Proc. Natl. Acad. Sci. USA Vol. 94, pp. 12604–12609, November 1997 Medical Sciences Transglutaminase-catalyzed inactivation of glyceraldehyde 3-phosphate dehydrogenase and a-ketoglutarate dehydrogenase complex by polyglutamine domains of pathological length ARTHUR J. L. COOPER*†‡§, K.-F. REX SHEU†‡,JAMES R. BURKE¶i,OSAMU ONODERA¶i, WARREN J. STRITTMATTER¶i,**, ALLEN D. ROSES¶i,**, AND JOHN P. BLASS†‡,†† Departments of *Biochemistry, †Neurology and Neuroscience, and ††Medicine, Cornell University Medical College, New York, NY 10021; ‡Burke Medical Research Institute, Cornell University Medical College, White Plains, NY 10605; and Departments of ¶Medicine, **Neurobiology, and iDeane Laboratory, Duke University Medical Center, Durham, NC 27710 Edited by Louis Sokoloff, National Institutes of Health, Bethesda, MD, and approved August 28, 1997 (received for review April 24, 1997) ABSTRACT Several adult-onset neurodegenerative dis- Q12-containing peptide (16). In the work of Kahlem et al. (16) the eases are caused by genes with expanded CAG triplet repeats largest Qn domain studied was Q18. We found that both a within their coding regions and extended polyglutamine (Qn) nonpathological-length Qn domain (n 5 10) and a longer, patho- domains within the expressed proteins. Generally, in clinically logical-length Qn domain (n 5 62) are excellent substrates of affected individuals n > 40. Glyceraldehyde 3-phosphate dehy- tTGase (17, 18). drogenase binds tightly to four Qn disease proteins, but the Burke et al. (1) showed that huntingtin, huntingtin-derived significance of this interaction is unknown. We now report that fragments, and the dentatorubralpallidoluysian atrophy protein purified glyceraldehyde 3-phosphate dehydrogenase is inacti- bind selectively to glyceraldehyde 3-phosphate dehydrogenase vated by tissue transglutaminase in the presence of glutathione (GAPDH) in human brain homogenates and to immobilized S-transferase constructs containing a Qn domain of pathological rabbit muscle GAPDH. Burke et al. also showed that GAPDH in length (n 5 62 or 81). The dehydrogenase is less strongly a brain homogenate is selectively bound to an immobilized Q60 inhibited by tissue transglutaminase in the presence of con- polypeptide, but not to an immobilized Q20 polypeptide. Koshy et structs containing shorter Qn domains (n 5 0 or 10). Purified al. (2) showed that the androgen receptor and ataxin-1 bind to a-ketoglutarate dehydrogenase complex also is inactivated by rabbit muscle GAPDH. When assayed in the yeast two-hybrid tissue transglutaminase plus glutathione S-transferase con- system, GAPDH interacts selectively with ataxin-1 and with the structs containing pathological-length Qn domains (n 5 62 or androgen receptor (2). Binding of both wild-type and mutant (i.e., 81). The results suggest that tissue transglutaminase-catalyzed pathological) ataxin-1 to GAPDH is very strong and cannot be covalent linkages involving the larger poly-Q domains may disrupted by 1 M NaCl (2). The region of ataxin-1 that binds to disrupt cerebral energy metabolism in CAGyQn expansion dis- GAPDH is the N terminus containing the Qn repeat sequence eases. (2). Moreover, ataxin-1 interacts with a GAPDH protein frag- ment that contains the NAD1-binding domain and the first 21 CAG expansion diseases include Huntington disease (HD), den- residues of the catalytic domain (2). Burke et al. (1) and Koshy et tatorubralpallidoluysian atrophy (Smith disease), spinobulbar al. (2) suggested that interactions with Qn tracts may disrupt muscular atrophy (Kennedy disease), and spinocerebellar ataxias GAPDH activity and thereby interfere with energy metabolism (SCA) type 1, 2, 3 (Machado–Joseph disease), and 7 (SCA-1, (2). Here we show that longer Qn domains by themselves do not SCA-2, SCA-3, and SCA-7, respectively) (1–12). These genetic inhibit GAPDH activity, but do so in the presence of tTGase. abnormalities lead to progressively worsening disorders of coor- dination or motor neuron degeneration with variable degrees of MATERIALS AND METHODS mental symptoms. The expanded CAG repeat disorders lead to Reagents. a-Ketoglutarate (sodium salt), glyceraldehyde 3- the expression of polyglutamine (Qn) expansions within the phosphate (GAP), NAD1, NADH, BSA, bovine milk a-casein, affected proteins. Qn length is inversely correlated with age of hydroxylamine-HCl, DTT, coenzyme A (CoASH), thiamin py- onset of symptoms (6–10). Different proteins are specific for each rophosphate, and N-a-carbobenzoxyglutaminylglycine (CGG) disease (e.g., huntingtin, androgen receptor, and ataxin-1 in HD, were obtained from Sigma. spinobulbar muscular atrophy, and SCA-1, respectively). Enzyme Activity Measurements. Rabbit muscle GAPDH CAGyQn expansions (where n $ 40) are believed to lead to a (crystalline suspension in 2.6 M ammonium sulfate; 1,000 ‘‘toxic gain of function’’ (13, 14), but the mechanism is unknown. unitsymg of protein; 31 mgyml) was purchased from Sigma. A Several authors (e.g., refs. 14 and 15) have hypothesized that single protein species (Mr of monomer '36,000) was detected by tissue transglutaminase (tTGase), an enzyme that catalyzes for- SDSyPAGE with Coomassie blue staining. The GAPDH assay mation of a crosslink (isopeptide) between the g-carboxamide mixture (0.8 ml) consisted of 50 mM TriszHCl (pH 7.6), 1 mM group of a protein Q residue and an « amino group of a protein NAD1, 1 mM GAP, 10 mM DTT, 12.5 mM sodium arsenate, K residue, may be involved in the toxic response. Kahlem et al. 0.2% (volyvol) Triton X-100, and enzyme at 30°C. The initial rate (16) showed that Qn domains are good substrates of tTGase and of increase of absorbance at 340 nm of NADH (« 5 6.23 3 103) that generally the longer the Qn domain the greater the activity was determined in a Cary 200 spectrophotometer. To ensure that (16). The authors also reported that R5Q18R5 forms highly insoluble aggregates in the presence of brain proteins and tTGase, 14 This paper was submitted directly (Track II) to the Proceedings office. and that brain homogenates catalyze incorporation of [ C]gly- Abbreviations: CoASH, coenzyme A; CGG, N-a-carbobenzoxyglu- cine ethyl ester (a nucleophile substrate of tTGase) into a taminylglycine; GAP, glyceraldehyde 3-phosphate; GAPDH, glycer- aldehyde 3-phosphate dehydrogenase; GST, glutathione S- The publication costs of this article were defrayed in part by page charge transferase; HD, Huntington disease; KGDHC, a-ketoglutarate de- hydrogenase complex; Q , polyglutamine; SCA, spinocerebellar payment. This article must therefore be hereby marked ‘‘advertisement’’ in n ataxia; tTGase, tissue transglutaminase. accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed at: Burke Medical © 1997 by The National Academy of Sciences 0027-8424y97y9412604-6$2.00y0 Research Institute, 785 Mamaroneck Avenue, White Plains, NY PNAS is available online at http:yywww.pnas.org. 10605. e-mail: [email protected]. 12604 Downloaded by guest on September 30, 2021 Medical Sciences: Cooper et al. Proc. Natl. Acad. Sci. USA 94 (1997) 12605 the reaction was linear with time the amount of enzyme added to mM glutathione (or DTT), guinea pig liver tTGase (20 milliunits; the assay mixture was adjusted so that the initial absorbance 2.6 unitsymg of protein), and '12.5 mg of GSTQn fusion protein change was no greater than 0.02ymin. GAP was obtained as a 200 (where indicated) in a final volume of 25 ml at 20°C. At intervals, mM frozen solution. Because GAP is unstable it was thawed just 2 ml-aliquots were withdrawn and assayed for GAPDH activity by before assay of GAPDH, and an aliquot then was added to the the standard procedure described above. Glutathione was chosen reaction mixture. as the reductant in the initial experiments because it is present in a-Ketoglutarate Dehydrogenase Complex (KGDHC). Pig solutions of the GSTQn fusion proteins purified by affinity batch heart (KGDHC) [0.5 unitsymg of protein (11.1 mgyml) in a chromatography, and glutathione was used as a reductant in the solution containing 50% glycerol, 25% sucrose, 5 mM EGTA, 5 earlier work of Folk and Cole (20). However, DTT gave more mM DTT, 1% Triton X-100, 0.01% sodium azide and 50 mM consistent results. potassium phosphate (pH 6.9)] was obtained from Sigma. The Measurement of KGDHC After Exposure to Qn Constructs assay mixture (0.8 ml) contained 1.0 mM a-ketoglutarate, 1.0 mM and tTGase. To determine if KGDHC is inhibited by GSTQn plus NAD1, 0.3 mM thiamin pyrophosphate, 0.13 mM CoASH, 0.18% tTGase, KGDHC (75 milliunits) was incubated at 20°C in a (volyvol) Triton X-100, 10 mM CaCl2, 1 mM EDTA, 1 mM reaction mixture (0.1 ml) containing 8.5 milliunits of guinea pig 2-mercaptoethanol, 1 mM MgCl2, and 50 mM TriszHCl buffer liver tTGase (specific activity of 2.6 unitsymg), 80 mM TriszHCl (pH 7.6) (19). The initial linear rate of appearance of absorption buffer (pH 7.4), 10 mM CaCl2, 10 mM DTT and GSTQn was measured at 340 nm in a Cary 200 spectrophotometer at construct. At intervals, 15-ml aliquots were removed and assayed 30°C. Except as noted, the volume of the standard GAPDH and for remaining activity. KGDHC assay mixtures was 0.8 ml. In some experiments, activ- Protein Measurement and Western blotting. Protein was mea- ities of GAPDH and KGDHC were determined in a SpectraMax sured by the method of Lowry et al. (22) by using BSA as a 250 96-well plate analyzer. In this case the standard assay volume standard. Immunoblotting was carried out by using a mouse mAb was 0.2 ml. against rabbit GAPDH (clone 65C) (Advanced Immunochemi- tTGase. Guinea pig liver tTGase was obtained from Sigma as cals, Long Beach, CA) at a dilution of 1:1,000 and a polyclonal a lyophilized powder [specific activity of 1.6–2.6 units (hydrox- antibody against GST (Pharmacia) at a dilution of 1:500.