[Show abstract][Hide abstract] ABSTRACT: Mercury (Hg) is a bioaccumulating trace metal that globally circulates the atmosphere and waters in its elemental, inorganic and organic chemical forms. While Hg represents a notorious neurotoxicant, the underlying cellular pathways are insufficiently understood. We identify amyloid protein aggregation in the cell nucleus as a novel pathway of Hg-bio-interactions. By mass spectrometry of purified protein aggregates, a subset of spliceosomal components and nucleoskeletal protein lamin B1 were detected as constituent parts of an Hg-induced nuclear aggregome network. The aggregome network was located by confocal imaging of amyloid-specific antibodies and dyes to amyloid cores within splicing-speckles that additionally recruit components of the ubiquitin-proteasome system. Hg significantly enhances global proteasomal activity in the nucleus, suggesting that formation of amyloid speckles plays a role in maintenance of protein homeostasis. RNAi knock down showed that lamin B1 for its part regulates amyloid speckle formation and thus likewise participates in nuclear protein homeostasis. As the Hg-induced cascade of interactions between the nucleoskeleton and protein homeostasis reduces neuronal signalling, amyloid fibrillation in the cell nucleus is introduced as a feature of Hg-neurotoxicity that opens new avenues of future research. Similar to protein aggregation events in the cytoplasm that are controlled by the cytoskeleton, amyloid fibrillation of nuclear proteins may be driven by the nucleoskeleton.
[Show abstract][Hide abstract] ABSTRACT: Xenobiotics, as well as intrinsic processes such as cellular aging, contribute to an environment that constantly challenges nuclear organization and function. While it becomes increasingly clear that proteasome-dependent proteolysis is a major player, the topology and molecular mechanisms of nuclear protein homeostasis remain largely unknown. We have shown previously that (1) proteasome-dependent protein degradation is organized in focal microenvironments throughout the nucleoplasm and (2) heavy metals as well as nanoparticles induce nuclear protein fibrillation with amyloid characteristics. Here, we describe methods to characterize the landscape of intranuclear amyloid on the global and local level in different systems such as cultures of mammalian cells and the soil nematode Caenorhabditis elegans. Application of discrete mathematics to imaging data is introduced as a tool to develop pattern recognition of intracellular protein fibrillation. Since stepwise fibrillation of otherwise soluble proteins to insoluble amyloid-like protein aggregates is a hallmark of neurodegenerative protein-misfolding disorders including Alzheimer's disease, CAG repeat diseases, and the prion encephalopathies, investigation of intracellular amyloid may likewise aid to a better understanding of the pathomechanisms involved. We consider aggregate profiling as an important experimental approach to determine if nuclear amyloid has toxic or protective roles in various disease processes.
[Show abstract][Hide abstract] ABSTRACT: In aging societies increasing cases of neurodegenerative protein deposit diseases urge for the identification of the underlying mechanisms. Expectations are that in 2050 the percentage of population over age 60 is 42% in Japan, 34% in China, and 27% in the US. The cell nucleus is a major target of amyloid-like protein fibrillation in a variety of disorders that are characterized by widespread aggregation of proteins with instable homopolymeric amino acid repeats, ubiquitin and other proteinaceous components. Additionally, accumulation of insoluble, SDS-resistant proteins has been identified as an intrinsic property of organismal aging. This review collects current knowledge about the composition and function of insoluble, nuclear protein inclusions from the protein homeostasis perspective. It discusses the occurrence and role of nuclear amyloid in the diseased as well as the healthy cell. Features of nuclear inclusions such as protein composition and locally active protein degradation may predict neural fitness and survival in a variety of health or disease settings.
[Show abstract][Hide abstract] ABSTRACT: Invertebrate animal models such as the nematode Caenorhabditis elegans (C. elegans) are increasingly used in nanotechnological applications. Research in this area covers a wide range from remote control of worm behavior by nanoparticles (NPs) to evaluation of organismal nanomaterial-safety. Despite of the broad spectrum of investigated NP-bio interactions, little is known about the role of nanomaterials with respect to aging processes in C. elegans. We trace NPs in single-cells of adult C. elegans and correlate particle-distribution with the worm's metabolism and organ function. By confocal microscopy analysis of fluorescently labelled NPs in living worms we identify two entry portals for the uptake of nanomaterials via the pharynx to the intestinal system and via the vulva to the reproductive system. NPs are localized throughout the cytoplasm and the cell nucleus in single intestinal, and vulval B and D cells. Silica NPs induce an untimely accumulation of insoluble ubiquitinated proteins, nuclear amyloid and reduction of pharyngeal pumping that taken together constitute a premature aging phenotype of C. elegans on the molecular and behavioral level, respectively. Screening of different nanomaterials for their effects on protein solubility shows that polystyrene or silver NPs do not induce accumulation of ubiquitinated proteins suggesting that alteration of protein homeostasis is a unique property of silica NPs. The nematode C. elegans represents an excellent model to investigate the effect of different types of nanomaterials on aging at the molecule, cellular, and whole organism level.
[Show abstract][Hide abstract] ABSTRACT: Understanding of nanoparticle-bio-interactions within living cells requires knowledge about the dynamic behavior of nanomaterials during their cellular uptake, intracellular traffic and mutual reactions with cell organelles. Here, we introduce a protocol of combined kinetic imaging techniques that enables investigation of exemplary fluorochrome-labelled nanoparticles concerning their intracellular fate. By time-lapse confocal microscopy we observe fast, dynamin-dependent uptake of polystyrene and silica nanoparticles via the cell membrane within seconds. Fluorescence recovery after photobleaching (FRAP) experiments reveal fast and complete exchange of the investigated nanoparticles at mitochondria, cytoplasmic vesicles or the nuclear envelope. Nuclear translocation is observed within minutes by free diffusion and active transport. Fluorescence correlation spectroscopy (FCS) and raster image correlation spectroscopy (RICS) indicate diffusion coefficients of polystyrene and silica nanoparticles in the nucleus and the cytoplasm that are consistent with particle motion in living cells based on diffusion. Determination of the apparent hydrodynamic radii by FCS and RICS shows that nanoparticles exert their cytoplasmic and nuclear effects mainly as mobile, monodisperse entities. Thus, a complete toolkit of fluorescence fluctuation microscopy is presented for the investigation of nanomaterial biophysics in subcellular microenvironments that contributes to develop a framework of intracellular nanoparticle delivery routes.
PLoS ONE 04/2013; 8(4):e62018. DOI:10.1371/journal.pone.0062018 · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Investigation of differential gene regulation by protein degradation requires analysis of the spatial and temporal association between proteolysis and transcription. Here, we describe the isochronal visualization of proteasomal proteolysis and transcription in cell culture or in vivo in the model organism Caenorhabditis elegans. This includes localization of proteasome-dependent proteolysis by fluorescent degradation products of model and endogenous substrates of the proteasome in combination with immunolabelling of RNA polymerase II and transcription in situ run-on assays.
[Show abstract][Hide abstract] ABSTRACT: Promyelocytic leukemia (PML) nuclear bodies selectively associate with transcriptionally active genomic regions, including
the gene-rich major histocompatibility (MHC) locus. In this paper, we have explored potential links between PML and interferon
(IFN)-γ–induced MHC class II expression. IFN-γ induced a substantial increase in the spatial proximity between PML bodies
and the MHC class II gene cluster in different human cell types. Knockdown experiments show that PML is required for efficient
IFN-γ–induced MHC II gene transcription through regulation of the class II transactivator (CIITA). PML mediates this function
through protection of CIITA from proteasomal degradation. We also show that PML isoform II specifically forms a stable complex
with CIITA at PML bodies. These observations establish PML as a coregulator of IFN-γ–induced MHC class II expression.
The Journal of Cell Biology 09/2012; 199(1):49-63. DOI:10.1083/jcb.201112015 · 9.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Stepwise fibrillation of otherwise soluble proteins to insoluble amyloid-like protein aggregates is a hallmark of neurodegenerative protein-misfolding diseases including Alzheimer's, polyglutamine diseases, and the prion encephalopathies. Investigation of protein aggregation mechanisms has considerably advanced in vitro due to recent technical innovation, whereas the development of analyses tools for intracellular protein fibrillation remains a major challenge. Here, we introduce a method that enables monitoring of the protein fibrillation status in the cell nucleus. We show that the amyloid indicator Congo red can be induced to bind to distinct nucleoplasmic microdomains that are describable by application of discrete mathematics on the image information. Since formation of Congo red-binding nuclear microdomains (CRBDs) correlates with increased amyloid formation and decreased solubility of endogenous proteins with homopolymeric polyglutamine (polyQ) stretches we introduce the idea that different protein fibrillation steps can be characterized intracellularly by graph theory-aided pattern recognition.
[Show abstract][Hide abstract] ABSTRACT: While it is widely acknowledged that the ubiquitin-proteasome system plays an important role in transcription, little is known concerning the mechanistic basis, in particular the spatial organization of proteasome-dependent proteolysis at the transcription site. Here, we show that proteasomal activity and tetraubiquitinated proteins concentrate to nucleoplasmic microenvironments in the euchromatin. Such proteolytic domains are immobile and distinctly positioned in relation to transcriptional processes. Analysis of gene arrays and early genes in Caenorhabditis elegans embryos reveals that proteasomes and proteasomal activity are distantly located relative to transcriptionally active genes. In contrast, transcriptional inhibition generally induces local overlap of proteolytic microdomains with components of the transcription machinery and degradation of RNA polymerase II. The results establish that spatial organization of proteasomal activity differs with respect to distinct phases of the transcription cycle in at least some genes, and thus might contribute to the plasticity of gene expression in response to environmental stimuli.
Nucleic Acids Research 02/2011; 39(11):4612-27. DOI:10.1093/nar/gkr069 · 8.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Caseins are major constituents of mammalian milks that are thought to be exclusively expressed in mammary glands and to function primarily as a protein source, as well as to ameliorate intestinal calcium uptake. In addition, proinflammatory and immunomodulatory properties have been reported for bovine caseins. Our aim was to investigate whether human casein α s1 (CSN1S1) is expressed outside the mammary gland and possesses immunomodulatory functions in humans as well. For this purpose, CSN1S1 mRNA was detected in primary human monocytes and CD4(+) and CD8(+) T cells, but not in CD19(+) B cells. CSN1S1 protein was traceable in supernatants of cultured primary human CD14(+) monocytes by ELISA. Similarly, CSN1S1 mRNA and protein were detected in the human monocytic cell lines HL60, U937, and THP1 but not in Mono Mac 6 cells. Moreover, permeabilized human monocytes and HL60 cells could be stained by immunofluorescence, indicating intracellular expression. Recombinant human CSN1S1 was bound to the surface of Mono Mac 6 cells and upregulated the expression of GM-CSF mRNA in primary human monocytes and Mono Mac 6 cells in a time- and concentration-dependent manner. A similar increase in GM-CSF protein was found in the culture supernatants. CSN1S1-dependent upregulation of GM-CSF was specifically blocked by the addition of the p38 MAPK inhibitor ML3403. Our results indicated that human CSN1S1 may possess an immunomodulatory role beyond its nutritional function in milk. It is expressed in human monocytes and stimulates the expression of the proinflammatory cytokine GM-CSF.
The Journal of Immunology 01/2011; 186(1):592-601. DOI:10.4049/jimmunol.1001461 · 5.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nuclear inclusions that contain proteins with expanded polyglutamine (polyQ) repeats are observed in neurodegenerative aggregation diseases and are, therefore, viewed as a pathologic feature. However, a summary of research indicates that polyQ repeats are inherently both toxic and functional at the same time. PolyQ motifs occur in proteins involved in gene expression and promote nuclear assemblies such as the transcription initiation complex. Transition of these functional complexes to insoluble protein aggregates is constitutively prevented by proteasomal proteolysis. Thus, conditions that exhaust the ubiquitin-proteasome system, such as the extensive production of expanded polyQ proteins, aging and xenobiotic stress, induce a congested state in which nuclear proteins, including those with polyQ stretches, form amyloid-like aggregates. Because protein aggregation is preceded by a series of protein misfolding steps termed polyQ fibrillation, the characterization of distinct fibrillation steps correlating with nuclear function and identification of the respective genetic modifiers is essential for understanding both the biology and pathology of polyQ. Thus, the comprehension of the physiological role of polyQ repeats is a prerequisite for uncovering the underlying mechanisms of neurodegenerative aggregation diseases.
[Show abstract][Hide abstract] ABSTRACT: Protein aggregates and nuclear inclusions containing components of the ubiquitin-proteasome system, expanded polyglutamine (polyQ) proteins, and transcriptional co-activators characterize cellular responses to stress and are hallmarks of neurodegenerative diseases. For instance, in Huntington's disease, an expansion of a polyQ region causes its aggregation into beta-sheet-containing amyloid fibrils. The biological function of polyQ-containing inclusions is unknown. By means of a silica nanoparticle (NP)-based strategy we induced intranuclear protein inclusions that form amyloid-like structures, recapitulating the protein composition and solubility of polyQ-induced nuclear protein aggregates exactly. We showed that global proteasomal proteolysis increases in silica-NP-treated nuclei and, on the local level, a subpopulation of nuclear inclusions overlaps with focal domains of proteasome-dependent protein degradation. The results suggest that inclusions in the nucleus constitute active proteolysis modules that may serve to concentrate and decompose damaged, mal-folded, or misplaced proteins. While nanoparticle-nucleus interactions turn out to be invaluable tools to study the molecular mechanisms of degenerative protein aggregation diseases, one also has to consider the other side of the coin, namely, emerging environmental risks posed by these very interactions.
[Show abstract][Hide abstract] ABSTRACT: Nanotechnology makes use of the special surface properties of extremely small particles. In this rapidly growing field, many different materials are produced for a multitude of diverse applications. Possible adverse health effects of these materials however are so far scarcely investigated and are therefore a special task of toxicology. Although strategies for risk assessment have been suggested, the authors of the current review emphasize the fact that on the cellular, subcellular and molecular levels, interactions between nanoparticles (NP) and target cells relevant for the induction of possible adverse health effects are poorly understood. On the basis of existing literature, the potentially most relevant cellular target sites of NP as well as the so far known major molecular events specifically induced by these xenobiotics are reviewed. Starting with NP uptake across the cell membrane, mechanisms of generation of reactive oxygen species and the activation of redox-sensitive signalling cascades are described. Besides the cell membrane, mitochondria and cell nucleus are considered as major cell compartments relevant for possible NP-induced toxicity. Finally, an integrated research protocol is proposed to identify fundamental cellular responses to NP in order to complement current toxicological screening strategies with a mechanism-based approach.
[Show abstract][Hide abstract] ABSTRACT: While expectations and applications of nanotechnologies grow exponentially, little is known about interactions of engineered nanoparticles with multicellular organisms. Here we propose the transparent roundworm Caenorhabditis elegans as a simple but anatomically and biologically well defined animal model that allows for whole organism analyses of nanoparticle-bio-interactions. Microscopic techniques showed that fluorescently labelled nanoparticles are efficiently taken up by the worms during feeding, and translocate to primary organs such as epithelial cells of the intestine, as well as secondary organs belonging to the reproductive tract. The life span of nanoparticle-fed Caenorhabditis elegans remained unchanged, whereas a reduction of progeny production was observed in silica-nanoparticle exposed worms versus untreated controls. This reduction was accompanied by a significant increase of the 'bag of worms' phenotype that is characterized by failed egg-laying and usually occurs in aged wild type worms. Experimental exclusion of developmental defects suggests that silica-nanoparticles induce an age-related degeneration of reproductive organs, and thus set a research platform for both, detailed elucidation of molecular mechanisms and high throughput screening of different nanomaterials by analyses of progeny production.
PLoS ONE 02/2009; 4(8):e6622. DOI:10.1371/journal.pone.0006622 · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Protein aggregates and nuclear inclusions (NIs) containing components of the ubiquitin-proteasome system (UPS), expanded polyglutamine (polyQ) proteins, and transcriptional coactivators characterize cellular responses to stress and are hallmarks of neurodegenerative diseases. The biological function of polyQ-containing aggregates is unknown. To analyze proteasomal activity within such aggregates, we present a nanoparticle (NP)-based method that enables controlled induction of sodium dodecyl sulfate-resistant inclusions of endogenous nuclear proteins while normal regulatory mechanisms remain in place. Consistent with the idea that the UPS maintains quality control, inhibition of proteasomal proteolysis promotes extra large protein aggregates (1.4-2 mum), whereas formation of NP-induced NIs is found to be inversely correlated to proteasome activation. We show that global proteasomal proteolysis increases in NP-treated nuclei and, on the local level, a subpopulation of NIs overlaps with focal domains of proteasome-dependent protein degradation. These results suggest that inclusions in the nucleus constitute active proteolysis modules that may serve to concentrate and decompose damaged, malfolded, or misplaced proteins.
The Journal of Cell Biology 03/2008; 180(4):697-704. DOI:10.1083/jcb.200708131 · 9.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The 20S proteasome is part of a larger complex, the 26S proteasome, that is implicated in the ATP-dependent degradation of multiubiquitin-conjugated proteins (1). About 80% of intracellular protein breakdown occurs via the ubiquitin-proteasome system (UPS). Key proteins such as transcription factors, nuclear receptors, cyclins, cyclin-dependent kinase inhibitors, p53, and NF-kappaB are regulated by this pathway. Thus, the UPS has been implicated to play a role in multiple cellular events including the cell cycle, signal transduction, antigen presentation, and DNA repair and transcription (2, 3). In 1984 Varshavsky and co-workers discovered that ubiquitin-dependent pathways play a role in cell cycle control, and suggested that protein degradation is instrumental in regulation of gene expression (4). Consistent with this idea, Franke and colleagues had shown that proteasomes localize to the nuclei of Xenopus laevis oocytes and HeLa cells (5, 6). Subsequent work confirmed that (i) all components of the UPS that are required for protein degradation indeed reside in the cell nucleus (7); (ii) nuclear proteins are substrates for proteasomal degradation (8); and (iii) proteasome-dependent proteolysis occurs in distinct nucleoplasmic foci (9). The intricate balance between nuclear function and quality control through proteolysis is exemplified by reports that show a correlation of aberrant nuclear protein aggregates with inhibition of transcription in neurodegenerative diseases such as Huntington's chorea and animal and cell culture models of polyglutamine repeat disorders (10,11).Considering the central role of the UPS in nuclear processes, a detailed knowledge of the time and place at which a substrate is ubiquitinylated and degraded will be essential to our understanding of the cellular mechanisms that orchestrate the expression of thousands of genes or development of subnuclear pathologies. Here, we describe fluorescence-based localization methods for proteasomes, protein aggregates, and proteasomal proteolysis in the cell nucleus that may aid to analyse the UPS in housekeeping and disease conditions.
[Show abstract][Hide abstract] ABSTRACT: Proteasomes are ATP-driven, multisubunit proteolytic machines that degrade endogenous proteins into peptides and play a crucial role in cellular events such as the cell cycle, signal transduction, maintenance of proper protein folding and gene expression. Recent evidence indicates that the ubiquitin-proteasome system is an active component of the cell nucleus. A characteristic feature of the nucleus is its organization into distinct domains that have a unique composition of macromolecules and dynamically form as a response to the requirements of nuclear function. Here, we show by systematic application of different immunocytochemical procedures and comparison with signature proteins of nuclear domains that during interphase endogenous proteasomes are localized diffusely throughout the nucleoplasm, in speckles, in nuclear bodies, and in nucleoplasmic foci. Proteasomes do not occur in the nuclear envelope region or the nucleolus, unless nucleoplasmic invaginations expand into this nuclear body. Confirmedly, proteasomal proteolysis is detected in nucleoplasmic foci, but is absent from the nuclear envelope or nucleolus. The results underpin the idea that the ubiquitin-proteasome system is not only located, but also proteolytically active in distinct nuclear domains and thus may be directly involved in gene expression, and nuclear quality control.
[Show abstract][Hide abstract] ABSTRACT: HAX1 is an ubiquitously expressed human gene. Though a number of cellular and viral proteins are known to interact with HAX1, its function is still not completely understood. On the basis of these identified interaction partners, HAX1 seems to play a role in apoptosis and the organization of the cytoskeleton. The cDNAs for human and mouse Hax1 share 86% identity and 80% identity at the protein level, suggesting a similar functional importance. To date, no conclusive data on the tissue specific expression of the murine Hax1 are available and only one interaction partner has been identified. Here, we show a detailed expression analysis for the murine ortholog by RT-PCR, Northern and Western blot. Furthermore, the distribution of Hax1 within different mouse tissues was studied by immunohistochemistry (IHC). In general, we found a good correlation between the results obtained from different detection techniques. Similar to its human counterpart, mouse Hax1 seems to be ubiquitously expressed. At the RNA level, we found the highest expression of Hax1 in liver, kidney and testis. In sharp contrast to the human HAX1 which is highly expressed in skeletal muscle, the mouse ortholog was detected only at very low levels. Using a specific antibody, we detected Hax1 in the majority of mouse tissues by IHC. Interestingly, the most prominent expression of Hax1 was found in epithelial, endothelial and muscle cells. Surprisingly, thymus, spleen and pancreas did not show detectable immunostaining. Furthermore, we have studied the subcellular localisation of Hax1 in a keratinocyte and a neuronal cell line by immunofluorescence. We found Hax1 to be localised mainly in the cytoplasm and detected a partial colocalisation with mitochondria. The results presented here summarize for the first time the expression of the murine Hax1 in different tissues and two cell lines. Further studies will elucidate the functional importance of this protein in individual cell types with respect to structural aspects, cell mobility and apoptosis.
[Show abstract][Hide abstract] ABSTRACT: In eukaryotes, thousands of genes have to be organized and expressed in the cell nucleus. Conformational and kinetic instability of nuclear structure and components appear to enable cells to use the encoded information selectively. The ubiquitin-proteasome system is active in distinct nuclear domains and plays a major role controlling the initial steps of gene expression, DNA repair and nuclear quality-control mechanisms. Recent work indicates that a tuned balance of ubiquitylation and proteasome-dependent protein degradation of nuclear proteins is instrumental in nuclear function and, when deregulated, leads to the development of diseases such as polyQ disorders and other neurodegenerative conditions.