Putting proteins in their place: palmitoylation in Huntington disease and other neuropsychiatric diseases

Post-translational modification of proteins by the lipid palmitate is critical for protein localization and function. Palmitoylation is regulated by the opposing enzymes palmitoyl acyltransferases (PATs) and acyl protein thioesterases, which add and remove palmitate from proteins, respectively. Palmitoylation is particularly important for a number of processes including neuronal development and synaptic activity in the central nervous system. Dysregulated palmitoylation contributes to neuropsychiatric disease. In total six PATs (HIP14, HIP14L, ZDHHC8, ZDHHC9, ZDHHC12, and ZDHHC15) and one thioesterase (PPT1) have been implicated in Huntington disease (HD), Alzheimer disease, schizophrenia, mental retardation, and infantile and adult onset forms of neuronal ceroid lipofuscinosis. Currently there is no genetic link between PATs and Alzheimer disease pathogenesis but palmitoylation of amyloid precursor protein-processing enzyme, γ-secretase, influences β-amyloid generation. Several lines of evidence point to a role for palmitoylation by HIP14 in the pathogenesis of HD; HIP14 is dysfunctional in the presence of the HD mutation and Hip14-deficient mice develop features of HD. Wildtype huntingtin (the protein mutated in HD) enhances the PAT activity of HIP14 and mutant HTT interacts less with HIP14. Therefore, it may be that loss of the positive modulation of HIP14 activity due to reduced interaction with huntingtin is important in the disease mechanism. Preliminary evidence suggests a closely related PAT to HIP14, HIP14L, may also play a role in the pathogenesis of HD. In order to design rational therapeutic approaches to restore palmitoylation in neuropsychiatric disease, it will be critical to better understand the relationships between PATs and thioesterases with their regulators and substrates.     

Wild-type HTT modulates the enzymatic activity of the neuronal palmitoyl transferase HIP14

Huntington disease (HD) is caused by polyglutamine expansion in the huntingtin (HTT) protein. Huntingtin-interacting protein 14 (HIP14), one of 23 DHHC domain-containing palmitoyl acyl transferases (PATs), binds to HTT and robustly palmitoylates HTT at cysteine 214. Mutant HTT exhibits reduced palmitoylation and interaction with HIP14, contributing to the neuronal dysfunction associated with HD. In this study, we confirmed that, among 23 DHHC PATs, HIP14 and its homolog DHHC-13 (HIP14L) are the two major PATs that palmitoylate HTT. Wild-type HTT, in addition to serving as a palmitoylation substrate, also modulates the palmitoylation of HIP14 itself. In vivo, HIP14 palmitoylation is decreased in the brains of mice lacking one HTT allele (hdh+/-) and is further reduced in mouse cortical neurons treated with HTT antisense oligos (HTT-ASO) that knockdown HTT expression by ～95%. Previously, it has been shown that palmitoylation of DHHC proteins may affect their enzymatic activity. Indeed, palmitoylation of SNAP25 by HIP14 is potentiated in vitro in the presence of wild-type HTT. This influence of HTT on HIP14 activity is lost in the presence of CAG expansion. Furthermore, in both brains of hdh+/- mice and neurons treated with HTT-ASO, we observe a significant reduction in palmitoylation of endogenous SNAP25 and GluR1, synaptic proteins that are substrates of HIP14, suggesting wild-type HTT also influences HIP14 enzymatic activity in vivo. This study describes an important biochemical function for wild-type HTT modulation of HIP14 palmitoylation and its enzymatic activity.     

Degenerative phenotypes caused by the combined deficiency of murine HIP1 and HIP1r are rescued by human HIP1

The members of the huntingtin-interacting protein-1 (HIP1) family, HIP1 and HIP1-related (HIP1r), are multi-domain proteins that interact with inositol lipids, clathrin and actin. HIP1 is over-expressed in a variety of cancers and both HIP1 and HIP1r prolong the half-life of multiple growth factor receptors. To better understand the physiological importance of the HIP1 family in vivo, we have analyzed a large cohort of double Hip1/Hip1r knockout (DKO) mice. All DKO mice were dwarfed, afflicted with severe vertebral defects and died in early adulthood. These phenotypes were not observed during early adulthood in the single Hip1 or Hip1r knockouts, indicating that HIP1 and HIP1r compensate for one another. Despite the ability of HIP1 and HIP1r to modulate growth factor receptor levels when over-expressed, studies herein using DKO fibroblasts indicate that the HIP1 family is not necessary for endocytosis but is necessary for the maintenance of diverse adult tissues in vivo. To test if human HIP1 can function similar to mouse HIP1, transgenic mice with 'ubiquitous' expression of the human HIP1 cDNA were generated and crossed with DKO mice. Strikingly, the compound human HIP1 transgenic DKO mice were completely free from dwarfism and spinal defects. This successful rescue demonstrates that the human HIP1 protein shares some interchangeable functions with both HIP1 and HIP1r in vivo. In addition, we conclude that the degenerative phenotypes seen in the DKO mice are due mainly to HIP1 and HIP1r protein deficiency rather than altered expression of neighboring genes or disrupted intronic elements.     

Cloning, expression analysis, and chromosomal localization of HIP1R, an isolog of huntingtin interacting protein (HIP1)

Huntington disease (HD) is an inherited neurodegenerative disorder which is associated with CAG expansion in the coding region of the gene for huntingtin protein. Recently, a huntingtin interacting protein, HIP1, was isolated by the yeast two-hybrid system. Here we report the isolation of a cDNA clone for HIP1R (huntingtin interacting protein-1 related), which encodes a predicted protein product sharing a striking homology with HIP1. RT-PCR analysis showed that the messenger RNA was ubiquitously expressed in various human tissues. Based on PCR-assisted analysis of a radiation hybrid panel and fluorescence in situ hybridization, HIP1R was localized to the q24 region of chromosome 12. Received: May 18, 1998 / Accepted July 13, 1998     

Huntingtin Interacting Protein 1 mutations lead to abnormal hematopoiesis, spinal defects and cataracts

Huntingtin Interacting Protein 1 (HIP1) binds clathrin and AP2, is overexpressed in multiple human tumors, and transforms fibroblasts. The function of HIP1 is unknown although it is thought to play a fundamental role in clathrin trafficking. Gene-targeted Hip1-/- mice develop premature testicular degeneration and severe spinal deformities. Yet, although HIP1 is expressed in many tissues including the spleen and bone marrow and was part of a leukemogenic translocation, its role in hematopoiesis has not been examined. In this study we report that three different mutations of murine Hip1 lead to hematopoietic abnormalities reflected by diminished early progenitor frequencies and resistance to 5-FU-induced bone marrow toxicity. Two of the Hip1 mutant lines also display the previously described spinal defects. These observations indicate that, in addition to being required for the survival/proliferation of cancer cells and germline progenitors, HIP1 is also required for the survival/proliferation of diverse types of somatic cells, including hematopoietic progenitors.     

Palmitoylation of huntingtin by HIP14 is essential for its trafficking and function

Post-translational modification by the lipid palmitate is crucial for the correct targeting and function of many proteins. Here we show that huntingtin (htt) is normally palmitoylated at cysteine 214, which is essential for its trafficking and function. The palmitoylation and distribution of htt are regulated by the palmitoyl transferase huntingtin interacting protein 14 (HIP14). Expansion of the polyglutamine tract of htt, which causes Huntington disease, results in reduced interaction between mutant htt and HIP14 and consequently in a marked reduction in palmitoylation. Mutation of the palmitoylation site of htt, making it palmitoylation resistant, accelerates inclusion formation and increases neuronal toxicity. Downregulation of HIP14 in mouse neurons expressing wild-type and mutant htt increases inclusion formation, whereas overexpression of HIP14 substantially reduces inclusions. These results suggest that the expansion of the polyglutamine tract in htt results in decreased palmitoylation, which contributes to the formation of inclusion bodies and enhanced neuronal toxicity.     

IFNγ contributes to the development of gastric epithelial cell metaplasia in Huntingtin interacting protein 1 related (Hip1r)-deficient mice

Huntingtin interacting protein 1 related (Hip1r) is an F-actin- and clathrin-binding protein involved in vesicular trafficking that is crucial for parietal cell function and epithelial cell homeostasis in the stomach. Gastric parietal cells in Hip1r-deficient mice are lost by apoptotic cell death, which leads to a progressive epithelial cell derangement, including glandular hypertrophy, zymogenic cell loss and expansion of a metaplastic mucous cell lineage known as spasmolytic polypeptide-expressing metaplasia (SPEM). The epithelial cell changes are associated with infiltration of inflammatory cells. As inflammatory mediators, such as IFNγ, have been shown to contribute to the development of the gastric epithelial cell metaplasia after Helicobacter infection, we tested whether IFNγ played a role in the spontaneous progressive epithelial metaplasia observed in Hip1r-deficient mice. Hip1r-deficient mice were crossed with IFNγ-deficient mice and single- and double-mutant mice were analyzed at 3 and 12 months of age. Histopathology scoring showed that loss of IFNγ tempered the spontaneous development of metaplastic lesions in Hip1r-deficient mice. Loss of IFNγ was observed to abrogate the glandular hypertrophy evident in Hip1r mutant stomach, although increased epithelial cell proliferation and elevated gastrin levels were not affected by the presence or absence of this pro-inflammatory cytokine. An analysis of cell lineage markers in the double-mutant mice demonstrated that IFNγ specifically affected the development of metaplastic mucous cells in the neck region, whereas the parietal cell, surface mucous cell and zymogenic cell alterations remained similar to the histopathology in the Hip1r mutant. Morphometric analysis showed that IFNγ was required for the mucous cell hypertrophy and hyperplasia observed in Hip1r-deficient mice. Together, these findings demonstrate that IFNγ is critical for the development of the gastric epithelial cell metaplasia that results from parietal cell atrophy in the Hip1r-deficient mice.     

Changes in the cytologic distribution of heparin/heparan sulfate interacting protein/ribosomal protein L29 (HIP/RPL29) during in vivo and in vitro mouse mammary epithelial cell expression and differentiation.

HIP/RPL29 is a small, highly basic, heparin/heparan sulfate interacting protein identical to ribosomal protein L29 and present in most adult epithelia. In the present study, we show that mouse HIP/RPL29 is ubiquitously present in adult mammary epithelia and is significantly increased during pregnancy and lactation. We observed for the first time that HIP/RPL29 intracellular expression and distribution varies, depending on the growth/differentiation state of the luminal epithelium. HIP/RPL29 was detected at low levels in mammary glands of virgin animals, increased markedly during lactation, and was lost again during involution. HIP/RPL29, preferentially found in the expanded cytoplasm of mature epithelial cells secreting milk, is present also in the nucleus of proliferating and differentiating ductal and alveolar elements. We used COMMA-D cells as an in vitro model for mammary-specific differentiation and examined similar intracellular redistribution of HIP/RPL29 associated with functional differentiation. However, no changes in HIP/RPL29 expression levels were detected in response to lactogenic hormones. Finally, the cellular distribution of HIP/RPL29 in both nuclear and cytoplasmic compartments was confirmed by transfecting a normal mammary epithelial cell line, NMuMG, with a fusion protein of HIP/RPL29 and EGFP. Collectively, these data support the idea that HIP/RPL29 plays more than one role during adult mammary gland development.     

The huntingtin interacting protein HIP1 is a clathrin and alpha-adaptin-binding protein involved in receptor-mediated endocytosis

The huntingtin interacting protein (HIP1) is enriched in membrane-containing cell fractions and has been implicated in vesicle trafficking. It is a multidomain protein containing an N-terminal ENTH domain, a central coiled-coil forming region and a C-terminal actin-binding domain. In the present study we have identified three HIP1 associated proteins, clathrin heavy chain and alpha-adaptin A and C. In vitro binding studies revealed that the central coiled-coil domain is required for the interaction of HIP1 with clathrin, whereas DPF-like motifs located upstream to this domain are important for the binding of HIP1 to the C-terminal 'appendage' domain of alpha-adaptin A and C. Expression of full length HIP1 in mammalian cells resulted in a punctate cytoplasmic immunostaining characteristic of clathrin-coated vesicles. In contrast, when a truncated HIP1 protein containing both the DPF-like motifs and the coiled-coil domain was overexpressed, large perinuclear vesicle-like structures containing HIP1, huntingtin, clathrin and endocytosed transferrin were observed, indicating that HIP1 is an endocytic protein, the structural integrity of which is crucial for maintenance of normal vesicle size in vivo.     

The HIP gene encoding a heparin/heparan sulfate interacting protein is mutated in metastatic human colorectal cancer

Heparin/heparan sulfate interacting protein (HIP) was initially identified as an adhesion molecule from a human uterine epithelial cell line. It was previously demonstrated that HIP was upregulated in human colorectal cancer. However, its expression was significantly lower in Dukes' D samples compared to earlier Dukes' stages suggesting that HIP was inversely correlated to metastasis. The present study shows the presence of mutations in human metastatic colorectal cancer tissue and a cell line. Interestingly, a 12-base deletion encoding the heparin/heparan sulfate binding motif was common between the metastatic tissue and cell line. There was no mutation in the primary carcinoma and normal tissue. The findings suggest an important role for HIP in colorectal cancer metastasis.     

经典型蛋白激酶Cγ及其相互作用蛋白14-3-3γ在原代培养小鼠脑皮层神经元氧-糖剥夺缺血损伤中的作用

目的 探讨经典型蛋白激酶Cγ (cPKCγ)及其相互作用蛋白14-3-3γ在原代培养小鼠脑皮层神经元氧-糖剥夺(OGD)缺血损伤中的作用。方法 利用cPKCγ基因敲除(cPKCγ^-/-)小鼠和原代培养脑皮层神经元1h OGD/24h复糖复氧(R)模拟离体细胞缺血模型,给予14-3-3γ抑制剂R18干扰,采用免疫共沉淀(Co-IP)、蛋白免疫印迹(Western blotting)和四甲基偶氮唑盐(MTT)比色法,验证cPKCγ与14-3-3γ的相互作用及其对原代培养小鼠脑皮层神经元1h OGD/24h R缺血损伤的影响。结果 Co-IP结果证明,14-3-3γ与cPKCγ在小鼠脑皮层中确实存在相互作用;1h OGD/24h R处理使原代培养野生型(cPKCγ ^+/+)小鼠脑皮层神经元内cPKCγ蛋白水平明显降低,而14-3-3γ蛋白水平显著增加(P<0.05,每组n=6);对于原代培养cPKCγ^-/-小鼠脑皮层神经元,尽管14-3-3γ表达水平明显高于cPKCγ^+/+小鼠脑皮层神经元,但1h OGD/24h R处理却使14-3-3γ表达水平显著降低(P<0.05, 每组n=6),提示cPKCγ可影响神经元内14-3-3γ的表达水平。此外,MTT结果表明,14-3-3γ抑制剂R18 (100μmol/L)可显著加重1h OGD/24h R处理原代培养cPKCγ^-/-和cPKCγ^+/+小鼠脑皮层神经元的缺血损伤(P<0.05,每组n=6)。结论 cPKCγ与14-3-3γ在小鼠脑皮层神经元内存在相互作用并影响14-3-3γ的蛋白表达水平,cPKCγ和14-3-3γ在原代培养小鼠脑皮层神经元缺血/低氧损伤中起保护作用。     

Loss of wild-type huntingtin influences motor dysfunction and survival in the YAC128 mouse model of Huntington disease

Huntington disease (HD) is an adult-onset neurodegenerative disease caused by a toxic gain of function in the huntingtin (htt) protein. The contribution of wild-type htt function to the pathogenesis of HD is currently uncertain. To assess the role of wild-type htt in HD, we generated YAC128 mice that do not express wild-type htt (YAC128-/-) but express the same amount of mutant htt as normal YAC128 mice (YAC128+/+). YAC128-/- mice perform worse than YAC128+/+ mice in the rotarod test of motor coordination (P = 0.001) and are hypoactive compared with YAC128+/+ mice at 2 months (P = 0.003). Striatal neuropathology was not clearly worse in YAC128-/- mice compared with YAC128+/+ mice. There was no significant effect of decreased wild-type htt on striatal volume, neuronal counts or DARPP-32 expression but a modest worsening of striatal neuronal atrophy was evident (6%, P = 0.03). The testis of YAC128+/+ mice showed atrophy and degeneration, which was markedly worsened in the absence of wild-type htt (P = 0.001). YAC128+/+ mice also showed a male specific deficit in survival compared with WT mice which was exacerbated by the loss of wild-type htt (12-month-male survival, P < 0.001). Overall, we demonstrate that the loss of wild-type htt influences motor dysfunction, hyperkinesia, testicular degeneration and impaired lifespan in YAC128 mice. The mild effect of wild-type htt on striatal phenotypes in YAC128 mice suggests that the characteristic striatal neuropathology in HD is caused primarily by the toxicity of mutant htt and that replacement of wild-type htt will not be an adequate treatment for HD.     

Global growth deficiencies in mice lacking the ribosomal protein HIP/RPL29.

Because of their deleterious effects on developing organisms, ribosomal protein (RP) mutations have been poorly described in mammals, and only a few heterozygous mutations have been shown to be viable. This observation is believed to be due to the fact that each RP is an essential component in the assembly of a functional stable ribosome. Here, we created gene targeted mutant mice lacking HIP/RPL29, an RP associated with translationally active ribosomes in eukaryotes. In contrast to other RP mutants, HIP/RPL29 null mice are viable but are up to 50% smaller than their control littermates at weaning age. In null embryos, delayed global growth is first observed around mid-gestation, and postnatal lethality due to low birth weight results in distortion of the Mendelian ratio. Prenatal growth defects are not fully compensated for during adulthood, and null animals display proportionately smaller organs and stature, and reach sexual maturity considerably later when compared with their control siblings. Additionally, HIP/RPL29 null embryonic fibroblasts have decreased rates of proliferation and protein synthesis and exhibit reduced steady state levels of core RPs. Altogether, our findings provide conclusive genetic evidence that HIP/RPL29 functions as an important regulator of global growth by modulating the rate of protein synthesis.     

Striatal phosphodiesterase mRNA and protein levels are reduced in Huntington's disease transgenic mice prior to the onset of motor symptoms

Inheritance of a single copy of the gene encoding huntingtin (HD) with an expanded polyglutamine-encoding CAG repeat leads to neuronal dysfunction, neurodegeneration and the development of the symptoms of Huntington's disease (HD). We have found that the steady-state mRNA levels of two members of the phosphodiesterase (PDE) multi-gene family decrease over time in the striatum of R6 transgenic HD mice relative to age-matched wild-type littermates. Phosphodiesterase 10A (PDE10A) mRNA and protein levels decline in the striatum of R6/1 and R6/2 HD mice prior to motor symptom development. The rate of reduction in PDE10A protein correlates with the rate of decline of the message and the decrease in PDE10A mRNA and protein is more rapid in R6/2 compared with R6/1 mice. Both PDE10A protein and mRNA, therefore, decline to minimum levels prior to the onset of overt physical symptoms in both strains of transgenic mice. Moreover, protein levels of PDE10A are decreased in the caudate-putamen of grade 3 HD patients compared with age-matched neuropathologically normal controls. Striatal PDE1B mRNA levels also decline in R6/1 and R6/2 HD mice; however, the decrease in striatal PDE10A levels (>60%) was greater than that observed for PDE1B and immediately preceded the onset of motor symptoms. In contrast, PDE4A mRNA levels are relatively low in the striatum and do not differ between age-matched wild-type and transgenic HD mice. This suggests that the regulation of PDE10A and PDE1B, but not PDE4A, mRNA levels is dependent on the relative expression of or number of CAG repeats within the human HD transgene. The loss of phosphodiesterase activity may lead to dysregulation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) levels in the striatum, a region of the brain that contributes to the control of movement and cognition.     

Hepatocarcinoma-Intestine-Pancreas/Pancreatic Associated Protein (HIP/PAP) Is Expressed and Secreted by Proliferating Ductules as well as by Hepatocarcinoma and Cholangiocarcinoma Cells

Hepatocarcinoma-intestine-pancreas/pancreatic associated protein (HIP/PAP) gene was identified because of its increased expression in 25% of human hepatocellular carcinoma. HIP/PAP protein, a C-type lectin, binds laminin, acts as an adhesion molecule for hepatocytes, and has also been described as an acute phase secretory protein during acute pancreatitis in humans and rats. We investigated HIP/PAP protein expression in patients with various liver diseases associated with ductular reaction. At the same time, we analyzed patients with hepatocellular carcinoma and cholangiocarcinoma, and tested HIP/PAP protein levels in sera to establish the pattern of secretion. Our data show that HIP/PAP expression was not restricted to hepatocellular carcinoma, but was also detected in cholangiocarcinoma cells as well as in reactive non-malignant bile ductules. In contrast, HIP/PAP protein expression was undetectable in normal mature hepatocytes, but some ductular cells localized at the interface of portal tracts with parenchyma were HIP/PAP immunoreactive in normal liver. Finally, we present evidence that HIP/PAP serum levels were increased in 21/28 (75%) patients with hepatocellular carcinoma, and in 25/51 (49%) patients with nonmalignant cirrhosis. Altogether, these results suggest that HIP/PAP protein may be implicated in hepatocytic and cholangiolar differentiation and proliferation.     

Polyglutamine length-dependent interaction of Hsp40 and Hsp70 family chaperones with truncated N-terminal huntingtin: their role in suppression of aggregation and cellular toxicity

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by polyglutamine expansion in the disease protein, huntingtin. In HD patients and transgenic mice, the affected neurons form characteristic ubiquitin-positive nuclear inclusions (NIs). We have established ecdysone-inducible stable mouse Neuro2a cell lines that express truncated N-terminal huntingtin (tNhtt) with different polyglutamine lengths which form both cytoplasmic and nuclear aggregates in a polyglutamine length- and inducer dose-dependent manner. Here we demonstrate that newly synthesized polyglutamine-expanded truncated huntingtin interacts with members of Hsp40 and Hsp70 families of chaperones in a polyglutamine length-dependent manner. Of these interacting chaperones, only Hdj-2 and Hsc70 frequently (Hdj-2 > Hsc70) co-localize with both the aggregates in the cellular model and with the NIs in the brains of HD exon 1 transgenic mice. However, Hdj-2 and Hsc70 do not co-localize with cytoplasmic aggregates in the brains of transgenic mice despite these chaperones being primarily localized in the cytoplasmic compartment. This strongly suggests that the chaperone interaction and their redistribution to the aggregates are two completely different phenomena of the cellular unfolded protein response. This unfolded protein response is also evident from the dramatic induction of Hsp70 on expression of polyglutamine-expanded protein in the cellular model. Transient overexpression of either Hdj-1 or Hsc70 suppresses the aggregate formation; however, suppression efficiency is much higher in Hdj-1 compared with Hsc70. Overexpression of Hdj-1 and Hsc70 is also able to protect cell death caused by polyglutamine-expanded tNhtt and their combination proved to be most effective.     

Compensatory changes in the ubiquitin-proteasome system, brain-derived neurotrophic factor and mitochondrial complex II/III in YAC72 and R6/2 transgenic mice partially model Huntington's disease patients

Intraneuronal protein aggregates of the mutated huntingtin in Huntington's disease (HD) brains suggest an overload and/or dysfunction of the ubiquitin-proteasome system (UPS). There is a general inhibition of the UPS in many brain regions (cerebellum, cortex, substantia nigra and caudate-putamen) and skin fibroblasts from HD patients. In the current experiment, the widely used mutant huntingtin-exon 1 CAG repeat HD transgenic mice model (R6/2) (with 144 CAG repeat and exon 1) during late-stage pathology, had increases in proteasome activity in the striatum. However, this discrepancy with HD patient tissue was not apparent in the mutant CAG repeat huntingtin full-length HD (YAC72) transgenic mouse model during post-symptomatic and late-stage pathology, which then also showed UPS inhibition similar to HD patients' brains. In both types of HD model mice, we determined biochemical changes, including expression of brain-derived neurotrophic factor (BDNF) and mitochondrial complex II/III (MCII/III) activities related to HD pathology. We found increases of both BDNF expression, and MCII/III activities in YAC72 transgenic mice, and no change of BDNF expression in R6/2 mice. Our data show that extreme CAG repeat lengths in R6/2 mice is paradoxically associated with increased proteasome activity, probably as a cellular compensatory biochemical change in response to the underlying mutation. Changes in HD patients for UPS function, BDNF expression and MCII/III activity are only partially modeled in R6/2 and YAC72 mice, with the latter at 16 months of age being most congruent with the human disease.