Polyubiquitylation targets multiple proteins for degradation by the proteasome. Typically, the first ubiquitin is linked to lysine residues in the substrate for degradation via an isopeptide bond, although rarely ubiquitin linkage to the N-terminal residue has also been observed. We have recently shown that Neurogenin (NGN), a basic helix-loop-helix transcription factor that plays a central role in regulating neuronal differentiation, is degraded by ubiquitin-mediated proteolysis. We have taken a biochemical and mutagenesis approach to investigate sites of ubiquitylation of NGN, initially using extracts of eggs from the frog Xenopus laevis as a source of ubiquitylation and degradation components. NGN can be targeted for destruction by ubiquitylation via lysines or the N terminus. However, we see that a modified NGN, where canonical lysine ubiquitylation and N-terminally linked ubiquitylation are prevented, is nevertheless ubiquitylated and degraded by the proteasome. We show that polyubiquitin chains covalently attach to non-canonical cysteine residues in NGN, and these non-canonical linkages alone are capable of targeting NGN protein for destruction. Importantly, canonical and non-canonical ubiquitylation occurs simultaneously in the native protein and may differ in importance for driving degradation in interphase and mitosis. We conclude that native NGN is ubiquitylated on multiple canonical and non-canonical sites by cellular ubiquitin ligases, and all types of linkage can contribute to protein turnover.The selective degradation of proteins by ubiquitin-mediated proteolysis is a crucial regulator of diverse cellular processes in eukaryotes. Ub 4 is classically conjugated to the ⑀-amino group of a substrate lysine by an isopeptide bond (1). This serves as an anchor point to build a polyubiquitin chain that results in subsequent targeting to the proteasome for destruction. In a small number of proteins, Ub has been shown to be conjugated to the N-terminal ␣-amino group of a protein via a peptide linkage used as an anchor site for assembly of a lysine-linked polyubiquitin chain (2).The conjugation of ubiquitin to substrate proceeds when a nucleophilic substrate amino group attacks the labile E2-ubiquitin thioester bond. This mechanism does not exclude the possibility that ubiquitylation can occur on other substrate nucleophilic groups on amino acid side chains, such as the thiol groups present on cysteines. The resultant thioester-anchored ubiquitin chains would, however, be predicted to be less stable than the isopeptide/peptide bonds formed between ubiquitin and lysine/N-terminal amino groups (3, 4). Based on this, cysteine residues have generally been considered to be unlikely targets for ubiquitin modification (4).However, there is recent evidence that ubiquitylation can occur on substrate cysteine residues, albeit in a very limited number of cases, largely regulating intracellular protein movements and usually driven by viral E3 ligases (3, 5-7). Only one substrate, the N-terminal fragment of the Bcl2-relat...

The ability to differentiate human ESCs (hESCs) to defined lineages in a totally controlled manner is fundamental to developing cell-based therapies and studying human developmental mechanisms. We report a novel, scaleable, and widely applicable system for deriving and propagating neural stem cells from hESCs without the use of animal products, proprietary formulations, or genetic manipulation. This system provides a definitive platform for studying human neural development and has potential therapeutic implications. STEM CELLS 2007;25:731-737