Ubiquitination of α-Synuclein Is Not Required for Formation of Pathological Inclusions in α-Synucleinopathies

Sampathu, Deepak M.; Giasson, Benoit I.; Pawlyk, Aaron C.; Trojanowski, John Q.; Lee, Virginia M.‐Y.

doi:10.1016/s0002-9440(10)63633-4

Cited by 128 publications

(95 citation statements)

References 65 publications

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“…This hypothesis is supported by previous studies showing that not all α-Syn within LBs is ubiquitinated (19), as well as our own findings that monoubiquitination stabilizes monomeric α-Syn and tetraubiquitination favors the formation of nonfibrillar aggregates. Furthermore, polyubiquitination of α-Syn by the E3 ligase carboxy-terminus of Hsp70-interacting protein (CHIP) in a cell-free system and in cells results in significant reduction of the amount of insoluble α-Syn species (43) and oligomers (21).…”

Section: Discussionsupporting

confidence: 90%

Synthetic polyubiquitinated α-Synuclein reveals important insights into the roles of the ubiquitin chain in regulating its pathophysiology

Haj‐Yahya

Fauvet

Herman-Bachinsky

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

139

115

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Ubiquitination regulates, via different modes of modifications, a variety of biological processes, and aberrations in the process have been implicated in the pathogenesis of several neurodegenerative diseases. However, our ability to dissect the pathophysiological relevance of the ubiquitination code has been hampered due to the lack of methods that allow site-specific introduction of ubiquitin (Ub) chains to a specific substrate. Here, we describe chemical and semisynthetic strategies for site-specific incorporation of K48-linked di-or tetra-Ub chains onto the side chain of Lys12 of α-Synuclein (α-Syn). These advances provided unique opportunities to elucidate the role of ubiquitination and Ub chain length in regulating α-Syn stability, aggregation, phosphorylation, and clearance. In addition, we investigated the cross-talk between phosphorylation and ubiquitination, the two most common α-Syn pathological modifications identified within Lewy bodies and Parkinson disease. Our results suggest that α-Syn functions under complex regulatory mechanisms involving cross-talk among different posttranslational modifications. eukaryotes is the covalent attachment of ubiquitin (Ub) to proteins. This reversible modification, which regulates a variety of biological processes, such as protein degradation, trafficking, and DNA damage response (1, 2), involves the attachment of the C terminus of Ub mainly to the side chain of a Lys residue in a protein substrate via an isopeptide linkage. The process is catalyzed by three enzymes that act in concert: the Ub-activating enzyme (E1), the Ub-conjugating enzyme (E2), and the Ub ligase (E3). The reaction is repeated, and a second Ub is attached to an internal Lys in the previously conjugated ubiquitin. Several repeats result in the synthesis of a poly-Ub chain that can be of varying lengths and internal linkages. The presence of seven Lys residues as possible anchoring sites within Ub in addition to the N-terminal amine results in a highly complex landscape of diverse Ub bioconjugates, which accounts for the diversity of the Ub signaling (3).Research in the Ub field, which aims at understanding the ubiquitination system at the molecular level, has been hampered by the difficulties of controlling ubiquitination in the cell and challenges associated with the preparation of specific Ub conjugates in vitro. These limitations have inspired the development of novel synthetic strategies to facilitate site-specific ubiquitination of proteins (4, 5). Recent advancements in the field have enabled the synthesis of relatively large amounts of highly complex Ub conjugates of defined covalent structure and provided novel insights into the structural, biochemical, and functional consequences of protein ubiquitination, along with unique opportunities to elucidate the molecular basis of Ub signaling. For example, monoubiquitinated α-Synuclein (α-Syn) and histone H2B bearing native isopeptide bonds were prepared and used to shed light on the role of monoubiquitination in regulating α-Syn aggregation a...

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Section: Discussionsupporting

confidence: 90%

Synthetic polyubiquitinated α-Synuclein reveals important insights into the roles of the ubiquitin chain in regulating its pathophysiology

Haj‐Yahya

Fauvet

Herman-Bachinsky

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

139

115

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“…These observations support the idea that oligoubiquitination occurs after SOD1 aggregation, as a secondary event, not necessary for aggregate formation. A similar mechanism has been proposed for ␣-synuclein, a major component of pathological inclusions in patients with Parkinson and Lewy body diseases, also found as mono-and oligoubiquitinated, but principally as nonubiquitinated (37)(38)(39). This is in agreement also with the work by Gilchrist et al (40), in which were observed ubiquitin-positive lesions appearing 30 days later than SOD1-positive inclusions in the spinal cords of SOD1 G93A mice crossed with epitopetagged ubiquitins.…”

Section: Microns (A-d F-i and N-q) And 20 Microns (E L M R And S)supporting

confidence: 91%

Insoluble Mutant SOD1 Is Partly Oligoubiquitinated in Amyotrophic Lateral Sclerosis Mice

Basso

Massignan

Giuseppina

et al. 2006

Journal of Biological Chemistry

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Mutations in the Cu,Zn-superoxide dismutase (SOD1) gene cause a familial form of amyotrophic lateral sclerosis (ALS) through an unknown gain-of-function mechanism. Mutant SOD1 aggregation may be the toxic property. In fact, proteinaceous inclusions rich in mutant SOD1 have been found in tissues from the familial form of ALS patients and in mutant SOD1 animals, before disease onset. However, very little is known of the constituents and mechanism of formation of aggregates in ALS. We and others have shown that there is a progressive accumulation of detergent-insoluble mutant SOD1 in the spinal cord of G93A SOD1 mice. To investigate the mechanism of SOD1 aggregation, we characterized by proteome technologies SOD1 isoforms in a Triton X-100-insoluble fraction of spinal cord from G93A SOD1 mice at different stages of the disease. This showed that at symptomatic stages of the disease, part of the insoluble SOD1 is unambiguously mono-and oligoubiquitinated, in spinal cord and not in hippocampus, and that ubiquitin branches at Lys 48 , the major signal for proteasome degradation. At presymptomatic stages of the disease, only insoluble unmodified SOD1 is recovered. Partial ubiquitination of SOD1-rich inclusions was also confirmed by immunohistochemical and electron microscopy analysis of lumbar spinal cord sections from symptomatic G93A SOD1 mice. On the basis of these results, we propose that ubiquitination occurs only after SOD1 aggregation and that oligoubiquitination may underline alternative mechanisms in disease pathogenesis.

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“…As to the question whether ubiquitination is crucial to inclusion body formation, a significant percentage of ␣-synuclein-containing inclusion bodies in human disease are ubiquitin negative, raising doubts about the necessity of ubiquitination for protein sequestration into Lewy body-like structures and fueling speculations that proteins other than ␣-synuclein might actually be ubiquitinated in Lewy bodies (Spillantini et al, 1998;Gomez-Tortosa et al, 2000). There has been significant (albeit indirect) evidence that mono-and di-ubiquitinated species of ␣-synuclein are present in the insoluble fraction of brain extracts from patients with Lewy body dementia (Hasegawa et al, 2002;Sampathu et al, 2003). In this study, we provide direct evidence that ␣-synuclein itself is ubiquitinated in a mouse model of synucleinopathy.…”

Section: Discussionmentioning

confidence: 99%

Inclusion Body Formation and Neurodegeneration Are Parkin Independent in a Mouse Model of α-Synucleinopathy

Coelln¹,

Thomas²,

Andrabi³

et al. 2006

J. Neurosci.

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Mutations in the genes coding for ␣-synuclein and parkin cause autosomal-dominant and autosomal-recessive forms of Parkinson's disease (PD), respectively. ␣-Synuclein is a major component of Lewy bodies, the proteinaceous cytoplasmic inclusions that are the pathological hallmark of idiopathic PD. Lewy bodies appear to be absent in cases of familial PD associated with mutated forms of parkin. Parkin is an ubiquitin E3 ligase, and it may be involved in the processing and/or degradation of ␣-synuclein, as well as in the formation of Lewy bodies. Here we report the behavioral, biochemical, and histochemical characterization of double-mutant mice overexpressing mutant human A53T ␣-synuclein on a parkin null background. We find that the absence of parkin does not have an impact on the onset and progression of the lethal phenotype induced by overexpression of human A53T ␣-synuclein. Furthermore, all major behavioral, biochemical, and morphological characteristics of A53T ␣-synuclein-overexpressing mice are not altered in parkin null ␣-synucleinoverexpressing double-mutant mice. Our results demonstrate that mutant ␣-synuclein induces neurodegeneration independent of parkin-mediated ubiquitin E3 ligase activity in nondopaminergic systems and suggest that PD caused by ␣-synuclein and parkin mutations may occur via independent mechanisms.

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Ubiquitination of α-Synuclein Is Not Required for Formation of Pathological Inclusions in α-Synucleinopathies

Cited by 128 publications

References 65 publications

Synthetic polyubiquitinated α-Synuclein reveals important insights into the roles of the ubiquitin chain in regulating its pathophysiology

Synthetic polyubiquitinated α-Synuclein reveals important insights into the roles of the ubiquitin chain in regulating its pathophysiology

Insoluble Mutant SOD1 Is Partly Oligoubiquitinated in Amyotrophic Lateral Sclerosis Mice

Inclusion Body Formation and Neurodegeneration Are Parkin Independent in a Mouse Model of α-Synucleinopathy

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