Controlling subcellular localization to alter function: Sending oncogenic Bcr-Abl to the nucleus causes apoptosis.

Altering the subcellular localization of signal transducing proteins is a novel approach for therapeutic intervention. Mislocalization of tumor suppressors, oncogenes, or factors involved in apoptosis results in aberrant functioning of these proteins, leading to disease. In the case of chronic myelogenous leukemia (CML), cytoplasmic Bcr-Abl causes oncogenesis/proliferation. On the other hand, nuclear entrapment of endogenous Bcr-Abl (in K562 human leukemia cells) causes apoptosis. The goal of this study was to determine whether ectopically expressed Bcr-Abl could cause apoptosis of K562 cells when specifically directed to the nucleus via strong nuclear localization signals (NLSs). A single NLS from SV40 large T-antigen or four NLSs were subcloned to Bcr-Abl (1NLS-Bcr-Abl or 4NLS-Bcr-Abl). When transfected into K562 cells, only 4NLS-Bcr-Abl translocated to the nucleus. Bcr-Abl alone was found to localize in the cell cytoplasm, colocalizing with actin due to its actin binding domain. 1NLS-Bcr-Abl also localized with actin. Apoptosis induced by 4NLS-Bcr-Abl was evaluated 24h post-transfection by morphologic determination, DNA staining, and caspase-3 assay. This is the first demonstration that altering the location of ectopically expressed Bcr-Abl can kill leukemia cells. Multiple NLSs are required to overcome Bcr-Abl binding to actin, thus driving it into the nucleus and causing apoptosis.

Optimizing the protein switch: altering nuclear import and export signals, and ligand binding domain.

Ligand regulated localization controllable protein constructs were optimized in this study. Several constructs were made from a classical nuclear export signal (HIV-rev, MAPKK, or progesterone receptor) in combination with a SV40 T-antigen type nuclear import signal. Different ligand binding domains (LBDs from glucocorticoid receptor or progesterone receptor) were also tested for their ability to impart control over localization of proteins. This study was designed to create constructs which are cytoplasmic in the absence of ligand and nuclear in the presence of ligand, and also to regulate the amount of protein translocating to the nucleus on ligand induction. The balance between the strengths of import and export signals was critical for overall localization of proteins. The amount of protein entering the nucleus was also affected by the dose of ligand (10-100 nM). However, the overall import characteristics were determined by the strengths of localization signals and the inherent localization properties of the LBD used. This study established that the amount of protein present in a particular compartment can be regulated by the use of localization signals of various strengths. These optimized localization controllable protein constructs can be used to correct for diseases due to aberrant localization of proteins.

Signal sequences for targeting of gene therapy products to subcellular compartments: the role of CRM1 in nucleocytoplasmic shuttling of the protein switch.

PURPOSE: The purpose of this study was to understand the mechanism of nuclear export of the protein switch, used for controlled intracellular delivery of gene products, by studying the involvement of classical export receptor CRM1. METHOD: Transient transfections of protein switch constructs, isolated nuclear export and import signals were carried out. Effect of leptomycin B (inhibitor of export receptor) and geldanamycin (inhibitor of Hsp90) on localization of these constructs was studied using fluorescence microscopy. Putative nuclear export signals in the glucocorticoid and progesterone receptor ligand binding domains were identified and studied. RESULTS: It was observed that treatment with leptomycin B caused nuclear accumulation of the protein switch constructs. However, geldanamycin did not have any pronounced effect on the localization. The isolated nuclear export signal from glucocorticoid receptor localized mostly in the cytoplasm, while its mutated version was present everywhere. CONCLUSION: The localization controlled protein switch constructs are exported out of the nucleus by the classical CRM1 receptors. The ligand binding domain of these protein switch constructs plays an important role in maintaining these constructs in the cytoplasm in the absence of ligand, as well the re-export back to the cytoplasm from the nucleus after ligand washout.

Controlling protein compartmentalization to overcome disease.

Over the past decade, considerable progress has been made to improve our understanding of the intracellular transport of proteins. Mechanisms of nuclear import and export involving classical receptors have been studied. Signal sequences required for directing a protein molecule to a specific cellular compartment have been defined. Knowledge of subcellular trafficking of proteins has also increased our understanding of diseases caused due to mislocalization of proteins. A specific protein on deviating from its native cellular compartment may result in disease due to loss of its normal functioning and aberrant activity in the "wrong" compartment. Mislocalization of proteins results in diseases that range from metabolic disorders to cancer. In this review we discuss some of the diseases caused due to mislocalization. We further focus on application of nucleocytoplasmic transport to drug delivery. Various rationales to treat diseases by exploiting intracellular transport machinery have been proposed. Although the pathways for intracellular movement of proteins have been defined, these have not been adequately utilized for management of diseases involving mislocalized proteins. This review stresses the need for designing drug delivery systems utilizing these mechanisms as this area is least exploited but offers great potential.

Geldanamycin, an inhibitor of Hsp90, blocks cytoplasmic retention of progesterone receptors and glucocorticoid receptors via their respective ligand binding domains.

Steroid hormone receptors (SHRs), such as glucocorticoid receptors (GR) and progesterone receptors (PR), are shuttling proteins that undergo continuous nuclear import and export. Various mechanisms have been proposed to explain the localization of SHRs. It has been suggested that the ligand-binding domain (LBD) of SHRs is important in determining the subcellular localization. We have studied the localization of GR-LBD and PR-LBD alone, as well as of full-length GR and PR in the presence of geldanamycin (GA), a benzoquinoid ansamycin that specifically inhibits heat shock protection (Hsp90), using transient transfections and fluorescent microscopy. Our studies have indicated that GR-LBD and PR-LBD are retained in the cytoplasm via interaction with Hsp90. It was observed that in the unliganded state, treatment with GA translocates these LBDs to the nucleus. Similar results were obtained for full-length PR and GR. Additionally, it was found that after ligand induction, GA accelerated reexport of SHRs after ligand washout, implicating Hsp90 in nuclear retention of SHRs in the washout state. We also propose that a recently found "export" signal present in the LBD of SHRs is involved in interactions with Hsp90 and hence cytoplasmic retention of these receptors. After ligand induction, Hsp90 also may play a role in nuclear retention of SHRs following hormone washout.