BIO 344 1st Edition Lecture 22 Outline of Last Lecture I. mRNA degradationa. 5’ capb. Premature termination codonc. Exon-exon junctionII. Translation and degradation are opposing pathwaysa. Xrn1III. NMDa. EJCIV. Treating Disease due to PTCsOutline of Current Lecture I. Protein Turnovera. N-end ruleb. Ubiquitini. Degradation signalc. E3d. Proteosomee. P53i. Mdm2 and HAUSPCurrent LectureProtein Turnover- Proteosome= degrades proteinso Ubiquitin and the N-end pathwayo P53 regulation- Defective proteins are rapidly degraded- Certain amino acids have longer or shorter half lives depending on residueo Half life correlates to N-terminal residue First amino acid—dictates stability, cis-actingThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.- Degradation is not just for misfoldso Chaperones help in folding proteins properly as peptide chain leaves the ribosome If too much time with chaperone and refolding degradationo Properly folded proteins that are no longer needed, need to be recycled- N-end Ruleo N- terminus residue—1st amino acid out of the ribosome Usually dictates stability which corresponds to half life Size of the side chain is an indicator- Smaller side chains are more stable and have longer half lives- Shorter side chains are unstable and targeted for degradation (shorter half lives) Trans-acting factor recognizes size when sorting proteins- Ubiquitino Highly conserved, very slow evolutionary rate implies its importance and that its structure is sensitive and crucial to its functiono Covalently attaches to protein targeted for degradation A fusion protein Attached to lysine of target protein- Linkage at its C-terminuso Isopeptide bond at lysine of target with carboxylate of ubiquitin- Degradation signal for ubiquitin= large N terminus side chain or exposed hydrophobic residues (typically situated at the core of properly folded proteins)o E1, E2, E3 enzymes covalently link ubiquitin to targeto E1= activates Ub, ATP-dependent E1-S-C(=O)-Oo E2= adapter for E1 and E3o E3= substrate recognition, specifico E2 and E3 receive Ub from E1 and E1 leaves Can now recruit the target to Ub Can be monoubiquitinated, one Ub on one lysine Polyubiquitinated, several Ub on one lysine Multiubiquitinated, one Ub on each of several lysineso Recruiting substrate to E3 Constitutive= N-end rule recognition Modification= phosphorylate post-translationally to regulate interaction with E3 Assisucate with anciallary protein= additional recognition with another protein- Proteosome= hydrolyzes Ub proteins to degradeo Large enzyme/protein 26S= whole 19s= regulatory cap control access to core (alpha) 20s= core, catalytic (beta)o Active beta catalytic core is situated inward to not destroy cellular proteins When proteosome is assembled, initially longer and only active when cleaved Binding of cap opens the channel to the coreo 11s cap= variant of proteosome that possess peptides for antigen presentation for antibody production immunity- Monoubiquitination and multio Endocytic pathway, regulate sorting, DNA repair, nuclear export, histone code- Polyubiquitination K63o DNA repair, endocytosis activation of protein kinases- Polyubiquitination K48o Proteosomal degradation- Diseases—problems with protein degradation or accumulation of misfoldso Parkinson’s—improper E3, accumulation of proteino Anglemano HPV- P53o Tumor suppressor, stops cell cycle progression when DNA mutationso Cell cycle arrest, apoptosis G1/S S-cdk regulationo P53 mutation often leads to cancer Causes an incorporation of mutations which can cause aneuploidy or an accumulation of mutationso P53 is a transcription factor that activates several gene pathwayso P53 activation occurs through stabilization of protein Activate by stabilizing it (normally unstable) Constitutively degraded by Mdm2o Mdm2= ubiquitin ligase (E3)o P53 phosphorylated post translationally to prevent Mdm2 binding Stabilizes/activateso P53 targeted to proteosome by E3 Labeling with phosphate kicks off E3 Mutation of phosphorylated sites does not prevent stabilization—must beanother additional factor- HAUSP= increasing p53 decreases ubiquitin p53 (removing Ub)- HAUSP and Mdm2 (antagonistic)= HAUSP wins out as HAUSP increases more and more p53o Balance between affects p53
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