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[24] Multicatalytic endopeptidase complex: Proteasome
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After the genes for the different Clp components were cloned and mutations introduced into the chromosomal genes, it was shown that the Clp proteases had only a small role in abnormal protein degradation. 3'7 However, a variety of specific protein targets for Clp proteases have been identified. ClpAP degrades several/3-galactosidase fusion proteins 5 and may show specificity for the amino-terminal amino acid in at least one class of such substrates. 34 CIpXP degrades the highly unstable O protein of h phage in vivo (tl/2 ~ 2 rain) 7 and in vitro. 8 CIpXP appears to be involved in plasmid maintenance 35 and in phage mu virulence. 36 Data indicate that CIpP also contributes to degradation of proteins synthesized during carbon starvation, 37 and it is possible that CIpP and other Clp family members may have broader roles in both specific and nonspecific protein degradation in E. coli than is currently appreciated. 34 j. W. Tobias, T. E. Shrader, G. Rocap, and A. Varshavsky, Science 254, 1374 (1991). 35 M. Yarmolinsky (personal communication). 36 V. Geuskens, A. Mhamrnedi-Alaoui, L. Desmet, and A. Toussaint, EMBO J. 11, 5121 (1992). 37 K. Darnerau and A. C. St. John, J. Bacteriol. 175, 53 (1993).
[24] M u l t i c a t a l y t i c E n d o p e p t i d a s e C o m p l e x : P r o t e a s o m e By A. JENNIFER RIVETT, PETER J. SAVORY, and HAKIM DJABALLAH
Introduction The multicatalytic endopeptidase complex (EC 3.4.99.46) is a 700-kDa multisubunit enzyme complex that is widely distributed in eukaryotic cells. It has been described as a high molecular mass protease under many different names ~ and is apparently identical to a variety of cylindrical particles of unknown function, 2 as well as to the prosome, a particle that was believed to play a role in the control of translation. 3 The complex is now commonly referred to as either the multicatalytic proteinase complex ~.4
I A. J. Rivett, Arch. Biochem. Biophys. 268, 1 (1989). 2 p. E. Falkenberg, P. C. Haass, P. M. Kloetzel, B. Niedel, F. Kopp, L. Kuehn, and B. Dahlrnann, Nature (London) 331, 190 (1988). 3 H. P. Schmid, O. Akhayat, C. Martins de Sa, F. Puvion, K. Koehler, and K. Scherrer, EMBO J. 3, 29 (1984). 4 M. Orlowski, Biochemistry 29, 10289 (1990).
METHODS IN ENZYMOLOGY, VOL. 244
Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.
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or the proteasome) -7 The multicatalytic endopeptidase complex, either by itself or as the catalytic core of the 26S proteinase complex, 8,9is believed to play an important role in ubiquitin-dependent as well as ubiquitinindependent nonlysosomal pathways of protein turnover, including the degradation of regulatory proteins and the processing of antigens for presentation by the major histocompatibility complex (MHC) class I pathway. 6,7
Assays of Multicatalytic Endopeptidase Activity The term "multicatalytic" has been applied to the complex because it was realized from early work 1° that the broad specificity of the mammalian enzyme could be attributed to distinct types of catalytic sites, the activities of which can be distinguished using a variety of protease inhibitors and other effectors (see below). Activities responsible for cleavage on the carboxyl side of basic (usually Arg), hydrophobic (Leu, Tyr, Phe), and acidic (Glu) residues have been referred to as trypsin-like, chymotrypsin-like, and peptidylglutamyl-peptide bond hydrolase activities, respectively, l° although these terms are not very accurate in describing the specificities. The enzyme can also catalyze cleavage after other residues, including Gin, Thr, and Ala.10-12 Different substrates have been used to assay the endopeptidase activities of the complex purified from many different sources. Its substrates include proteins, peptides, and synthetic peptides (Table I) and the enzyme is active over a range of neutral to weakly alkaline pH values in a variety of different buffers. 11,13-15However, there can be significant variations in activity in different buffers, with Tris and both Na + and K + ions having been found to inhibit some activities. 10 Assaying multicatalytic endopeptidase activity is complicated by the fact that there are multiple distinct catalytic sites and interactions between them. The complex has some unusual kinetic properties (Table II) and there may be differences depending on the source of the enzyme and the 5 K. Tanaka, T. Tamura, T. Yoshimura, and A. Ichihara, New Biol. 4, 173 (1992)~ 6 A. L. Goldbergand K. L. Rock, Nature (London) 357, 375 (1992). 7A. J. Rivett, Biochem. J. 291, 1 (1993). 8A. Hershko and A. Ciechanover,Annu. Rev. Biochem. 61, 761 (1992). 9 M. Rechsteiner, L. Hoffman,and W. Dubiel, J. Biol. Chem. 268, 6065 (1993). l0 S. Wilk and M. Orlowski,J. Neurochem. 35, 1172(1980). it A. J. Rivett, J. Biol. Chem. 2,60, 12600(1985). 12H. Djaballah and A. J. Rivett, unpublishedobservations, (1992). z3K. Tanaka, T. Yoshimura,A. Kumatori,A. Ichihara,A. Ikai, M. Nishigai,K. Kameyama, and T. Takagi,J. Biol. Chem. 263, 16209(1988). 14R. W. Mason,Biochem. J. 265, 479 (1990). 15H. Djaballahand A. J. Rivett, Biochemistry 31, 4133 (1992).
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TABLE I SUBSTRATES OF MULTICATALYTICENDOPEPTIDASE COMPLEX
Type of substrate Proteins Peptides Synthetic peptides-7-amido-4-methylcoumarin (AMC) derivatives
Synthetic peptides--2naphthylamides Other synthetic peptides p-Nitroanilide Methoxynaphthylamide Thiobenzyl p-Aminobenzoate (pAB)
Examples
Refs.
Casein, oxidized enzymes, a-crystallin, lysozyme, serum albumin, hemoglobin Insulin B chain, substance P, glucagon, angiotensin I, and many others Suc-Leu-Leu-Val-Tyr-AMC (Suc)-AIa-AIa-Phe-AMC Boc-Leu-Ser-Thr-Arg-AMC Boc-Phe-Ser-Arg-AMC Boc-Leu-Arg-Arg-AMC Z-GIy-GIy-Arg-AMC Z-Leu-Leu-Glu-2-naphthylamide Z-oAla-Leu-Arg-2-naphthylamide
a-d
Z-Gly-Gly-Leu-p-nitroanilide Z-Ala-Ala-Arg-methoxynaphthylamide Boc-Ala-Ala-Asp-S-benzyl Z-GIy-Pro-Ala-Ala-Gly-pAB
a, e - g
e. f, h. i
e,j k ! m
a A. J. Rivett, J. Biol. Chem. 260, 12600 (1985). b A. J. Rivett, Arch. Biochem. Biophys. 243, 624 (1985). c K. Ray and H. Harris, Proc. Natl. Acad. Sci. U.S.A. 82, 7545 (1985). d M. Orlowski and C. Michaud, Biochemistry 28, 9270 (1989). e S. Wilk and M. Orlowski, J. Neurochem. 35, 1172 (1980). fA. J. Rivett, J. Biol. Chem. 264, 12215 (1989). g J. R. McDermott, A. M. Gibson, A. E. Oakley, and J. A. Biggins, J. Neurochem. 56, 1509 (1991). h K. Tanaka, K. Ii., A. Ichihara, L. Waxman, and A. L. Goldberg, J. Biol. Chem. 261, 15197 (1986). i S. Ishiura, T. Tsukahara, T. Tabira, and H. Sugita, FEBS Lett. 257, 388 (1989). J T. Achstetter, C. Ehmann, A. Osaki, and D. H. Wolf, J. Biol. Chem. 259, 13344 (1984). k j. Arribas and J. G. Castafio, J. Biol. Chem. 265, 13969 (1990). i H. Djaballah and A. J. Rivett, Biochemistry 31, 4133 (1992). 0' M. Odowski, C. Cardozo, and C. Michaud, Biochemistry 32, 1563 (1993).
purification procedure. There are latent and active forms of the complex (for references, see Table II) as well as the possibility of different subpopulations. 16,17 A number of synthetic peptides have proved useful for assaying individual activities of the multicatalytic endopeptidase complex (examples in Table I). The 7-amino-4-methylcoumarin leaving group provides the most sensitive assay and these synthetic peptides are often used at concentra16p. E. Falkenburg and P. M. Kloetzel, J. Biol. Chem. 264, 6660 (1989). 17M. G. Brown, J. Driscoll, and J. J. Monaco, J. lmmunol. 115, 1193 (1993).
TABLE II KINETIC PROPERTIES OF PURIFIED EUKARYOTIC MULTICATALYTIC ENDOPEPTIDASE COMPLEXES
Kinetic property
Refs.
Broad specificity of bond cleavage due to multiple distinct catalytic components, which can be assayed using appropriate synthetic substrates and distinguished using inhibitors and other effectors (see Tables V-VII) Can be isolated in "latent" form; activation by removal of glycerol, by dialysis, by heat treatment, or by addition of polylysine Optimum pH range, pH 7-9, depending on substrate No protease inhibitor yet found to block all peptidase activities; different catalytic components have very different reactivity with some protease inhibitors Addition of inhibitors of one activity can affect kinetic parameters at other catalytic sites Activation (see Table VI for effectors) involves conformational changes, allosteric effects Some peptidase activities show positive cooperativity, possible substrate channeling Mechanism not established but believed to be an unusual type of serine peptidase Some differences in kinetic characteristics of enzyme isolated from different sources
a-d
e-h
f,i c, d; see also Table VII
j--l m-o
i, o, p q, r c,d,l
a S. Wilk and M. Orlowski, J. Neurochem. 35, 1172 (1980). b B. Dahlmann, L. Kuehn, M. Rutschmann, and H. Reinauer, Biochem. J. 228, 161 (1985). c H. Djaballah, J. A. Harness, P. J. Savory, and A. J. Rivett, Eur. J. Biochem. 209, 629 (1992). d M. Orlowski, C. Cardozo, and C. Michaud, Biochemistry 32, 1563 (1993). e K. Tanaka, K. Ii., A. Ichihara, L. Waxman, and A. L. Goldberg,, J. Biol. Chem. 261, 15197 (1986). f K. Tanaka, T. Yoshimura, A. Kumatori, A. Ichihara, A. Ikai, M. Nishigai, K. Kameyama, and T. Takagi, J. Biol. Chem. 263, 16209 (1988). g D. L. Mykles, Arch. Biochem. Biophys. 274, 216 (1989)o h D. Weitman and J. D. Etlinger, J. Biol. Chem. 267, 6977 (1992). i H. Djaballah and A. J. Rivett, Biochemistry 31, 4133 (1992). J S. Wilk and M. Orlowski, J. Neurochem. 40, 842 (1983). C. Cardozo, A. Vinitsky, M. C. Hidalgo, C. Michaud, and M. Orlowski, Biochemistry 31, 7373 (1992). t H. Djaballah, P. J. Savory, and A. J. Rivett, unpublished observations. m H. Djaballah, A. J. Rowe, S. E. Harding, and A. J. Rivett, Biochem. J. 292, 857 (1993). n j. Arribas and J. G. Castafio, J. Biol. Chem. 265, 13969 (1990). o M. Orlowski, C. Cardozo, M. C. Hidalgo, and C. Michaud, Biochemistry 30, 5999 (1991). P L. R. Dick, C. R. Moomaw, G. N. DeMartino, and C. A. Slaughter, Biochemistry 30, 2725 (1991). q M. Orlowski, Biochemistry 29, 10289 (1990). r A. J. Rivett, Biochem. J. 291, 1 (1993).
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)he-VaI-Asn-GIn- His-Leu- Cya-Gly-Ser-His-Leu- VaI-Glu- Ala-Leu- Tyr-Leu- VaI-Cya- Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-AI
¢ I
¢ ~
¢ I
¢ I
¢ ~
(a) Ra~,w~ (b)Human e~tthrocyte
FIG. 1. Multicatalytic endopeptidase complex cleavage sites in oxidized insulin B chain. (a) A. J. Rivett, J. Biol. Chem. 260, 12600 (1985); (b) L. R. Dick, C. R. Moomaw, G. N. DeMartino, and C. A. Slaughter, Biochemistry 30, 2725 (1991); and (c) T. Takahashi, T. Tokumoto, K. Ishikawa, and K. Takahashi, J. Biochem. (Tokyo) 113, 225 (1993).
tions below their K m values, which are in the range of 0.1-1 mM. t8 Other fluorogenic or chromogenic leaving groups are also suitable (Table I). In particular, Z-Leu-Leu-Glu-2-naphthylamide is commonly used to assay peptidylglutamyl-peptide hydrolase activity. For some activities of the bovine pituitary enzyme a coupled assay with an aminopeptidase has been u s e d ) 9 Although we describe below stopped-fluorimetric assay procedures, it is necessary to establish that the production of product is linear with both time and enzyme concentration, because this is not always the case. Fluorimetric assays can of course be carded out continuously using a water-jacketed cell holder to maintain a constant temperature. The degradation of peptide substrates such as oxidized insulin B chain (Table I) can be monitored by C18 reversed-phase high-performance liquid chromatography (HPLC). Some differences have been observed in the cleavage pattern obtained with multicatalytic endopeptidases from different sources (Fig. 1). 12 The degradation of protein substrates, of which casein has been the most widely used, is easily assayed by measuring acidsoluble counts released from radiolabeled protein (see below). Alternative methods for investigating protein degradation include sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and the use of fluorescamine to determine the level of acid-soluble products from unlabeled proteins. 2° Not all proteins are substrates for the complex but it is not yet entirely clear what structural features of proteins determine proteolytic susceptibility. 21,22 Those proteins which are substrates are usually degraded to peptides." 18 A. J. Rivett, J. Biol. Chem. 264, 12215 (1989). 19 M. Orlowski, C. Cardozo, and C. Michaud, Biochemistry 32, 1563 (1993). 2o A. J. Rivett, Arch. Biochem. Biophys. 243, 624 (1985). 21 A. J. Rivett and R. L. Levine, Arch. Biochem. Biophys. 2/8, 26 (1990). 22 R. E. Pacifici, Y. Kono, and K. J. A. Davies, J. Biol. Chem. 268, 15405 (1993).
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SERINE PEPTIDASES
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Assays with 7-Amido-4-methylcoumarin Substrates A variety of different commercially available synthetic peptides may be used to assay peptidase activities of the complex and the assay procedure is the same for all of them. Although the pH optimum varies for different substrates, we routinely use buffer of pH 7.5. Commonly used peptidyl7-amino-4-methylcoumarin substrates are listed in Table I. We routinely use Ala-Ala-Phe-7-amido-4-methylcoumarin, Suc-Leu-Leu-Val-Tyr-7amido-4-methylcoumarin, and Boc-Leu-Ser-Thr-Arg-7-amido-4-methylcoumarin.
Reagents Buffer: 100 mM HEPES/KOH, pH 7.5, diluted to 50 mM in the assay. Substrate stock solutions: These can be made at 2 or 10 mM in water, 50 mM acetic acid, or dimethyl sulfoxide (depending on the solubility of the substrate) and stored in aliquots at - 2 0 °. Dimethyl sulfoxide concentrations in the assays should not normally exceed 5%. Stop mix: Sodium acetate trihydrate (0.25 g) is added to 4.375 ml 1 M acetic acid and made up to a volume of 25 ml with water. Procedure. Assay mixtures containing approximately I-2/zg enzyme, substrate (at a fixed concentration, e.g., 20 or 50/zM), and 50 mM HEPES/ KOH, pH 7.5, are made up in a total volume of 200/zl in 4-ml disposable test tubes and then incubated at 37° for 15 or 30 min. Reactions are started by addition of either substrate or enzyme and are stopped by addition of 0.1 ml stop mix. H20 (2 ml) is added to each tube prior to measuring fluorescence (excitation 370 nm, emission 430 nm). Blanks are prepared without the addition of enzyme and a standard curve is prepared with 7amino-4-methylcoumarin.
Assay with Z-Leu-Leu-Glu-2-naphthylamide The substrate is hydrolyzed to give 2-naphthylamine, which can be either assayed colorimetrically by coupling with a diazonium salt or measured directly in a fluorimeter. This product is probably carcinogenic and cannot easily be purchased, so we make just enough by an enzymatic procedure to produce a standard curve.
Reagents Buffer: 100 mM HEPES/KOH, pH 7.5, diluted to 50 mM in the assay. Substrate stock solution: 10 mM Z-Leu-Leu-Glu-2-naphthylamide in dimethyl sulfoxide (DMSO), stored in aliquots at - 2 0 °.
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Procedure. Assay mixes are made to a volume of 200/zl in 4-ml disposable test tubes. Usually 1-2 /zg of enzyme is used with a final buffer concentration of 50 mM. The amounts of substrate and water are varied. The amount of sub strate is limited to a maximum of 0.6 mM by its solubility in aqueous solutions. 15 There are two distinct types of catalytic centers (LLE1 and LLE2), which possess peptidylglutamyl-peptide hydrolase activity? 5 We usually assay with substrate at concentrations of 0.1 mM (LLE1 activity) and 0.4 mM (LLE1 + LLE2 activity). Because the kinetic characteristics of the peptidylglutamyl-peptide hydrolase activities (see later) of the rat liver and bovine pituitary enzymes are not identical, 15'23 they should be tested in other systems. Assay mixtures are incubated at 37° usually for 30 min and are then stopped by addition of 0.3 ml ethanol. Water (2 ml) is added prior to reading samples in a fluorimeter (excitation 333 nm, emission 450 nm). Blank samples are prepared at the appropriate substrate concentrations with no enzyme. 2-Naphthylamine is produced by complete digestion of a known amount of Z-Leu-Leu-Glu-2-naphthylamide (e.g., 0.6 mM) by Staphylococcus aureus V8 proteinase in 50 mM ammonium bicarbonate buffer, pH 7.8. A standard curve can then be prepared using the 50 mM H E P E S / K O H , pH 7.5, assay buffer, and diluting samples with ethanol and water as described above. Assay with Casein as Substrate Protein substrates are usually degraded to low molecular mass trichloroacetic acid-soluble products. Therefore, assays with radiolabeled substrate are most convenient. Reagents Buffer: 100 mM H E P E S / K O H , pH 8.0, diluted to 50 mM in the assay. Substrate stock solution: Casein (14C, 3H, or lzsI labeled), stored in aliquots at - 2 0 °. Others: 10% (w/v) trichloroacetic acid (TCA); 5% (w/v) bovine serum albumin (BSA) in 0.1 N HCI. Procedure. Assays are carried out in a 100-/~1 volume using 1-2 ~g enzyme with 10/.~g casein in 50 mM H E P E S / K O H buffer, pH 8.0, in 1.5ml Eppendorf tubes. Incubations are at 37° for 1 hr. Assays are stopped by the addition of 0.5 ml of the 10% trichloroacetic acid followed by addition of 0.1 ml of the bovine serum albumin solution. Samples are left on ice for 10 rain and then centrifuged in a microfuge for 5 min. Scintillant 23 M. Orlowski, C. Cardozo, M. C. Hidalgo, and C. Michaud, Biochemistry 311,5999 (1991).
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TABLE III SOURCES USED FOR PURIFICATION OF MULTICATALYTIC ENDOPEPTIDASE COMPLEXa
Source
Ref.
Animal tissues and cells Liver (e.g., mouse, rat, human) Muscle (rat) Lung (human) Brain (human, bovine) Pituitary (bovine) Lens (bovine) Placenta (human) Erythroeytes (human) Plants (tobacco, potato, mung bean, pea seed) Frog (Xenopus laevis or Rana pipiens): oocytes Lobster: muscle Fish (carp, white croaker): muscle Sea urchin: eggs, sperm Yeast (Saccharomyces cerevisiae) Archaebacteria (Thermoplasma acidophilium)
b-e f g h, i j, k l m n o, p q, r s t, u o, w x y
a The enzyme has been described under many different names [reviewed in A. J. Rivett, Arch. Biochem. Biophys. 268, 1 (1989)]. b A. J. Rivett, J. Biol. Chem. 7,60, 12600 (1985). c K. Tanaka, K. Ii., A. Ichihara, L. Waxman, and A. L. Goldberg, J. Biol. Chem. 261, 15197 (1986). d R. W. Mason, Biochem. J. 265, 479 (1990). ' H. Djaballah, and A. J. Rivett, Biochemistry 31, 4133 (1992). f B. Dahlmann, L. Kuehn, M. Rutschmann, and H. Reinauer, Biochem. J. 228, 161 (1985). R. Zolfaghari, C. R. F. Baker, P. C. Canizaro, A. Amirgholami, and F. J. Behal, Biochem. J. 241, 129 (1987). h A. Azaryan, M. Banay-Schwartz, and A. Lajtha, Neurochem. Res. 14, 995 (1989). J J. R. McDermott, A. M. Gibson, A. E. Oakley, and J. A. Biggins, J. Neurochem. 56, 1509 (1991). YS. Wilk and M. Orlowski, J. Neurochem. 35, 1172 (1980). k M. Orlowski and C. Michaud, Biochemistry 28, 9270 (1989). t K. Ray and H. Harris, Proc. Natl. Acad. Sci. U.S.A. 82, 7545 (1985). ,n K. B. Hendil and W. Uerkvitz, J. Biochem. Biophys. Methods 22, 159 (1991). n M. J. McGuire and G. N. DeMartino, Biochim. Biophys. Acta. 873, 279 (1986). o M. Schliephacke, A. Kremp, H. P. Schmid, K. Kohler, and U. Kull, Eur. J. CellBiol. 55, 114 (1991).
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TABLE III (continued) p B. Skoda and L. Malek, Plant Physiol. 99, 1515 (1992). q K. Tanaka, T. Yoshimura, A. Kumatori, A. Ichihara, A. Ikai, M. Nishigai, K. Kameyama, and T. Takagi, J. Biol. Chem. 263, 16209 (1988). r y. Azuma, T. Tokumoto, and K. Ishikawa, Mol. Cell. Biochem. 100, 177 (1991). s D. L. Mykles, Arch. Biochem. Biophys. 274, 216 (1989). t M. Kinoshita, H. Toyohara, and Y. Shimizu, Comp. Biochem. Physiol. 96B, 565 (1990). u E. J. Folco, L, Busconi, C. B. Martone, and J. J. Sanchez, Arch. Biochem. Biophys. 267, 599 (1988), v j. L. Grainger and M. M. Winkler, J. Cell Biol. 109, 675 (1989). wK. Matsumura and K. Aketa, Mol. Reprod. Dev. 29, 189 (1991). x T. Achstetter, C. Ehmann, A. Osaki, and D. H. Wolf, J. Biol. Chem. 259, 13344 (1984). YB. Dahlmann, F. Kopp, L. Kuehn, B. Niedel, G. Pfeifer, R. Hegerl, and W. Baurneister, FEBS Lett. 251, 125 (1989). (10 ml) is added to an aliquot of the supernatant (0.63 ml) and samples are then counted in a liquid scintillation counter. Purification of the Multicatalytic Endopeptidase Complex The multicatalytic endopeptidase complex is an abundant protein and can constitute up to 1% of the soluble cellular protein, with the highest levels in animal cells and tissues being found in the liver. 24,25 The e n z y m e has b e e n purified f r o m a wide variety of sources (Table liD. Purification p r o c e d u r e s often involve a combination of anion-exchange chromatography and gel filtration, often p r e c e d e d by an a m m o n i u m sulfate fractionation, and usually involve additional chromatographic steps such as hydroxylapatite, h y d r o p h o b i c , h e p a r i n - S e p h a r o s e or Affi-Gel blue chrom a t o g r a p h y or a second ion-exchange or gel-filtration step. Purification b y immunoaffinity c h r o m a t o g r a p h y has also b e e n described. 26 The yield of multicatalytic endopeptidase c o m p l e x that can be e x p e c t e d depends on the source but is usually in the range of 1-10 rag/100 g tissue. 13,~5 A low a m o u n t obtained f r o m e r y t h r o c y t e s 27 reflects the relatively low level 24K. Tanaka, K. Ii., A. Ichihara, L. Waxman, and A. L. Goldberg, J. Biol. Chem. 261, 15197 (1986). 25A. J. Rivctt and S, T. Sweeney, Biochem. J. 278, 171 (1991). 26K. B. Hendi| and W. Uerkvitz, J. Biochem. Biophys. Methods 22, 159 (1991). 27 M. J. McGuire and G. N. DeMartino, Biochim. Biophys. Actu. 873, 279 (1986).
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SERINE PEPTIDASES
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of the complex in these cells. 25 The enzyme is usually prepared from soluble extracts, but purification from erythrocyte membranes has also been reported. 28 The enzyme can be purified in a latent form (references in Table II), the difference in procedure usually involving the inclusion 0f20% glycerol in all the buffers for the latent enzyme. There are only minor differences between latent and active forms in the molecular masses and pI values of the associated polypeptides? 9'3° In the majority of cases (Table I) the enzyme has been purified in the active state. It is, however, quite difficult to compare the specific activities of the final preparations obtained using different procedures, for seveal reasons. First, the purification procedure may have an effect on activity; second, the activity can be markedly influenced by the composition of the buffer (see below); and third, the activities have often not been assayed using the same substrates under identical conditions. The purification-fold can also be misleading. For example, it is not straightforward to measure activity in crude extracts of animal cells using protein substrates,1~,27 possibly due to the presence of endogenous inhibitor proteins 3L32 or competing protein substrates. With synthetic peptide substrates, the ratio of activity with different substrates varies at different stages of the purification 33 and some, if not all, of the usual substrates are also hydrolyzed by other peptidases. With apparently homogeneous enzyme preparations from different sources it is also difficult to make comparisons because of differences in assay conditions and possibly also some kinetic differences, the basis for which we do not yet understand. The kinetic characteristics of the purified multicatalytic endopeptidase complex (Table I) will be discussed in more detail below. Purification of the Multicatalytic Endopeptidase from Rat Liver The enyzme can be purified from fresh or frozen rat liver using the following procedure. All steps are carried out at 4 ° except for those involv28 M. Kinoshita, T. Hamakubo, I. Fukui, T. Murachi, and H. Toyohara, J. Biochem. (Tokyo) 107, 440 (1990). 29 L. W. Lee, C. R. Moomaw, K. Orth, M. J. McGuire, G. N. DeMartino, and C. A. Slaughter, Biochim. Biophys. Acta 1037, 178 (1990). 30 M. E. Pereira, T. Nguyen, B. J. Wagner, J. W. Margolis, B. Yu, and S. Wilk, J. Biol. Chem. 267, 7949 (1992). 31 X. Li, M. Gu, and J. D. Etlinger, Biochemistry 30, 9709 (1991). 32 M. Chu-Ping, C. A. Slaughter, and G. N. DeMartino, Biochim. Biophys. Acta 1119, 303 0992). 33 S. Wiik and M. Orlowski, J. Neurochem. 40, 842 (1983).
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ing fast protein liquid chromatography (FPLC), which are performed at room temperature. Column and buffer volumes are given for a preparation from 200 g rat liver. We recommend Ala-Ala-Phe-7-amino-4-methylcoumarin as the substrate for assays during the purification. Step I. Homogenization and Centrifugation. Rat livers are washed in homogenization buffer (20 mM HEPES, pH 8.0, containing 1 mM 2-mercaptoethanol and 1 mM EDTA) and then homogenized (3 × 30 sec in a Waring blendor) in 5 volumes of buffer. The homogenate is centrifuged at 27,000 g for 2.5 hr. Step 2. Ammonium Sulfate Fractionation. Solid ammonium sulfate to give 35% saturation is added to the supernatant from step 1. After stirring for 30 min on ice, the precipitate is removed by centrifugation and ammonium sulfate is added to the supernatant to give 60% saturation. Proteins precipitated during the second ammonium sulfate precipitation (left stirring for 45 rain after all the ammonium sulfate has dissolved) are collected by centrifugation and redissolved in approximately 200 ml of 10 mM TrisHCI buffer, pH 7.2. The preparation is dialyzed against 2 x 5 liters l0 mM Tris-HCl buffer, pH 7.2, containing 50 mM KCI, 0.1 mM EDTA. 1 mM 2-mercaptoethanol. Step 3. DEAE-Cellulose Chromatography. The dialyzed ammonium sulfate fraction is centrifuged at 45,000 g for 20 min and then loaded on to a DEAE-cellulose column (2.6 x 30 cm) equilibrated in l0 mM TrisHC1, pH 7.2, containing 50 mM KC1 at a flow rate of approximately 2 ml/ min. After washing the column with equilibration buffer, a gradient of 50-350 mM KCI (2 × 1.5 liters) is applied. The enzyme elutes at approximately 150 mM KCI. Active fractions are pooled (approximately 400 ml) and concentrated to a volume of about 100 ml using an Amicon (Danvers, MA) ultrafiltration cell with an XMS0 membrane. EDTA (0.1 mM) and 2-mercaptoethanol (1 mM) are added to the preparation after pooling fractions from this and all the subsequent chromatography steps. Step 4. Mono Q Anion-Exchange Chromatography. The DEAE-ceUulose pool is diluted with one-half volume of 20 mM Tris-HC1, pH 7.2, and then divided into at least two portions. The portions are loaded separately on to a Mono Q 10/10 FPLC column (Pharmacia, Piscataway, NJ) equilibrated in 20 mM Tris-HCl, pH 7.2, containing 100 mM KC1 at a flow rate of 4 ml/min. The enzyme is eluted on a gradient (200 ml) of 100-500 mM KCI at a KCI concentration of about 400 mM (usually peak fraction 19 out of 25 x 8 ml fractions). Active fractions are pooled and concentrated to less than l0 ml using an Amicon ultrafiltration cell (XMS0 membrane). Step 5. Superose 6 Gel Filtration. The sample is filtered and loaded onto a preparative-size Superose 6 FPLC column (Pharmacia), equilibrated in 50 mM potassium phosphate buffer, pH 7.0, containing 0.1 M
342
SERINE PEPTIDASES
[24]
KCI. Although the sample could be concentrated more and loaded for one column run, we find that the resolution and recovery are better if the sample is divided into two or three portions, loading 2 or 3 ml each time. The enzyme elutes at approximately the same volume as thyroglobulin. Step 6. Mono Q Ion-Exchange Chromatography. We usually find it helpful to carry out a final ion-exchange (Mono Q 5/5) chromatography step. The pool from the gel-filtration step is therefore dialyzed against 20 mM Tris-HCl buffer, pH 7.2, and then run (<5 mg/run) on the Mono Q 5/5 column equilibrated in the same buffer and eluted at approximately 0.35 - 0.4 M KCI by applying a linear gradient to 500 mM KCI. The active fractions from this column are pooled and dialyzed against 50 mM potassium phosphate, pH 7.0, containing 1 mM dithiothreitol, 0. I mM EDTA, and 10% glycerol. Protein Determination. The amount of enzyme can be quantitated using the Bradford method 34 for protein determination with bovine serum albumin as standard, because this gives a very good estimate of actual protein concentration determined by amino acid analysis) 5 Purity. The purity of the preparation is best assessed by PAGE carried out under nondenaturing conditions, ~ and the enzyme can be visualized under UV light in nondenaturing gels following incubation with fluorogenic peptide substrates (for example, at a concentration of 50-100 tzM in 50 mM HEPES buffer, pH 7.5, incubated at 37° for 1 hr). There has been a report suggesting multiple electrophoretic forms of the enzyme, 36but these are not always observed. 11SDS-PAGE gels can also be used to determine whether preparations contain any contaminating proteins in addition to the characteristic ladder of multicatalytic endopeptidase complex bands of M r of 20-34 kDa. 11'15 Specific Activity. The specific activities of the rat liver complex assayed with 50/zM Ala-Ala-Phe-7-amido-4-methylcoumarin and 0.4 mM Z-LeuLeu-Glu-2°naphthylamide are usually in the ranges of 12-25 nmol/min/mg protein and 100-150 nmol/min/mg, respectively. Storage. The purified enzyme is stable for several months when stored frozen at - 2 0 ° in 50 mM potassium phosphate buffer, pH 7.0, containing 10% glycerol, 1 mM DTT, 0.1 mM EDTA. Purification of 26S Proteinase The multicatalytic endopeptidase complex forms part of the 26S proteinase (also called ubiquitin-conjugate degrading proteinase), and several 34 M. M. Bradford, Anal. Biochem. 72, 248 (1976). 35 p. j. Savory and A. J. Rivett, Biochem. J. 289, 45 (1993). 36 L. Hoffman, G. Pratt, and M. Rechsteiner, J. Biol. Chem. 267, 22362 (1992).
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purification protocols have been reported for this larger proteinase complex. 9'37-4° The 26S proteinase, which is precipitated at lower ammonium sulfate concentrations than the multicatalytic endopeptidase complex alone, appears to require ATP for its stability. Also, unlike purified multicatalytic endopeptidase complex preparations, ~ it catalyzes ATP-dependent protein degradation. 26S proteinase activity, as well as multicatalytic endopeptidase activity, can conveniently be assayed with Suc-Leu-LeuVal-Tyr-7-amido-4-methylcoumarin or with casein as a substrate. Properties of Purified Multicatalytic Endopeptidase Complex The size, shape, and subunit structure of multicatalytic endopeptidase complexes from eukaryotic cells are broadly similar irrespective of the source of the enzyme, and are summarized in Table IV. The number of different types of polypeptide associated with the complex does seem to vary with the source and it is not yet clear whether these differences can be explained solely in terms of post-translational modification, such as phosphorylation, glycosylation, and proteolysis, cDNAs have been cloned for many yeast, rat, and human multicatalytic endopeptidase subunits. 5'7 All subunits of the complex are encoded by members of the same gene family, with different subunits from the same source having 18-40% identity. The individual subunit sequences are highly conserved between different mammalian species (rat/human 95-98% identity at the amino acid level) and are 40-70% identitical with the most closely related subunits in yeast. The multicatalytic endopeptidase complex isolated from the archaebacterium Thermoplasrna acidophilum is an interesting and useful one because it is a much simpler molecule than the eukaryotic complex. The overall structure is similar, 41but it is composed of only two different types of subunit, a and fl (a14f114 stoichiometry). The subunit sequences are related to those of all the eukaryotic multicatalytic endopeptidase complex subunits. 42 The a subunits are located on the outer rings of the cylinder 37 E. Eytan, D. Ganoth, T. Armon, and A. Hershko, Proc. Natl. Acad. Sci. U.S.A. 86, 7751 (1989). 38 j. Driscoll and A. L. Goldberg, J. Biol. Chem. 265, 4789 (1990). 39 H. Kanayama, T. Tamura, S. Ugai, S. Kagawa, N. Tanahashi, T. Yoshimura, K. Tanaka, and A. Ichihara, Eur. J. Biochem. 206, 567 (1992). 40 A. Azaryan, M. Banay-Schwartz, and A. Lajtha, Neurochem. Res. 14, 995 (1989). 41 G. P0hler, S. Weinkauf, L. Bachmann, S. MOiler, A. Engel, R. Heged, and W. Baumeister, EMBO J. 11, 1607 (1992). 42 p. Zwickl, A. Grziwa, G. Piihler, B. Dahlmann, F. Lottspeich, and W. Baumeister, Biochemistry 31, 964 (1992).
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SERINE PEPTIDASES
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TABLE IV PROPERTIES OF EUKARYOTICMULT1CATALYTICENDOPEPTIDASECOMPLEX Property
Comments
Refs.
Occurrence
Found in all types of cells (see also Table III); usually in nucleus as well as in cytoplasm Can constitute up to 1% of cellular protein Has a molecular mass of around 700,000 Da; also forms part of 26S proteinase Many (14-20) different polypeptides Mr of 20,000-34,000, pl 4-9; some may be related by post-translational modification Has very low amounts of small species (approximately 80 nucleotides) of RNA associated Has a cylindrical structure (approximately 11 x 17 nm) Pseudo-helical arrangement of subunits Possibly a complex dimer Subunits are encoded by members of same gene family, but can be divided into two groups, A (a) and B ~), related to archaebacterial a and fl subunits Genes are located on different chromosomes; some are essential for cell proliferation Mammalian complex contains at least five distinct peptidase sites (for substrates, see Table I; other kinetic properties, see Table II) Ubiquitin-dependent and ubiquitin-independent nonlysosomal pathways of protein breakdown, including the degradation of regulatory proteins, and probably antigen processing for presentation by MHC class I pathway
a-c
Level Size Subunits RNA Shape
Genes
Activity
Function
d, e f, g h, i j, k l m n o, p
i, q, r
i, o, p, s, t
a K. Tanaka, A. Kumatori, K. Ii, and A. Ichihara, J. Cell. Physiol. 139, 34 (1989). A. J. Rivett, A. Palmer, and E. Knecht, J. Histochem. Cytochem. 40, 1165 (1992). c A. J. Rivett and E. Knecht, Curr. Biol. 3, 127 (1993). d L. Tanaka, K. Ii., A. Ichihara, L. Waxman, and A. L. Goldberg, J. Biol. Chem. 261, 15197 (1986). e K. B. Hendil, Biochem. Int. 17, 471 (1988). [ M. Rechsteiner, L. Hoffman, and W. Dubiel, J. Biol. Chem. 268, 6065 (1993). g A. Hershko and A. Ciechanover, Annu. Rev. Biochem. 61, 761 (1992). h A. J. Rivett and S. T. Sweeney, Biochem. J. 278, 171 (1991). i W. Heinemeyer, J. A. Kleinschmidt, J. Saidowsky, C. Escher, and D. H. Wolf, EMBO J. 10, 555 (1991). J H. S. Skilton, I. C. Eperon, and A. J. Rivett, FEBS Lett. 279, 351 (1991). k H. G. Nothwang, O. Coux, G. Keith, I. Silva-Pereira, and K. Scherrer, Nucleic Acids Res. 20, 1959 (1992). t W. Baumeister, B. Dahlmann, R. Hegerl, F. Kopp, L. Kuehn, and G. Pfeifer, FEBS Lett. 241, 239 (1988). m H. Djaballah, A. J. Rowe, S. E. Harding, and A. J. Rivett, Biochem. J. 292, 857 (1993). n F. Kopp, B. Dahlmann and K. B. Hendil, J. Mol. Biol. 229, 14 (1993). o K. Tanaka, T. Tamura, T. Yoshimura, and A. Ichihara, New Biol. 4, 173 (1992).
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TABLE IV (continued) P A. J. Rivett, Biochem. J. 291, 1 (1993). q T. Fujiwara, K. Tanaka, E. Orino, T. Yoshimura, A. Kumatori, T. Tamura, C. H. Chung, T. Nakai, K. Yamaguchi, S. Shin, A. Kakizuka, S. Nakanishi, and A. Ichihara, J. Biol. Chem. 265, 16604 (1990). Y. Emori, T. Tsukahara, H. Kawasaki, S. Ishiura, H. Sugita, and K. Suzuki, Mol. Cell. Biol. 11, 344 (1991). M. Orlowski, Biochemistry 29, 10289 (1990). t A. L. Goldberg and K. L. Rock, Nature (London) 357, 375 (1992).
and the fl subunits are in the inner rings. 43 The subunits of the eukaryotic multicatalytic endopeptidase complex can be divided into two groups, A (a) and B (fl),6 depending on whether they resemble the archaebacterial o~or/3 subunit. The A group members are quite similar, with a very highly conserved region close to the N terminus, which is blocked. The B group members, on the other hand, are not so closely related to each other and have a variable region at the N terminus, which is often unblocked. 44
Catalytic Mechanism and Identification of Catalytic Components The enzyme is now widely believed to be an unusual type of serine peptidase based on its inhibitor specificity (Table V). However, a definite assignment must await the identification of catalytic residues. Although it seems likely that the different catalytic centers of the multicatalytic endopeptidase complex are mechanistically related, there are remarkable differences in reactivity of the different peptidase sites with some serine peptidase inhibitors.4S For example, a given inhibitor can rapidly inactivate one activity while having no effect on another. Such observations can explain why in some of the earliest studies the enzyme was not thought to be a serine protease. The inhibition observed with thiol-reactive reagents is probably nonspecific, because activity of the Thermoplasma enzyme is unaffected by even the mercury-containing thiol reagents. 46 Because the primary structures of the multicatalytic endopeptidase complex subunits bear no resemblence to any other known peptidases, the catalytic components cannot be identified from sequence information. Also, dissociated 43 A. Grziwa, W. Baumeister, B. Dahlmann, and F. Kopp, FEBS Left. 290, 186 (1991). 44 K. S. Lilley, M. D. Davison, and A. J. Rivett, FEBS Lett. 262, 327 (1990). 45 H. Djaballah, J. A. Harness, P. J. Savory, and A. J. Rivett, Eur. J. Biochem. 209, 629 (1992). B. Dahlmann, L. Kuehn, A. Grziwa, P. Zwickl, and W. Baumeister, Eur. J. Biochem. 208, 789 (1992).
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SERINE PEPTIDASES
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TABLE V PROTEASEINHIBITORSEFFECTIVEON MAMMALIANMULTICATALYTIC ENDOPEPTIDASEACTIVITIESa Type of inhibitor Organophosphorus compound Sulfonyl fluoride Isocoumarins Peptide aldehydes Peptidyl chloromethanes Peptidyl diazomethanes Thiol-reactive reagents
Examples Diisopropyl fluoropbosphate 4-(2-Aminoethyl)benzene sulfonyl fluoride 3,4-Dichloroisocoumarin; others Leupeptin, antipain Chymostatin and analogs Z-Leu-Leu-PheH Tyr-GIy-Arg-CH2CI (some have no effect) Z-Leu-Leu-Tyr-CHN2, Z-Phe-Gly-TyrCHN 2 (some have no effect) N-Ethylmaleimide, p-hydroxymercuribenzoate, others
Refs. b-d
c c, e. f
g, h c i j, k j g, l
a There are marked differences in reactivity of different peptidase sites. None of the inhibitors here inhibit all activities of the mammalian multicatalytic endopeptidase complex (see also Table VII). b K. Tanaka, K. Ii., A. Ichihara, L. Waxman, and A. L. Goldberg, J. Biol. Chem. 261, 15197 (1986). ¢ H. Djaballah, J. A. Harness, P. J. Savory, and A. J. Rivett, Eur. J. Biochem. 209, 629 (1992). M. Orlowski, C. Cardozo, and C. Michaud, Biochemistry 32, 1563 (1993). e M. Orlowski and C. Michaud, Biochemistry 28, 9270 (1989). f R. W. Mason, Biochem. J. 265, 479 (1990). g S. Wilk and M. Orlowski, J. Neurochem. 40, 842 (1983). h p. j. Savory and A. J. Rivett, Biochem. J. 289, 45 (1993). i A. Vinitsky, C. Michaud, J. C. Powers, and M. Orlowski, Biochemistry 31, 9421 (1992). i p. j. Savory, H. Djahallah, H. Angliker, E. Shaw, and A. J. Rivett, Biochem. J. 296, 601 (1993). k C. Cardozo, A. Vinitsky, M. C. Hidalgo, C. Michaud, and M. Orlowski, Biochemistry 31, 7373 (1992). t A. J. Rivett, J. Biol. Chem. 260, 12600 (1985).
subunits are inactive. It is possible that the e n z y m e has a n o v e l t y p e o f catalytic m e c h a n i s m . T h e distinction b e t w e e n different catalytic activities o f the e u k a r y o t i c c o m p l e x is b a s e d o n the use o f different synthetic peptide substrates with a v a r i e t y o f p r o t e a s e inhibitors a n d o t h e r effectors (Table VI). T h e effects o f a c t i v a t o r s such as low c o n c e n t r a t i o n s o f S D S can be quite variable d e p e n d i n g o n the s o u r c e o f the e n z y m e , and, at least in s o m e cases,
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347
TABLE VI OTHER EFFECTORS OF MULTICATALYTICENDOPEPT1DASE ACTIVITIES Tested effector
Concentration
Effect on activity a'b
EDTA MnCI2, MgCI2, CaCI2 ZnCI2 KCI ATP Polylysine Linoleic acid, oleic acid Sodium dodecyl sulfate Nonidet P-40 Guanidine hydrochloride
1 mM 1 mM 0.1 mM 25 mM 1-5 mM 0.1 mg/ml 0.2-5 mM 0.01% 0.5% 20 mM
Little or no effect Stimulation or inhibition Inhibition Inhibition or no effect No effect or slight inhibition Stimulation or no effect Stimulation Stimulation, inhibition, or no effect Stimulation, inhibition, or no effect Stimulation or inhibition
Depends on source of multicatalytic endopeptidase complex and substrate. b Many references, including the following sources: S. Wilk and M. Orlowski, J. Neurochem. 35, 1172 (1980); B. Dahlmann, M. Rutschmann, L. Kuehn, and H. Reinauer, Biochem. J. 228, 171 (1985); K. Tanaka, K. Ii., A. Ichihara, L. Waxman, and A. L. Goldberg, J. Biol. Chem. 261, 15197 (1986); Y. Saitoh, H. Yokosawa, and S.-I. Ishii, Biochem. Biophys. Res. Commun. 162, 334 (1989); J. Arribas and J. G. Castafio, J. Biol. Chem. 265, 13969 (1990); R. L. Mellgren, Biochim. Biophys. Acta 1040, 28 (1990); D. L. Mykles and M. F. Haire, Arch. Biochem. Biophys. 288, 543 (1991); H. Djaballah, J. A. Harness, P. J. Savory, and A. J. Rivett, Eur. J. Biochem. 209, 629 (1992); H. Djaballah, A. J. Rowe, S. E. Harding, and A. J. Rivett, Biochem. J, 292, 857 (1993).
involve conformational changes within the complex. 47,4s It is difficult to define precisely the specificity of the individual peptidase activities of the complex because of the possibility of overlapping specificity and lack of suitable substrates/inhibitors. The number of distinct peptidase sites of the mammalian multicatalytic endopeptidase complex is probably greater than the five shown in Table VII. The original nomenclature for the different activities is clearly inadequate because there are at least two distinct chymotrypsin-like activities and two peptidylglutamyl-peptide hydrolase activities. Additional names such as "branched-chain amino acid preferring" a n d " small neutral amino acid preferring" 19are cumbersome. Moreover, all of these names imply something about the specificity of the sites, of which we still know rather little. We therefore suggest a simple and short alternative nomenclature for the different sites (MEC 1, MEC 2, MEC 3, etc., as defined in Table VII), which can be added to as necessary. 47 H. Djaballah, A. J. Rowe, S. E. Harding, and A. J. Rivett, Biochem. J. 292, 857 (1993). 48 y. Saitoh, H. Yokosawa, and S.-I. Ishii, Biochem. Biophys. Res. Commun. 162, 334 (1989).
348
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Another difficulty in trying to define peptidase activities is that there are some substantial differences between the kinetic properties of the best studied, rat liver and bovine pituitary multicatalytic endopeptidase complexes.19'45 For example, the trypsinlike activity, which in many ways is the easiest to distinguish, is inhibited by 3,4-dichloroisocoumarin in the case of the bovine pituitary enzyme, but not in the case of the rat liver enzyme. Therefore, the precise definition of the activities of the mammalian multicatalytic endopeptidase complex may ultimately depend on the identification of the catalytic subunits and residues. Some useful reagents have been identified,45'49but work with peptidyl chloromethane and peptidyl diazomethane inhibitors has provided some surprising results. 49 For example, Ala-Ala-Phe-chloromethane inhibits MEC3 but not MEC2, for which the substrate is Ala-Ala-Phe-7-amido-4-methylcoumarin. The catalytic subunits of the yeast multicatalytic endopeptidase complex have been investigated by the production of mutants defective in chymotrypsinlike or peptidylglutamyl-peptide hydrolase activity 5°-52 and the data suggest that two subunits may be required to form a single catalytic site.
Functions of Multicatalytic Endopeptidase Complex and 26S Proteinase It appears that the multicatalytic endopeptidase complex and the 26S proteinase constitute a major nonlysosomal proteolytic system. The 26S proteinase is believed to play a role in the ubiquitin system of protein degradation. 9 Nonlysosomal pathways, which can be either ubiquitin-dependent8'53or ubiquitin-independent, seem to account for the degradation of short-lived regulatory proteins and for the breakdown of abnormal proteins and damaged proteins produced under stress conditions. 8,54'55The yeast mutants that are defective in multicatalytic endopeptidase complex activity have proved useful for establishing the role of the complex in ubiquitin-dependent proteolysis in vivo, 5° because these mutants have been found to be defective in the degradation of known substrates of the
49 p. j. Savory, H. Djaballah, H. Angliker, E. Shaw, and A. J. Rivett, Biochem. J. 296, 601 (1993). 50 W. Heinemeyer, J. A. Kleinschmidt, J. Saidowsky, C. Escher, and D. H. Wolf, EMBO J. 10, 555 (1991). 5J W. Hilt, C. Enenkel, A. Gruhler, T. Singer, and D. H. Wolf, J. Biol. Chem. 268, 3479 (1993). 52 W. Heinemeyer, A. Gruhler, V. M6hrle, Y. Mah6 and D. H. Wolf, J. Biol. Chem. 268, 5115 (1993). 53 S. Jentsch, Trends Cell Biol. 2, 98 (1992). 54 A. J. Rivett and E. Knecht, Curr. Biol. 3, 127 (1993). ~5 M. Rechsteiner, Cell (Cambridge, Mass.) 66, 615 (1991).
350
SERINE PROTEASES
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ubiquitin system. 56,57 Also, the discovery of two y-interferon-inducible multicatalytic endopeptidase complex genes in the MHC class II region58,59 has prompted the suggestion that the enzyme is involved in antigen processing for presentation by the MHC class I pathway. 56B. Richter-Ruoff, W. Heinemeyer, and D. H. Wolf, FEBS Lett. 302, 192 (1992). 57S. Seufert and S. Jentsch, EMBO J. 11, 3077 (1992). s8 R. Glynne, S. H. Powis, S. Beck, A. Kelly, L. A. Kerr, and J. Trowsdale, Nature (London) 353, 357 (1991). 59C. K. Martinez and J. J. Monaco, Nature (London) 353, 664 (1991).
[25] A T P - D e p e n d e n t P r o t e a s e La (Lon) from Escherichia coli B y ALFRED L. GOLDBERG, RICHARD P. MOERSCHELL,
CHIN HA CHUNG, and MICHAEL R. MAURIZI Introduction Protease L a (endopeptidase La, EC 3.4.21.53), the product of the lon gene in E s c h e r i c h i a coli, is an A T P - d e p e n d e n t cytosolic p r o t e a s e that plays an important role in intracellular protein degradation.l-4 In bacteria, as in eukaryotic cells, protein degradation is an energy-requiring p r o c e s s ) Studies o f the biochemical basis for this energy requirement led to the d i s c o v e r y o f p r o t e a s e La, a new type o f p r o t e o l y t i c e n z y m e , w h o s e activity is coupled to A T P hydrolysis. 6-9 This e n z y m e catalyzes the rate-limiting steps in the degradation of highly abnormal proteins in E. coli 7 and certain short-lived regulatory p r o t e i n s ) ° It functions independently o f ubiquitin; in fact, no factor similar to ubiquitin has b e e n found in E. coli or other bacteria. Unlike proteases described previously, this e n z y m e has A T P a s e i A. L, Goldberg, Eur. J. Biochem. 203, 9 (1992). 2 A. L. Goldberg, K, H. S. Swamy, C. H. Chung, and F. S. Larimore, this series, Vol. 80, p. 680. 3 S. Gottesman, in "Escherichia Coli and Salmonella Typhimurium" (F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger, eds.), p. 1308. American Society for Microbiology, Washinghton, D.C., 1987. 4 S. Gottesman and M. R. Maurizi, Microbiol. Reo. 36, 592 (1992). 5 K. Olden and A. L. Goldberg, Biochim. Biophys. Acta 542, 385 (1978). 6 K. S. Swamy and A. L. Goldberg, Nature (London) 292, 652 (1981). 7 C. H. Chung and A. L. Goldberg, Proc. Natl. Acad. Sci. U.S.A. 78, 4931 (1981). 8 M. Charette, G. W. Henderson, and A. Markovitz, Proc. Natl. Acad. Sci. U.S.A. 78, 4728 (1981). 9 F. S. Larimore, L. Waxman, and A. L. Goldberg, J. Biol. Chem. 257, 4187 (1982). l0 S. Gottesman, Annu. Rev. Genet. 23, 163 (1989).
METHODS IN ENZYMOLOGY, VOL. 244
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After the genes for the different Clp components were cloned and mutations introduced into the chromosomal genes, it was shown that the Clp proteases had only a small role in abnormal protein degradation. 3'7 However, a variety of specific protein targets for Clp proteases have been identified. ClpAP degrades several/3-galactosidase fusion proteins 5 and may show specificity for the amino-terminal amino acid in at least one class of such substrates. 34 CIpXP degrades the highly unstable O protein of h phage in vivo (tl/2 ~ 2 rain) 7 and in vitro. 8 CIpXP appears to be involved in plasmid maintenance 35 and in phage mu virulence. 36 Data indicate that CIpP also contributes to degradation of proteins synthesized during carbon starvation, 37 and it is possible that CIpP and other Clp family members may have broader roles in both specific and nonspecific protein degradation in E. coli than is currently appreciated. 34 j. W. Tobias, T. E. Shrader, G. Rocap, and A. Varshavsky, Science 254, 1374 (1991). 35 M. Yarmolinsky (personal communication). 36 V. Geuskens, A. Mhamrnedi-Alaoui, L. Desmet, and A. Toussaint, EMBO J. 11, 5121 (1992). 37 K. Darnerau and A. C. St. John, J. Bacteriol. 175, 53 (1993).
[24] M u l t i c a t a l y t i c E n d o p e p t i d a s e C o m p l e x : P r o t e a s o m e By A. JENNIFER RIVETT, PETER J. SAVORY, and HAKIM DJABALLAH
Introduction The multicatalytic endopeptidase complex (EC 3.4.99.46) is a 700-kDa multisubunit enzyme complex that is widely distributed in eukaryotic cells. It has been described as a high molecular mass protease under many different names ~ and is apparently identical to a variety of cylindrical particles of unknown function, 2 as well as to the prosome, a particle that was believed to play a role in the control of translation. 3 The complex is now commonly referred to as either the multicatalytic proteinase complex ~.4
I A. J. Rivett, Arch. Biochem. Biophys. 268, 1 (1989). 2 p. E. Falkenberg, P. C. Haass, P. M. Kloetzel, B. Niedel, F. Kopp, L. Kuehn, and B. Dahlrnann, Nature (London) 331, 190 (1988). 3 H. P. Schmid, O. Akhayat, C. Martins de Sa, F. Puvion, K. Koehler, and K. Scherrer, EMBO J. 3, 29 (1984). 4 M. Orlowski, Biochemistry 29, 10289 (1990).
METHODS IN ENZYMOLOGY, VOL. 244
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or the proteasome) -7 The multicatalytic endopeptidase complex, either by itself or as the catalytic core of the 26S proteinase complex, 8,9is believed to play an important role in ubiquitin-dependent as well as ubiquitinindependent nonlysosomal pathways of protein turnover, including the degradation of regulatory proteins and the processing of antigens for presentation by the major histocompatibility complex (MHC) class I pathway. 6,7
Assays of Multicatalytic Endopeptidase Activity The term "multicatalytic" has been applied to the complex because it was realized from early work 1° that the broad specificity of the mammalian enzyme could be attributed to distinct types of catalytic sites, the activities of which can be distinguished using a variety of protease inhibitors and other effectors (see below). Activities responsible for cleavage on the carboxyl side of basic (usually Arg), hydrophobic (Leu, Tyr, Phe), and acidic (Glu) residues have been referred to as trypsin-like, chymotrypsin-like, and peptidylglutamyl-peptide bond hydrolase activities, respectively, l° although these terms are not very accurate in describing the specificities. The enzyme can also catalyze cleavage after other residues, including Gin, Thr, and Ala.10-12 Different substrates have been used to assay the endopeptidase activities of the complex purified from many different sources. Its substrates include proteins, peptides, and synthetic peptides (Table I) and the enzyme is active over a range of neutral to weakly alkaline pH values in a variety of different buffers. 11,13-15However, there can be significant variations in activity in different buffers, with Tris and both Na + and K + ions having been found to inhibit some activities. 10 Assaying multicatalytic endopeptidase activity is complicated by the fact that there are multiple distinct catalytic sites and interactions between them. The complex has some unusual kinetic properties (Table II) and there may be differences depending on the source of the enzyme and the 5 K. Tanaka, T. Tamura, T. Yoshimura, and A. Ichihara, New Biol. 4, 173 (1992)~ 6 A. L. Goldbergand K. L. Rock, Nature (London) 357, 375 (1992). 7A. J. Rivett, Biochem. J. 291, 1 (1993). 8A. Hershko and A. Ciechanover,Annu. Rev. Biochem. 61, 761 (1992). 9 M. Rechsteiner, L. Hoffman,and W. Dubiel, J. Biol. Chem. 268, 6065 (1993). l0 S. Wilk and M. Orlowski,J. Neurochem. 35, 1172(1980). it A. J. Rivett, J. Biol. Chem. 2,60, 12600(1985). 12H. Djaballah and A. J. Rivett, unpublishedobservations, (1992). z3K. Tanaka, T. Yoshimura,A. Kumatori,A. Ichihara,A. Ikai, M. Nishigai,K. Kameyama, and T. Takagi,J. Biol. Chem. 263, 16209(1988). 14R. W. Mason,Biochem. J. 265, 479 (1990). 15H. Djaballahand A. J. Rivett, Biochemistry 31, 4133 (1992).
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TABLE I SUBSTRATES OF MULTICATALYTICENDOPEPTIDASE COMPLEX
Type of substrate Proteins Peptides Synthetic peptides-7-amido-4-methylcoumarin (AMC) derivatives
Synthetic peptides--2naphthylamides Other synthetic peptides p-Nitroanilide Methoxynaphthylamide Thiobenzyl p-Aminobenzoate (pAB)
Examples
Refs.
Casein, oxidized enzymes, a-crystallin, lysozyme, serum albumin, hemoglobin Insulin B chain, substance P, glucagon, angiotensin I, and many others Suc-Leu-Leu-Val-Tyr-AMC (Suc)-AIa-AIa-Phe-AMC Boc-Leu-Ser-Thr-Arg-AMC Boc-Phe-Ser-Arg-AMC Boc-Leu-Arg-Arg-AMC Z-GIy-GIy-Arg-AMC Z-Leu-Leu-Glu-2-naphthylamide Z-oAla-Leu-Arg-2-naphthylamide
a-d
Z-Gly-Gly-Leu-p-nitroanilide Z-Ala-Ala-Arg-methoxynaphthylamide Boc-Ala-Ala-Asp-S-benzyl Z-GIy-Pro-Ala-Ala-Gly-pAB
a, e - g
e. f, h. i
e,j k ! m
a A. J. Rivett, J. Biol. Chem. 260, 12600 (1985). b A. J. Rivett, Arch. Biochem. Biophys. 243, 624 (1985). c K. Ray and H. Harris, Proc. Natl. Acad. Sci. U.S.A. 82, 7545 (1985). d M. Orlowski and C. Michaud, Biochemistry 28, 9270 (1989). e S. Wilk and M. Orlowski, J. Neurochem. 35, 1172 (1980). fA. J. Rivett, J. Biol. Chem. 264, 12215 (1989). g J. R. McDermott, A. M. Gibson, A. E. Oakley, and J. A. Biggins, J. Neurochem. 56, 1509 (1991). h K. Tanaka, K. Ii., A. Ichihara, L. Waxman, and A. L. Goldberg, J. Biol. Chem. 261, 15197 (1986). i S. Ishiura, T. Tsukahara, T. Tabira, and H. Sugita, FEBS Lett. 257, 388 (1989). J T. Achstetter, C. Ehmann, A. Osaki, and D. H. Wolf, J. Biol. Chem. 259, 13344 (1984). k j. Arribas and J. G. Castafio, J. Biol. Chem. 265, 13969 (1990). i H. Djaballah and A. J. Rivett, Biochemistry 31, 4133 (1992). 0' M. Odowski, C. Cardozo, and C. Michaud, Biochemistry 32, 1563 (1993).
purification procedure. There are latent and active forms of the complex (for references, see Table II) as well as the possibility of different subpopulations. 16,17 A number of synthetic peptides have proved useful for assaying individual activities of the multicatalytic endopeptidase complex (examples in Table I). The 7-amino-4-methylcoumarin leaving group provides the most sensitive assay and these synthetic peptides are often used at concentra16p. E. Falkenburg and P. M. Kloetzel, J. Biol. Chem. 264, 6660 (1989). 17M. G. Brown, J. Driscoll, and J. J. Monaco, J. lmmunol. 115, 1193 (1993).
TABLE II KINETIC PROPERTIES OF PURIFIED EUKARYOTIC MULTICATALYTIC ENDOPEPTIDASE COMPLEXES
Kinetic property
Refs.
Broad specificity of bond cleavage due to multiple distinct catalytic components, which can be assayed using appropriate synthetic substrates and distinguished using inhibitors and other effectors (see Tables V-VII) Can be isolated in "latent" form; activation by removal of glycerol, by dialysis, by heat treatment, or by addition of polylysine Optimum pH range, pH 7-9, depending on substrate No protease inhibitor yet found to block all peptidase activities; different catalytic components have very different reactivity with some protease inhibitors Addition of inhibitors of one activity can affect kinetic parameters at other catalytic sites Activation (see Table VI for effectors) involves conformational changes, allosteric effects Some peptidase activities show positive cooperativity, possible substrate channeling Mechanism not established but believed to be an unusual type of serine peptidase Some differences in kinetic characteristics of enzyme isolated from different sources
a-d
e-h
f,i c, d; see also Table VII
j--l m-o
i, o, p q, r c,d,l
a S. Wilk and M. Orlowski, J. Neurochem. 35, 1172 (1980). b B. Dahlmann, L. Kuehn, M. Rutschmann, and H. Reinauer, Biochem. J. 228, 161 (1985). c H. Djaballah, J. A. Harness, P. J. Savory, and A. J. Rivett, Eur. J. Biochem. 209, 629 (1992). d M. Orlowski, C. Cardozo, and C. Michaud, Biochemistry 32, 1563 (1993). e K. Tanaka, K. Ii., A. Ichihara, L. Waxman, and A. L. Goldberg,, J. Biol. Chem. 261, 15197 (1986). f K. Tanaka, T. Yoshimura, A. Kumatori, A. Ichihara, A. Ikai, M. Nishigai, K. Kameyama, and T. Takagi, J. Biol. Chem. 263, 16209 (1988). g D. L. Mykles, Arch. Biochem. Biophys. 274, 216 (1989)o h D. Weitman and J. D. Etlinger, J. Biol. Chem. 267, 6977 (1992). i H. Djaballah and A. J. Rivett, Biochemistry 31, 4133 (1992). J S. Wilk and M. Orlowski, J. Neurochem. 40, 842 (1983). C. Cardozo, A. Vinitsky, M. C. Hidalgo, C. Michaud, and M. Orlowski, Biochemistry 31, 7373 (1992). t H. Djaballah, P. J. Savory, and A. J. Rivett, unpublished observations. m H. Djaballah, A. J. Rowe, S. E. Harding, and A. J. Rivett, Biochem. J. 292, 857 (1993). n j. Arribas and J. G. Castafio, J. Biol. Chem. 265, 13969 (1990). o M. Orlowski, C. Cardozo, M. C. Hidalgo, and C. Michaud, Biochemistry 30, 5999 (1991). P L. R. Dick, C. R. Moomaw, G. N. DeMartino, and C. A. Slaughter, Biochemistry 30, 2725 (1991). q M. Orlowski, Biochemistry 29, 10289 (1990). r A. J. Rivett, Biochem. J. 291, 1 (1993).
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)he-VaI-Asn-GIn- His-Leu- Cya-Gly-Ser-His-Leu- VaI-Glu- Ala-Leu- Tyr-Leu- VaI-Cya- Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-AI
¢ I
¢ ~
¢ I
¢ I
¢ ~
(a) Ra~,w~ (b)Human e~tthrocyte
FIG. 1. Multicatalytic endopeptidase complex cleavage sites in oxidized insulin B chain. (a) A. J. Rivett, J. Biol. Chem. 260, 12600 (1985); (b) L. R. Dick, C. R. Moomaw, G. N. DeMartino, and C. A. Slaughter, Biochemistry 30, 2725 (1991); and (c) T. Takahashi, T. Tokumoto, K. Ishikawa, and K. Takahashi, J. Biochem. (Tokyo) 113, 225 (1993).
tions below their K m values, which are in the range of 0.1-1 mM. t8 Other fluorogenic or chromogenic leaving groups are also suitable (Table I). In particular, Z-Leu-Leu-Glu-2-naphthylamide is commonly used to assay peptidylglutamyl-peptide hydrolase activity. For some activities of the bovine pituitary enzyme a coupled assay with an aminopeptidase has been u s e d ) 9 Although we describe below stopped-fluorimetric assay procedures, it is necessary to establish that the production of product is linear with both time and enzyme concentration, because this is not always the case. Fluorimetric assays can of course be carded out continuously using a water-jacketed cell holder to maintain a constant temperature. The degradation of peptide substrates such as oxidized insulin B chain (Table I) can be monitored by C18 reversed-phase high-performance liquid chromatography (HPLC). Some differences have been observed in the cleavage pattern obtained with multicatalytic endopeptidases from different sources (Fig. 1). 12 The degradation of protein substrates, of which casein has been the most widely used, is easily assayed by measuring acidsoluble counts released from radiolabeled protein (see below). Alternative methods for investigating protein degradation include sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and the use of fluorescamine to determine the level of acid-soluble products from unlabeled proteins. 2° Not all proteins are substrates for the complex but it is not yet entirely clear what structural features of proteins determine proteolytic susceptibility. 21,22 Those proteins which are substrates are usually degraded to peptides." 18 A. J. Rivett, J. Biol. Chem. 264, 12215 (1989). 19 M. Orlowski, C. Cardozo, and C. Michaud, Biochemistry 32, 1563 (1993). 2o A. J. Rivett, Arch. Biochem. Biophys. 243, 624 (1985). 21 A. J. Rivett and R. L. Levine, Arch. Biochem. Biophys. 2/8, 26 (1990). 22 R. E. Pacifici, Y. Kono, and K. J. A. Davies, J. Biol. Chem. 268, 15405 (1993).
336
SERINE PEPTIDASES
[24]
Assays with 7-Amido-4-methylcoumarin Substrates A variety of different commercially available synthetic peptides may be used to assay peptidase activities of the complex and the assay procedure is the same for all of them. Although the pH optimum varies for different substrates, we routinely use buffer of pH 7.5. Commonly used peptidyl7-amino-4-methylcoumarin substrates are listed in Table I. We routinely use Ala-Ala-Phe-7-amido-4-methylcoumarin, Suc-Leu-Leu-Val-Tyr-7amido-4-methylcoumarin, and Boc-Leu-Ser-Thr-Arg-7-amido-4-methylcoumarin.
Reagents Buffer: 100 mM HEPES/KOH, pH 7.5, diluted to 50 mM in the assay. Substrate stock solutions: These can be made at 2 or 10 mM in water, 50 mM acetic acid, or dimethyl sulfoxide (depending on the solubility of the substrate) and stored in aliquots at - 2 0 °. Dimethyl sulfoxide concentrations in the assays should not normally exceed 5%. Stop mix: Sodium acetate trihydrate (0.25 g) is added to 4.375 ml 1 M acetic acid and made up to a volume of 25 ml with water. Procedure. Assay mixtures containing approximately I-2/zg enzyme, substrate (at a fixed concentration, e.g., 20 or 50/zM), and 50 mM HEPES/ KOH, pH 7.5, are made up in a total volume of 200/zl in 4-ml disposable test tubes and then incubated at 37° for 15 or 30 min. Reactions are started by addition of either substrate or enzyme and are stopped by addition of 0.1 ml stop mix. H20 (2 ml) is added to each tube prior to measuring fluorescence (excitation 370 nm, emission 430 nm). Blanks are prepared without the addition of enzyme and a standard curve is prepared with 7amino-4-methylcoumarin.
Assay with Z-Leu-Leu-Glu-2-naphthylamide The substrate is hydrolyzed to give 2-naphthylamine, which can be either assayed colorimetrically by coupling with a diazonium salt or measured directly in a fluorimeter. This product is probably carcinogenic and cannot easily be purchased, so we make just enough by an enzymatic procedure to produce a standard curve.
Reagents Buffer: 100 mM HEPES/KOH, pH 7.5, diluted to 50 mM in the assay. Substrate stock solution: 10 mM Z-Leu-Leu-Glu-2-naphthylamide in dimethyl sulfoxide (DMSO), stored in aliquots at - 2 0 °.
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Procedure. Assay mixes are made to a volume of 200/zl in 4-ml disposable test tubes. Usually 1-2 /zg of enzyme is used with a final buffer concentration of 50 mM. The amounts of substrate and water are varied. The amount of sub strate is limited to a maximum of 0.6 mM by its solubility in aqueous solutions. 15 There are two distinct types of catalytic centers (LLE1 and LLE2), which possess peptidylglutamyl-peptide hydrolase activity? 5 We usually assay with substrate at concentrations of 0.1 mM (LLE1 activity) and 0.4 mM (LLE1 + LLE2 activity). Because the kinetic characteristics of the peptidylglutamyl-peptide hydrolase activities (see later) of the rat liver and bovine pituitary enzymes are not identical, 15'23 they should be tested in other systems. Assay mixtures are incubated at 37° usually for 30 min and are then stopped by addition of 0.3 ml ethanol. Water (2 ml) is added prior to reading samples in a fluorimeter (excitation 333 nm, emission 450 nm). Blank samples are prepared at the appropriate substrate concentrations with no enzyme. 2-Naphthylamine is produced by complete digestion of a known amount of Z-Leu-Leu-Glu-2-naphthylamide (e.g., 0.6 mM) by Staphylococcus aureus V8 proteinase in 50 mM ammonium bicarbonate buffer, pH 7.8. A standard curve can then be prepared using the 50 mM H E P E S / K O H , pH 7.5, assay buffer, and diluting samples with ethanol and water as described above. Assay with Casein as Substrate Protein substrates are usually degraded to low molecular mass trichloroacetic acid-soluble products. Therefore, assays with radiolabeled substrate are most convenient. Reagents Buffer: 100 mM H E P E S / K O H , pH 8.0, diluted to 50 mM in the assay. Substrate stock solution: Casein (14C, 3H, or lzsI labeled), stored in aliquots at - 2 0 °. Others: 10% (w/v) trichloroacetic acid (TCA); 5% (w/v) bovine serum albumin (BSA) in 0.1 N HCI. Procedure. Assays are carried out in a 100-/~1 volume using 1-2 ~g enzyme with 10/.~g casein in 50 mM H E P E S / K O H buffer, pH 8.0, in 1.5ml Eppendorf tubes. Incubations are at 37° for 1 hr. Assays are stopped by the addition of 0.5 ml of the 10% trichloroacetic acid followed by addition of 0.1 ml of the bovine serum albumin solution. Samples are left on ice for 10 rain and then centrifuged in a microfuge for 5 min. Scintillant 23 M. Orlowski, C. Cardozo, M. C. Hidalgo, and C. Michaud, Biochemistry 311,5999 (1991).
338
SEreNE PEPTIDASES
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TABLE III SOURCES USED FOR PURIFICATION OF MULTICATALYTIC ENDOPEPTIDASE COMPLEXa
Source
Ref.
Animal tissues and cells Liver (e.g., mouse, rat, human) Muscle (rat) Lung (human) Brain (human, bovine) Pituitary (bovine) Lens (bovine) Placenta (human) Erythroeytes (human) Plants (tobacco, potato, mung bean, pea seed) Frog (Xenopus laevis or Rana pipiens): oocytes Lobster: muscle Fish (carp, white croaker): muscle Sea urchin: eggs, sperm Yeast (Saccharomyces cerevisiae) Archaebacteria (Thermoplasma acidophilium)
b-e f g h, i j, k l m n o, p q, r s t, u o, w x y
a The enzyme has been described under many different names [reviewed in A. J. Rivett, Arch. Biochem. Biophys. 268, 1 (1989)]. b A. J. Rivett, J. Biol. Chem. 7,60, 12600 (1985). c K. Tanaka, K. Ii., A. Ichihara, L. Waxman, and A. L. Goldberg, J. Biol. Chem. 261, 15197 (1986). d R. W. Mason, Biochem. J. 265, 479 (1990). ' H. Djaballah, and A. J. Rivett, Biochemistry 31, 4133 (1992). f B. Dahlmann, L. Kuehn, M. Rutschmann, and H. Reinauer, Biochem. J. 228, 161 (1985). R. Zolfaghari, C. R. F. Baker, P. C. Canizaro, A. Amirgholami, and F. J. Behal, Biochem. J. 241, 129 (1987). h A. Azaryan, M. Banay-Schwartz, and A. Lajtha, Neurochem. Res. 14, 995 (1989). J J. R. McDermott, A. M. Gibson, A. E. Oakley, and J. A. Biggins, J. Neurochem. 56, 1509 (1991). YS. Wilk and M. Orlowski, J. Neurochem. 35, 1172 (1980). k M. Orlowski and C. Michaud, Biochemistry 28, 9270 (1989). t K. Ray and H. Harris, Proc. Natl. Acad. Sci. U.S.A. 82, 7545 (1985). ,n K. B. Hendil and W. Uerkvitz, J. Biochem. Biophys. Methods 22, 159 (1991). n M. J. McGuire and G. N. DeMartino, Biochim. Biophys. Acta. 873, 279 (1986). o M. Schliephacke, A. Kremp, H. P. Schmid, K. Kohler, and U. Kull, Eur. J. CellBiol. 55, 114 (1991).
[24]
MULTICATALYTIC ENDOPEPTIDASE COMPLEX
339
TABLE III (continued) p B. Skoda and L. Malek, Plant Physiol. 99, 1515 (1992). q K. Tanaka, T. Yoshimura, A. Kumatori, A. Ichihara, A. Ikai, M. Nishigai, K. Kameyama, and T. Takagi, J. Biol. Chem. 263, 16209 (1988). r y. Azuma, T. Tokumoto, and K. Ishikawa, Mol. Cell. Biochem. 100, 177 (1991). s D. L. Mykles, Arch. Biochem. Biophys. 274, 216 (1989). t M. Kinoshita, H. Toyohara, and Y. Shimizu, Comp. Biochem. Physiol. 96B, 565 (1990). u E. J. Folco, L, Busconi, C. B. Martone, and J. J. Sanchez, Arch. Biochem. Biophys. 267, 599 (1988), v j. L. Grainger and M. M. Winkler, J. Cell Biol. 109, 675 (1989). wK. Matsumura and K. Aketa, Mol. Reprod. Dev. 29, 189 (1991). x T. Achstetter, C. Ehmann, A. Osaki, and D. H. Wolf, J. Biol. Chem. 259, 13344 (1984). YB. Dahlmann, F. Kopp, L. Kuehn, B. Niedel, G. Pfeifer, R. Hegerl, and W. Baurneister, FEBS Lett. 251, 125 (1989). (10 ml) is added to an aliquot of the supernatant (0.63 ml) and samples are then counted in a liquid scintillation counter. Purification of the Multicatalytic Endopeptidase Complex The multicatalytic endopeptidase complex is an abundant protein and can constitute up to 1% of the soluble cellular protein, with the highest levels in animal cells and tissues being found in the liver. 24,25 The e n z y m e has b e e n purified f r o m a wide variety of sources (Table liD. Purification p r o c e d u r e s often involve a combination of anion-exchange chromatography and gel filtration, often p r e c e d e d by an a m m o n i u m sulfate fractionation, and usually involve additional chromatographic steps such as hydroxylapatite, h y d r o p h o b i c , h e p a r i n - S e p h a r o s e or Affi-Gel blue chrom a t o g r a p h y or a second ion-exchange or gel-filtration step. Purification b y immunoaffinity c h r o m a t o g r a p h y has also b e e n described. 26 The yield of multicatalytic endopeptidase c o m p l e x that can be e x p e c t e d depends on the source but is usually in the range of 1-10 rag/100 g tissue. 13,~5 A low a m o u n t obtained f r o m e r y t h r o c y t e s 27 reflects the relatively low level 24K. Tanaka, K. Ii., A. Ichihara, L. Waxman, and A. L. Goldberg, J. Biol. Chem. 261, 15197 (1986). 25A. J. Rivctt and S, T. Sweeney, Biochem. J. 278, 171 (1991). 26K. B. Hendi| and W. Uerkvitz, J. Biochem. Biophys. Methods 22, 159 (1991). 27 M. J. McGuire and G. N. DeMartino, Biochim. Biophys. Actu. 873, 279 (1986).
340
SERINE PEPTIDASES
[24]
of the complex in these cells. 25 The enzyme is usually prepared from soluble extracts, but purification from erythrocyte membranes has also been reported. 28 The enzyme can be purified in a latent form (references in Table II), the difference in procedure usually involving the inclusion 0f20% glycerol in all the buffers for the latent enzyme. There are only minor differences between latent and active forms in the molecular masses and pI values of the associated polypeptides? 9'3° In the majority of cases (Table I) the enzyme has been purified in the active state. It is, however, quite difficult to compare the specific activities of the final preparations obtained using different procedures, for seveal reasons. First, the purification procedure may have an effect on activity; second, the activity can be markedly influenced by the composition of the buffer (see below); and third, the activities have often not been assayed using the same substrates under identical conditions. The purification-fold can also be misleading. For example, it is not straightforward to measure activity in crude extracts of animal cells using protein substrates,1~,27 possibly due to the presence of endogenous inhibitor proteins 3L32 or competing protein substrates. With synthetic peptide substrates, the ratio of activity with different substrates varies at different stages of the purification 33 and some, if not all, of the usual substrates are also hydrolyzed by other peptidases. With apparently homogeneous enzyme preparations from different sources it is also difficult to make comparisons because of differences in assay conditions and possibly also some kinetic differences, the basis for which we do not yet understand. The kinetic characteristics of the purified multicatalytic endopeptidase complex (Table I) will be discussed in more detail below. Purification of the Multicatalytic Endopeptidase from Rat Liver The enyzme can be purified from fresh or frozen rat liver using the following procedure. All steps are carried out at 4 ° except for those involv28 M. Kinoshita, T. Hamakubo, I. Fukui, T. Murachi, and H. Toyohara, J. Biochem. (Tokyo) 107, 440 (1990). 29 L. W. Lee, C. R. Moomaw, K. Orth, M. J. McGuire, G. N. DeMartino, and C. A. Slaughter, Biochim. Biophys. Acta 1037, 178 (1990). 30 M. E. Pereira, T. Nguyen, B. J. Wagner, J. W. Margolis, B. Yu, and S. Wilk, J. Biol. Chem. 267, 7949 (1992). 31 X. Li, M. Gu, and J. D. Etlinger, Biochemistry 30, 9709 (1991). 32 M. Chu-Ping, C. A. Slaughter, and G. N. DeMartino, Biochim. Biophys. Acta 1119, 303 0992). 33 S. Wiik and M. Orlowski, J. Neurochem. 40, 842 (1983).
[24]
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341
ing fast protein liquid chromatography (FPLC), which are performed at room temperature. Column and buffer volumes are given for a preparation from 200 g rat liver. We recommend Ala-Ala-Phe-7-amino-4-methylcoumarin as the substrate for assays during the purification. Step I. Homogenization and Centrifugation. Rat livers are washed in homogenization buffer (20 mM HEPES, pH 8.0, containing 1 mM 2-mercaptoethanol and 1 mM EDTA) and then homogenized (3 × 30 sec in a Waring blendor) in 5 volumes of buffer. The homogenate is centrifuged at 27,000 g for 2.5 hr. Step 2. Ammonium Sulfate Fractionation. Solid ammonium sulfate to give 35% saturation is added to the supernatant from step 1. After stirring for 30 min on ice, the precipitate is removed by centrifugation and ammonium sulfate is added to the supernatant to give 60% saturation. Proteins precipitated during the second ammonium sulfate precipitation (left stirring for 45 rain after all the ammonium sulfate has dissolved) are collected by centrifugation and redissolved in approximately 200 ml of 10 mM TrisHCI buffer, pH 7.2. The preparation is dialyzed against 2 x 5 liters l0 mM Tris-HCl buffer, pH 7.2, containing 50 mM KCI, 0.1 mM EDTA. 1 mM 2-mercaptoethanol. Step 3. DEAE-Cellulose Chromatography. The dialyzed ammonium sulfate fraction is centrifuged at 45,000 g for 20 min and then loaded on to a DEAE-cellulose column (2.6 x 30 cm) equilibrated in l0 mM TrisHC1, pH 7.2, containing 50 mM KC1 at a flow rate of approximately 2 ml/ min. After washing the column with equilibration buffer, a gradient of 50-350 mM KCI (2 × 1.5 liters) is applied. The enzyme elutes at approximately 150 mM KCI. Active fractions are pooled (approximately 400 ml) and concentrated to a volume of about 100 ml using an Amicon (Danvers, MA) ultrafiltration cell with an XMS0 membrane. EDTA (0.1 mM) and 2-mercaptoethanol (1 mM) are added to the preparation after pooling fractions from this and all the subsequent chromatography steps. Step 4. Mono Q Anion-Exchange Chromatography. The DEAE-ceUulose pool is diluted with one-half volume of 20 mM Tris-HC1, pH 7.2, and then divided into at least two portions. The portions are loaded separately on to a Mono Q 10/10 FPLC column (Pharmacia, Piscataway, NJ) equilibrated in 20 mM Tris-HCl, pH 7.2, containing 100 mM KC1 at a flow rate of 4 ml/min. The enzyme is eluted on a gradient (200 ml) of 100-500 mM KCI at a KCI concentration of about 400 mM (usually peak fraction 19 out of 25 x 8 ml fractions). Active fractions are pooled and concentrated to less than l0 ml using an Amicon ultrafiltration cell (XMS0 membrane). Step 5. Superose 6 Gel Filtration. The sample is filtered and loaded onto a preparative-size Superose 6 FPLC column (Pharmacia), equilibrated in 50 mM potassium phosphate buffer, pH 7.0, containing 0.1 M
342
SERINE PEPTIDASES
[24]
KCI. Although the sample could be concentrated more and loaded for one column run, we find that the resolution and recovery are better if the sample is divided into two or three portions, loading 2 or 3 ml each time. The enzyme elutes at approximately the same volume as thyroglobulin. Step 6. Mono Q Ion-Exchange Chromatography. We usually find it helpful to carry out a final ion-exchange (Mono Q 5/5) chromatography step. The pool from the gel-filtration step is therefore dialyzed against 20 mM Tris-HCl buffer, pH 7.2, and then run (<5 mg/run) on the Mono Q 5/5 column equilibrated in the same buffer and eluted at approximately 0.35 - 0.4 M KCI by applying a linear gradient to 500 mM KCI. The active fractions from this column are pooled and dialyzed against 50 mM potassium phosphate, pH 7.0, containing 1 mM dithiothreitol, 0. I mM EDTA, and 10% glycerol. Protein Determination. The amount of enzyme can be quantitated using the Bradford method 34 for protein determination with bovine serum albumin as standard, because this gives a very good estimate of actual protein concentration determined by amino acid analysis) 5 Purity. The purity of the preparation is best assessed by PAGE carried out under nondenaturing conditions, ~ and the enzyme can be visualized under UV light in nondenaturing gels following incubation with fluorogenic peptide substrates (for example, at a concentration of 50-100 tzM in 50 mM HEPES buffer, pH 7.5, incubated at 37° for 1 hr). There has been a report suggesting multiple electrophoretic forms of the enzyme, 36but these are not always observed. 11SDS-PAGE gels can also be used to determine whether preparations contain any contaminating proteins in addition to the characteristic ladder of multicatalytic endopeptidase complex bands of M r of 20-34 kDa. 11'15 Specific Activity. The specific activities of the rat liver complex assayed with 50/zM Ala-Ala-Phe-7-amido-4-methylcoumarin and 0.4 mM Z-LeuLeu-Glu-2°naphthylamide are usually in the ranges of 12-25 nmol/min/mg protein and 100-150 nmol/min/mg, respectively. Storage. The purified enzyme is stable for several months when stored frozen at - 2 0 ° in 50 mM potassium phosphate buffer, pH 7.0, containing 10% glycerol, 1 mM DTT, 0.1 mM EDTA. Purification of 26S Proteinase The multicatalytic endopeptidase complex forms part of the 26S proteinase (also called ubiquitin-conjugate degrading proteinase), and several 34 M. M. Bradford, Anal. Biochem. 72, 248 (1976). 35 p. j. Savory and A. J. Rivett, Biochem. J. 289, 45 (1993). 36 L. Hoffman, G. Pratt, and M. Rechsteiner, J. Biol. Chem. 267, 22362 (1992).
[24]
MULTICATALYT1C ENDOPEPTIDASE COMPLEX
343
purification protocols have been reported for this larger proteinase complex. 9'37-4° The 26S proteinase, which is precipitated at lower ammonium sulfate concentrations than the multicatalytic endopeptidase complex alone, appears to require ATP for its stability. Also, unlike purified multicatalytic endopeptidase complex preparations, ~ it catalyzes ATP-dependent protein degradation. 26S proteinase activity, as well as multicatalytic endopeptidase activity, can conveniently be assayed with Suc-Leu-LeuVal-Tyr-7-amido-4-methylcoumarin or with casein as a substrate. Properties of Purified Multicatalytic Endopeptidase Complex The size, shape, and subunit structure of multicatalytic endopeptidase complexes from eukaryotic cells are broadly similar irrespective of the source of the enzyme, and are summarized in Table IV. The number of different types of polypeptide associated with the complex does seem to vary with the source and it is not yet clear whether these differences can be explained solely in terms of post-translational modification, such as phosphorylation, glycosylation, and proteolysis, cDNAs have been cloned for many yeast, rat, and human multicatalytic endopeptidase subunits. 5'7 All subunits of the complex are encoded by members of the same gene family, with different subunits from the same source having 18-40% identity. The individual subunit sequences are highly conserved between different mammalian species (rat/human 95-98% identity at the amino acid level) and are 40-70% identitical with the most closely related subunits in yeast. The multicatalytic endopeptidase complex isolated from the archaebacterium Thermoplasrna acidophilum is an interesting and useful one because it is a much simpler molecule than the eukaryotic complex. The overall structure is similar, 41but it is composed of only two different types of subunit, a and fl (a14f114 stoichiometry). The subunit sequences are related to those of all the eukaryotic multicatalytic endopeptidase complex subunits. 42 The a subunits are located on the outer rings of the cylinder 37 E. Eytan, D. Ganoth, T. Armon, and A. Hershko, Proc. Natl. Acad. Sci. U.S.A. 86, 7751 (1989). 38 j. Driscoll and A. L. Goldberg, J. Biol. Chem. 265, 4789 (1990). 39 H. Kanayama, T. Tamura, S. Ugai, S. Kagawa, N. Tanahashi, T. Yoshimura, K. Tanaka, and A. Ichihara, Eur. J. Biochem. 206, 567 (1992). 40 A. Azaryan, M. Banay-Schwartz, and A. Lajtha, Neurochem. Res. 14, 995 (1989). 41 G. P0hler, S. Weinkauf, L. Bachmann, S. MOiler, A. Engel, R. Heged, and W. Baumeister, EMBO J. 11, 1607 (1992). 42 p. Zwickl, A. Grziwa, G. Piihler, B. Dahlmann, F. Lottspeich, and W. Baumeister, Biochemistry 31, 964 (1992).
344
SERINE PEPTIDASES
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TABLE IV PROPERTIES OF EUKARYOTICMULT1CATALYTICENDOPEPTIDASECOMPLEX Property
Comments
Refs.
Occurrence
Found in all types of cells (see also Table III); usually in nucleus as well as in cytoplasm Can constitute up to 1% of cellular protein Has a molecular mass of around 700,000 Da; also forms part of 26S proteinase Many (14-20) different polypeptides Mr of 20,000-34,000, pl 4-9; some may be related by post-translational modification Has very low amounts of small species (approximately 80 nucleotides) of RNA associated Has a cylindrical structure (approximately 11 x 17 nm) Pseudo-helical arrangement of subunits Possibly a complex dimer Subunits are encoded by members of same gene family, but can be divided into two groups, A (a) and B ~), related to archaebacterial a and fl subunits Genes are located on different chromosomes; some are essential for cell proliferation Mammalian complex contains at least five distinct peptidase sites (for substrates, see Table I; other kinetic properties, see Table II) Ubiquitin-dependent and ubiquitin-independent nonlysosomal pathways of protein breakdown, including the degradation of regulatory proteins, and probably antigen processing for presentation by MHC class I pathway
a-c
Level Size Subunits RNA Shape
Genes
Activity
Function
d, e f, g h, i j, k l m n o, p
i, q, r
i, o, p, s, t
a K. Tanaka, A. Kumatori, K. Ii, and A. Ichihara, J. Cell. Physiol. 139, 34 (1989). A. J. Rivett, A. Palmer, and E. Knecht, J. Histochem. Cytochem. 40, 1165 (1992). c A. J. Rivett and E. Knecht, Curr. Biol. 3, 127 (1993). d L. Tanaka, K. Ii., A. Ichihara, L. Waxman, and A. L. Goldberg, J. Biol. Chem. 261, 15197 (1986). e K. B. Hendil, Biochem. Int. 17, 471 (1988). [ M. Rechsteiner, L. Hoffman, and W. Dubiel, J. Biol. Chem. 268, 6065 (1993). g A. Hershko and A. Ciechanover, Annu. Rev. Biochem. 61, 761 (1992). h A. J. Rivett and S. T. Sweeney, Biochem. J. 278, 171 (1991). i W. Heinemeyer, J. A. Kleinschmidt, J. Saidowsky, C. Escher, and D. H. Wolf, EMBO J. 10, 555 (1991). J H. S. Skilton, I. C. Eperon, and A. J. Rivett, FEBS Lett. 279, 351 (1991). k H. G. Nothwang, O. Coux, G. Keith, I. Silva-Pereira, and K. Scherrer, Nucleic Acids Res. 20, 1959 (1992). t W. Baumeister, B. Dahlmann, R. Hegerl, F. Kopp, L. Kuehn, and G. Pfeifer, FEBS Lett. 241, 239 (1988). m H. Djaballah, A. J. Rowe, S. E. Harding, and A. J. Rivett, Biochem. J. 292, 857 (1993). n F. Kopp, B. Dahlmann and K. B. Hendil, J. Mol. Biol. 229, 14 (1993). o K. Tanaka, T. Tamura, T. Yoshimura, and A. Ichihara, New Biol. 4, 173 (1992).
[24]
MULTICATALYTIC ENDOPEPTIDASE COMPLEX
345
TABLE IV (continued) P A. J. Rivett, Biochem. J. 291, 1 (1993). q T. Fujiwara, K. Tanaka, E. Orino, T. Yoshimura, A. Kumatori, T. Tamura, C. H. Chung, T. Nakai, K. Yamaguchi, S. Shin, A. Kakizuka, S. Nakanishi, and A. Ichihara, J. Biol. Chem. 265, 16604 (1990). Y. Emori, T. Tsukahara, H. Kawasaki, S. Ishiura, H. Sugita, and K. Suzuki, Mol. Cell. Biol. 11, 344 (1991). M. Orlowski, Biochemistry 29, 10289 (1990). t A. L. Goldberg and K. L. Rock, Nature (London) 357, 375 (1992).
and the fl subunits are in the inner rings. 43 The subunits of the eukaryotic multicatalytic endopeptidase complex can be divided into two groups, A (a) and B (fl),6 depending on whether they resemble the archaebacterial o~or/3 subunit. The A group members are quite similar, with a very highly conserved region close to the N terminus, which is blocked. The B group members, on the other hand, are not so closely related to each other and have a variable region at the N terminus, which is often unblocked. 44
Catalytic Mechanism and Identification of Catalytic Components The enzyme is now widely believed to be an unusual type of serine peptidase based on its inhibitor specificity (Table V). However, a definite assignment must await the identification of catalytic residues. Although it seems likely that the different catalytic centers of the multicatalytic endopeptidase complex are mechanistically related, there are remarkable differences in reactivity of the different peptidase sites with some serine peptidase inhibitors.4S For example, a given inhibitor can rapidly inactivate one activity while having no effect on another. Such observations can explain why in some of the earliest studies the enzyme was not thought to be a serine protease. The inhibition observed with thiol-reactive reagents is probably nonspecific, because activity of the Thermoplasma enzyme is unaffected by even the mercury-containing thiol reagents. 46 Because the primary structures of the multicatalytic endopeptidase complex subunits bear no resemblence to any other known peptidases, the catalytic components cannot be identified from sequence information. Also, dissociated 43 A. Grziwa, W. Baumeister, B. Dahlmann, and F. Kopp, FEBS Left. 290, 186 (1991). 44 K. S. Lilley, M. D. Davison, and A. J. Rivett, FEBS Lett. 262, 327 (1990). 45 H. Djaballah, J. A. Harness, P. J. Savory, and A. J. Rivett, Eur. J. Biochem. 209, 629 (1992). B. Dahlmann, L. Kuehn, A. Grziwa, P. Zwickl, and W. Baumeister, Eur. J. Biochem. 208, 789 (1992).
346
SERINE PEPTIDASES
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TABLE V PROTEASEINHIBITORSEFFECTIVEON MAMMALIANMULTICATALYTIC ENDOPEPTIDASEACTIVITIESa Type of inhibitor Organophosphorus compound Sulfonyl fluoride Isocoumarins Peptide aldehydes Peptidyl chloromethanes Peptidyl diazomethanes Thiol-reactive reagents
Examples Diisopropyl fluoropbosphate 4-(2-Aminoethyl)benzene sulfonyl fluoride 3,4-Dichloroisocoumarin; others Leupeptin, antipain Chymostatin and analogs Z-Leu-Leu-PheH Tyr-GIy-Arg-CH2CI (some have no effect) Z-Leu-Leu-Tyr-CHN2, Z-Phe-Gly-TyrCHN 2 (some have no effect) N-Ethylmaleimide, p-hydroxymercuribenzoate, others
Refs. b-d
c c, e. f
g, h c i j, k j g, l
a There are marked differences in reactivity of different peptidase sites. None of the inhibitors here inhibit all activities of the mammalian multicatalytic endopeptidase complex (see also Table VII). b K. Tanaka, K. Ii., A. Ichihara, L. Waxman, and A. L. Goldberg, J. Biol. Chem. 261, 15197 (1986). ¢ H. Djaballah, J. A. Harness, P. J. Savory, and A. J. Rivett, Eur. J. Biochem. 209, 629 (1992). M. Orlowski, C. Cardozo, and C. Michaud, Biochemistry 32, 1563 (1993). e M. Orlowski and C. Michaud, Biochemistry 28, 9270 (1989). f R. W. Mason, Biochem. J. 265, 479 (1990). g S. Wilk and M. Orlowski, J. Neurochem. 40, 842 (1983). h p. j. Savory and A. J. Rivett, Biochem. J. 289, 45 (1993). i A. Vinitsky, C. Michaud, J. C. Powers, and M. Orlowski, Biochemistry 31, 9421 (1992). i p. j. Savory, H. Djahallah, H. Angliker, E. Shaw, and A. J. Rivett, Biochem. J. 296, 601 (1993). k C. Cardozo, A. Vinitsky, M. C. Hidalgo, C. Michaud, and M. Orlowski, Biochemistry 31, 7373 (1992). t A. J. Rivett, J. Biol. Chem. 260, 12600 (1985).
subunits are inactive. It is possible that the e n z y m e has a n o v e l t y p e o f catalytic m e c h a n i s m . T h e distinction b e t w e e n different catalytic activities o f the e u k a r y o t i c c o m p l e x is b a s e d o n the use o f different synthetic peptide substrates with a v a r i e t y o f p r o t e a s e inhibitors a n d o t h e r effectors (Table VI). T h e effects o f a c t i v a t o r s such as low c o n c e n t r a t i o n s o f S D S can be quite variable d e p e n d i n g o n the s o u r c e o f the e n z y m e , and, at least in s o m e cases,
[24]
MULTICATALYTIC ENDOPEPTIDASE COMPLEX
347
TABLE VI OTHER EFFECTORS OF MULTICATALYTICENDOPEPT1DASE ACTIVITIES Tested effector
Concentration
Effect on activity a'b
EDTA MnCI2, MgCI2, CaCI2 ZnCI2 KCI ATP Polylysine Linoleic acid, oleic acid Sodium dodecyl sulfate Nonidet P-40 Guanidine hydrochloride
1 mM 1 mM 0.1 mM 25 mM 1-5 mM 0.1 mg/ml 0.2-5 mM 0.01% 0.5% 20 mM
Little or no effect Stimulation or inhibition Inhibition Inhibition or no effect No effect or slight inhibition Stimulation or no effect Stimulation Stimulation, inhibition, or no effect Stimulation, inhibition, or no effect Stimulation or inhibition
Depends on source of multicatalytic endopeptidase complex and substrate. b Many references, including the following sources: S. Wilk and M. Orlowski, J. Neurochem. 35, 1172 (1980); B. Dahlmann, M. Rutschmann, L. Kuehn, and H. Reinauer, Biochem. J. 228, 171 (1985); K. Tanaka, K. Ii., A. Ichihara, L. Waxman, and A. L. Goldberg, J. Biol. Chem. 261, 15197 (1986); Y. Saitoh, H. Yokosawa, and S.-I. Ishii, Biochem. Biophys. Res. Commun. 162, 334 (1989); J. Arribas and J. G. Castafio, J. Biol. Chem. 265, 13969 (1990); R. L. Mellgren, Biochim. Biophys. Acta 1040, 28 (1990); D. L. Mykles and M. F. Haire, Arch. Biochem. Biophys. 288, 543 (1991); H. Djaballah, J. A. Harness, P. J. Savory, and A. J. Rivett, Eur. J. Biochem. 209, 629 (1992); H. Djaballah, A. J. Rowe, S. E. Harding, and A. J. Rivett, Biochem. J, 292, 857 (1993).
involve conformational changes within the complex. 47,4s It is difficult to define precisely the specificity of the individual peptidase activities of the complex because of the possibility of overlapping specificity and lack of suitable substrates/inhibitors. The number of distinct peptidase sites of the mammalian multicatalytic endopeptidase complex is probably greater than the five shown in Table VII. The original nomenclature for the different activities is clearly inadequate because there are at least two distinct chymotrypsin-like activities and two peptidylglutamyl-peptide hydrolase activities. Additional names such as "branched-chain amino acid preferring" a n d " small neutral amino acid preferring" 19are cumbersome. Moreover, all of these names imply something about the specificity of the sites, of which we still know rather little. We therefore suggest a simple and short alternative nomenclature for the different sites (MEC 1, MEC 2, MEC 3, etc., as defined in Table VII), which can be added to as necessary. 47 H. Djaballah, A. J. Rowe, S. E. Harding, and A. J. Rivett, Biochem. J. 292, 857 (1993). 48 y. Saitoh, H. Yokosawa, and S.-I. Ishii, Biochem. Biophys. Res. Commun. 162, 334 (1989).
348
SERINE PEPTIDASES
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Another difficulty in trying to define peptidase activities is that there are some substantial differences between the kinetic properties of the best studied, rat liver and bovine pituitary multicatalytic endopeptidase complexes.19'45 For example, the trypsinlike activity, which in many ways is the easiest to distinguish, is inhibited by 3,4-dichloroisocoumarin in the case of the bovine pituitary enzyme, but not in the case of the rat liver enzyme. Therefore, the precise definition of the activities of the mammalian multicatalytic endopeptidase complex may ultimately depend on the identification of the catalytic subunits and residues. Some useful reagents have been identified,45'49but work with peptidyl chloromethane and peptidyl diazomethane inhibitors has provided some surprising results. 49 For example, Ala-Ala-Phe-chloromethane inhibits MEC3 but not MEC2, for which the substrate is Ala-Ala-Phe-7-amido-4-methylcoumarin. The catalytic subunits of the yeast multicatalytic endopeptidase complex have been investigated by the production of mutants defective in chymotrypsinlike or peptidylglutamyl-peptide hydrolase activity 5°-52 and the data suggest that two subunits may be required to form a single catalytic site.
Functions of Multicatalytic Endopeptidase Complex and 26S Proteinase It appears that the multicatalytic endopeptidase complex and the 26S proteinase constitute a major nonlysosomal proteolytic system. The 26S proteinase is believed to play a role in the ubiquitin system of protein degradation. 9 Nonlysosomal pathways, which can be either ubiquitin-dependent8'53or ubiquitin-independent, seem to account for the degradation of short-lived regulatory proteins and for the breakdown of abnormal proteins and damaged proteins produced under stress conditions. 8,54'55The yeast mutants that are defective in multicatalytic endopeptidase complex activity have proved useful for establishing the role of the complex in ubiquitin-dependent proteolysis in vivo, 5° because these mutants have been found to be defective in the degradation of known substrates of the
49 p. j. Savory, H. Djaballah, H. Angliker, E. Shaw, and A. J. Rivett, Biochem. J. 296, 601 (1993). 50 W. Heinemeyer, J. A. Kleinschmidt, J. Saidowsky, C. Escher, and D. H. Wolf, EMBO J. 10, 555 (1991). 5J W. Hilt, C. Enenkel, A. Gruhler, T. Singer, and D. H. Wolf, J. Biol. Chem. 268, 3479 (1993). 52 W. Heinemeyer, A. Gruhler, V. M6hrle, Y. Mah6 and D. H. Wolf, J. Biol. Chem. 268, 5115 (1993). 53 S. Jentsch, Trends Cell Biol. 2, 98 (1992). 54 A. J. Rivett and E. Knecht, Curr. Biol. 3, 127 (1993). ~5 M. Rechsteiner, Cell (Cambridge, Mass.) 66, 615 (1991).
350
SERINE PROTEASES
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ubiquitin system. 56,57 Also, the discovery of two y-interferon-inducible multicatalytic endopeptidase complex genes in the MHC class II region58,59 has prompted the suggestion that the enzyme is involved in antigen processing for presentation by the MHC class I pathway. 56B. Richter-Ruoff, W. Heinemeyer, and D. H. Wolf, FEBS Lett. 302, 192 (1992). 57S. Seufert and S. Jentsch, EMBO J. 11, 3077 (1992). s8 R. Glynne, S. H. Powis, S. Beck, A. Kelly, L. A. Kerr, and J. Trowsdale, Nature (London) 353, 357 (1991). 59C. K. Martinez and J. J. Monaco, Nature (London) 353, 664 (1991).
[25] A T P - D e p e n d e n t P r o t e a s e La (Lon) from Escherichia coli B y ALFRED L. GOLDBERG, RICHARD P. MOERSCHELL,
CHIN HA CHUNG, and MICHAEL R. MAURIZI Introduction Protease L a (endopeptidase La, EC 3.4.21.53), the product of the lon gene in E s c h e r i c h i a coli, is an A T P - d e p e n d e n t cytosolic p r o t e a s e that plays an important role in intracellular protein degradation.l-4 In bacteria, as in eukaryotic cells, protein degradation is an energy-requiring p r o c e s s ) Studies o f the biochemical basis for this energy requirement led to the d i s c o v e r y o f p r o t e a s e La, a new type o f p r o t e o l y t i c e n z y m e , w h o s e activity is coupled to A T P hydrolysis. 6-9 This e n z y m e catalyzes the rate-limiting steps in the degradation of highly abnormal proteins in E. coli 7 and certain short-lived regulatory p r o t e i n s ) ° It functions independently o f ubiquitin; in fact, no factor similar to ubiquitin has b e e n found in E. coli or other bacteria. Unlike proteases described previously, this e n z y m e has A T P a s e i A. L, Goldberg, Eur. J. Biochem. 203, 9 (1992). 2 A. L. Goldberg, K, H. S. Swamy, C. H. Chung, and F. S. Larimore, this series, Vol. 80, p. 680. 3 S. Gottesman, in "Escherichia Coli and Salmonella Typhimurium" (F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger, eds.), p. 1308. American Society for Microbiology, Washinghton, D.C., 1987. 4 S. Gottesman and M. R. Maurizi, Microbiol. Reo. 36, 592 (1992). 5 K. Olden and A. L. Goldberg, Biochim. Biophys. Acta 542, 385 (1978). 6 K. S. Swamy and A. L. Goldberg, Nature (London) 292, 652 (1981). 7 C. H. Chung and A. L. Goldberg, Proc. Natl. Acad. Sci. U.S.A. 78, 4931 (1981). 8 M. Charette, G. W. Henderson, and A. Markovitz, Proc. Natl. Acad. Sci. U.S.A. 78, 4728 (1981). 9 F. S. Larimore, L. Waxman, and A. L. Goldberg, J. Biol. Chem. 257, 4187 (1982). l0 S. Gottesman, Annu. Rev. Genet. 23, 163 (1989).
METHODS IN ENZYMOLOGY, VOL. 244
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