Ruth Rott

Monoubiquitylation of α-Synuclein by Seven in Absentia Homolog (SIAH) Promotes Its Aggregation in Dopaminergic Cells

α-Synuclein plays a major role in Parkinson disease. Unraveling the mechanisms of α-synuclein aggregation is essential to understand the formation of Lewy bodies and their involvement in dopaminergic cell death. α-Synuclein is ubiquitylated in Lewy bodies, but the role of α-synuclein ubiquitylation has been mysterious. We now report that the ubiquitin-protein isopeptide ligase seven in absentia homolog (SIAH) directly interacts with and monoubiquitylates α-synuclein and promotes its aggregation in vitro and in vivo, which is toxic to cells. Mass spectrometry analysis demonstrates that SIAH monoubiquitylates α-synuclein at lysines 12, 21, and 23, which were previously shown to be ubiquitylated in Lewy bodies. SIAH ubiquitylates lysines 10, 34, 43, and 96 as well. Suppression of SIAH expression by short hairpin RNA to SIAH-1 and SIAH-2 abolished α-synuclein monoubiquitylation in dopaminergic cells, indicating that endogenous SIAH ubiquitylates α-synuclein. Moreover, SIAH co-immunoprecipitated with α-synuclein from brain extracts. Inhibition of proteasomal, lysosomal, and autophagic pathways, as well as overexpression of a ubiquitin mutant less prone to deubiquitylation, G76A, increased monoubiquitylation of α-synuclein by SIAH. Monoubiquitylation increased the aggregation of α-synuclein in vitro. At the electron microscopy level, monoubiquitylated α-synuclein promoted the formation of massive amounts of amorphous aggregates. Monoubiquitylation also increased α-synuclein aggregation in vivo as observed by increased formation of α-synuclein inclusion bodies within dopaminergic cells. These inclusions are toxic to cells, and their formation was prevented when endogenous SIAH expression was suppressed. Our data suggest that monoubiquitylation represents a possible trigger event for α-synuclein aggregation and Lewy body formation.

Phosphorylation of Parkin by the cyclin-dependent kinase 5 at the linker region modulates its ubiquitin-ligase activity and aggregation.

Mutations in Parkin are responsible for a large percentage of autosomal recessive juvenile parkinsonism cases. Parkin displays ubiquitin-ligase activity and protects against cell death promoted by several insults. Therefore, regulation of Parkin activities is important for understanding the dopaminergic cell death observed in Parkinson disease. We now report that cyclin-dependent kinase 5 (Cdk5) phosphorylates Parkin both in vitro and in vivo. We found that highly specific Cdk5 inhibitors and a dominant negative Cdk5 construct inhibited Parkin phosphorylation, suggesting that a significant portion of Parkin is phosphorylated by Cdk5. Parkin interacts with Cdk5 as observed by co-immunoprecipitation experiments of transfected cells and rat brains. Phosphorylation by Cdk5 decreased the auto-ubiquitylation of Parkin both in vitro and in vivo. We identified Ser-131 located at the linker region of Parkin as the major Cdk5 phosphorylation site. The Cdk5 phosphorylation-deficient S131A Parkin mutant displayed a higher auto-ubiquitylation level and increased ubiquitylation activity toward its substrates synphilin-1 and p38. Additionally, the S131A Parkin mutant more significantly accumulated into inclusions in human dopaminergic cells when compared with the wild-type Parkin. Furthermore, S131A Parkin mutant increased the formation of synphilin-1/α-synuclein inclusions, suggesting that the levels of Parkin phosphorylation and ubiquitylation may modulate the formation of inclusion bodies relevant to the disease. The data indicate that Cdk5 is a new regulator of the Parkin ubiquitin-ligase activity and modulates its ability to accumulate into and modify inclusions. Phosphorylation by Cdk5 may contribute to the accumulation of toxic Parkin substrates and decrease the ability of dopaminergic cells to cope with toxic insults in Parkinson disease.

α-Synuclein fate is determined by USP9X-regulated monoubiquitination

α-Synuclein is central to the pathogenesis of Parkinson disease (PD). Mutations as well as accumulation of α-synuclein promote the death of dopaminergic neurons and the formation of Lewy bodies. α-Synuclein is monoubiquitinated by SIAH, but the regulation and roles of monoubiquitination in α-synuclein biology are poorly understood. We now report that the deubiquitinase USP9X interacts in vivo with and deubiquitinates α-synuclein. USP9X levels are significantly lower in cytosolic fractions of PD substantia nigra and Diffuse Lewy Body disease (DLBD) cortices compared to controls. This was associated to lower deubiquitinase activity toward monoubiquitinated α-synuclein in DLBD cortical extracts. A fraction of USP9X seems to be aggregated in PD and DLBD, as USP9X immunoreactivity is detected in Lewy bodies. Knockdown of USP9X expression promotes accumulation of monoubiquitinated α-synuclein species and enhances the formation of toxic α-synuclein inclusions upon proteolytic inhibition. On the other hand, by manipulating USP9X expression levels in the absence of proteolytic impairment, we demonstrate that monoubiquitination controls the partition of α-synuclein between different protein degradation systems. Deubiquitinated α-synuclein is mostly degraded by autophagy, while monoubiquitinated α-synuclein is preferentially degraded by the proteasome. Moreover, monoubiquitination promotes the degradation of α-synuclein, whereas deubiquitination leads to its accumulation, suggesting that the degradation of deubiquitinated α-synuclein by the autophagy pathway is less efficient than the proteasomal one. Lower levels of cytosolic USP9X and deubiquitinase activity in α-synucleinopathies may contribute to the accumulation and aggregation of monoubiquitinated α-synuclein in Lewy bodies. Our data indicate that monoubiquitination is a key determinant of α-synuclein fate.

The sequence and structure of the 3′-untranslated regions of chloroplast transcripts are important determinants of mRNA accumulation and stability

A general characteristic of the 3′-untranslated regions (3′ UTRs) of plastid mRNAs is an inverted repeat (IR) sequence that can fold into a stem-loop structure. These stem-loops are RNA 3′-end processing signals and determinants of mRNA stability, not transcription terminators. Incubation of synthetic RNAs corresponding to the 3′ UTRs of Chlamydomonas chloroplast genes atpB and petD with a chloroplast protein extract resulted in the accumulation of stable processing products. Synthetic RNAs of the petA 3′ UTR and the antisense strand of atpB 3′ UTR were degraded in the extract. To examine 3′ UTR function in vivo, the atpB 3′ UTR was replaced with the 3′ UTR sequences of the Chlamydomonas chloroplast genes petD, petD plus trnR, rbcL, petA and E. coli thrA by biolistic transformation of Chlamydomonas chloroplasts. Each 3′ UTR was inserted in both the sense and antisense orientations. The accumulation of both total atpB mRNA and ATPase β-subunit protein in all transformants was increased compared to a strain in which the atpB 3′ UTR had been deleted. However, the level of discrete atpB transcripts in transformants containing the antisense 3′ UTR sequences was reduced to approximately one-half that of transformants containing the 3′ UTRs in the sense orientation. These results imply that both the nucleotide sequences and the stem-loop structures of the 3′ UTRs are important for transcript 3′-end processing, and for accumulation of the mature mRNAs.

3'-PROCESSED MRNA IS PREFERENTIALLY TRANSLATED IN CHLAMYDOMONAS REINHARDTII CHLOROPLASTS

ABSTRACT 3′-end processing of nucleus-encoded mRNAs includes the addition of a poly(A) tail that is important for translation initiation. Since the vast majority of chloroplast mRNAs acquire their 3′ termini by processing yet are not polyadenylated, we asked whether 3′ end maturation plays a role in chloroplast translation. A general characteristic of the 3′ untranslated regions of chloroplast mRNAs is an inverted repeat (IR) sequence that can fold into a stem-loop structure. These stem-loops and their flanking sequences serve as RNA 3′-end formation signals. Deletion of theChlamydomonas chloroplast atpB 3′ IR in strain Δ26 results in reduced accumulation of atpB transcripts and the chloroplast ATPase β-subunit, leading to weakly photosynthetic growth. Of the residualatpB mRNA in Δ26, approximately 1% accumulates as a discrete RNA of wild-type size, while the remainder is heterogeneous in length due to the lack of normal 3′ end maturation. In this work, we have analyzed whether these unprocessed atpBtranscripts are actively translated in vivo. We found that only the minority population of discrete transcripts of wild-type size is associated with polysomes and thus accounts for the ATPase β-subunit which accumulates in Δ26. Analysis of chloroplast rbcLmRNA revealed that transcripts extending beyond the mature 3′ end were not polysome associated. These results suggest that 3′-end processing of chloroplast mRNA is required for or strongly stimulates its translation.

Glycogen Synthase Kinase 3β Modulates Synphilin-1 Ubiquitylation and Cellular Inclusion Formation by SIAH IMPLICATIONS FOR PROTEASOMAL FUNCTION AND LEWY BODY FORMATION

α-Synuclein is known to play a major role in the pathogenesis of Parkinson disease. We previously identified synphilin-1 as an α-synuclein-interacting protein and more recently found that synphilin-1 also interacts with the E3 ubiquitin ligases SIAH-1 and SIAH-2. SIAH proteins ubiquitylate synphilin-1 and promote its degradation through the ubiquitin proteasome system. Inability of the proteasome to degrade synphilin-1 promotes the formation of ubiquitylated inclusion bodies. We now show that synphilin-1 is phosphorylated by GSK3β within amino acids 550–659 and that this phosphorylation is significantly decreased by pharmacological inhibition of GSK3β and suppression of GSK3β expression by small interfering RNA duplex. Mutation analysis showed that Ser556 is a major GSK3β phosphorylation site in synphilin-1. GSK3β co-immunoprecipitated with synphilin-1, and protein 14-3-3, an activator of GSK3β activity, increased synphilin-1 phosphorylation. GSK3β decreased the in vitro and in vivo ubiquitylation of synphilin-1 as well as its degradation promoted by SIAH. Pharmacological inhibition and small interfering RNA suppression of GSK3β greatly increased ubiquitylation and inclusion body formation by SIAH. Additionally, synphilin-1 S556A mutant, which is less phosphorylated by GSK3β, formed more inclusion bodies than wild type synphilin-1. Inhibition of GSK3β in primary neuronal cultures decreased the levels of endogenous synphilin-1, indicating that synphilin-1 is a physiologic substrate of GSK3β. Using GFPu as a reporter to measure proteasome function in vivo, we found that synphilin-1 S556A is more efficient in inhibiting the proteasome than wild type synphilin-1, raising the possibility that the degree of synphilin-1 phosphorylation may regulate the proteasome function. Activation of GSK3β during endoplasmic reticulum stress and the specific phosphorylation of synphilin-1 by GSK3β place synphilin-1 as a possible mediator of endoplasmic reticulum stress and proteasomal dysfunction observed in Parkinson disease.

The 3′ untranslated regions of chloroplast genes inChlamydomonas reinhardtii do not serve as efficient transcriptional terminators

A general characteristic of the 3′ untranslated regions of plastid mRNAs is an inverted repeat sequence that can fold into a stem-loop structure. These stem-loops are superficially similar to structures involved in prokaryotic transcription termination, but were found instead to serve as RNA 3′ end processing signals in spinach chloroplasts, and in theatpB mRNA ofChlamydomonas reinhardtii chloroplasts. In order to carry out a broad study of the efficiency of the untranslated sequences at the 3′ ends of chloroplast genes inChlamydomonas to function as transcription terminators, we performed in vivo run-on transcription experiments usingChlamydomonas chloroplast transformants in which different 3′ ends were inserted into the chloroplast genome between apetD promoter and a reporter gene. The results showed that none of the 3′ ends that were tested, in either sense or antisense orientation, prevented readthrough transcription, and thus were not highly efficient transcription terminators. Therefore, we suggest that most or all of the 3′ ends of mature mRNAs inChlamydomonas chloroplasts are formed by 3′ end processing of longer precursors.

Synphilin-1A Inhibits Seven in Absentia Homolog (SIAH) and Modulates α-Synuclein Monoubiquitylation and Inclusion Formation

Parkinson disease (PD) is characterized by the presence of ubiquitylated inclusions and the death of dopaminergic neurons. Seven in absentia homolog (SIAH) is a ubiquitin-ligase that ubiquitylates α-synuclein and synphilin-1 and is present in Lewy bodies of PD patients. Understanding the mechanisms that regulate the ubiquitylation of PD-related proteins might shed light on the events involved in the formation of Lewy bodies and death of neurons. We show in this study that the recently described synphilin-1 isoform, synphilin-1A, interacts in vitro and in vivo with the ubiquitin-protein isopeptide ligase SIAH and regulates its activity toward α-synuclein and synphilin-1. SIAH promotes limited ubiquitylation of synphilin-1A that does not lead to its degradation by the proteasome. SIAH also increases the formation of synphilin-1A inclusions in the presence of proteasome inhibitors, supporting the participation of ubiquitylated synphilin-1A in the formation of Lewy body-like inclusions. Synphilin-1A/SIAH inclusions recruit PD-related proteins, such as α-synuclein, synphilin-1, Parkin, PINK1, and UCH-L1. We found that synphilin-1A robustly increases the steady-state levels of SIAH by decreasing its auto-ubiquitylation and degradation. In addition, synphilin-1A blocks the ubiquitylation and degradation of the SIAH substrates synphilin-1 and deleted in colon cancer protein. Furthermore, synphilin-1A strongly decreases the monoubiquitylation of α-synuclein by SIAH and the formation of α-synuclein inclusions, supporting a role for monoubiquitylation in α-synuclein inclusion formation. Our results suggest a novel function for synphilin-1A as a regulator of SIAH activity and formation of Lewy body-like inclusions.

Site-specific differences in proteasome-dependent degradation of monoubiquitinated α-synuclein.

The formation of toxic aggregates composed largely of the protein α-synuclein are a hallmark of Parkinsons disease. Evidence from both early-onset forms of the disease in humans and animal models has shown that the progression of the disease is correlated with the expression levels of α-synuclein, suggesting that cellular mechanisms that degrade excess α-synuclein are key. We and others have shown that monoubiquitinated α-synuclein can be degraded by the 26S proteasome; however, the contributions of each of the nine known individual monoubiquitination sites were unknown. Herein, we determined the consequences of each of the modification sites using homogenous, semisynthetic proteins in combination with an in vitro proteasome turnover assay. The data suggest that the site-specific effects of monoubiquitination support different levels of α-synuclein degradation.

Glycation potentiates α-synuclein-associated neurodegeneration in synucleinopathies

α-Synuclein misfolding and aggregation is a hallmark in Parkinsons disease and in several other neurodegenerative diseases known as synucleinopathies. The toxic properties of α-synuclein are conserved from yeast to man, but the precise underpinnings of the cellular pathologies associated are still elusive, complicating the development of effective therapeutic strategies. Combining molecular genetics with target-based approaches, we established that glycation, an unavoidable age-associated post-translational modification, enhanced α-synuclein toxicity in vitro and in vivo, in Drosophila and in mice. Glycation affected primarily the N-terminal region of α-synuclein, reducing membrane binding, impaired the clearance of α-synuclein, and promoted the accumulation of toxic oligomers that impaired neuronal synaptic transmission. Strikingly, using glycation inhibitors, we demonstrated that normal clearance of α-synuclein was re-established, aggregation was reduced, and motor phenotypes in Drosophila were alleviated. Altogether, our study demonstrates glycation constitutes a novel drug target that can be explored in synucleinopathies as well as in other neurodegenerative conditions.