2.1 The SCF E3 Ubiquitin Ligases
The SKP1-Cullin1-F-box (SCF) E3 ligases are the largest family of E3 ubiquitin ligases and regulate many cellular processes by promoting the ubiquitination of regulatory proteins, e.g. cell cycle regulators and transcription factors, targeting them for proteasome-mediated degradation or otherwise affecting their function/activity.59
Ubiquitination of a substrate is a multi-step process (Figure 3). Ubiquitin-activating enzyme E1 coordinates to ubiquitin through use of ATP and transfers it to ubiquitin- conjugating enzyme E2. The SCF ligase E3 recognises and coordinates to the protein substrate and catalyses ubiquitin transfer from E2 to the protein. Ubiquitin is conjugated to a protein by an isopeptide link formed between the carboxy group of the C-terminal glycine residue of ubiquitin and the ɛ-amino group of a lysine residue within the
substrate. This process can be repeated to assemble polyubiquitin chains by conjugating the carboxy group of the C-terminal glycine residue of the new ubiquitin molecule to the ɛ-amino group of one of seven internal lysines within the preceding ubiquitin. The fate of the ubiquitinated substrate depends on the type of isopeptide linkage formed; K48-linked polyubiquitination targets the substrate for proteasome-mediated
degradation, but K63-linked or monoubiquitination alters the protein’s function and its interactions with other proteins etc.60
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Figure 3: Protein ubiquitination pathway (Ub, ubiquitin); adapted from60
The SCF E3 ligases are multi-subunit complexes consisting of adaptor proteins, cullins, RING finger proteins and F-box (receptor) proteins (Figure 4). Crystal structures of SCF complexes reveal the cullins are scaffold proteins, which bind the adaptor and F- box proteins at its N-terminus and the RING finger proteins at its C-terminus.59 Seven cullins are known in the human genome (Cul-1, -2, -3, -4A, -4B, -5 and -7) and all are involved in the coordination of different F-box proteins. The RING finger proteins coordinate to E2 enzymes and facilitate ubiquitin transfer to the substrate. Two RING finger proteins are known in the human genome, RBX1 (RoC1) and RBX2 (RoC2), both of which have been shown to be functionally non-redundant in mouse-knockout studies.59, 61 SKP1 is an adaptor protein found in several SCF complexes and links the cullin with the F-box protein.59
Ub E1 E2 E3 Substrate ATP Mono-Ub K63 Ub K48 Ub Ub E1 Ub E2 E3 Substrate Ub E2 E3 Substrate Substrate Ub Substrate Ub Ub Ub Ub Substrate Ub Ub Ub Ub
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Figure 4: The SCFSKP2 E3 Ligase; adapted from62. Ubiquitin (Ub) is transferred from an E2 enzyme to a substrate that is bound to SKP2. The substrate must be phosphorylated before SKP2 can bind to it (P = phosphate). Once ubiquitinated, the substrate is degraded by the 26S proteasome with the release of ubiquitin. Substrates of SKP2 include p21, p27, p57, p103, TOB1, FOXO1 and c-Myc
F-box proteins are the substrate-recognising subunits that determine the selectivity of an SCF complex. One F-box protein can recognise multiple substrates, some of which have opposite biological functions, e.g. cell cycle suppressors and promoters. Whether a particular substrate will be targeted or not depends on the cell’s internal environment, highlighting the significance of SCF complexes in determining the life and death of a cell. In the human genome there are 69 F-box proteins,63 each of which binds to the adaptor protein via its F-box domain, and are classed into one of three categories depending on the nature of the binding domain that links them to the substrate.59 Those which have WD40 domains (i.e. domains consisting of a 40-amino acid motif ending in a tryptophan-aspartic acid dipeptide) are called FBXWs, those with domains consisting of leucine-rich repeats are called FBXLs and those which have other diverse domains are called FBXOs. To date, three F-box proteins have been well studied: Fbxw7,64 β- TrCP65 and SKP2.59, 65, 66
2.2 SCF E3 Ubiquitin Ligases as Anticancer Targets
Several subunits of the SCF complex have demonstrated oncogenic activity; of the seven cullin scaffold proteins, Cul-1 is overexpressed in 40% of lung cancers and Cul- 4A is overexpressed in breast cancer, HCC and mesotheliomas. Cul-4A overexpression is associated with cell cycle progression in breast cancer cells resulting in poor
prognosis and Cul-4A knockdown by siRNA causes accumulation of cell cycle suppressors p21 and p27, inducing G1 cell cycle arrest.59
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SCF-mediated ubiquitination must be activated by the coordination of a ubiquitin-like protein, Nedd8 activating enzyme (NAE), to the cullins of the SCF ligase; a process termed neddylation. This forms an isopeptide bond between the C-terminal glycine residue of Nedd8 and the ε-amino group of a conserved cullin lysine residue. Inhibitors of neddylation, e.g. MLN4924 (14), induce many SCF substrates, including p27, which can promote cell cycle arrest and cell death (Figure 5). This evidence suggests the significance of targeting Nedd8 as a form of cancer treatment.67
The accessory protein cyclin kinase subunit 1 (Cks1) is essential for efficient SKP2- mediated degradation of p27, as evidenced by the slow proliferation of p27 in Cks1- knockout mice. Cks1 levels have been shown to correlate positively with those of SKP2 and inversely with those of p27. As a result, Cks1 overexpression has been linked with tumour aggressiveness and a poor prognosis in several cancers including colorectal carcinoma.68
RBX1 is overexpressed in several human tumours including lung cancer. Knockdown of RBX1 by siRNA triggers a DNA damage response and subsequent G2/M cell cycle arrest, senescence and apoptosis. RBX2 is also overexpressed in several human cancers giving a poor prognosis. The RING finger proteins exert E3 ubiquitin ligase activity and promote the degradation of multiple cell cycle regulators including p27 and c-Jun.61 RBX2 siRNA silencing induces cancer cell apoptosis and sensitisation to
chemotherapeutics and radiation. This evidence suggests the significance of the RING finger proteins as anticancer targets.59
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Figure 5: Targeting the SCF E3 ligases to trigger multiple cancer cell killing mechanisms59
2.2.1 Targeting the F-Box Protein SKP2 for Cancer Therapy
The SCFSKP2 E3 ligase recognises and targets several cell cycle negative regulators, e.g. p21, p27, p57 and p130.69 Oncogenic SKP2 increases ubiquitination of these proteins and enables replicative immortality.63 Reduced p27 levels are common in many human cancers, being associated with an aggressive phenotype and poor prognosis.70, 71 In
Cullins Nedd8 88 RING finger protein Adaptor F-box protein Substrate E2 Ub Ub Ub Ub Si-RBX1/2 MLN 4924 Si-Cul-4A Si-SKP2
Accumulation of SCF E3 ligase substrates
Multiple cancer cell killing pathways