Inhibition of Calpain Cleavage of Huntingtin Reduces Toxicity

Self Cite

Caspase-7 is an executioner caspase that plays a key role in apoptosis, cancer, and a number of neurodegenerative diseases. The mechanism of caspase-7 activation by granzyme B and caspase-3 has been well characterized. However, whether other proteases such as calpains activate or inactivate caspase-7 is not known. Here, we present that recombinant caspase-7 is directly cleaved by calpain-1 within the large subunit of caspase-7 to produce two novel products, large subunit p18 and p17. This new form of caspase-7 has a 6-fold increase in V max when compared with the previously characterized p20/p12 form. Zymography revealed that the smaller caspase-7 product (p17) is 18-fold more active than either the caspase-3-cleaved product (p20) or the larger calpain-1 product of caspase-7 (p18). Mass spectrometry and site-directed mutagenesis identified the calpain cleavage sites within the caspase-7 large subunit at amino acid 36 and 45/47. These proteolysis events occur in vivo as indicated by the accumulation of caspase-7 p18 and p17 subunits in cortical neurons undergoing Ca 2؉ dysregulation. Further, cleavage at amino acid 45/47 of caspase-7 by calpain results in a reduction in nuclear localization when compared with the caspase-3 cleavage product of caspase-7 (p20). Our studies suggest the calpain-activated form of caspase-7 has unique enzymatic activity, localization, and binding affinity when compared with the caspase-activated form.Apoptosis is a well-defined cellular destruction pathway that primarily utilizes a family of cysteine proteases, the caspases (1, 2). This cell death program can be initiated by cell death receptor activation (extrinsic pathway) or a variety of drugs or cellular stresses (intrinsic pathway) leading to activation of apical caspase-8, -9, and/or -10 (1, 3, 4). These initiator caspases in turn directly activate the executioner caspases, caspase-3 and -7, which through proteolysis of defined substrates are responsible for the dismantling of the cell and subsequent death (3, 4). Granzyme B, released by cytotoxic T lymphocytes to protect the host from pathogens and tumor cells, can also initiate this apoptotic cascade and therefore is considered an apical caspase mimic (5-7). All caspases, as well as granzyme B, preferentially cleave after aspartic acid residues, with many having well-defined consensus sequences, making substrate cleavage sites easy to predict and establish (3,4,7,8).Caspases exist in a latent form prior to activation. Both the initiator and executioner caspases are synthesized as a single chain protein, which require proteolytic cleavage to become active. Procaspase-7 is expressed as a 303-amino acid residue polypeptide chain. The activation and regulation of executioner caspase-7 by caspases and granzyme B has been extensively studied. Caspase-7 requires cleavage by caspase-3 and caspase-8/-10 or granzyme B, for activation (6, 9). Current evidence suggests that caspase-3 initially cleaves off the first 23 amino acids (propeptide, 2 kDa), followed by caspase-8/-10 or granzyme B...

Section: Discussionsupporting

confidence: 72%

“…We and others (17,19) have previously shown that mutating a single amino acid is not adequate to prevent recognition of the substrate. Therefore we used deletion analysis in which we removed three amino acids in the region of cleavage.…”

Section: Confirmation Of the Three Calpain Cleavage Sites In Caspase-mentioning

confidence: 99%

Calpain-1 Cleaves and Activates Caspase-7

Gafni

Cong

Chen

et al. 2009

Self Cite

“…Caspase enzymes that can cleave Htt include caspase-2, -3, -6, and -7, and cleavage occurs between amino acids 513 and 587 (9,11,12). Htt is cleaved in several places by calpains as well (13,14) and this may also contribute to toxicity (15). The major calpain sites are located between amino acids 469 and 537 (13,15).…”

Section: Huntington Disease (Hd)mentioning

confidence: 99%

“…Htt is cleaved in several places by calpains as well (13,14) and this may also contribute to toxicity (15). The major calpain sites are located between amino acids 469 and 537 (13,15).…”

Section: Huntington Disease (Hd)mentioning

confidence: 99%

Huntingtin Phosphorylation Sites Mapped by Mass Spectrometry

Schilling

Gafni

Torcassi

et al. 2006

Self Cite

133

114

Huntingtin (Htt) is a large protein of 3144 amino acids, whose function and regulation have not been well defined. Polyglutamine (polyQ) expansion in the N terminus of Htt causes the neurodegenerative disorder Huntington disease (HD). The cytotoxicity of mutant Htt is modulated by proteolytic cleavage with caspases and calpains generating N-terminal polyQ-containing fragments. We hypothesized that phosphorylation of Htt may modulate cleavage and cytotoxicity. In the present study, we have mapped the major phosphorylation sites of Htt using cell culture models (293T and PC12 cells) expressing fulllength myc-tagged Htt constructs containing 23Q or 148Q repeats. Purified myc-tagged Htt was subjected to mass spectrometric analysis including matrix-assisted laser desorption/ionization mass spectrometry and nano-HPLC tandem mass spectrometry, used in conjunction with on-target alkaline phosphatase and protease digestions. We have identified more than six novel serine phosphorylation sites within Htt, one of which lies in the proteolytic susceptibility domain. Three of the sites have the consensus sequence for ERK1 phosphorylation, and addition of ERK1 inhibitor blocks phosphorylation at those sites. Other observed phosphorylation sites are possibly substrates for CDK5/CDC2 kinases. Mutation of amino acid Ser-536, which is located in the proteolytic susceptibility domain, to aspartic acid, inhibited calpain cleavage and reduced mutant Htt toxicity. The results presented here represent the first detailed mapping of the phosphorylation sites in full-length Htt.

“…The proteases of this family have been implicated previously in Htt proteolysis; calpain-mediated cleavage of Htt (at residues 469 and 536) may be linked to mutant Htt toxicity (10,(21)(22)(23)25). Our previous findings indicate that calpain may be also involved in degradation of cp-1/cp-2 fragments as calpain inhibitors caused increased fragment accumulation in PC12 cells (32).…”

Section: Discussionmentioning

confidence: 97%

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

Ratovitski

Chighladze

Waldron

et al. 2011

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...