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* Center of Neurology and Hertie-Institute for Clinical Brain Research, University of Tübingen, Tübingen, Germany;
Department of Medical Genetics, University of Tübingen, Tübingen, Germany;
Department of Pharmacology, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel;
Core Unit Chip Application, Institute of Human Genetics and Anthropology, Friedrich-Schiller-University, Jena, Germany;
|| Department of Pathology, Neuropathologic Institute, University Hospital Zürich, Zürich, Switzerland; and
¶ School of Biomedical Sciences, University of Nottingham, Nottingham, UK
2Correspondence: Laboratory of Functional Neurogenomics, Center of Neurology and Hertie-Institute for Clinical Brain Research, Hoppe-Seyler-Str. 3, 72076 Tübingen, Germany. E-mail: rejko.krueger{at}uni-tuebingen.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: neurodegeneration • ubiquitin • alpha-synuclein • proteasome
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Ubiquitin-mediated protein degradation involves a sequential addition of ubiquitin residues to selected proteins leading to the formation of polyubiquitin chains as a prerequisite for recognition of the protein by the 26S proteasome (2)
. The ubiquitination of proteins is mediated by the activation of monoubiquitin via an ubiquitin-activating enzyme (E1). The activated ubiquitin is transferred to an ubiquitin-conjugating enzyme (E2). Subsequent linkage of the ubiquitin residue to the respective protein is conferred by an ubiquitin-ligase (E3). In humans a wide variety of E3 enzymes are known that mediate ubiquitination of a single or a very limited number of substrates contributing to the specificity of the UPS. Nonubiquitinated proteins may be degraded via the 20S proteasome, whereas polyubiquitinated proteins are degraded via the 26S proteasome, that is characterized by the 20S particle and an additional 19S cap particle (3)
. The latter consists of 18 different subunits and is supposed to mediate ubiquitin-dependent proteasomal function.
Disturbance of the UPS in PD is reflected by the presence of proteins known to be linked to PD in characteristic protein aggregates in brains of familial and/or sporadic PD patients, known as Lewy bodies (4
5
6)
. Indeed even in sporadic PD patients, in which no disease-causing mutation was identified, proteasomal dysfunction involving proteolytic stress has been described (7
, 8)
. Synphilin-1 links two proteins involved in PD, alpha-synuclein and Parkin, suggesting a common pathogenic mechanism (9
, 10)
. We identified a novel R621C mutation in the synphilin-1 gene in two apparently sporadic PD patients that mediates toxicity in different paradigms of cellular stress in vitro (11)
. Thus, in order to further elucidate possible signaling pathways of synphilin-1, we decided to screen for novel synphilin-1-interacting proteins. A first direct link between the proteasome and proteins involved in PD pathogenesis has been established by the interaction between alpha-synuclein and S6' proteasomal protein in vitro (12)
. Structural analysis of relevant domains of the synphilin-1 protein revealed six ankyrin-like repeats, a motif that has been implicated in mediating interaction with the proteasomal subunits (13)
. Therefore, we performed coimmunoprecipitation assays of synphilin-1 with different proteasomal subunits. Using a candidate approach, we report for the first time the specific interaction between synphilin-1 and the 19S proteasomal protein S6 ATPase and functionally characterize this interaction in cell culture and in postmortem PD brains.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 13)
. FLAG-tagged R621 or C621 synphilin-1 constructs were subcloned from pAd-Track-CMV-FLAG-R621/C621 synphilin-1 vector into XhoI and HindIII sites of the pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA). Full-length cDNA of tbp1 was cloned into pET16b vector (Novagen, Berlin, Germany) between XhoI and Bpu1102I sites to generate pET16b-tbp1. Tbp1 was subcloned from pET16b vector, including the his-tag into XbaI and HindIII sites of the pcDNA3.1 vector (Invitrogen).
Cell culture, transfection, immunoprecipitation and pull-down assay
HEK293 cells were cultured in a 5% CO2 humidified atmosphere in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) containing penicillin and streptomycin (PAN, Hamburg, Germany) and 10% fetal bovine serum (FBS; Biochrom, Berlin, Germany). HEK293 cells were transiently transfected with 2 µg of each vector according to the FuGENE 6 transfection protocol (Roche Diagnostics, Mannheim, Germany). Forty-eight hours after transfection, cells were washed with cold phosphate-buffered saline (PBS; Invitrogen) and harvested in PBS containing 1% (v/v) Triton X-100 and Complete protease inhibitor (Roche, Germany). Precipitation was carried out using anti-FLAG M2 affinity gel (Sigma, Munich, Germany), c-myc monoclonal-antibody-agarose beads (Clontech, USA) or TALON metal affinity resin (Clontech, Palo Alto, CA, USA). The precipitates were resolved on SDS-PAGE gel and subjected to Western blot analysis using a rabbit anti-FLAG polyclonal antibody (Sigma), a rabbit anti-HA polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), a mouse antimyc monoclonal antibody (Santa Cruz Biotechnology), and a rabbit anti S6' ATPase polyclonal antibody (Affiniti, Buckinghamshire, UK).
To generate polyclonal stable cell lines, 500,000 HEK293 cells were transfected with 2 µg FLAG-tagged wt synphilin-1 in pDNA3.1 and subsequently selected using medium containing G418 (0.7 mg/ml).
Surface-enhanced laser desorption ionization-mass spectrometry (SELDI-MS)
For surface-enhanced laser desorption ionization-mass spectrometry (SELDI-MS) analyses, HEK293 cells were transiently transfected with 2 µg of FLAG-tagged wt synphilin in pcDNA3.1 using lipofectamine (Invitrogen). The protein-protein interaction assay and coupling of unspecific antibodies to Interaction Discovery Mapping (IDM) beads (Ciphergen Biosystems, Fremont, CA, USA) as a negative control were performed as described by Lehmann et al. (14)
. Agarose-conjugated FLAG antibodies were purchased from Sigma (Germany) and used to specifically immunoprecipitate FLAG-tagged synphilin-1 from 130 µl of cell extract, as described in the Immunoprecipitation section. Bound proteins were eluted from both the IDM beads and the agarose-conjugated FLAG antibodies with 10 µl 50% acetonitrile/0.5% trifluoroacetic acid and gently vortexed for 5 min. Five microliters of the eluted samples were applied to the activated reverse-phase surface of an NP20 ProteinChip array (Ciphergen Biosystems) and dried on air. After washing with 3 µl aqua bidest, 0.5 µl of sinapinic acid (saturated solution in 0.5% TFA/50% acetonitrile) were applied twice, and the array was analyzed in a ProteinChip reader series 4000 mass spectrometer (Ciphergen) according to an automated data collection protocol.
For the analysis of fragment masses, proteins eluted from IDM beads or agarose-conjugated FLAG antibodies underwent tryptic digestion, as described previously (14)
. The digest products were spotted on NP20 arrays and the size of the obtained fragments was determined by the ProteinChip reader series 4000 instrument. In parallel, a theoretical tryptic digestion of S6 ATPase was carried out using a public database (Expasy PeptideMass tool; http://us.expasy.org/tools/peptide-mass.html). Subsequently, theoretical S6 ATPase peptide fragments were marked in the spectra. Special attention was paid to assure that these peaks were not present in an approach using nonspecific antibodies. The S6 ATPase associated peaks were used to confirm the identity of the S6 ATPase protein by searching a publicly available database (http://129.85.19.192/profound_bin/WebProFound.exe).
Proteasomal activity assay
To determine chymotryptic proteasomal activity of transfected HEK293 cells, total cellular extracts were prepared 48 h after transfection using lysis buffer (PBS, 1% (v/v) Triton X-100). Total protein concentrations of the cell lysates were determined using Bio-Rad protein assay kit (Bio-Rad, Munich, Germany). Twenty microliters of total cellular extract were incubated at 37°C in assay buffer (30 mM Tris-HCl, pH 7.6, 2 mM MgCl2, 10 mM NaCl, 10 mM KCl, 0.5 mM DTT, 1 mM ATP) containing 100 µM of the fluorogenic substrate Suc-LLVY-AMC (Sigma). Samples were analyzed using an excitation wavelength of 360 nm and an emission wavelength of 450 nm with a Spectrafluor fluorescence spectrophotometer (Tecan, Crailsheim, Germany) after different time points (30, 60, 90, 120, and 150 min). This method allows the determination of the chymotryptic proteasomal activity, including 26S activity due to the presence of ATP in the reaction buffer.
Cell viability assay
Analysis of cellular toxicity induced by coexpression of synphilin-1 and S6 compared to toxicity of synphilin-1 expression alone in living cells was performed by a fluorescent-activated cell sorting (FACS)-based method. Stable polyclonal HEK293 cells overexpressing wt synphilin-1 were transfected with 2 µg of pCMV5L-S6 or empty vector (pcCMV-Tag4A). 48 h after transfection cells were analyzed by flow cytometry on a CyAnTM ADP apparatus (DakoCytomation, Carpinteria, CA, USA) using Yo-Pro-1 (Invitrogen). Therefore 24 h after transfection, cells were treated with 5 µM MG132 (Sigma) in a humidified 5% CO2 atmosphere at 37°C for 24 h. Cells were harvested with trypsin, pelleted, and washed in PBS. Cells were incubated in PBS, containing 100 nM Yo-Pro-1 (Invitrogen) for 30 min on ice, followed by three wash steps with cold PBS. For each sample, 100,000 cells were analyzed for Yo-Pro1 fluorescence on on a CyAnTM ADP apparatus (DakoCytomation) with a 488-nm argon laser. We counted more than 50,000 cells for each group, experiments were performed three times with similar results. To confirm efficient transfection and expression of S6 ATPase a subset of the cells, which were analyzed for viability, were lysed and subjected to Western blot analysis with antibodies against S6.
Steady state levels
HEK293 cells were transiently transfected with 2 µg of pAd-Track-CMV-FLAG-R621/C621 synphilin-1 and increasing amounts of pCMV5L-S6 (0, 1, or 2 µg). Empty pcDNA3.1 vector (Invitrogen) was added to each transfection experiment to achieve equal amounts of DNA. Forty eight hours after transfection, total cellular extracts were prepared using lysis buffer (PBS, 1% (v/v) Triton X-100 and complete protease inhibitor; Roche). Total protein concentrations of the cell lysates were determined using Bio-Rad protein assay kit (Bio-Rad). Equal amounts of protein (30 µg per lane) were resolved on SDS–PAGE gel and subjected to Western blot analysis using a mouse anti-FLAG polyclonal antibody (Sigma) and a rabbit anti-HA polyclonal antibody (Santa Cruz Biotechnology). (EGFP protein, which is coexpressed by pAd-Track-CMV-FLAG synphilin-1 vector, was detected by a rabbit anti-GFP polyclonal antibody (Santa Cruz Biotechnology) to show equal efficiency of transfection.
Immunocytochemistry and immunohistochemistry
For immunocytochemistry HEK293 were grown on poly-L-Lysine coated slides and cotransfected with a combination of either pcDNA3.1-FLAG-R621 synphilin-1 and pCMV5L-S6 or pcDNA3.1-FLAG-C621 synphilin-1 and pCMV5L-S6. Forty eight hours after transfection, cells were washed with PBS, fixed with 4% (w/v) PFA for 10 min, and permeabilized with 0.1% (v/v) Triton X-100 for 10 min. After washing with PBS, cells were incubated for 30 min in 10% (v/v) FCS in PBS for blocking. They were then labeled overnight at 4°C with a combination of either a mouse monoclonal anti-FLAG (Stratagene, La Jolla, CA, USA) and a rabbit polyclonal anti-human S6 (Affiniti) primary antibody diluted 1:300 in PBS containing 5% (v/v) FCS or a rabbit polyclonal anti-synphilin-1 (raised against amino acids 696–710) diluted 1:300 and a mouse monoclonal anti-gamma tubulin (Sigma) primary antibody diluted 1:5.000 in PBS containing 5% (v/v) FCS. For lysosomal staining, cells were incubated for 2 h at 37°C with polyclonal mouse 
-LAMP-1 antibody diluted 1:1000 in PBS containing 5% (v/v) FCS.
At the next day, cells were washed with PBS and labeled with a combination of either Cy3-conjugated anti-mouse and FITC-conjugated anti-rabbit or Cy3-conjugated anti-rabbit and FITC-conjugated anti-mouse secondary antibody (Dianova, Hamburg, Germany). Cell nuclei were stained with the fluorescent chromatin dye Hoechst 33258 (Molecular Probes, Eugene, OR, USA).
Immunohistochemistry was carried out on formalin-fixed, paraffin-embedded 8-µm-thick sections of substantia nigra of three patients with clinically and histopathologically proven PD using the avidin-biotin-immunoperoxidase technique. Sections were deparaffinized and microwaved for 15 min at 400 W. Endogenous peroxidase activity was blocked by incubating the sections with 2.4% (v/v) H2O2 in bidistilled water for 15 min. After rising in PBS, sections were brought into 10% (v/v) goat serum and 2% (w/v) bovine serum albumin (Biogenex, San Ramon, CA, USA) for 20 min at room temperature. Sections were then incubated with the rabbit anti-human S6 antibody (Affiniti), diluted 1:100 in PBS containing 0.1% (w/v) BSA overnight at 4°C. The next day, sections were washed in PBS, incubated in biotinylated goat anti-rabbit IgG diluted in PBS (StrAviGen multi-Link Kit, Biogenex) for 30 min at room temperature, rinsed again in PBS and then incubated for 30 min at room temperature in peroxidase-conjugated streptavidin in PBS (StrAviGen multi-Link Kit, Biogenex). After rinsing again in PBS, we carried out the enzymatic reaction for 2 min at room temperature with a solution containing 4% (w/v) 3-amino-9-ethylcarbazole (ACE) and H2O2 substrate buffer (Biogenex). Subsequently, sections were counterstained with hematoxylin. To confirm specificity of the immunostaining, the S6 antibody was omitted in control sections.
Quantitative analysis of synphilin-1 positive inclusions
We performed cotransfection of pEGFPN1-synphilin-1 with pCMV5L-S6 or pcDNA3.1-myc6 vector in HEK 293 cells. Forty eight hours after transfection, cells were examined for EGFP fluorescence without fixation on an inverted fluorescence microscope (DM IRBE, Leica, Leipzig, Germany). For quantitative analysis of protein inclusions observed in cells overexpressing synphilin-1 alone or with S6 ATPase, more than 300 cells in randomly selected microscopic fields were counted by an investigator blinded to the transfection in each of three independent cultures. Each experiment was replicated twice with similar results.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A). We recently identified a R621C mutation in the synphilin-1 gene as a susceptibility factor in PD. This mutation localizes to the fifth ankyrin repeat (aa 603–632) outside the central region known to mediate interaction with Parkin and alpha-synuclein. No differential interaction with S6 ATPase was observed comparing wt and C621 synphilin-1 (Fig. 1A
). We defined the interaction between S6 ATPase and synphilin-1 as specific, as no interaction with S6' ATPase (tbp1), another 19S proteasomal subunit, was found using the coimmunoprecipitation technique (Fig. 1B
).
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As a next step we investigated, whether the proteasomal subunit protein S6 interacts with the synphilin-1 interacting protein alpha-synuclein. The latter has been previously shown to interact with the proteasomal subunit S6' ATPase (tbp1). Lysates prepared from HEK293 cells cotransfected with myc-tagged alpha-synuclein (wt or A30P) and HA-tagged S6 ATPase were subjected to IP with antimyc and subsequently immunoblotted with anti-HA antibodies. The proteasomal subunit protein S6 ATPase could neither be coimmunoprecipitated with wt nor with A30P mutant alpha-synuclein (Fig. 1C
).
To confirm the interaction between synphilin-1 and S6 ATPase with an independent method and to define an interaction of synphilin-1 with the endogenous S6 ATPase protein, we subsequently performed a protein-protein interaction assay, applying SELDI-MS ProteinChip technology. For SELDI analyses, protein lysates from HEK cells transiently transfected with FLAG-tagged wt synphilin-1 were prepared. Agarose-conjugated FLAG antibodies were used to specifically immunoprecipitate FLAG-tagged synphilin-1. As a negative control, nonspecific antibodies coupled to IDM beads were used in parallel. Bound proteins were eluted and digested with trypsin. The respective digest products were spotted on NP20 ProteinChip arrays, and the size of the obtained fragments was determined by the ProteinChip reader series 4000 instrument. Subsequently, theoretical tryptic S6 ATPase peptide fragments were marked in the spectra (Fig. 2
). Database searches (Profound; http://129.85.19.192/profound _bin/WebProFound.exe) using the identified S6 ATPase fragments revealed endogenous S6 ATPase as an interactor of synphilin-1 (estimated Z score 1.09). The fragments covered 31% of the S6 ATPase protein.
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Colocalization of synphilin-1 with the proteasomal subunit protein S6 ATPase
To characterize the observed interaction of synphilin-1 and S6 ATPase in cell culture, we cotransfected HEK293 cells with a combination of pcDNA3.1-FLAG-R621-synphilin-1 and pCMV5L-S6. Forty eight hours after transfection, cells were double-labeled with a combination of anti-FLAG and anti-human S6 ATPase antibodies and analyzed with an Axioplan 2 microscope (Zeiss, Oberkochen, Germany). We and others found a spontaneous inclusion forming propensity of synphilin-1 on overexpression in neuronal and nonneuronal cell lines, that could be increased in number and size by cellular stress, i.e., treatment with the proteasomal inhibitors MG132 (11
, 16
, 17)
. We identified S6 ATPase as a component of synphilin-1-positive cytoplasmic inclusions (Fig. 3
A). Synphilin-1 inclusions fulfilled the criteria of aggresomes due to perinuclear localization and positive staining for tubulin (15; Fig. 3B
). Colocalization was not restricted to overexpression of recombinant S6 ATPase, because endogenous S6 ATPase was also sequestrated into synphilin-1 inclusions in HEK293 cells overexpressing wt or C621 mutant synphilin-1 alone (data not shown).
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Effects of coexpression of S6 ATPase and synphilin-1 on inclusion formation
To study a possible functional relevance of the observed interaction and colocalization of S6 ATPase with synphilin-1 in HEK293 cells, we performed cotransfection of pEGFPN1-synphilin-1 with pCMV5L-S6 or pcDNA3.1-myc6 vector. Quantitative analysis showed that cells expressing both synphilin-1-EGFP and S6 ATPase produced significantly more synphilin-1-positive inclusions than cells expressing synphilin-1-EGFP and myc6 as a control protein (Fig. 3C
). The same was true for cells coexpressing R621C mutant synphilin-1-EGFP and S6 ATPase (data not shown). These results were specific for S6 ATPase interaction with synphilin-1, because we found that cotransfection of synphilin-1 with another interacting protein, alpha-synuclein, did not result in increased inclusion formation (11)
.
Effects of S6 ATPase expression on cell viability in synphilin-1 overexpressing cells
In previous studies, we and others found an increased susceptibility of synphilin-1-overexpressing cells in terms of proteasomal stress (11
, 17)
. To test a potential effect of S6 ATPase coexpression on cell viability in HEK293 cells stably overexpressing physiological synphilin-1, we determined the proportion of apoptotic cells under established conditions of cellular stress by FACS analysis. After treatment with 5 µM MG132 for 24 h, cells expressing synphilin-1 alone were consistently more susceptible to toxic insult with an increased number of cell death of 41.8% (SD±5.21) compared to 30.2% (SD±10.77) cells, expressing synphilin-1 together with S6 ATPase (Fig. 3D
).
Effects of coexpression of S6 ATPase and synphilin-1 on proteasomal function and protein levels
S6 ATPase is an integral component of the 19S subunit of the 26S proteasome. Therefore, we speculated that coexpression of synphilin-1 and S6 might affect proteasomal function in HEK293 cells. To monitor proteasomal activity, we decided to analyze chymotryptic activity of the proteasome using Suc-LLVY-AMC as a substrate. Cells overexpressing both, S6 ATPase and synphilin-1, for 48 h displayed a significantly reduced proteasomal activity compared to cells expressing synphilin-1 and myc6 protein as control (Fig. 4
A). Overexpression of S6 ATPase alone did not decrease chymotryptic proteasomal function, whereas overexpression of C621 mutant synphilin-1 alone caused a decrease in proteasomal function that was not affected by cotransfection with S6 ATPase (data not shown). We then monitored the levels of synphilin-1 protein in different experiments with increasing amounts of coexpressed S6 ATPase. However, we found no evidence for alterations in the synphilin-1 steady state (Fig. 4B
). Recent studies indicate a potential involvement of lysosomal degradation pathways in the removal of aggresomes from the cell. To study an involvement of lysosomal degradation in the control of synphilin-1 protein levels we followed the subcellular localization of lysosomal structures and synphilin-1 after overexpression of FLAG-tagged synphilin-1 in HEK293 cells. We found colocalization of the lysosomal protein LAMP-1 with a subset of synphilin-1-positive inclusions (Fig. 4C
). Our results might indicate a way to dissolve and/or degrade intracytoplasmic synphilin-1 inclusions in HEK293 cells.
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Synphilin-1 interacting S6 ATPase is a component of Lewy bodies in brains of PD patients
Subsequently, we focused on the potential pathogenic relevance of the observed interaction of synphilin-1 with the proteasomal protein S6. In analogy to the situation in our cellular models of synphilin-1 aggregation in vitro, we identified the proteasomal subunit S6 as a component of Lewy bodies in brains of sporadic PD patients (Fig. 5
). The staining was positive in 
25% of Lewy bodies and predominantly present in the halo, indicating a similar distribution as observed for alpha-synuclein (18)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Indeed functional and structural deficits at different steps of the ubiquitin-mediated protein degradation pathway have been reported in patients with idiopathic PD (7)
. Our finding of physical interaction of synphilin-1 with S6 ATPase directly links its physiological function to the protein surveillance machinery. This association is defined by immunoprecipitation, colocalization studies in vitro, and by the presence of S6 in Lewy bodies in brains of idiopathic PD patients. Recently, a first direct link between proteins involved in PD and proteasomal subunits has been established by the interaction of alpha-synuclein with the proteasomal subunit S6' ATPase (tbp1; 12). S6 ATPase and S6' ATPase belong to the AAA ATPase superfamily that exhibit chaperone-like activity and are involved in diverse events, including 26S proteasomal functions, peroxisomal biogenesis, membrane docking, or fusion and transcription factor regulation (19
20
21
22)
. Degradation of misfolded proteins by the 26S proteasome is ATP dependent and requires unfolding and translocation of the substrates by six AAA ATPases present in the 19S particle, including S6 ATPase (23)
. Direct interactions of disease-related proteins with components of the 19S proteasome have been described also in other neurodegenerative disorders, including spinocerebellar ataxia (SCA) 7 (24)
. Ataxin-7 interacts with the ATPase subunit S4 of the proteasomal 19S regulatory complex and colocalizes to nuclear foci (24)
. Moreover, loss of function mutations in a novel AAA ATPase, spastin, cause neurodegeneration in autosomal dominant hereditary spastic paraplegia (25)
. Also, ataxin-3, which is mutated in SCA3, is a proteasome-associated factor (26
, 27)
. Together, these observations support the concept of abnormalities in either recognition or degradation of ubiquitinated proteins contributing to neurodegeneration.
Protein accumulation and aggregation is a common feature in many neurodegenerative disorders (28)
. As already shown for synphilin-1 (6)
, we defined S6 ATP-ase as a novel component of Lewy bodies in PD. In cellular models of neurodegenerative diseases, equivalents of characteristic protein aggregations found in brains of patients, i.e., Lewy bodies in PD, are qualified as aggresomes (15
, 29)
. Overexpression of synphilin-1 alone is sufficient to form inclusions in transfected cells in vitro that correspond to aggresomes (11
, 16
, 17
, 30)
. We show that inclusions formed by coexpression of synphilin-1 and S6 ATPase also fulfill criteria of aggresomes. We observe increased aggresome formation after cotransfection of synphilin-1 and S6 ATPase and find that this is linked to decreased proteasomal function. To date, it is not clear whether aggresomes cause or are reactive to proteasomal dysfunction (30)
. In previous studies, we and others identified a dissociation of aggresome formation and cell death overexpressing wt or R621C mutant synphilin-1 in human neuroblastoma and HEK293 cells (11
, 30)
. Thus we speculate that not the inclusion itself, but a possible toxic intermediate, might be responsible for increased susceptibility of cells toward different paradigms of cellular stress and the reduced proteasomal activity of mutant compared to wt synphilin-1 observed in the present study. This is in line with existing hypotheses on neurodegeneration that postulate that not the aggregated forms of mutant proteins, but misfolded oligomeric structures, might be involved in proteasomal dysfunction and subsequent neurodegeneration (reviewed in 31). In this context, the increased aggresome formation observed after coexpression of synphilin-1 with S6 ATPase might represent a cellular mechanism to sequester or more efficiently detoxify accumulating proteins. Indeed, the observed trend for increased cell viability under stress conditions in HEK293 cells overexpressing synphilin-1 together with S6 compared to synphilin-1 alone clearly argues against a toxic role of these inclusions.
Recently, an important alternative to K48-linked polyubiquitination that determines proteins for degradation via the 26S proteasome has been described for synphilin-1. It was shown that synphilin-1 is normally ubiquitinated by Parkin in a way that involves K63-linked polyubiquitin chain formation and only at high Parkin levels via K48-linked polyubiquitination (32)
. The K63-linked polyubiquitination is not typically associated with proteasomal degradation, but—in contrast—may interfere with proteasomal function and favor the aggregation of the respective substrate (33)
. Indeed K63-linked ubiquitination of synphilin-1 and alpha-synuclein was found to promote aggresome formation in cellular models of PD (32
, 34)
.
Therefore, we speculate that S6 ATPase, in addition to its suggested activity in proteasomal degradation, may propagate inclusion formation of synphilin-1 to sequestrate proteins that cannot be directed to the proteasome. These inclusions, in turn, might interfere with proteasomal function via 1) disturbance of K48-linked polyubiquitination or 2) sequestration of functional proteasomal subunits.
The fact that decreased proteasomal activity and increased aggresome formation in cells coexpressing synphilin-1 and S6 was not related to increased protein levels of synphilin-1, a protein known to be directed to the UPS, might indicate alternative degradation pathways mediated by aggresomes. Interestingly, in cellular models of other neurodegenerative disorders an association of aggresomes with accelerated turnover of misfolded proteins has been described involving multiple degradation pathways, including the lysosomal system (35)
. In previous studies, aggresomes were found to associate with lysosomal structures, implicating autophagy as a possible way of removal of accumulating proteins (36)
. Interestingly, it has been speculated, that diversion of proteins from the proteasome to alternative degradation pathways may be mediated by K63-linked ubiquitination (37)
. Indeed our findings that inclusions formed by overexpression of synphilin-1 and S6 colocalize in part with lysosomal markers underscore a potential lysosomal clearance of the respective protein inclusions and might explain the observed protein levels of synphilin-1.
In summary, our results further support a beneficial role of inclusion formation for the homeostasis of the cell. In brains of PD patients, aggresome-like structures have been documented that are rich in proteasomal proteins (7
, 8)
. Since these structures were found in brain regions that are relatively spared from pathogenic alterations, including neuronal loss, i.e., ventral tegmental area, and dorsal midbrain, it has been suggested, that aggresomes might be a response toward an unphysiological increase in certain proteins aiming for better clearance.
Data on a direct interaction between alpha-synuclein and synphilin-1 with regulatory components of the proteasome establish a novel direct link between the proteolytic system and pathogenesis of PD. On the basis of these interactions between regulatory proteasomal proteins and due to the presence of S6 ATPase and other proteasomal components in pathognomonic Lewy bodies in brains of PD patients, genetic analyses of components of the 19S proteasome in PD patients are highly warranted.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication July 16, 2006. Accepted for publication January 18, 2007.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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