Huntingtin phosphorylation sites mapped by mass spectrometry. Modulation of cleavage and toxicity

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 full-length 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. Dissection of phosphorylation modifications in Htt may provide clues to Huntington disease pathogenesis and targets for therapeutic development.     

N-Terminal Proteolysis of Full-Length Mutant Huntingtin in an Inducible PC12 Cell Model of Huntington’s Disease

Proteolytic cleavage of mutant huntingtin may play a key role in the pathogenesis of Huntington’s disease; however the steps in huntingtin proteolysis are not fully understood. Huntingtin was shown to be cleaved by caspases and calpains within a region between 460-600 amino acids from the N-terminus. Two smaller N-terminal fragments produced by unknown protease have been previously described as cp-A and cp-B. To further investigate the huntingtin proteolytic pathway, we used an inducible PC12 cell model expressing full-length huntingtin with either normal or expanded polyglutamine. This cell model recapitulates several steps of huntingtin proteolysis: proteolysis mediated by caspases within the region previously mapped for caspase cleavage, and cleavage generating two novel N-terminal fragments (cp-1 approximately 90-105 residues long and cp-2 extending beyond 115-129 epitope of huntingtin). Interestingly, the deletion of amino acids 105-114 (mapped previously as a cleavage site for cp-A) failed to affect the production of cp-1 or cp-2. Therefore, we conclude that these new fragments are distinct from previously described cp-A and cp-B. We demonstrate that cp-1 and cp-2 fragments are produced and accumulate within nuclear and cytoplasmic inclusions prior to huntingtin-induced cell toxicity, and these fragments can be formed by caspase-independent proteolytic cleavage of huntingtin in PC12 cells. In addition, inhibition of calpains leads to decreased subsequent degradation of cp-1 and cp-2 fragments, and accelerated formation of inclusions. Further delineation of huntingtin cleavage events may lead to novel therapeutic targets for HD.     

Expanded Polyglutamine-containing N-terminal Huntingtin Fragments Are Entirely Degraded by Mammalian Proteasomes

Huntington disease is a neurodegenerative disorder caused by an expanded polyglutamine (polyQ) repeat within the protein huntingtin (Htt). N-terminal fragments of the mutant Htt (mHtt) proteins containing the polyQ repeat are aggregation-prone and form intracellular inclusion bodies. Improving the clearance of mHtt fragments by intracellular degradation pathways is relevant to obviate toxic mHtt species and subsequent neurodegeneration. Because the proteasomal degradation pathway has been the subject of controversy regarding the processing of expanded polyQ repeats, we examined whether the proteasome can efficiently degrade Htt-exon1 with an expanded polyQ stretch both in neuronal cells and in vitro. Upon targeting mHtt-exon1 to the proteasome, rapid and complete clearance of mHtt-exon1 was observed. Proteasomal degradation of mHtt-exon1 was devoid of polyQ peptides as partial cleavage products by incomplete proteolysis, indicating that mammalian proteasomes are capable of efficiently degrading expanded polyQ sequences without an inhibitory effect on the proteasomal activity.     

Interaction with polyglutamine-expanded huntingtin alters cellular distribution and RNA processing of huntingtin yeast two-hybrid protein A (HYPA)

Huntington disease (HD) is an autosomal inherited disorder that causes the deterioration of brain cells. The polyglutamine (polyQ) expansion of huntingtin (Htt) is implicated in the pathogenesis of HD via interaction with an RNA splicing factor, Htt yeast two-hybrid protein A/forming-binding protein 11 (HYPA/FBP11). Besides the pathogenic polyQ expansion, Htt also contains a proline-rich region (PRR) located exactly in the C terminus to the polyQ tract. However, how the polyQ expansion influences the PRR-mediated protein interaction and how this abnormal interaction leads to the biological consequence remain elusive. Our NMR structural analysis indicates that the PRR motif of Htt cooperatively interacts with the tandem WW domains of HYPA through domain chaperoning effect of WW1 on WW2. The polyQ-expanded Htt sequesters HYPA to the cytosolic location and then significantly reduces the efficiency of pre-mRNA splicing. We propose that the toxic gain-of-function of the polyQ-expanded Htt that causes dysfunction of cellular RNA processing contributes to the pathogenesis of HD.     

Huntingtin proteolysis releases non‐polyQ fragments that cause toxicity through dynamin 1 dysregulation

Cleavage of mutant huntingtin (HTT) is an essential process in Huntington's disease (HD), an inherited neurodegenerative disorder. Cleavage generates N‐ter fragments that contain the polyQ stretch and whose nuclear toxicity is well established. However, the functional defects induced by cleavage of full‐length HTT remain elusive. Moreover, the contribution of non‐polyQ C‐terminal fragments is unknown. Using time‐ and site‐specific control of full‐length HTT proteolysis, we show that specific cleavages are required to disrupt intramolecular interactions within HTT and to cause toxicity in cells and flies. Surprisingly, in addition to the canonical pathogenic N‐ter fragments, the C‐ter fragments generated, that do not contain the polyQ stretch, induced toxicity via dilation of the endoplasmic reticulum (ER) and increased ER stress. C‐ter HTT bound to dynamin 1 and subsequently impaired its activity at ER membranes. Our findings support a role for HTT on dynamin 1 function and ER homoeostasis. Proteolysis‐induced alteration of this function may be relevant to disease.     

IKKα and IKKβ Regulation of DNA Damage-Induced Cleavage of Huntingtin

Background

Proteolysis of huntingtin (Htt) plays a key role in the pathogenesis of Huntington''s disease (HD). However, the environmental cues and signaling pathways that regulate Htt proteolysis are poorly understood. One stimulus may be the DNA damage that accumulates in neurons over time, and the subsequent activation of signaling pathways such as those regulated by IκB kinase (IKK), which can influence neurodegeneration in HD.

Methodology/Principal Findings

We asked whether DNA damage induces the proteolysis of Htt and if activation of IKK plays a role. We report that treatment of neurons with the DNA damaging agent etoposide or γ-irradiation promotes cleavage of wild type (WT) and mutant Htt, generating N-terminal fragments of 80–90 kDa. This event requires IKKβ and is suppressed by IKKα. Elevated levels of IKKα, or inhibition of IKKβ expression by a specific small hairpin RNA (shRNA) or its activity by sodium salicylate, prevents Htt proteolysis and increases neuronal resistance to DNA damage. Moreover, IKKβ phosphorylates the anti-apoptotic protein Bcl-xL, a modification known to reduce Bcl-xL levels, and activates caspases that can cleave Htt. When IKKβ expression is blocked, etoposide treatment does not decrease Bcl-xL and activation of caspases is diminished. Similar to silencing of IKKβ, increasing the level of Bcl-xL in neurons prevents etoposide-induced caspase activation and Htt proteolysis.

Conclusions/Significance

These results indicate that DNA damage triggers cleavage of Htt and identify IKKβ as a prominent regulator. Moreover, IKKβ-dependent reduction of Bcl-xL is important in this process. Thus, inhibition of IKKβ may promote neuronal survival in HD as well as other DNA damage-induced neurodegenerative disorders.     

Does Huntingtin play a role in selective macroautophagy?

The accumulation of protein aggregates in neurons appears to be a basic feature of neurodegenerative disease. In huntington disease (HD), a progressive and ultimately fatal neurodegenerative disorder caused by an expansion of the polyglutamine repeat within the protein huntingtin (Htt), the immediate proximal cause of disease is well understood. However, the cellular mechanisms which modulate the rate at which fragments of Htt containing polyglutamine accumulate in neurons is a central issue in the development of approaches to modulate the rate and extent of neuronal loss in this disease. We have recently found that Htt is phosphorylated by the kinase IKK on serine (s) 13, activating its phosphorylation on S16 and its acetylation and poly-SUMOylation, modifications that modulate its clearance by the proteasome and lysosome in cells.¹ In the discussion here I suggest that Htt may have a normal function in the lysosomal mechanism of selective macroautophagy involved in its own degradation which may share some similarity with the yeast cytoplasm to vacuole targeting (Cvt) pathway. Pharmacologic activation of this pathway may be useful early in disease progression to treat HD and other neurodegenerative diseases characterized by the accumulation of disease proteins.Key words: Huntington disease, Huntingtin, polyglutamine, autophagy, IKKAn age-related reduction in protein clearance mechanisms has been implicated in the pathogenesis of neurodegenerative diseases including the polyglutamine (polyQ) repeat diseases, Alzheimer disease (AD), Parkinson disease (PD) and Amyotrophic Lateral Sclerosis (ALS). These diseases are each associated with the accumulation of insoluble protein aggregates in diseased neurons. Huntington Disease (HD), caused by an expansion of the polyQ repeat in the protein Huntingtin (Htt), is one such disease of aging in which mutant Htt inclusions form in striatal and cortical neurons as disease progresses. Clarification of the mechanisms of Htt clearance is paramount to finding therapeutic targets to treat HD that may be broadly useful in the treatment of these currently incurable neurodegenerative diseases.     

The Role of Chaperone-Mediated Autophagy in Huntingtin Degradation

Huntington Disease (HD) is caused by an abnormal expansion of polyQ tract in the protein named huntingtin (Htt). HD pathology is featured by accumulation and aggregation of mutant Htt in striatal and cortical neurons. Aberrant Htt degradation is implicated in HD pathogenesis. The aim of this study was to investigate the regulatory role of chaperone-mediated autophagy (CMA) components, heat shock protein cognate 70 (Hsc70) and lysosome-associated protein 2A (LAMP-2A) in degradation of Htt fragment 1-552aa (Htt-552). A cell model of HD was produced by overexpression of Htt-552 with adenovirus. The involvement of CMA components in degradation of Htt-552 was determined with over-expression or silencing of Hsc70 and LAMP-2A. The results confirmed previous reports that both macroautophagy and CMA were involved in degradation of Htt-552. Changing the levels of CMA-related proteins affected the accumulation of Htt-552. The lysosomal binding and luminal transport of Htt-552 was demonstrated by incubation of Htt-552 with isolated lysosomes. Expansion of the polyQ tract in Htt-552 impaired its uptake and degradation by lysosomes. Mutation of putative KFERQ motif in wild-type Htt-552 interfered with interactions between Htt-552 and Hsc70. Endogenous Hsc70 and LAMP-2A interacted with exogenously expressed Htt-552. Modulating the levels of CMA related proteins degraded endogenous full-length Htt. These studies suggest that Hsc70 and LAMP-2A through CMA play a role in the clearance of Htt and suggest a novel strategy to target the degradation of mutant Htt.     

Calcium-dependent cleavage of endogenous wild-type huntingtin in primary cortical neurons

Huntington's disease (HD) is caused by a polyglutamine expansion in the amino-terminal region of huntingtin. Mutant huntingtin is proteolytically cleaved by caspases, generating amino-terminal aggregates that are toxic for cells. The addition of calpains to total brain homogenates also leads to cleavage of wild-type huntingtin, indicating that proteolysis of mutant and wild-type huntingtin may play a role in HD. Here we report that endogenous wild-type huntingtin is promptly cleaved by calpains in primary neurons. Exposure of primary neurons to glutamate or 3-nitropropionic acid increases intracellular calcium concentration, leading to loss of intact full-length wild-type huntingtin. This cleavage could be prevented by calcium chelators and calpain inhibitors. Degradation of wild-type huntingtin by calcium-dependent proteases thus occurs in HD neurons, leading to loss of wild-type huntingtin neuroprotective activity.     

Deranged neuronal calcium signaling and Huntington disease

Huntington disease (HD) is an autosomal-dominant neurodegenerative disorder that primarily affects medium spiny striatal neurons (MSN). HD is caused by polyglutamine (polyQ) expansion (exp) in the amino-terminal region of a protein huntingtin (Htt). The connection between polyQ expansion in Httexp and MSN neurodegeneration remains elusive. Here we discuss recent data that link polyQ expansion in Httexp and deranged Ca2+ signaling in MSN neurons. Experimental evidence indicates that (1) Ca2+ homeostasis is abnormal in mitochondria isolated from lymphoblasts of HD patients and from brains of the YAC72 HD mouse model; (2) Httexp leads to potentiation of NR1/NR2B NMDA receptor activity in heterologous expression systems and in MSN from YAC72 HD mouse model; and (3) Httexp binds to the type 1 inositol 1,4,5-trisphosphate receptor (InsP3R1) carboxy-terminus and causes sensitization of InsP3R1 to activation by InsP3 in planar lipid bilayers and in MSN. Based on these results we propose that Httexp-induced cytosolic and mitochondrial Ca2+ overload of MSN plays an important role in the pathogenesis of HD and that Ca2+ signaling blockers may play a beneficial role in treatment of HD.     

Calpain inhibition mediates autophagy-dependent protection against polyglutamine toxicity

Over recent years, accumulated evidence suggests that autophagy induction is protective in animal models of a number of neurodegenerative diseases. Intense research in the field has elucidated different pathways through which autophagy can be upregulated and it is important to establish how modulation of these pathways impacts upon disease progression in vivo and therefore which, if any, may have further therapeutic relevance. In addition, it is important to understand how alterations in these target pathways may affect normal physiology when constitutively modulated over a long time period, as would be required for treatment of neurodegenerative diseases. Here we evaluate the potential protective effect of downregulation of calpains. We demonstrate, in Drosophila, that calpain knockdown protects against the aggregation and toxicity of proteins, like mutant huntingtin, in an autophagy-dependent fashion. Furthermore, we demonstrate that, overexpression of the calpain inhibitor, calpastatin, increases autophagosome levels and is protective in a mouse model of Huntington''s disease, improving motor signs and delaying the onset of tremors. Importantly, long-term inhibition of calpains did not result in any overt deleterious phenotypes in mice. Thus, calpain inhibition, or activation of autophagy pathways downstream of calpains, may be suitable therapeutic targets for diseases like Huntington''s disease.Huntington''s disease (HD) is a currently incurable, autosomal dominant neurodegenerative disease resulting from the expansion of the trinucleotide (CAG) repeat region of the huntingtin (HTT) IT15 gene, encoding huntingtin protein (Htt). In mutant Htt, the polyglutamine tract encoded by this region contains over 35 glutamines and the length of the tract correlates inversely with the age of disease onset, with longer tracts resulting in earlier onset (reviewed in Imarisio et al.¹). HD is one of the 10 trinucleotide repeat disorders resulting from expansions of polyglutamine tracts in different proteins. These expansions cause disease by conferring toxic gain-of-function properties onto the mutant proteins. Hence, one strategy that has been considered for HD and related diseases is to find ways of decreasing the levels of the mutant protein, for instance by harnessing the cell''s capacity to degrade such aggregate-prone proteins via (macro)autophagy.^{2, 3, 4, 5} Autophagy involves the engulfment of cytoplasmic contents by double-membraned autophagosomes, which then traffic to lysosomes where their contents are degraded. Mutant huntingtin, some other polyglutamine expanded proteins like mutant ataxin 3, and proteins like tau (which mediates toxicity in Alzheimer''s disease and related dementias) are autophagy substrates and their clearance can be enhanced in Drosophila and mouse models by autophagy upregulation, which also reduces their toxicity.^{2, 3, 4,6}Calpains are a family of calcium-activated cysteine proteases (reviewed in Ono and Sorimachi⁷) that inhibit autophagy. Strategies that reduce calpain activity in cell culture increase autophagy and decrease levels of autophagy substrates, like mutant Htt. These effects are likely to be mediated by Gsα, a heterotrimeric G-protein subunit which is activated by calpain cleavage. Similar to calpain inhibition, siRNA knockdown of Gsα, or chemical inhibition by NF449, induces autophagy and decreases the number of aggregates resulting from the overexpression of exon-1 Htt with an expanded polyglutamine repeat region (HttQ74) in cell culture models.⁸ In addition to this mechanism of autophagy upregulation by calpains, the core autophagy protein ATG5 has also been demonstrated to be cleaved and inactivated by calpains,^9,10 suggesting that calpains may act on a number of substrates to negatively regulate autophagy.In mammals, the two most abundantly expressed calpains are μ-calpain and m-calpain, which differ in their affinity for calcium and therefore the calcium concentration required for their activation. As well as being regulated by calcium, they are also controlled by an endogenous inhibitor, calpastatin (CAST). Drosophila have four forms of calpain:¹¹ CalpA and CalpB are the conventional calpains formed by a recent duplication in the Drosophila insect lineage, CalpC is also an evolutionarily recent, but not highly conserved duplication (data not shown) and is thought to be catalytically inactive,¹¹ and CalpD (SOL) is a member of the unconventional family of calpains. Drosophila does not appear to have any obvious orthologs of CAST.A role for calpains in HD has been investigated previously. Following observations that shorter Htt fragments are more toxic than full-length Htt,¹² it was demonstrated that Htt can be cleaved by both caspases¹³ and calpains¹⁴ to generate these toxic, short fragments. Blocking Htt cleavage by calpains by mutating their calpain cleavage sites decreases Htt aggregation and toxicity.¹⁵ In addition, calpain activation has been shown to be increased in HD patients compared with controls.¹⁴In this study, we have investigated a role for calpain activity as a modulator of autophagy in both Drosophila and mouse models of HD. To avoid confounding effects from alterations in cleavage of Htt by calpain, we have used models expressing short fragments of Htt, which do not contain calpain cleavage sites and correspond to the shortest fragments of huntingtin seen in patients.¹⁶ We demonstrate that knockdown of CalpA in Drosophila is sufficient to both reduce the number of Htt aggregates and the toxicity associated with the expression of the mutant protein. Importantly, we show that these effects are autophagy-dependent. Furthermore, we show that overexpression of CAST in mice results in enhanced autophagy and improves locomotor function and delays tremor onset in a mouse model of HD, as well as decreasing the number of Htt aggregates seen in the brain. We extended the analysis of CAST overexpressing mice to investigate the possible adverse effects from long-term calpain inhibition or autophagy upregulation but did not observe any obvious deleterious effects.     

Huntington's Disease

Huntington''s disease (HD) is the most common inherited neurodegenerative disease and is characterized by uncontrolled excessive motor movements and cognitive and emotional deficits. The mutation responsible for HD leads to an abnormally long polyglutamine (polyQ) expansion in the huntingtin (Htt) protein, which confers one or more toxic functions to mutant Htt leading to neurodegeneration. The polyQ expansion makes Htt prone to aggregate and accumulate, and manipulations that mitigate protein misfolding or facilitate the clearance of misfolded proteins tend to slow disease progression in HD models. This article will focus on HD and the evidence that it is a conformational disease.     

Cdk5 phosphorylation of huntingtin reduces its cleavage by caspases: implications for mutant huntingtin toxicity

Huntington's disease (HD) is a neurodegenerative disorder caused by an expanded polyglutamine (polyQ) tract in the huntingtin (htt) protein. Mutant htt toxicity is exposed after htt cleavage by caspases and other proteases release NH(2)-terminal fragments containing the polyQ expansion. Here, we show htt interacts and colocalizes with cdk5 in cellular membrane fractions. Cdk5 phosphorylates htt at Ser434, and this phosphorylation reduces caspase-mediated htt cleavage at residue 513. Reduced mutant htt cleavage resulting from cdk5 phosphorylation attenuated aggregate formation and toxicity in cells expressing the NH(2)-terminal 588 amino acids (htt588) of mutant htt. Cdk5 activity is reduced in the brains of HD transgenic mice compared with controls. This result can be accounted for by the polyQ-expanded htt fragments reducing the interaction between cdk5 and its activator p35. These data predict that the ability of cdk5 phosphorylation to protect against htt cleavage, aggregation, and toxicity is compromised in cells expressing toxic fragments of htt.     

Wild-type huntingtin plays a role in brain development and neuronal survival

While the role of the mutated Huntington's disease (HD) protein in the pathogenesis of HD has been the focus of intensive investigation, the normal protein has received less attention. Nonetheless, the wild-type HD protein appears to be essential for embryogenesis, since deletion of the HD gene in mice results in early embryonic lethality. This early lethality is due to a critical role the HD protein, called huntingtin (Htt), plays in extraembryonic membrane function, presumably in vesicular transport of nutrients. Studies of mutant mice expressing low levels of Htt and of chimeric mice generated by blastocyst injection of Hdh-/- embryonic stem cells show that wildtype Htt plays an important role later in development as well, specifically in forebrain formation. Moreover, various lines of study suggest that normal Htt is also critical for survival of neurons in the adult forebrain. The observation that Htt plays its key developmental and survival roles in those brain areas most affected in HD raises the possibility that a subtle loss of function on the part of the mutant protein or a sequestering of wild-type Htt by mutant Htt may contribute to HD pathogenesis. Regardless of whether this is so, the prosurvival role of Htt suggests that HD therapies that block production of both wild-type and mutant Htt may themselves be harmful.     

Evidence for involvement of calpain in c-Myc proteolysis in vivo

Precise control of the level of c-Myc protein is important to normal cellular homeostasis, and this is accomplished in part by degradation through the ubiquitin-proteasome pathway. The calpains are a family of calcium-dependent proteases that play important roles in proteolysis of some proteins, and their possible participation in degradation of intracellular c-Myc was therefore investigated. Activation of calpain with the cell-permeable calcium ionophore A23187 in Rat1a-myc or ts85 cells in culture induced rapid cleavage of c-Myc. This degradation was both calpain- and calcium-dependent since it was inhibited by preincubation with either the calpain-inhibitory peptide calpeptin or the calcium-chelating agent EGTA. A23187-induced c-Myc cleavage occurred in a time-dependent manner comparable to that of FAK, a known calpain substrate, and while calpeptin was able to significantly protect c-Myc from degradation, inhibitors of the proteasome or caspase proteases could not. Exposure of Rat1a-myc or ts85 cells in culture to calpeptin, or to the thiol-protease inhibitor E64d, resulted in the accumulation of c-Myc protein without an impact on ubiquitin-protein conjugates. Using an in vitro assay, calpain-mediated degradation occurred rapidly with wild-type c-Myc as the substrate, but was significantly prolonged in some c-Myc mutants with increased transforming activity derived from lymphoma patients. Those mutants with a prolonged half-life in vitro were also more resistant to A23187-induced cleavage in intact cells. These studies support a role for calpain in the control of c-Myc levels in vivo, and suggest that mutations impacting on sensitivity to calpain may contribute to c-Myc-mediated tumorigenesis.     

Cleavage at the caspase-6 site is required for neuronal dysfunction and degeneration due to mutant huntingtin

Cleavage of huntingtin (htt) has been characterized in vitro, and accumulation of caspase cleavage fragments represents an early pathological change in brains of Huntington's disease (HD) patients. However, the relationship between htt proteolysis and the pathogenesis of HD is unknown. To determine whether caspase cleavage of htt is a key event in the neuronal dysfunction and selective neurodegeneration in HD, we generated YAC mice expressing caspase-3- and caspase-6-resistant mutant htt. Mice expressing mutant htt, resistant to cleavage by caspase-6 but not caspase-3, maintain normal neuronal function and do not develop striatal neurodegeneration. Furthermore, caspase-6-resistant mutant htt mice are protected against neurotoxicity induced by multiple stressors including NMDA, quinolinic acid (QA), and staurosporine. These results are consistent with proteolysis of htt at the caspase-6 cleavage site being an important event in mediating neuronal dysfunction and neurodegeneration and highlight the significant role of htt proteolysis and excitotoxicity in HD.     

Cysteine proteases bleomycin hydrolase and cathepsin Z mediate N-terminal proteolysis and toxicity of mutant huntingtin

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.