Ubiquitin dimers control the hydrolase activity of UCH-L3

doi:10.1016/j.neuint.2008.12.013

Neurochemistry International

Volume 54, Issues 5–6, May–June 2009, Pages 314-321

https://doi.org/10.1016/j.neuint.2008.12.013 Get rights and content

Abstract

Ubiquitin (Ub) carboxy terminal hydrolase (UCH)-L1 and UCH-L3 are two of the deubiquitinating enzymes expressed in the brain. Both gad mice, which lack UCH-L1 expression and Uchl3 knockout mice exhibit neurodegeneration, although at distinct areas. These phenotypes indicate the importance of UCH-L1 and UCH-L3 in the regulation of the central nervous system. However, molecular substrates and the molecular regulators of UCH-L1 and UCH-L3 remain poorly identified. Here we show that Ub dimers interact non-covalently with UCH-L3 in vitro and in cells. These interactions were not observed with UCH-L1 in cells. In vitro, K48-linked Ub dimers pronouncedly inhibited the hydrolase activity of UCH-L3, while mono-Ub, a previously identified interacting protein, inhibited the hydrolase activity of UCH-L1. These results indicate that mono-Ub and Ub dimers may regulate the enzymatic functions of UCH-L1 and UCH-L3, respectively, in vivo.

Introduction

The ubiquitin (Ub) system is involved in the regulation of various physiological phenomena including development, inflammatory response, and intracellular trafficking (Hershko and Ciechanover, 1998, Hicke, 2001, Glickman and Ciechanover, 2002, Mukhopadhyay and Riezman, 2007). Malfunction of the Ub system in the central nervous system (CNS) may lead to neurodegeneration or synaptic dysfunction (Rubinsztein, 2006, Hegde and Upadhya, 2007). Ub can be covalently conjugated to substrate proteins by the sequential action of E1, E2 and E3 enzymes forming an isopeptide bond between the carboxy terminus of Ub and the lysine residues of the substrates (Hershko and Ciechanover, 1998). The attachment of Ub to the substrates may occur either in the form of a single Ub or a poly-Ub chain, where the carboxy terminus of one Ub moiety is covalently linked to one of the seven Lys residues of an adjacent Ub molecule.

Deubiquitination involves the hydrolysis of the isopeptide bonds between the carboxy terminus glycine of the Ub and the lysine of the substrates or Ub, which requires the activity of deubiquitinating enzymes (DUBs) (Nijman et al., 2005). DUBs can be divided into six distinct families: the Ub-specific processing proteases, the Ub carboxyl-terminal hydrolases (UCHs), the Ataxin-3/Josephin domains, the ovarian tumor domain-containing proteases, the viral processing proteases and the JAMM proteases.

UCH is a cysteine protease with relatively small molecular weight. There is one UCH in yeast, and four UCH isozymes in mammals: UCH-L1, UCH-L3, UCH37 and BAP1. Of these, UCH-L1 and UCH-L3 are the most closely related family members with about 53% identity. These UCHs have also been shown to hydrolyze Nedd8 (neural precursor cell expressed developmentally down regulated 8), a Ub-like protein with 68% identity to Ub (Wada et al., 1998, Hemelaar et al., 2004). The expression of UCH-L3 is ubiquitous whereas that of UCH-L1 is mainly restricted to the brain and the testis/ovary (Wilkinson et al., 1989, Kurihara et al., 2000, Kurihara et al., 2001).

Previous studies using mutant mice of Uchl1 and Uchl3 indicated that these enzymes are involved in the regulation of the brain function (Setsuie and Wada, 2007). The I93M point mutation in UCH-L1 is associated with familial Parkinson's disease (Leroy et al., 1998) and we have previously shown that I93M UCH-L1 expression in the cell or in transgenic mice induces dopaminergic neuron death (Setsuie et al., 2007, Kabuta et al., 2008). We also reported that gad (gracile axonal dystrophy) mice, which lack Uchl1 expression, show axonal degeneration of the gracile tract, which results in sensory ataxia (Saigoh et al., 1999). Our analysis also indicated that UCH-L1 is involved in many biological aspects including neuronal apoptosis (Harada et al., 2004), neurotransmitter receptor activation (Manago et al., 2005), neural progenitor regulation (Sakurai et al., 2006), learning and memory (Sakurai et al., 2008). On the other hand, UCH-L3 knockout mice show photoreceptor cell degeneration (Semenova et al., 2003, Sano et al., 2006) and defects in memory function (Wood et al., 2005). These different phenotypes of mutant mice indicate that although both enzymes share similar properties in terms of biochemical identity (Johnston et al., 1997, Larsen et al., 1998, Misaghi et al., 2005, Das et al., 2006), UCH-L1 and UCH-L3 should be differentially controlled or be regulated by distinct molecules.

To identify the proteins that regulate UCH-L1 and UCH-L3 in a diverse manner, we performed immunoaffinity purification assay using UCH-L1 and UCH-L3 and found that UCH-L3 is inhibited by Ub dimers (di-Ub) while UCH-L1 is inhibited by mono-Ub.

Section snippets

Isolation and culture of mouse embryonic fibroblasts (MEFs) and their immortalization

Pregnant female Uchl3 heterozygote mice (Kurihara et al., 2000) were sacrificed at 14–15 days postcoitus. The head, the viscera and the bones of each embryo were removed under a dissecting microscope and a part of these samples were used for genotyping. The remaining portion of embryo was placed in 0.25% trypsin-EDTA (Invitrogen, Carlsbad, CA, USA), cut finely with razor blade, and incubated for 15 min at 37 °C. The cell suspension was passed several times through a 1 ml pipette tip until no

UCH-L3 but not UCH-L1 interacts with di-Ub

To identify the proteins that interact with UCH-L1 or UCH-L3, which might represent the potential substrates or the functional effecters, we performed immunoaffinity chromatography. Floating Hela cells that stably expressed human UCH-L3 tagged with the FLAG-HA epitope at its N-terminus (L3 WT), UCH-L3 C95S (active site mutant of UCH-L3), UCH-L1 (L1 WT), UCH-L1 C90S (active site mutant of UCH-L1), GFP and Mock were constructed (Fig. 1A). Western blotting using anti-HA and anti-FLAG antibodies

Discussion

Our immunoaffinity purification assay coupled with LC–MS/MS analysis of Hela cells stably expressing exogenous UCH-L3 and UCH-L1 revealed that UCH-L3, but not its closest isozyme UCH-L1, interacts with di-Ub. Interaction with mono-Ub, an important physiological interacting partner of UCH-L1, was not observed with WT UCH-L3 in cells (Fig. 1). Structure analysis by X-ray crystallography and NMR spectroscopy indicated that important amino acids for the interaction of Ub-aldehyde or

Acknowledgements

This work was supported by grants-in-aid for scientific research from the Japan Society for the Promotion of Science; a research grant in priority area research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan; grants-in-aid for scientific research from the Ministry of Health, Labor, and Welfare, Japan; and the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation and the New Energy and Industrial

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Ubiquitin C-terminal hydrolase (UCH)-L3 is an enzyme with a strongly suggested de-ubiquitinating function by in vitro studies, but has poorly been investigated in vivo. In this study, we show that skeletal muscles of Uchl3^−/− mice exhibit the up-regulation of cleaved ATF6, Grp78, and PDI as well as HSP27, HSP70, HSP90 and HSP110, which indicate the induction of stress responses. The prominent accumulation of polyubiquitinated proteins, one of the factors reported to induce stress responses, was observed in the skeletal muscle of Uchl3^−/− mice. Mouse embryonic fibroblasts (MEFs) from Uchl3^−/− mice also showed an accumulation of polyubiquitinated proteins. Moreover, the polyubiquitinated protein accumulation in Uchl3^−/− MEFs was attenuated by the exogenous expression of wild-type, but not hydrolase activity deficient, UCH-L3. In addition, wild-type, but not its hydrolase activity or ubiquitin binding activity deficient UCH-L3 showed the ability to cleave ubiquitin from polyubiquitinated lysozyme in vitro. These results suggest that UCH-L3 functions as a de-ubiquitinating enzyme in vivo where lack of its hydrolase activity may result in the prominent accumulation of ubiquitinated proteins and subsequent induction of stress responses in skeletal muscle.

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¹: Present address: Department of Pathology and Taub Institute, Columbia University, New York, NY 10032, USA.

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Ubiquitin dimers control the hydrolase activity of UCH-L3

Abstract

Introduction

Section snippets

Isolation and culture of mouse embryonic fibroblasts (MEFs) and their immortalization

UCH-L3 but not UCH-L1 interacts with di-Ub

Discussion

Acknowledgements

Am. J. Pathol.

Trends Neurosci.

Cell

Cell

J. Biol. Chem.

Cell

Biochem. Biophys. Res. Commun.

Am. J. Pathol.

Neurochem. Int.

Neurochem. Int.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Structural basis for conformational plasticity of the Parkinson's disease-associated ubiquitin hydrolase UCH-L1

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

The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction

Physiol. Rev.

Specific and covalent targeting of conjugating and deconjugating enzymes of ubiquitin-like proteins

Mol. Cell Biol.

The ubiquitin system

Annu. Rev. Biochem.

Protein regulation by monoubiquitin

Nat. Rev. Mol. Cell Biol.

Crystal structure of a deubiquitinating enzyme (human UCH-L3) at 1,8 A resolution

Embo J.

Aberrant molecular properties shared by familial Parkinson's disease-associated mutant UCH-L1 and carbonyl-modified UCH-L1

Hum. Mol. Genet.

A ubiquitin ligase complex assembles linear polyubiquitin chains

Embo J.

Weighing in on ubiquitin: the expanding role of mass-spectrometry-based proteomics

Nat. Cell Biol.