Publications by authors named "Wei-Ling Tsou"

16 Publications

  • Page 1 of 1

Targeting the VCP-binding motif of ataxin-3 improves phenotypes in Drosophila models of Spinocerebellar Ataxia Type 3.

Neurobiol Dis 2021 Dec 24;160:105516. Epub 2021 Sep 24.

Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI 48201, USA; Department of Neurology, Wayne State University School of Medicine, Detroit, MI 48201, USA. Electronic address:

Of the family of polyglutamine (polyQ) neurodegenerative diseases, Spinocerebellar Ataxia Type 3 (SCA3) is the most common. Like other polyQ diseases, SCA3 stems from abnormal expansions in the CAG triplet repeat of its disease gene resulting in elongated polyQ repeats within its protein, ataxin-3. Various ataxin-3 protein domains contribute to its toxicity, including the valosin-containing protein (VCP)-binding motif (VBM). We previously reported that VCP, a homo-hexameric protein, enhances pathogenic ataxin-3 aggregation and exacerbates its toxicity. These findings led us to explore the impact of targeting the SCA3 protein by utilizing a decoy protein comprising the N-terminus of VCP (N-VCP) that binds ataxin-3's VBM. The notion was that N-VCP would reduce binding of ataxin-3 to VCP, decreasing its aggregation and toxicity. We found that expression of N-VCP in Drosophila melanogaster models of SCA3 ameliorated various phenotypes, coincident with reduced ataxin-3 aggregation. This protective effect was specific to pathogenic ataxin-3 and depended on its VBM. Increasing the amount of N-VCP resulted in further phenotype improvement. Our work highlights the protective potential of targeting the VCP-ataxin-3 interaction in SCA3, a key finding in the search for therapeutic opportunities for this incurable disorder.
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http://dx.doi.org/10.1016/j.nbd.2021.105516DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8693084PMC
December 2021

Ubiquitin-interacting motifs of ataxin-3 regulate its polyglutamine toxicity through Hsc70-4-dependent aggregation.

Elife 2020 09 21;9. Epub 2020 Sep 21.

Department of Pharmacology, Wayne State University, Detroit, United States.

Spinocerebellar ataxia type 3 (SCA3) belongs to the family of polyglutamine neurodegenerations. Each disorder stems from the abnormal lengthening of a glutamine repeat in a different protein. Although caused by a similar mutation, polyglutamine disorders are distinct, implicating non-polyglutamine regions of disease proteins as regulators of pathogenesis. SCA3 is caused by polyglutamine expansion in ataxin-3. To determine the role of ataxin-3's non-polyglutamine domains in disease, we utilized a new, allelic series of . We found that ataxin-3 pathogenicity is saliently controlled by polyglutamine-adjacent ubiquitin-interacting motifs (UIMs) that enhance aggregation and toxicity. UIMs function by interacting with the heat shock protein, Hsc70-4, whose reduction diminishes ataxin-3 toxicity in a UIM-dependent manner. Hsc70-4 also enhances pathogenicity of other polyglutamine proteins. Our studies provide a unique insight into the impact of ataxin-3 domains in SCA3, identify Hsc70-4 as a SCA3 enhancer, and indicate pleiotropic effects from HSP70 chaperones, which are generally thought to suppress polyglutamine degeneration.
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http://dx.doi.org/10.7554/eLife.60742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7505662PMC
September 2020

Nanoplastics impact the zebrafish (Danio rerio) transcriptome: Associated developmental and neurobehavioral consequences.

Environ Pollut 2020 Nov 16;266(Pt 2):115090. Epub 2020 Jul 16.

Institute of Environmental Health Sciences, Wayne State University, 6135 Woodward Ave, Detroit, MI, 48202, USA; Department of Pharmacology - School of Medicine, Wayne State University, 540 E Canfield, Detroit, MI, 28201, USA. Electronic address:

Microplastics (MPs) are a ubiquitous pollutant detected not only in marine and freshwater bodies, but also in tap and bottled water worldwide. While MPs have been extensively studied, the toxicity of their smaller counterpart, nanoplastics (NPs), is not well documented. Despite likely large-scale human and animal exposure to NPs, the associated health risks remain unclear, especially during early developmental stages. To address this, we investigated the health impacts of exposures to both 50 and 200 nm polystyrene NPs in larval zebrafish. From 6 to 120 h post-fertilization (hpf), developing zebrafish were exposed to a range of fluorescent NPs (10-10,000 parts per billion). Dose-dependent increases in accumulation were identified in exposed larval fish, potentially coinciding with an altered behavioral response as evidenced through swimming hyperactivity. Notably, exposures did not impact mortality, hatching rate, or deformities; however, transcriptomic analysis suggests neurodegeneration and motor dysfunction at both high and low concentrations. Furthermore, results of this study suggest that NPs can accumulate in the tissues of larval zebrafish, alter their transcriptome, and affect behavior and physiology, potentially decreasing organismal fitness in contaminated ecosystems. The uniquely broad scale of this study during a critical window of development provides crucial multidimensional characterization of NP impacts on human and animal health.
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http://dx.doi.org/10.1016/j.envpol.2020.115090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7492438PMC
November 2020

Degron capability of the hydrophobic C-terminus of the polyglutamine disease protein, ataxin-3.

J Neurosci Res 2020 10 9;98(10):2096-2108. Epub 2020 Jul 9.

Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI, USA.

Ataxin-3 is a deubiquitinase and polyglutamine disease protein whose cellular properties and functions are not entirely understood. Mutations in ataxin-3 cause spinocerebellar ataxia type 3 (SCA3), a neurodegenerative disorder that is a member of the polyglutamine family of diseases. Two major isoforms arise from alternative splicing of ATXN3 and are differently toxic in vivo as a result of faster proteasomal degradation of one isoform compared to the other. The isoforms vary only at their C-termini, suggesting that the hydrophobic C-terminus of the more quickly degraded form of ataxin-3 (here referred to as isoform 2) functions as a degron-that is, a peptide sequence that expedites the degradation of its host protein. We explored this notion in this study and present evidence that: (a) the C-terminus of ataxin-3 isoform 2 signals its degradation in a proteasome-dependent manner, (b) this effect from the C-terminus of isoform 2 does not require the ubiquitination of ataxin-3, and (c) the isolated C-terminus of isoform 2 can enhance the degradation of an unrelated protein. According to our data, the C-terminus of ataxin-3 isoform 2 is a degron, increasing overall understanding of the cellular properties of the SCA3 protein.
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http://dx.doi.org/10.1002/jnr.24684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8693082PMC
October 2020

Differential toxicity of ataxin-3 isoforms in Drosophila models of Spinocerebellar Ataxia Type 3.

Neurobiol Dis 2019 12 13;132:104535. Epub 2019 Jul 13.

Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI, USA; Department of Neurology, Wayne State University School of Medicine, Detroit, MI, USA. Electronic address:

The most commonly inherited dominant ataxia, Spinocerebellar Ataxia Type 3 (SCA3), is caused by a CAG repeat expansion that encodes an abnormally long polyglutamine (polyQ) repeat in the disease protein ataxin-3, a deubiquitinase. Two major full-length isoforms of ataxin-3 exist, both of which contain the same N-terminal portion and polyQ repeat, but differ in their C-termini; one (denoted here as isoform 1) contains a motif that binds ataxin-3's substrate, ubiquitin, whereas the other (denoted here as isoform 2) has a hydrophobic tail. Most SCA3 studies have focused on isoform 1, the predominant version in mammalian brain, yet both isoforms are present in brain and a better understanding of their relative pathogenicity in vivo is needed. We took advantage of the fruit fly, Drosophila melanogaster to model SCA3 and to examine the toxicity of each ataxin-3 isoform. Our assays reveal isoform 1 to be markedly more toxic than isoform 2 in all fly tissues. Reduced toxicity from isoform 2 is due to much lower protein levels as a result of its expedited degradation. Additional studies indicate that isoform 1 is more aggregation-prone than isoform 2 and that the C-terminus of isoform 2 is critical for its enhanced proteasomal degradation. According to our results, although both full-length, pathogenic ataxin-3 isoforms are toxic, isoform 1 is likely the primary contributor to SCA3 due to its presence at higher levels. Isoform 2, as a result of rapid degradation that is dictated by its tail, is unlikely to be a key player in this disease. Our findings provide new insight into the biology of this ataxia and the cellular processing of the underlying disease protein.
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http://dx.doi.org/10.1016/j.nbd.2019.104535DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6834911PMC
December 2019

Interaction of the polyglutamine protein ataxin-3 with Rad23 regulates toxicity in Drosophila models of Spinocerebellar Ataxia Type 3.

Hum Mol Genet 2017 04;26(8):1419-1431

Department of Pharmacology, Wayne State University, Detroit MI, USA.

Polyglutamine (polyQ) repeat expansion in the deubiquitinase ataxin-3 causes neurodegeneration in Spinocerebellar Ataxia Type 3 (SCA3), one of nine inherited, incurable diseases caused by similar mutations. Ataxin-3's degradation is inhibited by its binding to the proteasome shuttle Rad23 through ubiquitin-binding site 2 (UbS2). Disrupting this interaction decreases levels of ataxin-3. Since reducing levels of polyQ proteins can decrease their toxicity, we tested whether genetically modulating the ataxin-3-Rad23 interaction regulates its toxicity in Drosophila. We found that exogenous Rad23 increases the toxicity of pathogenic ataxin-3, coincident with increased levels of the disease protein. Conversely, reducing Rad23 levels alleviates toxicity in this SCA3 model. Unexpectedly, pathogenic ataxin-3 with a mutated Rad23-binding site at UbS2, despite being present at markedly lower levels, proved to be more pathogenic than a disease-causing counterpart with intact UbS2. Additional studies established that the increased toxicity upon mutating UbS2 stems from disrupting the autoprotective role that pathogenic ataxin-3 has against itself, which depends on the co-chaperone, DnaJ-1. Our data reveal a previously unrecognized balance between pathogenic and potentially therapeutic properties of the ataxin-3-Rad23 interaction; they highlight this interaction as critical for the toxicity of the SCA3 protein, and emphasize the importance of considering protein context when pursuing suppressive avenues.
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http://dx.doi.org/10.1093/hmg/ddx039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6075452PMC
April 2017

Polyglutamine length-dependent toxicity from α1ACT in Drosophila models of spinocerebellar ataxia type 6.

Biol Open 2016 Dec 15;5(12):1770-1775. Epub 2016 Dec 15.

Department of Pharmacology, Wayne State University, Detroit, MI 48201, USA

Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disease that results from abnormal expansion of a polyglutamine (polyQ) repeat. SCA6 is caused by CAG triplet repeat expansion in the gene CACNA1A, resulting in a polyQ tract of 19-33 in patients. CACNA1A, a bicistronic gene, encodes the α1A calcium channel subunit and the transcription factor, α1ACT. PolyQ expansion in α1ACT causes degeneration in mice. We recently described the first Drosophila models of SCA6 that express α1ACT with a normal (11Q) or hyper-expanded (70Q) polyQ. Here, we report additional α1ACT transgenic flies, which express full-length α1ACT with a 33Q repeat. We show that α1ACT33Q is toxic in Drosophila, but less so than the 70Q version. When expressed everywhere, α1ACT33Q-expressing adults die earlier than flies expressing the normal allele. α1ACT33Q causes retinal degeneration and leads to aggregated species in an age-dependent manner, but at a slower pace than the 70Q counterpart. According to western blots, α1ACT33Q localizes less readily in the nucleus than α1ACT70Q, providing clues into the importance of polyQ tract length on α1ACT localization and its site of toxicity. We expect that these new lines will be highly valuable for future work on SCA6.
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http://dx.doi.org/10.1242/bio.021667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5200913PMC
December 2016

USP5 Is Dispensable for Monoubiquitin Maintenance in Drosophila.

J Biol Chem 2016 Apr 25;291(17):9161-72. Epub 2016 Feb 25.

From the Departments of Pharmacology and Neurology, Wayne State University School of Medicine, Detroit, Michigan 48201 and

Ubiquitination is a post-translational modification that regulates most cellular pathways and processes, including degradation of proteins by the proteasome. Substrate ubiquitination is controlled at various stages, including through its reversal by deubiquitinases (DUBs). A critical outcome of this process is the recycling of monoubiquitin. One DUB whose function has been proposed to include monoubiquitin recycling is USP5. Here, we investigated whether Drosophila USP5 is important for maintaining monoubiquitin in vivo We found that the fruit fly orthologue of USP5 has catalytic preferences similar to its human counterpart and that this DUB is necessary during fly development. Our biochemical and genetic experiments indicate that reduction of USP5 does not lead to monoubiquitin depletion in developing flies. Also, introduction of exogenous ubiquitin does not suppress developmental lethality caused by loss of endogenous USP5. Our work indicates that a primary physiological role of USP5 is not to recycle monoubiquitin for reutilization, but that it may involve disassembly of conjugated ubiquitin to maintain proteasome function.
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http://dx.doi.org/10.1074/jbc.M115.703504DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4861482PMC
April 2016

The deubiquitinase ataxin-3 requires Rad23 and DnaJ-1 for its neuroprotective role in Drosophila melanogaster.

Neurobiol Dis 2015 Oct 22;82:12-21. Epub 2015 May 22.

Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI 48201, USA; Cancer Biology Graduate Program, Wayne State University School of Medicine, Detroit, MI 48201, USA; Department of Neurology, Wayne State University School of Medicine, Detroit, MI 48201, USA.

Ataxin-3 is a deubiquitinase and polyglutamine (polyQ) disease protein with a protective role in Drosophila melanogaster models of neurodegeneration. In the fruit fly, wild-type ataxin-3 suppresses toxicity from several polyQ disease proteins, including a pathogenic version of itself that causes spinocerebellar ataxia type 3 and pathogenic huntingtin, which causes Huntington's disease. The molecular partners of ataxin-3 in this protective function are unclear. Here, we report that ataxin-3 requires its direct interaction with the ubiquitin-binding and proteasome-associated protein, Rad23 (known as hHR23A/B in mammals) in order to suppress toxicity from polyQ species in Drosophila. According to additional studies, ataxin-3 does not rely on autophagy or the proteasome to suppress polyQ-dependent toxicity in fly eyes. Instead this deubiquitinase, through its interaction with Rad23, leads to increased protein levels of the co-chaperone DnaJ-1 and depends on it to protect against degeneration. Through DnaJ-1, our data connect ataxin-3 and Rad23 to protective processes involved with protein folding rather than increased turnover of toxic polyQ species.
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http://dx.doi.org/10.1016/j.nbd.2015.05.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4710962PMC
October 2015

DnaJ-1 and karyopherin α3 suppress degeneration in a new Drosophila model of Spinocerebellar Ataxia Type 6.

Hum Mol Genet 2015 Aug 7;24(15):4385-96. Epub 2015 May 7.

Department of Pharmacology, Cancer Biology Graduate Program and Department of Neurology, Wayne State University School of Medicine, Detroit, MI 48201, USA and

Spinocerebellar ataxia type 6 (SCA6) belongs to the family of CAG/polyglutamine (polyQ)-dependent neurodegenerative disorders. SCA6 is caused by abnormal expansion in a CAG trinucleotide repeat within exon 47 of CACNA1A, a bicistronic gene that encodes α1A, a P/Q-type calcium channel subunit and a C-terminal protein, termed α1ACT. Expansion of the CAG/polyQ region of CACNA1A occurs within α1ACT and leads to ataxia. There are few animal models of SCA6. Here, we describe the generation and characterization of the first Drosophila melanogaster models of SCA6, which express the entire human α1ACT protein with a normal or expanded polyQ. The polyQ-expanded version of α1ACT recapitulates the progressively degenerative nature of SCA6 when expressed in various fly tissues and the presence of densely staining aggregates. Additional studies identify the co-chaperone DnaJ-1 as a potential therapeutic target for SCA6. Expression of DnaJ-1 potently suppresses α1ACT-dependent degeneration and lethality, concomitant with decreased aggregation and reduced nuclear localization of the pathogenic protein. Mutating the nuclear importer karyopherin α3 also leads to reduced toxicity from pathogenic α1ACT. Little is known about the steps leading to degeneration in SCA6 and the means to protect neurons in this disease are lacking. Invertebrate animal models of SCA6 can expand our understanding of molecular sequelae related to degeneration in this disorder and lead to the rapid identification of cellular components that can be targeted to treat it.
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http://dx.doi.org/10.1093/hmg/ddv174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4492400PMC
August 2015

An optimal ubiquitin-proteasome pathway in the nervous system: the role of deubiquitinating enzymes.

Front Mol Neurosci 2014 19;7:72. Epub 2014 Aug 19.

Department of Pharmacology, Wayne State University School of Medicine Detroit, MI, USA ; Department of Neurology, Wayne State University School of Medicine Detroit, MI, USA.

The Ubiquitin-Proteasome Pathway (UPP), which is critical for normal function in the nervous system and is implicated in various neurological diseases, requires the small modifier protein ubiquitin to accomplish its duty of selectively degrading short-lived, abnormal or misfolded proteins. Over the past decade, a large class of proteases collectively known as deubiquitinating enzymes (DUBs) has increasingly gained attention in all manners related to ubiquitin. By cleaving ubiquitin from another protein, DUBs ensure that the UPP functions properly. DUBs accomplish this task by processing newly translated ubiquitin so that it can be used for conjugation to substrate proteins, by regulating the "where, when, and why" of UPP substrate ubiquitination and subsequent degradation, and by recycling ubiquitin for re-use by the UPP. Because of the reliance of the UPP on DUB activities, it is not surprising that these proteases play important roles in the normal activities of the nervous system and in neurodegenerative diseases. In this review, we summarize recent advances in understanding the functions of DUBs in the nervous system. We focus on their role in the UPP, and make the argument that understanding the UPP from the perspective of DUBs can yield new insight into diseases that result from anomalous intra-cellular processes or inter-cellular networks. Lastly, we discuss the relevance of DUBs as therapeutic options for disorders of the nervous system.
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http://dx.doi.org/10.3389/fnmol.2014.00072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4137239PMC
September 2014

Ubiquitin-binding site 2 of ataxin-3 prevents its proteasomal degradation by interacting with Rad23.

Nat Commun 2014 Aug 21;5:4638. Epub 2014 Aug 21.

1] Department of Pharmacology, Wayne State University School of Medicine, 540 E Canfield, Scott Hall Room 3108, Detroit, Michigan 48201, USA [2] Department of Neurology, Wayne State University School of Medicine, 540 E Canfield, Scott Hall Room 3108, Detroit, Michigan 48201, USA [3] Cancer Biology Program, Wayne State University School of Medicine, 540 E Canfield, Scott Hall Room 3108, Detroit, Michigan 48201, USA.

Polyglutamine repeat expansion in ataxin-3 causes neurodegeneration in the most common dominant ataxia, spinocerebellar ataxia type 3 (SCA3). Since reducing levels of disease proteins improves pathology in animals, we investigated how ataxin-3 is degraded. Here we show that, unlike most proteins, ataxin-3 turnover does not require its ubiquitination, but is regulated by ubiquitin-binding site 2 (UbS2) on its N terminus. Mutating UbS2 decreases ataxin-3 protein levels in cultured mammalian cells and in Drosophila melanogaster by increasing its proteasomal turnover. Ataxin-3 interacts with the proteasome-associated proteins Rad23A/B through UbS2. Knockdown of Rad23 in cultured cells and in Drosophila results in lower levels of ataxin-3 protein. Importantly, reducing Rad23 suppresses ataxin-3-dependent degeneration in flies. We present a mechanism for ubiquitination-independent degradation that is impeded by protein interactions with proteasome-associated factors. We conclude that UbS2 is a potential target through which to enhance ataxin-3 degradation for SCA3 therapy.
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http://dx.doi.org/10.1038/ncomms5638DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4237202PMC
August 2014

Using membrane-targeted green fluorescent protein to monitor neurotoxic protein-dependent degeneration of Drosophila eyes.

J Neurosci Res 2014 Sep 2;92(9):1100-9. Epub 2014 May 2.

Graduate Program in Cancer Biology, Wayne State University School of Medicine, Detroit, Michigan; Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan.

Age-related neurodegeneration has been studied extensively through the use of model organisms, including the genetically versatile Drosophila melanogaster. Various neurotoxic proteins have been expressed in fly eyes to approximate degeneration occurring in humans, and much has been learned from this heterologous system. Although Drosophila expedites scientific research through rapid generational times and relative inexpensiveness, one factor that can hinder analyses is the examination of milder forms of degeneration caused by some toxic proteins in fly eyes. Whereas several disease proteins cause massive degeneration that is easily observed by examining the external structure of the fly eye, others cause mild degeneration that is difficult to observe externally and requires laborious histological preparation to assess and monitor. Here, we describe a sensitive fluorescence-based method to observe, monitor, and quantify mild Drosophila eye degeneration caused by various proteins, including the polyglutamine disease proteins ataxin-3 (spinocerebellar ataxia type 3) and huntingtin (Huntington's disease), mutant α-synuclein (Parkinson's disease), and Aβ42 (Alzheimer's disease). We show that membrane-targeted green fluorescent protein reports degeneration robustly and quantitatively. This simple yet powerful technique, which is amenable to large-scale screens, can help accelerate studies to understand age-related degeneration and to find factors that suppress it for therapeutic purposes.
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http://dx.doi.org/10.1002/jnr.23395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4144675PMC
September 2014

Ubiquitination regulates the neuroprotective function of the deubiquitinase ataxin-3 in vivo.

J Biol Chem 2013 Nov 8;288(48):34460-9. Epub 2013 Oct 8.

From the Departments of Pharmacology and Neurology and.

Deubiquitinases (DUBs) are proteases that regulate various cellular processes by controlling protein ubiquitination. Cell-based studies indicate that the regulation of the activity of DUBs is important for homeostasis and is achieved by multiple mechanisms, including through their own ubiquitination. However, the physiological significance of the ubiquitination of DUBs to their functions in vivo is unclear. Here, we report that ubiquitination of the DUB ataxin-3 at lysine residue 117, which markedly enhances its protease activity in vitro, is critical for its ability to suppress toxic protein-dependent degeneration in Drosophila melanogaster. Compared with ataxin-3 with only Lys-117 present, ataxin-3 that does not become ubiquitinated performs significantly less efficiently in suppressing or delaying the onset of toxic protein-dependent degeneration in flies. According to further studies, the C terminus of Hsc70-interacting protein (CHIP), an E3 ubiquitin ligase that ubiquitinates ataxin-3 in vitro, is dispensable for its ubiquitination in vivo and is not required for the neuroprotective function of this DUB in Drosophila. Our work also suggests that ataxin-3 suppresses degeneration by regulating toxic protein aggregation rather than stability.
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http://dx.doi.org/10.1074/jbc.M113.513903DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3843061PMC
November 2013

Systematic analysis of the physiological importance of deubiquitinating enzymes.

PLoS One 2012 24;7(8):e43112. Epub 2012 Aug 24.

Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan, United States of America.

Deubiquitinating enzymes (DUBs) are proteases that control the post-translational modification of proteins by ubiquitin and in turn regulate diverse cellular pathways. Despite a growing understanding of DUB biology at the structural and molecular level, little is known about the physiological importance of most DUBs. Here, we systematically identify DUBs encoded by the genome of Drosophila melanogaster and examine their physiological importance in vivo. Through domain analyses we uncovered 41 Drosophila DUBs, most of which have human orthologues. Systematic knockdown of the vast majority of DUBs throughout the fly or in specific cell types had dramatic consequences for Drosophila development, adult motility or longevity. Specific DUB subclasses proved to be particularly necessary during development, while others were important in adults. Several DUBs were indispensable in neurons or glial cells during developmental stages; knockdown of others perturbed the homeostasis of ubiquitinated proteins in adult flies, or had adverse effects on wing positioning as a result of neuronal requirements. We demonstrate the physiological significance of the DUB family of enzymes in intact animals, find that there is little functional redundancy among members of this family of proteases, and provide insight for future investigations to understand DUB biology at the molecular, cellular and organismal levels.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0043112PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3427330PMC
April 2013

Splice isoform-specific suppression of the Cav2.1 variant underlying spinocerebellar ataxia type 6.

Neurobiol Dis 2011 Sep 29;43(3):533-42. Epub 2011 Apr 29.

Institute of Neuroscience, School of Life Sciences, National Yang-Ming University, Taipei, Taiwan.

Spinocerebellar ataxia type 6 (SCA6) is an inherited neurodegenerative disease caused by a polyglutamine (polyQ) expansion in the Ca(V)2.1 voltage-gated calcium channel subunit (CACNA1A). There is currently no treatment for this debilitating disorder and thus a pressing need to develop preventative therapies. RNA interference (RNAi) has proven effective at halting disease progression in several models of spinocerebellar ataxia (SCA), including SCA types 1 and 3. However, in SCA6 and other dominantly inherited neurodegenerative disorders, RNAi-based strategies that selectively suppress expression of mutant alleles may be required. Using a Ca(V)2.1 mini-gene reporter system, we found that pathogenic CAG expansions in Ca(V)2.1 enhance splicing activity at the 3'end of the transcript, leading to a CAG repeat length-dependent increase in the levels of a polyQ-encoding Ca(V)2.1 mRNA splice isoform and the resultant disease protein. Taking advantage of this molecular phenomenon, we developed a novel splice isoform-specific (SIS)-RNAi strategy that selectively targets the polyQ-encoding Ca(V)2.1 splice variant. Selective suppression of transiently expressed and endogenous polyQ-encoding Ca(V)2.1 splice variants was achieved in a variety of cell-based models including a human neuronal cell line, using a new artificial miRNA-like delivery system. Moreover, the efficacy of gene silencing correlated with effective intracellular recognition and processing of SIS-RNAi miRNA mimics. These results lend support to the preclinical development of SIS-RNAi as a potential therapy for SCA6 and other dominantly inherited diseases.
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http://dx.doi.org/10.1016/j.nbd.2011.04.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3169420PMC
September 2011
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