Modulation of nuclear protein import: a novel means of regulating gene expression.

Eukaryotic cells depend upon the regulated exchange of proteins and RNA between the cytoplasm and the nucleus for survival. Various cytoplasmic and nuclear proteins play a fundamental role in this specific transport process. Over the last few years the components and stages of nuclear protein transport have been characterized in significant detail. Because many of the proteins that are transported into the nucleus are transcription factors, the import process is an interesting target for the manipulation of gene expression. Over time the eukaryotic cell has assembled a number of methods by which to regulate the nuclear localization of transcription factors. Within the last few years, there have been several reports of the pharmacologic manipulation of the localization of nuclear proteins as well. In addition, a recent study suggests that viruses are able to modulate host cell nuclear protein transport in vivo. This report will present an overview of nuclear protein import, describe the various in vivo mechanisms by which the cell regulates this process, and discuss recent attempts to manipulate the process with small molecule compounds. As nuclear import is a fundamental cellular process, potential opportunities for the future may arise from direct and specific ways to modulate this process and thereby treat diseases characterized by dysregulation of transcription factor activity.

Intranuclear targeted delivery of functional NF-kappaB by 70 kDa heat shock protein.

The 70 kDa heat shock protein (Hsp70) is a highly conserved, ubiquitous protein involved in chaperoning proteins to various cellular organelles. Here we show that when added exogenously to cells, Hsp70 is readily imported into both cytoplasmic and nuclear compartments in a cell-type-specific fashion. We exploited this ability of Hsp70 to deliver NF-kappaB, a key transcriptional regulator of inflammatory responses. We demonstrate that a fusion protein composed of a C-terminal Hsp70 peptide and the p50 subunit of NF-kappaB was directed into the nucleus of cells, could bind DNA specifically, and activated Igkappa expression and TNFalpha production. We therefore propose that Hsp70 can be used as a vehicle for intracytoplasmic and intranuclear delivery of proteins or DNA to modulate gene expression and thereby control immune responses.

Identification of a binding site on Hsc70 for the immunosuppressant 15-deoxyspergualin.

Hsc70, the constitutive form of the heat shock protein 70 family of proteins, is involved in a number of biological activities which include protein folding and molecular chaperoning. Previously, we had shown that the immunosuppressant 15-deoxyspergualin (DSG) specifically interacted with Hsc70, as well as the Hsp90 family of proteins. Although the exact binding site on Hsc70 for protein substrates is unknown, a recent study shows that the extreme C-terminal four amino acids 647EEVD650 play a role in regulating AT-Pase activity, substrate binding, and interaction with HDJ-1. These four amino acids are also found at the C-terminus of Hsp90 and may be involved in similar functions. In this study, we show that DSG binds specifically to this EEVD regulatory domain. Binding of DSG to Hsc70 did not affect its ability to bind peptides. These results suggest that in addition to the ATP binding domain, there are two additional substrate binding domains on Hsc70. DSG should provide a tool for understanding the role of the EEVD motif in biological processes.

A D-amino acid peptide inhibitor of NF-kappa B nuclear localization is efficacious in models of inflammatory disease.

The transcription factor NF-kappa B regulates many genes involved in proinflammatory and immune responses. The transport of NF-kappa B into the nucleus is essential for its biologic activity. We describe a novel, potent, and selective NF-kappa B inhibitor composed of a cell-permeable peptide carrying two nuclear localization sequences (NLS). This peptide blocks NF-kappa B nuclear localization, resulting in inhibition of cell surface protein expression, cytokine production, and T cell proliferation. The peptide is efficacious in vivo in a mouse septic shock model as well as a mouse model of inflammatory bowel disease, demonstrating that NF-kappa B nuclear import plays a role in these acute inflammatory disease models.