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 (MLLN320) and C-terminal 180kD (MLLC180) fragments. Processed MLL fragments form a complex to regulate the stability and availability of MLLN320 for downstream gene regulation. We subsequently purified and cloned the responsible protease and entitled it Taspase1 (Threonine Aspartase 1). The discovery of Taspase1 initiates a new class of proteases utilizing their N- terminal Threonine of mature ? subunit to cleave polypeptide substrates after P1 aspartate. Preliminary studies in HeLa cells indicated the importance of Taspase1-mediated MLL cleavage in HOX gene expression. Recently, we also identified a basal transcription factor, TFIIA, as a bona fide Taspase1 substrate. To investigate the physiological functions of Taspase1 in vivo, we generated Taspase1 knockout mice. Initial studies on Taspase1 deficient animals indicate the essential role of Taspase1 in body patterning, nervous system development, and cell cycle progression. With these unique reagents, we will further interrogate Taspase1 functions via the following specific aims:
Specific Aim 1 : We will characterize the role of Taspase1 in mouse embryonic development. It entails the creation of straight and conditional Taspase1 knockout mice to determine whether Taspase1 deficiency in mice results in embryonic lethality, homeotic transformations and/or other developmental abnormalities. We will dissect the mechanisms by which Taspase1 regulates Hox gene expression.
Specific Aim 2 : We will investigate the requirement of Taspase1 in normal cell cycle progression. We will start with studying cell cycle progression defects in Taspase1 deficient animals and cells, followed by dissecting the mechanisms by which Taspase1 regulates cell cycle progression and perform genetic reconstitutions of processed MLL family proteins into Taspase1 deficient cells to determine whether MLL proteolysis regulates cell proliferation.
Specific Aim 3 : We will perform studies to identify additional Taspase1 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 tumorigenesis, this combined genetic, biochemical, and proteomic approach to investigate Taspase1 functions will provide further insights regarding MLL leukemia and may lay the foundation for future development of Taspase1 inhibitors as anti-cancer therapeutics. ? ? ?
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