HERC3 directly targets RPL23A for ubiquitination degradation and further regulates Colorectal Cancer proliferation and the cell cycle

Abstract

Aims : Colorectal cancer (CRC) has high mortality and morbidity rates; however, the mechanism of CRC cells uncontrolled proliferation is unclarified, E3 ligases are widely reported to have crucial functions in cancers. HERC3 was once recognized as an important role in CRC, however its effects on CRC cell proliferation and cell cycle are blank. Methods : Correlation between HERC3 and clinical characteristics was analyzed. Coimmunoprecipitation, mass spectrometry analysis and GST-pull down were performed to identify interacting-proteins of HERC3. Expression pattern of RPL23A and its correlation between HERC3 was researched via qRT-PCR, western blot and immunohistochemistry. In vivo and vitro gain-and loss-of-function assays and rescue experiments concentrating HERC3-RPL23A axis in terms of cell proliferation and cell cycle were conducted. The ubiquitination regulatory mechanism between HERC3 and RPL23A were identified via vivo ubiquitylation assays, cycloheximide analysis and mass spectrometry analysis. GSEA aided to research the potential functional mechanism of RPL23A and validated by western blot and in vivo ubiquitylation assays. Results : HERC3 expression decreased gradually from colorectal tissues in healthy individuals to adjacent-tumors normal tissue in CRC patients, and to tumor tissues and HERC3 could inhibit CRC cell proliferation and arrest cells in the G0-G1 phase. RPL23A which was recognized as one potential target of HERC3 was identified to be overexpressed in CRC and could serve as a prognostic biomarker in CRC. RPL23A could also independently regulate the cell cycle and cell proliferation and attenuate the influence of HERC3 on CRC. In addition, HERC3 directly interacted with RPL23A and served as an E3 ligase to ubiquitination degrade RPL23A via K48-dependant manner through the HECT domain. Furthermore, HERC3 could regulate the ubiquitination of p21 and further modulate protein expression of c-Myc and p21 via regulating RPL23A. Conclusion : HERC3 controlled CRC proliferation, the cell cycle and regulated the c-Myc/p21 axis via directly targeting RPL23A for ubiquitination degradation.

HERC3 E3 ligase provides an ERAD branch eliminating select membrane proteins

“…Considering that HERC3 is primarily localized in the cytoplasm (Cruz et al, 2001), it is reasonable that HERC3 primarily acts on the misfolded CFTR dislocated to the cytoplasm. Moreover, this presumed function is in line with previous findings that HERC3 promotes the proteasomal degradation of cytosolic proteins such as RPL23A (Zhang et al, 2022b), EIF5A (Zhang et al, 2022a), and SMAD7 (Li et al, 2019). On the other hand, HERC3 appears to be at least partially involved in retrotranslocation, and its contribution may be particularly important when the function of RNF5/185 is insufficient.…”

E3 ubiquitin ligase Herc3 deficiency leads to accumulation of subretinal microglia and retinal neurodegeneration

“…Meanwhile, the HECT domain in Herc3 is functional and able to bind ubiquitin (and undergo ubiquitination) 61 . Within the last 2 years, this HECT domain of Herc3 has also been shown to ubiquitinate RPL23A 65 , EIF5A2 66 , and ERK2 67 and target them for degradation. Herc3 may also have important biological functions that are independent of the RLD and HECT domains.…”

HERC3 promotes YAP/TAZ stability and tumorigenesis independently of its ubiquitin ligase activity

“…Despite its suggested involvement in ubiquitination, HERC3 is an understudied member of the HERC family with limited functional implications, especially in cancer. HERC3 targets few substrates for ubiquitinationdependent degradation, including SMAD7, MM1, EIF5A2, and RPL23A (Chen et al, 2018;Li et al, 2019;Zhang et al, 2022aZhang et al, , 2022b. In addition, HERC3 can inhibit NF-jB signaling independent of its E3 activity (Hochrainer et al, 2015).…”

“…HERC3 could also facilitate ubiquitinationmediated degradation of SMAD7 to positively regulate TGF-b signaling in glioblastoma (Li et al, 2019). Recent studies found that HERC3 targets degradation of EIF5A2 and RPL23A to potentially block cell proliferation and metastasis in colorectal cancer (Zhang et al, 2022a(Zhang et al, , 2022b. In addition, HERC3 promotes RelA degradation independent of its E3 activity to inhibit NF-jB signaling (Hochrainer et al, 2015).…”