Role of SKP1-CUL1-F-Box-Protein (SCF) E3 Ubiquitin Ligases in Skin Cancer

Many biological processes such as cell proliferation, differentiation, and cell death depend precisely on the timely synthesis and degradation of key regulatory proteins. While protein synthesis can be regulated at multiple levels, protein degradation is mainly controlled by the ubiquitin-proteasome system （UPS）, which consists of two distinct steps： （1） ubiquitylation of targeted protein by E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme and E3 ubiquitin ligase, and （2） subsequent degradation by the 26S proteasome. Among all E3 ubiquitin ligases, the SCF （SKP1-CUL1-F-box protein） E3 ligases are the largest family and are responsible for the turnover of many key regulatory proteins. Aberrant regulation of SCF E3 ligases is associated with various human diseases, such as cancers, including skin cancer. In this review, we provide a comprehensive overview of all currently published data to define a promoting role of SCF E3 ligases in the development of skin cancer. The future directions in this area of research are also discussed with an ultimate goal to develop small molecule inhibitors of SCF E3 ligases as a novel approach for the treatment of human skin cancer. Furthermore, altered components or substrates of SCF E3 ligases may also be developed as the biomarkers for early diagnosis or predicting prognosis.

An engineered platform for reconstituting functional multisubunit SCF E3 ligase in vitro

Multisubunit SKP1/Cullin1/F-box(SCF)E3 ligases play essential roles in regulating the stability of crucial regulatory factors and controlling growth and development in eukaryotes.Detecting E3 ligase activity in vitro is important forexploring the molecular mechanism of protein ubiquitination.However,in vitro ubiquitination assay systems for multisubunit E3 ligases remain difficult to achieve,especially in plants,mainly owing to difficulties in achieving active components of multisubunit E3 ligases with high purity and characterizing specific E2 and E3 pairs.In this study,we characterized components of the rice ScFDiwARF3(SCFDs)E3 ligase,screened the coordinated E2,and reconstituted active ScFD3 E3 ligase in vitro.We further engineered SCFD3 E3 ligase using a fused SKP1-Cullin1-RBX1(eSCR)protein and found that both the wild-type SCFD3 E3 ligase and the engineered SCFD3 E3 ligase catalyzed ubiquitination of the substrate D53,which is the key transcriptional repressor in strigolactone signaling.Finally,we replaced D3 with other F-box proteins from rice and humans and reconstituted active escF E3 ligases,including escFaID2,escFBxL1s,and escFcDC4 E3 ligases.Our work reconstitutes functional SCF E3 ligases in vitro and generates an engineered system with interchangeable F-box proteins,providing a powerful platform for studying the mechanisms of multisubunit SCF E3 ligases in eukaryotes.

Functional characterization of SAG/RBX2/ROC2/RNF7, an antioxidant protein and an E3 ubiquitin ligase

SAG(Sensitive to Apoptosis Gene),also known as RBX2(RING box protein 2),ROC2(Regulator of Cullins 2),or RNF7(RING Finger Protein 7),was originally cloned in our laboratory as a redox inducible antioxi-dant protein and later characterized as the second member of the RBX/ROC RING component of the SCF(SKP1-CUL-F-box Proteins)E3 ubiquitin ligase.When acting alone,SAG scavenges oxygen radicals by forming inter-and intra-molecular disulfide bonds,whereas by forming a complex with other components of the SCF E3 ligase,SAG promotes ubiquitination and degradation of a number of protein substrates,includ-ing c-JUN,DEPTOR,HIF-1α,IκBα,NF1,NOXA,p27,and procaspase-3,thus regulating various signaling path-ways and biological processes.Specifically,SAG pro-tects cells from apoptosis,confers radioresistance,and plays an essential and non-redundant role in mouse embryogenesis and vasculogenesis.Furthermore,stress-inducible SAG is overexpressed in a number of human cancers and SAG overexpression correlates with poor patient prognosis.Finally,SAG transgenic expression in epidermis causes an early stage inhibi-tion,but later stage promotion,of skin tumorigenesis triggered by DMBA/TPA.Given its major role in pro-moting targeted degradation of tumor suppressive proteins,leading to apoptosis suppression and accel-erated tumorigenesis,SAG E3 ligase appears to be an attractive anticancer target.