Regulatory features of multicatalytic and 26S proteases

CURRENT TOPICS IN CELLULAR REGULATION, VOLUME 34

Regulatory Features of Multicatalytic and 26S Proteases LAURA HOFFMAN MARTIN RECHSTEINER Department of Biochemistry University of Utah School of Medicine Salt Lake City, Utah 84132

I. Introduction Intracellular proteolysis serves as an important regulatory mechanism (see Refs. 1-6 for reviews). Specific enzymes are subject to rapid degradation in response to changing nutritional conditions as seen, for example, during glucose repression in yeast (7) or polyaminestimulated degradation of ornithine decarboxylase (8). A host of eukaryotic transcription factors are naturally short-lived proteins (1). Moreover, cell cycle progression requires destruction of cyclins, the polypeptide activators of cell division cycle 2 (cdc2) kinase (9-11). The number of proteases involved in removing such rapidly degraded proteins is less clear. In fact, surprisingly few endoproteases have been localized to the nucleus and C5^osol of eukaryotic cells. Whereas there is an abundance of lysosomal cathepsins (12) and secreted proteases (13), only a handful of C3rtoplasmic endoproteases have been identified. These include the calpains (14), a 70-kDa metalloprotease (15), proline endopeptidase (16), a recently described interleukin 1(3 processing enzyme (17,18), and two large degradative enzymes, the multicatalytic protease (MCP), and the 26S ATP/ubiquitin-dependent protease (19,20). In this chapter, we focus on regulatory aspects of the latter two proteolytic complexes. The MCP and 26S complexes provide rich possibilities for regulation. Besides traditional mechanisms, such as control of enzyme levels or phosphorylation, potential combinatorial associations between families of MCP subunits and perhaps even larger families of ATPase subunits could generate a variety of specific proteolytic complexes. When the reported activators and inhibitors of MCP are considered, the regulatory capabilities are substantial (see Fig. 1). Furthermore, the nuclear/ cytoplasmic distribution of the two proteases varies with development and growth state. Changes in the locations of the proteases could well affect their access to substrates. Despite these possibilities, there are 1 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

LAURA HOFFMAN AND MARTIN RECHSTEINER

activated

MCP

FIG. 1. Schematic representation of the association between MCP and various regulatory complexes. MCP is shown as the central cylinder that can interact with several other protein complexes. The ATPase complex (AC) combines with MCP in an ATPdependent reaction to form the 26S protease. When the regulator (REG) interacts with MCP, the resulting MCP complex is activated for hydrolysis of small fluorogenic peptides. A second MCP complex with increased peptidase activity has an additional subunit with an apparent Mr of 160,000. Latent MCP may contain an additional protein of ~30 kDa that inhibits MCP's activity. Evidence supporting these interactions is presented in the text. Finally, a 15S ATPase complex is composed of a protein homologous to the S4 subfamily of ATPase (AC) subunits. It could conceivably interact with MCP although there are no data supporting this hypothesis.

only a few well-documented examples of regulation of MCP or the 26S enzyme. Consequently, several topics are covered in this chapter because they illustrate conceivable regulatory strategies, not because it has been demonstrated that such mechanisms operate within cells.

11. Structural and Enzymatic Properties of MCP During the past few years considerable progress has been made regarding the structural and functional aspects of the multicatalytic protease, and recent publications are available on the primary structures of MCP subunits, their arrangements in the proteolytic complex, and on various proteolytic activities of MCP (19-23). Although this chapter focuses on the regulatory aspects of MCP, a brief structural review should prove useful. Because the multicataljrtic protease is a large (700 kDa), complex (>10 subunits), potentially dangerous (broad

MULTICATALYTIC AND 26S PROTEASES

3

cleavage specificity), and ubiquitous protease, its existence in the cytoplasm has been somewhat of an enigma. However, studies have indicated that MCP is, in fact, a well-regulated and versatile enzyme. It has been identified as a component of several multisubunit complexes with varying specific activities toward fluorogenic peptides and varying abilities to degrade polypeptide substrates. Although there are two reports that an MCP-like protease may be present in prokaryotes (24,25), MCP proper appears to be restricted to eukaryotes and the archea. In these organisms, the multicatalytic protease is a cylindrical structure composed of four stacked rings each containing six or seven subunits (21). The archaebacterial Thermoplasma enzyme consists of only two subunits, termed a and p, whose sequences are related (26). Immunoelectron microscopy studies on the Thermoplasma enzjrme demonstrate that the j8 subunits, which may provide the enzjrme's active sites, are located in the inner two rings of the cylinder; the a subunits, which may serve regulatory rather than proteolytic functions, form rings at each end of the cylinder (27). Two-dimensional gels reveal more than a dozen MCP subunits in eukaryotes (see Fig. 2). Genes for 10 distinct MCP subunits have been cloned from a variety of organisms, including yeast (28), Drosophila (29), Xenopus (30), and mammals (31,32). The deduced sequences of eukaryotic MCP subunits, now classified as a or j8 according to their similarity to the Thermoplasma subunits, are unlike those of any previously identified proteases. MCP subunits are, however, related to each other and thus seem to represent a new family of proteases (22). There is little doubt that the eukaryotic multicatalytic protease contains several distinct proteolytic activities. Early studies demonstrated a very broad cleavage specificity for MCP, e.g., the enzyme cleaves peptides after basic, acidic, and hydrophobic residues (33). Protease inhibitors provided the key observation implicating distinct active sites in peptide bond breakage. Orlowski and Wilk (34) found that certain compounds inhibited cleavage of one peptide while actually stimulating hydrolysis of another. The existence of multiple genes encoding a and j8 subunits is consistent with the presence of distinct protease active sites in MCP. It also opens the possibility that MCP activities might be regulated by altering the subunit composition within the MCP particle. The catalytic mechanism employed by MCP has not been determined. Data on inhibition of MCPs isolated from various organisms are compatible with peptide bond cleavage being mediated by either serine or cysteine residues (see Table I). Analyses of MCP subunit sequences have not resolved the issue. Histidine, a key residue in the catalytic triad of serine proteases, is not conserved among MCP subunits, nor

r.

LAURA HOFFMAN AND MARTIN RECHSTEINER P*^

7 I

6

5 I

4 I

MW ^^^

7 I

I

6 I

5 I

4 I

ATPase complex

66-

45 29-

•

24 -

Regulator

26S prdlease •< 66 -

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— 0

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FIG. 2. 2D PAGE patterns of subunits from MCP, AC, 26S protease, and regulator. Proteins in the various complexes were separated in the first dimension by isoelectric focusing (pH 10-3) and in the second dimension by denaturing SDS-PAGE, and the gels were subsequently silver stained. Three major MCP subxinits are denoted by open arrowheads; MCP proteins are also found in the 26S protease, but not in the ATPase complex or regulator. The ATPase complex subunits between 30 and 110 kDa are shown, although resolution of the 100- and 110-kDa proteins was not obtained. Four of the AC proteins are denoted by closed arrowheads for orientation; the same set of proteins are also found in the 26S protease. The 30-kDa regulator protein typically resolves as a protein with a p/ of ~5.5 (open circle). Occasionally, an additional protein with a higher p/ can also be resolved. The region where regulator would migrate is circled on the AC and 26S protease gels. The regulator protein is clearly absent from the ATPase and 26S protease complexes.

are there patterns of histidine and cysteine characteristic of sulfhydryl proteases. At present, it seems hkely that MCP will prove to be an at3rpical serine protease (22). The enzyme is shown in Fig. 1 as a hollow cylinder, a structure much like that of microtubules. It is not clear whether the particle contains an aqueous central channel, nor is it clear where the protease active sites are located. However, it is attractive to

MULTICATALYTIC AND 26S PROTEASES TABLE I INHIBITORS OF SUC-LEU-LEU-VAL-TYR-MCA CLEAVAGE BY M C P

SoureeofMCP Rabbit reticuloc5d:e°'' Compound Chymostatin Antipain Pepstatin Leupeptin PMSF^ TPCK^ NEM'' EDTA EGTA Calpain inhibitor I Calpain inhibitor II

Sea urchin sperm^

Cone. ifjiM)

Inhib.

Cone. ifiM)

Inhib.

(%)

250 250 250 250 2500 250 2500

83 0 STIM 20 0 5 85

100 100 10 100 2000 100 1000

99 23 STIM 63 61 7 98

—

—

1000 250

4 87

— — —

— — —

250

65

—

—

(%)

Spinach*^ Cone. ifiM)

Inhib.

100 100 100 5000 100 500 5000 1000

92 6 0 57 23 0 95 0

— — —

(%)

Thermc)plasma^ Cone. ifiM) 16 18 18 25

Inhib.

(%) 0 0 0 11

— —

— —

— —

4000 5000 5000 26

0 97 100 100

—

25

73

Note. Cone., eoneentration; Inhib., inhibition. " Hough et al. (41). *Dubiele^a/. (100). 'Inahaietal. (185). ^Ozaki etai (186). ^ Dahlmann e^ a/. (187). ^ PMSF, phenylmethylsulfonyl fluoride. ^ TPCK, tosyl-phenylalanine ehloromethyl ketone. '' NEM, N-ethylmaleimide.

imagine that the active sites Hne a central canal. This would prevent indiscriminate proteolysis of cellular proteins by MCP.

III. Structural and Enzymatic Properties of 26S Protease A major advance in our understanding of intracellular proteolysis was the demonstration that ubiquitin (Ub) targets proteins for destruction. In two classic 1980 papers, Hershko and colleagues (35,36) showed that a 8.5-kDa protein, later identified as Ub (37), was required for ATP-dependent degradation of bovine serum albumin and RNase in rabbit reticulocyte lysate. They also found that Ub was covalently attached to the protein substrates. Based on these findings, they proposed that Ub marks proteins for destruction. Others have suggested

b

LAURA HOFFMAN AND MARTIN RECHSTEINER

that Ub has proteolytic activity (38) or that it stimulates proteolysis by inactivating an endogenous protease inhibitor (39). However, most who study Ub-mediated proteolysis would agree with some version of the pathway presented in Fig. 3. In this figure, the carboxyl terminus of Ub is shown to be activated by an enzyme (El), transferred as a high-energy thiol ester to Ub carrier proteins (E2s), and subsequently deposited in monomeric form on amino groups of histones (H2A) or as poly (Ub) chains on proteolytic substrates (S). The latter reaction often requires the participation of a ubiquitin protein ligase (E3). The marked substrates are then shown to be degraded and Ub is recycled. Experimental support for the scheme in Fig. 3 is considerable, and it has been reviewed on several occasions during the past few years (1-4).

l^

S peptides

FIG. 3. Schematic representation of the ubiquitin-mediated proteolytic pathway. Starting at the top of the diagram, the carboxyl terminus of ubiquitin (Ub) is shown to be activated by the E l enzyme and transferred as a reactive thiol ester to one of several small E2 carrier proteins. Ub is then conjugated directly to lysine-119 on histone H2A or to lysine amino groups on proteolytic substrates (S) by a Ub-ligase enzyme (E3). Monoubiquitinated proteins, such as H2A, are apparently not targeted for degradation. For other proteins though, Lys-48 of ubiquitin is used as a target site for building ubiquitin "chains." These polyubiquitinated proteins are substrates for degradation by the 26S protease (P) which hydrolyzes the substrate in an ATP-dependent reaction, generating small peptide products and recycled ubiquitin molecules.

MULTICATALYTIC AND 26S PROTEASES

7

A major feature of the marking hypothesis is the existence of a protease that specifically degrades ubiquitinated proteins. In 1986, Hough et al. (40) identified an enz3rme that degraded Ub-lysozyme conjugates in an ATP-dependent reaction; a year later they reported its purification (41). The 26S enzyme contains at least 20 different polypeptides (see Fig. 2), including a subset with molecular weights and isoelectric points characteristic of MCP subunits. Similarities in subunit composition led to the proposal that MCP subunits were integral parts of the 26S enzyme (42); the larger (>42 kDa) polypeptides were proposed to confer ATP dependence as well as Ub recognition on the 26S protease. Based on electron microscopy images obtained in 1970 (43) and subunit stoichiometries, Hough et aL (42) proposed a specific arrangement for MCP and other subunits in the 26S complex (see Fig. 1). Two lines of evidence support the shared subunit h5rpothesis and the idea that higher molecular weight polypeptides confer energy dependence. Whereas Hough et al. (41) isolated Ub-conjugate degrading activity as a 26S enzyme, Hershko and colleagues found that mixing three smaller components, termed CFl, CF2, and CF3, was required for degradation of Ub-lysozyme (44). Subsequent studies demonstrated that CF3 is the multicatalytic protease (45-47). Armon et al. (48) also showed that an NTPase activity is generated upon assembly of the CFs; this result has been confirmed (49). Recent cDNA cloning provides a second observation supporting the idea that the larger 26S subunits confer energy dependence. Dubiel et al. (50) reported the sequence for a 51-kDa polypeptide, subunit 4, from human red cell 26S protease. Subunit 4 belongs to a novel eukaryotic ATPase family that includes yeast CDC48p and yeast PASlp, Chinese hamster NSF, Xenopus p97, and four proteins—TBPl, TBP7, MSSl, and SUGl—very closely related to subunit 4 (50). The latter four proteins together with S4 constitute a subfamily of proteins, each about 440 residues, that contains one nucleotide binding site. The other members of the ATPase family are roughly twice as long and contain two ATP consensus sites per chain. Functionally, we believe that the S4 subfamily ATPase subunits serve to present substrates to the proteolytic core provided by MCP (see Ref. 22 and below for further discussion). In addition, two ATP-dependent proteases from Escherichia coli, Lon and Clp, are almost certainly members of this family (22). Given the structural and functional parallels between the 26S protease, Clp, and Lon, we proposed that the three enzymes are evolutionarily related (22). Structually, 26S and Clp appear to be closer relatives. Both are

8

LAURA HOFFMAN AND MARTIN RECHSTEINER

composed of hexameric rings of protease subunits (ClpP and MCP) that associate with separate ATPase subunits (ClpA and S4-Hke proteins). However, from an enzymatic perspective, the 26S enzyme is more Hke Lon. Both 26S and Lon can utiHze ATP, CTP, GTP, or UTP for proteolysis. In addition, both enzymes exhibit high affinity for nucleotides; the A'ni for proteolysis is about 30 fiM ATP (51). By contrast, Clp is specific for ATP and much higher levels of the nucleotide (K^n -1000 /xM) are needed to support protein breakdown (52). In this regard, the ATPase activity of ClpA resembles that of Xenopus p97. The latter may also be specific for ATP; it requires high levels of nucleotide for hydrolysis (53) and, interestingly, it also forms a hexamer (see Fig. 1 and below). IV. Regulation of MCP and 26S Protease A. Regulation of Protease Levels The concentration of MCP varies markedly among tissues. Values range from more than 2% of the total protein in rat thymus and testes (54) to as little as 0.01% of human lymphocyte proteins (55). Typically, however, MCP is a reasonably abundant cellular constituent present at about 0.5% of soluble proteins or roughly one MCP for every two ribosomes (see Table II). TABLE II LEVELS OF MCP

IN TISSUES OR CELL TYPES

MCP levels Tissue or cell type Human lymphocytes MOLT-10 HL60 Human renal cells Rat muscle Rat testis Rat th3mius Rat muscle Rat liver Rat kidney Sea urchin egg

jjLg per milligram cell protein 0.135 1.36 3.94 -7.0 3.27 23.6 28.5 1.1 9.4 2.0

—

Molecules per cell 1.2 5.8 1.7 2.9

X W X 10^ X W X W

— —

Ref.

55 56 54

1.2 X 10^

— 4 X 10^ 8.5 X W 2 X 10«

" For lymphocytes we assumed 1 mg protein = 10^ cells. For all other cell types we assumed 1 mg = 2 x 10^ cells.

57 58

MULTICATALYTIC AND 26S PROTEASES

9

The steady-state concentration of an enzyme is determined by its rates of synthesis and degradation (59). Three studies indicate that MCP is a relatively stable enzyme. It is now widely accepted that certain forms of "prosomes" are equivalent to MCP (6,22). Hence, some examples are drawn from the prosomes literature. Akhayat et al. (60) could not detect synthesis of prosomes (MCP) in developing sea urchins. Because the quantity of prosomes was unchanged after 48 hr of development, Akhayat et al, (60) concluded that MCP is metabolically stable. Hendil (61) examined MCP turnover in human HeLa cells and found that all MCP subunits exhibited half-lives of ~5 days. He also observed that MCP synthesis was not induced by heat shock or cell crowding. Tanaka and Ichihara (62) reported a significantly longer half-life for rat liver MCP. They found that MCP was roughly 1% of soluble liver proteins, and its apparent half-life was 12-15 days. If MCP generally proves to be as stable as it is in sea urchin, HeLa cells, or rat liver, then differential synthesis must account for the several hundred-fold range in concentrations shown in Table II. Several groups have demonstrated differential accumulation of MCP in developing tissues. Using antibodies against 28- and 35-kDa subunits of MCP, Klein et al (63) observed large differences in immunofluorescent staining ofDrosophila embryonic tissues. MCP was shown to be particularly concentrated in cells undergoing morphogenetic movements anterior and posterior of the cephalic furrow. The authors also reported transient accumulation of MCP in pole cells. This observation suggests that MCP subunits can be rapidly degraded in some tissues, although one cannot rule out masking of epitopes as an explanation for reduced staining in pole cells from older embryos. Tanaka and colleagues examined the synthesis of MCP subunits in human hematopoietic and renal tumor cells (64). Having established that the levels of MCP subunits and their mRNAs were much higher in malignant human hematopoietic cells (65), they compared the expression of MCP subunits in normal peripheral T lymphoc3rtes to MCP expression in human leukemia cells (66). Rapid synthesis of both MCP subunits and the higher-molecular-weight proteins characteristic of the 26S protease was observed after treating normal T lymphocytes with mitogens. By contrast, when various leukemic cell lines were induced to differentiate, there was reduced expression of mRNAs that encode MCP subunits and reduced synthesis of MCP proteins. Because the intracellular levels of MCP did not change markedly after either treatment, the authors proposed the existence of two pools of MCP—a larger metabolically stable pool and a small pool of rapidly degraded MCP subunits. However, the data presented are consistent with a

10

LAURA HOFFMAN AND MARTIN RECHSTEINER

single pool of MCP subunits. Since the specific activities of radiolabeled proteins were not measured, the extent to which absolute amounts of MCP protein should have increased cannot be determined. B. Expression of Specific MCP Subunits

As noted in the Introduction, the existence of multiple MCP subunits and numerous 26S ATPase subunits would allow eukaryotic cells, in principle, to generate large numbers of specific proteases by combinatorial association to these components. Do eukaryotic cells mix and match subunits? The answer is not clear, although three studies suggest that this may be a regulatory strategy. Haass and Hoetzel (67) examined Drosophila MCP by twodimensional PAGE and found changes during development. A relatively simple subunit pattern present in early embryos and Schneiders S3 cells became increasingly more complex in older embryos and adult flies. The authors proposed that the new protein species arose by posttranslational modification, but they recognized that synthesis of distinct subunits could account for the more complicated patterns at later developmental stages. Similarly, Ahn et al. (68) reported significant changes in the levels of five MCP subunits in developing chick muscle; the intensity of three species increased while that of two subunits declined. These investigators concluded that some subunits, at least, are under developmental control. A great deal of excitement has been generated by the possible role of MCP in antigen presentation (see Refs. 69 and 70 for reviews). MCP and/or the 26S protease are candidates for generating the peptides that bind major histocompatibility complex (MHC) class I receptors. Although circumstantial, there is a reasonable body of evidence supporting this idea. First, genes for MCP-like subunits, Ring 10 and Ring 12, are located in the MHC complex (71-73). Second, y-interferon (yIFN) stimulates production of many components in the antigen presentation pathway. In fact, expression of two MCP-like subunits whose genes are located in the major histocompatibility locus provides particularly strong evidence for tissue-specific expression of unique MCP subunits. Yang et al. (74) found that five new polypeptides were present in MCP complexes immunopurified from HeLa cells treated with yIFN. The use of human lymphoblastoid cell lines deleted for Ring 12 allowed these investigators to identify a specifically induced protein, subunit b, as the Ring 12 gene product. Using ammonium sulfate fractionation to separate MCP from 26S protease, Yang et al. provided evidence that y-IFN-induced MCP subunits partitioned uniquely between the two enzymes. That is, of five induced subunits, two were

MULTICATALYTIC AND 26S PROTEASES

11

found in the 26S enzyme and three in MCP. Finally, cell fractionation studies indicated that MCP particles isolated from microsomes were enriched in several y-IFN-induced subunits. This is consistent with an overall presentation scheme in which MCP generates peptides that enter microsomes by specific peptide transporters, also encoded in the MHC, and then subsequently bind class I receptors for movement to the cell surface. C. Regulation by Posttranslational Modification 1. PHOSPHORYLATION

Phosphorylation is, without a doubt, the major reaction by which eukaryotes regulate biochemical processes (75). Therefore, it is, surprising that so few examples of MCP or 26S protease regulation by protein kinases can be found. The only evidence that MCP subunits are phosphorylated in vivo was provided by Haass and Kloetzel (67) who showed that growing Drosophila S3 cells in medium containing ^^P-labeled phosphate resulted in labeled MCP subunits. Based on sequence analysis of MCP subunits, Haass et al. (76) and Tanaka et al. (77) proposed that a tyrosine in a src consensus site was the phosphorylated residue. However, this proposal has not yet been directly confirmed. Pereira and Wilk (78) reported that 27- and 28-kDa subunits of bovine pituitary MCP were major substrates for a copuripfying cAMPdependent kinase. Two additional subunits (31 and 24 kDa) were phosphorylated to a lesser extent. Unfortunately, evidence was not presented demonstrating that phosphorylation affected the proteolytic activity of MCP. Hough et al. (79) observed ^^P-labeled phosphate incorporation into at least two subunits (110 and 62 kDa) of the 26S ATPase complex. On pharmacological grounds, the responsible protein kinase appeared to be casein kinase II, Ferrell et al. (unpublished observations, 1988) extended these studies by showing that serine and, to a lesser degree, threonine are the phosphate-accepting residues. Moreover, the extent of phosphorylation is markedly affected by two inhibitors of Ub-conjugate degradation, hemin and aurintricarboxylic acid (see Fig. 4). 2. GLYCOSYLATION

Whether MCP subunits are covalently modified by sugars is a controversial subject. Using lectin blotting procedures, Schmid and colleagues reported the presence of glucosyl-, mannosyl- andN-acetylgalactosaminyl residues in plant MCP (80,81). Rivett and Sweeney (82) also claim that three subunits of rat liver MCP bind concanavalin. On the other

12

LAURA HOFFMAN AND MARTIN RECHSTEINER

B

A P2

Pi

PS

2 c

^ '^ if O

45 — 3629—

£

-•

<

O 0) »o i: <

^^^k^

• ^

^ PT

X

origin

24 — 20 —

-f-

pH 1.9

250Vx6h

(-)

FIG. 4. Phosphorylation of 26S protease subunits. (A) Rabbit reticulocyte 26S protease was purified to apparent homogeneity and incubated with 20 /JLM y-^^P-ATP in the presence or absence of hemin and aurintricarboxylic acid (ATA), both of which are inhibitors of conjugate degradation. The extent of phosphorylation was then determined by separation of subunits on SDS-PAGE gels and subsequent autoradiography. ATA severely depresses phosphorylation, whereas hemin promotes it. Pi and P2 represent different pools of 26S protease obtained from hydroxylapatite. (B) The 26S protease was incubated with 32PO4-ATP, and its subunits were separated on SDS-PAGE gels. The 110kDa subunit was excised from the gel and digested with pronase, and the phosphorylated amino acids were separated by electrophoresis. The resulting autoradiogram shows that serine is the predominant phosphorylated residue with minor labeling of threonine.

hand, Haass and Kloetzel (67) and Kaltoft et al. (83) did not find carbohydrate in Drosophila or human MCPs, respectively. 3. PROTEOLYTIC PROCESSING

Conversion of inactive precursors to active enzymes by peptide bond cleavage is a recurrent theme in the field of proteolysis. At the physiological level, it is the central control step in blood clotting (84), and it plays a key role in activating digestive enzymes (85) as well as in regulating blood pressure (86). The mechanism also operates at the cellular level. Some lysosomal cathepsins are activated by removal of prepro regions (87), as are processing enzmes in the secretory pathway (88). The same holds true for cytoplasmic proteases. Calpain is activated by cleavage of the 80-kDa heavy chain (89) and there is evidence

MULTICATALYTIC AND 26S PROTEASES

13

that the interleukin lj8 protease is activated by hydrolysis of an internal peptide bond (17,18). In this context, it is perhaps not surprising to find that j3 subunits of MCP are also processed. This has been shown clearly by studies on the archaebacterial enzyme from Thermoplasma. Zwickl et al. (90) expressed T. acidophilum a and /3 subunits in E. coli and found that 8 residues were removed from the N terminus of the f3 chain. This was apparently an MCP-mediated reaction since it was dependent on coexpression of the a subunit. Studies by Lilley et al. (19) on rat liver MCP indicate that removal of N-terminal extensions from /3 subunits occurs generally. Direct peptide sequencing revealed that numerous rat MCP (3 chains began at a threonine about 10-20 residues into the sequences deduced from cDNAs. As a further parallel between the 26S and Clp proteases, Maurizi and colleagues have shown that ClpP is also missing 14 residues from its N terminus (92). In all these cases, processing is thought to activate precursor subunits thereby ensuring that protease activity is confined to the assembled particle. Two studies employing Western blot analyses suggest that additional processing reactions may occur. Kreutzer-Schmid and Schmid (93) probed HeLa nuclear and cytoplasmic extracts with a monoclonal antibody to a prosomal 27-kDa protein. Surprisingly, the major immunoreactive species was a 38-kDa nuclear protein. On digestion with V8 protease, the 38-kDa protein produced a 27-kDa species and several smaller peptides with Mr values similar to those obtained from the 27-kDa prosomal protein. Since p27 has been identified as a member of the MCP a-subunit family (94), these studies raise the possibility that an MCP a subunit is produced as a 38-kDa precursor. Weitman and Etlinger (95) have also obtained a monoclonal antibody that reacts with a 32-kDa protein associated with latent MCP particles. The same monoclonal recognizes a 28-kDa protein in activated MCP particles and a 41-kDa protein in unpurified preparations of MCP. These authors also suggest that the 41-kDa protein may be a precursor to the 32/28-kDa species. There are several reports of self-digestion by MCP. Tanaka and Ichihara (96) found that rat liver MCP subunits disappeared on addition of high levels of urea, presumably by autocatalytic cleavages. Lee et al. (97) observed proteolytic degradation of certain subunits in active as opposed to latent forms of human MCP. Yu et al. (98) observed a more limited degradation following dialysis of bovine pituitary MCP against low ionic strength Tris buffers. It is doubtful that any of these manipulations reflect physiological control mechanisms. They do, how-

14

LAURA HOFFMAN AND MARTIN RECHSTEINER

ever, demonstrate that a large proteolytic particle can be activated or destroyed by self-cleavage reactions. D. Regulation of MCP by Associated Proteins

In Fig. 1, MCP is shown associating with a variety of other protein complexes that affect its activity. Only the ATPase complex (AC) has been demonstrated to influence substrate selection by conferring the ability to degrade Ub conjugates on MCP. It seems likely, however, that other proteins in the diagram will serve similar roles. Consequently, all are considered potential agents for regulating the stability of cellular proteins. 1. ACTIVATORS

Two protein complexes that activate peptide hydrolysis by MCP have been purified and characterized. One consists of a presumed hexamer of 30-kDa subunits (99,100). The other is a multisubunit complex containing at least 10 proteins (101). Because this more complicated protein complex is a central component of the 26S protease, it is considered first. Starting with non-ATP-depleted rabbit reticulocyte lysate, Hough et al. (41) isolated a single 26S proteolytic complex capable of degrading Ub-lysozyme conjugates. By contrast, Hershko and colleagues (44) observed breakdown of Ub-lysozyme conjugates only on combining three factors obtained from ATP-depleted lysate. The three factors, termed CFl, CF2, and CF3, had molecular masses of approximately 600, 250, and 650 kDa, respectively. When combined in the presence of Mg2+-ATP, the three factors disappeared, and a large (>1000 kDa) ATP-dependent protease formed. Ganoth et al. (44) concluded that CFl, CF2, and CF3 combine to form the 26S protease. The properties of CF3 were similar in many ways to those of the multicatalytic protease and, as noted, subsequent studies confirmed that CF3 was, indeed, MCP (45-47). Two groups have characterized complexes that either correspond to CFl or to CFl plus CF2. Hoffman et al. (101) discovered and purified a proteolytically inactive particle that contains subunits characteristic of the 26S protease (e.g., proteins with molecular masses between 30 and 110 kDa). Incubation of this particle with MCP and Mg^^-ATP resulted in its association with MCP, significant stimulation of peptide hydrolysis by MCP, and generation of a protease capable of degrading Ub-lysozyme conjugates. Based on the sedimentation characteristics of the protein complex, and its ability to form the 26S protease when combined with MCP, Hoffman

MULTICATALYTIC AND 26S PROTEASES

15

et al, suggested that it corresponds to CFl and CF2. A 51-kDa polypeptide in the particle belongs to a family of putative ATPases (50), and the protein complex has been found to exhibit ATPase activity (L. Hoffman et al, manuscript in preparation). For this reason, we now substitute the term, ATPase complex (AC), for a rather inelegant descriptor, "the ball," used previously. Udvardy (102) has also purified and characterized a multiprotein complex, the /x, particle, from Drosophila oocytes. Like the reticulocyte ATPase complex, the /x particle does not possess protease activity. However, in the presence of ATP, it combines with Drosphila MCP to form a 26S protease capable of degrading Ub-yolk protein conjugates. In contrast to the results of Hoffman et al. (101), a third component is required for assembly of /x and MCP, but this component is not incorporated into the 26S protease. Udvardy suggests that CF2 may not be incorporated into the 26S enzyme. In this scheme, the /x particle would be equivalent to CFl, MCP would equal CF3, and the unincorporated assembly factor would be CF2. If, on the other hand, the /x particle corresponds to CFl and CF2, then a fourth factor is needed to generate the Drosophila 26S protease. Several groups have identified a smaller protein complex that stimulates peptide hydrolysis by MCP. Yukawa et al, (103) described a factor from platelets that enhanced MCP's ch)rmotrypsin- and trypsin-like activities; ATP was not required for activation. More complete descriptions of this factor were published in 1992, when three groups characterized a ~200- to 300-kDa protein complex that activiates hydrolysis of certain fluorogenic peptides (99,100,104). In two cases, the activator or regulator was purified sufficiently to identify its subunit composition. Chu-Ping et al (99) reported that bovine red cell MCP activator has a native molecular weight of —180,000 and is composed of a single 28-kDa subunit. The activator, which is presumably a hexamer, stimulated three distinct peptidase activities by increasing Kiax and reducing K^. The activator did not stimulate hydrolysis of proteins. Dubiel et aL (100) obtained similar results for human red blood cell regulator. They found that the regulator sedimented at U S and was composed of two closely related 30-kDa subunits. When added to MCP, the regulator stimulated hydrolysis of two fluorogenic peptides by almost 60-fold, whereas hydrolysis of two other peptides was stimulated only 3- to 10-fold. The human regulator did not stimulate hydrolysis of Ub-lysozyme conjugates, bovine serine albumin, or lysozyme. Using glycerol gradients, native gels, and two-dimensional PAGE, this group presented evidence that activation results from the reversible association of

16

LAURA HOFFMAN AND MARTIN RECHSTEINER

regulator and MCP. That is, neither component appears to be permanently affected by activation. Cloning of the cDNA encoding one of the two 30-kDa subunits revealed that it is a 249-residue protein (Realini et ai, manuscript in preparation, 1993). Like the ATPase subunits, the MCP regulator sequence possesses a stretch of amino acids strongly predicted to form coiled coils. The potential significance of coiled coil structural motifs is discussed below. Figure 1 depicts a hexamer of p97 subunits associating with MCP to form a complex similar to the 26S protease. Although the diagrammed reaction between p97 and MCP is entirely hypothetical, it is included in the figure because of some evident parallels between p97 and members of the S4-like subfamily of putative ATPases. Before discussing those parallels, a brief review of p97 is in order. In 1976, White and Ralston (105) extracted red blood cell membranes in 0.1 mM EDTA and obtained a soluble Mg^^-ATPase. The enzyme appeared to be specific for ATP in that CTP or GTP were not hydrolyzed. Later studies on the purified red cell ATPase showed that it has a native molecular weight of about 500,000 and is composed of a single 100-kDa subunit (106). The K^r, of the enzyme for ATP is 1 mM, and it is inhibited by AT-ethylmaleimide, Cd^^, Zn^^, andp-chloromercuribenzoate. In 1990, Peters et al. (53) described a 15S ATPase present in extracts ofXenopus laevis oocytes. Their enzyme displays sixfold radial symmetry and is composed of a single subunit with an apparent Mr of 97,000 (e.g., p97). Peters et al. (53) prepared antibodies to p97 and demonstrated that it was present in a wide variety of organisms and tissues. They also obtained the sequence of p97 from cDNA clones and found that it was closely related to two proteins involved in secretion, NSF, or its yeast equivalent SeclSp (107,108). Subsequent studies showed that p97 is even more closely related to the protein encoded by a yeast cell cycle mutant, cdc48 (109). Regarding Fig. 1, the evidence that p97 may assemble with MCP is as follows: The putative S4-like ATPases are members of a family of larger proteins that contain two candidate ATP-binding sites. Within this extended family are two proteins, cdc48p and p97, that bear close resemblance to the S4 subfamily. CDC48p and p97 have predicted coiled coil regions spaced relative to one of their ATP-binding sites at positions equivalent to proteins in the S4 subfamily. Like S4, the larger proteins have conserved cysteine residues C-terminal to the ATPbinding site. Moreover, the sixfold radial symmetry of p97 (53) suggests that it might well interact with the six a subunits at each end of MCP. Thus, there is reason to suspect that p97 will be shown to associate with MCP.

MULTICATALYTIC AND 26S PROTEASES 2.

17

INHIBITORS

Proteins that inhibit MCP activity have also been reported. Almost a decade ago, Speiser and Etlinger (39) proposed that ATP stimulates proteolysis in reticulocyte extracts by repressing an endogenous protease inhibitor. Three years later, Murakami and Etlinger (110) purified a hexameric complex of 40-kDa subunits that inhibited both calpain and MCP. Because heating destroyed inhibitor activity against MCP, but not against calpain, the authors concluded that different domains on the inhibitor interacted with each protease. Two papers have implicated the 40-kDa inhibitor in 26S protease function. Li and Etlinger (111) report that a ubiquitinated derivative of the 40-kDa inhibitor is a component of the 26S protease. However, this claim should be viewed with extreme caution since the subunit pattern of their "26S protease" bears no resemblance to the polypeptide pattern of the 26S protease reported by four independent groups (41,45,47,112). DriscoU et al. (113) claim that a 250-kDa ATP-stabiHzed inhibitor of MCP is a 26S protease component. They propose that the 250-kDa native complex, which is composed of 40-kDa subunits, corresponds to the CF2 component identified by Hershko and colleagues. As previously mentioned, Udvardy (102) questioned the idea that CF2 is incorporated into the 26S protease. Clearly, further experimentation will be required to assess the importance of inhibitors in 26S protease function. In 1991, Etlinger and colleagues (114) reported the isolation of yet another MCP inhibitor from human erythrocytes. This factor is apparently a tetramer since its native molecular mass is 200 kDa, and it is composed of a single 50-kDa subunit. A specific monoclonal antibody and peptide sequencing distinguished this inhibitor from the 40-kDa inhibitor. Li et al. (114) suggested that the 50-kDa inhibitor plays a role in Ub-mediated proteolysis. Finally, DeMartino and colleagues (115) have purified an inhibitor that forms multimers under nondenaturing conditions. It appears to be composed of a single, self-associating 31-kDa polypeptide. The protein inhibits both the three distinct catalytic activities of MCP and the enzyme's ability to degrade casein, lysoz3nne, and bovine serum albumin. These authors also suggest that this inhibitor may play a role in ATP/Ub-mediated proteolysis. Whereas there seems to be little doubt that protein complexes exist which can stimulate or inhibit peptide bond hydrolysis by MCP, certain key questions remain unanswered. Do the inhibitors represent bona fide regulatory molecules or substrates? Are the activators subcompo-

18

LAURA HOFFMAN AND MARTIN RECHSTEINER

nents of larger protein assemblies (e.g., ATPase complexes) or independent regulators? We believe it is premature to assign specific roles to the various activators and inhibitors. 3. ACTIVATION AND INHIBITION BY SMALL MOLECULES

There are reports that MCP can be activated or inhibited by fatty acids (116), detergents (116-119), sulfated lactosylceramides (120), inorganic ions (121,122), polylysine (117,123), and heating (124). Some of these findings might reflect relevant physiological regulatory mechanisms, e.g., palmitylation or myristoylation of proteins could conceivably target them for destruction by MCP (125). However, in our view the observed effects of the various small molecules do not invoke plausible control mechanisms. Hence, they are not covered further in this essay. E. Subcellular Distribution of MCP and 26S Proteases Experiments performed a decade ago convincingly demonstrated that a Xenopus 22S cylinder particle, now known to be MCP, is present in both nucleus and cytoplasm. Kleinschmidt et al. (126) analyzed extracts from manually dissected oocyte germinal vesicles and cytoplasms on 2D PAGE gels. They found identical MCP subunits in each compartment. In a companion paper, Hiigle et al. (127) obtained antibodies to a 30-kDa subunit from Xenopus MCP and examined the distribution of the enzyme in various tissues by immunofluorescence microscopy. MCP was enriched in the nuclei of all cells examined, which included liver, muscle, a Xenopus culture cell line, and ovarian tissues. In liver and muscle a characteristic punctate or speckled pattern was observed within nuclei; nucleoli and heterochromatin were not significantly stained. During mitosis of Xenopus A^ cells, MCP was dispersed throughout the cell, but apparently was not present on metaphase chromosomes. There have been a number of additional studies on the location of MCP in the intervening 10 years (see Table III for a summary). Despite the fact that a wide variety of tissues and species have been examined, certain themes emerge. MCP is largely C3rtoplasmic in zygotes and early embryos, although it becomes increasingly nuclear as development proceeds. The nuclear/cytoplasmic distribution of MCP can vary among cells within a specific tissue. The enzyme is, however, more heavily concentrated in nuclei of dividing or cancerous cells. In some nondividing cells, e.g., Drosophila salivary glands, the protease is apparently absent from the nucleus. Immimofluorescence staining often reveals a speckled or clustered pattern for MCP in both nucleus and cytoplasm.

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20

LAURA HOFFMAN AND MARTIN RECHSTEINER

However, a clustered distribution of MCP was not reported in a recent electron microscopic study (137). These generalizations hold for almost all studies except those reported by Scherrer and colleagues (129-131,135,136). This group consistently finds "prosome" antigens associated with intermediate filaments. Presumably their antibodies identify MCP, although a clear connection between prosomal antigens and MCP subunits has not been documented by these investigators. Moreover, the fixation procedures used by Scherrer and colleagues are not traditional. In contrast to most protocols, cell membranes are disrupted by detergent prior to fixation. For these reasons, the reported association between prosomal proteins and intermediate filaments may not apply to MCP. Two members of the S4-like subfamily of ATPases have also been localized predominantly in the nucleus. Nelbock et al. (139) produced polyclonal rabbit antibodies to TBPl and found the protein mainly in the nucleus of COS cells. Similar results were obtained with TBP7, a homolog of TBPl (140). In addition, two other members of the subfamily, MSSl and SUGl (141,142), have apparent effects on transcription so they are also likely to be located in the nucleus. It has been proposed that TBPl, MSSl, and SUGl are ATP-dependent transcription factors (141). Although this possibility cannot be eliminated, the observed effects on gene expression can be explained by proteolytic mechanisms (50), and it is likely that all members of the S4-like subfamily are components of the 26S protease. In fact, MSSl has recently been identified as subunit 7 of the human 26S enzyme (143). Other components of the Ub-mediated proteolytic pathway are also concentrated in the nucleus. Cook and Chock (144) report that the Ubactivating enzyme, El, is largely nuclear. The yeast cdc34 gene product, a ubiquitin-carrier protein, is also localized in nuclei (145). Finally, there is evidence that ubiquitin conjugates may also be enriched in nuclei. After microinjecting ^^^I-labeled ubiquitin into HeLa cells, Carlson and Rechsteiner (146) found, as expected, Ub-H2A histone conjugates were exclusively nuclear. They also observed an abundance of larger conjugates in the nuclear fraction. Likewise, Beers et al. (147) found numerous high-molecular-weight conjugates in plant cell nuclei. Finally, immunolocalization of Ub conjugates in rat cardiomyocytes revealed a "speckled" nuclear pattern (148). Current evidence indicates that the multicatalytic protease, the 26S protease, the ubiquitin activating enzyme, and the Ub carrier proteins are often enriched in the nucleus. Moreover, high-molecular-weight Ub conjugates, the presumed substrates for the 26S protease, are also prominent within nuclei. This raises the possibility that a large portion

MULTICATALYTIC AND 26S PROTEASES

21

of selective intracellular proteolysis occurs within the nuclear compartment. There is, furthermore, a voluminous literature on regulated nuclear entry of transcription factors, such as N F - K B and dorsal (149152). Thus, one can imagine that certain proteins are stable in the cytoplasm and rapidly degraded after entry into the nucleus. Compartment-specific degradation would provide an effective mechanism for controlling the metabolic stability of proteins. F. Multicatalytic Protease and 26S Activities during Development, during Cell Cycle, and after Physiological Stress

Changes in MCP or 26S activities must be viewed with some caution for a variety of reasons. First, a number of activators and inhibitors of MCP have been described (see above). Hence, one cannot know whether apparent changes in protease activity reflect the enzjones proper or changes in regulatory factors. Of course, the latter possibility is still physiologically relevant. Second, the multicatalytic protease is a notoriously "sticky" particle, and one must determine that copurifying activities are integral components of the protease complex. Likewise, the 26S protease can be fragile so apparent changes may reflect handling. Readers should view the following section with these caveats in mind. Chung, Tanaka, and colleagues have measured MCP peptidase activities during chick development (68). In embryonic muscle, they observed 2- or 3-fold decreases in MCFs chymotrypsin- and trypsin-like activities between embryonic Days 8 and 20. By contrast, there was a 4-fold increase in polylysine-stimulated casein degradation over the same period. Chymotryptic activity was relatively stable in developing chick brain and liver, but trypsin-like activity, assayed with the fluorogenic peptide Cbz-ARR-MNA, increased 30-fold in liver between Days 11 and 14. As previously mentioned, these investigators found changes in the relative proportion of five MCP subunits in developing muscle. Two groups have reported decreased MCP and 26S protease activity in maturing erj^hroid cells. Using density purified rabbit reticulocytes and mature red cells, Di Cola et al, (153) observed coordinate threefold decreases in several MCP peptidase activities. This was paralleled by twofold lower degradation of Ub conjugates by the 26S protease. Tsukahara et al. (154) similarly observed a decrease in the 26S protease on dimethyl sulfoxide-induced differentiation of murine erythroleukemia cells. There is a single report that the 26S protease is activated at specific points in the cell cycle. Kawahara et al. (155) measured peptide hydrolysis in developing ascidian embryos and found two peaks of chymotryptic activity corresponding to prophase and metaphase of the third cleavage

22

LAURA HOFFMAN AND MARTIN RECHSTEINER

stage. On the other hand, Mahaffey et al. (iinpubUshed observations, 1993) observed no changes in peptide hydrolysis or Ub-conjugate degradation during the first two cleavage cycles in Xenopus egg extract. Finally, two papers describe changes in MCP activity following physiological stress. Kuehn et al. (156) examined MCP peptidase activities in muscles of fasting rats. Whereas the amount of MCP protein was unchanged over the 3 days of starvation, cleavage of the chymotryptic substrate, Suc-Leu-Leu-Val-Tyr-MCA, fell threefold. This decrease was not observed in testis or thymus of the fasted animals. On the other hand, Medina et al, (157) found enhanced ATP-dependent proteolysis in fasted rat muscle. Based on changes in poly(Ub) transcripts and levels of Ub conjugates, they attributed this increase to the Ubdependent pathway.

V. Regulation by MCP and 26S Proteases Interest in the regulation of multicatalytic and 26S proteases stems, in large part, from mounting evidence that they, in turn, exert important controls on other metabolic pathways. Almost two decades ago, Schimke (59) pointed out that regulatory proteins would be metabolically labile because rapid changes in their concentration demand a short half-life. The list of rapidly degraded intracellular proteins grows daily and now includes transcription factors, oncoproteins, protein kinase-associated subunits, and key metabolic enzymes (158). Given the expanding numbers of short-lived proteins, a major task is to identify the proteases responsible for their destruction. Some proteins, at least, appear to be substrates for the 26S enzyme. A. Natural Substrates for MCP and the 26S Protease In Fig. 1, MCP is associating with various factors thought to affect its activity. It is not clear whether the central core particle, MCP, is able to degrade intact proteins. In fact, we suspect that the regulator/ MCP complex may simply be a very efficient peptidase rather than an endoproteinase. At the same time, MCP is known to be a central component of the 26S enzyme (22), so discussions of 26S substrates include, perforce, substrates for MCP. There are, however, several papers that implicate MCP alone in the destruction of oxidized proteins. Thus, before considering substrates of the larger 26S enzyme, the potential role of MCP in removing oxidized proteins from cells is reviewed. Although Goldberg and Boches (159) claimed that oxidized red cell proteins are degraded by an ATP-dependent process, the same labora-

MULTICATALYTIC AND 26S PROTEASES

23

tory arrived at different conclusions in several later studies. Fagan et al. (160) reported that hemoglobin oxidized by nitrite or phenylhydrazine was rapidly degraded, and this process was not inhibited by ATP depletion. Likewise, Davies and Goldberg (161) observed that proteins damaged by oxygen radicals are degraded by red cell extracts lacking nucleoside triphosphates. Two studies identify MCP as the responsible enzyme. Pacifici et al. (162) found that 70-80% of the degradative activity against oxidatively modified proteins was exhibited by a 670kDa proteinase complex, which they called M.O.P. Since the subunit pattern of M.O.P. is virtually identical to the SDS-PAGE profile for MCP, the two enzymes are presumably the same. Sacchetta et al. (163) obtained similar results using phenylhydrazine-denatured hemoglobin as substrate. These investigators found that MCP was unable to hydrolyze native Hb. It did, however, produce peptides from denatured globin. Interestingly, free amino acids were not final products of the reaction. In contrast to these studies, Fagan and Waxman (164) have recently concluded that red cell MCP is not responsible for degrading oxidantdamaged hemoglobin in crude red cell extracts. Using a combination of protease inhibitors and antibodies, these investigators present reasonably convincing evidence that insulinase, a 100-kDa metalloprotease, is responsible for most ATP-independent proteolysis of oxidized hemoglobin. Thus, isolated MCP may be capable of degrading oxidized hemoglobin, but it does not appear to be the principal protease doing so in crude red cell extracts. Several studies by Rivett implicate rat liver MCP in the degradation of oxidized proteins. Using oxidized glutamine S5nithetase (glutamateammonia ligase) as a substrate, she identified four rat liver proteases that degrade the inactive bacterial enzyme (165). Two of the enz3rmes were calpains, one was cathepsin D, and one was a large, ~300-kDa, protease with an alkaline pH optimum. Subsequent purification of the larger enzyme identified it as MCP, and improved gel filtration showed its molecular weight to be 650,000 (166). As with the studies using hemoglobin, it is difficult to know the fraction of oxidized proteins degraded by MCP within rat liver cells. To date, the 26S protease is the only enzyme known to degrade proteins conjugated to Ub. It seems reasonable, therefore, to consider proteins whose degradation is mediated by Ub as 26S substrates. A variety of intracellular proteins are thought to be degraded by Ubmediated pathways (see Table IV). However, as noted in an earlier review (1), the evidence implicating Ub in the destruction of natural cellular constituents remains largely circumstantial. Except for detailed studies on artificial Ub-j8-galactosidase substrates, the evidence

24

LAURA HOFFMAN AND MARTIN RECHSTEINER TABLE IV NATURAL SUBSTRATES OF 26S PROTEASE

Protein

Ref.

Protein

Ref.

Phytochrome MATa2 repressor Cyclin p53 Myc, Fos Cytochrome P450 2E1

174 175 11,184 176 177 178

PDGF receptor Mos Cytochrome P450 3A Retinoblastoma Ribonucleotide reductase subunit M l Ornithine decarboxylase

179 180 181 182 183 167

is mainly a correlation between the appearance of Ub-conjugates to a specific protein and the protein's rapid disappearance. Because the proteins listed in Table IV are present at such low concentrations within cells, it has not been possible to isolate Ub-conjugated derivatives and directly assay their degradation by the 26S protease. No doubt, additional proteins will join those in Table IV, but newer approaches will be required to obtain conclusive evidence that any of the proteins are degraded by Ub-mediated (26S) pathways. Murakami et aL (167) have reported that ornithine decarboxylase (ODC), an extremely short-lived enzyme, is degraded directly by the 26S protease. Rapid proteolysis of ODC required the addition of antizyme which forms a noncovalent complex with ODC (168). However, ubiquitination was not required. This demonstration that the 26S protease can directly degrade certain proteins is consistent with several previous observations. Bercovich et al. (169) showed that immunoprecipitation of the Ub-activating enzyme from reticulocyte lysates did not prevent ODC degradation. Likewise, thermal inactivation of E l in the mutant cell line, ts85, did not inhibit ODC turnover (170). B. Postulated Molecular Mechanisms for Target Selection

It seems clear that the 26S protease is a versatile enzyme capable of degrading both ubiquitinated and nonconjugated proteins. Exactly how the enzyme recognizes substrates has not been elucidated. Presumably, a subunit(s) in the ATPase complex can recognize ubiquitin or poly(lJb) chains. However, repeated attempts to identify a Ub-binding subunit using photoaflfinity cross-linking approaches have not yet proved successful (Ustrell et aL, unpublished observations, 1993). Hershko and colleagues have identified two isopeptidases (171,172). One of the enzymes acts on poly(Ub) chains, but it is not associated with the 26S complex (171). The other ubiquitin C-terminal hydrolase

MULTICATALYTIC AND 26S PROTEASES

25

activity is associated with the 26S complex and, interestingly, it requires ATP, CTP, or GTP to hydrolyze ubiquitin-lysozyme isopeptide bonds (172). This isopeptidase activity is tightly coupled to proteolysis, and these investigators propose that the enzyme releases Ub from substrate amino groups in the final stages of proteolysis. It is well estabUshed that intertwined a hehces (e.g., leucine zippers or coiled coils) can play important roles in protein-protein associations. Lupas et al (173) have devised an algorithm for predicting the occurrence of coiled coil regions in proteins. Application of this algorithm to the various proteolytic components shown in Fig. 1 produced some intriguing patterns. Potential coiled coil regions are present in MCP subunits, in the U S regulator subunit, and in all members of the S4-like subfamily of putative ATPases (see Fig. 5A). Conceivably, these regions promote binding of regulator or ATPase subunits to MCP. However, based on sequence variability in the S4-like subfamily, we have suggested that the coiled coil domains on the ATPase subunits at least, serve to bind substrates of the 26S enzyme (22). This helix-shuffle hypothesis can account for the rapid degradation of those unassembled

100 F

Regulator

100

200

300

Residue number FIG. 5. Coiled coils as possible substrate recognition motifs. (A) The sequences of three proteins (regulator, subunit 9 of human MCP, and subunit 4 of the 26S protease) were analyzed for potential coiled coil motifs by the algorithm of Lupas et al (173). Regions with high coiled coil probabilities are shown in the diagram. (B) Coiled coils may be used to target proteins for proteolytic degradation as in the hypothetical scheme shown. A mechanism for proteolytic substrate recognition by the 26S protease is proposed by which unpaired a helices of Fos, which normally dimerize with similar regions on Jun, could dimerize with a helices present in ATPase subunits of the 26S protease. This interaction with S4 rather than Jun would result in degradation of Fos.

26

LAURA HOFFMAN AND MARTIN RECHSTEINER

proteins that possess leucine zippers as shown schematically in Fig. 5B. Whether correct or not, the idea has the virtue of being readily testable by site-directed mutagenesis.

VI. Summary It should be clear from the foregoing accounts that our understanding of MCP and 26S regulation is still rudimentary. Moreover, we have only recently identified about a dozen natural substrates of these two proteases. Those outside the field may view the situation with some dismay. Those who study the MCP and 26S enzymes are provided with rich opportunities to address fundamental questions of protein catabolism and metabolic regulation. NOTE ADDED IN PROOF. There have been several significant advances in the two years that have elapsed between the submission and publication of this manuscript. While it is not possible to bring the entire manuscript up to date, several references must be added. Cloning of the 30-kDa activating protein (188) and description of Ub-conjugate degradation in the Xenopus cell cycle system (189) were referred to here as unpublished. Also, the Ub-conjugate binding subunit of the 26S protease has been identified (190), activation of MCP by two regulatory complexes has been further detailed (191), and a protein-protein interaction hjrpothesis for subunits and substrates has been proposed (192). Finally, we now refer to the multisubunit complex which combines with MCP to form the 26S protease as the Regulatory Complex (RC).

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

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27

17. Thomberry, N. A., Bull, H. G., Calaycay, J. R., Chapman, K. T., Howard, A. D., Kostura, M. J., Miller, D. K., Molineaiix, S. M., Weidner, J. R., Aunins, J., Elliston, K. O., Ayala, J. M., Casano, F. J., Chin, J., Ding, G. J. F., Egger, L. A., Gaffney, E. P., Limjuco, G., Palyha, O. C , Raju, S. M., Rolando, A. M., Salley, J. P., Yamin, T. T., Lee, T. D., Shively, J. E., MacCoss, M., Mumford, R. A., Schmidt, J. A., and Tocci, M. J. (1992). Nature {London) 356, 768-774. 18. Ceretti, D. P., Kozlosky, C. J., Mosley, B., Nelson, N., Van Ness, K., Greenstreet, T. A., March, C. J., Kronheim, S. R., Druck, T., Cannizzaro, L. A., Huebner, K., and Black, R. A. (1992). Science 256, 97-100. 19. Orlowski, M. (1990). Biochemistry 29, 10289-10297. 20. Tanaka, K., Tamura, T., Yoshimura, T., and Ichihara, A. (1992). New Biol 4, 173-187. 21. Piihler, G., Weinkauf, S., Bachmann, L., Muller, S., Engel, A., Hegerl, R., and Baumeister, W. (1992). EMBO J. 11, 1607-1616. 22. Rechsteiner, M., Hoffman, L., and Dubiel, W. (1993). J. Biol. Chem. 268,6065-6069. 23. Orlowski, M., Cardozo, C , and Michaud, C. (1993). Biochemistry 32, 1563-1572. 24. Vaithilingam, I., and Cook, R. A. (1989). Biochem. Int. 19, 1297-1307. 25. Benoist, P., Muller, A., Diem, H. G., and Schwencke, J. (1992). J. Bacteriol. 174, 1495-1504. 26. Zwickl, P., Grziwa, A., Piihler, G., Dahlmann, B., Lottspeich, F., and Baumeister, W. (1992). Biochemistry 31, 964-972. 27. Grziwa, A., Baumeister, W., Dahlmann, B., and Kopp, F. (1991). FEBS Lett. 290, 186-190. 28. Emori, Y., Tsukahara, T., Kawasaki, H., Ishiura, S., Sugita, H., and Suzuki, K. (1991). Mol. Cell. Biol. 11, 344-353. 29. Frentzel, S., Troxell, M., Haass, C , Pesold-Hurt, B., Glatzer, K. H., and Kloetzel, P.-M. (1992). Eur. J. Biochem. 205, 1043-1051. 30. van Riel, M. C. H. M., and Martens, G. J. M. (1991). FEBS Lett. 291, 37-40. 31. Tamura, T., Lee, D. H., Osaka, F., Fujiwara, T., Shin, S., Chimg, C. H., Tanaka, K., and Ichihara, A. (1991). Biochim. Biophys. Acta 1089, 95-102. 32. Tamura, T., Shimbara, N., Aki, M., Ishida, N., Bey, F., Scherrer, K., Tanaka, K., and Ichihara, A. (1992). J. Biochem. (Tokyo) 112, 530-534. 33. Wilk, S., and Orlowski, M. (1980). J. Neurochem. 35, 1172-1182. 34. Orlowski, M., and Wilk, S. (1981). Biochem. Biophys. Res. Commun. 101, 814-822. 35. Ciechanover, A., Heller, H., EUas, S., Haas, A. L., and Hershko, A. (1980). Proc. Natl. Acad. Sci. U.S.A. 77, 1365-1368. 36. Hershko, A., Ciechanover, A., Heller, H., Haas, A. L., and Rose, I. A. (1980). Proc. Natl. Acad. Sci. U.S.A. 77, 1783-1786. 37. Wilkinson, K. D., and Audhya, T. (1981). J. Biol. Chem. 256, 9235-9241. 38. Fried, V. A., Smith, H. T., Hildebrandt, E., and Weiner, K. (1987). Proc. Natl. Acad. Sci. U.S.A. 84, 3685-3689. 39. Speiser, S., and Etlinger, J. D. (1983). Proc. Natl. Acad. Sci. U.S.A. 80, 3577-3580. 40. Hough, R., Pratt, G., and Rechsteiner, M. (1986). J. Biol. Chem. 261, 2400-2408. 41. Hough, R,, Pratt, G., and Rechsteiner, M. (1987). J. Biol. Chem. 262, 8303-8313. 42. Hough, R., Pratt, G., and Rechsteiner, M. (1988). In 'TJbiquitin" (M. Rechsteiner, ed.), pp. 101-134. Plenum, New York. 43. Shelton, E., Kuff", E. L., Maxwell, E. S., and Harrington, J. T. (1970). J. Cell Biol. 45, 1-8. 44. Ganoth, D., Leshinsky, E., Eytan, E., and Hershko, A. (1988). J. Biol. Chem. 263, 12412-12419.

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