Chromosome 11q23 translocations disrupting human Mixed Lineage Leukemia (MLL)gene are found in 80% of infantile leukemia and almost all cases of treatment induced secondary acute myeloid leukemia (AML). The MLL gene is required for proper HOX gene expression. Our prior studies demonstrated that full-length 500kD MLL protein undergoes proteolysis to generate N-terminal 320kD (MLLN32?) and C-terminal 180kD (MLLC18?) fragments. Processed MLL fragments form a complex to regulate the stability and availability of MLLN32? for downstream gene regulation. We subsequently purified and cloned the responsible protease and entitled it Taspasel (Threonine Aspartase 1). The discovery of Taspasel initiates a new class of proteases utilizing their N- terminal Threonine of mature (3 subunit to cleave polypeptide substratesafter P1 aspartate. Preliminary studies in HeLa cells indicated the importance of Taspasel-mediated MLL cleavage in HOX gene expression. Recently, we also identified a basal transcription factor, TFIIA, as a bona fide Taspasel substrate. To investigate the physiological functions of Taspasel in vivo, we generated Taspasel knockout mice. Initial studies on Taspasel deficient animals indicate the essential role of Taspasel in body patterning, nervous system development, and cell cycle progression. With these unique reagents, we will further interrogate Taspasel functions via the following specific aims:
Specific Aim 1 : We will characterize the role of Taspasel in mouse embryonic development. It entails the creation of straight and conditional Taspasel knockout mice to determine whether Taspasel deficiency in mice results in embryonic lethality, homeotic transformations and/or other developmental abnormalities. We will dissect the mechanisms by which Taspasel regulates Hox gene expression.
Specific Aim 2 : We will investigate the requirement of Taspasel in normal cell cycle progression. We will start with studying cell cycle progression defects in Taspasel deficient animals and cells, followed by dissecting the mechanisms by which Taspasel regulates cell cycle progression and perform genetic reconstitutions of processed MLL family proteins into Taspasel deficient cells to determine whether MLL proteolysis regulates cell proliferation.
Specific Aim 3 : We will perform studies to identify additional Taspasel substrates and will validate their importance in vitro and in vivo. We will utilize 2-D difference in-gel electrophoresis in conjunction with mass spectrometry for the initial discovery, followed by phenotypic analyses of individual non-cleavable substrates. Since deregulation of HOX genes and cell cycle genes contribute to tumorigensis, this combined genetic, biochemical, and proteomic approach to investigate Taspasel functions will provide further insights regarding MLL leukemia and may lay the foundation for future development of Taspasel inhibitors as anti-cancer therapeutics.
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