Proteases are found in both extracellular (digestive tract, blood, extracellular matrix), membrane, and and intracellular locations.; As mentioned previously, one role of intracellular proteases is to degrade "older" and chemically damaged proteins.;; One of the main proteases involved in such intracellular proteolysis is the large protein complex called the proteasome.; It consist of three large structures
one 700,000 MW 20S complex which contains 14 different subunits arranged in 4 rings of 7 subunits (2 copies of each subunit).; The rings are stacked on each other.; The outer two rings contain a subunits while the middle two contain b subunits.;; ATP-dependent cleavage of protein substrates occurs within the complex, with N terminal Ser or Thr OH groups acting as nucleophiles.;
two 19S complex which cap both ends of the 20S complex (not unlike the structure of the E. Coli chaperone complex GroEL/ES.;
Proteins destined for cleavage by the proteasome must first be chemically modified through attachment of multiple copies of the 8,500 MW protein ubiquitin, a highly conserved protein found ubiquitously in eukaryotes.; (We modeled this protein in the first lab using VMD and NAMD.); The carboxyl; group; of the C-terminal Gly residue of ubiquitin forms an amide link to the side chain amine group of Lys; residues in the protein targeted for degradation.; The resulting link is an isopeptide bond since the N terminal of the target protein is not used in the amide bond.; Three different proteins are involved in the ubiqutinylation of the target protein, including E1 (ubiquitin-activating enzyme which requires ATP), E2 (ubiquitin conjugating enzyme) and E3 (ubiquitin-protein lyase).;; Once attached, a side chain Lys of ubiquitin can form another isopeptide bond to a C-terminal carboxyl group of another ubiquitin, forming a growing ubiquitin chain on the target protein.; Proteins with 4 or more linked ubiquitins are better substrates for the proteasome.; Proteins with short half-lives (those with certain amino terminal amino acids like arginine or leucine, or enriched in Pro (P), Glu (E), Ser (S), and Thr (T) - (PEST) appear to be better targets for the ubiquitin pathway and subsequent degradation by the proteasome.
Proteasome activity is;intimately related to health and disease.; A major role of the proteasome occurs in immune recognition of self and nonself.; The immune system must be able to recognize a virally infected or tumor cell (both self cells expressing foreign or aberrant proteins) as well as foreign cells like bacteria, which can be engulfed by immune cells such as macrophages.; Proteasome involvement; occurs when viral, tumor, or bacterial proteins are degraded to short peptides, which bind intercellular major histocompatability proteins (MHC) proteins and are translocated to the cell membrane.; Peptide/MHC complexes are displayed on the cell surface and are recognized by receptors on immune cells (specifically T cells).; Self cells are not recognized by T cells since the peptides in the peptide:MHC complex are self peptides derived from normal proteins.;; The T cell receptor recognizes determinants on both the MHC protein and the presented; peptide.;
- animation of protein processing and display of peptides by MHC proteins on cell surface
- HHMI animation of the ubiquitin and the proteasome
Nonrecognition of self peptide:MHC complexes prevents the immune system from targeting normal healthy cells.; Autoimmune diseases arise when the T cell receptor recognizes presented self peptides.;
The ubiquitin/proteasome pathways have been linked to disease manifestation in many neurodegenerative diseases like Alzheimers, Huntington's disease (which involves the aberrant folding of the Huntington protein which contains an expanded poly-Glu domain), and Parkinsons.; The degradation pathway is involved in many other normal cellular functions including gene transcription and programmed cell death.