Hinderin, a five-domains protein including coiled-coil motifs that binds to SMC3
© Patel and Ghiselli; licensee BioMed Central Ltd. 2005
Received: 11 August 2004
Accepted: 18 January 2005
Published: 18 January 2005
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© Patel and Ghiselli; licensee BioMed Central Ltd. 2005
Received: 11 August 2004
Accepted: 18 January 2005
Published: 18 January 2005
The structural maintenance of chromosome proteins SMC1 and SMC3 play an important role in the maintenance of chromosomal integrity by preventing the premature separation of the sister chromatids at the onset of anaphase. The two proteins are constitutive components of the multimeric complex cohesin and form dimers by interacting at their central globular regions.
In order to identify proteins that by binding to SMC3 may interfere with the protein dimerization process, a human cDNA library was screened by the yeast two-hybrid system by using the hinge region of SMC3 as bait. This has lead to the identification of Hinderin, a novel five domains protein including two coiled-coil motifs and sharing a strikingly structural similarity to the SMC family of proteins. Hinderin is ubiquitously expressed in human tissues. Orthologue forms of the protein are present in other vertebrates but not in lower organisms. A mapping of the interaction sites revealed that the N- and C-terminal globular domains mediate the binding of Hinderin to SMC3. Hinderin/SMC3 complexes could be recovered by immunoprecipitation from cell lysates using an anti-SMC3 antibody, thus demonstrating that the two proteins interact in vivo. On the contrary, Hinderin did not interact with SMC1. In vivo the rate of SMC1/SMC3 interaction was decreased by the ectopic expression of Hinderin.
Hinderin is a novel binding partner of SMC3. Based on its ability to modulate SMC1/SMC3 interaction we postulate that Hinderin affects the availability of SMC3 to engage in the formation of multimeric protein complexes.
The structural maintenance of chromosome (SMC) proteins are involved in several aspects of chromosomal dynamic, in DNA recombination and in DNA repairs [1–3]. Two SMC proteins named SMC1 and SMC3 bind to and prevent the premature separation of sister chromatids at the end of mitosis [4, 5]. SMC1 and SMC3 directly interact through their central globular binding domains by forming an heterodimer [6, 7]. The protein complex encircles the sister chromatids and is stabilized though the interaction with two other cohesin proteins known as Scc1 and Scc3 in s. cereviasie [7, 8]. At anaphase, the ring-shaped complex is broken down when separase, a cysteine protease, cleaves Scc1, thus freeing the sister chromatids to move in opposite directions [6, 9]. Somatic cells with deranged separase activity or lacking Scc1 develop aneuploidy at increased rate. This suggests that the cohesin complex plays a major role in the maintenance of chromosomal stability [10–13]. The mechanism regulating the interaction between SMC1 and SMC3 is still poorly understood. It has however been established that a single point mutation of the central globular domain (known as hinge) of either one of these proteins strongly affects the dimerization rate and prevents the attachment of the cohesin complex to chromatid DNA . Conceivably, proteins that interact with the hinge domain of SMC1 or SMC3 can act as modulator of the cohesin complex formation and may thus affect chromosomal stability. In this paper we report the identification of a new SMC3-interacting protein that specifically binds to SMC3's central globular domain. The sequence of the identified gene product matched that of a previously discovered hypothetical new protein with no known function. We have named the protein Hinderin. The gene is expressed in all the human tissues analyzed thus far. Orthologue forms of this protein are expressed in vertebrates but not in lower organisms. Hinderin is a five-domain proteins and its structure resembles that of SMC proteins with N- and C-terminal globular domains that are joined by a coiled coil region interrupted at the center by a third globular domain. However, unlike the canonical SMC proteins, Hinderin does not harbor ABC-like ATPase sequences. We have found that the protein interacts with the hinge region of SMC3 but not with SMC1. Hinderin acts as a binding competitor of SMC1 and, as such, qualifies as a putative modulator of the SMC3 function.
The region of SMC3 encompassing the protein hinge domain (Gln465 to Gln807) was used as bait in a yeast two-hybrid system to identify interacting proteins expressed by a human fetal brain Matchmaker two-hybrid cDNA library (Clontech). About 3 × 106 library clones were screened. Forty blue colonies reaching 2 mm in size after one week were collected and 21 of the isolated plasmids with inserts greater than 500 bp were sequenced. Three of the sequences matched the same region of the published cDNA Genbank clones AB037749 (coding for the hypothetical protein KIAA1328) and AL832625 (corresponding to the hypothetical protein DKFZp451C1618). The inserts of ~2 kb included part of the gene 3'-UTR. However the 5'-end of the coding region was not present in the retrieved clones. The issue was complicated by the fact that the sequences of AB037749 and AL832625 diverged at their 5'-end. 5'-RACE was thus employed to identify the transcriptional start site of the interacting gene by using mRNA derived from fetal kidney 293, hepatoma HepG2, and cervical HeLa human cells. All the cloned sequence coincided with that of the AL832625 clone and matched in full the putative coding sequence obtained by automated computational analysis of the human genome (Genbank XM029429). The conceptually translated sequence coded for a protein of 578 amino acids.
In summary, we have identified a novel interacting partner of SMC3. The protein, named Hinderin, specifically interacts with the hinge domain of SMC3. The protein is ubiquitously expressed in human tissues. We speculate that when in a certain context, SMC3 association with Hinderin becomes favored compared to the association to SMC1, the availability of SMC3 to engage in the cohesin complex formation is reduced. The binding of SMC3 to proteins affecting its association to functional partners represents a new modality of regulation of SMC3 activity.
In order to identify proteins encoded by the human fetal brain cDNA library and interacting with the hinge domain of SMC3, a bait plasmid was generated by subcloning the SMC3-465/807 polypeptide coding region into the yeast two-hybrid system bait vector pGBKT7 (Clontech) (see fig. 2 for a diagram of the constructs used and their designation). The insert was generated by PCR using Pfu DNA polymerase and primers terminated with restriction sites that allowed the directional cloning of the products into the accepting vector (see Additional file 1 for a listing of the primers used in this study). Mouse full-lenght SMC3 cDNA was used as template. To identify the gene encoded by the interacting plasmid, the prey insert was sequenced by priming at the Gal4AD site (5'-AATACCACTACAATGGA-3'). The sequences were BLASTed against the nr and human dEST databases. DNA restriction digestions provided information on the size of the clones retrieved.
The total RNA was isolated using TRI-reagent from 293, HepG2 and HeLa cells. The 5' RACE assay was performed using a RLM-RACE Ambion kit. The generated cDNA was used as template for nested PCR. The inner PCR product was cloned into pCRII-Topo vector (Invitrogen) and sequenced using a T7 primer. The sequence matched that of the DKFZp451C1618 clone (AL832625) and extended 5' to the published sequence of KIAA1328 (AB037749). Based on this information, the complete Hinderin coding region was generated by RT-PCR utilizing mRNA extracted from 293 cells, and the product cloned in frame with the tag sequence in pcDNA3.1/V5-His TOPO (Invitrogen). When transfected into 293 cells, the expressed product detected with an anti-V5 monoclonal antibody had size 69 KDa, as expected for a protein encoded by the Hinderin-V5 fusion gene (fig. 3A).
In order to map the SMC3 interacting site(s) a series of truncated constructs were produced in pGBKT7 vector. The inserts required for the SMC3-1/186, SMC3-976/1217, SMC3-552/807 and SMC3-711/807 constructs were produced by PCR. SMC3-465/550 and SMC3-465/716 were generated by introducing stop codons in the sequence of SMC3-465/807 using a QuikChange XL kit (Stratagene). The mutated duplex oligonucleotides used had the forward sequence: 5'-CTTTCTATACTTGTGT AAGTCACTGCTGGTAAC-3' and 5'-GACCAGTTGATGAACTAAATGCAGATAGAG-3', respectively. SMC3-465/643 and SMC3-643/807 were obtained by digesting SMC3-465/807 at the Pst I or alternatively the Nde I restriction sites present in the vector multiple cloning site and at the Sma I site of the insert. The resulting linear constructs were blunt-ended and religated. pGADT7-SMC3-465/807 was generated by retriving the insert from the bait vector. SMC1-485/670 encoding the entire SMC1 hinge region, was generated by RT-PCR from 293 cells mRNA and cloned in pGADT7. Hinderin deletion constructs H-64/360 and H-360/578 were generated by restriction digestion of the pACT2-Hinderin-47/578 clone identified in the yeast two-hybrid system screening with Nco I/Bgl II and Bgl II/Xho I respectively, followed by religation of the blunt-ended vector. Hinderin in bait pGBKT7 vector was generated by retrieving the H-47/578 insert from the prey clone. H-177/360 and H-1/85 inserts were obtained respectively by PCR and by digestion of the Hinderin pcDNA3.1 expression vector with BamH I/EcoR I. Both were subsequently subcloned in pGADT7. To assess the strenght of interaction between different SMC3 and Hinderin domains, three colonies were randomly selected and grown overnight in 5 ml of selection media. After cell lysis by freeze thawing in 300 ml of 100 mM Na2HPO4 pH 7.0, 10 mM KCl, 1 mM MgSO4, buffer, β-galactosidase activity was assessed using ortho-nitrophenyl-β-D-galactopyranoside as substrate (1 mM) in the presence of 50 mM β-mercaptoethanol. After 2 h incubation at 37°C the color intensity was read at 420 nm.
Total mRNA was extracted with TRI-reagent from subconfluent HeLa and HCT116 cells and separated on 1% agarose. After transfection to a nitrocellulose filter, the Hinderin transcript was identified by hybridization to a 335 bp 32P-labeled cDNA probe annealing to the 5'-end region of the gene. In order to examine the expression of Hinderin in different human tissues, semiquantitative RT-PCR was performed by using 0.5 μg of Marathon-ready first strand cDNA (Clontech) from 16 tissues. The PCR reaction was monitored at 20 and 30 cycles and the product analyzed on 2% agarose. In order to normalize for possible differences in mRNA content, the expression of the housekeeping gene G3PDH was analyzed in each sample.
The human polypeptide sequence was analyzed with the COILS program to predict globular and coiled-coil domains. The scanning window was set at 21. The homology with other known protein family was examined by querying the NCBI Conserved Domain protein database. We used PSORT to scan for nuclear and other localization signal consensus sequences. To identify orthologue forms of Hinderin in other species, the human protein sequence was BLASTed against the translated EST database of m. fascicularis, mouse, rat, cow, sheep, dog, zebrafish, c. elegans, drosophila, and s. cereviasie. When a homologous sequence had been identified in lower organisms, we ran the COILS program to assess whether it displayed the same secondary structure as that of the matching human sequence.
293 cells were grown at ~80% confluence in 35 mm cm plates and transfected with 1 μg of Hinderin-V5 expression vector. After 48 h of incubation, the cells were washed in ice-cold phosphate-buffered saline and lysed in 1.2 ml of 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P40, 0.5% Na-deoxycholate buffer containing 100 mM NaF, 2 mM Na3VO4, and a cocktail of protease inhibitors. The cell lysate was centrifuged at 12,000 g and the recovered supernatant preabsorbed on protein G-agarose. Aliquots (500 μl) of the cell lysate were then incubated overnight at 4°C with either 25 μg of goat anti-human SMC3 antibody or goat anti-human SMC1 antibody (Santa Cruz Biotech) or alternatively with 10 μg mouse anti-V5 antibody (Invitrogen). The immunocomplexes were captured on Protein G-agarose by incubating 1 h at 4°C. After washing in immunoprecipitation buffer containing 300 mM NaCl, the protein immunocomplex was analyzed by SDS-PAGE and the proteins transferred to nitrocellulose membranes by electroblotting. After saturation in 4% dry milk/0.1% Tween 20 in PBS, the filter was incubated 1 h at RT with primary antibody. The anti-SMC1 and anti-SMC3 immunoprecipitate filter blots were incubated with anti-V5 monoclonal antibody (200 ng/ml) whereas the V5 immunoblots were incubated with either anti SMC1 (100 ng/ml) or anti-SMC3 (100 ng/ml) antibodies. After incubation with species-specific anti IgG HRP-conjugated secondary antibody (1:10,000), the immunocomplexes were visualized by enhanced chemiluminescence reaction (ECL).
Inserts corresponding to the SMC3-474/702 and SMC1-474/663 hinge domains were generated by PCR and ligated in the pBIND or the pACT vectors (Promega) respectively through ligation at the BamH I and Sal I sites. The resulting bait and prey DNA constructs (0.25 μg/ml each) together with the Hinderin-V5 expression vector (either 0.3 or 1 μg/ml or alternatively 1 μg/ml of empty pcDNA3.1 vector) and the reporter plasmid pG5/luc encoding the firefly luciferase (0.1 μg/ml), were co-transfected into 293 cells using Lipofectamine. Control experiments were conducted using pACT-Id and pBIND-MyoD vectors (Promega) encoding respectively GAL4:Id and VP16:MyoD fusion proteins known to interact in vivo . After 48 h incubation, cells were lysed and the expressed luciferase activity quantitated using a dual luciferase reporter assay kit (Promega) and a Lumat LB 9501 luminometer. Firefly luciferase values were corrected for the transfection efficiency using the values of the Renilla luciferase activity encoded by the pBIND vector under the control of a strong constitutive promoter. In order to assess the effect of Hinderin on the bait and prey expression, total RNA was extracted from a group of transfected cells and the specifc transcript levels quantitated by RT-PCR using primers annealing to the ends of the fusion protein coding sequence.
This work was supported by the NIH grants CA82290 to GG.
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