Protein Processing and Degradation Flashcards
Exam 2
Explain the mechanism of protein export in human cells
- Protein synthesis by membrane-bound ribosomes of the rough ER (involved in the export, plasma membrane, lysosome, endosomes, or the golgi apparatus.
- The lumen of the ER is topologically equivalent to the exterior of the cell.
Signal Peptide
* Usually at or near the N-terminus
* Positively charged N-terminal region and a core of 8-12 hydrophobic amino acids in an alpha-helix
* Followed by more polar C-terminal segment that serves as a cleavage site.
Signal Recognition particle (SRP)
1. SRP recognizes the signal peoptide, and the translation is stalled
2. SRP associates with the SRP receptor or docking protein
3. The ribosome associates with a translocon (ribosome receptor)
4. The translation resumes, and the synthesized peptides is extruded into ER.
5. The signal peptide is cleaved by a peptidase.
Identify the functions of the signal peptide, membrane anchoring sequences, the ER retention signal, the presequences, and the nuclear localization signals.
Signal peptide: brings the newly synthesized peptide to the ER
Membrane anchoring sequences:
* Membrane anchoring are inserted to the membranes through the translocon
* Multiple anchoring sequences result in the membrane proteins spanning the membrane multiple times
ER retention signal:
* C-terminal KDEL signal
* Retains teh soluble protein in the ER
Presequences:
* Synthesized in the cytosol as pre-proteins with N-terminal pre-sequences
* Presequences are recognized by a mitochondrial receptor
* Translocase of the outer membrane complex (TOM) and translocase of the inner membrane complex (TIM)
* Translocation requires protein unfolding
* Energy-dependent process
Nuclear localization signals:
* Transported by carrier proteins (importins) to nuclear pore complexes
* Required the GTPS Ran
* Phosphorylation of SPS or TPT sequence induces nuclear pore complexes
Distinguish the differences in N-linked glycosylation and O-linked glycosylation.
N-linked glycosylation
* Oligosaccharide is linked to the amide nitrogen of asparagine in Asn-X-Thr(Ser) sequence.
* Oligosaccharide is synthesized as an intermediate linked to dolichol phosphate first and then transferred to a newly synthesized pepride in ER (cotranslational)
* Step A is inhibited by the antibiotic tunicamycin
* Glycosidases and glycosyltransferases may modify further the newly transferred oligosaccharide.
O-linked glycosylation
* Oligosaccharide is bound to the hydroxyl groups of serine or threonine (no sequence specificity)
* Occurs in Golgi (posttranslational)
* Initiated by GalNAc-transferase
* Results in heterogenous oligosaccharides.
Identify the functions of E1, E3, and E3 in ubiquitination.
- E1: Activates ubiquitin to form a thioester with the C-terminus of ubiquitin. ATP is required
- E2: Transfers the activated ubiquitin to the substrate protein
- E3: Catalyzes the ligation between a lysine residue of the substrate protein and the C-terminus of ubiquitin (specific for substrate proteins)
Describe the steps necessary for protein degradation by the proteosome.
Steps:
1. Recognition of the polyubiquitin chain
2. Translocation of the substrate chain
3. Deubiquitination
4. Unfolding
5. Proteolysis
Degradation of Lysosome:
* Maintains pH 5
Substates of lysosomal protein degradation
* Extracellular substances that the cell takes up via endocytosis
* Intracellular constituents via autophagy (nonselective under normal conditions)
Explain the N-end rule and ERAD in protein degradation.
N-end rule
* The N-terminal amino acid of a protein determines its life time
* In mammalian systems, Arg-> 1 hour and Val-> 100 hr
* Part of the ubiquitin-dependent degradation system
* Not sole determinant controlling protein degradation
ER-associated degradation (ERAD)
* Misfolded proteins in ER translocated back to cytosol
* During translocon, substrate proteins are polyubiquitinated
* Proteasomes in cytosol degrade the resulting polyubiquitinated proteins
Explain the mechanism of p53 degradation by MDM2 and E6.
MDM2:
* E3 ligase for p53
* Expressed by p53
* Suppresses the activity of p53 through autoregulatory feedback
Phosphorylation of p53 upon DNA damage
* Activates p53 by blocking the binding of MDM2
* Suppresses the degradation of p53.
E6 in HPV
* Brings p53 to E6-associated protein, which is an E3 ligase
* Induces the degradation of p53 by ubiquitination
* Blocks apoptosis