Proteases Acting on Mutant Huntingtin Generate Cleaved Products that Differentially Build Up Cytoplasmic and Nuclear Inclusions

Lunkes, Astrid; Lindenberg, Katrin S.; Ben-Haı̈em, Léa; Weber, C. R.; Devys, Didier; Landwehrmeyer, G. Bernhard; Mandel, Jean‐Louis; Trottier, Yvon

doi:10.1016/s1097-2765(02)00602-0

Cited by 366 publications

(325 citation statements)

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“…Two short N-terminal fragments designated cp-A and cp-B had previously been observed in NG108 -15 (neuroblastoma-glioma) cells with proteasomes inhibition, and the cp-A site was mapped to residues 105-114 (13). In several cell models, we consistently detect two similar fragments.…”

supporting

confidence: 63%

Cysteine Proteases Bleomycin Hydrolase and Cathepsin Z Mediate N-terminal Proteolysis and Toxicity of Mutant Huntingtin

Ratovitski

Chighladze

Waldron

et al. 2011

Journal of Biological Chemistry

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N-terminal proteolysis of huntingtin is thought to be an important mediator of HD pathogenesis. The formation of short N-terminal fragments of huntingtin (cp-1/cp-2, cp-A/cp-B) has been demonstrated in cells and in vivo. We previously mapped the cp-2 cleavage site by mass spectrometry to position Arg 167 of huntingtin. The proteolytic enzymes generating short N-terminal fragments of huntingtin remain unknown. To search for such proteases, we conducted a genome-wide screen using an RNA-silencing approach and an assay for huntingtin proteolysis based on the detection of cp-1 and cp-2 fragments by Western blotting. The primary screen was carried out in HEK293 cells, and the secondary screen was carried out in neuronal HT22 cells, transfected in both cases with a construct encoding the N-terminal 511 amino acids of mutant huntingtin. For additional validation of the hits, we employed a complementary assay for proteolysis of huntingtin involving overexpression of individual proteases with huntingtin in two cell lines. The screen identified 11 enzymes, with two major candidates to carry out the cp-2 cleavage, bleomycin hydrolase (BLMH) and cathepsin Z, which are both cysteine proteases of a papain-like structure. Knockdown of either protease reduced cp-2 cleavage, and ameliorated mutant huntingtin induced toxicity, whereas their overexpression increased the cp-2 cleavage. Both proteases partially co-localized with Htt in the cytoplasm and within or in association with early and late endosomes, with some nuclear co-localization observed for cathepsin Z. BLMH and cathepsin Z are expressed in the brain and have been associated previously with neurodegeneration. Our findings further validate the cysteine protease family, and BLMH and cathepsin Z in particular, as potential novel targets for HD therapeutics.Huntington disease (HD) 3 is caused by an expansion of the CAG repeat coding for polyglutamine (polyQ) within the HD gene product huntingtin (Htt) (1, 2). It is not clear how mutant Htt causes neuronal cell death, but evidence is accumulating that N-terminal fragments of mutant Htt are important mediators of pathogenesis. N-terminal fragments of Htt are found in human postmortem brain (3-8). In cell model experiments, shorter N-terminal fragments of Htt are generally more toxic to cells than longer fragments, and mouse HD models expressing shorter fragments usually develop more severe pathology than the full-length Htt models (6, 9 -19). The proteolytic pathway for mutant Htt may include several cleavage events mediated by different enzymes involved in the generation of toxic fragments of various lengths. These enzymes may represent potential novel therapeutic targets for HD.One example of such an enzyme is caspase-6, which cleaves mutant Htt at residue 586 from its N terminus and generates a toxic fragment; YAC128 mice expressing Htt with an altered caspase-6 site have a substantially ameliorated HD phenotype (20), whereas transgenic mice expressing a caspase-6 fragment with an expanded polyQ repeat develop an HD phe...

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supporting

confidence: 63%

Cysteine Proteases Bleomycin Hydrolase and Cathepsin Z Mediate N-terminal Proteolysis and Toxicity of Mutant Huntingtin

Ratovitski

Chighladze

Waldron

et al. 2011

Journal of Biological Chemistry

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“…Total cellular extracts were subsequently fractionated by microcentrifugation as described in ref. 21. The supernatant of this fractionation contains soluble proteins (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Monoclonal 2B4 is described in ref. 21. Calnexin polyclonal serum and GRP94 antibody were purchased from Stressgen, and ubiquitin rabbit antiserum was from Sigma.…”

Section: Methodsmentioning

confidence: 99%

Targeting expression of expanded polyglutamine proteins to the endoplasmic reticulum or mitochondria prevents their aggregation

Rousseau¹,

Dehay²,

Ben-Haı̈em³

et al. 2004

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

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Aggregation of misfolded proteins is a characteristic of several neurodegenerative diseases. The huntingtin amino-terminal fragment with extended polyglutamine repeat forms aggregates closely associated with chaperones both in the cytoplasm and the nucleus. Because each cellular compartment contains distinct chaperones and because the molecular mechanisms controlling polyglutamine aggregation are largely unknown, we decided to investigate the influence of different cellular environments on the aggregation of this pathological protein. Here, we show that aggregation of a protein containing a polyglutamine stretch of pathological length is abolished when its expression is targeted to the endoplasmic reticulum. Once retrogradely transported outside the endoplasmic reticulum, the aggregation-prone polyglutaminecontaining protein recovers its ability to aggregate. When expressed in the mitochondria, a protein containing 73 glutamines is entirely soluble, whereas the nucleocytosolic equivalent has an extremely high tendency to aggregate. Our data imply that polyglutamine aggregation is a property restricted to the nucleocytosolic compartment and suggest the existence of compartmentspecific cofactors promoting or preventing aggregation of pathological proteins.

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“…Supernatants were collected and analysed on SDS-PAGE gels. To recover insoluble fractions, pellets were solubilized with formic acid, lyophilised, resuspended in SDS buffer (final concentration of 2% SDS, 5% β-mercaptoethanol, 15% glycerol) and analysed on SDS-PAGE, as described (Lunkes et al, 2002). Primary antibodies, rabbit polyclonal anti-Nrl , rabbit polyclonal anti-cJun sc45 (Santa Cruz Biotechnology), rabbit polyclonal anti-phospho-ser63-c-Jun antibody (Cell Signalling), rabbit polyclonal anti-phospho-JNK antibody (Cell Signalling), mouse monoclonal anti-polyQ 1C2 (Trottier et al, 1995), mouse monoclonal anti-ataxin-7 1C1 (Yvert et al, 2000), mouse monoclonal anti-rhodopsin 4D2 (gift from D. Hicks) and mouse monoclonal anti-β-tubulin (Chemicon), were used at 1:1000 dilutions and revealed with appropriate anti-mouse or anti-rabbit peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) and the ECL chemiluminescent reaction (Pierce).…”

Section: Western Blotting Analysismentioning

confidence: 99%

Preventing polyglutamine-induced activation of c-Jun delays neuronal dysfunction in a mouse model of SCA7 retinopathy

Mérienne¹,

Friedman²,

Akimoto³

et al. 2007

Neurobiology of Disease

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We have approached the role of cellular stress in neurodegenerative diseases caused by polyglutamine expansion (polyQ) in the context of Spinocerebellar ataxia type 7 (SCA7) that includes retinal degeneration. Using the R7E mouse, in which polyQ-ataxin-7 is specifically over-expressed in rod photoreceptors, we previously showed that rod dysfunction correlated to moderate and prolonged activation of the JNK/c-Jun stress pathway. SCA7 retinopathy was also associated with reduced expression of rod-specific genes, including the transcription factor Nrl, which is essential for rod differentiation and function. Here, we report that R7E retinopathy is improved upon breeding with the JunAA knock-in mice, in which JNK-mediated activation of c-Jun is compromised. Expression of Nrl and its downstream targets, which are involved in phototranduction, are partially restored in the JunAA-R7E mice. We further show that c-Jun can directly repress the transcription of Nrl. Our studies suggest that polyQ-induced cellular stress leads to repression of genes necessary for neuronal fate and function.

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Proteases Acting on Mutant Huntingtin Generate Cleaved Products that Differentially Build Up Cytoplasmic and Nuclear Inclusions

Cited by 366 publications

References 36 publications

Cysteine Proteases Bleomycin Hydrolase and Cathepsin Z Mediate N-terminal Proteolysis and Toxicity of Mutant Huntingtin

Cysteine Proteases Bleomycin Hydrolase and Cathepsin Z Mediate N-terminal Proteolysis and Toxicity of Mutant Huntingtin

Targeting expression of expanded polyglutamine proteins to the endoplasmic reticulum or mitochondria prevents their aggregation

Preventing polyglutamine-induced activation of c-Jun delays neuronal dysfunction in a mouse model of SCA7 retinopathy

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