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bioRxiv · 10.1101/2022.06.13.495268

The Role of UBE2-Conjugating Enzymes in the Mechanism of MuRF1 Ubiquitylation

Dawson, P. W. J.·Baehr, L. M.·Hughes, D. C.·Knowles, T. J.·Sridhar, P.·Bodine, S. C.·Lai, Y.-C.
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Abstract

MuRF1 (Muscle-specific RING finger protein 1; gene name TRIM63) is a ubiquitin E3 ligase, associated with the progression of muscle atrophy. As a RING (Really Interesting New Gene)-type E3 ligase, its unique activity of ubiquitylation is driven by a specific interaction with UBE2 (ubiquitin conjugating enzyme) to ubiquitylate its substrate protein. The understanding of MuRF1 function remains unclear as candidate UBE2 has not been elucidated and thus the mechanism of ubiquitylation is inconclusive. In the present study, we screened human ubiquitin dependent E2s using in-vitro ubiquitylation assays. We found that MuRF1 engages in ubiquitylation/auto-ubiquitylation with UBE2D, UBE2E, UBE2N/V families and UBE2W. Our result indicated that MuRF1 can cause mono-ubiquitylation, K48, and K63 specific poly-ubiquitin chains in a UBE2-dependent manner. Interestingly, we identified a two-step UBE2-dependent mechanism by which UBE2W allows MuRF1 to mono-ubiquitylate which then acts as an anchor for UBE2N/V and UBE2D generated poly-ubiquitin chain formation. Furthermore, MuRF1 was shown to cooperate with the identified interacting UBE2s to directly ubiquitylate substrates Titin (A168-A170), Desmin, and MYLPF (Myosin Light Chain, Phosphorylatable, Fast Skeletal Muscle; also called Myosin Light Regulatory Chain 2). Our work presents a novel insight into the mechanisms that underpin MuRF1 activity by highlighting the diversity of MuRF1 ubiquitylation enabled by different UBE2s.

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Dawson, P. W. J., Baehr, L. M., Hughes, D. C., Knowles, T. J., Sridhar, P., Bodine, S. C., Lai, Y.-C.. 2022-06-13. The Role of UBE2-Conjugating Enzymes in the Mechanism of MuRF1 Ubiquitylation. https://doi.org/10.1101/2022.06.13.495268

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