Evidence that S-phase kinase associated protein 2 (SKP2) is ubiquitinated and degraded via a Rho-related BTB domain containing 1 (RhoBTB1) and Cullin-3 mechanism in placenta.

Rho related BTB domain containing 1 (RhoBTB1) is highly expressed in placenta and functions to deliver protein targets to the Cullin-3 (CUL3) E3 ubiquitin ligase where they are targeted for ubiquitination and degradation. The targets of RhoBTB1 in placenta have not been identified. Using RNAscope, we show that RhoBTB1 is mainly expressed in syncytiotrophoblasts (SCT) in the human and mouse placenta. We employed ascorbate peroxidase 2-mediated targeted proteomics to identify RhoBTB1 binding proteins in immortalized human extravillous trophoblast (HTR8/SVneo) cells. We selected 9 RhoBTB1-interacting proteins to examine functionally. Two of these, S-Phase Kinase Associated Protein 2 (SKP2) and Rho GTPase-Activating Protein 29 (ArhGAP29), increased in abundance when Cullin activity was blocked by the neddylation inhibitor MLN4924 and co-immunoprecipitated with RhoBTB1. SKP2 increased in abundance in CRISPR-Cas9 HEK293 cells that lack CUL3, and in HTR8/SVneo cells after siRNA-mediated inhibition of RhoBTB1. SKP2 was ubiquitinated by a RhoBTB1- and CUL3-dependent mechanism providing evidence that its stability is regulated by RhoBTB1/CUL3. Like RhoBTB1, SKP2 is highly expressed in the placenta. Reanalysis of single cell RNA sequencing data sets revealed that SKP2 exhibits co-expression with RhoBTB1 in SCT precursor cells, SCTs, and cytotrophoblasts. These findings identify SKP2 as a RhoBTB1/CUL3 target in the placenta.