The Role of Cullin-RING Ligases in Striated Muscle Development, Function, and Disease.
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ABSTRACT:
SUBMITTER: Blondelle J
PROVIDER: S-EPMC7672578 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cullin-RING E3 ubiquitin ligases (CRL) control a myriad of biological processes by directing numerous protein substrates for proteasomal degradation. Key to CRL activity is the recruitment of the E2 ubiquitin-conjugating enzyme Cdc34 through electrostatic interactions between E3's cullin conserved basic canyon and the acidic C terminus of the E2 enzyme. This report demonstrates that a small-molecule compound, suramin, can inhibit CRL activity by disrupting its ability to recruit Cdc34. Suramin, an antitrypansomal drug that also possesses antitumor activity, was identified here through a fluorescence-based high-throughput screen as an inhibitor of ubiquitination. Suramin was shown to target cullin 1's conserved basic canyon and to block its binding to Cdc34. Suramin inhibits the activity of a variety of CRL complexes containing cullin 2, 3, and 4A. When introduced into cells, suramin induced accumulation of CRL substrates. These observations help develop a strategy of regulating ubiquitination by targeting an E2-E3 interface through small-molecule modulators.
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Project description:Tagging proteins by polyubiquitin is a key step in protein degradation. Cullin-RING E3 ubiquitin ligases facilitate ubiquitin transfer from the E2-conjugating enzyme to the substrate, yet crystallography indicates a large distance between the E2 and the substrate, raising the question of how this distance is bridged in the ubiquitin transfer reaction. Here, we demonstrate that the linker motions in the substrate binding proteins can allosterically shorten this distance to facilitate this crucial ubiquitin transfer step and increase this distance to allow polyubiquitination. We performed molecular dynamics simulations for five substrate binding proteins, Skp2, Fbw7, beta-TrCP1, Cdc4, and pVHL, in two forms: bound to their substrates and bound to both substrate and adaptor. The adaptor connects the substrate binding proteins to the cullin. In the bound-to-both forms of all cases, we observed rotations of the substrate binding domain, shortening the gap between the tip of the substrate peptide and the E2 active site by 7-12 A compared with the crystal structures. Overall, together with our earlier simulations of the unbound forms and the bound-to-adaptor forms, the emerging picture is that the maximum distance of 51-73 A between the substrate binding domain and the E2 active site in the modeled unbound forms of these five proteins shrinks to a minimum of 39-49 A in the bound-to-both forms. This large distance range, the result of allosterically controlled linker motions, facilitates ubiquitin transfer and polyubiquitination and as such argues that the cullin-RING E3 ubiquitin ligase is under conformational control. We further observed that substrate binding proteins with multiple substrate acceptor lysines have a larger distance range between the substrate and the E2 as compared with beta-TrCP1, with only one acceptor lysine.
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Project description:Ubiquitination is a highly conserved post-translational modification in eukaryotes, well known for targeting proteins for degradation by the 26S proteasome. Proteins destined for proteasomal degradation are selected by E3 ubiquitin ligases. Cullin-RING E3 ubiquitin ligases (CRLs) are the largest superfamily of E3 ubiquitin ligases, with over 400 members known in mammals. These modular complexes are tightly regulated in the cell. In this chapter, we highlight recent structural and biochemical advances shedding light on the assembly and architecture of cullin-RING ligases, their dynamic regulation by a variety of host factors, and their manipulation by viral pathogens and small molecules.
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Project description:BackgroundThe Muscle-specific RING-finger (MuRF) protein family of E3 ubiquitin ligases is important for maintenance of muscular structure and function. MuRF proteins mediate adaptation of striated muscles to stress. MuRF2 and MuRF3 bind to microtubules and are implicated in sarcomere formation with noticeable functional redundancy. However, if this redundancy is important for muscle function in vivo is unknown. Our objective was to investigate cooperative function of MuRF2 and MuRF3 in the skeletal muscle and the heart in vivo.MethodsMuRF2 and MuRF3 double knockout mice (DKO) were generated and phenotypically characterized. Skeletal muscle and the heart were investigated by morphological measurements, histological analyses, electron microscopy, immunoblotting, and real-time PCR. Isolated muscles were subjected to in vitro force measurements. Cardiac function was determined by echocardiography and working heart preparations. Function of cardiomyocytes was measured in vitro. Cell culture experiments and mass-spectrometry were used for mechanistic analyses.ResultsDKO mice showed a protein aggregate myopathy in skeletal muscle. Maximal force development was reduced in DKO soleus and extensor digitorum longus. Additionally, a fibre type shift towards slow/type I fibres occurred in DKO soleus and extensor digitorum longus. MuRF2 and MuRF3-deficient hearts showed decreased systolic and diastolic function. Further analyses revealed an increased expression of the myosin heavy chain isoform beta/slow and disturbed calcium handling as potential causes for the phenotype in DKO hearts.ConclusionsThe redundant function of MuRF2 and MuRF3 is important for maintenance of skeletal muscle and cardiac structure and function in vivo.
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