W7: Proteasome

• key systems: ubiquitin-proteasome system, protein unfolded response, chaperone system

• balance between protein synthesis and degradation/control; when one is out of control: problem

• homeostasis: maintain balance between protein synthesis and quality control

• unfolded proteins have high energy

• there are multiple states that are more stable

  • correctly folded protein, but also folding intermediates and potentially harmful foldings

  • chaperones enable the crossing of energy barriers between semi-folded and folded states and inhibit aggregation and misfolding

Proteasome/Ubiquitin system

Autophagy system

ATP: general energy transfer

GTP: protein synthesis, signal transduction

Chaperone system

Unfoldase/AAA ATPase:

• tunnel with (mostly) hydrophobic handles that pull the protein through a hole

• ATP hydrolysis!

  • 1 ATP -> 5-8 residues

• initiation (“Einfädeln”) is the difficult part

Amino acids in the pore-loop:

• mainly hydrophobic, often aromatic (hydrophobic “arms” of the unfoldase)

• Phe, Val, Tyr

• grab hydrophobic/aromatic amino acids of the peptide

• interactions mustn’t be too strong either (not only hydrophobic interactions)

ATPases Associated with diverse cellular Activities

• Protein familiy with conserved module

• use ATP hydrolysis for conformational changes which exerts mechanical force onto macromolecules

• diverse roles in the cell, for example involved in:

  • DNA replication

  • protein degradation

  • membrane fusion

  • peroxysome biogenesis

  • …

Abundant AAA ATPase family proteins:

• proteasome

• NSF (snare disassembly)

• Orc1 (DNA replication)

19S: regulatory particle

20S: catalytic chamber

• alpha: no catalytic activity, just control of entry of substrtes

• beta: catalytic core, 3 catalytic subunits (caspase-, trypsine- and chemotrypsine-like activities)

Number of ATPase subunits per proteasome ATPase motor: 6

caspase-like (cleavage after aspartate)

trypsine-like (cleavage after basic residues: His, Lys, Arg)

chymotrypsine-like (cleavage after large hydrophobic residues: Tyr, Phe, …)

Proteins are not cut into single amino acids.

Proteins are cleaved at protease cleaving sites.

With smaller peptides, affinity to ribosome decreases and not all sites are cleaved any more.

Product of proteasome reactions: Shorter peptides.

The shorter peptides are then cut down into single amino acids by tripeptidyl peptidase (-> 2-4 aa long peptides) and aminopeptidase (-> single aa).

Cancer therapy (proteasome inhibitors are very toxic)

Problem: cancer cells can get resistant by mutation of the proteasome.

Activity can be adjusted by exchanging catalytic subunits.

Example: Immunoproteasome

• is assembled when a virus infects the cell

• cuts viral proteins into smaller peptides

  • degradation of potentially harmful proteins

  • small peptides can be used in immune recognition (as antigens)