The interleukin-1 beta (IL-1 beta) converting enzyme (ICE) processes the inactive IL-1 beta precursor to the proinflammatory cytokine. Adherent monocytes from mice harboring a disrupted ICE gene (ICE-/-) did not export IL-1 beta or interleukin-1 alpha (IL-1 alpha) after stimulation with lipopolysaccharide. Export of tumor necrosis factor-alpha and interleukin-6 (IL-6) from these cells was also diminished. Thymocytes from ICE-/- mice were sensitive to apoptosis induced by dexamethasone or ionizing radiation, but were resistant to apoptosis induced by Fas antibody. Despite this defect in apoptosis, ICE-/- mice proceed normally through development.
Cyclophilin, a specific cytosolic binding protein responsible for the concentration of the immunosuppressant cyclosporin A by lymphoid cells, was purified to homogeneity from bovine thymocytes. Cation-exchange high-performance liquid chromatography resolved a major and minor cyclophilin species that bind cyclosporin A with a dissociation constant of about 2 X 10(-7) moles per liter and specific activities of 77 and 67 micrograms per milligram of protein, respectively. Both cyclophilin species have an apparent molecular weight of 15,000, an isoelectric point of 9.6, and nearly identical amino acid compositions. A portion of the NH2-terminal amino acid sequence of the major species was determined. The cyclosporin A-binding activity of cyclophilin is sulfhydryl dependent, unstable at 56 degrees C and at pH 4 or 9.5, and sensitive to trypsin but not to chymotrypsin digestion. Cyclophilin specifically binds a series of cyclosporin analogs in proportion to their activity in a mixed lymphocyte reaction. Isolation of cyclophilin from the cytosol of thymocytes suggests that the immunosuppressive activity of cyclosporin A is mediated by an intracellular mechanism, not by a membrane-associated mechanism.
The interleukin-1beta (IL-1beta) converting enzyme (ICE) processes the inactive IL-1beta precursor to the proinflammatory cytokine. ICE was also shown to cleave the precursor of interferon-gamma inducing factor (IGIF) at the authentic processing site with high efficiency, thereby activating IGIF and facilitating its export. Lipopolysaccharide-activated ICE-deficient (ICE-/-) Kupffer cells synthesized the IGIF precursor but failed to process it into the active form. Interferon-gamma and IGIF were diminished in the sera of ICE-/- mice exposed to Propionibacterium acnes and lipopolysaccharide. The lack of multiple proinflammatory cytokines in ICE-/- mice may account for their protection from septic shock.
The structurally novel macrolide FK506 (refs 1,2) has recently been demonstrated to have potent immunosuppressive activity at concentrations several hundredfold lower than cyclosporin A (CsA). Cyclosporin A, a cyclic peptide, has found widespread clinical use in the prevention of graft rejection following bone marrow and organ transplantation. The mechanisms of immunosuppression mediated by FK506 and CsA appear to be remarkably similar, suggesting that these unrelated structures act on a common receptor or on similar molecular targets, perhaps the CsA receptor, cyclophilin, which has recently been shown by Fischer et al. and Takahashi et al. to have cis-trans peptidyl-prolyl isomerase activity. We have prepared an FK506 affinity matrix and purified a binding protein for FK506 from bovine thymus and from human spleen. This FK506-binding protein (FKBP) has a relative molecular mass (Mr) of approximately 14,000(14K), a pI of 8.8-8.9, and does not cross-react with antisera against cyclophilin. The first 40 N-terminal residues of the bovine and 16 residues of the human FKBP were determined; the 16-residue fragments are identical to each other and unrelated to any known sequences. This protein catalyses the cis-trans isomerization of the proline amide in a tetrapeptide substrate and FK506 inhibits the action of this new isomerase. The FKBP and cyclophilin appear to be members of an emerging class of novel proteins that regulate T cell activation and other metabolic processes, perhaps by the recognition (and possibly the isomerization) of proline-containing epitopes in target proteins.
BiochemistryExpression and characterization of human FKBP52, an immunophilin that associates with the 90-kDa heat shock protein and is a component of steroid receptor complexes Communicated by Etienne-Emile Baulieu, August 17, 1992 ABSTRACT Using an FK506 affinity column to identify mammalian immunosuppressant-binding proteins, we identified an immunophilin with an apparent Mr 55,000, which we have named FKBP52. We used chemically determined peptide sequence and a computerized algorithm to search GenPept, the translated GenBank data base, and identified two cDNAs likely to encode the murine FKBP52 homolog. We amlifed a murine cDNA fragment, used it to select a human FKBP52 (hFKBP52) cDNA clone, and then used the done to deduce the hFKBP52 sequence (calculated Mr 51,810) and to express hFKBP52 in Escherichia coi. Recombinant hFKBP52 has peptidyl-prolyl cis-trans isomerase activity that is inhibited by FK506 and rapamycin and an FKBP12-like consensus sequence that probably defines the immunosuppressant-binding site. FKBP52 Is apparently common to several vertebrate species and associates with the 90-kDa heat shock protein (hsp90) in untransformed mammalian steroid receptor complexes. The putative immunosuppressant-binding site is probably distinct from the hsp90-binding site, and we predict that FKBP52 has different structural domains to accommodate these functions. hFKBP52 contains 12 protein kinase phosphorylation-site motifs MATERIALS AND METHODSPreparation of an FK506 Affinity Matrix and Isolation of FKBPs. An amino derivative of FK506 was prepared (10) and coupled to Affi-Gel 10 (Bio-Rad) in methanol overnight; unreacted groups were blocked with 50 mM ethanolamine. FKBPs from calf thymus cytosol were prepared by using the matrix as described (11), dialyzed at 40C against 10 mM Tris-HCl (pH 7.0), lyophilized, reconstituted in SDS sample buffer, and resolved in a 12.5% acrylamide gel. Proteins were stained (Fig. 1A) or electroblotted.Protein Sequence Determination of bFKBP52. The Mr 55,000 band, later called bFKBP52, was stained on and excised from Immobilon-P (Millipore) for automated aminoterminal sequencing (26). Peptides were generated with endoproteinase Lys-C (Wako Chemicals USA, Richmond, VA) and separated by microbore C18 HPLC and eluted at 200A.l/min from 5% B at 0 min to 33% B at 65 min, 60%6 B at 90 min, and 100% B at 105 min (solvent A, 0.09%o trifluoroacetic acid in water; solvent B, 0.06% trifluoroacetic acid in acetonitrile). Effluent fractions corresponding to absorption peaks at 214 nm were collected, stored immediately at -200C, and applied later to a Polybrene-precycled glass-fiber filter for sequencing.Isolation of a Human cDNA Encoding FKBP52. Using BLAST (27) to search GenPept (translated GenBank Release 64.3 with daily updates, searched on July 11, 1991) with the deduced hFKBP12 sequence (7, 8), we identified two related murine polypeptides (encoded by GenBank sequences X17068 and X17069) that shared significant sequence identity with our bFKBP52 sequence (Fig. 2). Two DNA oligomers...
VX-680 is a potent inhibitor of Aurora kinases that induces the accumulation of cells with z4N DNA content, followed by cell death. Here, we define the role of p53 and p21Waf1/Cip1 in cell cycle perturbations following exposure to VX-680. Endoreduplication and apoptosis in response to VX-680 are limited in A549 and MCF-7 cells expressing wild-type p53, and markedly enhanced in cells lacking p53, including those engineered to express the HPV16-E6 oncoprotein or short interfering RNA pools targeting p53. In contrast, endoreduplication and apoptosis occur in the p53 wild-type cell lines, RKO and U2OS. The difference in response to VX-680 among these cell lines correlates with the timing of induction of p21Waf1/Cip1 and its ability to inhibit cyclin E-cdk2 activity. In A549 cells, VX-680 induces the expression of p53 and p21Waf1/Cip1 within 24 hours, with consequent inhibition of cyclin E-cdk2, and reduction of retinoblastoma protein phosphorylation, limiting endoreduplication. In RKO and U2OS cells, the induction of p21 Waf1/Cip1 is delayed and associated with higher residual cyclin E-cdk2 kinase activity and retinoblastoma protein phosphorylation, followed by progressive endoreduplication and apoptosis. Abrogation of p21Waf1/Cip1 expression by short interfering RNA targeting in A549 cells results in a substantial increase in the degree of endoreduplication, whereas inducible expression of p21Waf1/Cip1 in p53-negative NCI-H1299 cells inhibits VX-680-induced endoreduplication and cell death. These data suggest that the integrity of the p53-p21Waf1/Cip1 -dependent postmitotic checkpoint governs the response to Aurora kinase inhibition. Although cells with intact checkpoint function arrest with 4N DNA content, those with compromised checkpoint function are more likely to undergo endoreduplication followed by eventual apoptosis. (Cancer Res 2006; 66(15): 7668-77)
FKBP, an 11.8 kD intracellular protein that binds the immunosuppressants FK506 (Kd = 0.4 nM) and rapamycin (Kd = 0.2 nM) with high affinity, was purified to homogeneity from calf thymus. The complete amino acid sequence has been determined by automated Edman degradation of the intact molecule and overlapping fragments generated by proteolytic and chemical cleavage. The analysis revealed a 107 amino acid peptide chain with the following sequence: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFV- LGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPNATLIFDVELLKLE. The molecular weight, calculated from the amino acid sequence to be 11,778 D, was confirmed by electrospray ionization mass spectrometry. Thus, naturally isolated bovine FKBP does not appear to have any residues modified by glycosylation, phosphorylation, or other posttranslational derivatization processes. Bovine FKBP has only three amino acid residues that differ from human FKBP, whose sequence was elucidated by cloning and sequencing complementary DNA (Standaert et al., 1990). The protein has a substantial number of hydrophilic peptide segments with prevalent beta-strand type of chain fold. Understanding the biological function of FKBP and other members of the immunophilin class and their respective complexes with immunosuppressive drugs may provide insights into cytoplasmic signalling mechanisms, protein folding and translocation, and other cellular processes.
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