Protein Acylation: from Mechanism to Drug Discovery
Aberrant palmitoylation in Huntington disease Shaun S. Sanders*1 and Michael R. Hayden* *Centre for Molecular Medicine and Therapeutics, Department of Medical Genetics, Child & Family Research Institute, University of British Columbia, Vancouver, British Columbia, V5Z 4H4, Canada
Abstract Huntington disease (HD) is an adult-onset neurodegenerative disease caused by a CAG expansion in the HTT gene. HD is characterized by striatal atrophy and is associated with motor, cognitive and psychiatric deficits. In the presence of the HD mutation, the interactions between huntingtin (HTT) and huntingtin interacting protein 14 (HIP14 or DHHC17) and HIP14-like (DHHC13, a HIP14 orthologue), palmitoyl acyltransferases for HTT, are disturbed, resulting in reduced palmitoylation of HTT. Genetic ablation of either Hip14 or Hip14l recapitulates many features of HD, including striatal atrophy and motor deficits. However, there are no changes in palmitoylation of HTT in either mouse model and, subsequently, the similarities between the phenotypes of these two mouse models and the HD mouse model are believed to result from underpalmitoylation of other HIP14 and HIP14L substrates. HTT acts as a modulator of HIP14 activity such that in the presence of the HD mutation, HIP14 is less active. Consequently, HIP14 substrates are less palmitoylated, leading to neuronal toxicity. This suggests that altered HIP14–HTT and HIP14L–HTT interactions in the presence of the HD mutation reduces palmitoylation and promotes mislocalization of HTT and other HIP14/HIP14L substrates. Ultimately, HD may be, in part, a disease of altered palmitoylation.
Huntington disease Huntington disease (HD) is an adult onset neurodegenerative disease characterized by progressive cognitive, psychiatric and motor deficits. The typical age of onset is 50 years of age and the disease progresses such that death occurs, on an average, 20 years later [1,2]. The striatum, a part of the basal ganglia responsible for controlling movement, is the first area of the brain to be affected with other brain regions, particularly the cortex, affected at later stages of disease progression [1,2]. HD is an autosomal dominant disease caused by a CAG repeat expansion in exon 1 of the HTT gene that leads to a poly-glutamine expansion in the huntingtin (HTT) protein and a toxic gain of function [3]. A recent Canadian study estimated the prevalence of HD at 13.7 in 100,000 in the general population with 81.6 in 100,000 at a 25–50 % risk [4]. There are currently no disease-modifying treatments available. Thus, HD research is aimed at validating and identifying novel drug targets.
The first evidence for a role of palmitoylation in HD The HTT protein undergoes many posttranslational modifications, including phosphorylation, ubiquitination, acetylation, proteolysis, SUMOylation and palmitoylation. These various modifications influence the functions of Key words: Huntington disease, HIP14, zDHHC17, HIP14L, palmitoylation, zDHHC13. Abbreviations: HD, Huntington disease; HTT, huntingtin; wtHTT, wild type HTT; mHTT, mutant HTT; YAC128, full-length human HTT transgenic mouse model of HD; HIP14, huntingtin interacting protein 14; HIP14L, huntingtin interacting protein 14-like; SNAP25, synaptosomal-associated protein 25; PSD-95, post-synaptic density protein 95; APT, acyl-protein thioesterase; MSN, medium spiny neuron. To whom correspondence should be addressed (email [email protected]).
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wild type HTT (wtHTT) and the toxicity and functions of mutant HTT (mHTT) (reviewed in Ref. [5]). Spalmitoylation (simply palmitoylation here) is the reversible addition of long-chain saturated fatty acids, typically the 16-carbon palmitate, to proteins via a labile thioester bond (Figures 1A and 1B) [6,7]. Palmitoylation is mediated by the DHHC-domain containing palmitoyl acyltransferases (PATs) of which humans have 23; named zDhhc1-9 and 11-24 [8]. Depalmitoylation is mediated by acyl-protein thioesterases (APTs), four of which have been identified: the cytosolic ATP1, APT2, APTL1 and the lysosomal PPT1 [9,10]. Palmitoylation controls localization to and within membranes, function, additional post-translation modifications and stability of proteins [11]. The first PATs were identified in yeast more than a decade ago and the identification of the DHHC-cysteine rich domain allowed the rapid identification of the rest [12,13]. At the same time in a screen for interactors of HTT, Huntingtin interacting protein 14 (HIP14, DHHC17) was identified and was shown to interact with wtHTT and to interact less with mHTT [14,15]. An in silico screen for HIP14 orthologues also identified HIP14-like (HIP14L, DHHC13) [15], which also interacted with HTT in a manner inversely correlated to CAG expansion [16]. It is this altered interaction in the presence of the HD mutation that made HIP14 and HIP14L interesting for further study in the context of HD. HIP14 was the first mammalian PAT identified and was shown to palmitoylate HTT and other neuronal proteins [17]. HTT was later shown to be palmitoylated at cysteine 214 (C214) by HIP14 [18] and, in an in cellulo study where all 23 human PATs were co-expressed with HTT, HIP14 and HIP14L were shown to be the only two PATs to significantly palmitoylate HTT [19]. C The
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Figure 1 The process of S-palmitoylation (A) The 16-carbon saturated fatty acid palmitoyl-CoA is added to the thiol group of internal cysteine residues of proteins via a thioester bond by DHHC palmitoyl acyltransferases (PATs). Palmitoylation is reversed by acyl protein thioesterases (APTs). (B) Palmitate inserts in the lipid bilayer of membranes.
Interestingly, HIP14 and HIP14L were found to be unique as they are the only two PATs that contain ankyrin repeat domains and six transmembrane domains (Figure 2) [8,11,20]. The ankyrin repeat domain is the domain of interaction with HTT [19]. The first real evidence of a role for palmitoylation in the pathogenesis of HD came when mHTT was shown to be less palmitoylated in the YAC128 transgenic mouse model of HD [18], which expresses full-length human HTT from the endogenous promoter and accurately recapitulates many features of the human disease [21,22]. Mutating C214 to serine lead to increased aggregate and nuclear inclusion formation, increased cell death and increased susceptibility to excitotoxicity [18]. Similarily, siRNA-mediated decrease in HIP14 expression also increased inclusion formation and susceptibility to excitotoxicity. Interestingly, overexpressing HIP14 in neurons expressing mHTT decreased aggregate formation, suggesting that increasing palmitoylation may be beneficial [18]. The modulation of disease phenotypes by altering HIP14 levels provided strong evidence that HIP14 may be involved in the disease process and that modulating the pathway may be beneficial. C The
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Hip14 deficient mice recapitulate features of HD The strongest piece of evidence implicating palmitoylation in the pathogenesis of HD came from the characterization of the Hip14 deficient mouse model (Hip14 − / − ) [23]. This mouse model was shown to recapitulate many features of the YAC128 mouse. Neuropathological assessments and MRI analysis revealed that there is selective degeneration of the areas of the brain affected in HD in Hip14 − / − mice, including the striatum, cortex, hippocampus and corpus callosum (white matter). Interestingly, it was found that the population of neurons affected in the striatum of the Hip14 − / − mice was the same population affected in HD, namely medium spiny projection interneurons (MSNs) of the striatal indirect pathway involved in preventing unwanted movement [23]. In addition to the neuropathological phenotype, the Hip14 − / − mice were shown to have HD-like motor dysfunction, exhibiting loss of motor function and of sensorimotor gating, the ability to inhibit unwanted motor responses to a stimulus, believed to be mediated by the striatum [23]. Hip14 − / − mice were also shown to exhibit motor inflexibility in a plus maze where Hip14 − / − mice are less
Protein Acylation: from Mechanism to Drug Discovery
Figure 2 DHHC PAT domain structure and membrane topology (A) The typical PAT contains four transmembrane domains and is oriented in the membrane such that the DHHC domain is on the cytosolic side. (B) HIP14 and HIP14L are the only two PATs that have ankyrin repeat domains and are oriented such that both the ankyrin repeat and DHHC domains are on the cytosolic side of the membrane. (C) DHHC 3 and 8 have PDZ-binding domains and are inserted into the membrane so that this domain and the DHHC domain face the cytosol. (D) DHHC6 is the only PAT that has an SH3 domain that also faces the cytosol along with the DHHC domain.
likely to turn and explore the perpendicular arm of a plus maze, something that occurs in HD mouse models as well [24]. Striatal electrophysiological characterization of the Hip14 − / − mice provided further evidence that HIP14 is involved in the pathogenesis of HD [25]. The MSNs of Hip14 − / − mice were shown to be less excitable, to fire fewer action potentials, and to have decreased neuron surface membrane area. The Hip14 − / − MSNs also had fewer excitatory synapses, as shown electrophysiologically and by electron microscopy. The remaining synapses had a lower probability of neurotransmitter release [25]. These MSN cellular and synaptic changes are similar to those in late-stage HD mouse models [25,26]. The phenotypes between Hip14 − / − and YAC128 mice are not completely overlapping. The biggest difference between the phenotype of the Hip14 − / − and YAC128 mice is that the neuropathological and behavioural phenotypes of the Hip14 − / − mice are developmental and not progressive, unlike the adult-onset progressive phenotypes in the YAC128 mice [23]. The phenotype may be much for severe in the Hip14 − / − mice because there is no HIP14, whereas in the YAC128 mice HIP14 is only partly dysfunctional. It will be interesting to determine what happens if HIP14 is lost after the developmental period, i.e. does an HD-like phenotype develop? The similar deficits in the neuropathological, behavioural and electrophysiological phenotypes of the Hip14 − / − mice to HD-mouse models suggest that HIP14 may play an important role in the pathogenesis of HD. Initially it was thought this was due to HTT palmitoylation, but no change in HTT palmitoylation or expression was observed in Hip14 − / − mice. Presumably, this was due to
HIP14L compensating for the loss of HIP14 to palmitoylate HTT. However, palmitoylation of other HIP14 substrates, including the post-synaptic density protein 95 (PSD-95) and the pre-synaptic protein SNAP25 (synaptosomal-associated protein 25), was reduced in brains of Hip14 − / − mice [23]. These data suggest that HD may be partly due to aberrant palmitoylation of HIP14 substrates other than HTT. In a study determining how the palmitoyl proteome was altered in YAC128 and Hip14 − / − mice, the palmitoyl proteomes were compared between the two mouse models [27]. Unexpectedly, the two palmitoyl proteomes were not well correlated. Some proteins did show similar changes in levels of palmitoylation in the two mouse models, but many did not. However, all proteins in the present study that were examined further had a similar level of decrease in expression as in palmitoylation and, unfortunately, there was no way to differentiate in the mass spectrometry experiment between true changes in palmitoylation and changes in the expression/input level of palmitoylated proteins [27]. Indeed, palmitoylation and protein stability are not mutually exclusive, in some cases, loss of palmitoylation leads to ubiquititination and protein degradation, which may further complicate the interpretation of these results [28]. It would be interesting to see what degree of overlap there is between the two palmitoyl proteomes of the two mouse models after correcting for protein expression and input levels. In addition, those proteins that were shown to be changed in the YAC128 mice but not in the Hip14 − / − mice may be altered due to HIP14L dysfunction. Regardless, it may be those proteins that show reduced palmitoylation in both animal models that really contribute to the similarities in disease phenotypes [27]. C The
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Figure 3 A schematic representation of the hypothetical neuronal localization of HIP14 and HIP14L substrates when wild type HTT is present (A) compared with when mutant HTT is present (B; mHTT) In the presence of wild type HTT (wtHTT), HIP14 and HIP14 are fully active and their following substrates are fully palmitoylated and normally localized to the synapse: GAD65, SNAP25, synaptotagmin 1 (SYT1), PSD-95, AMPAR and HTT. In the presence of mHTT, HIP14 and HIP14L do not interact with HTT and are thus less active. This may result in mislocalization of the above substrates and in aggregation of mHTT.
Wild type HTT acts as a regulator of HIP14 function The phenotypic overlap between the Hip14 − / − and YAC128 phenotypes without changes in HTT palmitoylation suggests an alternate mechanism. There is evidence to suggest that the relationship between HIP14 and HTT goes beyond that of just an enzyme-substrate pair. When C214 was mutated to a serine or when the DHHC domain of HIP14 was deleted, HIP14 and HTT still interacted indicating that their interaction is independent of palmitoylation. This is in contrast to the interaction of HIP14 with its other substrate PSD-95, which is dependent on both the palmitoylation sites of PSD-95 itself and on the DHHC domain of HIP14 [19]. Indeed, HIP14 and HTT form such a tight complex that the HIP14 co-immunoprecipitated with HTT from mouse brain lysates had as much activity towards SNAP25 as immunoprecipitated HIP14 alone [19]. However, when HIP14 was immunoprecipitated from YAC128 mouse brain lysate, there was a 50 % decrease in HIP14 in vitro activity towards SNAP25 [23]. This indicates that HIP14 is less active in YAC128 mice. PATs are believed to autopalmitoylate and then transfer the palmitate from themselves to their substrates, thus loss of autopalmitoylation of a PAT suggests reduced activity [12,13,29]. Interestingly, autopalmitoylation C The
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of HIP14 was shown to be decreased in the YAC128 mice [23] or when less wtHTT is present [19]. This suggests that mHTT inhibits HIP14 activity in the YAC128 mice, resulting in reduced palmitoylation and mislocalization of HIP14 substrates in the presence of the HD mutation (Figure 3).
High degree of overlap between HIP14 and HTT interactors The recent finding of a highly significant overlap between interactors of HIP14 and interactors of HTT further implicated the HIP14-HTT complex in the pathogenesis of HD [30]. A yeast 2-hybrid screen for HIP14 interactors identified 214 interactors, including HTT and HIP14 itself. Surprisingly, 36 of the 214 HIP14 interactors were also found to be HTT interactors and seventeen of those have been implicated in HD. This high degree of overlap suggests that HIP14 is critically linked to HTT through its interactions and this warrants further investigation [30]. It will be interesting to know if these shared interactors are substrates or regulators of HIP14 and to know how altered interaction with HIP14 and HTT may contribute to the pathogenesis of HD.
Protein Acylation: from Mechanism to Drug Discovery
Hip14l deficient mice recapitulate features of HD Further evidence for a role of aberrant palmitoylation in HD came from the characterization of the Hip14l deficient mouse model [16]. Although Hip14l − / − mice display peripheral phenotypes not observed in HD mouse models, the neuropathological and behavioural phenotypes closely resemble those of HD mice. Neuropathological assessment revealed a progressive reduction in brain weight and striatal and cortical volume from three to 12 months of age in Hip14l − / − mice [16]. Similar to the Hip14 − / − and YAC128 mice, there was a specific loss the subpopulation of MSNs in the striatum that are lost in HD [16]. These neuropathological phenotypes are more like the progressive late onset degeneration in the YAC128 HD mice than the non-progressive, developmental loss of striatal and cortical volume in the Hip14 − / − mice [23,31,32]. Reduced volume of the globus pallidus, thalamus and corpus callosum was also observed in the Hip14l − / − mice, whereas the hippocampus was unchanged [16]. These neuropathological changes are actually more similar to the changes in the brain of YAC128 mice than to those in the Hip14 − / − mice as they are progressive adult-onset alterations unlike the developmental changes in the Hip14 − / − mice [23,32]. Assessing behaviour of the Hip14l − / − mice revealed deficits similar to those observed in the YAC128 and Hip14 − / − mice [23,31]. Hip14l − / − mice also displayed deficits in motor learning and motor coordination [16]. Taken together, these data indicate that Hip14l − / − mice display HD-like behavioural deficits [16]. Features of HD displayed in Hip14l − / − mice could result from reduced palmitoylation of key neuronal HIP14L substrates. However, HTT palmitoylation was not altered in Hip14l − / − mice, as was the case in the Hip14 − / − mice [16,23]. It is possible that HIP14 and HIP14L compensate for loss of each other for palmitoylation of HTT and some but not all other substrates. Overlap in PAT-substrate specificity have been previously documented [33,34]. Determining the substrate profiles for the two enzymes and how they overlap would provide an explanation for the similarities and differences between these two mouse models.
temporal and tissue specific deletion of either or both genes. It will also be interesting to know what happens in the case where both Hip14 and Hip14l are lost. The present experiment may provide further insight into the relationship between HIP14/HIP14L and HTT. The fact that wtHTT acts as a modulator of HIP14 activity and that the relationship between these two proteins goes beyond that of just enzyme-substrate pair suggests that this relationship may be very important [19]. The overall hypothesis from the work described here is that disturbed HIP14–HTT and HIP14L–HTT interaction in the presence of the HD mutation reduces HIP14, and potentially HIP14L, function leading to the under-palmitoylation and mislocalization of HTT and key HIP14 and HIP14L substrates (Figure 3). It still remains unclear exactly what the nature of the interaction between HIP14 and HTT is. There are a number of possibilities. First, HTT may act as an allosteric activator of HIP14, inducing a conformational change when bound to HIP14, making it more active. Second, HTT may act as a scaffold to bring HIP14 into proximity with its substrates. Third, HTT may traffic HIP14 to specific subcellular sites to palmitoylate its substrates. It is also still unknown if HTT also modulates HIP14L activity as with HIP14. Given the evidence presented here for aberrant palmitoylation in HD, one therapeutic approach could involve restoring levels of palmitoylation. This would be achieved by targeting the relevant PATs, i.e. HIP14 and HIP14L, or thioesterase enzymes. We have just begun to understand the complex relationships between PATs and their substrates and to understand the regulation of this posttranslational modification. The complexity of the role that these enzymes play in disease processes is not yet fully understood. In order to appropriately target and predict potential side effects, a much better understanding of these processes is required.
Acknowledgements We thank Dr. Dale Martin for assistance with the figures and for providing feedback on the manuscript, and Mandi Schmidt for assistance with the figures.
Conclusions and outstanding questions The Hip14 − / − and Hip14l − / − mouse models, both of which have HD-like phenotypes resembling those of the YAC128 mouse model of HD, provide the strongest evidence for a role of palmitoylation in HD [16,23]. The Hip14l − / − mouse more closely resembles the YAC128 mouse than the Hip14 − / − mouse does as its phenotype is progressive and not developmental. Does this mean that HIP14L plays a greater role in the pathogenesis of HD or is it that HIP14 is so important that the phenotype is much more severe? Conditional deficient Hip14 and Hip14l mouse models will help to begin to answer these questions as they will allow
Funding The present work was supported by the Canadian Institutes for Health Research (CIHR operating grant to M.R.H. [grant number GPG102165; www.cihr-irsc.gc.ca]; and CHDI foundation (CHDI operating grant to M.R.H.; www.chdifoundation.org). M.R.H. is a University Killam Professor and the Canada Research Chair in Human Genetics and Molecular Medicine. The funders had no role in study design and analysis, decision to publish or preparation of the manuscript.
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Received 08 September 2014 doi:10.1042/BST20140242
Aberrant palmitoylation in Huntington disease Shaun S. Sanders*1 and Michael R. Hayden* *Centre for Molecular Medicine and Therapeutics, Department of Medical Genetics, Child & Family Research Institute, University of British Columbia, Vancouver, British Columbia, V5Z 4H4, Canada
Abstract Huntington disease (HD) is an adult-onset neurodegenerative disease caused by a CAG expansion in the HTT gene. HD is characterized by striatal atrophy and is associated with motor, cognitive and psychiatric deficits. In the presence of the HD mutation, the interactions between huntingtin (HTT) and huntingtin interacting protein 14 (HIP14 or DHHC17) and HIP14-like (DHHC13, a HIP14 orthologue), palmitoyl acyltransferases for HTT, are disturbed, resulting in reduced palmitoylation of HTT. Genetic ablation of either Hip14 or Hip14l recapitulates many features of HD, including striatal atrophy and motor deficits. However, there are no changes in palmitoylation of HTT in either mouse model and, subsequently, the similarities between the phenotypes of these two mouse models and the HD mouse model are believed to result from underpalmitoylation of other HIP14 and HIP14L substrates. HTT acts as a modulator of HIP14 activity such that in the presence of the HD mutation, HIP14 is less active. Consequently, HIP14 substrates are less palmitoylated, leading to neuronal toxicity. This suggests that altered HIP14–HTT and HIP14L–HTT interactions in the presence of the HD mutation reduces palmitoylation and promotes mislocalization of HTT and other HIP14/HIP14L substrates. Ultimately, HD may be, in part, a disease of altered palmitoylation.
Huntington disease Huntington disease (HD) is an adult onset neurodegenerative disease characterized by progressive cognitive, psychiatric and motor deficits. The typical age of onset is 50 years of age and the disease progresses such that death occurs, on an average, 20 years later [1,2]. The striatum, a part of the basal ganglia responsible for controlling movement, is the first area of the brain to be affected with other brain regions, particularly the cortex, affected at later stages of disease progression [1,2]. HD is an autosomal dominant disease caused by a CAG repeat expansion in exon 1 of the HTT gene that leads to a poly-glutamine expansion in the huntingtin (HTT) protein and a toxic gain of function [3]. A recent Canadian study estimated the prevalence of HD at 13.7 in 100,000 in the general population with 81.6 in 100,000 at a 25–50 % risk [4]. There are currently no disease-modifying treatments available. Thus, HD research is aimed at validating and identifying novel drug targets.
The first evidence for a role of palmitoylation in HD The HTT protein undergoes many posttranslational modifications, including phosphorylation, ubiquitination, acetylation, proteolysis, SUMOylation and palmitoylation. These various modifications influence the functions of Key words: Huntington disease, HIP14, zDHHC17, HIP14L, palmitoylation, zDHHC13. Abbreviations: HD, Huntington disease; HTT, huntingtin; wtHTT, wild type HTT; mHTT, mutant HTT; YAC128, full-length human HTT transgenic mouse model of HD; HIP14, huntingtin interacting protein 14; HIP14L, huntingtin interacting protein 14-like; SNAP25, synaptosomal-associated protein 25; PSD-95, post-synaptic density protein 95; APT, acyl-protein thioesterase; MSN, medium spiny neuron. To whom correspondence should be addressed (email [email protected]).
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wild type HTT (wtHTT) and the toxicity and functions of mutant HTT (mHTT) (reviewed in Ref. [5]). Spalmitoylation (simply palmitoylation here) is the reversible addition of long-chain saturated fatty acids, typically the 16-carbon palmitate, to proteins via a labile thioester bond (Figures 1A and 1B) [6,7]. Palmitoylation is mediated by the DHHC-domain containing palmitoyl acyltransferases (PATs) of which humans have 23; named zDhhc1-9 and 11-24 [8]. Depalmitoylation is mediated by acyl-protein thioesterases (APTs), four of which have been identified: the cytosolic ATP1, APT2, APTL1 and the lysosomal PPT1 [9,10]. Palmitoylation controls localization to and within membranes, function, additional post-translation modifications and stability of proteins [11]. The first PATs were identified in yeast more than a decade ago and the identification of the DHHC-cysteine rich domain allowed the rapid identification of the rest [12,13]. At the same time in a screen for interactors of HTT, Huntingtin interacting protein 14 (HIP14, DHHC17) was identified and was shown to interact with wtHTT and to interact less with mHTT [14,15]. An in silico screen for HIP14 orthologues also identified HIP14-like (HIP14L, DHHC13) [15], which also interacted with HTT in a manner inversely correlated to CAG expansion [16]. It is this altered interaction in the presence of the HD mutation that made HIP14 and HIP14L interesting for further study in the context of HD. HIP14 was the first mammalian PAT identified and was shown to palmitoylate HTT and other neuronal proteins [17]. HTT was later shown to be palmitoylated at cysteine 214 (C214) by HIP14 [18] and, in an in cellulo study where all 23 human PATs were co-expressed with HTT, HIP14 and HIP14L were shown to be the only two PATs to significantly palmitoylate HTT [19]. C The
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Figure 1 The process of S-palmitoylation (A) The 16-carbon saturated fatty acid palmitoyl-CoA is added to the thiol group of internal cysteine residues of proteins via a thioester bond by DHHC palmitoyl acyltransferases (PATs). Palmitoylation is reversed by acyl protein thioesterases (APTs). (B) Palmitate inserts in the lipid bilayer of membranes.
Interestingly, HIP14 and HIP14L were found to be unique as they are the only two PATs that contain ankyrin repeat domains and six transmembrane domains (Figure 2) [8,11,20]. The ankyrin repeat domain is the domain of interaction with HTT [19]. The first real evidence of a role for palmitoylation in the pathogenesis of HD came when mHTT was shown to be less palmitoylated in the YAC128 transgenic mouse model of HD [18], which expresses full-length human HTT from the endogenous promoter and accurately recapitulates many features of the human disease [21,22]. Mutating C214 to serine lead to increased aggregate and nuclear inclusion formation, increased cell death and increased susceptibility to excitotoxicity [18]. Similarily, siRNA-mediated decrease in HIP14 expression also increased inclusion formation and susceptibility to excitotoxicity. Interestingly, overexpressing HIP14 in neurons expressing mHTT decreased aggregate formation, suggesting that increasing palmitoylation may be beneficial [18]. The modulation of disease phenotypes by altering HIP14 levels provided strong evidence that HIP14 may be involved in the disease process and that modulating the pathway may be beneficial. C The
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Hip14 deficient mice recapitulate features of HD The strongest piece of evidence implicating palmitoylation in the pathogenesis of HD came from the characterization of the Hip14 deficient mouse model (Hip14 − / − ) [23]. This mouse model was shown to recapitulate many features of the YAC128 mouse. Neuropathological assessments and MRI analysis revealed that there is selective degeneration of the areas of the brain affected in HD in Hip14 − / − mice, including the striatum, cortex, hippocampus and corpus callosum (white matter). Interestingly, it was found that the population of neurons affected in the striatum of the Hip14 − / − mice was the same population affected in HD, namely medium spiny projection interneurons (MSNs) of the striatal indirect pathway involved in preventing unwanted movement [23]. In addition to the neuropathological phenotype, the Hip14 − / − mice were shown to have HD-like motor dysfunction, exhibiting loss of motor function and of sensorimotor gating, the ability to inhibit unwanted motor responses to a stimulus, believed to be mediated by the striatum [23]. Hip14 − / − mice were also shown to exhibit motor inflexibility in a plus maze where Hip14 − / − mice are less
Protein Acylation: from Mechanism to Drug Discovery
Figure 2 DHHC PAT domain structure and membrane topology (A) The typical PAT contains four transmembrane domains and is oriented in the membrane such that the DHHC domain is on the cytosolic side. (B) HIP14 and HIP14L are the only two PATs that have ankyrin repeat domains and are oriented such that both the ankyrin repeat and DHHC domains are on the cytosolic side of the membrane. (C) DHHC 3 and 8 have PDZ-binding domains and are inserted into the membrane so that this domain and the DHHC domain face the cytosol. (D) DHHC6 is the only PAT that has an SH3 domain that also faces the cytosol along with the DHHC domain.
likely to turn and explore the perpendicular arm of a plus maze, something that occurs in HD mouse models as well [24]. Striatal electrophysiological characterization of the Hip14 − / − mice provided further evidence that HIP14 is involved in the pathogenesis of HD [25]. The MSNs of Hip14 − / − mice were shown to be less excitable, to fire fewer action potentials, and to have decreased neuron surface membrane area. The Hip14 − / − MSNs also had fewer excitatory synapses, as shown electrophysiologically and by electron microscopy. The remaining synapses had a lower probability of neurotransmitter release [25]. These MSN cellular and synaptic changes are similar to those in late-stage HD mouse models [25,26]. The phenotypes between Hip14 − / − and YAC128 mice are not completely overlapping. The biggest difference between the phenotype of the Hip14 − / − and YAC128 mice is that the neuropathological and behavioural phenotypes of the Hip14 − / − mice are developmental and not progressive, unlike the adult-onset progressive phenotypes in the YAC128 mice [23]. The phenotype may be much for severe in the Hip14 − / − mice because there is no HIP14, whereas in the YAC128 mice HIP14 is only partly dysfunctional. It will be interesting to determine what happens if HIP14 is lost after the developmental period, i.e. does an HD-like phenotype develop? The similar deficits in the neuropathological, behavioural and electrophysiological phenotypes of the Hip14 − / − mice to HD-mouse models suggest that HIP14 may play an important role in the pathogenesis of HD. Initially it was thought this was due to HTT palmitoylation, but no change in HTT palmitoylation or expression was observed in Hip14 − / − mice. Presumably, this was due to
HIP14L compensating for the loss of HIP14 to palmitoylate HTT. However, palmitoylation of other HIP14 substrates, including the post-synaptic density protein 95 (PSD-95) and the pre-synaptic protein SNAP25 (synaptosomal-associated protein 25), was reduced in brains of Hip14 − / − mice [23]. These data suggest that HD may be partly due to aberrant palmitoylation of HIP14 substrates other than HTT. In a study determining how the palmitoyl proteome was altered in YAC128 and Hip14 − / − mice, the palmitoyl proteomes were compared between the two mouse models [27]. Unexpectedly, the two palmitoyl proteomes were not well correlated. Some proteins did show similar changes in levels of palmitoylation in the two mouse models, but many did not. However, all proteins in the present study that were examined further had a similar level of decrease in expression as in palmitoylation and, unfortunately, there was no way to differentiate in the mass spectrometry experiment between true changes in palmitoylation and changes in the expression/input level of palmitoylated proteins [27]. Indeed, palmitoylation and protein stability are not mutually exclusive, in some cases, loss of palmitoylation leads to ubiquititination and protein degradation, which may further complicate the interpretation of these results [28]. It would be interesting to see what degree of overlap there is between the two palmitoyl proteomes of the two mouse models after correcting for protein expression and input levels. In addition, those proteins that were shown to be changed in the YAC128 mice but not in the Hip14 − / − mice may be altered due to HIP14L dysfunction. Regardless, it may be those proteins that show reduced palmitoylation in both animal models that really contribute to the similarities in disease phenotypes [27]. C The
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Figure 3 A schematic representation of the hypothetical neuronal localization of HIP14 and HIP14L substrates when wild type HTT is present (A) compared with when mutant HTT is present (B; mHTT) In the presence of wild type HTT (wtHTT), HIP14 and HIP14 are fully active and their following substrates are fully palmitoylated and normally localized to the synapse: GAD65, SNAP25, synaptotagmin 1 (SYT1), PSD-95, AMPAR and HTT. In the presence of mHTT, HIP14 and HIP14L do not interact with HTT and are thus less active. This may result in mislocalization of the above substrates and in aggregation of mHTT.
Wild type HTT acts as a regulator of HIP14 function The phenotypic overlap between the Hip14 − / − and YAC128 phenotypes without changes in HTT palmitoylation suggests an alternate mechanism. There is evidence to suggest that the relationship between HIP14 and HTT goes beyond that of just an enzyme-substrate pair. When C214 was mutated to a serine or when the DHHC domain of HIP14 was deleted, HIP14 and HTT still interacted indicating that their interaction is independent of palmitoylation. This is in contrast to the interaction of HIP14 with its other substrate PSD-95, which is dependent on both the palmitoylation sites of PSD-95 itself and on the DHHC domain of HIP14 [19]. Indeed, HIP14 and HTT form such a tight complex that the HIP14 co-immunoprecipitated with HTT from mouse brain lysates had as much activity towards SNAP25 as immunoprecipitated HIP14 alone [19]. However, when HIP14 was immunoprecipitated from YAC128 mouse brain lysate, there was a 50 % decrease in HIP14 in vitro activity towards SNAP25 [23]. This indicates that HIP14 is less active in YAC128 mice. PATs are believed to autopalmitoylate and then transfer the palmitate from themselves to their substrates, thus loss of autopalmitoylation of a PAT suggests reduced activity [12,13,29]. Interestingly, autopalmitoylation C The
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of HIP14 was shown to be decreased in the YAC128 mice [23] or when less wtHTT is present [19]. This suggests that mHTT inhibits HIP14 activity in the YAC128 mice, resulting in reduced palmitoylation and mislocalization of HIP14 substrates in the presence of the HD mutation (Figure 3).
High degree of overlap between HIP14 and HTT interactors The recent finding of a highly significant overlap between interactors of HIP14 and interactors of HTT further implicated the HIP14-HTT complex in the pathogenesis of HD [30]. A yeast 2-hybrid screen for HIP14 interactors identified 214 interactors, including HTT and HIP14 itself. Surprisingly, 36 of the 214 HIP14 interactors were also found to be HTT interactors and seventeen of those have been implicated in HD. This high degree of overlap suggests that HIP14 is critically linked to HTT through its interactions and this warrants further investigation [30]. It will be interesting to know if these shared interactors are substrates or regulators of HIP14 and to know how altered interaction with HIP14 and HTT may contribute to the pathogenesis of HD.
Protein Acylation: from Mechanism to Drug Discovery
Hip14l deficient mice recapitulate features of HD Further evidence for a role of aberrant palmitoylation in HD came from the characterization of the Hip14l deficient mouse model [16]. Although Hip14l − / − mice display peripheral phenotypes not observed in HD mouse models, the neuropathological and behavioural phenotypes closely resemble those of HD mice. Neuropathological assessment revealed a progressive reduction in brain weight and striatal and cortical volume from three to 12 months of age in Hip14l − / − mice [16]. Similar to the Hip14 − / − and YAC128 mice, there was a specific loss the subpopulation of MSNs in the striatum that are lost in HD [16]. These neuropathological phenotypes are more like the progressive late onset degeneration in the YAC128 HD mice than the non-progressive, developmental loss of striatal and cortical volume in the Hip14 − / − mice [23,31,32]. Reduced volume of the globus pallidus, thalamus and corpus callosum was also observed in the Hip14l − / − mice, whereas the hippocampus was unchanged [16]. These neuropathological changes are actually more similar to the changes in the brain of YAC128 mice than to those in the Hip14 − / − mice as they are progressive adult-onset alterations unlike the developmental changes in the Hip14 − / − mice [23,32]. Assessing behaviour of the Hip14l − / − mice revealed deficits similar to those observed in the YAC128 and Hip14 − / − mice [23,31]. Hip14l − / − mice also displayed deficits in motor learning and motor coordination [16]. Taken together, these data indicate that Hip14l − / − mice display HD-like behavioural deficits [16]. Features of HD displayed in Hip14l − / − mice could result from reduced palmitoylation of key neuronal HIP14L substrates. However, HTT palmitoylation was not altered in Hip14l − / − mice, as was the case in the Hip14 − / − mice [16,23]. It is possible that HIP14 and HIP14L compensate for loss of each other for palmitoylation of HTT and some but not all other substrates. Overlap in PAT-substrate specificity have been previously documented [33,34]. Determining the substrate profiles for the two enzymes and how they overlap would provide an explanation for the similarities and differences between these two mouse models.
temporal and tissue specific deletion of either or both genes. It will also be interesting to know what happens in the case where both Hip14 and Hip14l are lost. The present experiment may provide further insight into the relationship between HIP14/HIP14L and HTT. The fact that wtHTT acts as a modulator of HIP14 activity and that the relationship between these two proteins goes beyond that of just enzyme-substrate pair suggests that this relationship may be very important [19]. The overall hypothesis from the work described here is that disturbed HIP14–HTT and HIP14L–HTT interaction in the presence of the HD mutation reduces HIP14, and potentially HIP14L, function leading to the under-palmitoylation and mislocalization of HTT and key HIP14 and HIP14L substrates (Figure 3). It still remains unclear exactly what the nature of the interaction between HIP14 and HTT is. There are a number of possibilities. First, HTT may act as an allosteric activator of HIP14, inducing a conformational change when bound to HIP14, making it more active. Second, HTT may act as a scaffold to bring HIP14 into proximity with its substrates. Third, HTT may traffic HIP14 to specific subcellular sites to palmitoylate its substrates. It is also still unknown if HTT also modulates HIP14L activity as with HIP14. Given the evidence presented here for aberrant palmitoylation in HD, one therapeutic approach could involve restoring levels of palmitoylation. This would be achieved by targeting the relevant PATs, i.e. HIP14 and HIP14L, or thioesterase enzymes. We have just begun to understand the complex relationships between PATs and their substrates and to understand the regulation of this posttranslational modification. The complexity of the role that these enzymes play in disease processes is not yet fully understood. In order to appropriately target and predict potential side effects, a much better understanding of these processes is required.
Acknowledgements We thank Dr. Dale Martin for assistance with the figures and for providing feedback on the manuscript, and Mandi Schmidt for assistance with the figures.
Conclusions and outstanding questions The Hip14 − / − and Hip14l − / − mouse models, both of which have HD-like phenotypes resembling those of the YAC128 mouse model of HD, provide the strongest evidence for a role of palmitoylation in HD [16,23]. The Hip14l − / − mouse more closely resembles the YAC128 mouse than the Hip14 − / − mouse does as its phenotype is progressive and not developmental. Does this mean that HIP14L plays a greater role in the pathogenesis of HD or is it that HIP14 is so important that the phenotype is much more severe? Conditional deficient Hip14 and Hip14l mouse models will help to begin to answer these questions as they will allow
Funding The present work was supported by the Canadian Institutes for Health Research (CIHR operating grant to M.R.H. [grant number GPG102165; www.cihr-irsc.gc.ca]; and CHDI foundation (CHDI operating grant to M.R.H.; www.chdifoundation.org). M.R.H. is a University Killam Professor and the Canada Research Chair in Human Genetics and Molecular Medicine. The funders had no role in study design and analysis, decision to publish or preparation of the manuscript.
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Received 08 September 2014 doi:10.1042/BST20140242
