The PHD fingers of the human MLL and Drosophila trx proteins have strong amino acid sequence conservation but their function is unknown. We have determined that these fingers mediate homodimerization and binding of MLL to Cyp33, a nuclear cyclophilin. These two proteins interact in vitro and in vivo in mammalian cells and colocalize at specific nuclear subdomains. Overexpression of the Cyp33 protein in leukemia cells results in altered expression of HOX genes that are targets for regulation by MLL. These alterations are suppressed by cyclosporine and are not observed in cell lines that express a mutant MLL protein without PHD fingers. These results suggest that binding of Cyp33 to MLL modulates its effects on the expression of target genes.
Material and methods
Cloning of a full-length eDNA for hCyP33PCR amplification of DNA-fragments from different human Jurkat T cell line cDNA libraries were performed with two degenerate primers, which correspond to amino acid sequences QGGDF and KHVVFG (boxed in Fig. 2b) in the highly conserved regions of known cyclophilins [3,13,[17][18][19][20]. The resulting PCR products were subcloned into pUC18 and sequenced. Nested PCR was performed to extend the sequence using two libraries: a Marathon cDNA library of the human Jurkat T cell line (Clontech) and a cDNA library made of mRNA from human T cells. Both libraries yielded as longest cDNA one with a length of 1.6 kb. The eDNA was sequenced on both strands.
Residues 1^9 of M(12^26) (GLPALISWIKRKRQ-Q-NH 2 ), the C-terminal 15-residue segment of melittin, were substituted individually to change the hydropathicities in these positions. Antimicrobial and hemolytic activities of these peptides were determined. The results showed increased antimicrobial activities with increased hydrophobicities at almost all the positions studied. The e¡ects at positions 2, 5, 8 and 9 were signi¢cant while the e¡ects at the other positions were small. These two groups of residues were located on the opposite faces of the K K-helix. In other words, the hydrophobicities of the two faces were favorable, but one face (the more favorable face) contributed more to the antimicrobial activities than the other (the less favorable face). The hydrophobicity, not the amphipathicity, seems to be crucial for antimicrobial activity. In contrast, the hydrophobicity of one face was favorable but the other was unfavorable for the hemolytic activity, indicating that the amphipathicity may be important for hemolysis. Interestingly, the more favorable face for antimicrobial activity was located opposite to the favorable face for hemolytic activity, indicating the direction of the hydrophobic face for the antimicrobial activity and direction of the amphipathicity for the hemolytic activity were also important.
Human nuclear cyclophilin 33 (hCyP33) was the first protein which was found to contain an RNA-binding motif and a PPIase domain. It was not known what cellular and physiological roles are played by the RNA-binding activity as well as the PPIase activity of hCyP33. In this paper, we investigated the binding specificity of hCyP33 to different cellular RNA using ion-exchange chromatography and affinity adsorption. Furthermore, the influence of different cellular RNAs to the PPIase activity of hCyP33 was investigated using a protease-coupled method. The results show that hCyP33 binds specifically to mRNA, namely poly(A) + RNA, and that binding stimulates the PPIase activity of hCyP33.
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