Wolfgang Heinemeyer

The Active Sites of the Eukaryotic 20 S Proteasome and Their Involvement in Subunit Precursor Processing

The 26 S proteasome is the central protease involved in ubiquitin-mediated protein degradation and fulfills vital regulatory functions in eukaryotes. The proteolytic core of the complex is the 20 S proteasome, a cylindrical particle with two outer rings each made of 7 different α-type subunits and two inner rings made of 7 different β-type subunits. In the archaebacterial 20 S proteasome ancestor proteolytically active sites reside in the 14 uniform β-subunits. Their N-terminal threonine residues, released by precursor processing, perform the nucleophilic attack for peptide bond hydrolysis. By directed mutational analysis of 20 S proteasomal β-type proteins of Saccharomyces cerevisiae, we identified three active site-carrying subunits responsible for different peptidolytic activities as follows: Pre3 for post-glutamyl hydrolyzing, Pup1 for trypsin-like, and Pre2 for chymotrypsin-like activity. Double mutants harboring only trypsin-like or chymotrypsin-like activity were viable. Mutation of two potentially active site threonine residues in the Pre4 subunit excluded its catalytic involvement in any of the three peptidase activities. The generation of different, incompletely processed forms of the Pre4 precursor in active site mutants suggested that maturation of non-active proteasomal β-type subunits is exerted by active subunits and occurs in the fully assembled particle. Thistrans-acting proteolytic activity might also account for processing intermediates of the active site mutated Pre2 subunit, which was unable to undergo autocatalytic maturation.

Immuno- and Constitutive Proteasome Crystal Structures Reveal Differences in Substrate and Inhibitor Specificity

Constitutive proteasomes and immunoproteasomes shape the peptide repertoire presented by major histocompatibility complex class I (MHC-I) molecules by harboring different sets of catalytically active subunits. Here, we present the crystal structures of constitutive proteasomes and immunoproteasomes from mouse in the presence and absence of the epoxyketone inhibitor PR-957 (ONX 0914) at 2.9 Å resolution. Based on our X-ray data, we propose a unique catalytic feature for the immunoproteasome subunit β5i/LMP7. Comparison of ligand-free and ligand-bound proteasomes reveals conformational changes in the S1 pocket of β5c/X but not β5i, thereby explaining the selectivity of PR-957 for β5i. Time-resolved structures of yeast proteasome:PR-957 complexes indicate that ligand docking to the active site occurs only via the reactive head group and the P1 side chain. Together, our results support structure-guided design of inhibitory lead structures selective for immunoproteasomes that are linked to cytokine production and diseases like cancer and autoimmune disorders.

Contribution of Proteasomal β-Subunits to the Cleavage of Peptide Substrates Analyzed with Yeast Mutants

Proteasomes generate peptides that can be presented by major histocompatibility complex (MHC) class I molecules in vertebrate cells. Using yeast 20 S proteasomes carrying different inactivated β-subunits, we investigated the specificities and contributions of the different β-subunits to the degradation of polypeptide substrates containing MHC class I ligands and addressed the question of additional proteolytically active sites apart from the active β-subunits. We found a clear correlation between the contribution of the different subunits to the cleavage of fluorogenic and long peptide substrates, with β5/Pre2 cleaving after hydrophobic, β2/Pup1 after basic, and β1/Pre3 after acidic residues, but with the exception that β2/Pup1 and β1/Pre3 can also cleave after some hydrophobic residues. All proteolytic activities including the “branched chain amino acid-preferring” component are associated with β5/Pre2, β1/Pre3, or β2/Pup1, arguing against additional proteolytic sites. Because of the high homology between yeast and mammalian 20 S proteasomes in sequence and subunit topology and the conservation of cleavage specificity between mammalian and yeast proteasomes, our results can be expected to also describe most of the proteolytic activity of mammalian 20 S proteasomes leading to the generation of MHC class I ligands.

The proteasome/multicatalytic—multifunctional proteinase In vivo function in the ubiquitin-dependent N-end rule pathway of protein degradation in eukaryotes

Proteinase yscE, the proteasome/multicatalytic—multifunctional proteinase of yeast had been shown to function in stress response and in the degradation of ubiquitinated proteins [(1991) EMBO J. 10, 555–562]. A well‐defined set of proteins degraded via ubiquitin‐mediated proteolysis are the substrates of the N‐end rule pathway [(1986) Science 234, 179–186; (1989) Science 243, 1576–1583]. We show that mutants defective in the chymotryptic activity of proteinase yscE fail to degrade substrates of the N‐end rule pathway. This gives further proof of the proteasome being a central catalyst in ubiquitin‐mediated proteolysis.

Cic1, an adaptor protein specifically linking the 26S proteasome to its substrate, the SCF component Cdc4

In eukaryotes, the ubiquitin–proteasome system plays a major role in selective protein breakdown for cellular regulation. Here we report the discovery of a new essential component of this degradation machinery. We found the Saccharomyces cerevisiae protein Cic1 attached to 26S proteasomes playing a crucial role in substrate specificity for proteasomal destruction. Whereas degradation of short‐lived test proteins is not affected, cic1 mutants stabilize the F‐box proteins Cdc4 and Grr1, substrate recognition subunits of the SCF complex. Cic1 interacts in vitro and in vivo with Cdc4, suggesting a function as a new kind of substrate recruiting factor or adaptor associated with the proteasome.

A unified mechanism for proteolysis and autocatalytic activation in the 20S proteasome.

Biogenesis of the 20S proteasome is tightly regulated. The N-terminal propeptides protecting the active-site threonines are autocatalytically released only on completion of assembly. However, the trigger for the self-activation and the reason for the strict conservation of threonine as the active site nucleophile remain enigmatic. Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad. Substitution of Thr1 by Cys disrupts the interaction with Lys33 and inactivates the proteasome. Although a Thr1Ser mutant is active, it is less efficient compared with wild type because of the unfavourable orientation of Ser1 towards incoming substrates. This work provides insights into the basic mechanism of proteolysis and propeptide autolysis, as well as the evolutionary pressures that drove the proteasome to become a threonine protease.

Studies on the yeast proteasome uncover its basic structural features and multiple in vivo functions.

Proteasomes are large multicatalytic protease complexes found in the cytoplasm and nucleus of all eukaryotic cells. 20S proteasomes are cylindrically shaped particles composed of a set of different subunits arranged in a stack of 4 rings with 7-fold symmetry. In yeast 14 different genes are known, which are proposed to code for the complete set of 20S proteasomal subunits. They can be divided in 7 alpha- and 7 beta-type subunits. 26S proteasomes are even larger proteinase complexes which contain the 20S proteasome as the functional proteolytic core. They degrade ubiquitinylated proteins in vitro. Several yeast 26S proteasome subunits have been characterized as members of a novel ATPase family. Studies with yeast 20S and 26S proteasome mutants uncovered the function of proteasomes in stress-dependent and ubiquitin-mediated proteolytic pathways. Proteasomes are important for cellular regulation, cell differentiation, adaptation to environmental changes and are involved in cell cycle control.

The Proteasome and Protein Degradation in Yeast

In 1984 a high molecular mass multisubunit protease complex was isolated from Saccharomyces cerevisiae [Achstetter et al. 1984] which proved to be the yeast homologue of the 20S proteasome complexes found in all eukaryotic cells [Kleinschmidt et al. 1988]. The yeast 20S proteasome is able to cleave chromo- and fluorogenic peptides at the carboxyterminus of hydrophobic, basic or acidic amino acids (chymotrypsin-like-, trypsin-like- and peptidyl-glutamyl-peptide hydrolyzing activity, respectively) [Heinemeyer et al. 1991]. The yeast 20S proteasome is composed of different subunits, showing a set of protein bands in the SDS-PAGE with molecular masses ranging from 20 to 35 kDa. They can be separated into 14 protein spots after two-dimensional gel electrophoresis [Heinemeyer et al. 1991]. Genes named Y7, Y13, PRS1 and PRS2 (independendly cloned as Y8 and SCL1) were cloned and sequenced on the basis of protein sequences of 20S proteasome subunits, genes named PRS3, PUP1, PUP2 and PUP3 were sequenced by chance [for summary see Hilt et al. 1993b]. We cloned the s-type genes PREI, PRE2, PRE3 and PRE4 by complementation of mutants defective in the chymotrypsin-like- (prel and pre2 mutants) or the PGPH-activity (pre3 and pre4 mutants) of the proteasome [Heinemeyer et al. 1991, Heinemeyer et al. 1993, Hilt et al. 1993a, Enenkel et al. 1994]. Additionally we cloned two α-type genes PRE5 and PRE6 using peptide sequences derived from purified proteasome subunits, extending the number of yeast 20S proteasome subunit genes to 14 [Heinemeyer et al. 1994].

Interactions of the natural product kendomycin and the 20S proteasome.

Natural products are a valuable source for novel lead structures in drug discovery, but for the majority of isolated bioactive compounds, the cellular targets are unknown. The structurally unique ansa-polyketide kendomycin (KM) was reported to exert its potent cytotoxic effects via impairment of the ubiquitin proteasome system, but the exact mode of action remained unclear. Here, we present a systematic biochemical characterization of KM-proteasome interactions in vitro and in vivo, including complex structures of wild type and mutant yeast 20S proteasome with KM. Our results provide evidence for a polypharmacological mode of action for KMs cytotoxic effect on cancer cells.

Structural Elucidation of a Nonpeptidic Inhibitor Specific for the Human Immunoproteasome.

Selective inhibition of the immunoproteasome is a promising approach towards the development of immunomodulatory drugs. Recently, a class of substituted thiazole compounds that combine a nonpeptidic scaffold with the absence of an electrophile was reported in a patent. Here, we investigated the mode of action of the lead compound by using a sophisticated chimeric yeast model of the human immunoproteasome for structural studies. The inhibitor adopts a unique orientation perpendicular to the β5i substrate‐binding channel. Distinct interactions between the inhibitor and the subpockets of the human immunoproteasome account for its isotype selectivity.