Degradation of Functional Triose Phosphate Isomerase Protein Underlies Sugarkill Pathology

Degradation of Functional Triose Phosphate Isomerase Protein Underlies sugarkill Pathology

Jacquelyn L. Seigle,1 Alicia M. Celotto1 and Michael J. Palladino2 Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261 and Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260 Manuscript received January 26, 2008 Accepted for publication April 12, 2008

ABSTRACT Triose phosphate isomerase (TPI) deficiency glycolytic enzymopathy is a progressive neurodegenerative condition that remains poorly understood. The disease is caused exclusively by specific missense mutations affecting the TPI protein and clinically features hemolytic anemia, adult-onset neurological impairment, degeneration, and reduced longevity. TPI has a well-characterized role in glycolysis, catalyzing the isom- erization of dihydroxyacetone phosphate (DHAP) to glyceraldehyde-3-phosphate (G3P); however, little is known mechanistically about the pathogenesis associated with specific recessive mutations that cause progressive neurodegeneration. Here, we describe key aspects of TPI pathogenesis identified using the TPIsugarkill mutation, a Drosophila model of human TPI deficiency. Specifically, we demonstrate that the mutant protein is expressed, capable of forming a homodimer, and is functional. However, the mutant protein is degraded by the 20S proteasome core leading to loss-of-function pathogenesis.

LYCOLYTIC enzymopathies are devastating hu- instability of the protein homodimer, aberrant protein G man diseases that result from heritable mutations complexes, and/or a decrease in isomerase activity con- in genes encoding certain glycolytic enzymes. The un- tribute to pathogenesis of the disease (Olah et al. 2002; derlying pathogenesis of these diseases remains poorly Orosz et al. 2006). In part, the difficulty arises from the understood. Triose phosphate isomerase (TPI) defi- fact that the disease has a strong neurological compo- ciency is a severe glycolytic enzymopathy caused by nent, yet patients’ erythrocytes are often used for studies specific missense mutations in the TPI gene that are due to their accessibility. Although these studies have associated with hemolytic anemia, progressive neuro- produced important information about the disease, muscular degeneration, increased susceptibility to in- they have not provided much insight into neurological fection, and premature death (Schneider et al. 1965; and degenerative aspects of TPI deficiency. Such studies Valentine 1966). The TPI protein exists as a homo- have measured reduced isomerase activity from patients’ dimer of 26.5-kDa monomers that functions to convert erythrocytes, platelets, and lympocytes (Schneider et al. dihydroxyacetone phosphate (DHAP) to glyceraldehyde- 1965; Zanella et al. 1985; Orosz et al. 2001; Olah et al. 3-phosphate (G3P) in glycolysis (Rieder and Rose 1959; 2002, 2005), as well as brain and muscle tissue (Ationu Mande et al. 1994). Human TPI deficiency results when et al. 1999). Studies have also documented markedly specific missense mutations are homozygous or are het- elevated DHAP levels, suggesting that the anemia may erozygous with a null allele resulting in insufficient TPI be caused by toxicity of DHAP or one of its catabolites function (Schneider 2000). TPI’s well-characterized (Zanella et al. 1985; Eber et al. 1991; Hollan et al. role in glycolysis suggests that bioenergetic impairment 1997; Karg et al. 2000; Olah et al. 2002, 2005). Inter- may underlie pathogenesis. estingly, DHAP levels in nonerythrocytes are only mod- Alternatively, others have hypothesized that DHAP estly increased, presumably owing to it being a substrate toxicity or its catabolite methylglyoxal may be integral to for phospholipid biosynthesis in other cell types, and a pathogenesis independent of a bioenergetic deficit yeast model of TPI deficiency reports that proteins (Karg et al. 2000; Ahmed et al. 2003; Ramasamy et al. bearing the human disease mutations maintain catalytic 2005). Indeed, data are not consistent from existing activity (Orosz et al. 2001, 2006; Ralser et al. 2006). reports to establish whether reduced protein levels, We have previously identified a recessive, hypomorphic, missense (M80T) mutation (sugarkill or sgk) in the TPI gene of Drosophila featuring reduced longevity, 1These authors contributed equally to this work. neurodegenration, and locomotor impairment (Celotto 2Corresponding author: Pittsburgh Institute for Neurodegenerative Dis- et al. 2006). These mutants, referred to as TPIsugarkill, serve eases and Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, 3501 5th Ave., BST3 7042, Pittsburgh, as an amendable genetic model for elucidating the PA 15260. E-mail: [email protected] mechanism of TPI deficiency in vivo (Celotto et al.

Genetics 179: 855–862 ( June 2008) 856 J. L. Seigle, A. M. Celotto and M. J. Palladino

2006; Gnerer et al. 2006; Orosz et al. 2006). The TPIsugarkill with an electric homogenizer (IKA Ultra-TURAX T8) on the locomotor impairment is conditional with mechanical highest setting. Protein complexes .100 kDa were removed stress or elevated temperatures inducing acute paralysis. from the extract using Amicon Ultra-4 Ultracel 100K columns (Millipore, Bedford, MA) to prevent damage to the HPLC The mutation was originally identified as a temperature- column. The extract was then concentrated using an Amicon sensitive paralytic mutant (Palladino et al. 2002). Each Ultra-4 Ultracel 5K column (Millipore). One hundred micro- phenotype examined—including onset of neuropathol- liters was injected into the HPLC column, using an auto- ogy, locomotor impairment, and reduced longevity—is injector. Each genotype was performed in duplicate at each significantly worsened by modest elevations in tempera- temperature. elotto Nondenaturing size-exclusion chromatography: Protein fractions ture between 20° and 29° (C et al. 2006). This were collected using a Shimadzu HPLC system and a Superdex suggests that TPIsugarkill animals may serve as a useful 75 10/300 GL column at a flow rate of 0.4 ml/min. The model of TPI impairment where varying temperatures temperature of the column was maintained at 22°,30°,or40°. m m can be utilized to study a series of hypomorphic condi- The mobile phase consisted of 0.025 NaH2PO4, 0.025 m tions from mild to severe. Furthermore, in vitro temper- Na2HPO4 pH 7.0, and 0.15 NaCl. One hundred microliters of protein standard (Amersham Biosciences, Arlington Heights, ature sensitivity has also been reported for the most IL) (at 5 mg/ml: ribonuclease A, 14.6 kDa; chymotrypsinogen common homozygous human mutant allele, Glu104Asp A, 20.3 kDa; ovalbumin, 46.7 kDa; albumin, 62.9 kDa) or (Daar et al. 1986; Arya et al. 1997), suggesting a shared Drosophila extract were injected and monitored at 280 nm for mechanism of pathogenesis exists with at least one of 40 min. Four fractions were collected: (1) 1–23 min, .67-kDa the common disease-causing missense mutations. proteins and complexes; (2) 23–28 min, corresponding to 31–67 kDa (53-kDa TPI dimer); (3) 28–34 min, corresponding There are many proposed mechanisms of TPI de- to 12–31 kDa (26.5-kDa TPI monomer); and (4) 34–40 min, ficiency pathogenesis, including (1) protein misfolding corresponding to 5- to 12-kDa proteins. and toxic aggregates (Olah et al. 2002; Ovadi et al. Western: Once fractions were collected, they were concen- 2004), (2) accumulation of a DHAP catabolite methyl- trated using Amicon Ultra-4 Ultracel 5K columns (Millipore) m glyoxal, a potentially toxic metabolite (Karg et al. 2000; down to 100 l, 1 ml H2O was added, and the samples were hmed amasamy reconcentrated to 50 ml. Sixteen microliters of loading dye A et al. 2003; R et al. 2005), (3) instability were added to each fraction and were loaded onto a 12% of the homodimer via thermoliablilty (Schneider 2000), protein gel. The gel was transferred to a nitrocellulose and (4) bioenergetic defects resulting from reduced en- membrane and blocked with 1% milk. TPI primary antibody zyme activity. Our model allows us to directly test the role (Protein Tech rabbit anti-humanTPI) was added at a concen- of these mechanisms in pathogenesis resulting from a tration of 1:1000 and incubated at room temperature over- night. The blot was washed extensively with PBST, secondary specific pathogenic missense mutation affecting TPI. antibody (HRP goat anti-rabbit IgG) was added at a concen- Previously we have shown TPIsugarkill markedly reduces tration of 1:4000, and the blot was incubated at room glycolytic flux, but this does not result in a bioenergetic temperature for 1 hr. The blot was washed again with PBST crisis, suggesting a compensatory mechanism and an and enhanced chemiluminescence (ECL) reaction (Pierce, alternative cause of pathogenesis (Celotto et al. 2006). Rockford, IL) was performed. Film was exposed for 1 min sugarkill before developing. Our data utilizing the TPI model of TPI deficiency Western blot analysis: Fly lysates were obtained from flies reveal that the mutant protein is capable of forming a aged for 24 or 48 hr as indicated and ground with a pestle in dimer and retains function; however, pathogenesis results 100 ml of a standard SDS/Bromophenol Blue cell lysis buffer. when the protein is targeted for proteasome degradation. After boiling for 1 min at 100°,20ml of extract were added per lane in a 12% SDS–PAGE gel at denaturing conditions. Signal detection was performed as above. ImageJ Version 10.2 software was used to analyze subsaturation images. Protein intensity represents the normalized TPI protein level (either to actin or MATERIALS AND METHODS b-tubulin, as indicated) in arbitrary pixel units (intensity). In all Drosophila stocks and culture: The TPIsugarkill mutation, cases independent protein extracts were used (n $ 3). unless noted otherwise, is maintained as a homozygous viable ve e TPIsugarkill strain. The TPIsugarkill mutation, also known as sugarkill, TPIsgk,orTPIsgk1, results from a missense mutation predicted to cause an M80T. This numbering uses the estab- RESULTS lished nomenclature for TPI mutations, where the start methionine is assumed to be removed following translation (see Recessive nature of the sugarkill mutation: The the recent review by Orosz et al. 2006). TPI transgenic strains sugarkill mutation was identified as a recessive temper- elotto were previously published (C et al. 2006). All other ature-sensitive paralytic mutant that causes progressive strains used were obtained from the Bloomington Stock alladino Center. Wild-type controls are ve e TPIsugarkill heterozygotes, neurodegeneration (P et al. 2002). The muta- ve e, Canton-S, or appropriate Gal4 controls, as noted. tion was positionally cloned and found to be a missense Life span and behavioral analysis: Life span analysis was mutation in the TPI (triose phosphate isomerase) gene performed as previously described (Palladino et al. 2003). (Celotto et al. 2006). TPIsugarkill (sgk) animals have pro- Stress sensitivity (a.k.a. bang sensitivity) was assayed as pre- gressive stress-sensitive locomotor impairment and re- viously described, and the time to recovery from paralysis was measured (Ganetzky and Wu 1982). duced longevity. Surprisingly, given its well-characterized Size-exclusion chromatography: Drosophila extract: Two role in glycolysis, biochemical analyses revealed that milliliters of flies were homogenized in 5 ml of mobile phase bioenergic impairment was not a likely proximate cause Glycolytic Enzymopathy in Drosophila 857

Figure 1.—Life span and behavioral analysis of animals overexpressing wild-type and mutant TPI transgenes. (A) Survival of transgenic animals at 25° overexpressing mutant and wild-type TPI. A modest reduction in longevity is observed, associated with overexpression of wild-type TPI protein. Flies overexpressing the mutant TPI, UAS-TPI½sgk;ActinTGAL4;1/1, do not show a significant life span deficit relative to either the control strain or animals overexpressing wild-type TPI. Longevity of TPIsgk mutant flies is shown for comparison and is significantly reduced compared to all genotypes. N ¼ 80–120 animals in four to six independent populations. Locomotor function in response to stress was examined in transgenic flies at 29° (B) and room temperature (C). TPIsugarkill mutants exhibit a significantly longer recovery rate from mechanical stress compared to controls when aged at 29° (day 6) or room temperature (day 18). Transgenic overexpression of wild-type or mutant TPI protein did not result in stress-induced locomotor impairment when aged at either temperature. A total of 10–20 animals per genotype were tested. In A–C the error given is SEM and statistics were calculated using Student’s t-test (**P , 0.01 and ***P , 0.001). of these phenotypes (Celotto et al. 2006; Gnerer et al. Dimer instability of TPI protein in sugarkill mutants: 2006). This surprising result led us to investigate alter- Several disease-associated TPI mutations and the TPIsugarkill native hypotheses to explain neuropathogenesis associ- mutation result in an amino acid substitution near the ated with TPI impairment, including the possibility that dimer interface of the protein, which has led us and pathogenesis of the M80T missense mutation in TPI may others to hypothesize that dimer instability may underlie in part result from a semidominant gain-of-function. pathogenesis (Celotto et al. 2006; Orosz et al. 2006). Although previous data suggested that TPIsugarkill was fully Human TPI deficiency has previously been reported to recessive (Celotto et al. 2006), we examined a trans- be associated with the destabilization of the dimer state genic overexpression assay to test for semidominance (Olah et al. 2002). To determine if TPIsugarkill pathogenesis effects. Utilizing the UAS-GAL4 system, we generated may be associated with structural defects in the TPI animals that overexpressed either the TPIsugarkill or the protein that destabilize the dimer, we directly examined wild-type TPI protein in a wild-type genetic background the stability of the TPI homodimer, using a nondenatur- and examined the effect on longevity and locomotor ing size-exclusion chromatography assay (Figure 2). TPI function. Specifically, we analyzed longevity of both UAS- is 26.5 kDa and exists physiologically as an 53-kDa TPIsgk;actinTGAL4;1/1 and UAS-TPI1;actinTGAL4;1/1 homodimer (Rieder and Rose 1959; Mande et al. 1994). animals compared to actinTGAL4;1/1 controls at 25° Native size-exclusion chromatography was standardized, (Figure 1A). There was no significant decrease in life span whole fly extracts from wild-type controls and TPIsugarkill of the UAS-TPIsgk;actinTGAL4 animals compared to the were fractionated, and Westerns were performed to control or the UAS-TPI1;actinTGAL4 animals. There was determine which fraction contained TPI. At both 22° a modest but significant decrease in median life span and 40° the mutant and wild-type TPI were observed only of the UAS-TPI1;actinTGAL4 animals compared to that in the dimer fraction (31–67 kDa). No protein was of the GAL4 controls. This is likely the result of trans- detected in monomer or large fractions corresponding genic overexpression toxicity or a background strain to 12–31 kDa and .67 kDa, respectively. We have noted effect shared between the transgenic animal strains. that increases in temperature from 20° to 37° exacerbate Further studies examined the effect of mechanical all described phenotypes in TPIsugarkill (Celotto et al. stress on locomotion of these transgenic animals at 29° 2006), yet even at 40° the dimer remained stable in this or room temperature (22°). This revealed that over- assay. These data demonstrate that TPIsugarkill is capable of expression of either mutant or wild-type TPI protein in a forming a dimer and that the protein is not observed as a wild-type background does not result in a detectable monomer in fly extracts under native conditions. Addi- stress-induced locomotor phenotype, such as is prom- tional studies were performed with more extensive inent in TPIsugarkill animals (Figure 1, B and C; Celotto fractionation, which corroborate these findings (supple- et al. 2006). Importantly, the transgenes used in these mental Figure 1). The TPIsugarkill protein exists as a dimer overexpression experiments are capable of rescuing in the ,100-kDa protein fraction, as is the case for wild- TPIsugarkill to a similar extent (see Figure 6). Together type TPI protein. these transgenic experiments are consistent with the Protein stability and expression in sugarkill mutants: TPIsugarkill mutation being fully recessive and suggest a We have examined TPI mRNA expression in TPIsugarkill simple loss-of-function model of TPI pathogenesis. mutants at room temperature and at 29°. Expression of 858 J. L. Seigle, A. M. Celotto and M. J. Palladino

Figure 2.—Size-exclusion chromatography: chromatography demonstrating that both the wild-type TPI and mutant TPIsugarkill form a dimer. (A) HPLC chromatograph using four standards (albumin, ovalbumin, chymotrypsinogen, and ribonuclease A) to determine size retention. (B) Standard curve used to determine time of elution from the column: fraction L (.67-kDa molecules), fraction D (TPI dimer that is 53 kDa), fraction M (TPI monomer that is 26.5 kDa), and fraction C (molecules between 5 and 12 kDa). (C) Western blots using TPI antibody: each fraction from wild-type and TPIsugarkill extracts was separated at 22° and 40°. mRNA is not reduced, suggesting that the pathogenesis (Keller et al. 2000), Parkinson’s (McNaught and is associated with expression of TPI protein bearing the Olanow 2006; Olanow and Mcnaught 2006), and M80T missense mutation (Celotto et al. 2006 and data Huntington’s (Seo et al. 2004) diseases. Surprisingly, not shown). Surprisingly, we discovered that TPI protein Pros261 TPIsgk double mutants did not have reduced lon- in TPIsugarkill mutants is reduced and that the extent of the reduction in protein is temperature dependent (Figure 3). At room temperature there is a significant (73%) reduction in total TPI protein, whereas age-matched flies shifted to 29° for 48 hr reveal a marked (98%) reduction in TPI protein (Figure 3). The severity of this reduction is antagonized by temperature, consistent with other phenotypes observed in TPIsugarkill mutants. TPI protein being reduced in the absence of a reduction in TPI mRNA strongly suggests that TPI protein is unstable as a result of the TPIsugarkill mutation. Proteasomal degradation of TPIsugarkill protein: Pros261 is a dominant, temperature-sensitive lethal mutation, formerly known as DTS5, which results in larval stage-three lethality at nonpermissive temperatures $25° (Saville and Belote 1993). Pros26 1 was shown to be a hypomorphic missense mutation in the Pros26 gene that encodes an altered B6 subunit of the 20S proteasome core (Saville and Belote 1993; Smyth and Belote 1999). In Drosophila, this mutation has been shown to be a useful genetic inhibitor of proteasome function (Fernandez-Funez et al. 2000; Shulman and Feany 2003; Speese et al. 2003; Neuburger et al. 2006). We 1 sgk generated Pros26 TPI animals to investigate the effect Figure 3.—Western blot analysis of TPI protein levels. (A) sugarkill of proteasome inhibition on TPI mutants, utilizing TPIsugarkill mutants show a reduced level of TPI protein when longevity as our most sensitive and quantitative pheno- aged for 48 hr at room temperature and 29° compared to ac- type. Proteasome impairment is itself toxic with respect tin controls. (B) Quantification of Western blot shows an to aging (Keller et al. 2002; Chondrogianni and 3.5-fold decrease in TPI protein levels at room temperature onos and an 48-fold decrease at 29°.*P , 0.05 (Student’s t-test). G 2005). Impairment also dramatically affects pro- Error reported is SEM. N ¼ 3 at each temperature. Intensity of tein synthesis (Ding et al. 2006) and has been impli- TPI was measured from a subsaturation blot and normalized cated in numerous disease states, including Alzheimer’s to the intensity of the actin control using ImageJ 10.2. Glycolytic Enzymopathy in Drosophila 859

Figure 5.—Proteasome mutation increases TPI½sgk protein levels. (A) Western blot of TPI and b-tubulin proteins from flies aged at 29° for 24 hr. Pros261 TPIsgk/sgk animals show an increase in TPI protein compared to TPIsgk/sgk mutants. (B) Quantitative analysis of TPI protein normalized to the tubulin Figure 4.—Survivorship of TPIsugarkill Pros261 double mu- loading control indicates a greater than twofold increase in tants: statistical analysis of median life span of mutants aged TPI protein in the Pros261 TPIsgk/sgk double mutants (*P , at (A) 29° and (B) 25°. (A) Pros261 TPIsgk/sgk have a modest but 0.05, n ¼ 3 each genotype, Student’s t-test). Both TPIsgk/sgk significant increase in longevity compared to TPIsgk/sgk animals and Pros261 TPIsgk/sgk animals show a marked reduction in at 29° (P , 0.05). (B) Flies aged at 25° show a nonsignificant TPI protein when compared to wild-type controls, consistent improvement in longevity. The median life span of Pros261 with previous data. Error reported is SEM. Protein intensity is TPIsgk/sgk double mutants is still much reduced from that of het- measured from a subsaturation image with ImageJ 10.2. erozygous controls at both temperatures examined (P , 0.0001). In A and B, the error given is SEM, statistical comparison uses Student’s t-test, and n ¼ 120 animals in six independent populations for each genotype. core is implicated in TPIsugarkill degradation. Further experiments have shown that the Pros261 mutation does not have a significant effect on wild-type TPI protein gevity compared to that of TPIsugarkill mutants. In fact, there levels (supplemental Figure 2). These data imply either is a modest but significant increase in longevity in the that the proteasome does not regulate wild-type TPI double-mutant strain (Figure 4). These data suggest that protein or that wild-type protein turnover is insufficient the TPIsugarkill protein is targeted for degradation by the 20S to detect a difference with these methods. The finding proteasomecoreandthatimpairmentoftheproteasome, that a loss-of-function proteasome mutation reduces though normally toxic, can partially suppress the TPIsugarkill TPIsugarkill degradation and improves the longevity phe- life span phenotype. notype also strongly suggests that the TPIsugarkill protein is To examine this more directly, we measured TPI pro- functional and that degradation of functional protein tein levels from the double mutant compared to control underlies pathogenesis in TPIsugarkill mutants. animals (Figure 5). This analysis revealed a significant Transgenic rescue with overexpression of TPIsugarkill increase in TPIsugarkill protein levels in the proteasome- protein: If pathogenesis in TPIsugarkill results from a loss- inhibited double mutant compared to TPIsugarkill animals. of-function due to enhanced proteasomal degradation Partial rescue of TPIsugarkill protein levels is consistent of functional protein, one would predict that over- with the modest improvement observed in longevity expression of the mutant protein may rescue or partially (Figure 4). This also indicates that the 20S proteasome rescue the TPIsugarkill mutation. Previous data demon- 860 J. L. Seigle, A. M. Celotto and M. J. Palladino

Numerous lines of evidence demonstrate that homozygous TPI null mutations do not result in disease and are developmentally lethal. Most notably, the high frequency of heterozygous null mutations in the pop- ulation and lack of homozygous null observations, as well as the stage of lethality observed in mouse and fly null animals, are all consistent with this assertion (Mohrenweiser and Fielek 1982; Mohrenweiser 1987; Merkle and Pretsch 1989; Celotto et al. 2006). Several hypotheses have been proposed to explain TPI-deficiency pathogenesis associated with specific missense mutations. We have utilized a Drosophila model of human TPI-deficiency disease to examine several of these hypotheses in vivo. The Drosophila TPIsugarkill missense mutation recapitulates numerous key features of this progressive neurodegenerative disease and pro- vides a unique system for investigating pathogenesis (Celotto et al. 2006; Orosz et al. 2006). Independently, two laboratories studying the same genetic model of glycolytic enzymopathy demonstrated

igure sugarkill that TPI pathogenesis was not simply the result of F 6.—Rescue with TPI overexpression. Trans- elotto nerer genic animals expressing wild-type and mutant TPI protein bioenergetic impairment (C et al. 2006; G were examined for longevity at 25° in a TPIsgk background. et al. 2006). TPI-deficiency pathogenesis has also been Both UAS-TPI1;ActTGal4; TPIsgk/sgk and UAS-TPIsgk;ActTGal4; proposed to result from protein unfolding, which could TPIsgk/sgk animals show a significant increase in life span com- result from a loss-of-function or a gain-of-function tox- T sgk/sgk , pared to Act Gal4; TPI mutants (***P 0.001, Student’s icity (Olah et al. 2002). We investigated the possibility of t-test). Neither transgene was able to fully rescue the longevity sugarkill to that of heterozygous control animals. Error given is SEM, TPI semidominance that may result from a gain-of- and n ¼ 120 animals in six independent populations. function protein toxicity. The TPIsugarkill mutation is com- pletely recessive and expression of transgenic TPIsugarkill protein does not result in any detectable toxicity beyond elotto strated that this is not the case at 29° (C et al. that of wild-type overexpression. Native HPLC size 2006). This is likely due to the marked instability of exclusion chromatography assays demonstrate that sugarkill TPI protein at 29° (Figure 3). We examined whether TPIsugarkill protein is capable of forming a dimer, suggest- sugarkill transgenic overexpression of TPI protein could ing the protein is properly folded. sugarkill rescue the TPI mutation at 25°,wherethereduction The TPIsugarkill strain has reduced life span, progressive sgk in protein stability was not as severe. Both UAS-TPI ; locomotor impairment, and neurodegeneration at room T sgk/sgk 1 T sgk/sgk actin GAL4; TPI and UAS-TPI ;actin GAL4; TPI temperature, 25°, and 29°, where phenotypic onset is animals were examined for longevity and found to dra- accelerated with increasing temperature (Celotto et al. sugarkill matically improve longevity of TPI mutants (Figure 2006). These data suggest that the dysfunction associ- 6). Interestingly, the improvement was similar for each ated with the mutation is temperature sensitive and the transgene, and neither transgenic strain fully rescued extent of dysfunction increases within the physiologi- longevity compared to heterozygous controls. These data cally relevant range for Drosophila (18°–30°). This sugarkill demonstrate that proteasome degradation of TPI interpretation is consistent with the fact that the muta- protein underlies pathogenesis and opens up the possi- tion was initialy isolated as a temperature-sensitive bility that one or more of the human pathogenic muta- paralytic mutant (Palladino et al. 2002). Western blot tions that cause TPI deficiency may result from a decrease analysis has demonstrated that the TPIsugarkill protein is in protein stability. reduced in the mutants and that this decrease is enhanced with increases in temperature within the normal physiological temperatures for Drosophila. These find- DISCUSSION ings suggest that temperature can be used to antago- Despite the well-characterized role of TPI in glycoly- nize TPIsugarkill function and allow a range of phenotypic sis, TPI-deficiency pathogenesis remains poorly under- severity to be studied, analogous to an allelic series of stood. Considerable attention has been focused on the mutations. fact that only specific missense mutations are patho- Our studies demonstrate a temperature-dependent genic; TPI enzymopathy results when these missense reduction in TPI protein: even an acute temperature mutations are homozygous or are heterozygous with a shift dramatically reduces cellular TPI protein (Figure known null allele (Schneider 2000; Orosz et al. 2006). 3). However, mRNA levels are not reduced in TPIsugarkill Glycolytic Enzymopathy in Drosophila 861 animals at any temperature examined (Celotto et al. and Tetzlaff 2007). Cytosolic protein quality-control 2006 and data not shown). These findings suggested pathways are often mediated by Hsp90 and various active proteolytic degradation of TPIsugarkill protein. In- cochaperones that form extensive networks with chap- deed, genetic inhibition of the 20S proteasome core erones specific for individual or groups of substrates with the Pros261 mutation results in modest increases in (Zhao et al. 2005). Further experimentation will be TPIsugarkill protein levels (Figure 5). These data suggest needed to determine the basis of the TPIsugarkill protein the TPIsugarkill protein is functional. Transgenic overex- degradation and whether the mutant protein is de- pression of TPIsugarkill protein was able to partially rescue graded in an Hsp90 and/or ubiquitin-dependent man- the longevity defect associated with TPIsugarkill, further ner. The TPIsugarkill mutant may prove useful at elucidating supporting the assertion that TPIsugarkill protein is func- cytosolic quality-control mechanisms required to prevent tional. The data are consistent with a model whereby the premature senescence and progressive neurodegenera- mutation results in the degradation of functional TPI tive disease. protein. Further experiments will be needed to de- Together, the data suggest that TPIsugarkill pathogenesis termine whether the protein is fully functional and to does not result from bioenergetic impairment, the assess the relative contributions of protein degradation encoded protein forms a stable dimer and retains and hypomorphic function. sufficient function to rescue the mutation upon trans- It is possible that the mutant protein has an essentially genic overexpression, and that pathogenesis results normal structure and retains function but that the from proteasomal degradation of the apparently func- methionine-to-threonine missense mutation destabil- tional protein. Alternatively, it is possible that mutations izes the protein by a mechanism independent of protein abrogating isomerase activity are lethal (do not cause function and structure. This could occur if the mutation disease) and that the specific mutations that result in created a signal stimulating the protein’s degradation disease disrupt a hitherto novel function of TPI. This via a regulatory or a cellular quality-control mechanism. interpretation is consistent with recent findings in a Such signals could include (1) an exposed hydrophobic yeast TPI model, where transgenic expression of multi- patch in the protein, (2) a degradation signal for an E3 ple human mutant TPI proteins in vivo demonstrated ubiquitin ligase subunit, (3) a post-translational covalent catalytic isomerase activity associated with the patho- modification, or (4) a functional PEST domain. Although genic mutant proteins (Ralser et al. 2006). the protein retains function and the ability to form a We thank Don DeFranco, Lesley Ashmore, and Stacy Hrizo for dimer, it remains possible that it is partially misfolded, helpful comments; Emily Trostel for editorial suggestions; and the stimulating known quality-control pathways and its deg- Bloomington Stock Center for fly strains. We thank the National radation. Alternatively, the mutation could create a deg- Institutes of Health (NIH) National Institute of Neurological Disor- radation signal. One such pathway known to increase the ders and Stroke (T32NS 07391-07) (A.M.C.), the American Heart Association (0630344N) (M.J.P.), the NIH National Institute on Aging rate of proteolytic degradation is the PEST pathway. (AG025046) (M.J.P.), The University of Pittsburgh Department of Amino acid sequences rich in proline (P), glutamic acid Pharmacology and Chemical Biology, and The University of Pittsburgh (E), serine (S), and threonine (T) residues form func- School of Medicine for financial support. tional PEST domains that can result in rapid degradation of proteins in vivo (Rogers et al. 1986; Rechsteiner and Rogers 1996). These are assigned PEST-FIND scores on LITERATURE CITED the basis of an established algorithm (Rogers et al. 1986). TPI proteins are not known to be regulated by the PEST Ahmed, N., S. Battah,N.Karachalias,R.Babaei-Jadidi,M.Horanyi et al., 2003 Increased formation of methylglyoxal and protein gly- pathway—consistent with this, the PEST algorithm does cation, oxidation and nitrosation in triosephosphate isomerase de- not find any PEST sequences in the protein (http:// ficiency. Biochim. Biophys. Acta 1639: 121–132. www.at.embnet.org/toolbox/pestfind/). The sequence Arya, R., M. R. Lalloz,A.J.Bellingham and D. M. Layton, 1997 Evidence for founder effect of the Glu104Asp substitution KGAFTGEISPAMLK is identified as a ‘‘poor PEST and identification of new mutations in triosephosphate isomer- domain’’ with a score of 22.8. Although the TPIsugarkill ase deficiency. Hum. Mutat. 10: 290–294. mutant adds an additional T within this sequence Ationu, A., A. Humphries,M.R.Lalloz,R.Arya,B.Wild et al., 1999 Reversal of metabolic block in glycolysis by enzyme re- (KGAFTGEISPATLK), the algorithm still rates the re- placement in triosephosphate isomerase-deficient cells. Blood gion as a ‘‘poor PEST domain’’ with a marginally 94: 3193–3198. improved score of 16.55 (Rogers et al. 1986). Barral, J. M., S. A. Broadley,G.Schaffar and F. U. Hartl, 2004 Roles of molecular chaperones in protein misfolding dis- Although it is not clear what stimulates the degrada- eases. Semin. Cell Dev. Biol. 15: 17–29. tion of TPIsugarkill protein, the efficient degradation of Celotto, A. M., A. C. Frank,J.L.Seigle and M. J. Palladino, mutant protein suggests a regulated quality-control 2006 Drosophila model of human inherited triosephosphate isomerase deficiency glycolytic enzymopathy. Genetics 174: 1237– mechanism. Quality-control pathways that degrade non- 1246. functional, misfolded, and unfolded proteins are ex- Chondrogianni,N.,andE.S.Gonos, 2005 Proteasome dysfunction tensive and required for the health and longevity of the in mammalian aging: steps and factors involved. Exp. Gerontol. 40: artl ayer artl arral 931–938. cell (H and H -H 2002; B et al. 2004; Daar, I. O., P. J. Artymiuk,D.C.Phillips and L. E. Maquat, Young et al. 2004; Zhang and Kaufman 2006; Outeiro 1986 Human triose-phosphate isomerase deficiency: a single 862 J. L. Seigle, A. M. Celotto and M. J. Palladino

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