Protein Folding and Processing The classic principle of protein folding is that all the information required for a protein to adopt the correct three-dimensional conformation is provided by its amino acid sequence. Molecular chaperones are proteins that facilitate the folding of other proteins. Two specific families of chaperone proteins act in a general pathway of protein folding in both prokaryotic and eukaryotic cells – Heat shock proteins and Chaperonins. Unfolded polypeptide chains are shielded from the cytosol within the chamber of the chaperonin.
Protein Folding and Processing. The classic principle of protein folding is that all the information required for a protein to adopt the correct three-dimensional conformation is provided by its amino acid sequence. - PowerPoint PPT Presentation
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Protein Folding and Processing The classic principle of protein folding is that all the information required for a
protein to adopt the correct three-dimensional conformation is provided by its amino acid sequence.
Molecular chaperones are proteins that facilitate the folding of other proteins. Two specific families of chaperone proteins act in a general pathway of protein
folding in both prokaryotic and eukaryotic cells – Heat shock proteins and Chaperonins.
Unfolded polypeptide chains are shielded from the cytosol within the chamber of the chaperonin.
Action of chaperones during translation and Transport chains that are still being
translated on ribosomes, thereby preventing incorrect folding or aggregation of the amino-terminal portion of the polypeptide before synthesis of the chain is finished.
• Chaperones also stabilize unfolded polypeptide chains during their transport into subcellular organelles.
The role of N-linked glycosylation in ER protein folding.
3
The unfolded protein response in yeast
The export and degradation of misfolded ER proteins
Protein translocation
ENDOSITOSis
Protein folding in the cell
Basics- cell compartments, molecular crowding: cytosol, ER, etc.
Folding on the ribosome- co-translational protein folding
Molecular chaperones- concepts, introduction- intramolecular chaperones- chemical chaperones- protein chaperones
Folding in vitro vs. in vivo
folding by dilutionin buffer
protein denaturedin a chaotrope
foldedprotein
in vitro in vivo
folding
foldedprotein
Problem: non-native proteins• non-native proteins expose hydrophobic residues that are
normally buried within the ‘core’ of the protein
• these hydrophobic amino acids have a strong tendency to interact with other hydrophobic (apolar) residues
• The UPR occurs when proteins are misfolded in the endoplasmic reticulum (ER).
• Reducing agents, such as DTT, interfere with disulfide bond formation while drugs can interfere with glycosylation; both agents cause proteins to misfold in the ER thus triggering the UPR.
• The product of the ire-1 gene is the sensor of misfolded proteins and when activated removes an intron from the pre mRNA from the xbp-1 gene.
• Active xbp-1 protein (from spliced mRNA) activates the genes that code for ER chaperones, such as hsp-4.
Hsp4 (grp78)grp170
PROTEIN TURNOVER AND AMINO ACID CATABOLISM
Degradation of proteins
1) dietary proteins- amino acids- pepsin in stomach- pancreatic proteases- aminopeptidase N- other peptidases
2) endogenous proteins- protein turnover: synthesis, degradation, resynthesis- damaged proteins- half-lives of proteins: depend on amino-terminal residues
Cellular Protein Degradation• Lysosomal
• Nonspecific• Endocytosis• Foreign proteins• Energy favorable to degrade proteins
• Non-lysosomal• Specificity, requires ATP• Conditions of stress• Ubiquitin-proteosomal pathway• 26S proteosome• Role in cellular processes/signaling
Protein turnover; selective degradation/cleavage Individual cellular proteins turn over (are degraded and re-synthesized) at different rates. E.g., half-lives of selected enzymes of rat liver cells range from 0.2 to 150 hours. N-end rule: On average, a protein's half-life correlates with its N-terminal residue. Proteins with N-terminal Met, Ser, Ala, Thr, Val, or
Gly have half lives greater than 20 hours. Proteins with N-terminal Phe, Leu, Asp, Lys, or Arg
have half lives of 3 min or less.PEST proteins having domains rich in Pro (P), Glu (E), Ser (S), Thr (T), are more rapidly degraded than other proteins.
Ubiquitinylation – Proteosome DegradationE3 determines protein substrate
8.42 The ubiquitin-proteasome pathway
Ubiquitination1) ubiquitin- a 8.5 kd protein (76 residues)- formation of an isopeptide bond with ε-amino group of lysine of the proteins - a tag for destruction - polyubiquitin: a strong signal for degradation 2) enzymes for ubiquitination- E1 (ubiquitin-activating enzyme) - E2 (ubiquitin-conjugating enzyme)- E3 (ubiquitin-protein ligase)- variation: E3 > E2 > E1: more finely tuned substrate discrimination- HPV (human papilloma virus) activates a specific E3 enzyme: tumor suppressor protein p53
Regulation of ubiquitination: Some proteins regulate or facilitate ubiquitin conjugation. Regulation by phosphorylation of some target proteins has been observed. E.g., phosphorylation of PEST domains activates ubiquitination of proteins rich in the PEST amino acids. Glycosylation of some PEST proteins with GlcNAc has the opposite effect, prolonging half-life of these proteins.