Cand1 promotes assembly of new SCF complexes through dynamic exchange of F box proteins.

The modular SCF (Skp1, cullin, and F box) ubiquitin ligases feature a large family of F box protein substrate receptors that enable recognition of diverse targets. However, how the repertoire of SCF complexes is sustained remains unclear. Real-time measurements of formation and disassembly indicate that SCF(Fbxw7) is extraordinarily stable, but, in the Nedd8-deconjugated state, the cullin-binding protein Cand1 augments its dissociation by one-million-fold. Binding and ubiquitylation assays show that Cand1 is a protein exchange factor that accelerates the rate at which Cul1-Rbx1 equilibrates with multiple F box protein-Skp1 modules. Depletion of Cand1 from cells impedes recruitment of new F box proteins to pre-existing Cul1 and profoundly alters the cellular landscape of SCF complexes. We suggest that catalyzed protein exchange may be a general feature of dynamic macromolecular machines and propose a hypothesis for how substrates, Nedd8, and Cand1 collaborate to regulate the cellular repertoire of SCF complexes.

Detection of sequential polyubiquitylation on a millisecond timescale.

The pathway by which ubiquitin chains are generated on substrate through a cascade of enzymes consisting of an E1, E2 and E3 remains unclear. Multiple distinct models involving chain assembly on E2 or substrate have been proposed. However, the speed and complexity of the reaction have precluded direct experimental tests to distinguish between potential pathways. Here we introduce new theoretical and experimental methodologies to address both limitations. A quantitative framework based on product distribution predicts that the really interesting new gene (RING) E3 enzymes SCF(Cdc4) and SCF(beta-TrCP) work with the E2 Cdc34 to build polyubiquitin chains on substrates by sequential transfers of single ubiquitins. Measurements with millisecond time resolution directly demonstrate that substrate polyubiquitylation proceeds sequentially. Our results present an unprecedented glimpse into the mechanism of RING ubiquitin ligases and illuminate the quantitative parameters that underlie the rate and pattern of ubiquitin chain assembly.

Exome sequencing of Bardet-Biedl syndrome patient identifies a null mutation in the BBSome subunit BBIP1 (BBS18).

BACKGROUND: Bardet-Biedl syndrome (BBS) is a recessive and genetically heterogeneous ciliopathy characterised by retinitis pigmentosa, obesity, kidney dysfunction, postaxial polydactyly, behavioural dysfunction and hypogonadism. 7 of the 17 BBS gene products identified to date assemble together with the protein BBIP1/BBIP10 into the BBSome, a protein complex that ferries signalling receptors to and from cilia. METHODS AND RESULTS: Exome sequencing performed on a sporadic BBS case revealed for the first time a homozygous stop mutation (NM_001195306: c.173T>G, p.Leu58*) in the BBIP1 gene. This mutation is pathogenic since no BBIP1 protein could be detected in fibroblasts from the patient, and BBIP1[Leu58*] is unable to associate with the BBSome subunit BBS4. CONCLUSIONS: These findings identify BBIP1 as the 18th BBS gene (BBS18) and suggest that BBSome assembly may represent a unifying pathomechanism for BBS.

Structural biology. Structural basis for Notch1 engagement of Delta-like 4.

Notch receptors guide mammalian cell fate decisions by engaging the proteins Jagged and Delta-like (DLL). The 2.3 angstrom resolution crystal structure of the interacting regions of the Notch1-DLL4 complex reveals a two-site, antiparallel binding orientation assisted by Notch1 O-linked glycosylation. Notch1 epidermal growth factor-like repeats 11 and 12 interact with the DLL4 Delta/Serrate/Lag-2 (DSL) domain and module at the N-terminus of Notch ligands (MNNL) domains, respectively. Threonine and serine residues on Notch1 are functionalized with O-fucose and O-glucose, which act as surrogate amino acids by making specific, and essential, contacts to residues on DLL4. The elucidation of a direct chemical role for O-glycans in Notch1 ligand engagement demonstrates how, by relying on posttranslational modifications of their ligand binding sites, Notch proteins have linked their functional capacity to developmentally regulated biosynthetic pathways.

Cilia grow by taking a bite out of the cell.

Autophagy and primary cilium assembly have long been known to be induced by the same conditions in cultured cells. Two recent studies in Nature-Tang et al. (2013) and Pampliega et al. (2013)-link the two processes, suggesting that a specialized autophagy pathway near the basal body regulates cilium assembly.