Lecture 18: Protein processing and degradation Flashcards
Protein Export
-protein synthsis by ribosomes of rough ER
-lumen of ER like the exterior of cell
-signal peptide
Signal peptide
-usually at or near N-terminus
-positively charged N-terminal region and a core of 8-12 hydrophobic amino acids in a-helix
-followed by more polar C-terminal segement that serves as cleavage site
Protein export
- SRP recognizes signal peptide = STALLS translation
- SRP associates with SRP receptor
- ribosome associates with translocon
- translation resumes and peptides extrude into ER
-signal cleaved by peptidase
Glycoproteins
-proteins with carb chains
-many exported proteins and extracellular domains of membrane proteins
-recognition signals
glycosylation
alters stablities, solubilities, and size of proteins
N-linked glycosylation
-oligosaccharide linked to amide nitrogen of asparagine in Asn-X-Thr sequence
-oligo is synthesized as intermediate with dolichol phosphate
-then transferred to new peptide in ER (cotranslational)
tunicamycin
-inhibits step A of N-linked glycosylation
glycosidases and glycosyltransferases
may further modify oligosaccharride
O-LINKED GLYCOSYLATION
-oligo bound to hydroxyl of serine/theronine
-occurs in golgi (posttranslational)
-initiated by GalNAc-transferase
-results in heterogenous oligos
Targeting to membranes
anchoring sequence
-hydrophobic
-inserted to the membranes through TRANSLOCON
-multiple sequence = protein spans membrane multiple times
Retention of SOLUBLE proteins in ER
-C-terminal KDEL sequence
Targeting to nucleus
-made in cytosol with nuclear localization signals (clusters of amino acids)
-PKKKRKV
-KR[PAATKKAGQA]KKKK
-transported by carrier proteins to nuclear pore complexes
-needs GTPase Ran
-phosphorylation of SPS or TPT seq includes nuclear uptake
Targeting to mitochondira
-made in cytosol as PREproteins with N-terminal presequences (+ charged helices)
-recognized by mitochondrial receptor
-TOM and TIM complex
-translocation requires unfolding
-energy DEPENDENT
TOM
-translocase of outer membrane
TIM
-translocase of inner membrane
protein turnover
-constant
-regulate metabolism
-eliminate bad proteins
-break down as carbon and nitrogen sources in time of need
protein half lives
-short for regulatory
-long for housekeeping
Lyosomal protein degradation
-contain ~50 hydrolytic enzymes
-ph 5
-cathepsins
lysosomal uptake
-endocytosis for extracellular
-autophagy for intracellular (nonselective)
Protein degradation
-to amino acids
-recycled for protein synthesis or metabolized
-proteolysis
Intracellular proteolysis
-POLYUBIQUINATION marks protein for degradation
-PROTEASOMEs recognize polyubiquinated proteins
-proteins unfolded and translocated into chamber of proteasomes USING ATP
-Proteasome active site inside chamber
Ubiquitin dependent protein degradation
-highly conserved
-posttranslational mod
-E1-E3
E1
-activates ubiquitin to form thioester withits C-term
-ATP REQUIRED
E2
-transfers activated ubiquitin to substrate protein
E3
catalyzes ligation between lysine of protein and C-term of ubiquitin
-SPECIFIC for protein
ATP-dependent unfolding
- recognition of polyubiquitin chain
- Translocation of substrate chain
- Deubiquination
- Unfolding
- Proteolysis
Regulation of protein degradation
-N-end rule
-ERAD
N-end rule
-N-term of amino acid determines lifetime
-Arg 1 hour
-Val 100 hour
-part of ubiquintin degradation
-not sole regulator
ER-associated degradation (ERAD)
-misfold proteins in ER moved back to cytosol
-protein polyubiquinated during translocation
-proteasomes in cytosol degrade polyubiquinated proteins
MDM2
-regulate p53
-E3 ligase
-expressed by p53
-suppress p53 activity
-p53 is low under normal conditions
regulation of p53
-MDM2
-phosphorylation
-E6
Phosphorylation of p53 upon DNA damage
-activates p53 by blocking MDM2 binding
-supresses degradation
E6 in HPV
-brings p53 to E6AP (E3 ligase)
-induces degradation by ubiquination
-blocks apoptosis
