Human Molecular Genetics, 2002, Vol. 11, No. 26 3299-3308
© 2002 Oxford University Press
Systematic mutagenesis of the functional domains of AIRE reveals their role in intracellular targeting
1Department of Human Genetics, UCLA School of Medicine, Gonda Center, University of California Los Angeles, Los Angeles, California, USA, 2Department of Human Molecular Genetics, National Public Health Institute, Helsinki, Finland and 3Department of Medical Genetics, University of Helsinki, Finland
Received August 11, 2002; Accepted October 10, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mutations in the human autoimmune regulator (AIRE ) gene cause a multi-systemic autoimmune syndrome that is known as autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy (APECED). To date more than 39 different disease mutations have been identified. They span the entire region of the AIRE gene that encodes a polypeptide with multiple functional domains: an N-terminal homogeneously staining region (HSR), a bipartied nuclear localization signal (NLS), a SAND domain, two PHD fingers and four nuclear receptor targeting motifs. The APECED mutations include insertions, deletions, substitutions and introduction of premature termination codons, while most mutations disrupt one of the functional domains. We have constructed a series of deletion mutants systematically removing one or more functional domain(s) and investigated the stability and sub-cellular compartmentalization of the corresponding polypeptides. Here we show that the first 188 amino acids, containing the HSR domain and the NLS proved necessary for both cytoplasmic filament formation and nuclear targeting. Deletion of the SAND domain and even point mutations in the SAND domain, resulted in the aggregation of the polypeptides in the cytoplasm and interfered with the proper nuclear targeting. The PHD fingers seemed to be necessary for the formation of characteristic dot-like complexes in the nucleus, but their deletion did not interfere with nuclear entry.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Autoimmune regulator (AIRE) protein is a putative transcription factor with a predicted molecular weight of 57.5 kDa. Disruption of this protein results in autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy (APECED) (1–4) with autoimmune-based symptoms of multiple organs (5–8). Over 39 APECED mutations have so far been identified, all resulting in the functional deficiency of AIRE (9). AIRE gene encodes a polypeptide chain of 545 amino acids and the corresponding mouse ortholog shares 71% amino acid homology with the human protein (10). The polypeptide consists of multiple functional domains: (a) N-terminal homogeneously staining region (HSR) that is similar to nuclear dot (ND) proteins of Sp100 families (11,12), and shown to be necessary for homodimerization (11,13); (b) a classical bi-partied nuclear localization signal (NLS) shown to be functional (14); (c) a SAND domain, thought to represent a DNA binding domain (15); (d) two plant homeodomains (PHD)-like zinc fingers separated by a proline-rich region (PRR) and four LXXLL nuclear receptor motifs (16,17).
Detailed characterization of the tissue and cell-specific expression of AIRE has been reported for both mouse and human (18–20). AIRE is especially prominent in the nucleus of thymic medullary epithelial and dendritic antigen presenting cells (18,19,21). In tissues, the AIRE expressing cells show nuclear staining (19), whereas when expressed in mammalian cell cultures AIRE localized in both the nucleus and cytoplasm. Evidently, the cytoplasmic filamentous-like AIRE protein co-localized with vimentin (21,22). We and others have shown that AIRE can activate transcription of a reporter gene when fused to a heterologous DNA binding domain (13,23), suggesting that AIRE participates in transcriptional regulation, at least in vitro.
APECED is characterized by a breakdown in self-tolerance to organ-specific antigens and AIRE gene product is thought to have a crucial role in thymic T-cell negative selection (20,24). Our recent studies showed that Aire knock-out mice develop circulating autoantibodies against several organs with multiple organ lymphocytic infiltration (25). No major defects were observed in the MHC class II recognition nor was any abnormality detected in the thymic development of T-cells in naive mice, but hyperproliferation of T-cell was evident in the draining lymph nodes of immunized Aire-/- mice. Furthermore, distinct differences were observed in peripheral, but not in the thymic T-cell receptor (TCR) repertoire. Taken together, these analyses of the Aire-deficient mice would imply that the loss of homeostasis in the immune system potentially reflects a breakdown in the peripheral rather than in the central tolerance. However, detailed characterization of the central tolerance in Aire-deficient mice has not been addressed yet.
Very little data exists for the cellular consequences of APECED mutations and of the role of functional domains in the sub-cellular targeting of AIRE. Here we have expressed a series of deletion mutants, systematically removing one or more of the domain(s), and monitored the stability and intracellular targeting of the corresponding AIRE polypeptides.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Deletion constructs
A total of 39 different APECED-causing mutations have been identified in the AIRE gene (9) and 30 of them affect one or more functional domains analysed here (9). To initially monitor the importance of individual domains for the expressed polypeptides, we designed 10 deletion mutant constructs of the AIRE gene into the mammalian expression vector containing a FLAG epitope tag (Fig. 1A). The constructs represent different combinations of SAND, HSR, PHD-1, PHD-2 and PRR domains, including a full-length wild-type construct free of the heterologous epitope tag. We also generated a construct with the deleted exon 7, which does not harbor any of the functional domains. However, this deletion contracts the interval between the SAND domain and the first PHD finger. From here on, we will refer to these constructs and the corresponding polypeptides as follows: FL, full-length wild-type; Ex7del, wild-type missing the exon seven; NSP, missing the proline-rich region, PHD-2 and the rest of the C-terminus; NS, retaining amino acids 1–280, containing only the HSR up to the end of the SAND domain; SP3, retaining the SAND, PHD-1, PRR, PHD-2 and the rest of the C-terminus; SP2, retaining the SAND, PHD-1 and the PRR; SP, retaining the SAND and PHD-1; S, SAND domain by itself; P3, retaining PHD-1, PRR and PHD-2; (-)SAND, with the removed SAND domain (Fig. 1A). All of the constructs were FLAG tagged N-terminally.
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To confirm the correct reading frame and relative stability of the translated polypeptides, we performed Western blot analysis of immunoprecipitated proteins (Fig. 2). All of the expressed AIRE polypeptides could be immunoprecipitated and displayed the expected molecular weights, suggesting a reasonable stability and sufficiently proper folding of these polypeptides.
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Half-life of mutant AIRE polypeptides
We performed pulse-chase analysis of the synthesized polypeptides by labeling the cells 48 h after transfection with [35S] methionine/cysteine and chased them for 12, 24, 36 and 48 h. The linear graph of signal intensity plotted against time estimated the half-life of the overexpressed wild-type AIRE to be about 20 h and the AIRE protein deleted for the exon seven (Ex7del) showed stability comparable to that seen in the wild-type (Table 1). The N-terminal FLAG tag had no significant disparity when compared with the non-tagged (nFL) wild-type construct (Table 1). Somewhat surprisingly, none of the overexpressed deletion mutants displayed drastic differences in their protein decay. All mutant AIRE polypeptides displayed half-lives that ranged between 14 and 20 h, suggesting that our deletion design did not render the polypeptides unstable in this overexpression system. The S construct (SAND domain alone) had the lowest stability followed by P3<SP<SP2<SP3<NSP<NS < (-)SAND, while the FL (full-length) exhibited the greatest stability.
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Interestingly, all of the deletion constructs that contained one or both PHD fingers, such as NSP, SP, SP2, SP3 and P3, produced two distinct polypeptides as judged by SDS–PAGE (Fig. 3). The reason for this feature is unclear, but the rate of decay for both bands was similar. Since the FLAG tag is at the N-terminus, the usage of an alternative methionine start site is unlikely, especially when the next methionine is 144 amino acids downstream of the initial start codon in the NSP construct. Differential glycosylation or proteolytic degradation is unlikely, because the similar pattern of double bands was also produced in in vitro transcription/translation assays, devoid of membranes and proteases (data not shown). Since the PHD domain binds to zinc, one likely explanation is that small proteins that contain PHD fingers may have different conformations based on their zinc-binding states, and therefore migrate in two size classes on SDS–PAGE.
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Targeting of the deletion mutants
To monitor the localization of the AIRE polypeptides synthesized from the deletion mutants, we transfected both COS1 and HeLa cells with each of the mutant constructs and performed confocal immunofluorescence microscopy. We first monitored the targeting of the full-length AIRE polypeptide tagged with the FLAG epitope (FL) and compared it to the non-tagged protein (nFL). Both the cytoplasmic (Fig. 4A and C) and nuclear (Fig. 4B and D) staining was comparable between the FLAG tagged and non-tagged polypeptide. The intracellular localization of the Ex7del polypeptide (Fig. 4E and F) was also comparable to that of the wild-type AIRE (Fig. 4A–D).
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Previous data suggest that the HSR domain would be necessary for filamentous cytoplasmic staining (21). In agreement with this finding, all of the polypeptides missing the HSR domain (S, SP, SP2 and P3) revealed diffuse staining patterns both in the nucleus and in the cytoplasm and no cytoplasmic filaments were observed in our experimental system (Fig. 5B; S, SP and SP2 looked similar to P3). A similar diffused pattern was evident for the SP3 polypeptide, which lacked both the HSR and the NLS signal, but this protein was exclusively targeted to the cytoplasm (Fig. 5A). Both, the NS and NSP proteins, lacking the carboxydomains, but containing the minimum of 298 amino acids from the amino-terminal, including the HSR and SAND domains, showed filamentous staining comparable to the wild-type AIRE in the cytoplasm (Fig. 4G and I). These polypeptides contained the NLS, were targeted to the nucleus, but failed to form nuclear speckles characteristic of the wild-type AIRE (Fig. 4H and J).
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Based on structural similarities, the SAND domain has been implicated in mediation of DNA binding. The polypeptides missing the SAND domain aggregated into multiple large dots that were about 10–20 times larger than the average AIRE nuclear dots (Fig. 5C). These aggregates were not localized in the nucleus but in the cytoplasm tucked next to the nuclear membrane. Only a few diffuse and unorganized nuclear dots were observed in the cells transfected with NS construct (Fig. 4H) missing both PHD domains. The polypeptides with SAND and the first PHD domain displayed a greater number of nuclear dots (Fig. 4J), but again disorganization of dots was evident when compared with the wild-type AIRE (Fig. 4B and 4D).
We generated more subtle point mutations in the SAND domain to further elucidate its role in intracellular targeting. Based on the sequence homology between the AIRE SAND domain and SAND domain in two other proteins, Sp100b and NUDR, we mutagenized five conserved amino acids (K221A, K222A, K222E, E237A, K253A, K253E and R257A) (Fig. 1B). These charged residues are positioned on the surface of the molecule, potentially interacting with yet unknown ligands. Again, we confirmed the stability of the synthesized polypeptides of the SAND mutants with Western blot analysis (Fig. 2) and the pulse-chase experiments displayed similar half-lives for all of these mutants, which did not deviate from that of the wild-type AIRE (Table 1).
Three SAND mutants K221A, K222A and K222E showed cytoplasmic filamentous staining comparable to the wild-type. However, like the polypeptides synthesized from SAND deletion constructs, these polypeptides revealed perinuclear aggregates instead of nuclear dots (Fig. 6B, D and F). Similar results were observed for R257A mutant (Fig. 6H). Also the K253E polypeptide, carrying negatively charged glutamic acid instead of positively charged lysine, revealed abnormal localization (Fig. 6G), whereas the sub-cellular localization of the K253A mutant revealed close resemblance to the wild-type (comparing Fig. 6E with 6A). R237A mutant did not display a significant change in localization pattern when compared with the wild-type (Fig. 6C). These results suggest that K221, K222, R257 and K253 residues in the SAND domain are crucial for correct nuclear compartmentalization. However, we also recognize that these mutations in the AIRE polypeptide may have caused a conformational change that led to protein–protein aggregation.
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The polypeptides lacking the PHD domains (NS and NSP) were targeted to the nucleus. However, they failed to form nuclear dots. Cells transfected with NS construct displayed diffused nuclear localization and the NSP construct missing the second PHD displayed unorganized nuclear dot formation (Fig. 4H and J). Nuclear dots were disorganized or missing for all of the constructs lacking the amino terminal HSR domain. These findings imply that the proper nuclear targeting and organization of the AIRE polypeptide is dependent both on HSR and NLS domains. Taken together, all of the AIRE deletion polypeptides that contained the HSR domain formed cytoplasmic filaments and, conversely, those polypeptides missing the HSR failed to form cytoplasmic filaments (Table 1). Furthermore, PHD domains seemed to be necessary for nuclear dot formations and the SAND domain was necessary, but not sufficient for correct nuclear compartmentalization.
Iranian Jewish mutation (Y85C)
A founder APECED mutation in the Iranian Jewish population is the Y85C, which changes a tyrosine to a cysteine in the HSR domain. Since the removal of the HSR region resulted in the loss of cytoplasmic filament structure, we wanted to investigate if this mutation alone would have a similar effect. The majority of the cells transfected with the Y85C mutant showed a diffused staining pattern in the nucleus (Fig. 5D, Table 1), which is consistent with the data obtained from the HSR deletion mutants and in accordance with the previous data (5). Although the western blot analysis revealed the expression of the correct size polypeptide, the Y85C mutant systematically displayed a lower level of expression than the rest of the mutants (Fig. 2). The pulse-chase experiments using the Y85C polypeptide displayed a significantly shorter half-life (1.5 h versus 14–20 h) than any other mutant polypeptides (Fig. 3), suggesting that this polypeptide is subjected to a more rapid decay.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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To reveal the physiological functions of AIRE, it is paramount to understand the determinants of the sub-cellular targeting as well as the functions of the different domains of AIRE. In this report we describe an in vitro expression analysis of a series of deletion mutants of the AIRE gene, designed to remove one or more of its functional domain(s). Our aim was to address the role of each functional domain with respect to correct sub-cellular targeting of AIRE protein.
To date, 10 point mutations in APECED patients have been identified that each disrupt the HSR domain. Among these, three deletion/insertion mutations result in a premature termination of the AIRE polypeptide. HSR region has been demonstrated to mediate homodimerization both in vitro (13) and in vivo (26). Recently it was shown that deletion mutants lacking the HSR or HSR and SAND domains failed to form filamentous structures in the cytoplasm (14).
In our experimental system, all of the AIRE constructs that lacked the first 188 amino acids (deleting the HSR domain and the NLS signal) showed similar intracellular distribution (Table 1); these polypeptides localize diffusely throughout the cytoplasm and the nucleus. In addition, they fail to form organized cytoplasmic filaments and nuclear dots. Since all of the mutant constructs missing the HSR domain failed to form nuclear dots (Results section), we postulate that the correct nuclear compartmentalization of the AIRE polypeptide is dependent on homo-oligomerization.
A previous report demonstrated that the N-terminal AIRE deletion constructs lacking the HSR and the NLS (aa 175–545 and aa 292–545) were transported into the nucleus and the authors concluded that AIRE has a second functional nuclear localization signal hidden within the carboxyterminus, either between the PHD fingers (proline rich region) or distal to the second PHD finger (14). The same report also showed that AIRE NLS–green fluorescent protein fusion construct was transported into the nucleus in similar transient transfection assays. To address the existence of other potential nuclear localization signals at the C-terminus, we deleted both the HSR and the NLS domains and also systematically removed C-terminal domains one at a time. Somewhat contradictory to the earlier results, we found that the S, SP, SP2 and P3, which either lacked the carboxyterminal end, the PRR or both, was still localized to the nucleus. In addition, our SP3 construct containing the entire C-terminal was not localized to the nucleus. Since we have used a similar in vitro expression system, we conclude that the existence of a second NLS at the C-terminal end of AIRE polypeptide is unlikely. Nuclear entry of these smaller polypeptides may be explained by passive diffusion through the nuclear pore complexes (27). Our findings imply that deletion of the HSR domain will both disrupt the cytoplasmic filament formation and also affect the nuclear dot formation.
Recent data has shown that recombinant AIRE can bind to DNA (26). Although it remains to be shown which, if any, AIRE domain mediates DNA binding, similar SAND domains in other proteins have affinity for DNA (28,29). Recently, a three-dimensional structure of the SAND domain in the Sp100b protein was determined using heteronuclear multidimensional NMR spectroscopy (29). In addition, the SAND domain in nuclear DEAF-1-related protein was also shown to mediate DNA binding (29). Consequentially, two point mutations in the SAND domain result in APECED; both mutations represent an early truncation of the AIRE polypeptide, one occurring in the middle of the SAND domain (R230X) and the other at the end (R257X). The latter mutation is the Finnish major mutation and closely resembles the NS construct, lacking the two PHD and the PRR domain. Our SAND deletion mutant formed perinuclear aggregates in 100% of the transfected cells. Although the half-life of synthesized polypeptides was comparable to that of the wild-type AIRE, nevertheless, it was mistargeted. To specify the amino acids in SAND that are essential for nuclear compartmentalization, we produced seven single amino acid substitution changes in the SAND domain. Six mutations in charged surface residues resulted in the mistargeting of the polypeptide. In all cases, the nuclear dot structure was abrogated and the synthesized polypeptides localized adjacent to the nuclear membrane, suggesting that the AIRE SAND domain does play a key role in nuclear compartmentalization. So far no AIRE domain has been assigned to DNA binding, although the recombinant AIRE protein has been shown to bind DNA (26). Since the SAND domain of other proteins mediates DNA binding, our data would imply that nuclear dot structures represent AIRE–DNA complexes mediated by amino acids on the surface of the SAND domain. However, just like the SAND deletion construct [(-)SAND], point mutations in the SAND domain could cause the misfolding of the AIRE polypeptide and/or promote protein aggregation. Since AIRE is overexpressed in in vitro expression systems, it is possible that AIRE dots serve as deposit sites for excess protein regulating the availability of soluble AIRE as was suggested for ND10/nuclear bodies of Sp100 protein (30,31).
We have earlier studied the naturally occurring mutants of AIRE that disrupt the PHD domains and shown that these mutants fail to form nuclear dots (23). These mutations also abolish the activation of reporter genes when fused to heterologous binding domain (13,23). A previous report also demonstrated that the polypeptide lacking both PHD domains failed to form nuclear dots; however, cytoplasmic filament structures remained unaffected (22). In total, three APECED point mutations affect the PHD domains and several premature termination mutations eliminate these domains. In our experimental system, the PHD domains seem to be necessary for nuclear organization, but not in the actual nuclear targeting of the AIRE polypeptide. The three-dimensional NMR structure of the Williams-Beuren Syndrome Transcription Factor (WSTF) PHD finger would indicate that this structural motif supports two zinc atoms and the most intriguing aspect of it is its native structure that is globular, which does not resemble a typical zinc finger (32). The authors concluded that the PHD domains are unlikely to participate in the DNA binding, but probably play a role in protein–protein interaction. The decreased transactivation potential of APECED patient mutations Cys311Tyr and C1313del (23), disrupting the first and deleting the second PHD finger, respectively, and Cys437Pro that disrupt the second PHD (13), suggests that the PHD fingers are needed for transactivation. Perhaps, the AIRE PHD fingers are involved in recruitment of transcription repressors or co-activators and not directly involved in DNA binding. This argument would be supported by the interaction found between AIRE and the common co-activator creb binding protein (CBP) (13). However, it remains to be shown whether the PHD fingers interact with CBP.
Based on our findings we were able to pair up the mutants that share similar intracellular targeting properties. For example, NS and NSP polypeptides, missing either the carboxyterminal PRR and both PHD fingers or just the PRR and the second PHD finger, form cytoplasmic filaments comparable to that of the wild-type protein (Table 1). However, the observed nuclear structures were either not formed (NS) or disorganized (NSP), indicating that, although the PHD fingers are not necessary for nuclear targeting, they are crucial for correct organization of AIRE protein in the nucleus. Subsequently, those constructs that excluded the HSR domain clustered together (S, SP, SP2, P3) and all of them showed diffuse nuclear staining, indicating that the PHD domains rely on HSR and the NLS domains for nuclear entry.
We have previously characterized the transactivation competence of the Iranian Jewish mutation (Y85C) using a reporter assay (23). To our surprise, at least in vitro, this mutant activated transcription comparable to that of the wild-type. Since the mutation lies within the HSR domain, we predicted that the sub-cellular localization would resemble the HSR deleted constructs. Here we show that the Y85C polypeptide gets targeted to the nucleus in the majority of the cells, but the localization is diffuse. Interestingly, the stability of the Y85C polypeptide was significantly reduced with an estimated half-life of one-thirteenth compared with the wild-type. Since AIRE is expressed in antigen presenting cells (APCs) in low levels, perhaps the long half-life of AIRE compensates for its low abundance, especially in APCs that do not divide much in a naive state. It is also important to note that the Y85C mutation results in a milder clinical phenotype than the Finnish mutation. For example, Iranian Jewish patients do not develop chronic cases of candidiasis. Perhaps the Y85C mutation does not completely render the AIRE protein dysfunctional, but rather the short intracellular half-life of the protein results in APECED.
In conclusion, the experimental data obtained from in vitro expression of a series of deletion mutants of AIRE suggest that the HSR domain is necessary both for cytoplasmic filament staining and, together with NLS, for the entry of the AIRE protein into the nucleus. The SAND domain most likely participates in orchestrating the nuclear organization and its absence results in mislocalization of the AIRE polypeptide. Although the PHD fingers do not participate in intracellular targeting, their function is most likely specific in the nucleus since their deletion results in an unorganized arrangement of the AIRE polypeptide and the lack of nuclear foci. This type of information of the function of different domains of the polypeptide is important for the detailed understanding of the molecular pathogenesis of APECED.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Expression constructs
Human AIRE cDNA was previously isolated from a human adult thymus cDNA library (3). This cDNA was cloned into EcoRI sites of the mammalian expression vector pCMV5 (from Dr Jouni Vesa). For immunofluorescence labeling and western blotting of full-length protein (1–545), NS (1–298), NSP (1–345), P3 (286–545), S (175–298), SP (175–345), SP2 (175–433), SP3 (175–545), AIRE cDNA was amplified by PCR with specific primers and cloned into N-terminal FLAG tag (Tag2a; Stratagene, San Diego, CA, USA) mammalian expression vectors via EcoRI cloning site. Ex7del and (-)SAND were generated using the ExSite PCR-based Site-Directed Mutagenesis Kit (Stratagene, San Diego, CA, USA) and single amino acid substitutions (Y85C, K221A/K222A, K222E, E237A, K253A, K253E and R257A) were generated using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, San Diego, CA, USA) according to the manufacturer's protocol. All constructs were sequence verified.
Immunoprecipitation
All AIRE constructs along with vector-only control were transfected into COS1 cells using the Lipofectamine kit (Invitrogen, Carlsbad, CA, USA). Forty-eight hours after transfection cells were washed twice in cold PBS and lysed in RIPA buffer containing 1x complete protease inhibitors (Roche Molecular Biochemicals, Indianapolis, IN, USA) and allowed to incubate at 4°C for 20 min. Cells were scraped off and the entire lysate was spun down at 16000 g for 10 min at 4°C and the pellet was discarded. To each lysate, 1 µg of monoclonal anti-FLAG antibody (M2; Sigma, St Louis, MO, USA) was added followed by 30 µl of 50% slurry of protein A/G agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Lysate slurry was incubated at 4°C overnight while shaking. Lysate was centrifuged at 8000 rpm for 1 min and the supernatant was discarded. Pellets were resuspended in 25 µl of 2x Lamliae loading buffer and heat denatured at 95°C for 5 min. Samples were run on a 12% SDS–PAGE and blotted onto Protran Pure Nitocellulose membrane (Schleicher & Schuell, Dassel, Germany) using the Semi-Dry Transfer Cell (Bio-Rad) at 15 V for 30 min. Membranes were blocked for 30 min in PBST and 5% low-fat milk and primary monoclonal anti-FLAG antibody was added to the membrane for 1 h. Membranes were washed three times in PBST 5 min each and anti-mouse conjugated to alkaline phosphatase (AP; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as a secondary antibody for 1 h. Membranes were washed three times in PBS 5 min each and twice in AP buffer (100 mM Tris–HCl pH 9.4, 100 mM NaCl, 5 mM MgCl2) 5 min each. AP substrate was added to the membranes and the reaction was stopped with double-distilled water.
Pulse-chase
COS-1 cells were plated on six-well cell culture plates at 3x105 cells per well and incubated at 37°C overnight in MEM media supplemented with 10% fetal calf serum, L-glutamine and penicillin/streptomycin. Individual wells were washed twice with 1x PBS and transfected with each of the AIRE constructs using the Lipofectamine kit (Invitrogen, Carlsbad, CA, USA). Forty-eight hours after transfection, asynchronized cells were starved for 1 h with media lacking methionine and cysteine. To the cells were added [35S] labeled methionine and cysteine (Amersham Pharmacia Biotech, Piscataway, NJ, USA) for 1 h at 37°C. At each time point, cells were washed twice in cold PBS and lysed in RIPA buffer containing 1x complete protease inhibitors (Roche Molecular Biochemicals, Indianapolis, IN, USA) and incubated at 4°C for 20 min. Cells were scraped off and the entire lysate was spun down at 13 000 rpm for 10 min at 4°C and the pellet was discarded. To each lysate, 1 µg of monoclonal anti-FLAG antibody (M2) was added followed by 30 µl of 50% slurry of protein A/G agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Lysate slurry was incubated at 4°C overnight while shaking. Lysate was centrifuged at 6000 g for 1 min and the supernatant was discarded. Pellets were resuspended in 25 µl of 2x Lamliae loading buffer and heat denatured at 95°C for 5 min. The entire material from each sample was loaded on a 12% SDS–PAGE. Gels were run to completion then fixed in 30% ethanol/10% acetic acid and dried. Gels were exposed against a film and later were subjected to radiography. The films were scanned using a densitometer and analysed using NIH image program.
Immunofluorescence microscopy
Round cover slips were placed in six-well cell culture plates and coated with poly D-lysine (0.5 mg/ml) for 1 h. Cells were plated at 3x105 cells per well and incubated at 37°C for 24 h. All AIRE constructs along with vector-only control were transfected into COS1 cells using the Lipofectamine kit (Invitrogen, Carlsbad, CA, USA). Forty-eight hours after transfection cells were washed twice in PBS and fixed in 4% paraformaldehyde/PBS for 30 min. Then cells were washed twice in PBS and blocked solution (0.5% BSA, 0.2% saponin in PBS) for 30 min. Primary antibody M2 (1 : 100) was added to each appropriate coverslip and incubated for 1 h at room temperature. Next, cells were washed three times with blocking buffer and secondary antibody, anti-mouse conjugated to FITC (1 : 200; Sigma, St Louis, MO, USA) was added to each coverslip and incubated at room temperature for 1 h. Cells were washed three times in PBS and cover slips were mounted onto glass slides ready for Leica-inverted confocal microscopy. Sections of all samples were taken at 0.5 µm intervals.
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ACKNOWLEDGEMENTS |
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We would like to thank Dr Jouni Vesa for his expert advice and thorough critique of this manuscript. We would also like to thank the MacDonald Family Foundation for their generous support for this project.
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FOOTNOTES |
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* To whom correspondence should be addressed at: Department of Medical Genetics, University of Helsinki and Department of Molecular Medicine, NPHI, Biomediceem, Haartmoninkatu 8 00290 Helsinki, Finland. Tel: 358 947448393; Fax: 358 947448480; Email: leena.peltonen{at}ktl.gi
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REFERENCES |
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