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Research Interests
The nuclear envelope (NE) is a specialized domain of the endoplasmic reticulum that forms the boundary of the eukaryotic cell nucleus. It consists of inner and outer nuclear membranes, the nuclear lamina and nuclear pore complexes (NPCs). The nuclear lamina, a protein meshwork lining the inner nuclear membrane, provides a structural scaffold for the NE and an anchoring site at the nuclear periphery for interphase chromatin. NPCs are large supramolecular assemblies that span the NE and serve as channels for molecular transport between the nucleus and the cytoplasm. We are using a combination of biochemical, structural and functional approaches to investigate NPCs and the lamina.
Nucleocytoplasmic transport mechanisms.
Transport of protein and RNA through the NPC is an energy-dependent process mediated by nucleocytoplasmic shuttling receptors of the karyopherin b family. Karyopherins bind to specific transport signals on protein or RNA cargoes, and the receptor-cargo complexes are translocated through the NPC by receptor binding to a group of NPC proteins (nucleoporins) containing Phe-Gly (FG) amino acid motifs. The directionality of nuclear transport is determined in large part by the small GTPase Ran, which exists in the GTP-bound form in the nucleus and the GDP-bound form in the cytoplasm. Ran-GTP in the nucleus directly binds to karyopherins, and promotes association of cargo with export receptors and dissociation of cargo from import receptors. Conversely, hydrolysis of Ran-GTP in the cytoplasm triggers disassembly of export complexes. Conformational flexibility of karyopherins is thought to be fundamental to their dynamic interactions with cargo, Ran and nucleoporins.
We are using in vitro assays with digitonin-permeabilized cells to analyze the molecular events specifying translocation of cargo-receptor complexes through the NPC. Our recent work involving site-directed mutagenesis of importin b, the prototypical nuclear import receptor, has characterized two distinct binding sites for FG repeat nucleoporins in importin b, and has defined mutational hotspots for cargo binding. Our eventual goal is to determine how the conformational dynamics of importin b are linked to discrete transport steps. To this end, we are complementing structure-function studies with analysis involving small molecule inhibitors. A second line of work is directed at understanding movement of cargo-receptor complexes though the NPC's central channel, which forms the major permeability barrier of the pore. For this we are focusing on the transport of large protein complexes, which have more stringent Ran and energy requirements for translocation than small proteins. Analysis of large cargo import is expected to reveal new properties of the transport channel and to elucidate kinetically limiting transport steps.
In a related project, we are analyzing nuclear import of the adenovirus genome, which comprises a 36 kb double-stranded DNA molecule. Results from our in vitro transport studies indicate that adenovirus DNA transport is driven by import signals on DNA-associated protein(s). We have identified adenovirus protein VII as a strong candidate for the protein adaptor involved in the DNA import, based on our identification of multiple import signals in protein VII and on its tight association with the genome. Further analysis with this system may help to define more general themes used by viruses for uncoating at the NPC, and for transport of their nucleic acids into the nucleus.
We also are analyzing nuclear export of HIV-1 mRNA that is mediated by the viral regulatory protein Rev. Rev polymerizes on a cis-acting sequence of viral mRNAs, providing a platform for assembly of nuclear export factors. We are implementing proteomics combined with a permeabilized cell assay for Rev-dependent HIV mRNA export to functionally characterize the proteins assembled on the Rev platform. This project is part of a larger collaboration with a research team at TSRI that is working to identify small molecules inhibitors of Rev function, with the goal of finding lead compounds for developing new drugs to inhibit HIV replication in humans.
Nuclear lamina, higher level nuclear organization and disease.
The nuclear lamina in vertebrates contains a polymer of 2-4 related intermediate filament proteins called lamins, associated with a number of transmembrane proteins of the inner nuclear membrane. The lamina plays essential roles in nuclear structure and functions, as demonstrated by the recent findings that over 15 inherited human diseases, including a range of muscular dystrophies, are caused by mutations in lamins or lamina-associated transmembrane proteins. The involvement of the lamina in disease could stem from its importance for nuclear integrity or from a role in chromatin structure and gene expression. Until recently, only ~10 transmembrane proteins specific to the NE had been identified.
To survey the complete complement of NE proteins, we carried out a proteomics analysis of the NE of rodent liver in collaboration with the Yates laboratory at TSRI. From this analysis we identified 67 novel putative nuclear envelope transmembrane proteins (NETs). Analysis of a subset of these proteins indicates that the great majority are authentic NE components. We currently are carrying out a systems-level analysis of the lamina in the process of muscle differentiation. This work involves transcriptional microarray analysis, quantitative proteomics, and gene silencing by RNA interference, to characterize lamina components that are important for muscle cells. We expect that this work will identify novel candidate genes with a potential role in human muscular dystrophies, and further elucidate how the protein network consisting of lamins and associated transmembrane proteins acts in nuclear structure and functions.
