Publications by authors named "Jessica R Blount"

15 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
December 2021

Unanchored Ubiquitin Chains, Revisited.

Front Cell Dev Biol 2020 26;8:582361. Epub 2020 Oct 26.

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

The small modifier protein, ubiquitin, holds a special place in eukaryotic biology because of its myriad post-translational effects that control normal cellular processes and are implicated in various diseases. By being covalently conjugated onto other proteins, ubiquitin changes their interaction landscape - fostering new interactions as well as inhibiting others - and ultimately deciding the fate of its substrates and controlling pathways that span most cell physiology. Ubiquitin can be attached onto other proteins as a monomer or as a poly-ubiquitin chain of diverse structural topologies. Among the types of poly-ubiquitin species generated are ones detached from another substrate - comprising solely ubiquitin as their constituent - referred to as unanchored, or free chains. Considered to be toxic byproducts, these species have recently emerged to have specific physiological functions in immune pathways and during cell stress. Free chains also do not appear to be detrimental to multi-cellular organisms; they can be active members of the ubiquitination process, rather than corollary species awaiting disassembly into mono-ubiquitin. Here, we summarize past and recent studies on unanchored ubiquitin chains, paying special attention to their emerging roles as second messengers in several signaling pathways. These investigations paint complex and flexible outcomes for free ubiquitin chains, and present a revised model of unanchored poly-ubiquitin biology that is in need of additional investigation.
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http://dx.doi.org/10.3389/fcell.2020.582361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7659471PMC
October 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
October 2020

Isoleucine 44 Hydrophobic Patch Controls Toxicity of Unanchored, Linear Ubiquitin Chains through NF-κB Signaling.

Cells 2020 06 22;9(6). Epub 2020 Jun 22.

Department of Pharmacology, Wayne State University School of Medicine, 540 East Canfield St., Scott Hall Rm. 3108, Detroit, MI 48201, USA.

Ubiquitination is a post-translational modification that regulates cellular processes by altering the interactions of proteins to which ubiquitin, a small protein adduct, is conjugated. Ubiquitination yields various products, including mono- and poly-ubiquitinated substrates, as well as unanchored poly-ubiquitin chains whose accumulation is considered toxic. We previously showed that transgenic, unanchored poly-ubiquitin is not problematic in . In the fruit fly, free chains exist in various lengths and topologies and are degraded by the proteasome; they are also conjugated onto other proteins as one unit, eliminating them from the free ubiquitin chain pool. Here, to further explore the notion of unanchored chain toxicity, we examined when free poly-ubiquitin might become problematic. We found that unanchored chains can be highly toxic if they resemble linear poly-ubiquitin that cannot be modified into other topologies. These species upregulate NF-κB signaling, and modulation of the levels of NF-κB components reduces toxicity. In additional studies, we show that toxicity from untethered, linear chains is regulated by isoleucine 44, which anchors a key interaction site for ubiquitin. We conclude that free ubiquitin chains can be toxic, but only in uncommon circumstances, such as when the ability of cells to modify and regulate them is markedly restricted.
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http://dx.doi.org/10.3390/cells9061519DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7348737PMC
June 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

Unanchored ubiquitin chains do not lead to marked alterations in gene expression in .

Biol Open 2019 May 30;8(5). Epub 2019 May 30.

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

The small protein modifier ubiquitin regulates various aspects of cellular biology through its chemical conjugation onto proteins. Ubiquitination of proteins presents itself in numerous iterations, from a single mono-ubiquitination event to chains of poly-ubiquitin. Ubiquitin chains can be attached onto other proteins or can exist as unanchored species, i.e. free from another protein. Unanchored ubiquitin chains are thought to be deleterious to the cell and rapidly disassembled into mono-ubiquitin. We recently examined the toxicity and utilization of unanchored poly-ubiquitin in We found that free poly-ubiquitin species are largely innocuous to flies and that free poly-ubiquitin can be controlled by being degraded by the proteasome or by being conjugated onto another protein as a single unit. Here, to explore whether an organismal defense is mounted against unanchored chains, we conducted RNA-Seq analyses to examine the transcriptomic impact of free poly-ubiquitin in the fly. We found ∼90 transcripts whose expression is altered in the presence of different types of unanchored poly-ubiquitin. The set of genes identified was essentially devoid of ubiquitin-, proteasome-, or autophagy-related components. The seeming absence of a large and multipronged response to unanchored poly-ubiquitin supports the conclusion that these species need not be toxic and underscores the need to re-examine the role of free ubiquitin chains in the cell.
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http://dx.doi.org/10.1242/bio.043372DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6550069PMC
May 2019

Expression and Regulation of Deubiquitinase-Resistant, Unanchored Ubiquitin Chains in Drosophila.

Sci Rep 2018 05 31;8(1):8513. Epub 2018 May 31.

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

The modifier protein, ubiquitin (Ub) regulates various cellular pathways by controlling the fate of substrates to which it is conjugated. Ub moieties are also conjugated to each other, forming chains of various topologies. In cells, poly-Ub is attached to proteins and also exists in unanchored form. Accumulation of unanchored poly-Ub is thought to be harmful and quickly dispersed through dismantling by deubiquitinases (DUBs). We wondered whether disassembly by DUBs is necessary to control unanchored Ub chains in vivo. We generated Drosophila melanogaster lines that express Ub chains non-cleavable into mono-Ub by DUBs. These chains are rapidly modified with different linkages and represent various types of unanchored species. We found that unanchored poly-Ub is not devastating in Drosophila, under normal conditions or during stress. The DUB-resistant, free Ub chains are degraded by the proteasome, at least in part through the assistance of VCP and its cofactor, p47. Also, unanchored poly-Ub that cannot be cleaved by DUBs can be conjugated en bloc, in vivo. Our results indicate that unanchored poly-Ub species need not be intrinsically toxic; they can be controlled independently of DUB-based disassembly by being degraded, or through conjugation onto other proteins.
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http://dx.doi.org/10.1038/s41598-018-26364-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5981470PMC
May 2018

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

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

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

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

Ubiquitin-specific protease 25 functions in Endoplasmic Reticulum-associated degradation.

PLoS One 2012 9;7(5):e36542. Epub 2012 May 9.

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

Endoplasmic Reticulum (ER)-associated degradation (ERAD) discards abnormal proteins synthesized in the ER. Through coordinated actions of ERAD components, misfolded/anomalous proteins are recognized, ubiquitinated, extracted from the ER and ultimately delivered to the proteasome for degradation. It is not well understood how ubiquitination of ERAD substrates is regulated. Here, we present evidence that the deubiquitinating enzyme Ubiquitin-Specific Protease 25 (USP25) is involved in ERAD. Our data support a model where USP25 counteracts ubiquitination of ERAD substrates by the ubiquitin ligase HRD1, rescuing them from degradation by the proteasome.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0036542PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3348923PMC
September 2012

Activity and cellular functions of the deubiquitinating enzyme and polyglutamine disease protein ataxin-3 are regulated by ubiquitination at lysine 117.

J Biol Chem 2010 Dec 13;285(50):39303-13. Epub 2010 Oct 13.

Department of Neurology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.

Deubiquitinating enzymes (DUbs) play important roles in many ubiquitin-dependent pathways, yet how DUbs themselves are regulated is not well understood. Here, we provide insight into the mechanism by which ubiquitination directly enhances the activity of ataxin-3, a DUb implicated in protein quality control and the disease protein in the polyglutamine neurodegenerative disorder, Spinocerebellar Ataxia Type 3. We identify Lys-117, which resides near the catalytic triad, as the primary site of ubiquitination in wild type and pathogenic ataxin-3. Further studies indicate that ubiquitin-dependent activation of ataxin-3 at Lys-117 is important for its ability to reduce high molecular weight ubiquitinated species in cells. Ubiquitination at Lys-117 also facilitates the ability of ataxin-3 to induce aggresome formation in cells. Finally, structure-function studies support a model of activation whereby ubiquitination at Lys-117 enhances ataxin-3 activity independent of the known ubiquitin-binding sites in ataxin-3, most likely through a direct conformational change in or near the catalytic domain.
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http://dx.doi.org/10.1074/jbc.M110.181610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2998082PMC
December 2010

Ubiquitination directly enhances activity of the deubiquitinating enzyme ataxin-3.

EMBO J 2009 Feb 15;28(4):372-82. Epub 2009 Jan 15.

Department of Neurology, University of Michigan, Ann Arbor, MI 48109, USA.

Deubiquitinating enzymes (DUBs) control the ubiquitination status of proteins in various cellular pathways. Regulation of the activity of DUBs, which is critically important to cellular homoeostasis, can be achieved at the level of gene expression, protein complex formation, or degradation. Here, we report that ubiquitination also directly regulates the activity of a DUB, ataxin-3, a polyglutamine disease protein implicated in protein quality control pathways. Ubiquitination enhances ubiquitin (Ub) chain cleavage by ataxin-3, but does not alter its preference for K63-linked Ub chains. In cells, ubiquitination of endogenous ataxin-3 increases when the proteasome is inhibited, when excess Ub is present, or when the unfolded protein response is induced, suggesting that the cellular functions of ataxin-3 in protein quality control are modulated through ubiquitination. Ataxin-3 is the first reported DUB in which ubiquitination directly regulates catalytic activity. We propose a new function for protein ubiquitination in regulating the activity of certain DUBs and perhaps other enzymes.
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http://dx.doi.org/10.1038/emboj.2008.289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2646149PMC
February 2009
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