Biomedical subjects

Carol S Lim

Publications and source records attributed to Carol S Lim.

5 recordsLinked to original sources

PubMed2006

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.

Animals↗

PubMed2006

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.

Animals↗

PubMed2003

Correlation among agonist dose, rate of import, and transcriptional activity of liganded progesterone receptor B isoform in living cells.

PURPOSE: To determine the correlation among progesterone dose, rate of import of progesterone-occupied progesterone receptor (PR) complexes into the nucleus of cells, and transcriptional activity of progesterone-PR complexes. METHODS: Live cell imaging and time-lapse microscopy of green fluorescent protein-tagged PR were performed to measure the rate of import into the nucleus of progesterone-PR complexes. To measure transcriptional activity, a progesterone-PR-sensitive luciferase reporter gene assay was used. RESULTS: For low doses of progesterone, there was a correlation among dose, import into the nucleus, and transcriptional activity. At higher doses of progesterone (beyond 12.5 nM), transcriptional activity increased, but there was no further increase in the rate of import, indicating a saturation of the import machinery. In both cases, a simple one-compartment model was sufficient to describe the import data. CONCLUSIONS: At low doses, progesterone dose correlates well with rate of import and transcriptional activity. At high doses, more progesterone can get into the nucleus and can activate unoccupied receptors already in the nucleus, leading to higher transactivation than would be expected from rates of import of progesterone-PR complexes into the nucleus.

Algorithms↗

PubMed2002

Model system to study classical nuclear export signals.

Signal-mediated protein transport through the nuclear pore complex is of considerable interest in the field of molecular pharmaceutics. Nuclear localization signals can be used to target genes/antisense delivery systems to the nucleus. Studying nuclear export is useful in enhancing the expression and the efficiency of action of these therapeutic agents. The mechanism of nuclear import has been well studied and most of the proteins participating in this mechanism have been identified. The subject of nuclear export is still in the initial stages, and there is a considerable amount of uncertainty in this area. Two main export receptors identified so far are Exportin 1 (Crm1) and Calreticulin. Crm1 recognizes certain leucine-rich amino acid sequences in the proteins it exports called classical nuclear export signals. This paper describes a model system to study, identify, and establish these classical nuclear export signals using green fluorescent protein (GFP). Two putative export signals in the human progesterone receptor (PR) and the strongest nuclear export signal known (from mitogen activated protein kinase kinase [MAPKK]) were studied using this model system.

Active Transport, Cell Nucleus↗

PubMedDate not supplied

Effect of initial subcellular localization of progesterone receptor on import kinetics and transcriptional activity.

Progesterone receptors (PR) are ligand-activated transcription factors that modulate transcription by activating genes. There are two isoforms of PR, PRA and PRB. In most cell contexts, the PRA isoform is a repressor of the PRB isoform. Without hormone induction, PRA is mostly located in the nucleus whereas PRB distributes both in the nucleus and in the cytoplasm. In this paper, a new model system has been used to study the impact of initial subcellular localization, and import rate of progesterone receptor on transcriptional activity. This new model system involves using a mutant version of PRB which is found only in the cytoplasmic compartment of cells in the unliganded state, making the distribution of the receptor more homogeneous to start with compared with the previous model, wild type (wt) PRB, which has a more heterogeneous distribution (nuclear and cytoplasmic even without ligand). Import kinetics has been shown to be one of the major means by which to regulate PR transcriptional activity. Fluorescence microscopy was used to measure green fluorescent protein tagged PRB import rate into the nucleus. Luciferase reporter gene assay was used to measure transcriptional activity of PRB. In addition, a two-hybrid assay was performed to measure the interaction between PRB and importin alpha. Mutant versions of PRA and PRB with the constitutively active nuclear localization signal removed were created (PRA-NLSc mutant and PRB-NLSc mutant). These PR mutants were found to localize mainly in the cytoplasm in the absence of hormone. With addition of hormone, PR mutants translocated to the nucleus, although at a slower rate compared to wt PRB. Our results show that the activation of reporter gene transcription is proportional to the nuclear import rate of PRB-NLSc mutant, and the difference in import kinetics between wt PRB and the PRB-NLSc mutant is due to a stronger interaction of wt PRB with importin alpha. We also show that the hormone inducible NLS in PR, NLSh, is a weak nuclear localization signal even without hormone and can act as a weak hormone dependent nuclear localization signal when combined with the ligand binding domain of PR. In addition, by changing the initial subcellular localization of PRA from the nucleus to the cytoplasm, this diminished PRA's ability to act as an inhibitor of PRB.

Adenocarcinoma↗