1. Ubiquitin and Ub-like proteins Ubiquitin and ubiquitin-like proteins - background - the ubiquitin fold - ThiS, molybdopterin synthase Degradation - degradation of newly-synthesized proteins 18-1

2.  Schlesinger and Goldstein (1975) “Molecular conservation of the 74 amino acid sequence of ubiquitin between cattle and man” Nature 255, 42304.  note: it is actually 76 amino acids in length  Wolf et al. (1993) “Ubiquitin found in the archaebacterium Thermoplasma acidophilum” FEBS Lett. 326, 42.  Sequencing of the first bacteria and archaea (1995/1996): no sign of ubiquitin!  ubiquitin is the most highly conserved protein in eukaryotes and is not found in prokaryotes  how can such a protein arise in eukaryotes only? Is there not an ancestral ubiquitin-like protein in prokaryotes?  ubiquitinated proteins are recognized and degraded by the 26S proteasome in eukaryotes after a complex ubiquitination pathway - archaea possess a proteasome as well (20S) but no regulatory particle which recognizes ubiquitin Ubiquitin 18-2 Ubiquitin NC GG fusion protein (often a ribosomal subunit)  ubiquitin is always made as a fusion protein: - ubiquitin cleaved after GlyGly by enzyme  Ubiquitin-like proteins were first identified and characterized ~5 years ago

3. Ubiquitin-fold structures  ubiquitin, Nedd8, UBX and ThiS all have ubiquitin folds (i.e., similar structures)  ThiS is the most divergent, with only 14% sequence identity with ubiquitin; in comparison, Nedd8 is >50% identical  all of the sequences contain C-terminal Gly-Gly residues that is used for conjugation; this Gly-Gly terminus is perfectly conserved ubiquitin Nedd8 UBX ThiS 18-3

4. ThiS  ThiS is a sulfur ‘carrier’ protein that plays a central role in thiamin biosynthesis in E. coli  during thiamin biosynthesis, sulfur from Cysteine is transferred to thiazole, which is then incorporated into thiamin ThiS ThiF ATP ThiS O AMP ThiF Cys ThiS O SH S N ThiF, ThiH, Thil, ThiG S N thiazole N N NH 2 + thiamin OP reaction scheme for the biosynthesis of thiamin 18-4

5. Molybdopterin synthase  Molybdopterin (MPT) synthase is an evolutionarily-conserved enzyme that is present in bacteria, archaea and eukaryotes; it is a dimeric protein that consists of MoaD and MoaE, and together with MoeB catalyzes the formation of Molypdopterin  Molypdopterin plays a role in sulfite detoxification and the metabolism of xenobiotics  defects in molydopterin biosynthesis results in human disease MoaD (Ub-like fold) MoaE ubiquitin for comparison O P O OH precursor z O MPT synthase (MoaD, MoaE) and MoeB OH molybdopterin SH OPO 3 - SH O O 18-5

6. Ubiquitin/Ub-like activation pathways  all proteins in ubiquitin superfamily are used in a very similar activation pathway, with the activating enzyme being homologous GlyC O O ubiquitin Uba1 (E1) ATPPPi GlyC O AMP E1 GlyC O S-E1 (1) GlyC O O ThiS *ThiF* ATPPPi GlyC O AMP GlyC O S-ThiF (2) GlyC O O MoaD ATPPPi GlyC O AMP *MoeB* GlyC O S-MoeB (3) (1) E2 GlyC O S-E2 (3) X-SY GlyC O SH molybdopterin (2) Thil, cys GlyC O SH thiamin E3 GlyC O NH-protein multi- Ub ’ d protein 18-6

7. transports iron JAB domain: related to de-ubiquitinating isopeptidase

8. Degradation of newly-synthesized proteins  proteins are degraded down to 8- 10-mer peptides by the proteasome  the peptides are then imported into the ER by the peptide transporter associated with antigen processing (TAP)  MHC class I receptors containing bound peptides are then presented on the cell surface First: one important use of proteasome degradation products 18-7

9. DRiPs  DRiPs represent polypeptides that never attain native structure owing to errors in translation or post-translational processes necessary for the proper biogenesis of the proteins  Schubert et al.* found that upwards of 30% of all newly-synthesized proteins from various cell types are degraded by the proteasome  at least some of the DRiPs are ubiquitinated  a ubiquitinated DRiP is formed from HIV Gag polyprotein, a long-lived viral protein that serves as a source of antigenic peptides  presentation of MHC class I molecules require continuing protein translation, implying that peptides that are presented are derived from newly-made proteins DRiPs, Defective Ribosomal products - does it make sense that the cell should degrade 30% of its proteins before they have a chance to function? - might the degradation of a large fraction of DRiPs represent a defence mechanism against viral intrusions? 18-8 *Schubert et al. (2000) Rapid degradation of a large fraction of newly synthesized proteins by proteasomes. Nature 404, 770-774.