Proteasome inhibition induces α-synuclein SUMOylation and aggregate formation
Introduction
Parkinson's disease (PD) and Dementia with Lewy Bodies (DLB) are progressive neurodegenerative disorders characterized by the presence of cytoplasmic inclusions in surviving neurons called Lewy bodies (LBs) and Lewy neurites [1]. A major component of these inclusions is the protein α-synuclein which accumulates as insoluble fibrillar aggregates. Multiple factors promote the aggregation of this natively unfolded protein [2], [3], [4], [5] including post-translational modifications. These include phosphorylation [6], [7], nitration [8], [9] and ubiquitination [10], [11]. These modifications affect the ability of α-synuclein to form aggregates and its sub-cellular localization.
The intracellular 26S proteasome is the major proteolytic complex responsible for ubiquitin-mediated substrate degradation in eukaryotic cells. Impaired proteasome activity is implicated in the pathogenesis of several neurodegenerative disorders including PD [12]. Postmortem analyses of brains from sporadic PD patients have demonstrated relatively low proteasomal activity in the substantia nigra, which bears the brunt of pathology in this disease [13], [14], [15]. The origin of proteasomal dysfunction in these neurodegenerative disorders remains largely unknown.
Small ubiquitin-like modifier (SUMO) belongs to a family of ubiquitin-related proteins and is covalently conjugated to lysine residues in its substrates. SUMOylation occurs through a series of enzymatic reactions analogous to that of the ubiquitination pathway, and this process leads to the modification of the biochemical and functional properties of substrates [16]. To date, four mammalian SUMO isoforms, a single heterodimeric SUMO-activating E1 enzyme SAE1/SAE2, a single SUMO-conjugating E2 enzyme UBE2I (also known as Ubc9 in yeast), and several families of SUMO E3 ligases have been identified [16].
The implications of SUMOylation have been studied in relation to the pathogenesis of several neurodegenerative disorders [17], [18], [19], [20], [21], [22], [23]. Although α-synuclein has been reported to be SUMOylated in cultured cells [24], the contribution of this posttranslational modification to the pathological picture of α-synucleinopathies was not established. Here, we provide evidence that impaired proteasomal function significantly promotes the formation of SUMOylated α-synuclein containing aggregates in aggresome-like structures in cultured cells as well as in the LBs of PD and DLB affected brains.
Section snippets
Chemicals and antibodies
MG-132 was purchased from EMD Chemicals (Gibbstown, NJ, USA). The mouse monoclonal anti-α-synuclein (Syn-1) antibody was purchased from BD Biosciences (San Jose, CA, USA), and mouse monoclonal anti-SUMO1 (GMP-1) from Zymed Laboratories (San Francisco, CA, USA). Rabbit polyclonal anti-SUMO1, anti-ubiquitin, and anti-HA antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Invitrogen
α-Synuclein co-localizes with SUMO1 in aggresome-like structures and in LBs
To gain insight into the physiological relevance of α-synuclein SUMOylation, we investigated whether SUMO modification is closely associated with the formation of α-synuclein aggregates. After co-transfection with plasmids encoding α-synuclein and HA-SUMO1-GG, COS-7 cells were treated with the non-specific proteasome inhibitor MG-132. Immunofluorescence staining with antibodies specific to α-synuclein and HA revealed that MG-132 treatment promoted the formation of α-synuclein- and
Discussion
Accumulating evidence suggests that posttranslational modifications of α-synuclein are important regulators of its pathogenetic role in neurodegenerative diseases. For example, proteasomal impairment leads to the accumulation of ubiquitinated α-synuclein [11], and phosphorylation promotes its aggregation [7], [29]. Here we demonstrate that insoluble α-synuclein aggregates are also SUMOylated both in cultured cells subjected to proteasomal impairment in the form of perinuclear aggresomes as well
Acknowledgements
This study was supported in part by the US NIH intramural program (Z01NS002826) to M. M. M. who is currently the William Dow Lovett Professor of Neurology.
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Current address: Department of Biochemistry, College of Medicine, Inje University, Busan 614-735, Republic of Korea.