Abstract
The HECT-type ubiquitin ligase Smurf1 (Smad ubiquitination regulatory factor-1) plays the prominent role in regulation of bone formation, embryonic development, and tumorigenesis by directing the ubiquitin-proteasomal degradation of specific targets. In contrast with RING-type E3s, the catalytic HECT domain of Smurf1 firstly binds to and then transfers ubiquitin (Ub) molecules onto the substrates. The Smurf1-Ub interaction is required for Smurf1 catalytic ligase activity to promote substrate degradation. However, so far specific regulators or compounds controlling Smurf1-Ub interaction and the ligase activity have not been identified. Here we report two small molecule compounds targeting Ub binding region of HECT domain interrupt Smurf1-Ub contact, inhibit Smurf1 ligase activity and stabilize BMP signal components Smad1/5 protein level. Furthermore, these compounds increase BMP signal responsiveness and enhance osteoblastic activity in cultured cells. These findings provide a novel strategy through targeting Smurf1 ligase activity to potentially treat bone disorders such as osteoporosis.
Highlights
Ubiquitination, which involves in the intracellular protein turnover of various biological processes, is essential for maintaining physiological function of organism and cellular homeostasis
The structure of Smurf1 WW domains (WW1 and WW2) and a phospholipid binding C2 domain have been identified and analyzed, the three-dimensional structure of Smurf1 catalytic HECT domain remains unclear. Given that both Smurf1 and Smurf2 belong to Nedd4 family, and their amino acid sequence homology of HECT domain is more than 90%, the structure of this domain was obtained by protein modeling performed on PyMOL and referred to the counterpart domain of Smurf2 (Figure 1A)
The results showed that B06, B07, B10, B11 and B75 improved alkaline phosphatase (ALP) activity compared with negative control DMSO (Figure 2A)
Summary
Ubiquitination, which involves in the intracellular protein turnover of various biological processes, is essential for maintaining physiological function of organism and cellular homeostasis. A series of studies have shown that certain activators could enhance the E3 activity of Smurf1and augment Smurf1-mediated ubiquitination, such as casein kinase 2 interacting protein-1 (CKIP-1) [22, 24] and Cdh, which is identified as the activator of anaphase-promoting complex(APC) [10] These activators reduce cellular responses to TGF-β/BMP signaling, and depress the bone formation process and attenuate osteogenetic activity. We simulated the Ub binding region of Smurf and identified two small molecule compounds via computer virtual screening They target the HECT domain of Smurf and interrupt Ub-Smurf contact, inhibit the degradation of Smad1/5. As we know, this is the first time to identify small molecule compound which targets the HECT domain of Smurf ligase
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