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The cell's nucleus represents a major control unit. In eukaryotes, thousands of genes have to be organized and expressed in the nucleus. Hundreds of macromolecules including chromatin modulators, transcription factors and ribonucleoprotein particles (RNPs), cluster to form the molecular machineries for replication, DNA repair, transcription, RNA splicing, and ribosome biogenesis. Thus, a major question of current research concerns the spatial distribution and regulation of nuclear processes. Growing evidence implicates direct engagement of a nuclear ubiquitin-proteasome system (nUPS) in gene expression. Consistent with this idea, the von Mikecz group has shown that nuclear proteins are subjected to proteasome-dependent protein degradation, nucleoplasmic protein fractions contain proteasomal activity, and proteasomal proteolysis occurs in transient focal domains of the cell nucleus.Microinjection of ectopic fluorogenic protein DQ-ovalbumin revealed that this substrate is degraded in distinct nucleoplasmic foci which partially overlap with signature proteins of subnuclear domains such as splicing factor compartments, or promyelocytic leukemia bodies, ubiquitin, nucleoplasmic proteasomes, and RNA polymerase II. The results clearly establish proteasome-dependent proteolysis as an intrinsic function of the cell nucleus.
Protein aggregates and intranuclear inclusions containing a variety of cellular proteins such as components of the UPS, heat shock proteins, and transcriptional co-activator CREB-binding protein (CBP) are hallmarks of neurodegenerative diseases that are caused by expanding CAG repeats coding for polyglutamine (polyQ). The mechanisms of polyQ toxicity are largely unknown, however, two molecular processes are discussed: (1) sequestration and degradation of proteins that are essential for gene expression by the UPS or (2) inhibition of proteasomal degradation. We recently developed a nanoparticle(NP)-based method that enables controlled induction of protein aggregation in the cell nucleus. Such aggregates are composed of signature proteins of nuclear domains, CBP, UPS components, cellular polyQ proteins and huntingtin. Formation of NP-induced intranuclear protein aggregates is correlated with inhibition of gene expression and induction of cellular senescence. We reported recently that proteasomal proteolysis is induced in NP-treated nuclei and a subpopulation of intranuclear aggregates represents focal domains of proteasome-dependent protein degradation. These results favor the sequestration model (see (1)) and suggest that protein aggregation in the cell nucleus serves as a protective quality control system.
In order to characterize the role of the nUPS in nuclear quality control projects are proposed that aim to characterize transcriptional processes during protein aggregation and neurodegeneration. A tiered approach comprises analyses of ubiquitination and proteasomal degradation of proteins that are essential components of the transcription machinery, and segregate in protein aggregates. Most of the experiments will be performed in primary neuronal cells, neuronal cell lines, and the nematode C. elegans. Protein aggregation will be induced by NPs or transfection of polyQ-containing plasmids.
Selected publications
A. von Mikecz: J. Cell Sci. 119, 1977-1984 (2006)
M. Chen, A. von Mikecz: Exp. Cell Res. 305, 51-62 (2005)
M. Chen et al.: J. Cell Biol. 18, 697-704 (2008)
M. Chen et al.: Arthritis Rheum. 52, 877-884 (2005).
T.D. Rockel et al.: J. Cell Sci. 118, 5231-5242 (2005)
A. Scharf et al.: Histochem. Cell Biol. 127, 591-601 (2007) |
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