Mass spectrometric identification of novel posttranslational modification sites in Huntingtin

Huntington's disease (HD) is caused by a CAG triplet repeat expansion in exon 1 of the Huntingtin (Htt) gene, encoding an abnormal expanded polyglutamine (polyQ) tract that confers toxicity to the mutant Htt (mHtt) protein. Recent data suggest that posttranslational modifications of mHtt modulate its cytotoxicity. To further understand the cytotoxic mechanisms of mHtt, we have generated HEK293 cell models stably expressing Strep- and FLAG-tagged Htt containing either 19Q (wild-type Htt), 55Q (mHtt), or 94Q (mHtt) repeats. Following tandem affinity purification, the tagged Htt and associated proteins were subjected to tandem mass spectrometry or 2D nano-LC tandem mass spectrometry and several novel modification sites of mHtt containing 55Q or 94Q were identified. These were phosphorylation sites located at Ser431 and Ser432, and ubiquitination site located at Lys444. The two phosphorylation sites were confirmed by Western blot analysis using phosphorylation site-specific antibodies. In addition, prevention of phosphorylation at the two serine sites altered mHtt toxicity and accumulation. These modifications of mHtt may provide novel therapeutic targets for effective treatment of the disorder.     

Mechanisms of neuronal cell death in Huntington's disease

Huntington's disease (HD) is a genetically dominant neurodegenerative condition caused by an unique mutation in the disease gene huntingtin. Although the Huntington protein (Htt) is ubiquitously expressed, expansion of the polyglutamine tract in Htt leads to the progressive loss of specific neuronal subpopulations in HD brains. In this article, we will summarize the current understanding on mechanisms of how mutant Htt can elicit cytotoxicity, as well as how the selective sets of neuronal cell death occur in HD brains.     

Mass spectrometric identification of novel lysine acetylation sites in huntingtin

Huntingtin (Htt) is a protein with a polyglutamine stretch in the N-terminus and expansion of the polyglutamine stretch causes Huntington's disease (HD). Htt is a multiple domain protein whose function has not been well characterized. Previous reports have shown, however, that post-translational modifications of Htt such as phosphorylation and acetylation modulate mutant Htt toxicity, localization, and vesicular trafficking. Lysine acetylation of Htt is of particular importance in HD as this modification regulates disease progression and toxicity. Treatment of mouse models with histone deacetylase inhibitors ameliorates HD-like symptoms and alterations in acetylation of Htt promotes clearance of the protein. Given the importance of acetylation in HD and other diseases, we focused on the systematic identification of lysine acetylation sites in Htt23Q (1-612) in a cell culture model using mass spectrometry. Myc-tagged Htt23Q (1-612) overexpressed in the HEK 293T cell line was immunoprecipitated, separated by SDS-PAGE, digested and subjected to high performance liquid chromatography tandem MS analysis. Five lysine acetylation sites were identified, including three novel sites Lys-178, Lys-236, Lys-345 and two previously described sites Lys-9 and Lys-444. Antibodies specific to three of the Htt acetylation sites were produced and confirmed the acetylation sites in Htt. A multiple reaction monitoring MS assay was developed to compare quantitatively the Lys-178 acetylation level between wild-type Htt23Q and mutant Htt148Q (1-612). This report represents the first comprehensive mapping of lysine acetylation sites in N-terminal region of Htt.     

Degradation of mutant huntingtin via the ubiquitin/proteasome system is modulated by FE65

An unstable expansion of the polyglutamine repeat within exon 1 of the protein Htt (huntingtin) causes HD (Huntington's disease). Mounting evidence shows that accumulation of N-terminal mutant Htt fragments is the source of disruption of normal cellular processes which ultimately leads to neuronal cell death. Understanding the degradation mechanism of mutant Htt and improving its clearance has emerged as a new direction in developing therapeutic approaches to treat HD. In the present study we show that the brain-enriched adaptor protein FE65 is a novel interacting partner of Htt. The binding is mediated through WW-polyproline interaction and is dependent on the length of the polyglutamine tract. Interestingly, a reduction in mutant Htt protein level was observed in FE65-knockdown cells, and the process requires the UPS (ubiquitin/proteasome system). Moreover, the ubiquitination level of mutant Htt was found to be enhanced when FE65 is knocked down. Immunofluroescence staining revealed that FE65 associates with mutant Htt aggregates. Additionally, we demonstrated that overexpression of FE65 increases mutant Htt-induced cell death both in vitro and in vivo. These results suggest that FE65 facilitates the accumulation of mutant Htt in cells by preventing its degradation via the UPS, and thereby enhances the toxicity of mutant Htt.     

Inhibition of calpain cleavage of huntingtin reduces toxicity: accumulation of calpain/caspase fragments in the nucleus

Huntington's disease (HD) is a neurodegenerative disorder caused by a polyglutamine (polyQ) tract expansion near the N terminus of huntingtin (Htt). Proteolytic processing of mutant Htt and abnormal calcium signaling may play a critical role in disease progression and pathogenesis. Recent work indicates that calpains may participate in the increased and/or altered patterns of Htt proteolysis leading to the selective toxicity observed in HD striatum. Here, we identify two calpain cleavage sites in Htt and show that mutation of these sites renders the polyQ expanded Htt less susceptible to proteolysis and aggregation, resulting in decreased toxicity in an in vitro cell culture model. In addition, we found that calpain- and caspase-derived Htt fragments preferentially accumulate in the nucleus without the requirement of further cleavage into smaller fragments. Calpain family members, calpain-1, -5, -7, and -10, have increased levels or are activated in HD tissue culture and transgenic mouse models, suggesting they may play a key role in Htt proteolysis and disease pathology. Interestingly, calpain-1, -5, -7, and -10 localize to the cytoplasm and the nucleus, whereas the activated forms of calpain-7 and -10 are found only in the nucleus. These results support the role of calpain-derived Htt fragmentation in HD and suggest that aberrant activation of calpains may play a role in HD pathogenesis.     

Huntingtin's neuroprotective activity occurs via inhibition of procaspase-9 processing

Huntington's Disease is an inherited neurodegenerative disease that affects the medium spiny neurons in the striatum. The disease is caused by the expansion of a polyglutamine sequence in the N terminus of Huntingtin (Htt), a widely expressed protein. Recently, we have found that Htt is an antiapoptotic protein in striatal cells and acts by preventing caspase-3 activity. Here we report that Htt overexpression in other CNS-derived cells can protect them from more than 20 days exposure to fatal stimuli. In particular, we found that cytochrome c continues to be released from mitochondria into the cytosol of cells that overexpress normal Htt. However, procaspase-9 is not processed, indicating that wild-type Htt (wtHtt) acts downstream of cytochrome c release. These data show that Htt inhibits neuronal cell death by interfering with the activity of the apoptosome complex.     

Inhibition of metabotropic glutamate receptor signaling by the huntingtin-binding protein optineurin

Huntington disease is caused by a polyglutamine expansion in the huntingtin protein (Htt) and is associated with excitotoxic death of striatal neurons. Group I metabotropic glutamate receptors (mGluRs) that are coupled to inositol 1,4,5-triphosphate formation and the release of intracellular Ca(2+) stores play an important role in regulating neuronal function. We show here that mGluRs interact with the Htt-binding protein optineurin that is also linked to normal pressure open angled glaucoma and, when expressed in HEK 293 cells, optineurin functions to antagonize agonist-stimulated mGluR1a signaling. We find that Htt is co-precipitated with mGluR1a and that mutant Htt functions to facilitate optineurin-mediated attenuation of mGluR1a signaling. In striatal cell lines derived from Htt(Q111/Q111) mutant knock-in mice mGluR5-stimulated inositol phosphate formation is also severely impaired when compared with striatal cells derived from Htt(Q7/Q7) knock-in mice. In addition, we show that a missense single nucleotide polymorphism optineurin H486R variant previously identified to be associated with glaucoma is selectively impaired in mutant Htt binding. Although optineurin H486R retains the capacity to bind to mGluR1a, optineurin H486R-dependent attenuation of mGluR1a signaling is not enhanced by the expression of mutant Htt. Because G protein-coupled receptor kinase 2 (GRK2) protein expression is relatively low in striatal tissue, we propose that optineurin may substitute for GRK2 in the striatum to mediate mGluR desensitization. Taken together, these studies identify a novel mechanism for mGluR desensitization and an additional biochemical link between altered glutamate receptor signaling and Huntington disease.     

ROCK-phosphorylated vimentin modifies mutant huntingtin aggregation via sequestration of IRBIT

ABSTRACT: BACKGROUND: Huntington's Disease (HD) is a fatal hereditary neurodegenerative disease caused by the accumulation of mutant huntingtin protein (Htt) containing an expanded polyglutamine (polyQ) tract. Activation of the channel responsible for the inositol-induced Ca2+ release from ensoplasmic reticulum (ER), was found to contribute substantially to neurodegeneration in HD. Importantly, chemical and genetic inhibition of inositol 1,4,5-trisphosphate (IP3) receptor type 1 (IP3R1) has been shown to reduce mutant Htt aggregation. RESULTS: In this study, we propose a novel regulatory mechanism of IP3R1 activity by type III intermediate filament vimentin which sequesters the negative regulator of IP3R1, IRBIT, into perinuclear inclusions, and reduces its interaction with IP3R1 resulting in promotion of mutant Htt aggregation. Proteasome inhibitor MG132, which causes polyQ proteins accumulation and aggregation, enhanced the sequestration of IRBIT. Furthermore we found that IRBIT sequestration can be prevented by a rho kinase inhibitor, Y-27632. CONCLUSIONS: Our results suggest that vimentin represents a novel and additional target for the therapy of polyQ diseases.     

Dictyostelium huntingtin controls chemotaxis and cytokinesis through the regulation of myosin II phosphorylation

Abnormalities in the huntingtin protein (Htt) are associated with Huntington's disease. Despite its importance, the function of Htt is largely unknown. We show that Htt is required for normal chemotaxis and cytokinesis in Dictyostelium discoideum. Cells lacking Htt showed slower migration toward the chemoattractant cAMP and contained lower levels of cortical myosin II, which is likely due to defects in dephosphorylation of myosin II mediated by protein phosphatase 2A (PP2A). htt(-) cells also failed to maintain myosin II in the cortex of the cleavage furrow, generating unseparated daughter cells connected through a thin cytoplasmic bridge. Furthermore, similar to Dictyostelium htt(-) cells, siRNA-mediated knockdown of human HTT also decreased the PP2A activity in HeLa cells. Our data indicate that Htt regulates the phosphorylation status of myosin II during chemotaxis and cytokinesis through PP2A.     

Scalable Production in Human Cells and Biochemical Characterization of Full-Length Normal and Mutant Huntingtin

Huntingtin (Htt) is a 350 kD intracellular protein, ubiquitously expressed and mainly localized in the cytoplasm. Huntington’s disease (HD) is caused by a CAG triplet amplification in exon 1 of the corresponding gene resulting in a polyglutamine (polyQ) expansion at the N-terminus of Htt. Production of full-length Htt has been difficult in the past and so far a scalable system or process has not been established for recombinant production of Htt in human cells. The ability to produce Htt in milligram quantities would be a prerequisite for many biochemical and biophysical studies aiming in a better understanding of Htt function under physiological conditions and in case of mutation and disease. For scalable production of full-length normal (17Q) and mutant (46Q and 128Q) Htt we have established two different systems, the first based on doxycycline-inducible Htt expression in stable cell lines, the second on “gutless” adenovirus mediated gene transfer. Purified material has then been used for biochemical characterization of full-length Htt. Posttranslational modifications (PTMs) were determined and several new phosphorylation sites were identified. Nearly all PTMs in full-length Htt localized to areas outside of predicted alpha-solenoid protein regions. In all detected N-terminal peptides methionine as the first amino acid was missing and the second, alanine, was found to be acetylated. Differences in secondary structure between normal and mutant Htt, a helix-rich protein, were not observed in our study. Purified Htt tends to form dimers and higher order oligomers, thus resembling the situation observed with N-terminal fragments, although the mechanism of oligomer formation may be different.     

Huntingtin cleavage induced by thrombin in vitro

Huntingtin (Htt) mutation causes Huntington's disease.Sequence analysis of Htt revealed apossible thrombin cleavage site in the N-terminal region of Htt.In order to investigate if thrombin can eleaveHtt,we expressed the N-terminal fragment (1-969) of wild-type (wt) Htt (Htt 1-969) in MCF-7 cells andstudied its cleavage pattern by thrombin in vitro.An expression plasmid pcDNA3-Htt-18Q-969 was used totransfect MCF-cells and Htt 1-969 expression was confirmed with immunofluorescence.Cell lysates wereincubated with thrombin (1 U/ml, 10 U/ml,and 30 U/ml) for 1 h in the presence or absence of hirudin,athrombin inhibitor.Htt fragments were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and detected with anti-Htt antibodies. An Htt fragment with molecular mass of approximately80 kDa was produced after incubation with thrombin.The size of this Htt fragment was anticipated bymolecular mass generated from thrombin-mediated cleavage at the amino acid 183 in the Htt.Production ofan 80 kDa fragment was inhibited by hirudin. This study provides the first evidence that Htt is cleaved bythrombin in vitro at amino acid 183.If endogenous thrombin cleaves Htt in vivo,the physiological significanceof thrombin-mediated cleavage of Htt should be further investigated.     

Network organization of the huntingtin proteomic interactome in mammalian brain

We used affinity-purification mass spectrometry to identify 747 candidate proteins that are complexed with Huntingtin (Htt) in distinct brain regions and ages in Huntington's disease (HD) and wild-type mouse brains. To gain a systems-level view of the Htt interactome, we applied Weighted Correlation Network Analysis to the entire proteomic data set to unveil a verifiable rank of Htt-correlated proteins and a network of Htt-interacting protein modules, with each module highlighting distinct aspects of Htt biology. Importantly, the Htt-containing module is highly enriched with proteins involved in 14-3-3 signaling, microtubule-based transport, and proteostasis. Top-ranked proteins in this module were validated as Htt interactors and genetic modifiers in an HD Drosophila model. Our study provides a compendium of spatiotemporal Htt-interacting proteins in the mammalian brain and presents an approach for analyzing proteomic interactome data sets to build in vivo protein networks in complex tissues, such as the brain.     

HAP1 can sequester a subset of TBP in cytoplasmic inclusions via specific interaction with the conserved TBP<Subscript>CORE</Subscript>

Background  

Huntington's disease, spinal and bulbar muscular atrophy, and spinocerebellar ataxia 17 (SCA17) are caused by expansions in the polyglutamine (polyQ) repeats in Huntingtin protein (Htt), androgen receptor protein (AR), and TATA-binding protein (TBP), respectively. Htt-associated protein 1 (HAP1), a component of neuronal cytoplasmic stigmoid bodies (STBs), can sequester polyQ-expanded Htt and AR in STBs, thereby antagonizing formation of the nuclear aggregates associated with apoptotic neuron loss and disease progression.     

Huntington's disease: roles of huntingtin-interacting protein 1 (HIP-1) and its molecular partner HIPPI in the regulation of apoptosis and transcription

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-interacting 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.     

Polyglutamine expansion in huntingtin increases its insertion into lipid bilayers

An expanded polyglutamine (Q) tract (>37Q) in huntingtin (htt) causes Huntington disease. Htt associates with membranes and polyglutamine expansion in htt may alter membrane function in Huntington disease through a mechanism that is not known. Here we used differential scanning calorimetry to examine the effects of polyQ expansion in htt on its insertion into lipid bilayers. We prepared synthetic lipid vesicles composed of phosphatidylcholine and phosphatidylethanolamine and tested interactions of htt amino acids 1-89 with 20Q, 32Q or 53Q with the vesicles. GST-htt1-89 with 53Q inserted into synthetic lipid vesicles significantly more than GST-htt1-89 with 20Q or 32Q. We speculate that by inserting more into cell membranes, mutant huntingtin could increase disorder within the lipid bilayer and thereby disturb cellular membrane function.