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• W1966773864 abstract "Mutations in the PARK7/DJ-1 gene cause autosomal-recessive Parkinson's disease. In some patients the gene is deleted. The molecular basis of disease in patients with point mutations is less obvious. We have investigated the molecular properties of [L166P]DJ-1 and the novel variant [E64D]DJ-1. When transfected into non-neuronal and neuronal cell lines, steady-state expression levels of [L166P]DJ-1 were dramatically lower than wild-type [WT]DJ-1 and [E64D]DJ-1. Cycloheximide and pulse-chase experiments revealed that the decreased expression levels of [L166P]DJ-1 were because of accelerated protein turnover. Proteasomal degradation was not the major pathway of DJ-1 breakdown because treatment with the proteasome inhibitor MG-132 caused only minimal accumulation of DJ-1, even of the very unstable [L166P]DJ-1 mutant. Because of the structural resemblance of DJ-1 with bacterial cysteine proteases, we considered an autoproteolytic mechanism. However, neither pharmacological inhibition nor site-directed mutagenesis of the putative active site residue Cys-106 stabilized DJ-1. To gain further insight into the structural defects of DJ-1 mutants, human [WT]DJ-1 and both mutants were expressed in Escherichia coli. As in eukaryotic cells, expression levels of [L166P]DJ-1 were dramatically reduced compared with [WT]DJ-1 and [E64D]DJ-1. Circular dichroism spectrometry revealed that the solution structures of [WT]DJ-1 and [E64D]DJ-1 are rich in β-strand and α-helix conformation. α-Helices were more susceptible to thermal denaturation than the β-sheet, and [WT]DJ-1 was more flexible in this regard than [E64D]DJ-1. Thus, structural defects of [E64D]DJ-1 only become apparent upon denaturing conditions, whereas the L166P mutation causes a drastic defect that leads to excessive degradation. Mutations in the PARK7/DJ-1 gene cause autosomal-recessive Parkinson's disease. In some patients the gene is deleted. The molecular basis of disease in patients with point mutations is less obvious. We have investigated the molecular properties of [L166P]DJ-1 and the novel variant [E64D]DJ-1. When transfected into non-neuronal and neuronal cell lines, steady-state expression levels of [L166P]DJ-1 were dramatically lower than wild-type [WT]DJ-1 and [E64D]DJ-1. Cycloheximide and pulse-chase experiments revealed that the decreased expression levels of [L166P]DJ-1 were because of accelerated protein turnover. Proteasomal degradation was not the major pathway of DJ-1 breakdown because treatment with the proteasome inhibitor MG-132 caused only minimal accumulation of DJ-1, even of the very unstable [L166P]DJ-1 mutant. Because of the structural resemblance of DJ-1 with bacterial cysteine proteases, we considered an autoproteolytic mechanism. However, neither pharmacological inhibition nor site-directed mutagenesis of the putative active site residue Cys-106 stabilized DJ-1. To gain further insight into the structural defects of DJ-1 mutants, human [WT]DJ-1 and both mutants were expressed in Escherichia coli. As in eukaryotic cells, expression levels of [L166P]DJ-1 were dramatically reduced compared with [WT]DJ-1 and [E64D]DJ-1. Circular dichroism spectrometry revealed that the solution structures of [WT]DJ-1 and [E64D]DJ-1 are rich in β-strand and α-helix conformation. α-Helices were more susceptible to thermal denaturation than the β-sheet, and [WT]DJ-1 was more flexible in this regard than [E64D]DJ-1. Thus, structural defects of [E64D]DJ-1 only become apparent upon denaturing conditions, whereas the L166P mutation causes a drastic defect that leads to excessive degradation. Although hereditary parkinsonism is very rare compared with sporadic Parkinson's disease (PD), 1The abbreviations used are: PD, Parkinson's disease; WT, wild type; CD, circular dichroism; PH, Pyrococcus horikoshii. 1The abbreviations used are: PD, Parkinson's disease; WT, wild type; CD, circular dichroism; PH, Pyrococcus horikoshii. the identification of PARK genes has greatly expanded the molecular understanding of the most common neurodegenerative movement disorders (1Dawson T.M. Dawson V.L. J. Clin. Investig. 2003; 111: 145-151Crossref PubMed Scopus (197) Google Scholar). The discovery of PARK1/α-SYNUCLEIN has led to the identification of α-synuclein fibrils as the neuropathological hallmark of PD, and the investigation of α-synuclein has provided important clues to the molecular mechanisms of PD (2Kahle P.J. Haass C. Kretzschmar H.A. Neumann M. J. Neurochem. 2002; 82: 449-457Crossref PubMed Scopus (76) Google Scholar, 3Dev K.K. Hofele K. Barbieri S. Buchman V.L. van der Putten H. Neuropharmacology. 2003; 45: 14-44Crossref PubMed Scopus (225) Google Scholar). Moreover, two enzymes involved in ubiquitin metabolism were found to be encoded by PARK2 (the ubiquitin ligase parkin) and PARK5 (ubiquitin C-terminal hydrolase-L1), suggesting that failure of the ubiquitin-proteasome system generally contributes to PD (4McNaught K.S.P. Olanow C.W. Ann. Neurol. 2003; 53: S73-84Crossref PubMed Scopus (188) Google Scholar). More recently, DJ-1 was identified to be the gene mutated in the PARK7 locus (5Bonifati V. Rizzu P. van Baren M.J. Schaap O. Breedveld G.J. Krieger E. Dekker M.C.J. Squitieri F. Ibanez P. Joosse M. van Dongen J.W. Vanacore N. van Swieten J.C. Brice A. Meco G. van Duijn C.M. Oostra B.A. Heutink P. Science. 2003; 299: 256-259Crossref PubMed Scopus (2171) Google Scholar). Loss-of-function mutations of DJ-1 are compatible with the recessive inheritance of PARK7 (6van Duijn C.M. Dekker M.C.J. Bonifati V. Galjaard R.J. Houwing-Duistermaat J.J. Snijders P.J.L.M. Testers L. Breedveld G.J. Horstink M. Sandkuijl L.A. van Swieten J.C. Oostra B.A. Heutink P. Am. J. Hum. Genet. 2001; 69: 629-634Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). In the Dutch kindred, protein-coding sequences are deleted altogether (5Bonifati V. Rizzu P. van Baren M.J. Schaap O. Breedveld G.J. Krieger E. Dekker M.C.J. Squitieri F. Ibanez P. Joosse M. van Dongen J.W. Vanacore N. van Swieten J.C. Brice A. Meco G. van Duijn C.M. Oostra B.A. Heutink P. Science. 2003; 299: 256-259Crossref PubMed Scopus (2171) Google Scholar). Complete loss of functional DJ-1 was also predicted for a young-onset PD patient with compound frameshift and splice mutations (7Hague S. Rogaeva E. Hernandez D. Gulick C. Singleton A. Hanson M. Johnson J. Weiser R. Gallardo M. Ravina B. Gwinn-Hardy K. Crawley A. St. George-Hyslop P.H. Lang A.E. Heutink P. Bonifati V. Hardy J. Singleton A. Ann. Neurol. 2003; 54: 271-274Crossref PubMed Scopus (206) Google Scholar). It remains to be shown what physiological function of DJ-1 is depleted. Previous studies have implicated DJ-1 with tumor progression, RNA binding, male fertility, androgen receptor signaling, and cellular management of oxidative stress (8Nagakubo D. Taira T. Kitaura H. Ikeda M. Tamai K. Iguchi-Ariga S.M.M. Ariga H. Biochem. Biophys. Res. Commun. 1997; 231: 509-513Crossref PubMed Scopus (655) Google Scholar, 9Wagenfeld A. Gromoll J. Cooper T.G. Biochem. Biophys. Res. Commun. 1998; 251: 545-549Crossref PubMed Scopus (94) Google Scholar, 10Hod Y. Pentyala S.N. Whyard T.C. El-Maghrabi M.R. J. Cell. Biochem. 1999; 72: 435-444Crossref PubMed Scopus (170) Google Scholar, 11Mitsumoto A. Nakagawa Y. Takeuchi A. Okawa K. Iwamatsu A. Takanezawa Y. Free Radic. Res. 2001; 35: 301-310Crossref PubMed Scopus (232) Google Scholar, 12Takahashi K. Taira T. Niki T. Seino C. Iguchi-Ariga S.M.M. Ariga H. J. Biol. Chem. 2001; 276: 37556-37563Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 13Okada M. Matsumoto K. Niki T. Taira T. Iguchi-Ariga S.M.M. Ariga H. Biol. Pharm. Bull. 2002; 25: 853-856Crossref PubMed Scopus (81) Google Scholar, 14Niki T. Takahashi-Niki K. Taira T. Iguchi-Ariga S.M.M. Ariga H. Mol. Cancer Res. 2003; 1: 247-261PubMed Google Scholar). Furthermore, there is structural similarity of DJ-1 with bacterial proteases and hydroperoxidases (15Wilson M.A. Collins J.L. Hod Y. Ringe D. Petsko G.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9256-9261Crossref PubMed Scopus (251) Google Scholar, 16Tao X. Tong L. J. Biol. Chem. 2003; 278: 31372-31379Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 17Honbou K. Suzuki N.N. Horiuchi M. Niki T. Taira T. Ariga H. Inagaki F. J. Biol. Chem. 2003; 278: 31380-31384Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 18Huai Q. Sun Y. Wang H. Chin L.-S. Li L. Robinson H. Ke H. FEBS Lett. 2003; 549: 171-175Crossref PubMed Scopus (99) Google Scholar). However, the function of DJ-1 in the brain, where it is expressed at rather moderate levels (5Bonifati V. Rizzu P. van Baren M.J. Schaap O. Breedveld G.J. Krieger E. Dekker M.C.J. Squitieri F. Ibanez P. Joosse M. van Dongen J.W. Vanacore N. van Swieten J.C. Brice A. Meco G. van Duijn C.M. Oostra B.A. Heutink P. Science. 2003; 299: 256-259Crossref PubMed Scopus (2171) Google Scholar, 8Nagakubo D. Taira T. Kitaura H. Ikeda M. Tamai K. Iguchi-Ariga S.M.M. Ariga H. Biochem. Biophys. Res. Commun. 1997; 231: 509-513Crossref PubMed Scopus (655) Google Scholar, 9Wagenfeld A. Gromoll J. Cooper T.G. Biochem. Biophys. Res. Commun. 1998; 251: 545-549Crossref PubMed Scopus (94) Google Scholar), is completely unknown. A second PARK7 family from Italy (19Bonifati V. Breedveld G.J. Squitieri F. Vanacore N. Brustenghi P. Harhangi B.S. Montagna P. Cannella M. Fabbrini G. Rizzu P. van Duijn C.M. Oostra B.A. Meco G. Heutink P. Ann. Neurol. 2002; 51: 253-256Crossref PubMed Scopus (68) Google Scholar) was found to bear a point mutation in the DJ-1 gene, leading to replacement of leucine 166 with proline (5Bonifati V. Rizzu P. van Baren M.J. Schaap O. Breedveld G.J. Krieger E. Dekker M.C.J. Squitieri F. Ibanez P. Joosse M. van Dongen J.W. Vanacore N. van Swieten J.C. Brice A. Meco G. van Duijn C.M. Oostra B.A. Heutink P. Science. 2003; 299: 256-259Crossref PubMed Scopus (2171) Google Scholar). The L166P mutation is predicted to break a characteristic α-helical fold in the DJ-1 structure. More recently, a second point mutation (E64D) was found in a small kindred of Turkish ancestry. 2R. Hering, C. Strauss, X. Tao, A. Bauer, E.-M. Mietz, S. Petrovic, P. Bauer, W. Schaible, D. Woitalla, T. Müller, L. Schöls, C. Klein, D. Berg, P. T. Meyer, J. B. Schulz, B. Wollnik, L. Tong, R. Krüger, and O. Riess, submitted for publication. 2R. Hering, C. Strauss, X. Tao, A. Bauer, E.-M. Mietz, S. Petrovic, P. Bauer, W. Schaible, D. Woitalla, T. Müller, L. Schöls, C. Klein, D. Berg, P. T. Meyer, J. B. Schulz, B. Wollnik, L. Tong, R. Krüger, and O. Riess, submitted for publication. The homozygous index patient had early-onset PD along with significant depletion of striatal dopamine receptors, as evidenced by the reduced [18F]FP-CIT uptake. Subclinical PD was indicated by reduction of [18F]FP-CIT positron emission in a homozygous sister, whereas a heterozygous brother had normal [18F]FP-CIT uptake. Five more siblings as well as the heterozygous mother were unaffected. To elucidate the molecular basis of the deficits of DJ-1 point mutations, the expression, processing, and turnover of WT and mutant DJ-1 were examined. Expression levels of mutant [L166P]DJ-1 were significantly reduced compared with [WT]DJ-1 (20Miller D.W. Ahmad R. Hague S. Baptista M.J. Canet-Aviles R. McLendon C. Carter D.M. Zhu P.-P. Stadler J. Chandran J. Klinefelter G.R. Blackstone C. Cookson M.R. J. Biol. Chem. 2003; 278: 36588-36595Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 21Macedo M.G. Anar B. Bronner I.F. Cannella M. Squitieri F. Bonifati V. Hoogeveen A. Heutink P. Rizzu P. Hum. Mol. Genet. 2003; 12: 2807-2816Crossref PubMed Scopus (126) Google Scholar) as well as [E64D]DJ-1, both in prokaryotic and eukaryotic cells. Cycloheximide and pulse-chase experiments with transiently transfected human embryonic kidney HEK293 cells revealed that [L166P]DJ-1 was degraded much more rapidly than [WT]DJ-1 and [E64D]DJ-1. WT and mutant DJ-1 minimally accumulated in the presence of MG-132, suggesting the existence of a major non-proteasomal DJ-1 degradation pathway. Because of structural resemblance of DJ-1 with Pyrococcus horikoshii PH1704 class cysteine proteases (17Honbou K. Suzuki N.N. Horiuchi M. Niki T. Taira T. Ariga H. Inagaki F. J. Biol. Chem. 2003; 278: 31380-31384Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar), we considered an autoproteolytic mechanism of [L166P]DJ-1 breakdown. However, neither pharmacological inhibition nor site-directed mutagenesis of the putative active site cysteine 106 to alanine stabilized DJ-1. Furthermore, we found that the solution structures of [WT]DJ-1 and [E64D]DJ-1 were rich in α-helix and β-sheet, but α-helical elements were more susceptible to thermal denaturation in [WT]DJ-1 than in [E64D]DJ-1. Thus, the L166P mutation enhances cellular breakdown and depletion of DJ-1, whereas the structural defects of [E64D]DJ-1 are more subtle and might become apparent only under stress conditions. We conclude that Parkinson's disease in PARK7 patients is caused by a depletion of functionally active DJ-1 protein. Antibodies—DJ-1 was probed with mouse monoclonal anti-V5 (Invitrogen), mouse monoclonal Penta-His (Qiagen, Hilden, Germany), and mouse monoclonal anti-DJ-1 (Medical & Biological Laboratories, Nagoya, Japan), or polyclonal antiserum against DJ-1 (gift of H. Ariga, Hokkaido University, Sapporo, Japan), followed by peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG, respectively (Sigma). Western blots were normalized by reprobing with anti-β-actin (Sigma). Efficacy of proteasome inhibitors was demonstrated with mouse monoclonal anti-β-catenin (Transduction Laboratories, Lexington, KY). Site-directed Mutagenesis—[WT]DJ-1 and [L166P]DJ-1 (5Bonifati V. Rizzu P. van Baren M.J. Schaap O. Breedveld G.J. Krieger E. Dekker M.C.J. Squitieri F. Ibanez P. Joosse M. van Dongen J.W. Vanacore N. van Swieten J.C. Brice A. Meco G. van Duijn C.M. Oostra B.A. Heutink P. Science. 2003; 299: 256-259Crossref PubMed Scopus (2171) Google Scholar) cDNAs were used as a template for two independent polymerase chain reactions (PCR) with the following primers: NcoI forward primer 5′-AACCATGGGAATGGCTTCCAAAAGAGCTCTG-3′ and C106A-REV reverse primer 5′-AGGACCTGCAGCGATGGCGG-3′ and the second PCR with C106A-FWD forward primer 5′-CCGCCATCGCTGCAGGTCCT-3′ and BamHI stop reverse primer 5′-CGCGGATCCGATGTCTTTAAGAACAAGTGGAGCC-3′. The two resulting PCR products were then used as templates for a PCR using NcoI forward primer (see above) and BamHI stop reverse primer (see above). The mutagenized PCR products were subcloned into pcDNA3.1/V5-His TOPO (Invitrogen) and sequenced (GATC Biotech, Konstanz, Germany). Generation of the [E64D]DJ-1 construct in the same vector was described elsewhere. 2R. Hering, C. Strauss, X. Tao, A. Bauer, E.-M. Mietz, S. Petrovic, P. Bauer, W. Schaible, D. Woitalla, T. Müller, L. Schöls, C. Klein, D. Berg, P. T. Meyer, J. B. Schulz, B. Wollnik, L. Tong, R. Krüger, and O. Riess, submitted for publication. Eukaryotic DJ-1 Expression and Inhibitor Treatments—HEK293 cells, SY5Y cells, and MN9D cells were grown to near confluence and transiently transfected with eukaryotic expression constructs using LipofectAMINE 2000 in opti-minimum essential medium (Invitrogen) (DNA:lipid ratio 1:2.5). Two days after transfection, cells were treated with 100 μg/ml cycloheximide (Sigma), 10 μm MG-132 (Calbiochem), 50 μm leupeptin (Roche Diagnostics), or directly lysed in 1% Triton X-100, 150 mm sodium chloride, 1 mm EDTA, 10 mm Tris-HCl (pH 7.5) plus Cømplete protease inhibitor mixture (Roche Diagnostics). Protein content in the lysates was determined with the Bio-Rad protein assay. After denaturing 15% polyacrylamide gel electrophoresis, proteins were electroblotted onto polyvinylidene fluoride membranes (Millipore, Eschborn, Germany). Western blots were probed with the antibodies listed below, and immunoreactive bands were visualized by enhanced chemiluminescence. Pulse-Chase Labeling—HEK293 cells were transiently transfected as above. One day after transfection, the cells were starved in methionine/cysteine-free minimum essential medium (Sigma) plus 1% penicillin/streptomycin and 1% l-glutamine for one hour. They were then pulsed for 3 h with 75 μCi/ml [35S]Met/[35S]Cys (Promix; Amersham Biosciences), rinsed, and chased with Dulbecco's modified Eagle's medium (PAA, Pasching, Austria) plus 1% penicillin/streptomycin, 1% l-glutamine, 1 mml-methionine, and 10% fetal calf serum. The cells were lysed in 1% Triton X-100, 150 mm NaCl, 2 mm EDTA, 50 mm Tris (pH 7,6) plus Cømplete protease inhibitor mixture. Immunoprecipitation was performed with anti-V5 and protein G-Sepharose (Amersham Biosciences). After denaturing 15% polyacrylamide gel electrophoresis, the gels were fixed, soaked in Amplify (Amersham Biosciences), and dried. The radiolabeled proteins were visualized on BioMax film (Kodak, Rochester, New York). RT-PCR Determination of DJ-1 mRNA—Total RNA was extracted from transfected cells with Trizol (peQLab, Erlangen, Germany) and reverse-transcribed using superScript II and oligo(dT) primers (Invitrogen). The resulting cDNAs were PCR-amplified using NcoI forward primer (see above) and BamHI V5 tag reverse primer (see below). Amplification rates were linear at 25 cycles, as visualized on ethidium bromide-stained agarose gels. Bacterial DJ-1 Expression and Purification—[WT]DJ-1 and mutant DJ-1 cDNAs (see above) were amplified in polymerase chain reactions using the following primers: NcoI forward primer (see above), BamHI V5 tag reverse primer 5′-ATCTGGATCCCGTAGAATCGAGACCGAGGAG-3′, and BamHI reverse primer 5′-ATCTGGATCCGTCTTTAAGAACAAGTGGAGC-3′. After subcloning into pQE-60 (Qiagen), the prokaryotic expression constructs were used to transform E. coli BL21/RIL. All constructs were sequenced (GATC Biotech). Bacterial cultures were induced with 100 μm isopropyl-β-d-thiogalactoside for 5 h. The pelleted bacteria were French-pressed into phosphate-buffered saline containing Cømplete protease inhibitors. Imidazole was added to a final concentration of 20 mm, and this lysate was loaded onto a nickel-nitrilotriacetic acid Superflow column (Qiagen). The HIS-tagged DJ-1 proteins eluted in a linear gradient (20-500 mm) around 0.1 m imidazole as broad peaks that were concentrated in Centriprep-3 devices (Millipore). The concentrates were loaded onto a HiLoad Superdex 75 column (Amersham Biosciences) and eluted in 5% glycerol, 3 mm dithiothreitol, 150 mm NaCl, 20 mm Tris (pH 8.0). DJ-1/HIS proteins eluted earlier than expected for globular proteins with molecular mass 21.3 kDa (low molecular weight gel filtration calibration markers were purchased from Amersham Biosciences), consistent with the dimeric structure of native DJ-1. Peak integrations of analytical Source 5 reverse phase HPLC runs revealed at least 90% purity of the DJ-1 preparations. Mass Spectrometry—DJ-1 was diluted to 0.01-0.5 mg/ml with 0.1% trifluoroacetic acid in water. Two μl of this solution were mixed with 1 μl of sinapinic acid (Fluka) saturated in 0.4% trifluoroacetic acid in 30% acetonitrile as matrix. One μl was deposited on a stainless steel target plate. Matrix-assisted laser desorption ionization mass spectra were recorded in the linear mode on a Voyager DE STR mass spectrometer (Applied Biosystems) with 25,000 V accelerating voltage. 3-5 × 100 laser shots were collected from different positions and added. CD Spectrometry—Protein solutions were scanned in a 0.1-cm quartz cuvette using a J-810 CD spectrophotometer (Jasco). Temperature in the measuring cell was gradually increased, and at defined temperature intervals CD spectra were taken from 300-190 nm. Secondary structure was calculated using the Jasco JWSSE-480 software (least squares method Yang). Expression of WT and Mutant DJ-1 in Eukaryotic Cells—To study the cellular expression of DJ-1, plasmids encoding [WT]DJ-1 and mutant DJ-1 with a C-terminal-fused V5/HIS tag were transfected into HEK293 cells. Two days after transfection, the expression levels relative to β-actin were found to be much lower for [L166P]DJ-1 than [WT]DJ-1 and [E64D]DJ-1, as evidenced from Western blots probed with anti-V5 (Fig. 1). Probing with polyclonal or monoclonal antibodies against DJ-1 revealed that the expression levels of transfected [WT]DJ-1 were comparable with endogenous DJ-1 levels, whereas [L166P]DJ-1 levels were dramatically reduced (Fig. 1). RT-PCR analysis of the transfected DJ-1 mRNAs revealed that all constructs were robustly expressed. Thus, [L166P]DJ-1 was lost at the protein level and not because of inefficient transcription or mRNA lability. [E64D]DJ-1 was expressed slightly less than [WT]DJ-1. Surprisingly, monoclonal 3E8 anti-DJ-1 did not recognize the [E64D]DJ-1 protein (Fig. 1). Longer exposures of higher resolution blots provided evidence for cell type-specific processing of transfected DJ-1. The antibody against the C-terminal V5 tag visualized two truncated DJ-1 species (ΔN1 and ΔN2) in non-neuronal HEK293 cells but not in neuroblastoma SY5Y cells and immortalized dopaminergic neuronal MN9D cells (Fig. 2). The ratio of these N-terminal-truncated DJ-1 fragments to full-length DJ-1 was much higher for [L166P]DJ-1. In addition, the C-terminal V5 epitope could be cleaved off from transfected DJ-1 to produce band ΔC, which was visualized only with antibodies against DJ-1 (see Figs. 3 and 4).Fig. 3Accelerated turnover of [L166P]DJ-1. V5-tagged [WT]DJ-1 (left panels), [L166P]DJ-1 (middle panels), and [E64D]DJ-1 (right panels) were transiently expressed in HEK293 cells. A, for pulse-chase experiments, cells were incubated with 35S-labeled cysteine/methionine for 3 h and chased with non-radioactive medium for the times indicated at the bottom. Whole cell lysates were immunoprecipitated with anti-V5. The immunoprecipitated DJ-1/V5/HIS proteins were separated on denaturing 15% polyacrylamide gels and visualized by autoradiography. B, for translational inhibition, cells were treated with 100 μg/ml cycloheximide for the times indicated at the bottom. Cell lysates (20 μg for [WT]DJ-1 and [E64D]DJ-1, 50 μg for [L166P]DJ-1) were sequentially Western probed with monoclonal antibodies against the V5 epitope (upper panels), DJ-1 (middle panels), and β-actin (lower panels). Note that a long (15-min) exposure is shown to match the t = 0 V5 signal from the low-expressing [L166P]DJ-1 with those from [WT]DJ-1 and [E64D]DJ-1 (3-s exposure). This experiment was repeated three times with similar results, although this particular experiment showed the fastest V5 decay of [E64D]DJ-1. Molecular mass standards are indicated to the right.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4Minimal accumulation of DJ-1 upon proteasomal inhibition. V5/HIS-tagged [WT]DJ-1 (left panels), [L166P]DJ-1 (middle panels), and [E64D]DJ-1 (right panels) were transiently expressed in HEK293 cells. Two days post-transfection, the cells were treated with 10 μm MG-132 for the times indicated at the bottom. DJ-1 in cell lysates (20 μg for [WT]DJ-1 and [E64D]DJ-1, 50 μg for [L166P]DJ-1) was detected with anti-V5 as well as with polyclonal antiserum and 3E8 monoclonal against DJ-1, as indicated to the left. Efficacy of proteasomal inhibition was demonstrated by the accumulation of β-catenin, equal loading, and cellular viability by even β-actin immunoreactivity (lower panels). A long (10-min) exposure is shown to match the t = 0 V5 signal from the low-expressing [L166P]DJ-1 with those from [WT]DJ-1 and [E64D]DJ-1 (3-s exposure). Molecular mass standards are indicated to the right.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Turnover of WT and Mutant DJ-1 in HEK293 Cells—Despite the detection of similar mRNA levels, the steady-state levels of mutant DJ-1 proteins were reduced. To test whether rapid degradation could account for the loss of mutant DJ-1 protein, pulse-chase experiments were performed. Transiently transfected HEK293 cells were pulse-labeled with a mixture of [35S]cysteine/methionine and chased with non-radioactive medium up to 24 h. Radiolabeled transfected DJ-1 proteins were immunoprecipitated with anti-V5. [WT]DJ-1 was found to be a very stable protein. There was practically no breakdown of [WT]DJ-1 for 6-8 h, with little further decay up to 24 h (Fig. 3A). [E64D]DJ-1 was an equally stable protein. In contrast, the degradation of [L166P]DJ-1 protein was dramatically accelerated. [L166P]DJ-1 was degraded within 4-8 h, although it was translated as efficiently as [WT]DJ-1 as evidenced by the comparable incorporation of 35S-labeled amino acids at the 0-h chase time point. Thus, the dramatic reduction of [L166P]DJ-1 steady-state levels is not because of poor translation. Moreover, the 0-h chase time point showed only full-length [L166P]DJ-1. Both N-terminal-truncated [L166P]DJ-1 fragments ΔN1 and ΔN2 were cleaved from the full-length precursor protein within 4-6 h after translation, demonstrating that the N-terminal processing of [L166P]DJ-1/V5/HIS is a rapid event. To confirm that the reduced steady-state expression of [L166P]DJ-1 was because of accelerated protein degradation and to measure the turnover of endogenous DJ-1, protein synthesis was blocked with cycloheximide and the decay of DJ-1 proteins was monitored over a time course of 12 h. Like the endogenous DJ-1, transfected [WT]DJ-1 and [E64D]DJ-1 remained fully stable for 6 h with little further decay (Fig. 3B). In striking contrast, full-length [L166P]DJ-1 immunoreactivity completely vanished within 4 h (Fig. 3B), but the N-terminal-truncated DJ-1 species ΔN1 and ΔN2 remained stable throughout the 12-h time course. Expression of mutant DJ-1 had no significant influence on the turnover of endogenous DJ-1. Proteasomal and Autoproteolytic DJ-1 Degradation Pathways—Our results indicate that [WT]DJ-1 is a rather stable protein with relatively slow turnover. Indeed, blocking the major cellular protein degradation machinery (the proteasome) (22Glickman M.H. Ciechanover A. Physiol. Rev. 2002; 82: 373-428Crossref PubMed Scopus (3275) Google Scholar) with MG-132 did not lead to an accumulation of [WT]DJ-1 in transiently transfected HEK293 cells (Fig. 4). MG-132 treatment did not cause DJ-1 accumulation in stably transfected cells either (data not shown). Furthermore, accumulation of the endogenous DJ-1 was not found in MG-132-treated cells (Fig. 4). Likewise, the relatively stable mutant [E64D]DJ-1 did not accumulate in MG-132-treated HEK293 cells (Fig. 4). It has been proposed that the enhanced turnover of [L166P]DJ-1 was (at least in part) because of proteasomal degradation (20Miller D.W. Ahmad R. Hague S. Baptista M.J. Canet-Aviles R. McLendon C. Carter D.M. Zhu P.-P. Stadler J. Chandran J. Klinefelter G.R. Blackstone C. Cookson M.R. J. Biol. Chem. 2003; 278: 36588-36595Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). However, MG-132 treatment only minimally increased the levels of [L166P]DJ-1, although the proteasome was effectively blocked in our system as evidenced by the massive accumulation of β-catenin, a protein that is rapidly turned over in the proteasome (23Aberle H. Bauer A. Stappert J. Kispert A. Kemler R. EMBO J. 1997; 16: 3797-3804Crossref PubMed Scopus (2122) Google Scholar). Rather, the N-terminal-truncated [L166P]DJ-1 fragments ΔN1 and ΔN2 appeared to be stabilized upon proteasomal inhibition. Thus, there must exist proteolytic pathways for DJ-1 in addition to the proteasome. To study alternative, non-proteasomal DJ-1 breakdown pathways, we inhibited cellular cysteine proteases in HEK293 cells transiently transfected with DJ-1 with leupeptin. However, leupeptin treatment did not lead to significant accumulation of DJ-1 (Fig. 5A), although probing for co-transfected amyloid precursor protein revealed the expected accumulation of C-terminal stubs (not shown) because of efficient cysteine protease inhibition (24Haass C. Koo E.H. Mellon A. Hung A.Y. Selkoe D.J. Nature. 1992; 357: 500-503Crossref PubMed Scopus (767) Google Scholar). Specifically, the highly unstable [L166P]DJ-1 protein was not stabilized by inhibition of cellular cysteine proteases. Another cysteine protease inhibitor, E64, did not stabilize DJ-1 either (results not shown). To assess the autoproteolytic potential of DJ-1 more directly, we replaced the putative active-site cysteine 106 (17Honbou K. Suzuki N.N. Horiuchi M. Niki T. Taira T. Ariga H. Inagaki F. J. Biol. Chem. 2003; 278: 31380-31384Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar) with alanine by site-directed mutagenesis. When transiently transfected into HEK293 cells, the steady-state levels of [C106A]DJ-1 were not significantly elevated compared with [WT]DJ-1 (Fig. 5B). Likewise, introduction of the C106A active-site mutation did not stabilize [L166P]DJ-1. The steady-state expression levels of the double mutant [C106A/L166P]DJ-1 were as low as those of the single mutant [L166P]DJ-1 despite the strong expression of their mRNAs (Fig. 5B). Thus, the imperfectly conserved catalytic cleft around cysteine 106 of DJ-1, which is topologically equivalent to the catalytic cysteine residue of PH1704-type proteases, does not contribute to the rapid degradation of mutant [L166P]DJ-1. In other words, the fast breakdown of [L166P]DJ-1 is not because of derepressed self-digestion. Interestingly, however, the C106A mutation increased the appearance of a DJ-1 immunoreactive band that migrated at an apparent molecular mass expected for a dimer (Fig. 5B). Bacterial Expression of WT and Mutant DJ-1—A" @default.
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• W1966773864 title "Differential Effects of Parkinson's Disease-associated Mutations on Stability and Folding of DJ-1" @default.
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