Localization controllable protein constructs: application in Chronic Myelogenous Leukemia

Controlling intracellular localization of proteins to alter their function offers great potential for treatment of diseases caused due to mislocalization, as well as regulating cellular functions. Mislocalization of proteins is manifested in diseases ranging from cancer to metabolic disorders. In this study localization controllable constructs capable of changing localization on ligand induction were designed and optimized for efficiency of translocation. These constructs contain a nuclear export signal (NES), a nuclear localization signal (NLS) and a ligand binding domain (LBD). The constructs localize in the cytoplasm in the absence of ligand and translocate to the nucleus on ligand induction. The mechanism of nuclear export of the localization controllable constructs was studied and it was established that the classical nuclear export receptor Crml is involved in export of these constructs. Further, the cytoplasmic retention of glucocorticoid and progesterone receptors was investigated. The results indicated that GR and PR LBDs were involved in cytoplasmic retention of these receptors via Hsp90. Finally, controllable protein constructs for regulating localization of oncogene Bcr-Abl were constructed and tested. The Bcr-Abl fusion protein is causative agent for chronic myelogenous leukemia (CML). It is predominantly localized in the cytoplasm initiating signal transduction pathways involved in cell-proliferation and anti-apoptosis. However, nuclear localization of Bcr-Abl results in apoptosis. Localization controllable Bcr-Abl protein constructs were tested in human leukemia cell line (K562). These constructs localized in the cytoplasm in the absence of ligand, however, showed varied localization pattern on addition of ligand depending on the presence/absence of actin binding domain (ABD). The localization controllable Bcr-Abl constructs were unable to induce apoptosis either due to inactivation of certain critical domains or due to their inability to drive an adequate amount of Bcr-Abl into the nucleus necessary for apoptosis. Further investigation is required to constructs and test localization controllable versions of Bcr-Abl and its domains capable of inducing apoptosis in regular CML cells, as well as cells resistant to current standard treatment therapies. In summary, this dissertation explores the possibility of manipulating NES, NLS and LBD to construct regulatable protein constructs for correcting intracellular localization of mislocalized proteins and altering their functionality.