The coiled-coil mediated interaction of Leukemia-Associated AF10 and hDOT1L : : new insights and interfering options
Year: 2011
Type: dissertation
Abstract: The MLL (mixed lineage leukemia or myeloid/lymphoid leukemia) gene is frequently involved in chromosomal translocations leading to acute leukemia. More than 60 different translocation partner genes have been identified so far. Among them, only two genes have been reported to be part of another leukemia-relevant chromosomal rearrangement. One of them is the putative transcription factor AF10 (ALL-1 fused gene from chromosome 10), which was found in fusion to MLL in t(10;11)(q23;p12) as well as to CALM (clathrin associated lymphoid myeloid leukemia) in t(10;11)(p13;p14) in leukemia patients. The resulting fusion proteins, MLL-AF10 and CALM-AF10, were both demonstrated to recruit the methyltransferase DOT1L (disruptor of telomeric silencing 1 like) leading to disturbed Hox gene regulation and finally resulting in leukemic transformation. The interaction of the human DOT1L protein with AF10 is mediated by two closely linked α-helical domains of AF10 comprising a so called octapeptide motif (OM) and a leucine zipper region (LZ). Both domains were shown to be necessary and sufficient for leukemic transformation and were also reported to interact with hDOT1L as part of the AF10 homologue, AF17. Due to the involved leucine zipper, a coiled coil mediated interaction of hDOT1L and AF10 or AF17, respectively, was assumed in this study with the goal to investigate the unknown interaction region of hDOT1L in detail. Applying the coiled coil prediction program PairCoil, three possible coiled-coil regions of hDOT1L (hDcc1-3) were predicted and examined for their binding capabilities towards the coiled-coil domain of AF10 (AF10cc) and its homologue AF17 (AF17cc). The respective sequences were cloned and analyzed in different in vitro and in vivo interaction assays. The strongest affinity towards AF10cc was observed for the third coiled-coil domain and therein in the second register (hDcc3R2). The results of the interaction assays with AF17cc were less definite, since in addition to hDcc3R2, the overlapping third register (hDcc3R3) as well as the first coiled-coil domain (hDcc1) showed interaction tendencies with AF17cc. In order to interfere with the leukemia-relevant recruitment of hDOT1L by AF10, a semirational peptide library targeting AF10cc was designed, based on the hDcc3R2 sequence with randomized interface positions. From this library, peptides with increased affinities towards AF10cc were initially selected by phage display. For further affinity maturation, a second peptide library was designed considering previously selected amino acids. Subsequently, phage display was applied followed by a novel selection system based on bacterial hitchhiker translocation. Three inhibitory peptides (iPepAF10#1-3) were identified and characterized for their interactions with AF10cc. All three selected peptides formed parallel coiled coils with AF10cc and demonstrated increased solubility and thermal stability in heteromeric complexes with AF10cc compared to wild-type hDcc3R2. Notably, monomeric inhibitory peptides themselves also revealed increased solubility and thermal stability. Moreover, they performed significantly better than hDcc3R2 in all tested in vitro and in vivo interaction assays with AF10cc and were able to outcompete the wild-type interaction in the low nanomolar range. The availability of selective inhibitors of the hDOT1L-AF10 interaction by iPepAF10-peptides is expected to provide valuable insights into the physiologic and pathogenic role of the interplay of both proteins and may even form the basis for the development of therapeutic peptides specifically targeting MLL-AF10 or CALM-AF10 fusion constructs in acute leukemia.
Author: Christina Räuber
Language: English
Subfield: Hematology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W3026564176