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. 2025 Sep;126(9):e70066.
doi: 10.1002/jcb.70066.

RING-Box E3 Ligase Target N-Terminal Lysine 55 to Regulate Turnover of Sp7 Protein

Abeera Sikandar  1 , Hani Ali  1 , Anabia Javed  1 , Javeed Farooqi  1 , Amjad Javed  1
Affiliations

RING-Box E3 Ligase Target N-Terminal Lysine 55 to Regulate Turnover of Sp7 Protein

Abeera Sikandar et al. J Cell Biochem. 2025 Sep.

Abstract

Specificity protein 7 (Sp7) is essential for osteoblast differentiation and bone formation. In humans, Sp7 gene mutations are associated with skeletal abnormalities, including osteogenesis imperfecta. Ubiquitylation regulates the cellular levels of Sp7 protein. However, the role of the largest class of E3 ubiquitin ligases in the turnover of Sp7 protein remains unknown. Here, we report for the first time that both catalytic subunits of multi-subunit RING-box E3 ligase, Rbx1 and Rbx2, are expressed in the skeletal tissues and during osteoblast differentiation. In situ immunofluorescence and biochemical fractionation revealed that in osteoblasts, Rbx1 and Rbx2 E3 ligase reside in both the cell cytoplasm and nucleus. The coimmunoprecipitation experiment in primary osteoblasts showed that endogenous Rbx1 and Rbx2 E3 ligase form a molecular complex with the Sp7 protein. Both Rbx1 and Rbx2 enzymes target Sp7 protein for ubiquitination. Sp7 protein is degraded by Rbx1 and Rbx2 enzymes in a dose-dependent manner in both osseous and non-osseous cells. Chemical inhibition established the requirement of Rbx1 and Rbx2-mediated ubiquitination and degradation of Sp7 protein by the proteasomal pathway. In-silico analysis identified three evolutionarily conserved lysines, K-55, K-227, and K-229, in the Sp7 protein as potential targets for ubiquitination. A panel of Sp7 deletion and point mutants was generated that established the critical requirement of lysine-55 for Rbx1 and Rbx2-mediated ubiquitination and degradation. Deleting the Rbx2 gene in osteoprogenitors led to a significant accumulation of Sp7 protein, enhanced expression of osteoblast marker genes, and accelerated matrix mineralization.

Keywords: RING‐box E3 ligase; Rbx1 and Rbx2 cellular distribution; Sp7 ubiquitination; osteoblast differentiation; protein degradation.

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