L6 ubiquitin proteasome system (PF4)

• The ubiquitin proteasome system is crucial for maintaining a balance between the amount of proteins degraded and produced
• Ubiquitin, a protein, marks other proteins for degradation when covalently linked to lysine side chains
• For a protein to be degraded by the proteasome, it needs to be poly-ubiquitinated by three enzymes: E1, E2, and E3
• The ubiquitin proteasome cycle involves E1 recognizing ubiquitin in the cytosol, transferring it to E2, and then E3 transferring the ubiquitin to the substrate to create a poly-ubiquitin tail
• There are different types of ubiquitin ligases: E1, E2, and E3, with E3 being substrate-specific and providing the specificity of which substrate is going to be degraded
• Lysine plays a crucial role in the ubiquitin system, with lysine 48 attached to ubiquitin signaling a protein to be degraded
• The proteasome, a large molecular machine, degrades proteins into amino acids and recognizes the polyubiquitin tail for degradation
• The UPS client proteins include misfolded proteins, proteins degraded in response to a signal, and those with short half-lives
• Quality control degradation of misfolded proteins involves E3 ligases recognizing misfolded proteins and targeting them for degradation
• The N-end rule targets proteins without methionine as their first amino acid for degradation, recognizing proteins with short half-lives
• Regulated degradation by SCF E3 involves phosphorylation of a protein, recognition by the SCF E3 complex, and subsequent degradation
• The proteasomal degradation cycle involves a balance between proteins created and degraded, with E1, E2, and E3 enzymes playing crucial roles
• the ubiquitin proteasome system is the major mechanism for the cytosol (but this system is also able to degrade proteins in the ER, because proteins get kicked out of ER and into the cytosol)
• Ubiquitin proteasome cycle:

  • E1 recognizes ubiquitin in the cytosol, binds to it, and takes it to E2
  • E2 transfers the ubiquitin to the substrate
  • E3 is the ligase (scaffold protein) with one domain recognizing E2 and the other domain recognizing the substrate itself, creating a tail
  • With only one ubiquitin, this can be considered a PTM to the protein
  • Once a chain is added, two things can occur:

  1. The ubiquitin is removed from the protein by deubiquitinating proteins

  2. The ubiquitin tail is recognized by ubiquitin binding proteins, and the protein is taken for degradation; the ubiquitins are cut out and recycled in the cytosol
• E1 don't require specificity, 12 genes encode for E1, E1 attaches a Ub to itself via a cys side chain, creating a thioester bond, transfers Ub to the E2
• E2

  • we have 50 genes, so they need more specificity
  • also recieves the Ub via a thioester bond it has with a Cysteine
• E3

  • there are 600 genes, because there are many different E3s. these are substrate specific
  • already bound to substrate, another domain recognizes E2 that is ubiquitinalted
• • transfer of substrate for E3 can be direct or indirect
  • direct: undertake by E3 ligase that belongs to the ring family (most E3 belong to ring family) E3 transfers ubiquitin directly to the substrate. E3 is never ub itself, it puts the E2 with the substrate so the ubiquitin can be transferred.
  • indirect: undertake by E3 ligase that belongs to the head family (not as many E3). E2 transfers ubiquitin to the E3 (through a cysteine), then E3 transfers ubiquitin to the substrate.
  • at the end, the substrate is poly-ubiquinited
• carboxyl end of the ubiquitin is bound to the amino group of the lysine (forms an isopeptide bond). what you are leaving at the end is the n-terminus of the ubiquitin. meaning you get a protein that has 2 n-terminus and 1 c-terminus.
• ubis have many different lysines! the lysine 48 attached to ubiquitin is the signal of a protein to be degraded. lysine 63 and 11 signal for other things.
• the degradation of a substrate is controlled by selectivity of E3 ligase, not of the proteasome
• the proteins to be degraded by the ubiquitin proteasome system are: misfolded proteins, short-half life proteins, proteins signaled to be degraded
• degradation of misfolded proteins:

  • this E3 ligase recognizes
  • TPRs have a domain to recognize HSC70 or HSP90, but they have another domain that binds to E3 ligase. this domain brings the E2 that is ubiquitinaltyed
  • the chaperones are always trying to save proteins because protein synthesis is very expensive.
  • by action of the HSP70 or HSP90, the protein can get refolded. however, if this has been done over and over again, then the CHIP and the unfolded substrate can bind, which would lead that protein to be ubiqutuinlyated and be degraded
• n-end rule: a way to recognize proteins with short half life. methionine gets removed as first amino acid, if the second amino acid is arg, lys, his, phe, trp, tyr, leu, or Ile, then there are E3 ligases that will recognize these amino acids and degrade them. if these are the second amino acid, then they are a short lived protein
• n-end rule: if the second amino acid is aspartic acid or glutamic acid, the cell will add arginine to the n-terminus making arg the first amino acid. asparagine and glutamine undergo deamination to make them aspartic acid or glutamic acid, changing them to arginine, making them targeted by n-end rule
• regulated degradation: done by SCF-E3 complex. F-box brings the substrate to make E2 inc lose proximity.

  • phosphorylation by kinase is used as a signal for degradation
  • cullin is going to bind to the ubiquitinalted E2. then the F-box will bind to the substrate. there is a pocket of the SCF E3 that only allows the substrate that is phosphorylated to connect, allowing E2 to add ubiquitin
• the proteasome has a cap that recognizes the polyubiquitin chain. the core has the proteases
• • the 20s core has 4 rings. two on the outside and two on the inside.
  • those of the exterior have 7 alpha units. bound to the 19s cap
  • those of the interior have 7 beta units. 3 of those beta units per ring have protease activity. so 6 total proteins will degrade the substrate.
  • will cut where they identify basic, acidic or hydrophobic amino acids. if they are small polypeptides, they will be degraded by peptidases in the cytosol.
  • if a polypeptide only has polar residues, the peptides will diffuse out.
• • the 19s cap
  • the base of the cap has 6 AAA-family ATPase subunits, allows for the substrate to unfold before entering the chamber or the core.
  • the lid of the cap has non-ATPase subunits. has the receptors for the ubiquitin tail, and it has deubiquitinases (DUB) which will take off the poly-ubiquitin tail
  • ub receptors recognize the poly-ubiquitin tail. the receptors only recognize the tail only if it is on lysine 48. this protects against premature ubiquitin degradation.
• the 19s cap has two different ub receptors:

  • has intrinsic receptors → ones in the cap, there are two of them the RPN10 and the RPN13, recognize the poly ubiquitin tail.
  • extrinsic receptors → recognize the polyubqituinated protein. has the domain hat recognizes the tail (UBA - associated domain) and another domain that recognizes the cap