Lecture 8: Post-translational Regulation- Regulating Protein Functiono Post-translational regulation can range from: Simple, covalent addition/removal of functional groups like phosphorylation andglycosylation Degradation of specific proteins, like regulators (e.g. Ubiquitin) Stabilize proteins – SUMO Large changes in cellular landscape architecture through autophagy- Ubiquitin and the 26S Proteasome Systemo Reasons for degradation Proteins made, folded, assembled wrong- Caused by stress, chaperones not working- Stabilization: SUMO Controlled destruction for purpose of change/maintenance- Proteasome: selective removal of regulatory proteins- Bulk removal/recycling  autophagyo 26S Proteasome Proteasome is essential complex- comprised of 20S catalytic core and 19S regulatory particle- Ring of AAA-ATPase hydrolyzes ATPo Energy produced used to unfold targets and feed them into 20S core- Alpha dictates aperture of 20S- Beta rings have proteolytic active sites  cleave hydrophobic, acidic, and basic residues- Ub-binding proteins associate with 19S- De-ubiquitinating (DUBs) associate with 19So Ubiquitin Proteasomal degradation is facilitated by Ubiquitin Mode/number/location determines the outcome Conjugation Pathway Three distinct steps:- Activation of ubiquitin activating enzyme (E1) by addition of ubiquitin- Transfer of ubiquitin to a cysteine residue in a ubiquitin-conjugating enzyme (E2)- Formation of covalent bond between glycine 76 of ubiquitin bound to E2and amino group of lysine residue in the target protein (catalyzed by ubiquitin-protein ligase (E3) Mutation in an E3 Ub-ligase stabilize its target- Breast Cancer susceptibility proteino Mutation causes genomic instability and DNA lesionso Small Ubiquitin-like Modifier (SUMO) Plays role in protein stability and complex formation Transcriptional regulation; modification of histones, nuclear proteins Localized to transcriptional start sites Stress responses- Autophagyo Bulk degradation of cytosolic componentso Fuses with lysosome to deposit contents for hydrolysis and recyclingo Autophagy conjugation pathway Begins with formation of flattened double-membrane that envelops a region of cytosol or an entire organelle  results in autophagosome Outer membrane of autophagosome fuses with lysosome; lipases and proteases within the lysosome degrade the autophagosome and its contents o Evidence that autophagosome is initially derived from a fragment of the golgio Animals Acid hydrolases: nucleases, proteases, glycosidases, lipases, phosphatases, sulfatases, phospholipaseso Plants Helps plants resist starvation  recycle what is not essential to make what is required to survive  degrades dysfunctional organelles Regulates aging and cell deatho Disease associated with autophagy Neurodegeneration, cancer, aging, liver/heart disease, infection and immunity,