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Keywords:

  • DJ-1;
  • homo-dimerization;
  • PARK7 locus;
  • Parkinson's disease;
  • L166P;
  • M26I

Abstract

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Plasmids
  5. DJ-1 antibodies
  6. Cell culture and transfection
  7. Co-immunoprecipitation and western blotting
  8. [35S]-Methionine Pulse-chase experiments
  9. Northern blot analysis
  10. Results
  11. The L166P mutation markedly reduces the steady-state levels of the DJ-1 protein
  12. The L166P mutation does not reduce the expression of DJ-1 mRNA
  13. The L166P mutation reduces the stability of the DJ-1 protein
  14. The L166P mutation enhances DJ-1 degradation by the 20S/26S proteasome
  15. The L166P mutation impairs the homo-oligomerization of DJ-1
  16. Discussion
  17. Acknowledgments
  18. References

The identification of genetic mutations responsible for rare familial forms of Parkinson's disease (PD) have provided tremendous insight into the molecular pathogenesis of this disorder. Mutations in the DJ-1 gene cause autosomal recessive early onset PD in two European families. A Dutch kindred displays a large homozygous genomic deletion encompassing exons 1–5 of the DJ-1 gene, whereas an Italian kindred harbors a single homozygous L166P missense mutation. A homozygous M26I missense mutation was also recently reported in an Ashkenazi Jewish patient with early onset PD. Mutations in DJ-1 are predicted to be loss of function. The recent determination of the crystal structure of human DJ-1 demonstrates that it exists in a homo-dimeric form in vitro, whereas the L166P mutant exists only as a monomer. Here, we examine the in vivo effects of the pathogenic L166P and M26I mutations on the properties of DJ-1 in cell culture. We report that the L166P mutation confers markedly reduced protein stability to DJ-1, which results from enhanced degradation by the 20S/26S proteasome but not from a loss of mRNA expression. Furthermore, the L166P mutant protein exhibits an impaired ability to self-interact to form homo-oligomers. In contrast, the M26I mutation does not appear to adversely affect either protein stability, turnover by the proteasome, or the capacity of DJ-1 to form homo-oligomers. These properties of the L166P mutation may contribute to the loss of normal DJ-1 function and are likely to be the underlying cause of early onset PD in affected members of the Italian kindred.

Abbreviations used
DJBP

DJ-1 binding protein

HMW

high molecular weight

IP

immunoprecipitation

ORF

open reading frame

PD

Parkinson's disease

PIASxα

protein inhibitor of activated STAT

UPS

ubiquitin-proteasome system

Parkinson's disease (PD) is the second most common progressive neurodegenerative disorder, which results directly from the relatively selective loss of nigrostriatal dopaminergic neurons (Lang and Lozano 1998a, 1998b). PD is a heterogeneous disorder of unclear etiology, and although the majority of cases appear sporadic in nature, specific genetic defects have recently been identified in rare familial cases of PD (Cookson 2003; Dawson and Dawson 2003; Gasser 2003). To date, mutations in four genes are linked to familial PD, α-synuclein (Polymeropoulos et al. 1997), parkin (Kitada et al. 1998), ubiquitin carboxy-terminal hydrolase L1 (Lui et al. 2002), and more recently DJ-1 (Bonifati et al. 2003).

Mutations in the DJ-1 gene are clearly linked with autosomal recessive early onset PD in two consanguineous European families (van Duijn et al. 2001; Bonifati et al. 2002, 2003). The DJ-1 gene is localized to chromosome 1p36 (PARK7 locus) and consists of eight exons; exons 1A and 1B are non-coding and alternatively spliced, while exons 2–7 comprise the open reading frame (ORF) which encodes a 189 amino acid protein of approximately 20 kDa (Taira et al. 2001; Bonifati et al. 2003). In one affected Dutch family, a 14 kb homozygous genomic deletion encompassing the entire promoter region and exons 1A/B to 5 of the DJ-1 gene appears to abolish DJ-1 expression. In an Italian family, a homozygous missense mutation (T[RIGHTWARDS ARROW]C transition at position 497 relative to the ORF start site) results in the substitution of a highly conserved leucine residue for a proline at position 166 (L166P) of the DJ-1 protein. This L166P mutant appears to adopt an altered cytoplasmic distribution, whereby it partially mislocalizes or is sequestered to mitochondria, displays reduced protein stability, and is predicted to be functionally inactive (Bonifati et al. 2003; Macedo et al. 2003; Miller et al. 2003). Critically, in both families, individuals heterozygous for DJ-1 gene mutations appear to be unaffected, while those with homozygous mutations all have PD. Thus, disruptions of the DJ-1 gene are thought to result in the loss of normal function of DJ-1, which is probably the cause of familial PD.

More recently, additional rare genetic mutations in DJ-1 have been reported in several unrelated cases of early onset PD (Moore et al. 2003). A pathogenic homozygous missense mutation (G[RIGHTWARDS ARROW]A transition at position 78 relative to the ORF start site) was identified in an Ashkenazi Jewish patient, which results in a methionine to isoleucine substitution at position 26 (M26I) of the DJ-1 protein (Abou-Sleiman et al. 2003). A compound heterozygous mutation (IVS6–1G[RIGHTWARDS ARROW]C and c.56delC c.57G[RIGHTWARDS ARROW]A) has also been described in a Hispanic patient (Hague et al. 2003). The c.56delC c.57G[RIGHTWARDS ARROW]A mutation is predicted to result in a truncated protein of 18 amino acids, followed by a premature stop, while the IVS6–1G[RIGHTWARDS ARROW]C mutation results in a single nucleotide substitution in a conserved sequence of the invariant AG splice acceptor site of intron 6 that is predicted to drastically effect DJ-1 RNA. Several heterozygous mutations in DJ-1 have also been identified in PD subjects, including D149A, R98Q and A104T, although it is not yet clear whether they represent disease-causing mutations.

DJ-1 is a highly conserved protein present in a diverse number of organisms from humans to bacteria, and belongs to the DJ-1/ThiJ/PfpI protein superfamily. While the precise biological function of DJ-1 remains elusive it may play a role in the oxidative stress response. DJ-1 exhibits a pI change from 6.2 to 5.8 in response to oxidative stimuli such as hydrogen peroxide, bacterial endotoxin, or the herbicide paraquat (Mitsumoto and Nakagawa 2001; Mitsumoto et al. 2001a, 2001b). Furthermore, the transcription of YDR533C, a putative yeast DJ-1 homolog, is induced together with other oxidative stress-responsive genes (de Nobel et al. 2001). DJ-1 was also identified as a novel oncogene that can transform mouse NIH-3T3 cells in cooperation with activated ras (Nagakubo et al. 1997), and it appears to be a positive regulator of androgen receptor transcriptional activity by sequestering its negative regulators protein inhibitor of activated STAT (PIASxα) and the DJ-1 binding protein (DJBP) (Takahashi et al. 2001; Niki et al. 2003). PIASxα , a SUMO-1 E3 ligase interacts with and potentially sumoylates DJ-1 at lysine 130 (Takahashi et al. 2001). Additionally, in rodents, DJ-1 (also termed CAP1 or SP22) plays a role in the fertilization process (Klinefelter et al. 2002; Okada et al. 2002). Finally, DJ-1 may be a subunit of a 400 kDa RNA-binding protein complex where it appears to negatively regulate RNA binding by the complex (Hod et al. 1999).

The crystal structure of human DJ-1 has recently been resolved by several groups (Honbou et al. 2003; Tao and Tong 2003; Wilson et al. 2003). Structurally, the monomer of DJ-1 adopts α/β sandwich folds, with 8 α-helices and 11 β-strands. The DJ-1 protein appears to exist in a homo-dimeric form in vitro by X-ray crystallography and other methods. In the dimer, two DJ-1 monomers make extensive contacts mainly through interactions between α-helices 1, 7, and 8 from each monomer. Intriguingly, the familial PD-linked L166P mutant exists only in monomeric form but not as a dimer, whereas the sumoylation deficient Lys130Arg (K130R) mutant exists normally as a dimer (Tao and Tong 2003). The L166P mutation is located in the center of α-helix 7 and is predicted to lead to the unfolding of the C-terminal portion of DJ-1 due to the potent helix breaking properties of the substituted proline. Consistent with these observations, recent reports suggest that native DJ-1 may exist as an oligomer in mammalian cells, whereas the L166P mutant forms higher-order protein complexes (Macedo et al. 2003; Miller et al. 2003). In a yeast expression system, the L166P mutant shows a reduced ability to self-interact compared to normal DJ-1 (Miller et al. 2003).

To investigate the specific effects of the L166P and M26I mutations on the properties of DJ-1 in vivo we have examined these mutants in cultured human cells. Here, we report that the L166P mutant exhibits markedly reduced protein stability resulting directly from enhanced degradation by the 20S/26S proteasome but not from a reduction in mRNA expression. Furthermore, the L166P mutant protein exhibits an impaired ability to self-interact and form homo-oligomers. These properties of the L166P mutant may contribute to the predicted loss of normal function of DJ-1 and may underlie its linkage with familial PD.

Plasmids

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Plasmids
  5. DJ-1 antibodies
  6. Cell culture and transfection
  7. Co-immunoprecipitation and western blotting
  8. [35S]-Methionine Pulse-chase experiments
  9. Northern blot analysis
  10. Results
  11. The L166P mutation markedly reduces the steady-state levels of the DJ-1 protein
  12. The L166P mutation does not reduce the expression of DJ-1 mRNA
  13. The L166P mutation reduces the stability of the DJ-1 protein
  14. The L166P mutation enhances DJ-1 degradation by the 20S/26S proteasome
  15. The L166P mutation impairs the homo-oligomerization of DJ-1
  16. Discussion
  17. Acknowledgments
  18. References

Full length human DJ-1 cDNA was amplified by PCR from a human DJ-1 cDNA clone (2901102; Invitrogen, Carlsbad, CA, USA) using primers 5′-AAGCTTCCACCATGGCTTCCA-3′ (forward) and 5′-CTCGAGGTCTTTAAGAACAAGT-3′ (reverse) and cloned into pCMV-Tag vector (Stratagene, La Jolla, CA, USA) between HindIII and XhoI restriction sites. The L166P missense mutation of DJ-1 was introduced by PCR-mediated site-directed mutagenesis using the QuickChange kit (Stratagene) by replacing a T with a C at position 497 relative to the ORF start site, and the M26I missense mutation was introduced similarly by replacing a G with an A at position 78. Full length DJ-1 cDNAs were then subcloned into pcDNA3.1-Myc-His vector (Invitrogen) between BamHI and XhoI sites. All constructs were sequenced to confirm their integrity. Mammalian expression constructs containing human FLAG-tagged DJ-1 (wild-type or K130R mutant) and HA-tagged DJ-1 were kindly provided by H. Ariga (Hokkaido University, Japan). A similar vector containing full length β-galactosidase cDNA was used as a control plasmid in all experiments.

DJ-1 antibodies

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Plasmids
  5. DJ-1 antibodies
  6. Cell culture and transfection
  7. Co-immunoprecipitation and western blotting
  8. [35S]-Methionine Pulse-chase experiments
  9. Northern blot analysis
  10. Results
  11. The L166P mutation markedly reduces the steady-state levels of the DJ-1 protein
  12. The L166P mutation does not reduce the expression of DJ-1 mRNA
  13. The L166P mutation reduces the stability of the DJ-1 protein
  14. The L166P mutation enhances DJ-1 degradation by the 20S/26S proteasome
  15. The L166P mutation impairs the homo-oligomerization of DJ-1
  16. Discussion
  17. Acknowledgments
  18. References

Rabbit polyclonal antibodies were raised against peptides NH2-MASKRALVILAKG(C)-CO2H and NH2-(C)QVKAPLVLKD-CO2H corresponding to N-terminal residues 1–13 and C-terminal residues 179–189 of human DJ-1, respectively. Antibodies were raised in New Zealand white rabbits using peptide conjugated to KLH via an additional cysteine residue (noted in brackets). N-terminal (anti-DJ-1-N) and C-terminal (anti-DJ-1-C) DJ-1 antibodies were affinity-purified from crude serum using peptide antigen immobilized on a Sulfolink coupling gel matrix (Pierce Biotechnology, Rockford, IL, USA) according to manufacturer's instructions.

Co-immunoprecipitation and western blotting

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Plasmids
  5. DJ-1 antibodies
  6. Cell culture and transfection
  7. Co-immunoprecipitation and western blotting
  8. [35S]-Methionine Pulse-chase experiments
  9. Northern blot analysis
  10. Results
  11. The L166P mutation markedly reduces the steady-state levels of the DJ-1 protein
  12. The L166P mutation does not reduce the expression of DJ-1 mRNA
  13. The L166P mutation reduces the stability of the DJ-1 protein
  14. The L166P mutation enhances DJ-1 degradation by the 20S/26S proteasome
  15. The L166P mutation impairs the homo-oligomerization of DJ-1
  16. Discussion
  17. Acknowledgments
  18. References

For co-immunoprecipitation from cell cultures, SH-SY5Y cells were transfected with 2 µg of each plasmid. After 48 h, cells were washed with phosphate-buffered saline (PBS) and harvested in immunoprecipitation (IP) buffer (0.5% Triton X-100, 1 X Complete mini protease inhibitor cocktail (Roche, Indianapolis, IN, USA) in PBS). Lysates were then rotated at 4°C for 1 h followed by centrifugation at 17 500 g for 15 min. The supernatant fractions were then combined with 50 µL Protein G sepharose 4 fast flow (Amersham Biosciences, Piscataway, NJ, USA) precomplexed with 5 µg mouse monoclonal anti-myc antibody (clone 9E10; Roche) followed by rotating overnight at 4°C. The Protein G sepharose complex was pelleted and washed three times using IP buffer followed by three washes with PBS. Immunoprecipitates or inputs (1% total lysate) were resolved by 12.5% SDS-PAGE and subjected to western blot analysis with mouse monoclonal anti-FLAG (M2; Sigma, St Louis, MO, USA), anti-HA (Roche), or anti-myc (Roche) antibodies directly conjugated to HRP. Bands were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences).

For proteasome inhibitor studies, SH-SY5Y cells were transfected with 1 µg of plasmid. Cells were treated with MG132 (5 µm), clasto-Lactacystin β-lactone (20 µm) (Affiniti Research, Exeter, UK), or DMSO as a control, for 24 h prior to harvesting in IP buffer, and soluble lysates were prepared as above. Lysates were quantitated using the BCA kit (Pierce Biotechnology) with BSA standards. Where necessary, detergent-insoluble pellet fractions were solubilized in 0.1 mL 2 X SDS sample buffer containing β-mercaptoethanol with sonication and boiling at 95°C for 10 min. Proteins (20 µg protein/lane) were resolved by SDS-PAGE and subjected to western blot analysis with mouse monoclonal anti-myc-HRP (Roche), or rabbit polyclonal anti-DJ-1-N, anti-DJ-1-C or anti-actin (Sigma) antibodies. Bands were detected with anti-rabbit antibodies conjugated to HRP (Amersham Biosciences) and enhanced chemiluminescence.

For cycloheximide studies, SH-SY5Y cells were transfected with 1 µg plasmid. After 48 h, cells were treated with 100 µg/mL cycloheximide (Sigma) for the indicated time points prior to harvesting in IP buffer, and soluble lysates were prepared and quantitated as described above. Lysates (20 µg protein/lane) were subjected to western blot analysis with anti-myc-HRP (Roche) or anti-actin (Sigma) antibodies.

[35S]-Methionine Pulse-chase experiments

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Plasmids
  5. DJ-1 antibodies
  6. Cell culture and transfection
  7. Co-immunoprecipitation and western blotting
  8. [35S]-Methionine Pulse-chase experiments
  9. Northern blot analysis
  10. Results
  11. The L166P mutation markedly reduces the steady-state levels of the DJ-1 protein
  12. The L166P mutation does not reduce the expression of DJ-1 mRNA
  13. The L166P mutation reduces the stability of the DJ-1 protein
  14. The L166P mutation enhances DJ-1 degradation by the 20S/26S proteasome
  15. The L166P mutation impairs the homo-oligomerization of DJ-1
  16. Discussion
  17. Acknowledgments
  18. References

SH-SY5Y cells were transfected with 2 µg myc-tagged DJ-1(wild-type, L166P or M26I) plasmid. After 24 h, cells were washed and starved in methionine-free DMEM medium (ICN) for 1 h, pulsed with 80 µCi of [35S]-methionine (ICN) for 3 h, washed and chased in normal complete DMEM medium for 24 h. At different time points (0, 3, 6 and 24 h) cells were harvested in IP buffer for immunoprecipitation with anti-myc antibody (Roche), as described above. Immunoprecipitates were resolved by 12.5% SDS-PAGE and visualized with a phosphoimager.

Northern blot analysis

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Plasmids
  5. DJ-1 antibodies
  6. Cell culture and transfection
  7. Co-immunoprecipitation and western blotting
  8. [35S]-Methionine Pulse-chase experiments
  9. Northern blot analysis
  10. Results
  11. The L166P mutation markedly reduces the steady-state levels of the DJ-1 protein
  12. The L166P mutation does not reduce the expression of DJ-1 mRNA
  13. The L166P mutation reduces the stability of the DJ-1 protein
  14. The L166P mutation enhances DJ-1 degradation by the 20S/26S proteasome
  15. The L166P mutation impairs the homo-oligomerization of DJ-1
  16. Discussion
  17. Acknowledgments
  18. References

Total RNA was isolated from human SH-SY5Y cells transfected with 2 µg myc-tagged DJ-1 (wild-type or L166P) or control plasmid, using Trizol Reagent (Invitrogen) according to manufacturers instructions. Total RNA (30 µg/lane) was separated by denaturing agarose gel electrophoresis, transferred onto nylon membranes, hybridized with 32P-labeled DNA probes derived from human DJ-1 cDNA or bovine growth hormone (BGH) polyadenylation signal (from pcDNA3.1-Myc-His vector), and washed under high stringency conditions. As a control for RNA loading and integrity, agarose gels were stained with ethidium bromide prior to blotting to visualize 28S and 18S ribosomal RNAs (rRNAs).

The L166P mutation markedly reduces the steady-state levels of the DJ-1 protein

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Plasmids
  5. DJ-1 antibodies
  6. Cell culture and transfection
  7. Co-immunoprecipitation and western blotting
  8. [35S]-Methionine Pulse-chase experiments
  9. Northern blot analysis
  10. Results
  11. The L166P mutation markedly reduces the steady-state levels of the DJ-1 protein
  12. The L166P mutation does not reduce the expression of DJ-1 mRNA
  13. The L166P mutation reduces the stability of the DJ-1 protein
  14. The L166P mutation enhances DJ-1 degradation by the 20S/26S proteasome
  15. The L166P mutation impairs the homo-oligomerization of DJ-1
  16. Discussion
  17. Acknowledgments
  18. References

To begin to investigate the basic properties of pathogenic L166P and M26I mutant DJ-1 proteins in vivo we transfected SH-SY5Y cells with plasmids containing C-terminal myc-tagged wild-type, L166P, or M26I DJ-1, or control plasmid followed by western blotting of equivalent cell lysates with anti-myc, anti-DJ-1-N, anti-DJ-1-C or anti-actin antibodies. Unexpectedly, while myc-tagged wild-type and M26I mutant DJ-1 migrate as dominant bands at 29 kDa, the L166P mutant exhibits markedly reduced protein levels and migrates as a band of 29 kDa and numerous less abundant smaller bands (Fig. 1). We also observe similar results using polyclonal anti-DJ-1-N or anti-DJ-1-C antibodies raised against N- and C-terminal epitopes of human DJ-1, respectively (Fig. 1). The anti-DJ-1-N and anti-DJ-1-C antibodies do not reliably detect the L166P mutant proteins smaller than 29 kDa, perhaps suggesting that they may represent myc tag-specific cleavage products or proteolytic fragments not adequately recognized by these antibodies. Both anti-DJ-1 antibodies also detect endogenous DJ-1 as an approximate 23 kDa protein in lysates from SH-SY5Y cells. Equivalent loading of proteins in each lane is confirmed by probing with an anti-actin antibody. Collectively, these results indicate that L166P mutant DJ-1 exhibits reduced steady-state protein levels compared to the wild-type or M26I mutant proteins. This appears to be a general feature of the L166P mutant as we could replicate these results in HEK-293 cells (data not shown). In contrast, the M26I mutation does not adversely affect the steady-state levels of the DJ-1 protein.

image

Figure 1. L166P mutant DJ-1 exhibits reduced steady-state protein levels. Equivalent lysates prepared from SH-SY5Y cells transfected with myc-tagged wild-type, L166P, or M26I DJ-1, or control plasmid, were immunoblotted with anti-myc, anti-DJ-1-N, anti-DJ-1-C or anti-actin antibodies as indicated. Notice reduced levels of the L166P mutant compared to wild-type or M26I mutant DJ-1. Bands corresponding to myc-tagged DJ-1 (Myc) and endogenous DJ-1 (endo) are indicated where necessary on the right. Molecular weight markers are indicated in kDa on the right. All experiments were replicated 3 times with similar results.

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The L166P mutation does not reduce the expression of DJ-1 mRNA

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Plasmids
  5. DJ-1 antibodies
  6. Cell culture and transfection
  7. Co-immunoprecipitation and western blotting
  8. [35S]-Methionine Pulse-chase experiments
  9. Northern blot analysis
  10. Results
  11. The L166P mutation markedly reduces the steady-state levels of the DJ-1 protein
  12. The L166P mutation does not reduce the expression of DJ-1 mRNA
  13. The L166P mutation reduces the stability of the DJ-1 protein
  14. The L166P mutation enhances DJ-1 degradation by the 20S/26S proteasome
  15. The L166P mutation impairs the homo-oligomerization of DJ-1
  16. Discussion
  17. Acknowledgments
  18. References

The L166P mutation results from a single T[RIGHTWARDS ARROW]C transition at nucleotide position 497 relative to the ORF start site of DJ-1 in members of the Italian kindred with familial PD (Bonifati et al. 2003). Therefore, to determine whether the L166P mutation can influence the expression of DJ-1 mRNA, we performed northern blot analysis on total RNA preparations derived from SH-SY5Y cells transfected with myc-tagged DJ-1 (wild-type or L166P), or control plasmid, followed by hybridization with probes specific for total DJ-1, or myc-tagged DJ-1 (BGH polyadenylation signal) (Fig. 2). Both endogenous and myc-tagged DJ-1 mRNA appear to co-migrate as transcripts of approximately 1 kb. As expected, we detect more pronounced expression of total DJ-1 mRNA in cells transfected with myc-tagged DJ-1 (wild-type or L166P) compared to control plasmid. Furthermore, we observe no significant reduction of L166P mutant mRNA expression compared to wild-type DJ-1 using probes to detect either total or myc-tagged DJ-1 mRNA. In fact, we observe marginally increased mRNA expression of the L166P mutant compared to wild-type DJ-1, which may reflect transfection efficiencies in these experiments. Equivalent loading and integrity of RNA in each lane is demonstrated by staining of agarose gels with ethidium bromide prior to blotting to visualize 28S and 18S rRNAs (Fig. 2). Together, these data suggest that the L166P mutation resulting from a single nucleotide transition does not reduce the expression of DJ-1 mRNA.

image

Figure 2. Effects of the L166P mutation on DJ-1 mRNA expression. Northern blots containing total RNA isolated from SH-SY5Y cells transfected with myc-tagged DJ-1 (wild-type or L166P) or control plasmid, were hybridized with labeled probes specific for human myc-tagged DJ-1 (upper panel) or total DJ-1 (middle panel). The location of 18S rRNA is indicated for size comparison (upper panel). Equivalent loading of RNA is indicated by ethidium bromide staining of agarose gels prior to blotting to visualize 28S and 18S rRNAs, as indicated (lower panel). This experiment was replicated with similar results.

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The L166P mutation reduces the stability of the DJ-1 protein

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Plasmids
  5. DJ-1 antibodies
  6. Cell culture and transfection
  7. Co-immunoprecipitation and western blotting
  8. [35S]-Methionine Pulse-chase experiments
  9. Northern blot analysis
  10. Results
  11. The L166P mutation markedly reduces the steady-state levels of the DJ-1 protein
  12. The L166P mutation does not reduce the expression of DJ-1 mRNA
  13. The L166P mutation reduces the stability of the DJ-1 protein
  14. The L166P mutation enhances DJ-1 degradation by the 20S/26S proteasome
  15. The L166P mutation impairs the homo-oligomerization of DJ-1
  16. Discussion
  17. Acknowledgments
  18. References

L166P mutant DJ-1 exhibits markedly reduced steady-state protein levels compared to wild-type, suggesting that this mutation reduces the stability of the DJ-1 protein. To examine possible differences in protein stability between wild-type or mutant DJ-1, we performed pulse-chase experiments to determine the half-life of these proteins. To this end, SH-SY5Y cells were transfected with myc-tagged DJ-1 (wild-type, L166P or M26I), and 24 h later newly synthesized DJ-1 protein was metabolically labeled with [35S]-methionine for 3 h, collected at different time points, and immunoprecipitated with anti-myc antibody. Both wild-type and M26I mutant DJ-1 are relatively stable over the 24 h time course examined, demonstrating comparative protein levels (Fig. 3a). In marked contrast, L166P mutant DJ-1 appears to decrease rapidly and is undetectable following 3 h of chase time (Fig. 3a), suggesting a greatly reduced protein half-life. To further examine DJ-1 protein stability, we performed cycloheximide assays. SH-SY5Y cells were transfected with myc-tagged DJ-1 (wild-type, L166P or M26I), and 48 h later cells were treated with cycloheximide to inhibit protein synthesis, harvested at different time points, and resulting lysates were probed with anti-myc antibody to monitor DJ-1 protein turnover, or anti-actin antibody as a loading control. Again, both wild-type and M26I mutant DJ-1 proteins appear highly stable over the 4 h time course examined (Fig. 3b). However, we observe a rapid decline over time in the level of L166P mutant DJ-1 protein, decreasing to barely detectable levels by 4 h (Fig. 3b). Equivalent loading of proteins is confirmed by probing with an anti-actin antibody (Fig. 3b). This data is highly consistent with our findings from pulse-chase experiments and collectively demonstrates that L166P mutant DJ-1 protein is highly unstable and exhibits a markedly reduced half-life compared to wild-type or M26I mutant DJ-1. In contrast, the M26I mutation appears to have no obvious effect on DJ-1 protein stability.

image

Figure 3. The L166P mutation reduces the stability of the DJ-1 protein. (a) [35S]-Methionine Pulse-chase analysis of SH-SY5Y cells transfected with myc-tagged DJ-1 (wild-type, L166P or M26I). Following metabolic labeling of newly synthesized proteins for 3 h with [35S]-methionine, cells were harvested at different time points (0, 3, 6 and 24 h) and lysates were immunoprecipitated with anti-myc antibody. Immunoprecipitates were resolved by 12.5% SDS-PAGE and visualized with a phosphoimager. (b ) SH-SY5Y cells were transfected with myc-tagged DJ-1 (wild-type, L166P or M26I) and 48 h later were treated with cycloheximide (100 µg/mL) for different time points (0, 0.5, 1, 2 and 4 h). Equivalent lysates were immunoblotted with anti-myc or anti-actin antibodies as indicated. Molecular weight markers are indicated in kDa on the right. This experiment was replicated with similar results.

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The L166P mutation enhances DJ-1 degradation by the 20S/26S proteasome

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Plasmids
  5. DJ-1 antibodies
  6. Cell culture and transfection
  7. Co-immunoprecipitation and western blotting
  8. [35S]-Methionine Pulse-chase experiments
  9. Northern blot analysis
  10. Results
  11. The L166P mutation markedly reduces the steady-state levels of the DJ-1 protein
  12. The L166P mutation does not reduce the expression of DJ-1 mRNA
  13. The L166P mutation reduces the stability of the DJ-1 protein
  14. The L166P mutation enhances DJ-1 degradation by the 20S/26S proteasome
  15. The L166P mutation impairs the homo-oligomerization of DJ-1
  16. Discussion
  17. Acknowledgments
  18. References

To determine the exact cause of the reduced stability of the L166P mutant protein, we reasoned that the L166P mutant might be degraded by the 20S/26S proteasomal complex. Therefore, SH-SY5Y cells were transfected with myc-tagged DJ-1 (wild-type or L166P) or control plasmid and treated for 24 h in the absence or presence of the specific 20S/26S proteasome inhibitors MG132 (5 µm) or clasto-Lactacystin β-lactone (20 µm), and resulting cell lysates were probed with anti-myc, anti-DJ-1-N, anti-DJ-1-C or anti-actin antibodies. Treatment of cells with MG132 dramatically restores the levels of the 29 kDa myc-tagged L166P mutant protein to near wild-type levels, whereas the levels of the 29 kDa myc-tagged wild-type protein are largely unaffected by this treatment (Fig. 4a). Both the major 29 kDa and smaller L166P mutant proteins tend to accumulate upon MG132 treatment, implying that all L166P proteins are targets of proteasomal degradation. Myc-tagged wild-type DJ-1 proteins smaller than 29 kDa also appear to accumulate upon MG132 treatment, although these smaller protein species are not readily detected with anti-DJ-1-N or anti-DJ-1-C antibodies perhaps suggesting that they are myc tag-specific proteolytic fragments. MG132 treatment does not appreciably affect the levels of endogenous DJ-1 protein in SH-SY5Y cell lysates as assessed by probing with anti-DJ-1-N and anti-DJ-1-C antibodies, suggesting that endogenous DJ-1 protein is relatively stable and does not undergo significant 20S/26S proteasomal turnover during the 24 h time period examined. Equivalent loading of proteins is confirmed by probing with an anti-actin antibody (Fig. 4a). Similar results are obtained using clasto-Lactacystin β-lactone to inhibit the proteasome in SH-SY5Y cells (Fig. 4a), although this treatment has a less pronounced effect on the level of L166P mutant protein accumulation and is less effective at concentrations below 20 µm. Collectively, these results effectively demonstrate that L166P mutant DJ-1 displays a greatly enhanced rate of degradation by the 20S/26S proteasome compared to wild-type or endogenous DJ-1.

image

Figure 4. L166P mutant DJ-1 displays enhanced degradation by the 20S/26S proteasome. (a ) Equivalent lysates prepared from SH-SY5Y cells transfected with myc-tagged DJ-1 (wild-type or L166P) or control plasmid, following treatment with or without the proteasome inhibitors MG132 (5 µm) or clasto-Lactacystin β-lactone (20 µm) for 24 h, were immunoblotted with anti-myc, anti-DJ-1-N, anti-DJ-1-C or anti-actin antibodies as indicated. (b ) Equivalent lysates prepared from SH-SY5Y cells transfected with myc-tagged M26I mutant DJ-1, following treatment with or without MG132 (5 µm) or clasto-Lactacystin β-lactone (20 µm) for 24 h, were immunoblotted with anti-myc, anti-DJ-1-N, antiDJ-1-C or anti-actin antibodies as indicated. (c) Equivalent 0.5% Triton X-100-soluble or -insoluble (pellet) protein fractions prepared from SH-SY5Y cells transfected with myc-tagged DJ-1 (wild-type or L166P), following treatment with MG132 (5 µm) for 24 h, were immunoblotted with anti-myc antibody. Bands corresponding to myc-tagged DJ-1 (Myc) and endogenous DJ-1 (endo) are indicated where necessary on the right. Molecular weight markers are indicated in kDa on the right. All experiments were replicated 3 times with similar results.

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In similar experiments we examined the degradation of M26I mutant DJ-1 by the 20S/26S proteasome. SH-SY5Y cells were transfected with myc-tagged M26I mutant DJ-1 and treated for 24 h in the absence or presence of MG132 (5 µm) or clasto-Lactacystin β-lactone (20 µm), and resulting cell lysates were probed as before. Treatment with either inhibitor results in an extremely small level of accumulation of the major 29 kDa myc-tagged M26I mutant protein together with the appearance of smaller myc-tagged protein species, similar to those observed for wild-type DJ-1 (Fig. 4b). As before, these smaller proteins are not appreciably detected with anti-DJ-1-N or anti-DJ-1-C antibodies. Equivalent loading is confirmed by probing with an anti-actin antibody. Thus, M26I mutant DJ-1 appears to exhibit an extremely small level of turnover by the 20S/26S proteasome, being only marginally less stable than wild-type DJ-1.

To determine the nature of L166P mutant degradation by the 20S/26S proteasome, we examined both detergent-soluble and insoluble protein fractions for the presence of high molecular weight (HMW) aggregates or ladders of the L166P mutant, indicative of ubiquitin-protein conjugates that would accumulate upon proteasome inhibition. SH-SY5Y cells were transfected with myc-tagged DJ-1 (wild-type or L166P) and treated for 24 h with MG132 (5 µm), and equivalent 0.5% Triton X-100-soluble or -insoluble (pellet) protein fractions were probed with anti-myc antibody (Fig. 4c). Myc-tagged wild-type and L166P mutant DJ-1 migrate predominantly in the detergent-soluble fraction as major 29 kDa proteins together with less abundant smaller proteins, while similar less abundant proteins are also observed in the detergent-insoluble pellet fraction. The pellet fraction additionally contains a HMW protein of approximately 75 kDa, although this does not differ between wild-type and L166P mutant DJ-1. Importantly, there is a general absence of specific HMW aggregates or ladders of the L166P mutant protein when compared to wild-type DJ-1. Together, this data suggests that the L166P mutant does not appear to specifically form ubiquitin-protein conjugates and is therefore likely to be degraded by the proteasomal complex in an ubiquitin-independent manner.

The L166P mutation impairs the homo-oligomerization of DJ-1

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Plasmids
  5. DJ-1 antibodies
  6. Cell culture and transfection
  7. Co-immunoprecipitation and western blotting
  8. [35S]-Methionine Pulse-chase experiments
  9. Northern blot analysis
  10. Results
  11. The L166P mutation markedly reduces the steady-state levels of the DJ-1 protein
  12. The L166P mutation does not reduce the expression of DJ-1 mRNA
  13. The L166P mutation reduces the stability of the DJ-1 protein
  14. The L166P mutation enhances DJ-1 degradation by the 20S/26S proteasome
  15. The L166P mutation impairs the homo-oligomerization of DJ-1
  16. Discussion
  17. Acknowledgments
  18. References

It has recently been reported that the DJ-1 protein exists in homo-dimeric form in vitro whereas the L166P mutant appears to exist in monomeric form (Honbou et al. 2003; Tao and Tong 2003). To examine the effects of the L166P mutation on the dimerization of DJ-1 in vivo, we examined the ability of DJ-1 to self interact and form homo-oligomers in cell culture. We performed co-immunoprecipitation experiments with soluble lysates from SH-SY5Y cells transfected with myc-tagged DJ-1 (wild-type or L166P) or control plasmid together with either N-terminal FLAG-tagged DJ-1 (wild-type or K130R) (Fig. 5a) or C-terminal HA-tagged DJ-1 (wild-type) (Fig. 5b). Western blot analysis of anti-myc immunoprecipitates with anti-FLAG or anti-HA antibodies indicates that myc-tagged wild-type DJ-1 but not the L166P mutant can specifically and robustly interact with FLAG- or HA-tagged wild-type DJ-1 (Figs 5a.b). Furthermore, the DJ-1 point mutant, K130R also strongly interacts with wild-type DJ-1 (Fig. 5a), indicating that the effects of the L166P mutation are specific. The use of both N-terminal (FLAG) and C-terminal (HA) tags indicates that tagging of DJ-1 at either terminus does not affect its ability to form homo-oligomers. Immunoprecipitates were also probed with anti-myc antibody to demonstrate equivalent levels of either wild-type or L166P mutant DJ-1 and input lysates were probed with anti-FLAG or anti-HA antibodies to demonstrate equivalent starting levels of wild-type DJ-1 (Figs 5a.b). We obtain identical results when performing these co-immunoprecipitation experiments from cells treated with MG132 (5 µm) for 24 h to restore the level of L166P mutant protein to near wild-type levels (Figs 5a.b), suggesting that the amount of L166P mutant DJ-1 immunoprecipitated does not influence its inability to interact with wild-type DJ-1. Taken together, these results indicate that wild-type DJ-1 can robustly self interact to form homo-oligomers, whereas the L166P mutation, but not an alternative mutation (K130R), completely impairs homo-oligomerization.

image

Figure 5. The L166P mutation impairs the homo-oligomerization of DJ-1. (a) Lysates prepared from SH-SY5Y cells transfected with myc-tagged DJ-1 (wild-type or L166P) or control plasmid, together with FLAG-tagged DJ-1 (wild-type or K130R), were subjected to IP with anti-myc followed by immunoblotting with anti-FLAG (top, middle panel) or anti-myc (top, lower panel) antibodies. Input lysates (1%) were probed with anti-FLAG antibody (top, upper panel). Similar experiments were performed using transfected cells treated with the proteasome inhibitor MG132 (5 µm) for 24 h, as described above (bottom panels). (b) Lysates prepared from SH-SY5Y cells transfected with myc-tagged DJ-1 (wild-type or L166P) or control plasmid, together with HA-tagged wild-type DJ-1, were subjected to IP with anti-myc followed by immunoblotting with anti-HA (top, middle panel) or anti-myc (top, lower panel) antibodies. Input lysates (1%) were probed with anti-HA antibody (top, upper panel). Similar experiments were performed using transfected cells treated with MG132 (5 µm) for 24 h, as described above (bottom panels). (c) Lysates prepared from SH-SY5Y cells transfected with myc-tagged DJ-1 (wild-type or M26I) or control plasmid, together with FLAG-tagged wild-type DJ-1, were subjected to IP with anti-myc followed by immunoblotting with anti-FLAG (middle panel) or anti-myc (lower panel) antibodies. Inputs were probed with anti-FLAG antibody (upper panel). Molecular weight markers are indicated in kDa on the right. All experiments were replicated 3 times with similar results.

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To additionally determine the effects of the pathogenic M26I mutation on the ability of DJ-1 to form homo-oligomers, we performed similar co-immunoprecipitation experiments with soluble lysates from SH-SY5Y cells transfected with myc-tagged DJ-1 (wild-type or M26I) or control plasmid together with FLAG-tagged wild-type DJ-1 (Fig. 5c). Western blot analysis of anti-myc immunoprecipitates with anti-FLAG antibody indicates that myc-tagged wild-type and M26I mutant DJ-1 can both robustly and equivalently interact with FLAG-tagged wild-type DJ-1. Immunoprecipitates were also probed with anti-myc antibody to demonstrate equivalent levels of either wild-type or M26I mutant DJ-1, while probing input lysates with anti-FLAG antibody demonstrates equivalent starting levels of wild-type DJ-1 (Fig. 5c). Therefore, this data effectively demonstrates that the pathogenic M26I mutation does not affect the capacity of DJ-1 to form homo-oligomers.

Discussion

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Plasmids
  5. DJ-1 antibodies
  6. Cell culture and transfection
  7. Co-immunoprecipitation and western blotting
  8. [35S]-Methionine Pulse-chase experiments
  9. Northern blot analysis
  10. Results
  11. The L166P mutation markedly reduces the steady-state levels of the DJ-1 protein
  12. The L166P mutation does not reduce the expression of DJ-1 mRNA
  13. The L166P mutation reduces the stability of the DJ-1 protein
  14. The L166P mutation enhances DJ-1 degradation by the 20S/26S proteasome
  15. The L166P mutation impairs the homo-oligomerization of DJ-1
  16. Discussion
  17. Acknowledgments
  18. References

The major findings of this study are that a PD causing missense mutation in DJ-1, L166P, confers markedly reduced protein stability that leads to enhanced degradation by the 20S/26S proteasomal complex, whereas the expression of DJ-1 mRNA is not affected by the L166P mutation. Wild-type DJ-1 forms homo-dimers, whereas L166P mutant DJ-1 has an impaired capacity to dimerize. In contrast, M26I mutant DJ-1 is a relatively stable protein and can dimerize normally. The reduced stability of the L166P mutant protein is probably due to abnormal or misfolded DJ-1 as it is predicted that disruption of α-helix 7 by the L166P mutation would lead to the unfolding of the C-terminal portion of DJ-1 (Bonifati et al. 2003; Honbou et al. 2003; Tao and Tong 2003). The inability of the L166P mutant to dimerize suggests that normal DJ-1 functions as a dimer or homo-oligomer. Instability and the failure to dimerize may contribute to the loss of normal DJ-1 function and these properties are likely to be one of the underlying causes of early onset PD in patients with DJ-1 mutations.

The observation that the L166P mutant, but not the wild-type or M26I mutant proteins, displays greatly enhanced degradation by the 20S/26S proteasome implies that the L166P mutant protein adopts an undesirable abnormal or misfolded conformation which, if allowed to accumulate, might be potentially damaging to the cell. In contrast, the M26I mutation has a comparatively small effect on DJ-1 turnover by the proteasome, suggesting that this mutation does not adversely affect normal protein folding or conformation. It is unlikely that the L166P mutant protein itself contributes to the neuronal degeneration observed in DJ-1-linked familial PD patients through a toxic gain of function, as family members heterozygous for this mutation appear to be normal (Bonifati et al. 2003). Furthermore, the L166P mutation is not absolutely required to cause early onset PD as a homozygous deletion of the DJ-1 gene is sufficient to cause disease in affected members of the Dutch kindred (van Duijn et al. 2001; Bonifati et al. 2003). The L166P mutation is therefore likely to lead to a loss of the normal cellular function of DJ-1 and this may directly result from its inability to form homo-dimers rather than perturbation of a particular functional domain within the protein. Indeed, both the pathogenic M26I and synthetic K130R mutant proteins retain full ability to form homo-dimers and also do not appear to exhibit enhanced proteasomal degradation. Collectively, this suggests that homo-dimerization of DJ-1 is critical for its normal cellular function.

The exact molecular mechanism by which L166P mutant DJ-1 is targeted for enhanced degradation by the 20S/26S proteasomal complex remains to be clarified. The observed absence in our experiments of HMW aggregates or ladders of the L166P mutant protein, indicative of ubiquitin-protein conjugates, which would accumulate upon proteasome inhibition, suggests that ubiquitinylation is unlikely to play a major role in targeting L166P mutant DJ-1 for proteasomal degradation. The 20S proteasome is capable of proteolytically processing small, unfolded or oxidatively modified target proteins directly in an ubiquitin-independent manner, including natively unfolded proteins such as α-synuclein and tau (Orlowski and Wilk 2003; Tofaris et al. 2001; David et al. 2002). Although, the 26S proteasomal complex, comprised of the 20S proteolytic core and an ATPase-containing 19S regulatory particle, preferentially degrades poly-ubiquitinylated target proteins, it can also degrade some non-ubiquitinylated proteins in an ATP-dependent manner (McNaught and Olanow 2003; Benaroudj et al. 2001; Orlowski and Wilk 2003). Therefore, we suggest that the L166P mutant protein is potentially degraded by the 20S/26S proteasome in an ubiquitin-independent manner due to its newly acquired misfolded or partially unfolded conformation. In the absence of obvious ubiquitinylation, misfolded L166P mutant DJ-1 may be capable of directly entering the 20S proteolytic core possibly with further unfolding assistance by molecular chaperones or other cofactors (McNaught and Olanow 2003; Orlowski and Wilk 2003).

The specific effects of the pathogenic M26I mutation on the properties of DJ-1 in vivo or in vitro still remain to be clarified. In contrast to the L166P mutation, the M26I mutation does not appear to adversely affect either protein stability, turnover by the proteasome, or the capacity of DJ-1 to form homo-dimers. The M26I mutation is a relatively conservative point mutation located within α-helix 1 of the DJ-1 protein. Although α-helix 1 is intimately involved in forming the dimerization interface between two DJ-1 monomers along with α-helices 7 and 8, our data suggests that the M26I mutation is unlikely to have major effects on the structural conformation of the protein as the capacity to dimerize appears to be sensitive to structural changes. Instead, it is possible that the M26I mutation modifies some other fundamental property of DJ-1, such as enzymatic activity or protein–protein interactions, which would result in the predicted loss of normal function of DJ-1. However, such a role for the M26I mutation awaits further clarification.

During the preparation of this manuscript, two reports that describe similar findings to those presented here were published. Consistent with our data, L166P mutant DJ-1 appears to be a highly unstable protein with a reduced half-life that tends to show enhanced degradation by the proteasome in cultured mammalian cells (Macedo et al. 2003; Miller et al. 2003). Similarly, lymphoblast cells derived from an Italian PD patient carrying the homozygous L166P point mutation also show a marked reduction in DJ-1 protein stability (Macedo et al. 2003), implying that our findings are especially relevant to the molecular pathogenesis of PD caused by this DJ-1 mutation. Miller and coworkers suggest that L166P mutant DJ-1 is degraded through the ubiquitin-proteasome system (UPS), however, in contrast, we find no evidence for the involvement of ubiquitinylation in targeting DJ-1 to the proteasome and suggest that degradation is likely mediated by an ubquitin-independent mechanism. This is based on our observation that L166P mutant DJ-1 does not form HMW protein aggregates or ladders upon proteasome inhibition, which would be predicted to accumulate if ubiquitinylation played a role in targeting DJ-1 for proteasomal degradation.

Native DJ-1 appears to migrate at a size consistent with the predicted size of a homo-dimer, while the L166P mutant forms higher-order protein complexes (Macedo et al. 2003). Furthermore, the L166P mutation impairs the formation of homo-oligomers in a yeast expression system (Miller et al. 2003). These data are highly consistent with our own observations in cultured human cells by using co-immunoprecipitation of epitope-tagged DJ-1 proteins. However, while we demonstrate that the interaction between wild-type and L166P mutant DJ-1 is fully impaired, studies in yeast show only a reduction in this particular interaction but not full impairment. These discrepancies can be ascribed to differences in the cellular expression systems employed and the method of assay for protein–protein interaction analysis. Based on our data, the higher-order protein complexes recently described by Macedo and coworkers for L166P mutant DJ-1 are unlikely to contain more than one DJ-1 monomer as we would have detected this complex in our co-immunoprecipitation assays as a homo-oligomer. Collectively, these combined data suggest that L166P mutant DJ-1 exhibits impaired homo-oligomerization but likely forms complexes with proteins other than DJ-1 that are yet be identified, such as components of the UPS and/or molecular chaperones.

In conclusion, we report that an L166P missense mutation in DJ-1, recently linked to early onset PD in members of an Italian kindred, confers reduced protein stability, enhanced degradation by the 20S/26S proteasome, and an inability to form homo-oligomers, presumably homo-dimers. These features are probably a direct consequence of the predicted abnormal or misfolded conformation of L166P mutant DJ-1 compared to the wild-type protein. We suggest that homo-dimerization of DJ-1 is probably critical for its normal cellular function, and impairment of this ability by the L166P mutation, and hence loss of normal function, is likely to be the underlying cause of early onset PD in family members homozygous for this DJ-1 missense mutation.

References

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Plasmids
  5. DJ-1 antibodies
  6. Cell culture and transfection
  7. Co-immunoprecipitation and western blotting
  8. [35S]-Methionine Pulse-chase experiments
  9. Northern blot analysis
  10. Results
  11. The L166P mutation markedly reduces the steady-state levels of the DJ-1 protein
  12. The L166P mutation does not reduce the expression of DJ-1 mRNA
  13. The L166P mutation reduces the stability of the DJ-1 protein
  14. The L166P mutation enhances DJ-1 degradation by the 20S/26S proteasome
  15. The L166P mutation impairs the homo-oligomerization of DJ-1
  16. Discussion
  17. Acknowledgments
  18. References
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