Martin Rechsteiner

PEST sequences and regulation by proteolysis

In 1986, we proposed that polypeptide sequences enriched in proline (P), glutamic acid (E), serine (S) and threonine (T) target proteins for rapid destruction. For much of the past decade there were only sporadic experimental tests of the hypothesis. This situation changed markedly during the past two years with a number of papers providing strong evidence that PEST regions do, in fact, serve as proteolytic signals. Here, we briefly review the properties of PEST regions and some interesting examples of the conditional nature of such signals. Most of the article, however, focuses on recent experimental support for the hypothesis and on mechanisms responsible for the rapid degradation of proteins that contain PEST regions.

Recognition of the polyubiquitin proteolytic signal

Polyubiquitin chains linked through Lys48 are the principal signal for targeting substrates to the 26S proteasome. Through studies of structurally defined, polyubiquitylated model substrates, we show that tetraubiquitin is the minimum signal for efficient proteasomal targeting. The mechanism of targeting involves a simple increase in substrate affinity that is brought about by autonomous binding of the polyubiquitin chain. Assigning the proteasomal signaling function to a specific polymeric unit explains how a single ubiquitin can act as a functionally distinct signal, for example in endocytosis. The properties of the substrates studied here implicate substrate unfolding as a kinetically dominant step in the proteolysis of properly folded proteins, and suggest that extraproteasomal chaperones are required for efficient degradation of certain proteasome substrates.

Characterization of Two Polyubiquitin Binding Sites in the 26 S Protease Subunit 5a

Ubiquitylated proteins are degraded by the 26 S protease, an enzyme complex that contains 30 or more unique subunits. One of these proteins, subunit 5a (S5a), has been shown to bind ubiquitin-lysozyme conjugates and free polyubiquitin chains. Using deletional analysis, we have identified in the carboxyl-terminal half of human S5a, two independent polyubiquitin binding sites whose sequences are highly conserved among higher eukaryotic S5a homologs. The sites are approximately 30-amino acids long and are separated by 50 intervening residues. When expressed as small fragments or when present in full-length S5a molecules, the sites differ at least 10-fold in their apparent affinity for polyubiquitin chains. Each binding site contains 5 hydrophobic residues that form an alternating pattern of large and small side chains, e.g. Leu-Ala-Leu-Ala-Leu, and this pattern is essential for binding ubiquitin chains. Based on the importance of the alternating hydrophobic residues in the binding sites and previous studies showing that a hydrophobic patch on the surface of ubiquitin is essential for proteolytic targeting, we propose a model for molecular recognition of polyubiquitin chains by S5a.

Introduction of macromolecules into cultured mammalian cells by osmotic lysis of pinocytic vesicles

We have developed a new procedure for introducing macromolecules into cultured mammalian cells based on osmotic lysis of pinocytic vesicles. Cells are first incubated in culture medium containing 0.5 M sucrose, 10% polyethylene glycol 1000 and the macromolecule to be transferred. Cells are then placed in medium diluted with 0.66 parts water. Most pinocytic vesicles formed in the presence of sucrose burst in hypotonic medium, thereby releasing the enclosed macromolecule. L929 cells remain fully viable after a single hypertonic sucrose treatment, and a majority survives four successive rounds of osmotic lysis. This procedure, termed osmotic lysis of pinosomes, has been used to transfer substantial amounts of horseradish peroxidase, antiricin antibodies and dextran 70,000 into the cytosol of L929 cells. Direct comparison of the degree of ricin resistance conferred by transfer of antiricin antibodies revealed pinosome lysis to be equal, if not superior, to injection mediated by red blood cells.

PA200, a nuclear proteasome activator involved in DNA repair

We have identified a novel 200 kDa nuclear protein that activates the proteasome. The protein, which we call PA200, has been purified to homogeneity from bovine testis and has been shown to activate proteasomal hydrolysis of peptides, but not proteins. Following γ‐irradiation of HeLa cells the uniform nuclear distribution of PA200 changes to a strikingly punctate pattern, a behavior characteristic of many DNA repair proteins. Homologs of PA200 are present in worms, plants and yeast. Others have shown that mutation of yeast PA200 results in hypersensitivity to bleomycin, and exposure of yeast to DNA damaging agents induces the PA200 message. Taken together, these findings implicate PA200 in DNA repair, possibly by recruiting proteasomes to double strand breaks.

Natural substrates of the Ubiquitin proteolytic pathway

Martin Rechsteiner Department of Biochemistry University of Utah School of Medicine Salt Lake City, Utah 84112 To many, control of gene expression will conjure up the synthesis of new DNA-binding proteins. Yet, almost two decades ago, Schimke (1973) pointed out that rapid prote- olysis would be central to certain regulatory mechanisms. He deduced that the time required to alter levels of a regu- latory protein is tightly coupled to the protein’s half-life. Rapid changes in concentration necessitate short-lived proteins, and a growing list of rapidly degraded transcrip- tion factors and oncoproteins confirms Schimke’s analy- sis. However, it is not transcription per se, but rather a striking connection between proteolysis and control of the cell cycle that has sparked the recent interest in protein degradation. Mutations that inactivate components of the ubiquitin (Ub) proteolytic pathway (see below), such as cdc34 in budding yeast (Goebl et al., 1988) and ts85 in mouse (Finley et al., 1984) arrest cells at specific stages in the mitotic cycle. These observations, coupled with the demonstration that a single protein, cyclin B, must be de- graded for cells to exit mitosis (Murray et al., 1989) ac- count for the growing interest in protein degradation. Despite increased research in the area of intracellular proteolysis, a number of key questions remain unan- swered. For example, how many pathways do cells pos- sess for destroying their constituent proteins-5,50,500? Is an individual protein a substrate for several degradative pathways, or is its level typically controlled by a single pathway? In general, are proteins modified before being degraded? What features of a protein determine its aver- age lifetime within a cell? Although answering these questions will take us well into the future, recent studies on Ub-mediated proteolysis promise that some answers will be forthcoming. Ub is a highly conserved, 78 residue protein found only in eukary- otes. The macromolecule is unique in its ability to become reversibly cross-linked to other cellular proteins. Forma- tion of isopeptide bonds between the carboxyl terminus of Ub and lysine amino groups on acceptor proteins is associated with various biological processes ranging from insect flight-muscle organization to chromatin structure and lymphocyte homing (reviewed by Rechsteiner, 1988). In these situations Ub may function as a movable binding site promoting the association of proteins that do not have complementary surfaces. Ub is most fully characterized, however, as an essential component of a major cellular proteolytic pathway(s). Its widely accepted role as a proteolytic cofactor was discov- ered by Hershko and his colleagues (reviewed in Hershko, 1988) who elucidated most of the biochemical reactions presented in the figure. Ub is activated by El, an enzyme that hydrolyzes ATP to form a reactive thiol ester with the carboxyl terminus of Ub. A family of carrier proteins, the

KEKE MOTIFS. PROPOSED ROLES IN PROTEIN-PROTEIN ASSOCIATION AND PRESENTATION OF PEPTIDES BY MHC CLASS I RECEPTORS

A stretch of 28 ‘alternating’ lysine (K) and glutamate (E) residues is found in an activator of the multicatalytic protease. Such ‘KEKE sequences’ are also present in subunits of the multicatalytic protease, in subunits of the 26S protease and in a variety of chaperonins. We propose that KEKE regions promote association between protein complexes. Furthermore, they may contribute to the selection of peptides presented on MHC Class I receptors.

Structure of the proteasome activator REGα (PA28α)

The specificity of the 20S proteasome, which degrades many intracellular proteins, is regulated by protein complexes that bind to one or both ends of the cylindrical proteasome structure. One of these regulatory complexes, the 11S regulator (known as REG or PA28), stimulates proteasome peptidase activity, and enhances the production of antigenic peptides for presentation by class I molecules of the major histocompatibility complex (MHC),. The three REG subunits that have been identified, REGα, REGβ and REGγ (also known as the Ki antigen), share extensive sequence similarity, apart from a highly variable internal segment of 17–34 residues which may confer subunit-specific properties. REGα and REGβ preferentially form a heteromeric complex, although purified REGα forms a heptamer in solution and has biochemical properties similar to the heteromeric REGα/REGβ complex,. We have now determined the crystal structure of human recombinant REGα at 2.8 Å resolution. The heptameric barrel-shaped assembly contains a central channel that has an opening of 20 Å diameter at one end and another of 30 Å diameter at the presumed proteasome-binding surface. The binding of REG probably causes conformational changes that open a pore in the proteasome α-subunits through which substrates and products can pass.

Characterization of recombinant REGα, REGβ, and REGγ proteasome activators

Full-length cDNAs for three human proteasome activator subunits, called REGα, REGβ, and REGγ, have been expressed in Escherichia coli, and the purified recombinant proteins have been characterized. Recombinant α or γ subunits form heptameric species; recombinant β subunits are found largely as monomers or small multimers. Each recombinant REG stimulates cleavage of fluorogenic peptides by human red cell proteasomes. The pattern of activated peptide hydrolysis is virtually identical for REGα and REGβ. These two subunits, alone or in combination, stimulate cleavage after basic, acidic, and most hydrophobic residues in many peptides. Recombinant α and β subunits bind each other with high affinity, and the REGα/β heteromeric complex activates hydrolysis of LLVY-methylcoumaryl-7-amide (LLVY-MCA) and LLE-β-nitroanilide (LLE-βNA) more than REGα or REGβ alone. Using filter binding and gel filtration assays, recombinant REGγ subunits were shown to bind themselves but not α or β subunits. REGγ differs from REGα and REGβ in that it markedly stimulates hydrolysis of peptides with basic residues in the P1 position but only modestly activates cleavage of LLVY-MCA or LLE-βNA by the proteasome. REGγ binds the proteasome with higher affinity than REGα or REGβ yet with lower affinity than complexes containing both REGα and REGβ. In summary, each of the three REG homologs is a proteasome activator with unique biochemical properties.

Protein surveillance machinery in brains with spinocerebellar ataxia type 3: redistribution and differential recruitment of 26S proteasome subunits and chaperones to neuronal intranuclear inclusions.

Intracellular aggregates commonly forming neuronal intranuclear inclusions are neuropathological hallmarks of spinocerebellar ataxia type 3 and of other disorders characterized by expanded polyglutamine‐(poly‐Q) tracts. To characterize cellular responses to these aggregates, we performed an immunohistochemical analysis of neuronal intranuclear inclusions in pontine neurons of patients affected by spinocerebellar ataxia type 3, using a panel of antibodies directed against chaperones and proteasome subunits. A subset of the neuronal intranuclear inclusions stained positively for the chaperones Hsp90α and HDJ‐2, a member of the Hsp40 family. Most neuronal intranuclear inclusions were ubiquitin positive, suggesting degradation by ubiquitin‐dependent proteasome pathways. Surprisingly, only a fraction of neuronal intranuclear inclusions were immunopositive for antibodies directed against subunits of the 20S proteolytic core, whereas most inclusions were stained by antibodies directed against subunits of the 11S and 19S regulatory particles. These results suggest that the proteosomal proteolytic machinery that actively degrades neuronal intranuclear inclusions is assembled in only a fraction of pontine neurons in end stage spinocerebellar ataxia type 3. The dissociation between regulatory subunits and the proteolytic core and the changes in subcellular subunit distribution suggest perturbations of the proteosomal machinery in spinocerebellar ataxia type 3 brains.