L10 quality control in ER (MP4)

• ER chaperones:

  • All proteins in the secretory pathway are synthesized in the ER
  • Quality control mechanisms exist
  • BiP (HSP70) in the ER needs co-chaperones to assist in folding in the cytosol
  • ERdj proteins (DNAJ co-chaperones) like ERdj3 play a role in the folding process
  • NEF co-chaperones include GRP94 (HSP90) without co-chaperones
• Thioredoxin family - PDI and ERp57:

  • Responsible for disulfide bonding in the ER
  • PDIs and ERp57s direct which cysteines should bond together to ensure proper folding
  • PDI can correct incorrect disulfide bonding during folding
• ER N-linked glycosylation:

  • A post-translational modification that occurs to all proteins in the secretory pathway
  • Calnexin and calreticulin are important proteins in this process
  • Calnexin recognizes proteins by binding the polysaccharide and giving time to fold properly
• ER misfolded protein degradation (ERAD):

  • Degradation occurs on cytosolic proteasomes
  • ERAD degrades misfolded proteins and can do regulated degradation in response to signaling
  • Proteins involved in ERAD include BIP, lectin, ERdjs, ERLIN1, ERLIN2, E3 ligase, and p97 receptor
• ERdj5 and BIP form a complex protein
• Disulfide bonds are broken by a thioredoxin DNAJ
• Adaptors bring misfolded proteins to E3 ligases
• Membrane proteins IV include lectins, chaperones, lumenal and TM adaptors
• Proteins undergo ubiquitination by E3 ubiquitin ligases in the ER membrane
• p97 ATPase helps extract substrates from the membrane
• DUBs and p97 remove the poly-ubiquitin tail from proteins
• Proteins are ubiquitinated again to be sent to the proteasome
• Folding is necessary for proteins to exit the ER to the secretory pathway
• Cystic fibrosis transmembrane regulator (CFTR) is a large transmembrane protein with the N-terminus in the cytosol
• ER stress response is also known as the unfolded protein response (UPR)
• IRE1 and PERK pathways are studied to reverse neurodegenerative diseases
• IRE1 pathway involves dimerization and phosphorylation to activate transcription of ER components
• PERK pathway phosphorylates eIF2alpha, inhibiting translation of most proteins
• ATF6 is a transcription factor that activates transcription of ER components related to ER proteas
• ER chaperones include BiP (HSP70 version in ER that need co-chaperones), GRP94 (HSP90 version in ER that doesn't need co-chaperones), and thioredoxin family (called PDI and ERp57)
• BiP has co-chaperones called ERdj and NEFs

  • another ERdj called ERdj3
  • when nascent peptide enters into ER the OST adds the glycan, and the Sec63 is the co-chaperone that recruits BiP to help with the folding without interacting with the polypeptide.
• thioredoxin family proteins (PDI and ERp57) have cysteines that allow them to direct disulfide bondings. can be done through either PDI becoming reduced (and reoxidized through Ero1 with cofactor FAD) or PDI reduced that aids rearrangement of disulfide bonding during folding (PDI breaks disulfide bonds by recreating these bonds until native state is restored)
• calnexin and calrecitulin are proteins that recognize the polysaccharide on the secretory pathway proteins during n-linked glycosylation. calnexin is always attached to the membrane due to its transmembrane alpha helix, while calrecitulin is soluble in the lumen. calnexin and calreticulin recognize a glycan with one glucose, giving the protein time to fold by itself. if the protein continues being trimmed to have a mannose exposed, it will get sent to the cytosol
• UGGT (UDP-glucose:glycoprotein glycotransferase) will identify the protein with mannoses exposed, adding another glucose to allow calnexin cycle to start on these proteins again
• EDEM (mannose binding lectins) will recognize the proteins with no mannose to let them be degraded. glucosidases, mannosidases all remove the manose from the polysaccharide
• ER-associated degradation (ERAD) degrades lumenal and transmembrane proteins (steps 1-4)

  1. Misfolded proteins are recognized by BiP with ERdj5. EDEM recognizes polysaccharides with no mannoses
  2. ERdj5 has a J domain and the thioredoxin domain that breaks disulfide bonds in substrates
  3. Adaptors bring misfolded proteins to E3 ligases, which target a retrotranslocon (a pore that allows proteins from ER to cytosol)
  4. Transmembrane E3 ligases (HRD1 and gp78) polyubiquitinate substrates and start retro-translocation
• ER-associated degradation (ERAD) degrades lumenal and transmembrane proteins (steps 5-7)

  1. p97/VCP ATPase helps pull/unfold substrates to take them out of the membrane, aiding in retrotranslocation
  2. Substrates are de-glycosylated by PNGase, then deubiquitinated via DUBs and p97 to fit into an exomer that pulls the protein out of ER
  3. Proteins get ubiquitinated again to be sent to the proteasome
• cystic fibrosis transmembrane regulator (CFTR) is a chloride channel transporter, it is a type 2 transporter mutated in cystic fibrosis. mutation is deletion of a phenylalanine (hydrophobic amino acid). hydrophobic collapse doesn’t happen in the cytosol, so it doesn’t fold properly.
• unfolded protein response (UPR) has three parallel pathways occuring when the ER has too many misfolded proteins. IRE1, PERK and ATF6 pathways all happen simultaneously
• UPR pathway: IRE1

  • IRE1 is a transmembrane protein that stays a monomer when not needed, dimerizes and phosphorylates itself when active.
  • domain in IRE1 leads XBP1 unspliced to be translated at low levels. the only mRNA spliced in the cytosol
  • IRE1 splices out intron, frameshift allows XBP1 spliced to be translated efficiently, activating transcription of chaperones, synthesized lipids, and ERAD proteins
• UPR pathway: PERK

  • PERK is kept as a monomer, phosphorylates itself and eIF2alpha. when eIF2alpha is phosphorylated, it cannot begin translation (called integrated stress response). this will effect both cytosol and ER
  • mRNA ATF4 escapes this mechanism in the cytosol, triggering expression of chaperones, ERAD components.
• UPR pathway: ATF6

  • stays a monomer but becomes cleaved in the golgi via motif exposed under heat shock. motif for ATF6 to go to the golgi is usually hidden by chaperones.
  • ATF6 once cleaved is a transcription factor, activates transcription of ER components related to ER proteases.