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Carol Lim

Assistant Professor of Pharmaceutics and Pharmaceutical Chemistry

B.S. Purdue University

Ph.D. University of California, San Francisco

Research

References

carol.lim@pharm.utah.edu

Research

Our laboratory focuses on the study of signal sequences and their application to drug delivery and therapy.   As a model system, we have been studying steroid hormone receptor (SHR) subcellular compartmentalization, including their import and export mechanisms.   These import and export signals can be harnessed for use in the next phase of gene therapy:   localization controllable proteins.   These localization controllable proteins are currently targeted towards reproductive cancers and chronic myelogenous leukemia.

Import and Export Mechanisms: We have previously developed a model system to study nuclear import (NLS) and export signals (NES), and have defined a consensus NES, which consists of a leucine-rich stretch of amino acids:   L (X) 1-3 L (X) 2-3 L (X) L, where L= leucine and X= any amino acid; the last leucine can be replaced by conservative substitutions (such as isoleucine or valine).   Strong NESs have been identified in MAP kinase kinase and HIV-1 rev proteins.   It is known that SHRs contain classical NLSs which are short stretches of basic amino acids such as lysine or arginine.   However, the subcellular localization of steroid receptors varies, despite the presence of NLSs.   For example, the glucocorticoid receptor (GR) has a NLS, but is present predominantly in the cytoplasm (in the absence of ligand).   When hormone ligand is added, GR translocates to the nucleus.   The current theory assumes that molecular chaperone proteins such as heat shock protein 90 (hsp 90) retain these receptors in the cytoplasm, and when hormone is added, the receptors undergo a conformational change that allows nuclear translocation.   We are currently studying the role of hsp90 in SHR import.   We are particularly interested in possible masking/unmasking of SHR import signals by proteins like hsp90.   It is also known that the ligand binding domains (LBDs) of SHRs, when bound to ligand, cause a change in the conformation of SHRs which then allow nuclear localization.   The precise mechanism of ligand-driven nuclear localization is unknown, and is currently under study.

Cellular kinetics: Another area of research involves the import kinetics of the human progesterone receptor (PR) into the nucleus which involves time-lapse microscopy.   We have shown that the rate of import of PR into the nucleus correlates with agonist dose and activation of a reporter gene in the nucleus.   We are currently studying a version of PR with its nuclear localization signal knocked out, and have found that this version of the receptor, while still able to translocate to the nucleus, has a much slower import rate.   We are currently trying to determine if import occurs by the classical importin a/b mediated pathway or by some other mechanism.  

Application to Drug Delivery: Import and export signal sequences can be harnessed and controlled for use in the next phase of gene therapy:   controlled targeting and delivery of gene products. To achieve this, the inherent signals present in a protein will be removed and replaced with a switchable mechanism.   Gene products are directed to a specific subcellular compartment where they are exclusively active, and removed from the compartment in a controlled manner.   Localization controllable proteins could be used to correct for mislocalization of endogenous proteins that occurs in some cancers and other disease state.   We have shown already that a hormone inducible nuclear import signal and an export signal can be used as a bidirectional "on/off switch" for controlled targeting of proteins to subcellular compartments.   This switch consists of the LBD of PR (which acts as a NLS), a strong NES, and a gene of interest (green fluorescent protein, GFP, was first chosen as a model).   After transfection into mammalian cells, the protein (GFP) localizes in the cytoplasm of the cells.   After addition of hormone, the protein translocates to the nucleus in a dose-dependent fashion (see figure below).

One protein selected for delivery to the nucleus is the repression domain of the nuclear corepressor NCoR, which has a therapeutic use in inhibiting progesterone-receptor-responsive reproductive cancers.   NCoR is only active in the nucleus of cells, so it is an ideal candidate for regulated therapeutic delivery.

The other protein we are interested in is the causative agent in chronic myelogenous leukemia (CML):   BCR-ABL.   BCR-ABL proteins are oncogenic in the cytoplasmic compartment only, and must multimerize in order to be active.   Localization-controllable versions of BCR-ABL could multimerize with wild-type BCR-ABL, then be directed from the cytoplasm to the nucleus by the addition of ligand, resulting in loss of oncogenic activity.   BCR-ABL, when directed to the nucleus, indeed becomes apoptotic, so this is a possible new way to treat CML.

Protein switch construct is cytoplasmic in the "off" state (A: no ligand) and translocates to the nucleus in the "on" state (B: ligand added)

References