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. 2002 Oct 14:3:15.
doi: 10.1186/1471-2202-3-15.

The predominantly HEAT-like motif structure of huntingtin and its association and coincident nuclear entry with dorsal, an NF-kB/Rel/dorsal family transcription factor

Hiroki Takano  1 , James F Gusella
Affiliations

The predominantly HEAT-like motif structure of huntingtin and its association and coincident nuclear entry with dorsal, an NF-kB/Rel/dorsal family transcription factor

Hiroki Takano et al. BMC Neurosci. .

Abstract

Background: Huntington's disease (HD) pathogenesis is due to an expanded polyglutamine tract in huntingtin, but the specificity of neuronal loss compared with other polyglutamine disorders also implies a role for the protein's unknown inherent function. Huntingtin is moderately conserved, with 10 HEAT repeats reported in its amino-terminal half. HD orthologues are evident in vertebrates and Drosophila, but not in Saccharomyces cerevisiae, Caenorhabditis elegans or Arabidopsis thaliana, a phylogenetic profile similar to the NF-kB/Rel/dorsal family transcription factors, suggesting a potential functional relationship.

Results: We initially tested the potential for a relationship between huntingtin and dorsal by overexpression experiments in Drosophila S2 cells. Drosophila huntingtin complexes via its carboxyl-terminal region with dorsal, and the two enter the nucleus concomitantly, partly in a lipopolysaccharide (LPS)- and Nup88-dependent manner. Similarly, in HeLa cell extracts, human huntingtin co-immunoprecipitates with NF-kB p50 but not with p105. By cross-species comparative analysis, we find that the carboxyl-terminal segment of huntingtin that mediates the association with dorsal possesses numerous HEAT-like sequences related to those in the amino-terminal segment. Thus, Drosophila and vertebrate huntingtins are composed predominantly of 28 to 36 degenerate HEAT-like repeats that span the entire protein.

Conclusion: Like other HEAT-repeat filled proteins, huntingtin is made up largely of degenerate HEAT-like sequences, suggesting that it may play a scaffolding role in the formation of particular protein-protein complexes. While many proteins have been implicated in complexes with the amino-terminal region of huntingtin, the NF-kB/Rel/dorsal family transcription factors merit further examination as direct or indirect interactors with huntingtin's carboxyl-terminal segment.

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Figures

Figure 1
Figure 1
Concomitant nuclear accumulation of the carboxyl-terminal fragment of Drosophila huntingtin and dorsal. a,b, When pPac-FLAG-Dorsal encoding FLAG-tagged dorsal was transfected into S2 cells, approximately half of the transfected cells had predominantly cytoplasmic distribution of FLAG-Dorsal (a) and the remaining half showed predominantly nuclear distribution (b). FLAG-Dorsal was detected by anti-FLAG M5 antibody and Alexa Fluor 488 goat anti-mouse IgG. DNA staining with TOTO-3 determined the nuclei. c, When pIZ-Dhd2194C-myc encoding the myc-tagged carboxyl-terminal fragment of Drosophila huntingtin, Dhd2194C-myc, was introduced, the product was predominantly distributed to the cytoplasm. As a control, nuclei were determined by co-transfection of pIZ-NLS-GFP-lacZ encoding a SV40 nuclear localization signal-green fluorescence protein-lacZ chimeric protein [49] detected by anti-lacZ antibody and Alexa Fluor 488 goat anti-mouse IgG. Dhd2194C-myc was detected by anti-myc antiserum A-14 and Alexa Fluor 568 goat anti-rabbit IgG. d, Co-expression of FLAG-Dorsal and Dhd2194C-myc causes prominent nuclear accumulation of both proteins. The same observations were made using pIZ-HA-Dorsal and pIZ-FLAG-Dorsal (data not shown).
Figure 2
Figure 2
LPS signal-dependent, concomitant nuclear accumulation of the prenylated carboxyl-terminal fragment of Drosophila huntingtin and dorsal. a, Dhd2194C-myc-CKML has a prenylation-mediated membrane-targeting motif, Cys-Lys-Met-Leu sequence, derived from Drosophila ras protein, at the end of Dhd2194C-myc. Co-transfection of pIZ-Dhd2194C-myc-CKML and pIZ-NLS-GFP-lacZ showed cytoplasmic distribution of Dhd2194C-myc-CKML. b, Co-expression of Dhd2194C-myc-CKML and FLAG-Dorsal causes dot-like staining without apparent nuclear accumulation of both proteins. c, LPS stimulation to the cells expressing Dhd2194C-myc-CKML and FLAG-Dorsal caused nuclear accumulation of both proteins. The same phenomena were observed using another prenylation motif construct, pIZ-Dhd2194C-myc-CAAC (data not shown).
Figure 3
Figure 3
Gal4 mediated-inhibition of concomitant nuclear translocation of the carboxyl-terminal fragment of Drosophila huntingtin and dorsal. a,b, Dhd2194C-myc and FLAG-Dorsal were expressed under the control of Gal4-UAS system [31] by co-transfection of pUAS-Dhd2194C-myc, pUAS-FLAG-Dorsal, and actin5C-Gal4. The concomitant nuclear accumulation of Dhd2194C-myc and FLAG-Dorsal was not observed. Although a fraction of FLAG-Dorsal was distributed to the nucleus (a), it was mostly distributed to the cytoplasm (b). Apparent nuclear accumulation was not observed. c, LPS stimulation caused concomitant nuclear accumulation of FLAG-Dorsal and Dhd2194C-myc even in the presence of Gal4. d, Co-immunoprecipitation experiments were performed using proteins extracted from S2 cells expressing FLAG-Dorsal (lane 1: input, and lane 3: immunoprecipitated products by anti-myc A14 antiserum), and those expressing FLAG-Dorsal and Dhd2194C-myc (lane 2: input, and lane 4: immunoprecipitated products by anti-myc A14 antiserum). The proteins were expressed by Gal4-UAS system. Detection was performed with anti-FLAG M2 antibody. FLAG-Dorsal were co-immunoprecipitated with anti-myc A14 antiserum in the presence of Dhd2194C-myc (lane 4), but not in the absence of Dhd2194C-myc (lane 3).
Figure 4
Figure 4
Antisera, raised against Drosophila huntingtin, detected increased signals in the nucleus of S2 cells with nuclear dorsal accumulation after LPS treatment. a,b, Western blot analysis was performed using transfected and non-transfected S2 cells. Full-length and amino-terminal fragment of Drosophila huntingtin were expressed by Gal4-UAS system [31] using pUAS-DhdcDNA (lanes 3 and 5), pUAS-Dhdminigene (lane 7), pUAS-N605 (lanes 1 and 6), and actin5C-Gal4. Antisera 1893 (a) and 1894 (b), raised against the amino-terminal region of Drosophila huntingtin, recognized the products of the transgenes (arrow: full-length huntingtin, and arrow head: N605), and also detected an approximately 400kDa native protein corresponding to over-expressed full-length Drosophila huntingtin in size which is thought to be native Drosophila huntingtin. c, Antiserum 1894 recognized mainly cytoplasmic immunoreactivity with minor nuclear signals. Transfected NLS-GFP-lacZ detected by anti-lacZ antibody and Alexa Fluor 488 goat anti-mouse IgG was used for a control nuclear maker. d, Antiserum1894 recognized apparent nuclear immunoreactivity, when FLAG-Dorsal was accumulated in the nucleus after LPS treatment. FLAG-Dorsal was detected by anti-FLAG M5 antibody and Alexa Fluor 488 goat anti-mouse IgG.
Figure 5
Figure 5
TNF-alpha-enhanced co-immunoprecipitation of NF-kB with anti-human huntingtin antibodies in HeLa cell extracts. a, Immunoprecipitated products with mouse monoclonal IgG1 anti-human huntingtin antibodies MAB2166 (lane 2) and MAB2168 (lane 4), and unrelated mouse monoclonal IgG1 antibodies anti-FLAG M5 (lane 3) and anti-myc 9E10 (lane 5) were probed by anti-NF-kB p50 antiserum, sc-114. Human NF-kB p50, but not p105, was co-immunoprecipitated with MAB2166 and MAB2168, but not with M5 or 9E10. Input protein lysate was in lane 1. b, Before lysis, HeLa cells were treated with TNF-alpha (lane 6). This increased the amount of the p50 co-immunoprecipitated with MAB2166 (lane 8). Control input and immunoprecipitation were in lanes 7 and 9, respectively. Detection by anti-NF-kB antiserum, ZK50, generated the same result (data not shown).
Figure 6
Figure 6
Drosophila and vertebrate huntingtins as HEAT proteins a, The consensus cDNA sequences of the Drosophila HD gene predicted a 3584 amino acid polypeptide with four regions of greatest sequence identity with human huntingtin (noted by horizontal lines, aa residues and %identity). The polyglutamine and polyproline stretches present in the amino terminal region of human huntingtin (noted as a red bar) are absent from Drosophila huntingtin. The positions of 28 HEAT-like sequences are shown as vertical lines in Drosophila, with the first and last numbered as D1 and D28, respectively. The positions of 36 HEAT-like sequences in human huntingtin are similarly designated, from H1 to H36, with corresponding locations in other vertebrate huntingtins shown below. These sequences are referred to here as HEAT-like, as they were not defined by the same homology considerations originally used to define HEAT repeats and do not always precisely match their reported start and end-points. Vertebrate huntingtin HEAT-like sequences were identified by iterative MAST searches of the nr protein database, beginning with a MEME motif of 38 amino acids trained with the 10 published human huntingtin HEAT repeats (corresponding with the regions of H3-5, H9-12, H18-19, and H21). All matches in vertebrate huntingtins with position p values < 10-4 were used to create 6 species-specific MEME huntingtin motifs, along with one combined cross-species MEME motif, each of which was used in the next round. Shuffling the sequences in the training sets or attempting an iterative search process with random segments of proteins not reported to contain HEAT motifs produced either no motif or no significant additional matches. MEME motifs were also created using 436 HEAT repeats from a wide variety of proteins [36,37] as well as from subsets of these representing importin (HEAT_IMB), adaptin (HEAT_ADB) and PP2A (HEAT_AAA) families. The vertebrate huntingtin HEAT-like regions (detected in one or more species) by these motifs were: HEAT: 2–6, 9–12, 16, 19, 30, 36; HEAT_IMB: 2–4, 6, 12, 17; HEAT_ADB: none, HEAT_AAA 2–5, 9, 10, 12, 16, 17, 28, 30, 34–36. Drosophila huntingtin HEAT-like sequences were identified by similar iterative searches, seeding the initial species-specific MEME motif with 4 Drosophila huntingtin segments (HEAT-like sequences 1, 10, 13, 19) that showed significant matches with the HEAT_IMB importin MEME motif. During the iterative searching, additional MEME motifs were also generated using the combination of Drosophila and fish HEAT-like sequences. Individual MEME motifs created from each group of 6 vertebrate HEAT-like sequences revealed a direct correspondence (noted in green) between vertebrate segments 2, 12, 16 and 35 and Drosophila segments 1, 13, 16 and 28, respectively. It is likely that Drosophila huntingtin contains additional undetected HEAT-like sequences, as our search process could not benefit from comparison with more closely related species, as was possible among the vertebrates. b, Consensus secondary structures for both human and Drosophila HEAT-like sequences (probability of helical structure, pH_sec, for amino acids 1–38) were predicted using PhD without alignment and revealed a pair of helical regions separated by a non-helical region.
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