika D, Fischmeister R, Leblais V, Manoury B (2023). Eur J Pharmacol. 944:175562. - Influence of cell confluence on the cAMP signalling pathway in vascular smooth muscle cells. Belacel-Ouari M, Zhang L, Hubert F, Assaly R, Gerbier R, Jockers R, Dauphin F, Lechêne P, Fischmeister R, Manoury B, Leblais V (2017). Cell Signal. 35:118-128.
-
A high-throughput screening identifies ZNF418 as a novel regulator of the ubiquitin-proteasome system and the autophagy-lysosomal pathway. Singh SR, Meyer-Jens M, Alizoti E, Bacon WC, Davis G, Osinska H, Gulick J, Reischmann-Düsener S, Orthey E, McLendon PM, Molkentin JD, Schlossarek S, Robbins J, Carrier L (2021). Autophagy. 17:3124-3139. -
Desmin and Cardiac Disease: An Unfolding Story. Singh SR, Robbins J (2018). Circ Res. 122:1324-1326.