UBIQUITIN ● 76 Amino Acid polypeptide ● Highly conserved in evolution: 3 Amino acid differences between yeast and human homologues
Degradation of a Protein Via the Ubiquitin-Proteasome Involves Two Successive Steps ● 1. Covalent attachment of multiple ubiquitin molecules to a protein substrate. ● 2. Degradation of the tagged protein by the 26s proteasome. (ubiquitin is recycled)
UBIQUITINATION In general, multiple ubiquitin units are arranged in polyubiquitin chains linked via Lys 48 of ubiquitin, targeting the protein for degradation
Ubiquitin Conjugation: A 3 Step Mechanism Ubiquitin (Ub) activating enzyme E 1 High energy thiol ester is formed between C-terminal Gly of ubiqutin and a Cys in the E 1 active site (ATP/AMP) Ubiquitin conjugating enzymes E 2 Ub is transferred to a Cys of E 2 forming a new thiol ester Ubiquitin ligase E 3 Ub forms isopeptide bond between C-terminal Gly of Ub and ε-amino group of Lys on a target protein
Classes of E3 ligases ● N-end Rule E3 ● Skp/Cullin/F-box (SCF) ● Anaphase Promoting Complex ● HECT family ● RING finger domain
Phosphorylation of I leads to ubiquitination and degradation of the inhibitor and activation of the transcription factor.
ATP consuming process
Small Ubiquitin Related Modifier (SUMO) ● SUMO does not have the Lys-48 found in ubiquitin ● SUMO does not make multi-chain forms ● SUMO-1,2,3 are the mammalian forms ● SUMO-1, 101 amino acids, C-terminal Gly, 18% identical to ubiquitin.
MASS-SPEC Analysis Allows determination of peptide sequences and modified peptides (by P~) Mass spectrometers separate gas-phase ions according to their mass to charge ratios The analyzer uses a combination of magnetic and electric fields to move ions from the regions that they are produced to the detector.