Bivalency as a principle for proteasome inhibition

Abstract

The proteasome, a multicatalytic protease, is known to degrade unfolded polypeptides with low specificity in substrate selection and cleavage pattern. This lack of welldefined substrate specificities makes the design of peptidebased highly selective inhibitors extremely difficult. However, the x-ray structure of the proteasome from Saccharomyces cerevisiae reveals a unique topography of the six active sites in the inner chamber of the protease, which lends itself to strategies of specific multivalent inhibition. Structure-derived active site separation distances were exploited for the design of homo-and heterobivalent inhibitors based on peptide aldehyde head groups and polyoxyethylene as spacer element. Polyoxyethylene was chosen as a f lexible, linear, and proteasome-resistant polymer to mimic unfolded polypeptide chains and thus to allow access to the proteolytic chamber. Spacer lengths were selected that satisfy the inter-and intra-ring distances for occupation of the active sites from the S subsites. X-ray analysis of the proteasome͞bivalent inhibitor complexes confirmed independent recognition and binding of the inhibitory head groups. Their inhibitory potencies, which are by 2 orders of magnitude enhanced, compared with pegylated monovalent inhibitors, result from the bivalent binding. The principle of multivalency, ubiquitous in nature, has been successfully applied in the past to enhance affinity and avidity of ligands in molecular recognition processes. The present study confirms its utility also for inhibition of multicatalytic protease complexes.The proteasome is a multicatalytic protease complex that is involved in intracellular protein turnover in all three kingdoms of life. The proteasome is located in both the cytosol and the nucleus and acts in the degradation of abnormal, misfolded, or improperly assembled proteins, in stress response, cell cycle control, cell differentiation, metabolic adaptation, and cellular immune response. It also is involved in many pathophysiological processes like inflammation and cancer and constitutes a promising target for drug design. In mammals the proteasomes also are responsible for the production of the bulk of antigenic peptides, which are presented via MHC class I molecules on the cell surface to cytotoxic T lymphocytes. The antiviral cytokine INF-␥ induces transcription of three additional ␤ subunits (LMP2, MECL-1, and LMP7), which can replace their constitutive homologs (␤1, ␤2, and ␤5) in newly assembled proteasomes. The resulting immuno-proteasomes show altered cleavage patterns in vitro; these are thought to be essential for the proteasomal antigen processing (1). Most of these functions are linked to an ubiquitin-and ATP-dependent protein degradation pathway involving the 26S proteasome whose core and proteolytic chamber is formed by the 20S proteasome (2-7). The eukaryotic 20S proteasome consists of seven different ␣-type and seven different ␤-type subunits, all of which have been cloned and sequenced and can be grouped by sequence homology (8)....

Interactions of Inositol 1,4,5-Trisphosphate (IP3) Receptors with Synthetic Poly(ethylene glycol)-linked Dimers of IP3 Suggest Close Spacing of the IP3-binding Sites

“…Applications of this approach to multi-subunit proteins have generally employed synthetic bivalent constructs in which two molecules of a ligand are linked by a spacer in which the length is customized to span the distance between two binding sites within a protein multimer. Successful examples include the development of selective -opioid antagonists such as norbinaltorphine (18), potent muscarinic agonists (19), and inhibitors for ␤-tryptase (20) and the proteasome (21). Bivalent ligands may show enhanced affinity and selectivity for multimeric proteins, and they can also be used to investigate the separation of ligand-binding sites.…”

Ester Bond-containing Tea Polyphenols Potently Inhibit Proteasome Activity in Vitro and in Vivo

“…There are three major proteasomal activities: chymotrypsin-like, trypsin-like, and peptidyl-glutamyl peptide hydrolyzing (PGPH) activities (16,21). The ubiquitin-proteasome system plays a critical role in the specific degradation of cellular proteins (22), and two of the proteasome functions are to allow tumor cell cycle progression and to protect tumor cells against apoptosis (23).…”

BCR-ABL Prevents c-Jun-Mediated and Proteasome-Dependent FUS (TLS) Proteolysis through a Protein Kinase CβII-Dependent Pathway

“…In mammalian cells, the 26S proteasome is a specialized multisubunit enzyme with different catalytic activities (22). It is the predominant intracellular, nuclear, and cytoplasmic (3) nonlysosomal proteolytic mechanism involved in the regulation of a broad range of processes, such as cell cycle progression, antigen presentation, and gene expression (6).…”