Abstract
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 Arg167 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.
Highlights
Gene product huntingtin (Htt) [1, 2]
As the result of the above screen, we found that two cysteine proteases of papain-like structure, bleomycin hydrolase (BLMH) and cathepsin Z, are likely candidates to carry out the cp-2 cleavage of Htt in HEK293 and HT22 cells
We developed a direct assay for Htt proteolysis based on the detection of cp-1 and cp-2 fragments, generated from a portion of expanded Htt (N511–52Q, Fig. 1A)
Summary
Plasmids and Mutagenesis—Truncated Htt expression constructs N511– 8Q/82Q were generated as described previously [31] from the full-length Htt constructs (HD-FL-23/82Q) by an introduction of a stop codon after amino acid 511 of Htt. All cells were transfected with constructs encoding Htt or with selected proteases using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s protocol. For BLMH, cleavage lysates (50 g of total protein) were incubated for 1 h at 37 °C with equal volumes of recombinant human BLMH (0.8 g/reaction; R&D Systems) prepared in the assay buffer according to manufacturer’s protocol (50 mM HEPES, 5 mM EDTA, 10 mM DTT, pH 7.0). Cell lysates (50 g of total protein) were incubated for 1 h at 37 °C with equal volumes of the enzyme (0.4 or 0.6 g/reaction) prepared in the assay buffer according to manufacturer’s protocol (25 mM sodium acetate, pH 3.5, 5 mM DTT). Caspase-3 Activation Cytotoxicity Assay—HT22 cells were plated in chamber slides and co-transfected with indicated Htt constructs and siRNA pools to BLMH or cathepsin Z as described above. Ͼ200 – 400 transfected cells were counted per condition, and the number of cells expressing both Htt and active caspase-3 were presented as a percentage of the total number of Htt-positive cells
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