MINIREVIEW Huntington’s disease: roles of huntingtin-interacting protein 1 (HIP-1) and its molecular partner HIPPI in the regulation of apoptosis and transcription Nitai P. Bhattacharyya, Manisha Banerjee and Pritha Majumder* Crystallography and Molecular Biology Division and Structural Genomics Section, Saha Institute of Nuclear Physics, Kolkata, India Huntington’s disease (HD, OMIM 143100) is an auto- somal dominant progressive neurodegenerative disease caused by the expansion of polymorphic CAG (coding for glutamine) repeats beyond 36 at exon 1 of the huntingtin (htt) gene, localized at chromosome 4p16.3. Age at onset (AO) of the disease varies widely (1–90 years, mean  35 years). There is an inverse cor- relation between AO and expanded CAG repeat numbers, but it is not the only determinant of variation in AO [1]. HD is fatal within 10–15 years after appearance of the first symptom. The symptoms include uncontrolled movement, emotional distur- bances, psychiatric abnormalities, cognitive deficits, and dementia. The gene htt encodes a protein [hunting- tin (Htt),  348 kDa] with a polyglutamine stretch starting from the 18th amino acid. Also, two proline- rich regions adjacent to the polyglutamine domain and several HEAT repeats, known to be involved in Keywords apoptosis; HIP-1; HIPPI; huntingtin- interacting proteins; transcription Correspondence N. P. Bhattacharyya, Crystallography and Molecular Biology Division and Structural Genomics Section, Saha Institute of Nuclear Physics, 1 ⁄ AF Bidhan Nagar, Kolkata 700 064, India Fax: +91 033 23374637 Tel: +91 033 23375345–49 (5 lines), ext. 1301 E-mail: nitaipada.bhattacharya@saha.ac.in or nitai_sinp@yahoo.com *Present address Roswell Park Cancer Institute, Cell Stress Biology Department, Buffalo, New York, USA (Received 29 February 2008, revised 15 May 2008, accepted 18 June 2008) doi:10.1111/j.1742-4658.2008.06563.x Huntingtin protein (Htt), whose mutation causes Huntington’s disease (HD), interacts with large numbers of proteins that participate in diverse cellular pathways. This observation indicates that wild-type Htt is involved in various cellular processes and that the mutated Htt alters these processes in HD. The roles of these interacting proteins in HD pathogenesis remain largely unknown. In the present review, we present evidence that Htt-inter- acting protein 1 (HIP-1), an endocytic protein, together with its interacting partner HIPPI, regulates apoptosis and gene expression, both processes being implicated in HD. Further studies are necessary to establish whether the HIPPI–HIP-1 complex or other interacting partners of HIPPI regulate apoptosis and gene expression that are relevant to HD. Abbreviations ANTH, AP180 N-terminal homology domain; AO, age at onset; AP, adaptor protein; AR, androgen receptor; BLOC1S2, biogenesis of lysosome-related organelles complex-1 subunit 2; CLH1, clathrin heavy chain 1; CLH2, clathrin heavy chain 2; CLTA, clathrin light chain A; CLTB, clathrin light chain B; ENTH, Epsin N-terminal homology; HD, Huntington’s disease; HIP-1, huntingtin-interacting protein 1; HIPPI, huntingtin-interacting protein 1 interactor; Htt, huntingtin protein; NMDAR, N-methyl- D-aspartate receptor; pDED, pseudo-death effector domain; Shh, Sonic hedgehog. FEBS Journal 275 (2008) 4271–4279 ª 2008 The Authors Journal compilation ª 2008 FEBS 4271 protein–protein interactions, are present at the N-ter- minal region of the protein. Wild-type Htt is localized at the endoplasmic reticulum, Golgi complex, mito- chondria, and synaptic vesicles. Htt is ubiquitously expressed, although the neurodegeneration caused by the mutated Htt shows region specificity [2,3]. The expanded polyglutamine domain of mutant Htt is highly self-associative, resulting in aggregates ⁄ neuro- nal intranuclear inclusions. Aggregates ⁄ neuronal intra- nuclear inclusions are observed in cell models, brains of transgenic animals, and post-mortem brains of HD patients [2]. Aggregate formation is enhanced with the increase in the number of glutamines in vitro and in vivo, and is believed to cause neurodegeneration [4]. Although a contradictory finding, that visible aggre- gates are protective to neurons, has also been made [5]. The autosomal dominant nature of the disease sug- gests a toxic gain-of-function of the mutated protein that disrupts normal cellular functions and causes neuronal death [3]. Loss-of-function of the wild-type protein may also contribute, at least partially, to the disease pathology [6]. Over the years, various cellular events, such as excitotoxicity, oxidative stress, mito- chondrial dysfunction, stress in the endoplasmic reticu- lum, formation of channels through membranes, axonal transport, protein degradation, autophagy, transcriptional dysregulation, and apoptosis, have been implicated in HD. These processes may not be inde- pendent of each other. Detailed descriptions of these processes are beyond the scope of this review. In the present review, we specifically focus on the role of Htt-interacting protein HIP-1 and its molecular part- ner HIPPI in the regulation of apoptosis and transcrip- tion, the two processes that are altered in HD [7,8]. HIP-1 – its interacting partners and endocytosis Large numbers of proteins have been identified, by different techniques that interact with Htt [9–11]. These studies reveal that Htt may function as a scaffold and coordinate diverse cellular functions [9– 13]. Some of the Htt-interacting proteins also alter the pathogenicity in the Drosophila model of HD [13]. Among  300 Htt-interacting proteins described so far, HIP-1 is one of the most studied. The possible involvement of HIP-1 in various cancers has been reviewed recently [14] and will not be discussed here. HIP-1 has been shown by yeast two-hybrid assays to interact with N-terminal Htt. HIP-1 is orthologous to yeast Sla2p, which is known to be involved in endocy- tosis and regulation of the actin cytoskeleton. HIP-1 and Htt colocalize in neuronal cells [15,16]. The inter- action of HIP-1 with wild-type Htt is stronger than that observed with mutated Htt [17]. In addition to Htt, HIP-1 interacts with its paralog HIP1-R, subunits of clathrin-associated adaptor protein (AP) complex AP2A1 and AP2A2, clathrin heavy chain 1 (CLH1), and clathrin heavy chain 2 (CLH2), clathrin light chain A (CLTA), and clathrin light chain B (CLTB), and N-methyl-d-aspartate receptor (NMDAR) subun- its NR2A and NR2B. Various domains, such as the AP180 N-terminal homology domain (ANTH), also known as the Epsin N-terminal homology (ENTH) domain, the central coiled-coil region and a C-terminal talin homology domain are present at HIP-1. The coiled-coil domain contains a leucine-zipper motif and mediates heterodimerization with HIP-1R. Consensus binding sites for the endocytic adaptor protein AP2 (DPF motif), clathrin heavy chain (LMDMD clathrin- box motif) and a phosphatidylinositol 4,5-biphosphate- binding motif at its ANTH ⁄ ENTH domain are also present [14,18]. Various domains of HIP-1 are shown in Fig. 1. Direct evidence that HIP-1 is involved in endocytosis comes from HIP-1 knock-out (HIP-1 ) ⁄ ) ) mice, which show defects in assembly of endocytic protein complexes on liposomal membranes and a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor trafficking [19]. The similarities in amino acid sequences and domains between HIP-1, HIP-1R and yeast ortholog Sla2p, the interacting partners of HIP-1 with known functions and results with knockout mice Fig. 1. Various domains of HIP-1. The ANTH ⁄ ENTH domain (38–160), coiled-coil domain (371–610), and talin-like domain (814–1112) were predicted with the SMART tool (http://smart.embl-heidelberg.de/). Binding sites for HIPPI (422–503), AP2 (262–266 and 358–360), CLH1, CLH2 (332–336), CLTA, CLTB (484–489) and other domains are taken from the published literature and mentioned in the text. The positions of the amino acids are not to scale. HIP-1 & HIPPI mediated apoptosis and transcription N. P. Bhattacharyya et al. 4272 FEBS Journal 275 (2008) 4271–4279 ª 2008 The Authors Journal compilation ª 2008 FEBS show that HIP-1 participates in the regulation of cytoskeletal and endocytic processes. HIP-1 and its interacting partners – roles in apoptosis and survival Various pathways followed during apoptosis have been reviewed recently [20]. In the ‘extrinsic pathway’, acti- vation of caspase-8 ⁄ caspase-10, mostly through trans- membrane death receptors, leads to activation of downstream caspase-3 and cleavage of other down- stream substrates, leading to nucleosomal ladders, a hallmark of apoptosis. In the ‘intrinsic pathway’, signal factors released from mitochondria activate caspase-9 and then caspase-3, leading to cell death. These two pathways may crosstalk via caspase-8-medi- ated cleavage of Bid. In the caspase-independent path- way, apoptosis-inducing factors or endonuclease G, normally present in the mitochondria, are released and translocated to the nucleus, where they cleave the genome into nucleosomal ladders directly. Several experimental findings indicate that HIP-1 is a proapoptotic protein. Exogenous expression of HIP- 1 in neuronal and non-neuronal cells induces apoptosis following the intrinsic pathway [17,21]. The pseudo- death effector domain (pDED) of HIP-1 (Fig. 1) alone is able to induce apoptosis; Phe398 of HIP-1 (within the pDED) is critical for increased apoptosis. Coex- pression of wild-type N-terminal Htt (encoded by exon 1 of htt) and HIP-1 reduces HIP-1-induced apop- tosis [17,21]. Wild-type N-terminal Htt, being able to interact with HIP-1 strongly, may reduce the amount of HIP-1 that is available to interact with other pro- tein(s) and reduce apoptosis. In rat cells, HIP-1 is cleaved in response to drugs that are known to induce apoptosis, as well as in cells expressing exogenous HIP-1, although the relevance of such cleavages in apoptosis remains unknown. HIP-1 interacts directly with procaspase-9 and activates it. Direct interaction of HIP-1 with Apaf1 increases recruitment of cyto- chrome c to the apotosome complex, resulting in increased apoptosis [21]. Depending on the status of phosphorylation of HIP-1 by Dyrk1, HIP-1 interacts with caspase-3 and enhances apoptosis, in a condition where interaction and phosphorylation of HIP-1 by Dyrk1 are reduced [22]. Exogenous expression of HIPPI (HIP-1 protein interactor), a molecular partner of HIP-1, increases apoptosis through the extrinsic pathway. The HIP-1– HIPPI heterodimer recruits procaspase-8 and activates it [23]. Enhancement of apoptosis by exogenous HIPPI in the presence of endogenous HIP-1 is mediated through activation of caspase-8, caspase-1, caspase-9 ⁄ caspase-6, and caspase-3. Cleavage of Bid and release of cytochrome c and apoptosis-inducing factors from the mitochondria are also observed. Coexpression of wild-type htt exon 1 and Hippi decreases apoptosis and increases survival in comparison with that obtained in cells expressing Hippi only. In such a condition, inter- action of HIPPI with HIP-1 is reduced. This result fur- ther shows that freely available HIP-1 is necessary to induce apoptosis [24]. Contradictory findings that HIP-1 may act as a prosurvival ⁄ antiapoptotic protein and may not influ- ence apoptosis at all are also available. Expression of full-length HIP-1 does not increase apoptosis, whereas deletion of the N-terminal ANTH ⁄ ENTH domain increases apoptosis [25]. Deletion of murine HIP-1 in vivo increases testicular degeneration by apoptosis, indicating a protective role of HIP-1 in apoptosis [26]. Mice deficient in both HIP-1 and its paralog HIP-1R exhibit neurodegeneration at adulthood and can be rescued by human HIP-1 [27]. Reduced sperm count and defects in reproduction have been observed in HIP-1 ) ⁄ ) mice, due to apparent loss of postmeiotic spermatids [28]. These results show that HIP-1 in dif- ferent conditions may act as an antiapoptotic protein. Overexpression of HIP-1 in brain tumors is correlated with the increased expression of epidermal growth factor receptor and platelet-derived growth factor b-receptor [29]. The ANTH ⁄ ENTH domain of HIP-1 interacts with 3-phosphate containing inositol lipids and stabilizes the growth factor receptor tyrosine kinases by increasing their half-life following ligand induced endocytosis. Such interaction affects cell growth and survival [30]. This observation supports the contention that the ANTH⁄ ENTH domain of HIP-1 protects cells from death by apoptosis, as mentioned earlier [25]. Taken together, these results show that HIP-1 may act as a prosurvival protein in different conditions. Contradictory results showing that HIP-1 is a proa- poptotic protein [17,21–24] or an antiapoptotic protein [25–30] in different conditions could be due to the presence or absence of HIP-1-interacting partners. The decrease in HIP-1–HIPPI-mediated apoptosis, either by overexpression of Homer 1c, an interactor of HIP- PI (for details see the next section), or by the wild-type N-terminal Htt, which strongly interacts with HIP-1 [24,31], supports this contention. In such cases, the amounts of freely available HIPPI or HIP-1 may decrease, resulting in reduced apoptosis. Exogenous expression of HIP-1 (cloned in pcDNA3 and kindly provided to us by T. S. Ross, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA) in HeLa cells, where endoge- N. P. Bhattacharyya et al. HIP-1 & HIPPI mediated apoptosis and transcription FEBS Journal 275 (2008) 4271–4279 ª 2008 The Authors Journal compilation ª 2008 FEBS 4273 nous HIPPI is undetectable [24], did not increase apoptosis. However, in Neuro2A and K562 cells, where endogenous HIPPI is present [24], exogenous expression of HIP-1 increased apoptosis. Additionally, expression of HIPPI in HeLa cells, where HIP-1 was knocked down, decreased apoptosis (M. Banerjee and N. P. Bhattacharyya, unpublished observations) rela- tive to that obtained in HeLa cells with endogenous HIP-1 [24]. The proapoptotic activity of HIPPI or HIP-1 may thus be dependent on the presence or absence of its interacting partner HIP-1 and HIPPI respectively. HIPPI and its interacting partners – regulation of apoptosis HIPPI, also known as estrogen-related receptor b-like 1, is a homolog of Chlamydomonas intraflagellar transport 57. HIPPI does not have any known domain except a pDED and a myosin-like domain. Interaction of HIPPI with HIP-1 takes place through the pDED, specifically through 409 K, present in helix 5 of HIPPI, although other regions have influence over such inter- actions [23]. We mentioned above that HIP-1 and HIPPI together induce apoptosis [23,24]. Identification of additional proteins such as Homer1c ⁄ Homer1 [31], BAR ⁄ BFAR [32], RybP [33], BLOC1S2 [34] and apop- tin [35] that interact with HIPPI further indicates that HIPPI may regulate apoptosis. Homer1c ⁄ Homer1 belongs to the homer family of proteins and is known to participate, in neuronal signaling. HIPPI interacts with Homer1c and colocalizes in the postsynaptic region of hippocampus. It has been shown that Homer1c completely abolishes HIP-1–HIPPI-mediated apoptosis in striatal neurons, the specific region of neuronal loss in HD [31]. The bifunctional apoptosis inhibitor BAR, also known as BFAR, is expressed pre- dominantly in neurons, and interacts with HIP-1 as well as HIPPI. BAR inhibits neuronal apoptosis in response to diverse stimuli [32]. It is not known whether BAR can regulate HIP-1–HIPPI-mediated apoptosis. Recently, Rybp has been shown to interact with HIPPI and increase HIPPI-mediated apoptosis through the caspase-8-mediated pathway. Rybp also interacts with ubiquitin-binding protein, procaspase-8, procaspase-10, and the HIPPI interactor apotin. Inter- action of HIPPI with Rybp is involved in murine neural development, although the significance of such an interaction in apoptosis regulation or HD patho- genesis remains elusive [33]. Apotin, a chicken anemia virus-encoded protein, has been shown to colocalize with HIPPI in the cytoplasm of normal cells, whereas in tumor cells, they localize separately in the nucleus and cytoplasm. The HIPPI–apoptin interaction may suppress apoptosis [35]. The functional relevance of such interactions in HIP-1- or HIPPI-mediated apop- tosis also remains unknown. Biogenesis of lysosome- related organelles complex-1 subunit 2 (BLOC1S2) specifically interacts with HIPPI, but not with HIP-1. Coexpression of HIPPI and BLOC1S2 does not increase apoptosis but sensitizes apoptosis induction by stauro- sporin or death ligand. In addition, the expression of BLOC1S2 is increased in some tumors [34]. Information on the interacting partners of HIPPI such as HIP-1, Homer 1c, BAR and apoptin indicates that HIPPI may also be a proapoptotic protein. Rybp, an interactor of HIPPI, interacts with caspase-8 and caspase-10, indicating that HIPPI might also be involved in the regulation of apoptosis. Even though the exact function of HIPPI remains unknown, knockout mice for Hippi (HIPPI ) ⁄ ) ) exhi- bit downregulation of the Sonic hedgehog (Shh) pathway and developmental abnormalities [36]. However, the exact molecular defects in the Shh pathway in HIPPI ) ⁄ ) mice are unknown. It may be worthwhile to mention that Dyrk1, an HIP-1 inter- actor, is also involved in the Shh pathway by regu- lating Gli1 [37]. The specific role of the Shh pathway in apoptosis or HD remains unknown. Role of HIP-1 and HIPPI in transcriptional regulation There are not many reports on the transcriptional activity of HIP-1 or HIPPI. It has been shown that HIP-1 interacts with androgen receptor (AR) through its coiled-coil domain and increases the transcriptional activity of AR on known AR-inducible promoter. Treatment with androgen increases the nuclear fraction of AR as well as that of HIP-1, indicating that forma- tion of the HIP-1–AR heterodimer is involved in trans- location of AR to the nucleus. Facilitation of nuclear transport of AR by HIP-1 depends on its C-terminal nuclear localization signal for HIP-1. In addition to that of AR, mediation of transcriptional activity of genes by other nuclear hormone receptors, such as estrogen and glucocorticoid receptors, is also enhanced by HIP-1. All these results demonstrate a nonconven- tional function of HIP-1 as a transcriptional regulator [38], in addition to its endocytosis and proapoptotic or prosurvival ⁄ antiapoptotic functions described in the preceding sections. The evidence that HIPPI directly or indirectly alters gene expression comes from the observations that caspase-1, caspase-3, caspase-7, caspase-8 and caspase-10 expression is increased in cells expressing exogenous HIP-1 & HIPPI mediated apoptosis and transcription N. P. Bhattacharyya et al. 4274 FEBS Journal 275 (2008) 4271–4279 ª 2008 The Authors Journal compilation ª 2008 FEBS Hippi. Also, expression of the mitochondrial-coded genes ND1 and ND4, the nuclear genome-coded mitochondrial genes SDHA and SDHB and the antia- poptotic genes BCL-2 and survivin is decreased in Hippi-expressing cells [24]. Decreased expression of ND1, ND4, SDHA, SDHB and BCL-2 may cause mitochondrial dysfunctions and contribute towards the increased apoptosis by HIPPI as mentioned earlier. Decreased expression of the antiapoptotic gene survivin may also enhance apoptosis. HIPPI interacts with the putative promoter of caspase-1 in vitro and in vivo [39]. On the basis of in vitro interactions of various mutants of the sequence 5¢-AAAGACATG-3¢ ()101 to )93) present at the caspase-1 putative promoter sequence, where HIPPI can bind, it has been predicted that HIPPI will interact with AAAGA[GC][ATC][TG] [40]. The presence of other sequence motifs around the HIPPI binding site where transcription factors p53, p73 and ETS1 can bind and influence the expression of caspase-1 [41–43] indicate that these or other unknown transcription factors may cooperate with HIPPI for the regulation of caspase-1 expression. HIPPI also interacts with putative promoter sequences of caspase-8 and caspase-10 [40] and PARP-1 ()579 to )149) (P. Majumder and N. P. Bhattacharyya, unpublished results) in vitro. However, to determine whether HIPPI binds with a similar motif present at the putative promoters will require further studies. It will be of interest to determine whether such a motif is also present at the putative promoter regions of other genes, especially those that are altered in HD models, as observed in several high-throughput gene expression studies [8], and investigate whether HIPPI alters the expression of some of them. It is not clear how HIPPI, being a cytoplasmic protein, enters into the nucleus and interacts with the putative promoter sequences and eventually increases the expression of the genes. A similar mechanism to that described above for AR translocation by HIP-1 [38] may also operate for HIPPI translocation. We are presently investigating whether a similar mechanism of translocation of HIPPI may take place, using HIP-1 knocked down or HIP-1 overexpressed cells. However, indirect evidence that HIP-1 is necessary for the increased expression of caspase-1 has been obtained. Exogenous expression of Hippi and exon 1 of htt with 16 CAG repeats reduces the interaction of HIP-1 with HIPPI and reduces apoptosis [24]. In such a condition, expression of caspase-1 was decreased, as shown in Fig. 2. This result indicates that a similar mechanism of translocation of HIPPI by HIP-1 as observed with AR and HIP-1 may also act in this condition, but this requires further confirmation. Rybp, also known as death effector domain-associated factor, belongs to a family of small zinc finger-containing proteins that participate in transcriptional regulation by binding with other transcription factors such as YY1 and E2F or transcription repressors [44]. Additionally, the Rybp-related protein Yaf2 interacts with HIPPI. Inter- action of HIPPI with Rybp is proposed to be involved in murine neural development, although the signifi- cance of such an interaction in HD pathogenesis remains elusive [33]. It is speculative that Rybp, a molecular interactor of HIPPI, cooperates with HIPPI to augment transcription of caspase-1, and this war- rants further studies. A summary of the findings that HIPPI increases apoptosis and alters gene expression is shown in Fig. 3. wH16-Hi Pro-caspase-1 Caspase-1 Beta actin 45 kDa 20 kDa 42 kDa Hi Fig. 2. Western blot analysis for the expression of caspase-1 in HeLa cells expressing green fluorescent protein (GFP)-tagged HIPPI (GFP– Hippi, lane denoted by Hi) and HeLa cells coexpressing GFP–HIPPI and the red fluorescent protein-tagged wild-type exon 1 of the htt gene with 16 CAG repeats (DsRed–wH16, lane denoted by wH16-Hi). The lower panel shows the result with antibody to b-actin (42 kDa) as load- ing control. The sizes of procaspase-1 (45 kDa) and the activated caspase-1 (20 kDa) are shown by the arrows on the left. The bar diagram (right panel) shows the average (n = 3) of integrated optical density (IOD) of the bands obtained with antibody to caspase-1 in western blot analysis using GFP–HIPPI-expressing cells (unfilled) and cells coexpressing GFP–HIPPI and DsRed–wH16 (filled bar). N. P. Bhattacharyya et al. HIP-1 & HIPPI mediated apoptosis and transcription FEBS Journal 275 (2008) 4271–4279 ª 2008 The Authors Journal compilation ª 2008 FEBS 4275 Possible role of HIP-1 and HIPPI in the pathogenesis of HD Various cellular processes, such as apoptosis and tran- scriptional dysregulation, are altered in HD, leading to neuronal dysfunction and ⁄ or neurodegenaration. HIP-1 and HIPPI may participate in some of these processes. HIP-1 modulates aggregate formation, and mutant Htt induced neuronal dysfunction in the Caenorhabditis elegans model of HD [45]. The increased functional role of NMDAR in HD and the involvement of HIP-1 in NMDAR-mediated phos- phorylation of Htt [18] further indicate the possible participation of HIP-1 in HD pathogenesis. HIP-1– HIPPI-mediated apoptosis is observed in the striatal neuron, the specific target for neurodegeneration in HD. The role of HIPPI, if any, in either the increased apoptosis observed in animal and cell models and post-mortem brains of HD patients [3,7] or in the increased expression of caspase-1, caspase-3 and PARP-1 [46–48] remains unclear. There is are similari- ties in the apoptotic pathway and altered gene expres- sion observed in Hippi-expressing cells and cellular models, animal models or brains of HD patients. The transcriptional deregulation observed in a variety of HD model systems is possibly due to interactions of transcription factors ⁄ repressors with the mutated Htt [8]; whether HIP-1–HIPPI contributes, at least for the subset of genes altered in HD, needs further investiga- tions. The mechanism by which caspase-1 expression is increased in HD is not well understood, although the protein is implicated in the progression of HD [47,48]. The regulation of caspase-1 by HIPPI observed in cul- tured cells provides an explanation for the increased caspase-1 expression in HD. In HD, owing to weaker interactions of HIP-1 with the mutated Htt, the free HIP-1 pool might increase, and this in turn would lead to the formation of more HIP1–HIPPI, initiating apoptosis by caspase-8 activation and its downstream pathway, and might also increase the transcription of caspase-1. Further studies using animal models are necessary to confirm this. HIPPI Freely available HIP1 2 HIPPI 1 3 Strong interaction HIP1 6 4 5 Caspase-8 Nucleus Weak interaction pDED of HIPPI N-terminal of HIPPI Mutant Htt ? ? Caspase1 7 Normal Htt Pro-caspase 8 Apoptosis Caspase 8 Pro-caspase 3 Caspase 3 1. Heterodimerization of HIPPI and HIP1 2 Recruitment of Pro-caspase 8 Nuclear pore complex HIP1 2. 3. Activation of caspase 8 that leads to activation of caspase 3 either by extrinsic or intrinsic pathways 4. Activation of caspase 3 5. Entry of caspase 3 to nucleus 6. Entry of the HIP-1-HIPPI heterodimer into the nucleus 7. Regulation of gene expression by HIPPI Fig. 3. Possible mechanisms of regulation of transcription and apoptosis by HIPPI and HIP-1 in HD. Interaction of HIP-1 with the wild-type Htt allele is stronger than that of the mutated Htt [17]. In HD, one of the alleles of Htt is mutated and thus likely to release free HIP-1. Free HIP-1 then interacts with HIPPI through its C-terminal pDED domain and recruits caspase-8, and activates caspase-8 and its downstream effector proteins, resulting in apoptosis. On the other hand, HIPPI–HIP-1 heterodimer may translocate to the nucleus, interact with the putative promoters of caspase-1, caspase-8 and caspase-10, and increase their expressions. In turn, increased pro-caspase-8 is recruited to HIPPI–HIP-1 heterodimer and increases apoptosis. The role of caspase-1 in apoptosis is not known, but in some conditions it may increase apoptosis. Different symbols representing different proteins are shown in the box. Numbers representing different processes are also shown. HIP-1 & HIPPI mediated apoptosis and transcription N. P. Bhattacharyya et al. 4276 FEBS Journal 275 (2008) 4271–4279 ª 2008 The Authors Journal compilation ª 2008 FEBS Conclusions HIP-1 and its interacting partner HIPPI together induce apoptosis by the intrinsic and extrinsic pathways. Homer 1c, an interactor of HIPPI, and the wild-type N-terminal Htt, which interacts strongly with HIP-1, reduce HIP-1–HIPPI-mediated apoptosis. In the presence of Homer 1c, HIPPI may interact preferentially with it, resulting in a decrease of the amount of HIP-1–HIPPI heterodimer and apoptosis induction. The effect of HIP-1 may depend not only on the amount of the interacting proteins but also on the affinities of interacting proteins. The uncreased expression of caspase-1 observed in HD may be medi- ated through HIPPI. The role of HIP-1 in transloca- tion of HIPPI into the nucleus and that of other transcriptional regulators cooperating with HIPPI are yet to be determined. If the observations in cell culture are replicated in HD models or post-mortem brains, an explanation of the increased expression of caspase-1 and other subset of genes altered in HD may be available. Acknowledgements We acknowledge Professors A. Mukherjee, D. Mukho- padhyay and M. S. Moumita Datta for critically read- ing the manuscript and their valuable suggestions. References 1 Squitieri F, Cannella M, Giallonardo P, Maglione V, Mariotti C & Hayden MR (2001) Onset and pre-onset studies to define the Huntington’s disease natural history. Brain Res Bull 56, 233–238. 2 Young AB (2003) Huntingtin in health and disease. J Clin Invest 111, 299–302. 3 Cowan CM & Raymond LA (2006) Selective neuronal degeneration in Huntington’s disease. Curr Top Dev Biol 75, 25–71. 4 Ross CA & Poirier MA (2004) Protein aggregation and neurodegenerative disease. Nat Med 10, S10–S17. 5 Arrasate M, Mitra S, Schweitzer ES, Segal MR & Finkbeiner S (2004) Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death. Nature 431 , 805–810. 6 Cattaneo E, Rigamonti D, Goffredo D, Zuccato C, Squitieri F & Sipione S (2001) Loss of normal hunting- tin function: new developments in Huntington’s disease research. Trends Neurosci 24, 182–188. 7 Hickey MA & Chesselet MF (2003) Apoptosis in Huntington’s disease. Prog Neuropsychopharmacol Biol Psychiatry 27, 255–265. 8 Cha JH (2007) Transcriptional signatures in Hunting- ton’s disease. Prog Neurobiol 83, 228–248. 9 Harjes P & Wanker EE (2003) The hunt for huntingtin function: interaction partners tell many different stories. Trends Biochem Sci 28, 425–433. 10 Li SH & Li XJ (2004) Huntingtin–protein interactions and the pathogenesis of Huntington’s disease. Trends Genet 20, 146–154. 11 Li XJ, Friedman M & Li S (2007) Interacting proteins as genetic modifiers of Huntington disease. Trends Genet 23, 531–533. 12 Goehler H, Lalowski M, Stelzl U, Waelter S, Stroedicke M, Worm U, Droege A, Lindenberg KS, Knoblich M, Haenig C et al. (2004) A protein interaction network links GIT1, an enhancer of huntingtin aggregation, to Huntington’s disease. Mol Cell 15, 853–865. 13 Kaltenbach LS, Romero E, Becklin RR, Chettier R, Bell R, Phansalkar A, Strand A, Torcassi C, Savage J, Hurlburt A et al. (2007) Huntingtin interacting proteins are genetic modifiers of neurodegeneration. PLoS Gene 3, 689–708. 14 Hyun TS & Ross TS (2004) HIP1: trafficking roles and regulation of tumorigenesis. Trends Mol Med 10, 194– 199. 15 Kalchman MA, Koide HB, McCutcheon K, Graham RK, Nichol K, Nishiyama K, Kazemi-Esfarjani P, Lynn FC, Wellington C, Metzler M et al. (1997) HIP1, a human homologue of S. cerevisiae Sla2p, interacts with membrane-associated huntingtin in the brain. Nat Genet 16, 44–53. 16 Wanker EE, Rovira C, Scherzinger E, Hasenbank R, Wa ¨ lter S, Tait D, Colicelli J & Lehrach H (1997) HIP- I: a huntingtin interacting protein isolated by the yeast two-hybrid system. Hum Mol Genet 6, 487–495. 17 Hackam AS, Yassa AS, Singaraja R, Metzler M, Gut- ekunst CA, Gan L, Warby S, Wellington CL, Vaillan- court J, Chen N et al. (2000) Huntingtin interacting protein 1 induces apoptosis via a novel caspase-depen- dent death effector domain. J Biol Chem 275, 41299– 41308. 18 Metzler M, Gan L, Wong TP, Liu L, Helm J, Liu L, Georgiou J, Wang Y, Bissada N, Cheng K et al. (2007) NMDA receptor function and NMDA receptor-depen- dent phosphorylation of huntingtin is altered by the endocytic protein HIP1. J Neurosci 27, 2298–2308. 19 Metzler M, Li B, Gan L, Georgiou J, Gutekunst CA, Wang Y, Torre E, Devon RS, Oh R, Legendre- Guillemin V et al. (2003) Disruption of the endocytic protein HIP1 results in neurological deficits and decreased AMPA receptor trafficking. EMBO J 22, 3254–3266. 20 Chowdhury I, Tharakan B & Bhat GK (2006) Current concepts in apoptosis: the physiological suicide program revisited. Cell Mol Biol Lett 11, 506–525. N. P. Bhattacharyya et al. HIP-1 & HIPPI mediated apoptosis and transcription FEBS Journal 275 (2008) 4271–4279 ª 2008 The Authors Journal compilation ª 2008 FEBS 4277 21 Choi SA, Kim SJ & Chung KC (2006) Huntingtin-inter- acting protein 1-mediated neuronal cell death occurs through intrinsic apoptotic pathways and mitochondrial alterations. FEBS Lett 580, 5275–5282. 22 Kang JE, Choi SA, Park JB & Chung KC (2005) Regu- lation of the proapoptotic activity of huntingtin inter- acting protein 1 by Dyrk1 and caspase-3 in hippocampal neuroprogenitor cells. J Neurosci 81, 62–72. 23 Gervais FG, Singaraja R, Xanthoudakis S, Gutekunst C, Leavitt BR, Metzler M, Hackam AS, Tam J, Vai- llancourt JP, Houtzager V et al. (2002) Recruitment and activation of caspase8 by the Huntingtin-interacting protein HIP1 and a novel partner Hippi. Nat Cell Biol 4, 95–105. 24 Majumder P, Chattopadhyay B, Mazumder A, Das P & Bhattacharyya NP (2006) Induction of apoptosis in cells expressing exogenous Hippi, a molecular partner of huntingtin-interacting protein Hip1. Neurobiol Dis 22, 242–256. 25 Rao DS, Hyun TS, Kumar PD, Mizukami IF, Rubin MA, Lucas PC, Sanda MG & Ross TS (2002) Hunting- tin-interacting protein 1 is overexpressed in prostate and colon cancer and is critical for cellular survival. J Clin Invest 110, 351–360. 26 Rao DS, Chang JC, Kumar PD, Mizukami I, Smith- son GM, Bradley SV, Parlow AF & Ross TS (2001) Huntingtin interacting protein 1 is a clathrin coat binding protein required for differentiation of late spermatogenic progenitors. Mol Cell Biol 21, 7796– 7806. 27 Bradley SV, Hyun TS, Oravecz-Wilson KI, Li L, Wald- orff EI, Ermilov AN, Goldstein SA, Zhang CX, Drubin DG, Varela K et al. (2007) Degenerative phenotypes caused by the combined deficiency of murine HIP1 and HIP1r are rescued by human HIP1. Hum Mol Genet 16, 1279–1292. 28 Khatchadourian K, Smith CE, Metzler M, Gregory M, Hayden MR, Cyr DG & Hermo L (2007) Struc- tural abnormalities in spermatids together with reduced sperm counts and motility underlie the repro- ductive defect in HIP1 ) ⁄ ) mice. Mol Reprod Dev 74, 341–359. 29 Bradley SV, Holland EC, Liu GY, Thomas D, Hyun TS & Ross TS (2007) Huntingtin interacting protein 1 is a novel brain tumor marker that associates with epidermal growth factor receptor. Cancer Res 67, 3609–3615. 30 Hyun TS, Rao DS, Saint-Dic D, Michael LE, Kumar PD, Bradley SV, Mizukami IF, Oravecz-Wilson KI & Ross TS (2004) HIP1 and HIP1r stabilize receptor tyro- sine kinases and bind 3-phosphoinositides via epsin N-terminal homology domains. J Biol Chem 279, 14294–14306. 31 Sakamoto K, Yoshida S, Ikegami K, Minakami R, Kato A, Udo H & Sugiyama H (2007) Homer1c inter- acts with Hippi and protects striatal neurons from apoptosis. Biochem Biophys Res Commun 352, 1–5. 32 Roth W, Kermer P, Krajewska M, Welsh K, Davis S, Krajewski S & Reed JC (2003) Bifunctional apoptosis inhibitor (BAR) protects neurons from diverse cell death pathways. Cell Death Differ 10, 1178–1187. 33 Stanton SE, Blanck JK, Locker J & Schreiber-Agus N (2007) Rybp interacts with Hippi and enhances Hippi- mediated apoptosis. Apoptosis 12, 2197–2206. 34 Gdynia G, Lehmann-Koch J, Sieber S, Tagscherer KE, Fassl A, Zentgraf H, Matsuzawa SI, Reed JC & Roth W (2008) BLOC1S2 interacts with the HIPPI protein and sensitizes NCH89 glioblastoma cells to apoptosis. Apoptosis 13, 437–447. 35 Cheng CM, Huang SP, Chang YF, Chung WY & Yuo CY (2003) The viral death protein Apoptin interacts with Hippi, the protein interactor of Huntingtin-inter- acting protein 1. Biochem Biophys Res Commun 305, 359–364. 36 Houde C, Dickinson RJ, Houtzager VM, Cullum R, Montpetit R, Metzler M, Simpson EM, Roy S, Hayden MR, Hoodless PA et al. (2006) Hippi is essential for node cilia assembly and Sonic hedgehog signaling. Dev Biol 300, 523–533. 37 Mao J, Maye P, Kogerman P, Tejedor FJ, Toftgard R, Xie W, Wu G & Wu D (2002) Regulation of Gli1 tran- scriptional activity in the nucleus by Dyrk1. J Biol Chem 277, 35156–35161. 38 Mills IG, Gaughan L, Robson C, Ross T, McCracken S, Kelly J & Neal DE (2005) Huntingtin interacting protein 1 modulates the transcriptional activity of nuclear hormone receptors. J Cell Biol 70, 191–200. 39 Majumder P, Chattopadhyay B, Sukanya S, Ray T, Banerjee M, Mukhopadhyay D & Bhattacharyya NP (2007) Interaction of HIPPI with putative promoter sequence of caspase-1 in vitro and in vivo. Biochem Biophys Res Commun 353, 80–85. 40 Majumder P, Choudhury A, Banerjee M, Lahiri A & Bhattacharyya NP (2007) Interactions of HIPPI, a molecular partner of Huntingtin interacting protein HIP1, with the specific motif present at the putative promoter sequence of the caspase-1, caspase-8 and caspase-10 genes. FEBS J 274, 3886–3899. 41 Gupta S, Radha V, Furukawa Y & Swarup G (2001) Direct transcriptional activation of human caspase1 by tumor suppressor p53. J Biol Chem 276, 10585–10588. 42 Jain N, Gupta S, Sudhakar C, Radha V & Swarup G (2005) Role of p73 in regulating human caspase-1 gene transcription induced by interferon-{gamma} and cisplatin. J Biol Chem 280, 36664–36673. 43 Pei H, Li C, Adereth Y, Hsu T, Watson DK & Li R (2005) Caspase-1 is a direct target gene of ETS1 and HIP-1 & HIPPI mediated apoptosis and transcription N. P. Bhattacharyya et al. 4278 FEBS Journal 275 (2008) 4271–4279 ª 2008 The Authors Journal compilation ª 2008 FEBS plays a role in ETS1-induced apoptosis. Cancer Res 65, 7205–7213. 44 Garcı ´ a E, Marcos-Gutie ´ rrez C, del MarLorente M, Moreno JC & Vidal M (1999) RYBP, a new repressor protein that interacts with components of the mamma- lian Polycomb complex, and with the transcription factor YY1. EMBO J 18, 3404–3418. 45 Parker JA, Metzler M, Georgiou J, Mage M, Roder JC, Rose AM, Hayden MR & Neri C (2007) Hunting- tin-interacting protein 1 influences worm and mouse presynaptic function and protects Caenorhabditis ele- gans neurons against mutant polyglutamine toxicity. J Neurosci 27, 11056–11064. 46 Majumder P, Raychaudhuri S, Chattopadhyay B & Bhattacharyya NP (2007) Increased caspase-2, calpain activations and decreased mitochondrial complex II activity in cells expressing exogenous huntingtin exon 1 containing CAG repeat in the pathogenic range. Cell Mol Neurobiol 27, 1127–1145. 47 Ona VO, Li M, Vonsattel JP, Andrews LJ, Khan SQ, Chung WM, Frey AS, Menon AS, Li XJ, Stieg PE et al. (1999) Inhibition of caspase1 slows disease pro- gression in a mouse model of Huntington’s disease. Nature 399, 263–267. 48 Vis JC, Schipper E, de Boer-van Huizen RT, Verbeek MmM, de Waal RM, Wesseling P, Ten Donkelaar HJ & Kremer B (2005) Expression pattern of apoptosis- related markers in Huntington’s disease. Acta Neuropa- thol (Berl) 109, 321–328. N. P. Bhattacharyya et al. HIP-1 & HIPPI mediated apoptosis and transcription FEBS Journal 275 (2008) 4271–4279 ª 2008 The Authors Journal compilation ª 2008 FEBS 4279 . MINIREVIEW Huntington’s disease: roles of huntingtin-interacting protein 1 (HIP -1) and its molecular partner HIPPI in the regulation of apoptosis and transcription Nitai. chain A; CLTB, clathrin light chain B; ENTH, Epsin N-terminal homology; HD, Huntington’s disease; HIP -1, huntingtin-interacting protein 1; HIPPI, huntingtin-interacting