Interaction of Human HSP22 (HSPB8) with Other Small Heat Shock Proteins
2004; Elsevier BV; Volume: 279; Issue: 4 Linguagem: Inglês
10.1074/jbc.m311324200
ISSN1083-351X
AutoresXiankui Sun, Jean‐Marc Fontaine, Joshua S. Rest, Eric A. Shelden, Michael J. Welsh, Rainer Benndorf,
Tópico(s)Cardiovascular Effects of Exercise
ResumoMammalian small heat shock proteins (sHSP) are abundant in muscles and are implicated in both muscle function and myopathies. Recently a new sHSP, HSP22 (HSPB8, H11), was identified in the human heart by its interaction with HSP27 (HSPB1). Using phylogenetic analysis we show that HSP22 is a true member of the sHSP superfamily. sHSPs interact with each other and form homo- and hetero-oligomeric complexes. The function of these complexes is poorly understood. Using gel filtration HPLC, the yeast two-hybrid method, immunoprecipitation, cross-linking, and fluorescence resonance energy transfer microscopy, we report that (i) HSP22 forms high molecular mass complexes in the heart, (ii) HSP22 interacts with itself, cvHSP (HSPB7), MKBP (HSPB2) and HSP27, and (iii) HSP22 has two binding domains (N- and C-terminal) that are specific for different binding partners. HSP22 homo-dimers are formed through N-N and N-C interactions, and HSP22-cvHSP hetero-dimers through C-C interaction. HSP22-MKBP and HSP22-HSP27 hetero-dimers involve the N and C termini of HSP22 and HSP27, respectively, but appear to require full-length protein as a binding partner. Mammalian small heat shock proteins (sHSP) are abundant in muscles and are implicated in both muscle function and myopathies. Recently a new sHSP, HSP22 (HSPB8, H11), was identified in the human heart by its interaction with HSP27 (HSPB1). Using phylogenetic analysis we show that HSP22 is a true member of the sHSP superfamily. sHSPs interact with each other and form homo- and hetero-oligomeric complexes. The function of these complexes is poorly understood. Using gel filtration HPLC, the yeast two-hybrid method, immunoprecipitation, cross-linking, and fluorescence resonance energy transfer microscopy, we report that (i) HSP22 forms high molecular mass complexes in the heart, (ii) HSP22 interacts with itself, cvHSP (HSPB7), MKBP (HSPB2) and HSP27, and (iii) HSP22 has two binding domains (N- and C-terminal) that are specific for different binding partners. HSP22 homo-dimers are formed through N-N and N-C interactions, and HSP22-cvHSP hetero-dimers through C-C interaction. HSP22-MKBP and HSP22-HSP27 hetero-dimers involve the N and C termini of HSP22 and HSP27, respectively, but appear to require full-length protein as a binding partner. In a previous publication we identified mammalian HSP22 (other names: HSPB8, H11, E2IG1), a protein similar to known small heat shock proteins (sHSP), 1The abbreviations used are: sHSP, small mammalian heat shock protein; HSP22 (HSPB8, H11, E2IG1), heat shock protein 22; HSP27 (HSPB1), heat shock protein 27; MKBP (HSPB2), myotonic dystrophy kinase-binding protein; CL, cross-linking; co-IP, co-immunoprecipitation; FRET, fluorescence resonance energy transfer; TH, two hybrid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PBS, phosphate-buffered saline; HPLC, high performance liquid chromatography. by a two-hybrid (TH) screen using HSP27 (HSPB1, HSP25), the classic heat-inducible sHSP, as "bait," and a heart cDNA library (1.Benndorf R. Sun X. Gilmont R.R. Biederman K.J. Molloy M.P. Goodmurphy C.W. Cheng H. Andrews P.C. Welsh M.J. J. Biol. Chem. 2001; 276: 26753-26761Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). While in this and other reports HSP22 was characterized as an sHSP (2.Charpentier A.H. Bednarek A.K. Daniel R.L. Hawkins K.A. Laflin K.J. Gaddis S. MacLeod M.C. Aldaz C.M. Cancer Res. 2000; 60: 5977-5983PubMed Google Scholar, 3.Kappé G. Verschuure P. Philipsen R.L. Staalduinen A.A. Van de Boogaart P. Boelens W.C. De Jong W.W. Biochim. Biophys. Acta. 2001; 1520: 1-6Crossref PubMed Scopus (107) Google Scholar), in some reports the newly described protein was classified as a protein kinase with similarity to the Herpes simplex protein kinase ICP10 (4.Smith C.C. Yu Y.X. Kulka M. Aurelian L. J. Biol. Chem. 2000; 275: 25690-25699Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 5.Aurelian L. Smith C.C. Winchurch R. Kulka M. Gyotoku T. Zaccaro L. Chrest F.J. Burnett J.W. J. Investig. Dermatol. 2001; 116: 286-295Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 6.Depre C. Hase M. Gaussin V. Zajac A. Wang L. Hittinger L. Ghaleh B. Yu X. Kudej R.K. Wagner T. Sadoshima J. Vatner S.F. Circ. Res. 2002; 91: 1007-1014Crossref PubMed Scopus (82) Google Scholar). HSP22 occurs preferentially in striated and smooth muscles, but also in brain (1.Benndorf R. Sun X. Gilmont R.R. Biederman K.J. Molloy M.P. Goodmurphy C.W. Cheng H. Andrews P.C. Welsh M.J. J. Biol. Chem. 2001; 276: 26753-26761Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), estrogen receptor-positive breast cancer cells (2.Charpentier A.H. Bednarek A.K. Daniel R.L. Hawkins K.A. Laflin K.J. Gaddis S. MacLeod M.C. Aldaz C.M. Cancer Res. 2000; 60: 5977-5983PubMed Google Scholar), melanoma cells (4.Smith C.C. Yu Y.X. Kulka M. Aurelian L. J. Biol. Chem. 2000; 275: 25690-25699Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), and keratinocytes (5.Aurelian L. Smith C.C. Winchurch R. Kulka M. Gyotoku T. Zaccaro L. Chrest F.J. Burnett J.W. J. Investig. Dermatol. 2001; 116: 286-295Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). In addition to HSP22, also abundant in muscles are other sHSPs including HSP27, myotonic dystrophy kinase-binding protein (MKBP, HSPB2), HSPB3, αB-crystallin (αB-Cry, HSPB5), HSP20 (HSPB6), and cvHSP (HSPB7) (7.Krief S. Faivre J.F. Robert P. Le Douarin B. Brument-Larignon N. Lefrere I. Bouzyk M.M. Anderson K.M. Greller L.D. Tobin F.L. Souchet M. Bril A. J. Biol. Chem. 1999; 274: 36592-36600Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 8.Sugiyama Y. Suzuki A. Kishikawa M. Akutsu R. Hirose T. Waye M.M. Tsui S.K.W. Yoshida S. Ohno S. J. Biol. Chem. 2000; 275: 1095-1104Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). It is now widely accepted that sHSPs play a major role in muscles, although their precise role is not understood. A point mutation in the αB-Cry gene causes desmin-related cardiomyopathy in humans (9.Vicart P. Caron A. Guicheney P. Li Z. Prevost M.C. Faure A. Chateau D. Chapon F. Tome F. Dupret J.M. Paulin D. Fardeau M. Nat. Genet. 1998; 20: 92-95Crossref PubMed Scopus (975) Google Scholar), and MKBP binds to and activates the myotonic dystrophy protein kinase, an enzyme that when absent results in myotonic dystrophy (10.Suzuki A. Sugiyama Y. Hayashi Y. Nyu-I N. Yoshida M. Nonaka I. Ishiura S. Arahata K. Ohno S. J. Cell Biol. 1998; 140: 1113-1124Crossref PubMed Scopus (127) Google Scholar). Overexpression of HSP22 in heart was recently shown to be associated with hypertrophy (6.Depre C. Hase M. Gaussin V. Zajac A. Wang L. Hittinger L. Ghaleh B. Yu X. Kudej R.K. Wagner T. Sadoshima J. Vatner S.F. Circ. Res. 2002; 91: 1007-1014Crossref PubMed Scopus (82) Google Scholar). Also, the specific location of sHSPs in the sarcomers (11.Lutsch G. Vetter R. Offhauss U. Wieske M. Gröne H.J. Klemenz R. Schimke I. Stahl J. Benndorf R. Circulation. 1997; 96: 3466-3476Crossref PubMed Scopus (124) Google Scholar) and the protection of myocytes by sHSPs from ischemic stress (12.Martin J.L. Mestril R. Hilal-Dandan R. Brunton L.L. Dillmann W.H. Circulation. 1997; 96: 4343-4348Crossref PubMed Scopus (323) Google Scholar) suggest an important role for these proteins in muscles. Phosphorylation of HSP27 has been implicated in the contraction (13.Yamboliev I.A. Hedges J.C. Mutnick J.L. Adam L.P. Gerthoffer W.T. Am. J. Physiol. Heart Circ. Physiol. 2000; 278: H1899-H1907Crossref PubMed Google Scholar), and phosphorylation of HSP20 in the relaxation (14.Beall A. Bagwell D. Woodrum D. Stoming T.A. Kato K. Suzuki A. Rasmussen H. Brophy C.M. J. Biol. Chem. 1999; 274: 11344-11351Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) of smooth muscle cells. sHSPs have been shown to form two types of hetero-oligomeric complexes in striated muscles: type I complex consisting of HSP27, αB-Cry, and HSP20, and type II complex consisting of MKBP and HSPB3 (8.Sugiyama Y. Suzuki A. Kishikawa M. Akutsu R. Hirose T. Waye M.M. Tsui S.K.W. Yoshida S. Ohno S. J. Biol. Chem. 2000; 275: 1095-1104Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). sHSP complexes are heterogeneous in size and composition. The molecular mass of cellular sHSP complexes varies over a wide range (∼50–1000 kDa) and changes under stress conditions leading usually to smaller complexes (15.Kato K. Hasegawa K. Goto S. Inaguma Y. J. Biol. Chem. 1994; 269: 11274-11278Abstract Full Text PDF PubMed Google Scholar). The crucial role of these complexes for cell survival under stress conditions has been demonstrated (16.Mehlen P. Kretz-Remy C. Briolay J. Fostan P. Mirault M.E. Arrigo A.P. Biochem. J. 1995; 1312: 367-375Crossref Scopus (80) Google Scholar). Based on these findings it is believed that the formation of homo- and hetero-oligomeric complexes of sHSPs is essential for their function. Most available data concerning formation and structure of sHSP complexes are based on studies of αA-crystallin, αB-Cry, and HSP27. These three sHSPs form dynamic homo- and hetero-oligomeric complexes which may have a micellar structure and rapidly exchange subunits (17.Augusteyn R.C. Ghiggino K.P. Putilina T. Biochim. Biophys. Acta. 1993; 1162: 61-71Crossref PubMed Scopus (27) Google Scholar, 18.Sun T.X. Liang J.J. J. Biol. Chem. 1998; 273: 286-290Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 19.Bova M.P. Mchaourab H.S. Han Y. Fung B.K. J. Biol. Chem. 2000; 275: 1035-1042Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). HSP27 dimerizes, and two such dimers form tetramers, which are in equilibrium with larger complexes (20.Ehrnsperger M. Lilie H. Gaestel M. Buchner J. J. Biol. Chem. 1999; 274: 14867-14874Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Human HSP27 is phosphorylated at three sites (Ser15, Ser78, Ser82) by the protein kinase MAP-KAPK-2 resulting in the disassembly of large oligomeric structures predominantly into tetramers (15.Kato K. Hasegawa K. Goto S. Inaguma Y. J. Biol. Chem. 1994; 269: 11274-11278Abstract Full Text PDF PubMed Google Scholar, 21.Rogalla T. Ehrnsperger M. Preville X. Kotlyarov A. Lutsch G. Ducasse C. Paul C. Wieske M. Arrigo A.P. Buchner J. Gaestel M. J. Biol. Chem. 1999; 274: 18947-18956Abstract Full Text Full Text PDF PubMed Scopus (625) Google Scholar). Interactions within HSP27 complexes involve at least two sites, one site within the C-terminal α-crystallin domain and the other site at the far N terminus (22.Lambert H. Charette S.J. Bernier A.F. Guimond A. Landry J. J. Biol. Chem. 1999; 274: 9378-9385Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). The site in the α-crystallin domain has been proposed to be involved in dimer formation (23.Liu C. Welsh M.J. Biochem. Biophys. Res. Commun. 1999; 255: 256-261Crossref PubMed Scopus (45) Google Scholar), and these dimers are thought to further multimerize into larger complexes using the N-terminal dimerization site. If HSP22 belongs to this group of proteins, we hypothesized that it interacts with itself and with other sHSPs which may result in the formation of homo- and hetero-oligomeric complexes, similarly to other sHSPs. In an initial effort to characterize HSP22, we have studied its phylogenetic relationship among Bilateria proteins and confirmed that it is a member of the superfamily of sHSPs. By gel filtration high performance liquid chromatography (HPLC) we show that HSP22 forms high molecular mass complexes in the heart, similarly to other sHSPs. By yeast TH experiments, co-immunoprecipitation (co-IP), cross-linking (CL), and fluorescence resonance energy transfer (FRET) microscopy we show that HSP22 interacts with itself and with the other tested sHSPs. We show also that HSP22 has two binding domains that are specific for individual sHSPs. Phylogeny of sHSPs—A profile hidden Markov model in HMMR 2.2 g was employed to characterize, search for and align diverse sHSP superfamily members. The non-redundant protein data base (GenBank™ CDS translations+PDB+SwissProt+PIR) was queried with sHSP-HMM for significant matches (proteins with bit scores of 4 or greater and expectation values of 0.05 or less were retained). The resulting sequences were aligned with the sHSP-HMM. To estimate phylogeny and to approximate the posterior probabilities of the tree, a Bayesian inference approach with Metropolis-coupled Markov chain Monte Carlo, or MC3, was used. More details are given elsewhere (24.Fontaine J.M. Rest J.S. Welsh M.J. Benndorf R. Cell Stress Chaperones. 2003; 8: 62-69Crossref PubMed Scopus (134) Google Scholar). Origin of sHSP cDNAs and Plasmid Constructs—All relevant data on origin of sHSP cDNAs, plasmid constructs, used cloning methods and PCR primers are given in Table I. The PCR cycle conditions for cvHSP cDNA amplification from a human heart cDNA library (Clontech) were 50 s 95 °C, 50 s 63 °C, and 60 s 72 °C. For all TH experiments, the vectors pAS2–1 (abbreviated: pAS) and pACT2 (abbreviated: pACT) (Clontech) were used. For eukaryotic expression of Myc- and FLAG-tagged sHSPs the vectors pcDNA3.1-myc (Invitrogen) and pFlag-CMV2 (Sigma) were used, and for eukaryotic expression of YFP- and CFP-sHSP fusion proteins the vectors pEYFP and pECFP (Clontech) were used. All plasmid constructs were verified by sequencing. sHSP cDNAs were separated into N- and C-terminal fragments in the central region or beginning of the α-crystallin domain as indicated in Fig. 1A.Table IDesignation of the constructs, origin of the sHSP sequences, and cloning methodsConstruct no.Construct designationSource of sHSP cDNA/method of cloningUsed restriction sitesPrimers1pAS-HSP22-Fsubcloning of pcDNA-HSP22-FaDescribed in 1.EcoRI, XhoI/SalIdUsed restriction sites in the PCR product and in the vector are compatible.—2pACT-HSP22-FPCR of pcDNA-HSP22-FaDescribed in 1.SfiI, ECL136/SmaIdUsed restriction sites in the PCR product and in the vector are compatible.1, 23pAS-HSP22-NPCR of pcDNA-HSP22-F/TopoTAEcoRI, EcoRI3, 44pAS-HSP22-CPCR of pcDNA-HSP22-F/TopoTAEcoRI, EcoRI5, 65pACT-HSP22-NPCR of pcDNA-HSP22-F/TopoTANcoI, XhoI7, 86pACT-HSP22-CPCR of pcDNA-HSP22-F/TopoTAEcoRI, EcoRI9, 107pcDNA-HSP22-F-mycPCR of pcDNA-HSP22-F/TopoTAKpnI, XbnI11, 128pFlag-CMV2-HSP22-FPCR of pcDNA-HSP22-F/TopoTAEcoRI, XbaI13, 149pEYFPN1-HSP22-FPCR of pcDNA-HSP22-F/TopoTAEcoRI, KpnI15, 1610pAS-wtHSP27-FPCR of pBS-wtHSP27-FbwtHSP27, 3DHSP27, and 3AHSP27 cDNAs (in pBluescript) were obtained from L. A. Weber (Reno, NV).BspHI/NcoI, XhoI/SalIdUsed restriction sites in the PCR product and in the vector are compatible.17, 1811pAS-3DHSP27-FPCR of pBS-3DHSP27-FbwtHSP27, 3DHSP27, and 3AHSP27 cDNAs (in pBluescript) were obtained from L. A. Weber (Reno, NV).BspHI/NcoI, XhoI/SalIdUsed restriction sites in the PCR product and in the vector are compatible.17, 1812pAS-wtHSP27-NPCR of pcDNA-wtHSP27-F/TopoTAEcoRI, EcoRI19, 2013pAS-3DHSP27-NPCR of pcDNA-3DHSP27-F/TopoTAEcoRI, EcoRI19, 2014pAS-HSP27-CPCR of pcDNA-wtHSP27-F/TopoTAEcoRI, EcoRI21, 2215pACT-wtHSP27-FPCR of pcDNA-wtHSP27-F/TopoTANcoI, XmaI23, 2416pACT-3DHSP27-FPCR of pcDNA-3DHSP27-F/TopoTANcoI, XmaI23, 2417pECFPN1-wtHSP27-FPCR of pcDNA-wtHSP27-F/TopoTAEcoRI, BamHI25, 2618pECFPN1-3AHSP27-FPCR of pcDNA-3AHSP27-FbwtHSP27, 3DHSP27, and 3AHSP27 cDNAs (in pBluescript) were obtained from L. A. Weber (Reno, NV)./TopoTAEcoRI, BamHI25, 2619pECFPN1-3DHSP27-FPCR of pcDNA-3DHSP27-F/TopoTAEcoRI, BamHI25, 2620pAS-MKBP-FPCR of pGAD424-MKBPcMKBP (in pGAD424) and HSPB3 (in pGBT9) cDNAs were obtained from A. Suzuki (Yokohama, Japan)./TopoTAEcoRI, EcoRI27, 2821pAS-MKBP-NPCR of pAS-MKBP-F/TopoTAEcoRI, EcoRI27, 2922pAS-MKBP-CPCR of pAS-MKBP-F/TopoTAEcoRI, EcoRI30, 2823pcDNA-MKBP-F-mycPCR of pAS-MKBP-F/TopoTAKpnI, XbaI31, 3224pECFPN1-MKBP-FPCR of pcDNA-MKBP-F/TopoTAEcoRI, BamHI33, 3425pAS-cvHSP-FPCR of cDNA libraryNdeI, XmaI35, 3626pAS-cvHSP-NPCR of pAS-cvHSP-F/TopoTAEcoRI, EcoRI37, 3827pAS-cvHSP-CPCR of pAS-cvHSP-F/TopoTAEcoRI, EcoRI39, 4028pFlag-CMV2-cvHSP-FPCR of pAS-cvHSP-F/TopoTAKpnI, XbaI41, 4229pECFPC1-cvHSP-FPCR of pECFPC1-cvHSP-F/TopoTAEcoRI, BamHI43, 44a Described in 1.b wtHSP27, 3DHSP27, and 3AHSP27 cDNAs (in pBluescript) were obtained from L. A. Weber (Reno, NV).c MKBP (in pGAD424) and HSPB3 (in pGBT9) cDNAs were obtained from A. Suzuki (Yokohama, Japan).d Used restriction sites in the PCR product and in the vector are compatible. Open table in a new tab Two Hybrid Experiments—Small scale sequential transformation of the yeast strain Y190 was performed as described in the manufacturer's instructions (Clontech). Interactions between full-length (F), N-terminal (N), and C-terminal halves (C) of sHSPs were analyzed. Briefly, yeast was first transformed with the constructs 2, 5, or 6 and grown on -Leu medium, or with construct 1 and grown on -Trp medium. In the second step, the yeast was transformed with vectors as specified in the Figs., and plated on -Trp, -Leu, -His selective medium. Y190 has two reporter genes (his+, growth of colonies; gal+, blue colonies), and two proteins were considered as interacting partners only if both reporter genes were activated. In order to reduce false positive results, every used vector with an insert was tested with an empty partner vector. In some of these controls the his+ reporter gene was moderately activated resulting in between 10 and 100 colonies/10-cm Petri dish; however, the gal+ reporter gene was never activated (not shown). Cell Culture, Transfection, Co-immunoprecipitation, and Cross-linking—293T and COS-7 cells (ATCC) were maintained at 37 °C in a 5% CO2 humidified atmosphere in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. To prevent detachment of 293T cells, tissue culture plates were treated with poly-l-lysine before seeding the cells. For transient transfection FuGENE 6 (Roche Applied Science) was used according to the manufacturer's protocol. 48 h later, cells were harvested and processed for co-IP, CL, or FRET analysis. For co-IP, 2 μg of a rabbit anti-FLAG polyclonal antibody (Sigma) or mouse anti-Myc monoclonal antibody (Roche Applied Science) were bound to 50 μl of protein G-Sepharose (20% slurry, Sigma) by incubating for 2 h at 4 °C in 500 μl of buffer A (50 mm Tris-HCl, pH 8.0; 1% Triton X-100; 5 mm EDTA; 1 mm EGTA; 1×protease inhibitor mix, Roche Applied Science). Transfected and non-transfected COS-7 cells were rinsed with ice-cold PBS and lysed in buffer A on ice for 30 min. The lysate was centrifuged at 14,000 × g for 10 min at 4 °C, and the supernatant was then precleared by incubation with protein G-Sepharose for 2 h at 4 °C before it was incubated with the antibody-coated beads overnight at 4 °C on a rotating platform. Then the beads were collected by brief centrifugation and washed three times with buffer A. Bound immunocomplexes were released from the beads by a 3-min boiling in 100 μl of buffer B (62.5 mm Tris-HCl, pH 6.8; 2% SDS; 10% glycerol; 200 mm dithiothreitol; 0.01% bromphenol blue; 1×protease inhibitor mix). Thereafter, samples were analyzed using SDS-PAGE followed by Western blotting. Negative controls were run with rabbit and mouse preimmune sera instead of anti-FLAG or anti-Myc antibodies (not shown). For CL, transfected 293T cells grown in 6-well plates were washed with cold PBS. Glutaraldehyde (Sigma) was diluted with PBS (0.0001, 0.0002, 0.0005, 0.001, 0.002% final concentrations) immediately before use, and 1 ml of these solutions was added per well. Cells were incubated for 1 h at room temperature, and then cells were cooled on ice and washed with ice-cold PBS. Cells were lysed with 1 ml of buffer B. The lysates were sonicated for 10 s in order to break down DNA, and then boiled for 5 min. These samples were then analyzed by SDS-PAGE and Western blotting. Size Exclusion HPLC—0.5 g of the left ventricle of a Rhesus monkey heart was homogenized in 2 ml ice-cold buffer C (50 mm Tris-HCl, pH 7.4; 150 mm NaCl; 5 mm KCl; 1 mm EDTA; 10% glycerol; 1% CHAPS; 1× protease inhibitor mix) using a PowerGen 125 homogenizer. The homogenate was centrifuged at 14,000 × g for 20 min at 4 °C, and 100 μl of the supernatant was used for HPLC chromatography. Before loading the sample, the column (Protein PAK 300 sw, 0.75 × 30 cm, Waters) was equilibrated with buffer D (50 mm Tris-HCl, pH 7.4; 150 mm NaCl; 5 mm KCl; 1 mm EDTA), and separation of the proteins was at a flow rate of 1 ml/min. Fractions were collected as indicated in Fig. 2. Proteins in all fractions were precipitated overnight by 2 vol of ethanol/1% β-mercaptoethanol at –20 °C, and the precipitates were collected by centrifugation. The air-dried pellets were dissolved in 50 μl of buffer B, briefly sonicated and boiled for 5 min. These samples were then analyzed for the presence of HSP22, HSP27, and MKBP by SDS-PAGE and Western blotting using specific antibodies. The column resolved proteins with molecular masses ranging from 670 kDa and was calibrated using the molecular mass markers Dextran blue (>1000 kDa), thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B12 (1.35 kDa) (Fig. 2). Electrophoretic Methods and Western Blotting—SDS-PAGE and transfer of the separated proteins onto polyvinylidene difluoride membranes (Western blotting) were performed as described (1.Benndorf R. Sun X. Gilmont R.R. Biederman K.J. Molloy M.P. Goodmurphy C.W. Cheng H. Andrews P.C. Welsh M.J. J. Biol. Chem. 2001; 276: 26753-26761Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). For Western blotting, rabbit anti-FLAG antibody (1:5,000) (Sigma), mouse anti-Myc antibody (1:5000) (Roche Applied Science), and sheep anti-HSP22 (1.Benndorf R. Sun X. Gilmont R.R. Biederman K.J. Molloy M.P. Goodmurphy C.W. Cheng H. Andrews P.C. Welsh M.J. J. Biol. Chem. 2001; 276: 26753-26761Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) were used as primary antibodies. As secondary antibodies, goat anti-rabbit IgG (Fc fragment-specific), goat anti-mouse IgG (Fc fragment-specific) and rabbit anti-sheep IgG antibodies conjugated to horseradish peroxidase (Jackson) were used at a dilution of 1:10,000. The membranes were processed for detection using the ECL plus reagent (Amersham Biosciences). Fluorescence Resonance Energy Transfer Measurements—COS-7 cells grown on glass coverslips were transfected with various YFP-sHSP and CFP-sHSP constructs as listed in Table I coding for the corresponding fusion proteins. Two days later, cells were fixed with 4% formaldehyde in PBS, pH 7.4, for 30 min at room temperature. The cells were then washed with PBS, and the coverslips were mounted on slides with Prolong antifade mounting medium (Molecular Probes) and used for microscopy. For FRET microscopy, an Axiovert 135TV microscope equipped with a ×40 FLUAR 1.3NA oil immersion objective lens and a CFP/YFP filter set was used (Zeiss). Images were recorded at room temperature using a cooled integrated CCD camera DAGE RT 3000 (DAGE-MTI Inc.). NIH Image software was used to acquire 8-bit gray scale images from a Scion LG3 video capture board and to quantify the image pixel intensities within user selected regions of each cell. The same region was selected for analysis in each of the images obtained from an individual cell. Individual images were exported in TIFF format and image montages created using Adobe Photoshop. No post-processing of images was conducted after acquisition. FRET was measured by determining the CFP emission before and after an 8-min photobleaching step of YFP as described (25.Siegel R.M. Ka-Ming Chan F. Zacharias D.A. Swofford R. Holmes K.L. Tsien R.Y. Lenardo M.J. Science's Stke. 2000; 38: 1-6Google Scholar). The increase or dequenching of CFP emission is a direct measure of the FRET due to interaction. Interaction as measured by FRET is expressed as FRET Factor (FF) in Equation 1. FF=Ic-post-BpostIc-ante-BanteCF-1-1(Eq. 1) The symbols are defined as: Ic-post, Ic-ante, intensity of the CFP-signals in the region of interest after and before photobleaching, respectively; Bpost, Bante, background signals, outside of cell areas, after and before photobleaching, respectively; CF, correction factor (the ratio of the intensities of the CFP signals after and before photobleaching of an image area with cells which were not photobleached). The CF is used to eliminate fluctuations of the CFP signal during the course of the experiment. Ideally, if two partners do not interact, the FF-value would be 0, and any value above 0 would indicate interaction. However, individually expressed CFP and YFP control proteins are known to interact to a certain extent (26.Zacharias D.A. Violin J.D. Newton A.C. Tsien R.Y. Science. 2002; 296: 913-916Crossref PubMed Scopus (1803) Google Scholar). The FF value of this negative control (FF–C) was determined to be 0.096 ± 0.023 under the applied experimental conditions. Every FF value measured for interacting CFP/YFP-sHSP fusion proteins must be significantly higher than this value. For positive control, a CFP-YFP fusion protein was expressed (27.Hoppe A. Christensen K. Swanson J.A. Biophys. J. 2002; 83: 3652-3664Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). The measured FF value of 0.384 ± 0.037 is significantly different from FF–C (p < 0.0001) and indicates the range in which FF values for interacting proteins can be expected. For both, the interacting pairs of sHSPs and the controls, the CFP signal intensities before and after photobleaching of 10 randomly selected cells were measured. This allowed determining the significance of the differences of the FF values to the FF–C by the Student's t test. Phylogenetic Relationship of HSP22 among the Bilateria sHSPs—In the light of the divergent roles proposed for HSP22 (see Introduction and "Discussion"), we sought to gain additional information by a phylogenetic analysis. HSP22, like the other human sHSPs, consists of the conserved α-crystallin domain, a less conserved N-terminal domain, a variable central region, and variable N- and C-terminal tails (Fig. 1A). Based on the multiple alignment of all 10 human sHSPs, a hidden Markov model (sHSP-HMM) was built that was used to search the entire non-redundant protein data base (24.Fontaine J.M. Rest J.S. Welsh M.J. Benndorf R. Cell Stress Chaperones. 2003; 8: 62-69Crossref PubMed Scopus (134) Google Scholar). This sHSP-HMM identified 167 Bilateria sHSP-like proteins, including all known mammalian sHSPs as proteins with significant sequence similarity. The sHSP-HMM was used to align these 167 proteins, and the alignment was used to build a Byesian inference phylogeny. An abbreviated version of this phylogeny illustrating the overall topology of Bilateria sHSPs while detailing the relationship of the HSP22 and HSP27 clades, is shown in Fig. 1B. The mammalian HSP22 form a monophyletic group which is supported by the posterior probability of 1.0. The HSP22 clade is sister to the clade of HSP25/27 of Euteleostomi including mammals, birds and fish. Thus, HSP22 is most closely related to HSP27 among all proteins contained in the databases, while protein kinases or proteins related to the Herpex simplex protein kinase ICP10 were not identified. Based on this analysis, HSP22 clearly is classified as a member of the superfamily of sHSPs. Therefore, we analyzed HSP22 for the property, which is characteristic for this superfamily: the ability to interact with itself and other sHSPs, which results in the formation of complexes. Size Distribution of HSP22 Complexes in Monkey Heart—In order to determine the possible involvement of HSP22 in sHSP complex formation in tissues, a protein extract of a piece of Rhesus monkey heart was analyzed by gel filtration HPLC followed by SDS-PAGE and Western blotting. The proteins were extracted under moderate stringency conditions in order to leave sHSP complexes intact. HSP22 partitioned into all fractions corresponding to molecular masses between 25 kDa and >670 kDa with most of the proteins being found in the high molecular mass fractions (Fig. 2). For comparison, the size distribution of two further sHSPs, HSP27 and MKBP, was also analyzed. HSP27 showed a molecular mass partition, which is very similar to that of HSP22 and also to previous analyses (28.Arrigo A.P. Suhan J.P. Welch W.J. Mol. Cell. Biol. 1988; 8: 5059-5071Crossref PubMed Scopus (302) Google Scholar), while MKBP partitioned into fractions with smaller molecular masses approximately between 25 and 200 kDa. These data suggest that (i) different sHSPs may have different size distributions, and (ii) HSP22 may interact with itself and/or other sHSPs, such as HSP27 and MKBP, which partition completely or partially into the same fractions. HSP22-HSP22 Interaction—Earlier biochemical data suggested that HSP22 forms homo-dimers (1.Benndorf R. Sun X. Gilmont R.R. Biederman K.J. Molloy M.P. Goodmurphy C.W. Cheng H. Andrews P.C. Welsh M.J. J. Biol. Chem. 2001; 276: 26753-26761Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Here we examined the HSP22-HSP22 interaction in greater detail. Yeast TH assays using the constructs 1 and 2 (Table I) were performed to determine the interaction of HSP22 with itself (Fig. 3A). Both reporter genes (his+, gal+) clearly indicated interaction of HSP22 with itself. This interaction was verified biochemically by co-IP using Myc- and FLAG-tagged HSP22 (constructs 7, 8) expressed in COS-7 cells (Fig. 3B). As expected, after transfection with HSP22-myc the anti-FLAG antibody did not precipitate HSP22-myc, while after transfection with Flag-HSP22 the anti-FLAG antibody did precipitate Flag-HSP22 (controls). After co-transfection with both HSP22-myc and Flag-HSP22, the anti-FLAG antibody pulled down both Flag-HSP22 a
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