Abstract
Cullin 4B (CUL4B) is a scaffold protein that assembles cullin-RING ubiquitin ligase (E3) complexes. Recent studies have revealed that germ-line mutations in CUL4B can cause mental retardation, short stature, and many other abnormalities in humans. Identifying specific CUL4B substrates will help to better understand the physiological functions of CUL4B. Here, we report the identification of peroxiredoxin III (PrxIII) as a novel substrate of the CUL4B ubiquitin ligase complex. Two-dimensional gel electrophoresis coupled with mass spectrometry showed that PrxIII was among the proteins up-regulated in cells after RNAi-mediated CUL4B depletion. The impaired degradation of PrxIII observed in CUL4B knockdown cells was confirmed by Western blot. We further demonstrated that DDB1 and ROC1 in the DDB1-CUL4B-ROC1 complex are also indispensable for the proteolysis of PrxIII. In addition, the degradation of PrxIII is independent of CUL4A, a cullin family member closely related to CUL4B. In vitro and in vivo ubiquitination assays revealed that CUL4B promoted the polyubiquitination of PrxIII. Furthermore, we observed a significant decrease in cellular reactive oxygen species (ROS) production in CUL4B-silenced cells, which was associated with increased resistance to hypoxia and H(2)O(2)-induced apoptosis. These findings are discussed with regard to the known function of PrxIII as a ROS scavenger and the high endogenous ROS levels required for neural stem cell proliferation. Together, our study has identified a specific target substrate of CUL4B ubiquitin ligase that may have significant implications for the pathogenesis observed in patients with mutations in CUL4B.
Highlights
E3 ubiquitin ligases, the cullin-RING ubiquitin ligases [2]
We identified peroxiredoxin III (PrxIII) as a novel substrate of Cullin 4B (CUL4B) ubiquitin ligase
We found that CUL4B silencing resulted in the accumulation of PrxIII and that CUL4B mediates the polyubiquitination of PrxIII in vitro and in vivo
Summary
Cell Cultures, Plasmids, and Protein Extracts—HEK293 and HeLa cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) plus penicillin and streptomycin in a humidified incubator at 37 °C with 5% CO2. The protein samples prepared from cell cultures were quantitated using the Bradford assay [24], subjected to 10% SDSPAGE, and electrotransferred onto PVDF membranes (GE Healthcare) for 1 h at 100 V using a standard transfer solution. For CHX chase analysis, cells were harvested at specified time points after adding CHX (50 g/ml) to the medium and lysed in buffer, separated on SDS-PAGE, and analyzed by immunoblotting with specific antibodies. For the in vitro PrxIII ubiquitination, the CUL4B immunocomplex was mixed with His-tagged PrxIII substrate, and this mixture was added to a ubiquitin ligation reaction (final volume of 50 l, ubiquitinylation kit from Enzo Life Sciences) containing the following components: ubiquitinylation buffer, 20 units/ml inorganic pyrophosphatase solution, 1 mM dithiothreitol, 5 mM Mg-ATP, 100 mM E1, 2.5 M E2 (hUbc5c), 1 M PrxIII, and 2.5 M bovine ubiquitin. The data were analyzed using the FCSExpress V3 program (DeNovo Software)
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