Article |
Address correspondence to Felix Wieland, Biochemie Zentrum Heidelberg (BZH), Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany. Tel.: 49-6221-544150. Fax: 49-6221-544366. E-mail: felix.wieland{at}urz.uni-heidelberg.de
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: heat shock protein receptor; immune response; cross priming; co-chaperone; CD40; antigen-presenting cell
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Various cell surface proteins on immune cells are thought to play a role in the induction of cellular responses to heat shock proteins. One group of proteins, including the toll-like receptors 2 and 4 with their cofactor CD14 (Asea et al., 2002), and CD36, are reported to induce cytokine secretion in an Hsp70-dependent manner. So far, only one surface protein (CD91) has been implicated in eliciting a CD8+ T cell response upon administration of peptide bound to heat shock protein (Grp94; Binder et al., 2000), but an essential role of CD91 in this process has been questioned (Berwin et al., 2002). A recent report by Wang et al. (2001) described a direct interaction between bacterial Hsp70 (DnaK) with CD40. However, this interaction is mediated by the substrate-binding domain of Hsp70, making a role in uptake of antigenic peptides unlikely.
The list of heat shock proteins and molecular chaperones implicated as immune adjuvants includes Grp94, Hsp60, calreticulin, and mammalian cytosolic Hsp90 and Hsp70 (Srivastava et al., 1986; Udono and Srivastava, 1993; Basu and Srivastava, 1999; Kol et al., 2000), as well as DnaK (Wang et al., 2001). In general, these chaperones bind to peptide segments of nonnative polypeptides either during synthesis or in conditions of cellular stress, preventing protein aggregation and mediating proper folding (Hartl and Hayer-Hartl, 2002). The highly conserved members of the Hsp70 family are the best studied among this class of components (Bukau and Horwich, 1998). Hsp70 consists of an NH2-terminal nucleotide-binding (ATPase) domain of 44 kD and a COOH-terminal 25-kD domain that binds peptide or polypeptide substrate. In its ATP-bound state, Hsp70 binds and releases peptide rapidly, whereas after hydrolysis, in the ADP state, bound peptide is held tightly (Flynn et al., 1989). Hsp70 recognizes heptapeptide segments with a broad specificity but with a preference for hydrophobic residues, such as leucine or isoleucine (Flynn et al., 1989; Blond-Elguindi et al., 1993; Rudiger et al., 1997). Based on this broad range of peptides recognized, Hsp70 would be especially suited to serve as a carrier of antigenic peptides for cross priming. Hsp70peptide complexes may reach the extracellular space from necrotic cells or on viral cell lysis (Basu et al., 2000; Berwin and Nicchitta, 2001). Various cell lines have been investigated for their ability to bind heat shock proteins, including mostly professional APCs such as dendritic cells (Reed and Nicchitta, 2000), macrophages, and peripheral blood monocytes (Sondermann et al., 2000).
Here, we show that binding of Hsp70 to ANA-1 macrophages is markedly increased after stimulation with bacterial lipopolysaccharide (LPS). LPS treatment results in increased expression of CD40 (Tone et al., 2001), a member of the TNF receptor family with a crucial role in B cell function and autoimmunity (Bodmer et al., 2002). We find that human Hsp70 binds to the exoplasmic domain of CD40. Interestingly, this interaction is mediated by the NH2-terminal ATPase domain of human Hsp70 in its ADP-bound state. It is strongly enhanced by the presence of substrate peptide in the COOH-terminal domain of Hsp70 (C70) and is inhibited by Hip, a co-chaperone known to stabilize the Hsp70 ATPase domain in the ADP state. These surprising mechanistic features explain why the binding of human Hsp70 has remained undetected in a recent report describing the interaction of the COOH-terminal chaperone domain of bacterial Hsp70 (DnaK) with CD40 (Wang et al., 2001). We show further that binding of human Hsp70peptide complex to cells that express CD40 leads to peptide uptake and induction of signaling via p38. Thus, CD40 is an extracellular receptor for peptide-loaded human Hsp70 and mediates the internalization of Hsp70-bound peptides.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
In light of recent findings that the bacterial Hsp70 homologue DnaK associates with CD40 via the COOH-terminal peptidebinding domain (Wang et al., 2001), experiments were performed to directly determine the specificity of CD40 for the domains of human Hsp70. Recombinant, full-length Hsp70 was incubated in the presence of ADP and a fivefold molar excess of either recombinant Hsp70 NH2-terminal domain (residues 1381; Sondermann et al., 2001) or COOH-terminal domain (residues 382641; Scheufler et al., 2000), or of recombinant, full-length DnaK. The NH2-terminal domain of Hsp70 (N70) efficiently competed for the binding of full-length Hsp70 to CD40, as detected with an antibody against the NH2-terminal His6 tag on recombinant Hsp70 (Fig. 4 A). The ATPase domain itself bound to CD40 in the presence of ADP but not ATP (Fig. 4 C). In contrast, neither the COOH-terminal Hsp70 domain (C70) nor DnaK had a significant effect on this binding (Fig. 4 A). To analyze whether under these conditions DnaK binds directly to CD40, a pulldown experiment was performed with DnaK. As shown in Fig. 4 B, DnaK indeed binds to CD40, and this interaction is enhanced in the presence of ADP when compared with ATP. This establishes that Hsp70 and DnaK bind to different sites on CD40, and confirms that DnaK binds via its COOH-terminal domain as reported earlier (Wang et al., 2001). This binding strongly suggested that it is the substrate-binding site of DnaK that mediates the interaction with CD40. To address this possibility, a competition experiment was performed with an excess of peptide C. As shown in Fig. 4 B, a 10-fold molar excess of the peptide almost completely abolishes the binding of DnaK to CD40. Thus, it is the substrate binding of DnaK that interacts with CD40. In summary, Hsp70 interacts with CD40 dependent on ADP, and via its ATPase domain, as shown with the endogenous proteins in cell extracts and the recombinant proteins in vitro. In contrast, DnaK binds to CD40 via its substrate binding site proper.
|
The known cochaperone function of Hip in stabilizing Hsp70 in its substrate-bound ADP state (Höhfeld et al., 1995) raised the interesting possibility that the interaction between CD40 and Hsp70 may not only be ADP-regulated, but may also depend on Hsp70 substrate. Strikingly, binding of Hsp70-ADP to CD40 increased dramatically in the presence of peptide C (Fig. 5, A and B). The effect of peptide was saturable (ka 30 µM) in a range corresponding to the affinity of peptide C for Hsp70 (510 µM; Greene et al., 1995). These results demonstrate that it is the peptide- and ADP-bound state of Hsp70 that is recognized preferentially by CD40 and suggest that, similar to Hip, CD40 has a regulatory function in stabilizing the Hsp70substrate complex. This effect would ensure that CD40 binds Hsp70 predominantly when it is in complex with peptide.
|
Binding of Hsp70peptide complex to CD40 results in intracellular signaling and peptide uptake
Binding of CD40 ligand to CD40 induces signal transduction via phosphorylation of p38, a component of the signal cascade between activated CD40 and NFB, which eventually results in the release of TNF
and subsequent secretion of interferon-
(Pullen et al., 1999). Binding of the COOH-terminal domain of DnaK to CD40 was reported to have a similar effect (Wang et al., 2001). Therefore, we investigated whether binding of human Hsp70peptide complex to CD40 also stimulates this signaling pathway. These experiments were performed in HEK293T cells stably transfected with human CD40 cDNA. After incubation with either Hsp70peptide complex, recombinant Hsp70 domains, or DnaK, in the presence of ADP or the nonhydrolysable ATP analogue AMPPNP, cells were lysed and lysates were analyzed by immunoblotting with an antibody directed against active (i.e., phosphorylated) p38. Indeed, human Hsp70 and its ATPase domain caused an increase in phosphorylated p38 to an extent comparable to that observed with DnaK (Fig. 6 A, top panel). Although modest when compared with the signal induced by the same molar concentration of CD40 ligand, activation of p38 by Hsp70 was significant, and depended on the presence of ADP. As a control, HEK293T cells stably transfected with the same vector, but containing the cDNA for an unrelated membrane protein, murine cationic amino acid transporter (MCAT), did not show a detectable response to the various stimuli (Fig. 6 A, bottom panel). Thus, Hsp70peptide complex and the Hsp70 ATPase domain activate signaling via CD40, dependent on the presence of ADP and in a manner comparable to the effect of DnaK, although the latter binds to CD40 via its COOH-terminal domain (Wang et al., 2001).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Clearly, the most remarkable feature of Hsp70 binding to CD40 is the dramatic dependence of the interaction on substrate peptide bound to C70. Our results suggest that CD40 interacts exclusively with the ATPase domain of Hsp70, in the presence of ADP. Thus, the free COOH-terminal domain apparently masks the ATPase domain, either directly or by an allosteric conformational change, preventing recognition by CD40. This inhibitory effect is released when peptide is bound (Fig. 5, A and B). Conversely, it follows that CD40 stabilizes the ATPase domain in the ADP state that holds bound peptide stably. A similar effect in stabilizing Hsp70 in the ADP state has previously been described for the Hsp70 cochaperone Hip (Höhfeld et al., 1995). Indeed, binding of Hip to the ATPase domain of Hsp70 blocks the interaction of Hsp70 with CD40, suggesting that Hip and CD40 recognize overlapping regions on the ATPase domain.
The functional features of the CD40Hsp70 interaction may be adapted to a role in the uptake of Hsp70peptide complexes into APCs for cross priming. Fig. 7 describes a model for the binding of peptide antigen to Hsp70 in a tumor cell, followed by release after necrotic cell lysis and CD40-mediated uptake of the Hsp70peptide complex by an APC. Peptide binding to Hsp70 would be facilitated by the high intracellular concentration of ATP and the activity of the Hsp70 cochaperone Hsp40 in catalyzing peptide loading (Minami and Minami, 1999). During cell necrosis, the internal concentration of ATP relative to ADP drops markedly (Bradbury et al., 2000). A further dilution of ATP (and of Hsp70 cochaperones) would occur upon lysis and release of cytosol content into the extracellular medium. As a result, peptide-bound Hsp70 remains in its ADP state, the stability of which determines the half-life of the Hsp70peptide complex. Importantly, although peptide loading onto Hsp70 is possible in the absence of nucleotide with low efficiency (Minami et al., 1996), low nucleotide concentration would prohibit the reformation of an Hsp70ADPpeptide complex in the extracellular space. Thus, the strong preference of CD40 for Hsp70ADPpeptide ensures not only the binding of peptide-loaded Hsp70, but would also guarantee that intracellular peptide antigen is made available for cross priming. Thus, the uptake of circulating extracellular peptides, potentially triggering autoimmune reactions, would be avoided. Future work will be directed toward testing this model, with a focus on the fate of Hsp70-bound peptide after uptake into APCs, including possible peptide representation on the cell surface via MHC I.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
For Hsp70peptide complex formation, Hsp70 was incubated with a fivefold excess of peptide C, biotinylated peptide C, or peptide C-FITC for 30 min at 37°C in binding buffer consisting of the following (mM): 10 MOPS-KOH, pH 7.2, 150 KCl, 2 ADP, and 3 MgCl2. Excess of unbound peptide Cbiotin was removed by gel filtration on Sephadex G-50 (Amersham Biosciences). Quantification of Hsp70 complex formation by ELISA using a streptavidinperoxidase conjugate (Molecular Probes, Inc.) indicated yields of 2030%. All proteins were centrifuged at 100,000 g at 4°C for 1 h before the experiments in order to remove aggregates.
Cell culture and fluorescence microscopy
The murine macrophage cell line ANA-11 was provided by H. Wagner (Technische Universität München, Munich, Germany). ANA-1 cells were cultured in VLE-RPMI 1640 (Biochrom Ltd.), supplemented with 10% FCS, 2 mM L-glutamine, and antibiotics. For fluorescence microscopy, ANA-1 cells were either stimulated with LPS (20 µg/ml; Sigma-Aldrich), or kept in an LPS-free medium (mock treatment) for 7 h. After stimulation, cells were harvested and incubated with 100 nM biotinylated Hsp70, biotinylated GST, or Hsp70peptide Cbiotin complex for 30 min at 0°C in VLE-RPMI 1640. After incubation, cells were processed for staining with streptavidin-TRITC (Sigma-Aldrich), fixed with PFA, and analyzed by fluorescence microscopy (Axiovert 35; Carl Zeiss MicroImaging, Inc.) as described previously (Sondermann et al., 2000). Cos-7 cells were cultured in DME supplemented with 10% FCS, 2 mM L-glutamine, and antibiotics (Biochrom Ltd.), grown on coverslips in 24-well plates, and transiently transfected with human CD40 cDNA (inserted into the pIRES2-EGFP vector; Invitrogen) by the calcium phosphate method (Chen and Okayama, 1987). 24 h after transfection, cells were incubated with 250 nM biotinylated Hsp70, biotinylated GST, or Hsp70peptide Cbiotin complex for 30 min at 0°C in DME, fixed, stained with streptavidin-TRITC, and analyzed by fluorescence microscopy as described previously (Sondermann et al., 2000). HEK293T cells were grown on collagen-coated dishes or coverslips, and cultured in DME supplemented with 10% FCS, 2 mM L-glutamine, and antibiotics. HEK293T cells were stably transfected (Chen and Okayama, 1987) with CD40 cDNA cloned into pcDNA3.1/Zeo (Invitrogen), and selected with ZeocinTM (Invitrogen). Stably transfected cell lines were analyzed by immunoblotting for CD40 expression using an anti-CD40 antibody (CSA180; StressGen Biotechnologies). HEK293T-MCAT cells expressing the MCAT cloned into pcDNA3.1/Zeo vector were provided by W. Nickel (Biochemie Zentrum Heidelberg, Heidelberg, Germany). S-HeLa cells were cultured in -MEM supplemented with 8% FCS, L-glutamine, and antibiotics, and grown in spinner flasks.
For experiments to study uptake of the Hsp70peptide CFITC complex, HEK293T-CD40 and HEK293T-MCAT cells were seeded on collagen-coated coverslips 24 h before the experiment. For complex formation Hsp70, N70, or C70 were preincubated with a 10-fold molar excess of peptide C-FITC for 30 min at 37°C, as described above for the biotinylated proteins. HEK293T-CD40 and HEK293T-MCAT were incubated with 0.5 µM of Hsp70, N70, or C70, and preincubated with 2 mM ADP and 5 µM peptide C-FITC, or 5 µM peptide C-FITC alone for 30 min on ice. Thereafter, the cells were washed three times with medium, incubated for 15 min at 37°C, fixed, embedded in Fluoromount-G, and analyzed by confocal microscopy (LSM 510; Carl Zeiss MicroImaging, Inc.).
Analysis of CD40 expression
For immunoblot analysis, ANA-1 cells were stimulated with LPS or mock treated as described under Cell culture and fluorescence microscopy, harvested, lysed by repeated passage through a needle (0.40.8 mm; Braun) in the presence of protease inhibitors (complete, EDTA free; Roche), and fractionated by centrifugation at 100,000 g at 4°C for 1 h. The pellet was resuspended in 0.1% Triton X-100/PBS, and protein concentrations were determined in total lysate; supernatant and pellet fractions were determined by the Bradford assay (Bio-Rad Laboratories). Equal amounts of protein were subjected to SDS-PAGE, and were analyzed by immunoblotting with an anti-CD40 antibody (CSA180; StressGen Biotechnologies).
Binding assays with GST-CD40
For binding of endogenous Hsp70 and Hsc70 from HeLa cell lysate, HeLa cells were lysed as described above and centrifuged at 100,000 g at 4°C for 1 h. 20-µl bed volume of Glutathione Sepharose 4 Fast Flow (Amersham Biosciences) per sample was preincubated with 80 or 400 pmol of GST or GSTCD40 fusion protein in 50 µl binding buffer. Unbound protein was removed by washing three times with PBS. The beads were treated with 1% BSA-PBS to avoid nonspecific binding, and then incubated with 150 µl of HeLa cell lysate in the presence of 2 mM DTT for 20 min at 16°C. The immobilized proteins were washed three times with 1 ml PBS and eluted with 20 µl elution buffer (50 mM Tris-HCl, pH 8.0, 10 mM reduced glutathione) for 10 min at 37°C. 10 µl of the eluates was subjected to SDS-PAGE and analyzed by immunoblotting using an anti-Hsc/Hsp70 antibody and an anti-Hsp90 antibody (SPA822 and SPA835; StressGen Biotechnologies).
For binding assays with recombinant His6-tagged Hsp70 and its His6-tagged domains or DnaK, 2.8 µM of each protein was incubated with 2 mM ATP, 2 mM ADP, or 2 mM ADP and 28 µM peptide C in 50 µl binding buffer consisting of the following (mM): 10 MOPS-KOH, pH 7.2, 150 KCl, and 3 MgCl2 for 30 min at 37°C. In competition experiments, binding was probed with either a 10-fold excess of Hsp70, the two domains, DnaK, or with a fivefold excess of Hip or Bag-1 for 10 min at 0°C. To this end, Hsp70, N70, C70, and DnaK were preincubated with ADP, ATP, and peptide C as described under Plasmids and protein preparation. For comparison of CD40 binding of Hsp70 and its NH2-terminal domain, Hsp70 was preincubated in the presence or absence (mock treatment) of a 30-fold molar excess of peptide C. 3 µM GST or GST-CD40 was added to the samples and incubated for 20 min at 16°C. For titration of peptide C, 550-fold excess of peptide C (15150 µM) was added to the preincubation of Hsp70. Thereafter, the samples were combined with 20 µl of 1% BSA-treated Glutathione Sepharose 4 Fast Flow and incubated in the presence of 2 mM DTT for another 20 min at 16°C. Unbound proteins were removed by washing the beads three times with 1 ml PBS, and immobilized proteins were eluted with 30 µl elution buffer for 20 min at 22°C. 10 µl of the eluates were subjected to SDS-PAGE and analyzed by immunoblotting using a Penta-His antibody (QIAGEN). Blot signals were quantified by Quantity One software (Bio-Rad Laboratories).
Binding assay with His6-tagged Hsp70 and Ni-NTA agarose
ANA-1 cells were stimulated with LPS as described above and lysed in lysis buffer (150 mM Tris-HCl, pH 7.5, 1% CHAPS) for 45 min at 4°C. Cell lysate was centrifuged at 100,000 g for 15 min at 4°C. 500 µl of the supernatant was incubated with 10 µg His6-tagged Hsp70, preincubated with ADP and a 30-fold excess of peptide C, or mock-treated, as described above for biotinylated proteins, for 30 min at 4°C. Thereafter, the samples were added to 20 µl of 1% BSA-treated Ni-NTA agarose (QIAGEN) and incubated for 30 min at 4°C. Unbound protein was removed by washing the beads three times with 1 ml lysis buffer, and immobilized proteins were eluted with 10 µl SDS-PAGEsample buffer by incubation for 5 min at 95°C. Eluates were analyzed by immunoblotting using an anti-CD40 antibody (CSA-180; StressGen Biotechnologies).
p38 kinase assay
24 h before p38 kinase assays, HEK293T-CD40 and HEK293T-MCAT cells were seeded into a 24-well plate coated with collagen and incubated with 100 nM CD40L (Alexis Biochemicals Corp.), HSP70, N70-domain, C70-domain, or DnaK for 20 min at 37°C. DnaK, Hsp70, N70, and C70 were preincubated with either 2 mM ADP and 30 µM peptide C or 40 µM AMPPNP (Sigma-Aldrich). On stimulation, cells were washed twice with ice-cold PBS and lysed in SDSsample buffer. Equal amounts of protein were analyzed by immunoblotting with antibodies directed against phosphorylated p38 (Promega), and with a monoclonal antitubulin antibody (J. Wehland, German Research Centre for Biotechnology, Braunschweig, Germany).
![]() |
Footnotes |
---|
![]() |
Acknowledgments |
---|
Submitted: 14 August 2002
Revised: 26 August 2002
Accepted: 27 August 2002
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arnold-Schild, D., D. Hanau, D. Spehner, C. Schmid, H.G. Rammensee, H. de la Salle, and H. Schild. 1999. Cutting edge: receptor-mediated endocytosis of heat shock proteins by professional antigen-presenting cells. J. Immunol. 162:37573760.
Asea, A., M. Rehli, E. Kabingu, J.A. Boch, O. Bare, P.E. Auron, M.A. Stevenson, and S.K. Calderwood. 2002. Novel signal transduction pathway utilized by extracellular HSP70: role of toll-like receptor (TLR) 2 and TLR4. J. Biol. Chem. 277:1502815034.
Basu, S., and P.K. Srivastava. 1999. Calreticulin, a peptide-binding chaperone of the endoplasmic reticulum, elicits tumor- and peptide-specific immunity. J. Exp. Med. 189:797802.
Basu, S., R.J. Binder, R. Suto, K.M. Anderson, and P.K. Srivastava. 2000. Necrotic but not apoptotic cell death releases heat shock proteins, which deliver a partial maturation signal to dendritic cells and activate the NF-B pathway. Int. Immunol. 12:15391546.
Berwin, B., J.P. Hart, S.V. Pizzo, and C.V. Nicchitta. 2002. Cutting edge: CD91-independent cross-presentation of GRP94(gp96)-associated peptides. J. Immunol. 168:42824286.
Blond-Elguindi, S., S.E. Cwirla, W.J. Dower, R.J. Lipshutz, S.R. Sprang, J.F. Sambrook, and M.J. Gething. 1993. Affinity panning of a library of peptides displayed on bacteriophages reveals the binding specificity of BiP. Cell. 75:717728.[Medline]
Bradbury, D.A., T.D. Simmons, K.J. Slater, and S.P. Crouch. 2000. Measurement of the ADP:ATP ratio in human leukaemic cell lines can be used as an indicator of cell viability, necrosis and apoptosis. J. Immunol. Methods. 240:7992.[CrossRef][Medline]
Castellino, F., P.E. Boucher, K. Eichelberg, M. Mayhew, J.E. Rothman, A.N. Houghton, and R.N. Germain. 2000. Receptor-mediated uptake of antigenheat shock protein complexes results in major histocompatibility complex class I antigen presentation via two distinct processing pathways. J. Exp. Med. 191:19571964.
Chan, F.K., H.J. Chun, L. Zheng, R.M. Siegel, K.L. Bui, and M.J. Lenardo. 2000. A domain in TNF receptors that mediates ligand-independent receptor assembly and signaling. Science. 288:23512354.
Flynn, G.C., T.G. Chappell, and J.E. Rothman. 1989. Peptide binding and release by proteins implicated as catalysts of protein assembly. Science. 245:385390.[Medline]
Greene, L.E., R. Zinner, S. Naficy, and E. Eisenberg. 1995. Effect of nucleotide on the binding of peptides to 70-kDa heat shock protein. J. Biol. Chem. 270:29672973.
Hartl, F.U., and M. Hayer-Hartl. 2002. Molecular chaperones in the cytosol: from nascent chain to folded protein. Science. 295:18521858.
Höhfeld, J., and S. Jentsch. 1997. GrpE-like regulation of the hsc70 chaperone by the anti-apoptotic protein BAG-1. EMBO J. 16:62096216.
Kirchhausen, T., J.S. Bonifacino, and H. Riezman. 1997. Linking cargo to vesicle formation: receptor tail interactions with coat proteins. Curr. Opin. Cell Biol. 9:488495.[CrossRef][Medline]
Kol, A., A.H. Lichtman, R.W. Finberg, P. Libby, and E.A. Kurt-Jones. 2000. Cutting edge: heat shock protein (HSP) 60 activates the innate immune response: CD14 is an essential receptor for HSP60 activation of mononuclear cells. J. Immunol. 164:1317.
Minami, Y., and M. Minami. 1999. Hsc70/Hsp40 chaperone system mediates the Hsp90-dependent refolding of firefly luciferase. Genes Cells. 4:721729.
Minami, Y., J. Höhfeld, K. Ohtsuka, and F.U. Hartl. 1996. Regulation of the heat shock protein 70 reaction cycle by the mammalian DnaJ homolog, Hsp40. J. Biol. Chem. 271:1961719624.
Pullen, S.S., T.T. Dang, J.J. Crute, and M.R. Kehry. 1999. CD40 signaling through tumor necrosis factor receptor-associated factors (TRAFs). Binding site specificity and activation of downstream pathways by distinct TRAFs. J. Biol. Chem. 274:1424614254.
Rudiger, S., L. Germeroth, J. Schneider-Mergener, and B. Bukau. 1997. Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries. EMBO J. 16:15011507.
Sondermann, H., T. Becker, M. Mayhew, F. Wieland, and F.U. Hartl. 2000. Characterization of a receptor for heat shock protein 70 on macrophages and monocytes. Biol. Chem. 381:11651174.[Medline]
Sondermann, H., C. Scheufler, C. Schneider, J. Höhfeld, F.U. Hartl, and I. Moarefi. 2001. Structure of a Bag/Hsc70 complex: convergent functional evolution of Hsp70 nucleotide exchange factors. Science. 291:15531557.
Srivastava, P.K., A.B. DeLeo, and L.J. Old. 1986. Tumor rejection antigens of chemically induced sarcomas of inbred mice. Proc. Natl. Acad. Sci. USA. 83:34073411.[Abstract]
Szabo, A., T. Langer, H. Schroder, J. Flanagan, B. Bukau, and F.U. Hartl. 1994. The ATP hydrolysis-dependent reaction cycle of the Escherichia coli Hsp70 system DnaK, DnaJ, and GrpE. Proc. Natl. Acad. Sci. USA. 91:1034510349.
Tone, M., Y. Tone, P.J. Fairchild, M. Wykes, and H. Waldmann. 2001. Regulation of CD40 function by its isoforms generated through alternative splicing. Proc. Natl. Acad. Sci. USA. 98:17511756.
Udono, H., and P.K. Srivastava. 1993. Heat shock protein 70-associated peptides elicit specific cancer immunity. J. Exp. Med. 178:13911396.[Abstract]
Related Article