PROTEASOME - a protein digesting "organelle?"
                         - an enzyme complex for non-lysosomal protein degradation     

Protein degradation is essential to the cell...  
          to supply amino acids for fresh protein synthesis
          to remove excess enzymes
          to remove transcription factors (gene action) that are no longer needed.
There are two major intracellular organelles to digest damaged or unneeded proteins
                  1)  lysosomes :       for extracellular proteins from endocytosis, 
                                                receptor mediated endocytosis & phagocytosis
         and    2)  proteasomes:   for endogenous proteins (proteins synthesized within cell)
                                                 transcription factors, cell cycle cyclins, virus coded proteins,
                                                 improperly folded proteins due to translation errors (made by
                                                 faulty genes) and proteins damaged by cytosol molecules.
                                  [cystic fibrosis is due to the accelerated degradation of Cl transporter]

 Proteasomes degrade proteins to short peptides, followed by
     --> hydrolysis of these peptides via cytoplasmic exopeptidases    
                back 150                                                                   back 255





  were discovered by Alfred Goldberg (Harvard Med) & Martin Rechsteiner (Utah) in 1980's
            are large multi-enzyme complexes that digest proteins
            average human cell* holds about 20,000 to 30,000 proteasomes.
            they can lead to disease via overzealous degradation (CF)  or  neglecting to digest proteins
 Proteins digested by proteasomes include...
            90% of all abnormal, misfolded proteins
            all short-lived (2-3 hr life) regulatory proteins (ala --> transcription factors)
            also digests many longer-lived proteins of cells:
                                in all 80% to 90% of intracellular are degraded by proteasomes.
 Protein Digestion...
          begins when cells add small polypeptide (ubiquitin) to a protein to be digested
              Ubiquitin is a globular protein [76 aa] with a C-terminus that sticks into aqueous space of cytosol
                     conserved in all cell types;  is virtually identical in sequence in bacteria, yeast, or mammals

         3 enzymes of cells [ E1, E2, E3 ] add ubiquitin to proteins to be digested...

                 E1  (Ub-activating enzyme) modify Ub so that its C-terminal GLY reacts w LYS on protein
                 E2  (Ub-conjugating enzyme) attach Ub to the protein targeted for digestion
E3  (Ub-ligases) play a role in recognizing the substrate protein & linking Ub to it.

addition of uniquitin targets a protein's entry into a Proteasome complex




 STRUCTURE of PROTEASOME complex...                             
       the active proteasome has a MW of 2,400kD   [ electron micrograph   ref-1 ]
       each complex consists of a base  and  top & bottom lids,  with binding sites for
                           ubquitinized proteins & 6 ATPases to unfold proteins for entry
       it's a barrel shaped structure* made of 4 separate parts
               1.   a lid (cap) of 9 polypeptides, with binding specificity for ubiquitinzed proteins
               2.  a regulatory cap on top (& sometimes bottom) made of lid & regulator proteins
                           called PA700 (19s) is gatekeeper allowing only uniquitinized proteins in
                           made of 14 different proteins with 6 of them being ATPases
               3.  a base of 4 stacked rings (like a stack bagels ) of 7 proteins each...
                           two central
β rings with threonine protease catalytic activity sites
                                   -  2 sites with chymotrypsin activity which digests hydrophobic aa
                                   -  2 sites with trypsin activity that digests basic aa
                                   -  2 sites with caspase cleaving acidic aa
                           2 outer
α rings - with no known catalytic activity
       4.  a small base cap (a 11s piece called PA28)



 Sometimes blocking proteasome digestion of cellular proteins can help protect a cell.
           blocking protein degradation using Proteasome Inhibitors cause ubiquitin-tagged
           misfolded & damaged proteins to accumulate within cells which triggers a HEAT-SHOCK
           response thus protecting the cell from toxic agents and high temps.

 Drug companies are looking for proteasome inhibitors to bolster a cell's ability to
        1. withstand some injury protecting cells against ischemia or maintain organs after transplant
        2. modulate amount and life span of cyclins & transcription factors, etc...
        3. to treat cancers with drugs as Velcade that inhibity ubiquitin-mediated proteolysis

Some links to Proteasome Biology -
            Proteasomes  by Kimball
            Groll et al, Yeast Proteasome in Nature 1997
            Proteasome Research Products - Affiniti, Inc.

	 1.  Human proteasomes reacted with a monoclonal antibody with specificity for a subunit protein. 
	     F. Kopp et al, J. Mol. Biol., 1995, 248:264-272.