Protein Degradation by the 26S Proteasome

Cecile Pickart
Department of Biochemistry
Johns Hopkins University
615 North Wolfe St.
Baltimore, MD 21205

The 26S proteasome is the enzyme principally responsible for the turnover of short-lived proteins in eukaryotic cells. As shown through genetic and chemical inhibition studies, the substrates of the proteasome include a host of important regulatory proteins: in many cases, the degradation of these proteins is an important component of their regulatory functions. The 28-subunit catalytic core of the 26S proteasome, known as the 20S proteasome, has the ability to hydrolyze small peptides with broad side chain specificity. However, the multiple proteolytic active sites of the 20S proteasome are sequestered in a cavity to which access is highly restricted. This assures that unregulated protein degradation is avoided in both cytoplasm and nucleus, but at the same time necessitates the existence of some kind of targeting mechanism. The ability of the proteasome to degrade folded proteins is contingent upon the association of the 20S proteasome with the 19S or regulatory complex, to form the 26S proteasome. Moreover, degradation of folded proteins by the 26S proteasome usually requires that the substrate be covalently ligated to ubiquitin. Ubiquitination is accomplished through the formation of an isopeptide bond between the C-terminal carboxyl group of ubiquitin, and an internal lysine residue of the substrate. Ubiquitin attachment is the primary site of specificity generation in degradation catalyzed by the proteasome, but the number and organization of the ubiquitin molecules attached to the substrate appears to represent an additional site of discrimination.

Recent studies indicate that targeting to the proteasome is usually accomplished through the ligation of a homopolymeric ubiquitin chain, rather than through the ligation of one or several single ubiquitins. The chain is assembled through the formation of isopeptide bonds between K48 and G76 of successive ubiquitins. Determining the mode by which such chains interact with the 26S proteasome provides a starting point in unraveling the mechanistic coordination of (poly)ubiquitin recognition, substrate unfolding, substrate translocation, and de-ubiquitination during proteasome catalyzed degradation. We used the crystal structure of K48-linked tetraubiquitin as a starting point to identify features of the chain that are important for recognition by the proteasome. The results of studies in which residue 8 of ubiquitin was mutated to a series of smaller resides confirm a hydrophobic contact involving a patch on the ubiquitin surface is critical for recognition of K48-linked chains by the proteasome. This conclusion is based primarily the relative potencies with which different (unanchored) mutant polyubiquitin chains inhibit the purified proteasome. A similar approach was also used to measure the relative affinities of different length chains for the 26 S proteasome (di- through octaubiquitin). The mutagenic and length dependence data support a model in which the structure of the chain creates and amplifies a minimum binding element which includes two spatially adjacent hydrophobic patches. Using a chain as the proteasomal targeting signal provides documented advantages at the levels of signal generation, recognition, and persistence. Using a chain for proteolytic targeting may also allow mono-ubiquitination to serve non-proteolytic function(s).

(Support NIH DK46984)