- Email: [email protected]
Proteasome inhibition enhances the stability of mouse CuZn superoxide dismutase with mutations linked to familial amyotrophic lateral sclerosis
JOURNAL OF THE
NEUROLOGICAL SCIENCES
ELSEVIER
Journal of the Neurological Sciences 139 (1996) 15-20
Proteasome inhibition enhances the stability of mouse Cu/Zn superoxide dismutase with mutations linked to familial amyotrophic lateral sclerosis Eric K. Hoffman a3* , Heide M. Wilcox a, Richard W. Scott a, Robert Siman b ’ Department of Molecular Biology, Cephalon, Inc., 145 Brandyvine Parkway, West Chester, PA 19380, USA b Department of Biochemisty, Cephalon, Inc., 145 Brandyvine Parkway. West Chester, PA 19380, USA
Received 7 November 1995; accepted 9 January 1996
Abstract Point mutations occurring within the Cu/Zn superoxide dismutase (SODI) gene have been implicated in the etiology of some cases of familial amyotrophic lateral sclerosis (FALS). In order to better understand the functional consequences of these mutations, we have introduced FALS mutations into the mouse SODI gene and studied the expression of the mutant templates in stably transformed cell
lines. Pulse-chase analyses of lysates derived from cell lines stably expressing the Cu/Zn SOD isoforms indicate that the FALS mutant Cu/Zn SOD proteins are turned over more rapidly than wild-type SOD. Protease inhibitors specific for the major intracellular proteolytic activities were used to characterize the degradative pathways involved in the turnover of mutant Cu/Zn SOD. Inhibition of the chymotrypsin-like activity of the proteasome (also known as multicatalytic proteinase or ubiquitin, ATP-dependent proteinase) by a synthetic dipeptide aldehyde led to a significant increase in levels of the mutant Cu/Zn SOD implicating this proteolytic pathway in the turnover of the FALS mutant SOD proteins. Keywords:
Cu/Zn
superoxide dismutase; Proteasome; Amyotrophic
lateral sclerosis: Proteolytic degradation; Proteasome inhibitor; SOD1 mutations
1. Introduction
sents a principal oxidative
Amyotrophic lateral sclerosis (ALS) is a progressive paralytic disorder caused by degeneration of large motor neurons of the spinal cord and brain. Approximately 5- 10% of ALS cases are familial (FALS) and are inherited as an autosomal dominant trait. Recently, more than 20 different missense mutations have been identified in a subset of families with FALS and these have been shown to occur within the SODI gene encoding the anti-oxidant enzyme
Cu/Zn superoxide dismutase (Cu/Zn SOD) (Rosen et al., 1993). These mutations map to highly conserved structural domains and are thought to alter the normal folding or dimerization of the protein (Deng et al., 1993). Cu/Zn SOD catalyzes the dismutation of the superoxide free radical to form hydrogen peroxide and in so doing repre-
defense in protecting the cell against
damage. Erythrocyte
lysate samples from FALS
patients bearing mutations in the SOD1 gene show a mean reduction of approximately 50% decrease in total enzymatic activity suggesting the possibility that oxidative damage may play a causative role in this disorder (Deng et al., 1993). However, the available evidence provides little indication for increased oxidative stress or damage in FALS patients (Bowling
et al., 1993) and implies
that the
mutant Cu/Zn SOD proteins likely contribute to the onset or progression of FALS through an as yet undefined gain-of-function The majority
mechanism. of the mutant Cu/Zn
SOD proteins retain
near normal levels of specific activity, indicating that a reduction in SOD1 concentration or an increase in the rate of turnover, rather than enzymatic inactivation, may account for the observed near 50% decrease in Cu/Zn SOD activity seen in FALS patients (Bowling et al., 1995).
Abbreviations: Cu/Zn SOD, copper/zinc superoxide dismutase; FALS, familial amyotrophic lateral sclerosis * Corresponding author. Tel.: (610) 344-0200; Fax: (610) 344-0065. 0022-510X/96/$15.00 0 (1996) El sevier Science All rights reserved PII SOO22-5 10X(96)0003 1-7
Although it has been demonstrated that the human FALS mutant SOD proteins are less stable than wild-type SOD in transient expression assays (Borchelt et al., 19941, no study
I6
E.K. Hofitm
et al. /Journal
of the Neurolo&ul
to date has addressed the specific mechanism(s) responsible for the degradation of these proteins. Our goal in the present study was to measure the relative stability of the mutant Cu/Zn SOD proteins in stably transformed cell lines and to characterize the proteolytic pathway(s) involved in their degradation. Using protease inhibitors specific for major intracellular proteolytic activities, in conjunction with steady-state and pulse-chase methodologies, we have identified the proteasome as the major proteolytic pathway responsible for the degradation of several representative FALS mutant Cu/Zn SOD proteins.
2. Materials
and methods
2.1. Cell culture
The human embryonic kidney cell line designated 293 was obtained from ATCC (#CRL 1573) and grown in Dulbecco’s modified Eagle’s medium with 4.6 g/l glucose, 10% heat-inactivated horse serum (Gibco/Life Technologies). Cells were maintained at 37°C in an atmosphere of 5% CO,. Cell lines stably expressing mouse native and FALS mutant Cu/Zn SOD proteins were derived by calcium phosphate transfection of 293 cells (Chen and Okayama, 1988) with Cu/Zn SOD cDNA expression constructs followed by selection for neomycin resistance by growth in the presence of 1 mg/ml G418 (Gibco/Life Technologies). Cells were maintained in G418 selection for 3 weeks at which time drug selection was removed and resistant colonies were pooled for further growth. The proteasome and other protease inhibitors were solubilized in dimethyl sulfoxide (DMSO) at the indicated concentrations. Cells were treated with an equal volume of DMSO or left untreated as negative controls. Approximately 48 h prior to treatment, 5 X lo5 cells were seeded in 36 mm dishes and maintained at 37°C in an atmosphere of 5% CO,. Cells were treated with protease inhibitors (CEP1508, CEP 160 1, calpeptin (Calbiochem), DK-3 (Enzyme Systems Products)) or with DMSO as a vehicle control for a duration of 24 h. 2.2. Cloning and mutagenesis The mouse SOD1 cDNA clone was obtained from Jim Mahaffey at North Carolina State University (Bewley, 1988). This clone was modified by PCR methodologies to incorporate a Kozak translation initiation consensus signal (5’-GCCGCCACC-3’) directly upstream of the ATG start codon as well as a Hind111 restriction site 5’ of this consensus signal and an XhoI restriction site at the 3’ terminus of the cDNA. The FALS point mutations at amino acid 4 (Ala4Val) (GCG > GTG) and amino acid 113 (Ilel I3Thr) (ATT > ACT) were introduced into the SOD I cDNA clone using the overlap extension PCR mutagenesis procedure (Ho et al., 1989).
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139 (19961 15-20
2.3. lmmunoblot detection of Cu/Zn
SOD
Cell lysates were prepared by lysing the cell pellets from 36 mm dishes in 75 ~1 of phosphate-buffered saline (PBS) by freeze/thaw cycling. Protein concentrations of the cell lysates were determined using the BCA method (Pierce) and 2-2.5 p,g of each sample were electrophoresed on a 4-20% polyacrylamide gel using a Tris/glycine/SDS (25 mM Tris/192 mM glycine/O.l% SDS) buffer system. Proteins were transferred to nitrocellulose filters by electro-elution and filters were blocked by incubation in blotto solution (5% dry milk in 25 mM Tris-buffered saline (1 X TBS)) for 30 min. Filters were transferred to primary antibody solution (1 : 10,000 dilution in blotto solution) and incubated for 2-18 h. The primary antibody used in these studies was a polyclonal rabbit antiserum raised against purified mouse Cu/Zn SOD produced in Escherichia. coli (Hazelton Research Products). The secondary antibody was a goat anti-rabbit IgG conjugated to alkaline phosphatase (Bio-Rad). Stained bands corresponding to Cu/Zn SOD protein were quantitated using a DocuGel V image analysis system and RFLPscan software (Scanalytics). 2.4. Pulse-chase methodology Cu/Zn SOD was metabolically labeled and immunoprecipitated using the method of Firestone and Winguth (1990). Briefly, cells were incubated in medium lacking methionine for 45 min, then pulsed with a mixture of
23 8
h,h.
i *
1 wild-type
* lo!
[email protected]
Ilcl13Tlu
.--a--
Alarlval
, 0
, 10
,
, 20
,T --oh., 1 -..J 1
,
, 30
,
, 40
*
, 50
,
, 60
TIME IN HOURS Fig. I. Pulse-chase analysis of wild-type and FALS mutant Cu/Zn SOD proteins. Human 293 embryonic kidney cells expressing the indicated SOD isoform were pulse-labeled with “S-methionine/ ” S-cysteine and chased with cold amino acids. Cell lysates were prepared at various times after radiolabeling and mouse Cu/Zn SOD protein was immunoprecipitated with polyclonal anti-Cu/Zn SOD serum specific for mouse Cu/Zn SOD. Protein samples were fractionated by electrophoresis and SOD levels were quantitated by phosphor-imaging. Each data point was derived from experiments performed in triplicate.
E.K. Hoffman
et al. /Journal
of the Neurological
35S-methionine and 35S-cysteine (ICN) for 1 h. Cells were rinsed with DMEM and chased with medium containing cold methionine/cysteine for the indicated times. Labeled mouse Cu/Zn SOD was immunoprecipitated using antiserum raised against mouse Cu/Zn SOD and electrophoresed on a 4-20% polyacrylamide gel using a 25 mM Tris/ 192 mM glycine/O. 1% SDS buffer system. Gels were dryed and exposed for 24-48 h. Immunoprecipitated protein was quantitated by phosphor imaging (Molecular Diagnostics). Under immunoprecipitation conditions, the Cu/Zn SOD antiserum used in these studies specifically precipitates mouse Cu/Zn SOD and does not cross-react with endogenous human Cu/Zn SOD.
(a)
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3. Results To investigate the relative rates of turnover of the FALS mutant and wild-type Cu/Zn SOD proteins, we utilized sublines of human 293 embryonic kidney cells stably transformed with cDNA expression vectors encoding mouse wild-type or FALS mutant Cu/Zn SOD (Ala4Val or Ilel13Thr) in pulse-chase labeling studies. The results of these studies demonstrate that the wild-type SOD protein is extremely stable, having a half-life greater than 100 h, while the FALS mutant proteins show significant reductions in half-lives (approximately 45 h for Ile113Thr and 14 h for Ala4Val) (Fig. 1). The profiles of mutant Cu/Zn
TREATMENT
Cu/Zn
SOD
Wild-type
+ f-
Human Mouse
Ala4Val
f+
Human Mouse
q q
DMSO
CEP1508
CEP1601
Wild-type Ala4Val
CALPEPTIN
DK-3
TREATMENT Fig. 2. Effect of protease inhibitors on steady-state Cu/Zn SOD protein levels. Human 293 cells expressing the indicated SOD isoforms were treated for 24 h with protease inhibitors solubilized in DMSO. Inhibitor concentrations were as follows: 20 pM CEP1508, 20 p,M CEP1601, 10 pM calpeptin, and 5 p,M DK-3. Cell lysates were prepared 24 h after treatment, fractionated by electrophoresis and proteins were transferred to nitrocellulose. Cu/Zn SOD proteins were detected using polyclonal anti-Cu/Zn SOD serum. A: Cu/Zn SOD immunoblots of cell lysates following treatment with protease inhibitors. B: Quantitation of immunoblots by densitometric scanning. Protein levels were quantitated by densitometric scanning of immunoblots using a DocuGel V image analysis system and RFLPscan (Scanalytics) software. The levels of Cu/Zn protein are expressed as units of induction relative to DMSO-treated control samples. Each data point was derived from experiments performed in quadruplicate.
18
E.K. Hoffman et al./Journal
of the Neurological Sciences 139 (1996) 15-20
SOD turnover display a slight biphasic nature suggesting the possible presence of distinct pools of SOD protein which are degraded at different rates. The estimated halflives were extrapolated from data points taken early in the decay curve (I 20 h) and therefore represent rates of the most rapidly degraded pools of protein. As a complement to the pulse-chase analyses, we were interested in identifying the protease responsible for the preferential degradation of the mutant Cu/Zn SOD isoforms. To detect the SOD proteins, immunoblot assays were performed using polyclonal Cu/Zn SOD antiserum. Under these immunoblotting conditions, the antiserum recognizes both mouse and human Cu/Zn SOD species which can be distinguished by differences in electrophoretic mobility (Fig. 2A). Steady-state levels of wildtype and mutant Cu/Zn SOD (Ala4Val) were measured in the presence of protease inhibitors specific to major intracellular degradative pathways. The compound CEPl508 is a synthetic dipeptide aldehyde that specifically inhibits the chymotrypsin-like activity of the proteasome and CEP 1601 is a far less active semicarbazone analog of CEPl508 (Harding et al., 1995; Iqbal et al., 1995). Calpeptin (Calbiochem) inhibits the calcium-activated protease calpain and DK-3 (Enzyme Systems Products) inhibits lysosomal cysteine proteases. Cell lines stably expressing the indicated Cu/Zn SOD isoforms were treated with the various protease inhibitors or with DMSO as a vehicle control and cell lysates were prepared 24 h after treatment. The results of these studies indicate that the proteolytic
T
-.-.. --+ -
3.5
turnover of Ala4Val SOD isoform is clearly a function of proteasome activity. Specific inhibition of the proteasome by CEP1508 leads to an approximate 3-fold increase in the accumulation of mutant Cu/Zn SOD protein (Fig. 2B). Similar results were obtained when cells expressing the Ilell3Thr SOD isoform were treated with CEP1508 (data not shown). Neither treatment with the inactive analog CEP1601 or inhibitors of calpain or lysosomal cysteine proteases had a significant effect upon SOD turnover in these studies. To further establish the relation between proteasome activity and Cu/Zn SOD turnover, dose response studies were conducted on cell lines stably producing the wild-type mouse SOD or the FALS mutant proteins Ala4Val and Ilell3Thr. Cell lines expressing the indicated SOD isoforms were treated with various doses of CEP1508 for 24 h and SOD levels were measured by densitometric scanning of immunoblots. Levels of Cu/Zn SOD are expressed as fold increase in SOD levels relative to vehicle-treated samples. Dose-response analysis indicated that treatment of cells with CEP1508 led to significant accumulations of mutant SOD protein levels with essentially maximal effects occurring at approximately 5 pM (Fig. 3). CEP1508 also exhibits a modest effect on turnover of the wild-type SOD isoform; however the extreme stability of this protein precludes a more firm conclusion of this observation. To better quantitate the magnitude of proteasome inhibition on Cu/Zn SOD turnover, pulse-chase studies were done with the Ala4Val mutant SOD protein in the presence or absence of 20 p.M CEP1508. An approximate 2-fold increase
-_._ ---1.
-..
+ .-.. .+.“..
Wild-type Ald”gd
----o--m-
Ilell3Thr
T T
51 I E
+
UNTREATED
---o----
20 UM cEP1508
100 0
1 0
10
20
30
40
5
10
1
15
20
50
TIME IN HOURS CEP1508 CONCENTRATION
(ti)
Fig. 3. Dose-dependent increases in levels of wild-type and mutant Cu/Zn SOD proteins after treatment with CEP1508. Sublines of 293 fibroblasts expressing wild-type or mutant SOD isoforms were pretreated for 24 h with the indicated concentrations of CEP1508. Levels of Cu/Zn SOD protein in cell lysates were measured by densitometric scanning of immunoblots. Protein levels are expressed as units relative to vehicletreated samples. Shown are the results of three separate experiments + SD.
Fig. 4. Effect of the proteasome inhibitor CEP1508 on the turnover rate of the FALS mutant Ala4Val Cu/Zn SOD. Cells stably expressing the Ala4Val SOD isoform were pretreated for 24 h with 20 pM CEP1508 or DMSO prior to pulse-labeling with ‘SS-methionine/ “S-cysteine. Cells were chased with cold amino acids and Cu/Zn SOD was immunoprecipitated from cell lysates. Proteins were fractionated by electrophoresis and levels of Cu/Zn SOD were quantitated by phosphor-imaging. The data shown are the results of three separate experiments i SD.
E.K. Hofjinan
et al. /Journal
of the Neurological
in the half-life of the mutant protein was found after proteasome inhibition (Fig. 4). These results support a primary role for the proteasome degradative pathway in turnover of the FALS mutant Cu/Zn SOD protein in the stably transfected 293 cell line.
4. Discussion Despite the recent correlation between point mutations occurring in the SOD1 gene and a subset of familial ALS cases, the mechanism(s) by which the mutant Cu/Zn SOD isoforms contribute to the onset or progression of this disorder remain undefined. A previous study has used transient expression assays to determine that the mutations lead to a dimunition of protein half-life (Borchelt et al., 1994). The present study confirms these findings using stably transformed cell lines. Our results indicate that isoforms of mouse Cu/Zn SOD bearing point mutations associated with FALS show a marked increase in the rate of turnover when compared to wild-type protein. Moreover, the data implicate the proteasome in the preferential degradation of the FALS mutant Cu/Zn SOD proteins. This protease complex is an essential component of the ubiquitin, ATP-dependent degradative pathway known to be involved in the turnover of short-lived cell cycle regulatory proteins as well as misfolded or oxidized proteins (Ciechanover, 1994). Our data support the hypothesis that the point mutations are disrupting the structural integrity of the SOD protein which contributes to the premature degradation of the misfolded protein. This conclusion is in agreement with accumulated evidence indicating that recognition and degradation of substrates by the proteasome is enhanced by the exposure of hydrophobic amino acid side chains as a consequence of misfolding or oxidative damage (Grune et al., 1995). Doses of CEP1508 which elevate mutant Cu/Zn SOD levels correspond well with doses which inhibit intracellular proteasome activity and block other proteasome-mediated biological processes (Harding et al., 1995; Iqbal et al., 1995). These findings suggest that enhanced degradation through the proteasome pathway is responsible for the near 50% decrease in Cu/Zn SOD activity seen in FALS patients bearing these mutations. The present observations indicating that the mutant Cu/Zn SOD proteins display reduced stability parallel those reported by others in studies measuring the half-lives of human FALS mutant SOD proteins (Borchelt et al., 1994). The rank order with respect to the rate of turnover of the mutant mouse proteins is in good agreement with those published for the mutant human proteins (Ala4Val > Be 113Thr > wild-type). However, a consistent difference between the two reports is the relative longevity of wildtype and mutant isoforms. These discrepancies may reflect the different methods used to measure protein half-lives (transient vs. stable expression) or may represent inherent
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139 (1996)
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19
differences in stability between human and mouse Cu/Zn SOD proteins. While the reduction in Cu/Zn SOD activity seen in FALS patients with mutations in the SOD1 gene is a common feature of this disorder, there exists little evidence that these individuals are predisposed to increased oxidative damage (Bowling et al., 1993). Studies conducted with transgenic mice overexpressing select mutant SOD1 genes argue that it is most likely a novel gain-of-function attributable to the mutant enzyme that underlies the neurotoxicity seen in FALS (Gurney et al., 1994; Ripps et al., 1995; Wong et al., 1995) One potential detrimental attribute incurred by the FALS mutations is based on the proposal that Cu/Zn SOD represents a significant factor in the binding and sequestration of free copper (Brown, 1995). Due to its ability to participate in reactions with hydrogen peroxide to form the highly toxic hydroxyl radical, free copper could act as a potent cytotoxic agent if not bound properly to a protein carrier. Recent studies support this hypothesis by demonstrating that Cu/Zn SOD proteins bearing selective FALS mutations exhibit a reduction in metal binding capacity (Nishida et al., 1994; Carri et al., 1994). In view of our findings, it is conceivable that the elevated turnover of the mutant Cu/Zn SOD proteins may represent a cytotoxic insult in that the rapid turnover of this metalloprotein may liberate free copper that could potentiate metal toxicity. This raises the possibility that stabilization of the protein, by proteasome inhibition or other means, could be therapeutically beneficial. On the other hand, if the proposed gain-of-function is associated with a novel activity unrelated to normal Cu/Zn SOD function, then enhancing the stability of mutant protein could exacerbate the disease phenotype. Such questions can be addressed directly in FALS SOD1 transgenic mice that exhibit motor neuron degeneration. The proteasome inhibitors described in this report represent useful tools in the study of the role of Cu/Zn SOD turnover in the FALS neurodegenerative process. Moreover, the compounds provide a means to further characterize the involvement of the proteasome and ubiquitin-protein conjugates in other neuropathological processes. Acknowledgements The authors would like to thank Drs. Mohamed Iqbal and Sankar Chatterjee for synthesis and provision of proteasome inhibitors CEP1508 and CEP1601. We thank Todd Levins for expert technical assistance and Dr. Dorothy G. Flood for careful reading of the manuscript. We are indebted to Drs. Jeffry Vaught and Frank Baldino for their continued support of this research. References Bewley, B.C. (1988) cDNA and deduced amino acid sequence of murk Cu-Zn superoxide dismutase Nucl. Acids Res., 16: 2728.
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Borchelt, D.R., Lee, M.K., Slunt, H.S., Guamieri, M., Xu, Z-S., Wong, P.C., Brown Jr., R.H., Price, D.L., Sisodia, S.S. and Cleveland, D.W. (1994) Superoxide dismutase 1 with mutations linked to familial amyotrophic lateral sclerosis possesses significant activity. Proc. Natl. Acad. Sci. USA, 91: 8292-8296. Bowling, A.C., Schulz, J.B., Brown Jr., R.H. and Beal, M.F. (1993) Superoxide dismutase activity, oxidative damage, and mitochondrial energy metabolism in familial and sporadic amyotrophic lateral sclerosis. J. Neurochem., 61: 2322- 2325. Bowling, A.C., Barkowski, E.E., McKenna-Yasek, D., Sapp, P., Horvitz, H.R., Beal, M.F. and Brown Jr., R.H. (1995) Superoxide dismutase concentration and activity in familial amyotrophic lateral sclerosis, J. Neurochem., 64: 2366-2369. Brown Jr., R.H. (1995) Amyotrophic lateral sclerosis: recent insights from genetics and transgenic mice. Cell, 80: 687-692. Carri, M.T., Battistoni, A., Polizio, F., Desideri, A. and Rotilio, Cl. (1994) Impaired copper binding by the H46R mutant of human Cu,Zn superoxide dismutase, involved in amyotrophic lateral sclerosis. FEBS Lett., 356: 314-316. Chen, CA. and Okayama, H. (1988) Calcium phosphate-mediated gene transfer: a highly efficient transfection system for stably transforming cells with plasmid DNA. BioTechniques, 6: 632-637. Ciechanover, A. (1994) The ubiquitin-proteasome proteolytic pathway, Cell, 79: 13-21. Deng, H.-X., Hentati, A., Tainer, J.A., Iqbal, Z., Cayabyab, A., Hung, W.-Y., Getzoff, E.D., Hu, P., Herzfeldt, B., Roos, R.P., Warner, C., Deng, G., Soriano, E., Smyth, C., Parge, H.E., Ahmed, A., Roses, A.D., Hallewell, R.A., Pericak-Vance, M.A. and Siddique, T. (1993) Amyotrophic lateral sclerosis and structural defects in Cu,Zn superoxide dismutase. Science, 261: 1047-1051. Firestone, G.L. and Winguth, SD. (1990) In: M.P. Deutscher (Ed.), Guide to Protein Purification, Methods in Enzymology, Vol. 182, Academic Press, San Diego, CA, pp. 688-700. Grune, T., Reinheckel, T., Joshi, M. and Davies, K.J.A. (1995) Proteolysis in cultured liver epithelial cells during oxidative stress. J. Biol. Chem., 270: 2344-235 1. Gurney, M.E., Pu, H., Chiu, A.Y., Dal Canto, M.C., Polchow, C.Y.,
Alexander, D.D., Caliendo, J., Hentati, A., Kwon, Y.W., Deng, H., Chen, W., Zhai, P., Sufit, R.L. and Siddique, T. (1994) Motor neuron degeneration in mice that express a human Cu, Zn superoxide dismutase mutation. Science, 264: 1772- 1775. Harding, C.V., France, J., Song, R., Farah, J.M., Chatterjee, S., Iqbal, M. and Siman, R. (1995) Novel dipeptide aldehydes are proteasome inhibitors and block the MHC-1 antigen-processing pathway. J. Immunol., 155: 1767-1775. Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K. and Pease, L.R. (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene, 77: 51-59. Iqbal, M., Chatterjee, S., Kauer, J.C., Das, M., Messina, P., Freed, B., Biazzo, B. and Siman, R. (1995) Potent inhibitors of proteasome. J. Med. Chem., 39: 2276-2277. Nishida, C.R., Grail, E.B. and Valentine, J.S. (1994) Characterization of three yeast copper-zinc superoxide dismutase mutants analogous to those coded for in familial amyotrophic lateral sclerosis, Proc. Natl. Acad. Sci. USA, 91: 9906-9910. Ripps, M.E., Huntley, G.W., Hof, P.R., Morrison, J.H. and Gordon, J.W. (1995) Transgenic mice expressing an altered murine superoxide dismutase gene provide an animal model of amyotrophic lateral sclerosis. Proc. Natl. Acad. Sci. USA, 92: 689-693. Rosen, D.R., Siddique, T., Patterson, D., Figlewicz, D.A., Sapp, P., Hentati, A., Donaldson, D., Goto, J., O’Regan, J.P., Deng, H.-X., Rahmani, Z., Krius, A., McKenna-Yasek, D., Cayabyab, A., Gaston, S.M., Berger, R., Tanzi, R.E., Halperin, J.J., Herzfeldt, B., van den Bergh, R., Hung, W.-Y., Bird, T., Deng, G., Mulder, D.W., Smyth, C., Laing, N.G., Soriano, E., Pericak-Vance, M.A., Hains, J., Rouleau, G.A., Gusella, J.S., Horvitz, H.R. and Brown Jr., R.H. (1993) Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature, 362: 59-62. Wong, P.C., Pardo, CA., Borchelt, D.R., Lee, M.K., Copeland, N.G., Jenkins, N.A., Sisodia, S.S., Cleveland, D.W. and Price, D.L. (1995) An adverse property of a familial ALS-linked SOD1 mutation causes motor neuron disease characterized by vacuolar degeneration of mitochondria. Neuron, 14: 1105-l 116.
NEUROLOGICAL SCIENCES
ELSEVIER
Journal of the Neurological Sciences 139 (1996) 15-20
Proteasome inhibition enhances the stability of mouse Cu/Zn superoxide dismutase with mutations linked to familial amyotrophic lateral sclerosis Eric K. Hoffman a3* , Heide M. Wilcox a, Richard W. Scott a, Robert Siman b ’ Department of Molecular Biology, Cephalon, Inc., 145 Brandyvine Parkway, West Chester, PA 19380, USA b Department of Biochemisty, Cephalon, Inc., 145 Brandyvine Parkway. West Chester, PA 19380, USA
Received 7 November 1995; accepted 9 January 1996
Abstract Point mutations occurring within the Cu/Zn superoxide dismutase (SODI) gene have been implicated in the etiology of some cases of familial amyotrophic lateral sclerosis (FALS). In order to better understand the functional consequences of these mutations, we have introduced FALS mutations into the mouse SODI gene and studied the expression of the mutant templates in stably transformed cell
lines. Pulse-chase analyses of lysates derived from cell lines stably expressing the Cu/Zn SOD isoforms indicate that the FALS mutant Cu/Zn SOD proteins are turned over more rapidly than wild-type SOD. Protease inhibitors specific for the major intracellular proteolytic activities were used to characterize the degradative pathways involved in the turnover of mutant Cu/Zn SOD. Inhibition of the chymotrypsin-like activity of the proteasome (also known as multicatalytic proteinase or ubiquitin, ATP-dependent proteinase) by a synthetic dipeptide aldehyde led to a significant increase in levels of the mutant Cu/Zn SOD implicating this proteolytic pathway in the turnover of the FALS mutant SOD proteins. Keywords:
Cu/Zn
superoxide dismutase; Proteasome; Amyotrophic
lateral sclerosis: Proteolytic degradation; Proteasome inhibitor; SOD1 mutations
1. Introduction
sents a principal oxidative
Amyotrophic lateral sclerosis (ALS) is a progressive paralytic disorder caused by degeneration of large motor neurons of the spinal cord and brain. Approximately 5- 10% of ALS cases are familial (FALS) and are inherited as an autosomal dominant trait. Recently, more than 20 different missense mutations have been identified in a subset of families with FALS and these have been shown to occur within the SODI gene encoding the anti-oxidant enzyme
Cu/Zn superoxide dismutase (Cu/Zn SOD) (Rosen et al., 1993). These mutations map to highly conserved structural domains and are thought to alter the normal folding or dimerization of the protein (Deng et al., 1993). Cu/Zn SOD catalyzes the dismutation of the superoxide free radical to form hydrogen peroxide and in so doing repre-
defense in protecting the cell against
damage. Erythrocyte
lysate samples from FALS
patients bearing mutations in the SOD1 gene show a mean reduction of approximately 50% decrease in total enzymatic activity suggesting the possibility that oxidative damage may play a causative role in this disorder (Deng et al., 1993). However, the available evidence provides little indication for increased oxidative stress or damage in FALS patients (Bowling
et al., 1993) and implies
that the
mutant Cu/Zn SOD proteins likely contribute to the onset or progression of FALS through an as yet undefined gain-of-function The majority
mechanism. of the mutant Cu/Zn
SOD proteins retain
near normal levels of specific activity, indicating that a reduction in SOD1 concentration or an increase in the rate of turnover, rather than enzymatic inactivation, may account for the observed near 50% decrease in Cu/Zn SOD activity seen in FALS patients (Bowling et al., 1995).
Abbreviations: Cu/Zn SOD, copper/zinc superoxide dismutase; FALS, familial amyotrophic lateral sclerosis * Corresponding author. Tel.: (610) 344-0200; Fax: (610) 344-0065. 0022-510X/96/$15.00 0 (1996) El sevier Science All rights reserved PII SOO22-5 10X(96)0003 1-7
Although it has been demonstrated that the human FALS mutant SOD proteins are less stable than wild-type SOD in transient expression assays (Borchelt et al., 19941, no study
I6
E.K. Hofitm
et al. /Journal
of the Neurolo&ul
to date has addressed the specific mechanism(s) responsible for the degradation of these proteins. Our goal in the present study was to measure the relative stability of the mutant Cu/Zn SOD proteins in stably transformed cell lines and to characterize the proteolytic pathway(s) involved in their degradation. Using protease inhibitors specific for major intracellular proteolytic activities, in conjunction with steady-state and pulse-chase methodologies, we have identified the proteasome as the major proteolytic pathway responsible for the degradation of several representative FALS mutant Cu/Zn SOD proteins.
2. Materials
and methods
2.1. Cell culture
The human embryonic kidney cell line designated 293 was obtained from ATCC (#CRL 1573) and grown in Dulbecco’s modified Eagle’s medium with 4.6 g/l glucose, 10% heat-inactivated horse serum (Gibco/Life Technologies). Cells were maintained at 37°C in an atmosphere of 5% CO,. Cell lines stably expressing mouse native and FALS mutant Cu/Zn SOD proteins were derived by calcium phosphate transfection of 293 cells (Chen and Okayama, 1988) with Cu/Zn SOD cDNA expression constructs followed by selection for neomycin resistance by growth in the presence of 1 mg/ml G418 (Gibco/Life Technologies). Cells were maintained in G418 selection for 3 weeks at which time drug selection was removed and resistant colonies were pooled for further growth. The proteasome and other protease inhibitors were solubilized in dimethyl sulfoxide (DMSO) at the indicated concentrations. Cells were treated with an equal volume of DMSO or left untreated as negative controls. Approximately 48 h prior to treatment, 5 X lo5 cells were seeded in 36 mm dishes and maintained at 37°C in an atmosphere of 5% CO,. Cells were treated with protease inhibitors (CEP1508, CEP 160 1, calpeptin (Calbiochem), DK-3 (Enzyme Systems Products)) or with DMSO as a vehicle control for a duration of 24 h. 2.2. Cloning and mutagenesis The mouse SOD1 cDNA clone was obtained from Jim Mahaffey at North Carolina State University (Bewley, 1988). This clone was modified by PCR methodologies to incorporate a Kozak translation initiation consensus signal (5’-GCCGCCACC-3’) directly upstream of the ATG start codon as well as a Hind111 restriction site 5’ of this consensus signal and an XhoI restriction site at the 3’ terminus of the cDNA. The FALS point mutations at amino acid 4 (Ala4Val) (GCG > GTG) and amino acid 113 (Ilel I3Thr) (ATT > ACT) were introduced into the SOD I cDNA clone using the overlap extension PCR mutagenesis procedure (Ho et al., 1989).
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139 (19961 15-20
2.3. lmmunoblot detection of Cu/Zn
SOD
Cell lysates were prepared by lysing the cell pellets from 36 mm dishes in 75 ~1 of phosphate-buffered saline (PBS) by freeze/thaw cycling. Protein concentrations of the cell lysates were determined using the BCA method (Pierce) and 2-2.5 p,g of each sample were electrophoresed on a 4-20% polyacrylamide gel using a Tris/glycine/SDS (25 mM Tris/192 mM glycine/O.l% SDS) buffer system. Proteins were transferred to nitrocellulose filters by electro-elution and filters were blocked by incubation in blotto solution (5% dry milk in 25 mM Tris-buffered saline (1 X TBS)) for 30 min. Filters were transferred to primary antibody solution (1 : 10,000 dilution in blotto solution) and incubated for 2-18 h. The primary antibody used in these studies was a polyclonal rabbit antiserum raised against purified mouse Cu/Zn SOD produced in Escherichia. coli (Hazelton Research Products). The secondary antibody was a goat anti-rabbit IgG conjugated to alkaline phosphatase (Bio-Rad). Stained bands corresponding to Cu/Zn SOD protein were quantitated using a DocuGel V image analysis system and RFLPscan software (Scanalytics). 2.4. Pulse-chase methodology Cu/Zn SOD was metabolically labeled and immunoprecipitated using the method of Firestone and Winguth (1990). Briefly, cells were incubated in medium lacking methionine for 45 min, then pulsed with a mixture of
23 8
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i *
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* lo!
[email protected]
Ilcl13Tlu
.--a--
Alarlval
, 0
, 10
,
, 20
,T --oh., 1 -..J 1
,
, 30
,
, 40
*
, 50
,
, 60
TIME IN HOURS Fig. I. Pulse-chase analysis of wild-type and FALS mutant Cu/Zn SOD proteins. Human 293 embryonic kidney cells expressing the indicated SOD isoform were pulse-labeled with “S-methionine/ ” S-cysteine and chased with cold amino acids. Cell lysates were prepared at various times after radiolabeling and mouse Cu/Zn SOD protein was immunoprecipitated with polyclonal anti-Cu/Zn SOD serum specific for mouse Cu/Zn SOD. Protein samples were fractionated by electrophoresis and SOD levels were quantitated by phosphor-imaging. Each data point was derived from experiments performed in triplicate.
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35S-methionine and 35S-cysteine (ICN) for 1 h. Cells were rinsed with DMEM and chased with medium containing cold methionine/cysteine for the indicated times. Labeled mouse Cu/Zn SOD was immunoprecipitated using antiserum raised against mouse Cu/Zn SOD and electrophoresed on a 4-20% polyacrylamide gel using a 25 mM Tris/ 192 mM glycine/O. 1% SDS buffer system. Gels were dryed and exposed for 24-48 h. Immunoprecipitated protein was quantitated by phosphor imaging (Molecular Diagnostics). Under immunoprecipitation conditions, the Cu/Zn SOD antiserum used in these studies specifically precipitates mouse Cu/Zn SOD and does not cross-react with endogenous human Cu/Zn SOD.
(a)
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3. Results To investigate the relative rates of turnover of the FALS mutant and wild-type Cu/Zn SOD proteins, we utilized sublines of human 293 embryonic kidney cells stably transformed with cDNA expression vectors encoding mouse wild-type or FALS mutant Cu/Zn SOD (Ala4Val or Ilel13Thr) in pulse-chase labeling studies. The results of these studies demonstrate that the wild-type SOD protein is extremely stable, having a half-life greater than 100 h, while the FALS mutant proteins show significant reductions in half-lives (approximately 45 h for Ile113Thr and 14 h for Ala4Val) (Fig. 1). The profiles of mutant Cu/Zn
TREATMENT
Cu/Zn
SOD
Wild-type
+ f-
Human Mouse
Ala4Val
f+
Human Mouse
q q
DMSO
CEP1508
CEP1601
Wild-type Ala4Val
CALPEPTIN
DK-3
TREATMENT Fig. 2. Effect of protease inhibitors on steady-state Cu/Zn SOD protein levels. Human 293 cells expressing the indicated SOD isoforms were treated for 24 h with protease inhibitors solubilized in DMSO. Inhibitor concentrations were as follows: 20 pM CEP1508, 20 p,M CEP1601, 10 pM calpeptin, and 5 p,M DK-3. Cell lysates were prepared 24 h after treatment, fractionated by electrophoresis and proteins were transferred to nitrocellulose. Cu/Zn SOD proteins were detected using polyclonal anti-Cu/Zn SOD serum. A: Cu/Zn SOD immunoblots of cell lysates following treatment with protease inhibitors. B: Quantitation of immunoblots by densitometric scanning. Protein levels were quantitated by densitometric scanning of immunoblots using a DocuGel V image analysis system and RFLPscan (Scanalytics) software. The levels of Cu/Zn protein are expressed as units of induction relative to DMSO-treated control samples. Each data point was derived from experiments performed in quadruplicate.
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SOD turnover display a slight biphasic nature suggesting the possible presence of distinct pools of SOD protein which are degraded at different rates. The estimated halflives were extrapolated from data points taken early in the decay curve (I 20 h) and therefore represent rates of the most rapidly degraded pools of protein. As a complement to the pulse-chase analyses, we were interested in identifying the protease responsible for the preferential degradation of the mutant Cu/Zn SOD isoforms. To detect the SOD proteins, immunoblot assays were performed using polyclonal Cu/Zn SOD antiserum. Under these immunoblotting conditions, the antiserum recognizes both mouse and human Cu/Zn SOD species which can be distinguished by differences in electrophoretic mobility (Fig. 2A). Steady-state levels of wildtype and mutant Cu/Zn SOD (Ala4Val) were measured in the presence of protease inhibitors specific to major intracellular degradative pathways. The compound CEPl508 is a synthetic dipeptide aldehyde that specifically inhibits the chymotrypsin-like activity of the proteasome and CEP 1601 is a far less active semicarbazone analog of CEPl508 (Harding et al., 1995; Iqbal et al., 1995). Calpeptin (Calbiochem) inhibits the calcium-activated protease calpain and DK-3 (Enzyme Systems Products) inhibits lysosomal cysteine proteases. Cell lines stably expressing the indicated Cu/Zn SOD isoforms were treated with the various protease inhibitors or with DMSO as a vehicle control and cell lysates were prepared 24 h after treatment. The results of these studies indicate that the proteolytic
T
-.-.. --+ -
3.5
turnover of Ala4Val SOD isoform is clearly a function of proteasome activity. Specific inhibition of the proteasome by CEP1508 leads to an approximate 3-fold increase in the accumulation of mutant Cu/Zn SOD protein (Fig. 2B). Similar results were obtained when cells expressing the Ilell3Thr SOD isoform were treated with CEP1508 (data not shown). Neither treatment with the inactive analog CEP1601 or inhibitors of calpain or lysosomal cysteine proteases had a significant effect upon SOD turnover in these studies. To further establish the relation between proteasome activity and Cu/Zn SOD turnover, dose response studies were conducted on cell lines stably producing the wild-type mouse SOD or the FALS mutant proteins Ala4Val and Ilell3Thr. Cell lines expressing the indicated SOD isoforms were treated with various doses of CEP1508 for 24 h and SOD levels were measured by densitometric scanning of immunoblots. Levels of Cu/Zn SOD are expressed as fold increase in SOD levels relative to vehicle-treated samples. Dose-response analysis indicated that treatment of cells with CEP1508 led to significant accumulations of mutant SOD protein levels with essentially maximal effects occurring at approximately 5 pM (Fig. 3). CEP1508 also exhibits a modest effect on turnover of the wild-type SOD isoform; however the extreme stability of this protein precludes a more firm conclusion of this observation. To better quantitate the magnitude of proteasome inhibition on Cu/Zn SOD turnover, pulse-chase studies were done with the Ala4Val mutant SOD protein in the presence or absence of 20 p.M CEP1508. An approximate 2-fold increase
-_._ ---1.
-..
+ .-.. .+.“..
Wild-type Ald”gd
----o--m-
Ilell3Thr
T T
51 I E
+
UNTREATED
---o----
20 UM cEP1508
100 0
1 0
10
20
30
40
5
10
1
15
20
50
TIME IN HOURS CEP1508 CONCENTRATION
(ti)
Fig. 3. Dose-dependent increases in levels of wild-type and mutant Cu/Zn SOD proteins after treatment with CEP1508. Sublines of 293 fibroblasts expressing wild-type or mutant SOD isoforms were pretreated for 24 h with the indicated concentrations of CEP1508. Levels of Cu/Zn SOD protein in cell lysates were measured by densitometric scanning of immunoblots. Protein levels are expressed as units relative to vehicletreated samples. Shown are the results of three separate experiments + SD.
Fig. 4. Effect of the proteasome inhibitor CEP1508 on the turnover rate of the FALS mutant Ala4Val Cu/Zn SOD. Cells stably expressing the Ala4Val SOD isoform were pretreated for 24 h with 20 pM CEP1508 or DMSO prior to pulse-labeling with ‘SS-methionine/ “S-cysteine. Cells were chased with cold amino acids and Cu/Zn SOD was immunoprecipitated from cell lysates. Proteins were fractionated by electrophoresis and levels of Cu/Zn SOD were quantitated by phosphor-imaging. The data shown are the results of three separate experiments i SD.
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in the half-life of the mutant protein was found after proteasome inhibition (Fig. 4). These results support a primary role for the proteasome degradative pathway in turnover of the FALS mutant Cu/Zn SOD protein in the stably transfected 293 cell line.
4. Discussion Despite the recent correlation between point mutations occurring in the SOD1 gene and a subset of familial ALS cases, the mechanism(s) by which the mutant Cu/Zn SOD isoforms contribute to the onset or progression of this disorder remain undefined. A previous study has used transient expression assays to determine that the mutations lead to a dimunition of protein half-life (Borchelt et al., 1994). The present study confirms these findings using stably transformed cell lines. Our results indicate that isoforms of mouse Cu/Zn SOD bearing point mutations associated with FALS show a marked increase in the rate of turnover when compared to wild-type protein. Moreover, the data implicate the proteasome in the preferential degradation of the FALS mutant Cu/Zn SOD proteins. This protease complex is an essential component of the ubiquitin, ATP-dependent degradative pathway known to be involved in the turnover of short-lived cell cycle regulatory proteins as well as misfolded or oxidized proteins (Ciechanover, 1994). Our data support the hypothesis that the point mutations are disrupting the structural integrity of the SOD protein which contributes to the premature degradation of the misfolded protein. This conclusion is in agreement with accumulated evidence indicating that recognition and degradation of substrates by the proteasome is enhanced by the exposure of hydrophobic amino acid side chains as a consequence of misfolding or oxidative damage (Grune et al., 1995). Doses of CEP1508 which elevate mutant Cu/Zn SOD levels correspond well with doses which inhibit intracellular proteasome activity and block other proteasome-mediated biological processes (Harding et al., 1995; Iqbal et al., 1995). These findings suggest that enhanced degradation through the proteasome pathway is responsible for the near 50% decrease in Cu/Zn SOD activity seen in FALS patients bearing these mutations. The present observations indicating that the mutant Cu/Zn SOD proteins display reduced stability parallel those reported by others in studies measuring the half-lives of human FALS mutant SOD proteins (Borchelt et al., 1994). The rank order with respect to the rate of turnover of the mutant mouse proteins is in good agreement with those published for the mutant human proteins (Ala4Val > Be 113Thr > wild-type). However, a consistent difference between the two reports is the relative longevity of wildtype and mutant isoforms. These discrepancies may reflect the different methods used to measure protein half-lives (transient vs. stable expression) or may represent inherent
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differences in stability between human and mouse Cu/Zn SOD proteins. While the reduction in Cu/Zn SOD activity seen in FALS patients with mutations in the SOD1 gene is a common feature of this disorder, there exists little evidence that these individuals are predisposed to increased oxidative damage (Bowling et al., 1993). Studies conducted with transgenic mice overexpressing select mutant SOD1 genes argue that it is most likely a novel gain-of-function attributable to the mutant enzyme that underlies the neurotoxicity seen in FALS (Gurney et al., 1994; Ripps et al., 1995; Wong et al., 1995) One potential detrimental attribute incurred by the FALS mutations is based on the proposal that Cu/Zn SOD represents a significant factor in the binding and sequestration of free copper (Brown, 1995). Due to its ability to participate in reactions with hydrogen peroxide to form the highly toxic hydroxyl radical, free copper could act as a potent cytotoxic agent if not bound properly to a protein carrier. Recent studies support this hypothesis by demonstrating that Cu/Zn SOD proteins bearing selective FALS mutations exhibit a reduction in metal binding capacity (Nishida et al., 1994; Carri et al., 1994). In view of our findings, it is conceivable that the elevated turnover of the mutant Cu/Zn SOD proteins may represent a cytotoxic insult in that the rapid turnover of this metalloprotein may liberate free copper that could potentiate metal toxicity. This raises the possibility that stabilization of the protein, by proteasome inhibition or other means, could be therapeutically beneficial. On the other hand, if the proposed gain-of-function is associated with a novel activity unrelated to normal Cu/Zn SOD function, then enhancing the stability of mutant protein could exacerbate the disease phenotype. Such questions can be addressed directly in FALS SOD1 transgenic mice that exhibit motor neuron degeneration. The proteasome inhibitors described in this report represent useful tools in the study of the role of Cu/Zn SOD turnover in the FALS neurodegenerative process. Moreover, the compounds provide a means to further characterize the involvement of the proteasome and ubiquitin-protein conjugates in other neuropathological processes. Acknowledgements The authors would like to thank Drs. Mohamed Iqbal and Sankar Chatterjee for synthesis and provision of proteasome inhibitors CEP1508 and CEP1601. We thank Todd Levins for expert technical assistance and Dr. Dorothy G. Flood for careful reading of the manuscript. We are indebted to Drs. Jeffry Vaught and Frank Baldino for their continued support of this research. References Bewley, B.C. (1988) cDNA and deduced amino acid sequence of murk Cu-Zn superoxide dismutase Nucl. Acids Res., 16: 2728.
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Borchelt, D.R., Lee, M.K., Slunt, H.S., Guamieri, M., Xu, Z-S., Wong, P.C., Brown Jr., R.H., Price, D.L., Sisodia, S.S. and Cleveland, D.W. (1994) Superoxide dismutase 1 with mutations linked to familial amyotrophic lateral sclerosis possesses significant activity. Proc. Natl. Acad. Sci. USA, 91: 8292-8296. Bowling, A.C., Schulz, J.B., Brown Jr., R.H. and Beal, M.F. (1993) Superoxide dismutase activity, oxidative damage, and mitochondrial energy metabolism in familial and sporadic amyotrophic lateral sclerosis. J. Neurochem., 61: 2322- 2325. Bowling, A.C., Barkowski, E.E., McKenna-Yasek, D., Sapp, P., Horvitz, H.R., Beal, M.F. and Brown Jr., R.H. (1995) Superoxide dismutase concentration and activity in familial amyotrophic lateral sclerosis, J. Neurochem., 64: 2366-2369. Brown Jr., R.H. (1995) Amyotrophic lateral sclerosis: recent insights from genetics and transgenic mice. Cell, 80: 687-692. Carri, M.T., Battistoni, A., Polizio, F., Desideri, A. and Rotilio, Cl. (1994) Impaired copper binding by the H46R mutant of human Cu,Zn superoxide dismutase, involved in amyotrophic lateral sclerosis. FEBS Lett., 356: 314-316. Chen, CA. and Okayama, H. (1988) Calcium phosphate-mediated gene transfer: a highly efficient transfection system for stably transforming cells with plasmid DNA. BioTechniques, 6: 632-637. Ciechanover, A. (1994) The ubiquitin-proteasome proteolytic pathway, Cell, 79: 13-21. Deng, H.-X., Hentati, A., Tainer, J.A., Iqbal, Z., Cayabyab, A., Hung, W.-Y., Getzoff, E.D., Hu, P., Herzfeldt, B., Roos, R.P., Warner, C., Deng, G., Soriano, E., Smyth, C., Parge, H.E., Ahmed, A., Roses, A.D., Hallewell, R.A., Pericak-Vance, M.A. and Siddique, T. (1993) Amyotrophic lateral sclerosis and structural defects in Cu,Zn superoxide dismutase. Science, 261: 1047-1051. Firestone, G.L. and Winguth, SD. (1990) In: M.P. Deutscher (Ed.), Guide to Protein Purification, Methods in Enzymology, Vol. 182, Academic Press, San Diego, CA, pp. 688-700. Grune, T., Reinheckel, T., Joshi, M. and Davies, K.J.A. (1995) Proteolysis in cultured liver epithelial cells during oxidative stress. J. Biol. Chem., 270: 2344-235 1. Gurney, M.E., Pu, H., Chiu, A.Y., Dal Canto, M.C., Polchow, C.Y.,
Alexander, D.D., Caliendo, J., Hentati, A., Kwon, Y.W., Deng, H., Chen, W., Zhai, P., Sufit, R.L. and Siddique, T. (1994) Motor neuron degeneration in mice that express a human Cu, Zn superoxide dismutase mutation. Science, 264: 1772- 1775. Harding, C.V., France, J., Song, R., Farah, J.M., Chatterjee, S., Iqbal, M. and Siman, R. (1995) Novel dipeptide aldehydes are proteasome inhibitors and block the MHC-1 antigen-processing pathway. J. Immunol., 155: 1767-1775. Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K. and Pease, L.R. (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene, 77: 51-59. Iqbal, M., Chatterjee, S., Kauer, J.C., Das, M., Messina, P., Freed, B., Biazzo, B. and Siman, R. (1995) Potent inhibitors of proteasome. J. Med. Chem., 39: 2276-2277. Nishida, C.R., Grail, E.B. and Valentine, J.S. (1994) Characterization of three yeast copper-zinc superoxide dismutase mutants analogous to those coded for in familial amyotrophic lateral sclerosis, Proc. Natl. Acad. Sci. USA, 91: 9906-9910. Ripps, M.E., Huntley, G.W., Hof, P.R., Morrison, J.H. and Gordon, J.W. (1995) Transgenic mice expressing an altered murine superoxide dismutase gene provide an animal model of amyotrophic lateral sclerosis. Proc. Natl. Acad. Sci. USA, 92: 689-693. Rosen, D.R., Siddique, T., Patterson, D., Figlewicz, D.A., Sapp, P., Hentati, A., Donaldson, D., Goto, J., O’Regan, J.P., Deng, H.-X., Rahmani, Z., Krius, A., McKenna-Yasek, D., Cayabyab, A., Gaston, S.M., Berger, R., Tanzi, R.E., Halperin, J.J., Herzfeldt, B., van den Bergh, R., Hung, W.-Y., Bird, T., Deng, G., Mulder, D.W., Smyth, C., Laing, N.G., Soriano, E., Pericak-Vance, M.A., Hains, J., Rouleau, G.A., Gusella, J.S., Horvitz, H.R. and Brown Jr., R.H. (1993) Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature, 362: 59-62. Wong, P.C., Pardo, CA., Borchelt, D.R., Lee, M.K., Copeland, N.G., Jenkins, N.A., Sisodia, S.S., Cleveland, D.W. and Price, D.L. (1995) An adverse property of a familial ALS-linked SOD1 mutation causes motor neuron disease characterized by vacuolar degeneration of mitochondria. Neuron, 14: 1105-l 116.