Identifying Determinants of Cullin Binding Specificity Among the Three Functionally Different Drosophila melanogaster Roc Proteins via Domain Swapping

BACKGROUND: Cullin-dependent E3 ubiquitin ligases (CDL) are key regulators of protein destruction that participate in a wide range of cell biological processes. The Roc subunit of CDL contains an evolutionarily conserved RING domain that binds ubiquitin charged E2 and is essential for ubiquitylation. Drosophila melanogaster contains three highly related Roc proteins: Roc1a and Roc2, which are conserved in vertebrates, and Roc1b, which is specific to Drosophila. Our previous genetic data analyzing Roc1a and Roc1b mutants suggested that Roc proteins are functionally distinct, but the molecular basis for this distinction is not known. METHODOLOGY/PRINCIPAL FINDINGS: Using co-immunoprecipitation studies we show that Drosophila Roc proteins bind specific Cullins: Roc1a binds Cul1-4, Roc1b binds Cul3, and Roc2 binds Cul5. Through domain swapping experiments, we demonstrate that Cullin binding specificity is strongly influenced by the Roc NH(2)-terminal domain, which forms an inter-molecular beta sheet with the Cullin. Substitution of the Roc1a RING domain with that of Roc1b results in a protein with similar Cullin binding properties to Roc1a that is active as an E3 ligase but cannot complement Roc1a mutant lethality, indicating that the identity of the RING domain can be an important determinant of CDL function. In contrast, the converse chimeric protein with a substitution of the Roc1b RING domain with that of Roc1a can rescue the male sterility of Roc1b mutants, but only when expressed from the endogenous Roc1b promoter. We also identified mutations of Roc2 and Cul5 and show that they cause no overt developmental phenotype, consistent with our finding that Roc2 and Cul5 proteins are exclusive binding partners, which others have observed in human cells as well. CONCLUSIONS: The Drosophila Roc proteins are highly similar, but have diverged during evolution to bind a distinct set of Cullins and to utilize RING domains that have overlapping, but not identical, function in vivo

Identification of Distinctive Patterns of USP19-Mediated Growth Regulation in Normal and Malignant Cells

We previously reported that the USP19 deubiquitinating enzyme positively regulates proliferation in fibroblasts by stabilizing KPC1, a ubiquitin ligase for p27Kip1. To explore whether this role of USP19 extends to other cellular systems, we tested the effects of silencing of USP19 in several human prostate and breast models, including carcinoma cell lines. Depletion of USP19 inhibited proliferation in prostate cancer DU145, PC-3 and 22RV1 cells, which was similar to the pattern established in fibroblasts in that it was due to decreased progression from G1 to S phase and associated with a stabilization of the cyclin-dependent kinase inhibitor p27Kip1. However, in contrast to previous findings in fibroblasts, the stabilization of p27Kip1 upon USP19 depletion was not associated with changes in the levels of the KPC1 ligase. USP19 could also regulate the growth of immortalized MCF10A breast epithelial cells through a similar mechanism. This regulatory pattern was lost, though, in breast cancer MCF7 and MDA-MB-231 cells and in prostate carcinoma LNCaP cells. Of interest, the transformation of fibroblasts through overexpression of an oncogenic form of Ras disrupted the USP19-mediated regulation of cell growth and of levels of p27Kip1 and KPC1. Thus, the cell context appears determinant for the ability of USP19 to regulate cell proliferation and p27Kip1 levels. This may occur through both KPC1 dependent and independent mechanisms. Moreover, a complete loss of USP19 function on cell growth may arise as a result of oncogenic transformation of cells

Homo-PROTACs:bivalent small-molecule dimerizers of the VHL E3 ubiquitin ligase to induce self-degradation

E3 ubiquitin ligases are key enzymes within the ubiquitin proteasome system which catalyze the ubiquitination of proteins, targeting them for proteasomal degradation. E3 ligases are gaining importance as targets to small molecules, both for direct inhibition and to be hijacked to induce the degradation of non-native neo-substrates using bivalent compounds known as PROTACs (for 'proteolysis-targeting chimeras'). We describe Homo-PROTACs as an approach to dimerize an E3 ligase to trigger its suicide-type chemical knockdown inside cells. We provide proof-of-concept of Homo-PROTACs using diverse molecules composed of two instances of a ligand for the von Hippel-Lindau (VHL) E3 ligase. The most active compound, CM11, dimerizes VHL with high avidity in vitro and induces potent, rapid and proteasome-dependent self-degradation of VHL in different cell lines, in a highly isoform-selective fashion and without triggering a hypoxic response. This approach offers a novel chemical probe for selective VHL knockdown, and demonstrates the potential for a new modality of chemical intervention on E3 ligases.Targeting the ubiquitin proteasome system to modulate protein homeostasis using small molecules has promising therapeutic potential. Here the authors describe Homo-PROTACS: small molecules that can induce the homo-dimerization of E3 ubiquitin ligases and cause their proteasome-dependent degradation

The Arabidopsis thaliana F-Box Protein FBL17 Is Essential for Progression through the Second Mitosis during Pollen Development

In fungi and metazoans, the SCF-type Ubiquitin protein ligases (E3s) play a critical role in cell cycle regulation by degrading negative regulators, such as cell cycle-dependent kinase inhibitors (CKIs) at the G1-to-S-phase checkpoint. Here we report that FBL17, an Arabidopsis thaliana F-box protein, is involved in cell cycle regulation during male gametogenesis. FBL17 expression is strongly enhanced in plants co-expressing E2Fa and DPa, transcription factors that promote S-phase entry. FBL17 loss-of-function mutants fail to undergo pollen mitosis II, which generates the two sperm cells in mature A. thaliana pollen. Nonetheless, the single sperm cell-like cell in fbl17 mutants is functional but will exclusively fertilize the egg cell of the female gametophyte, giving rise to an embryo that will later abort, most likely due to the lack of functional endosperm. Seed abortion can, however, be overcome by mutations in FIE, a component of the Polycomb group complex, overall resembling loss-of-function mutations in the A. thaliana cyclin-dependent kinase CDKA;1. Finally we identified ASK11, as an SKP1-like partner protein of FBL17 and discuss a possible mechanism how SCFFBL17 may regulate cell division during male gametogenesis