Publications by authors named "Josef Gotzmann"

22 Publications

  • Page 1 of 1

Direct measurement of protein-protein interactions by FLIM-FRET at UV laser-induced DNA damage sites in living cells.

Nucleic Acids Res 2020 12;48(21):e122

Department of Biochemistry, Max Perutz Labs, University of Vienna, Vienna Biocenter (VBC), Dr Bohr-Gasse 9, 1030 Vienna, Austria.

Protein-protein interactions are essential to ensure timely and precise recruitment of chromatin remodellers and repair factors to DNA damage sites. Conventional analyses of protein-protein interactions at a population level may mask the complexity of interaction dynamics, highlighting the need for a method that enables quantification of DNA damage-dependent interactions at a single-cell level. To this end, we integrated a pulsed UV laser on a confocal fluorescence lifetime imaging (FLIM) microscope to induce localized DNA damage. To quantify protein-protein interactions in live cells, we measured Förster resonance energy transfer (FRET) between mEGFP- and mCherry-tagged proteins, based on the fluorescence lifetime reduction of the mEGFP donor protein. The UV-FLIM-FRET system offers a unique combination of real-time and single-cell quantification of DNA damage-dependent interactions, and can distinguish between direct protein-protein interactions, as opposed to those mediated by chromatin proximity. Using the UV-FLIM-FRET system, we show the dynamic changes in the interaction between poly(ADP-ribose) polymerase 1, amplified in liver cancer 1, X-ray repair cross-complementing protein 1 and tripartite motif containing 33 after DNA damage. This new set-up complements the toolset for studying DNA damage response by providing single-cell quantitative and dynamic information about protein-protein interactions at DNA damage sites.
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http://dx.doi.org/10.1093/nar/gkaa859DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7708043PMC
December 2020

Comparative Interactome Analysis of Emerin, MAN1 and LEM2 Reveals a Unique Role for LEM2 in Nucleotide Excision Repair.

Cells 2020 02 18;9(2). Epub 2020 Feb 18.

Max Perutz Labs, Center of Medical Biochemistry, Medical University of Vienna, Vienna Biocenter (VBC), 1030 Vienna, Austria.

LAP2-Emerin-MAN1 (LEM) domain-containing proteins represent an abundant group of inner nuclear membrane proteins involved in diverse nuclear functions, but their functional redundancies remain unclear. Here, using the biotinylation-dependent proximity approach, we report proteome-wide comparative interactome analysis of the two structurally related LEM proteins MAN1 () and LEM2 (), and the more distantly related emerin (). While over 60% of the relatively small group of MAN1 and emerin interactors were also found in the LEM2 interactome, the latter included a large number of candidates (>85%) unique for LEM2. The interacting partners unique for emerin support and provide further insight into the previously reported role of emerin in centrosome positioning, and the MAN1-specific interactors suggest a role of MAN1 in ribonucleoprotein complex assembly. Interestingly, the LEM2-specific interactome contained several proteins of the nucleotide excision repair pathway. Accordingly, LEM2-depleted cells, but not MAN1- and emerin-depleted cells, showed impaired proliferation following ultraviolet-C (UV-C) irradiation and prolonged accumulation of γH2AX, similar to cells deficient in the nucleotide excision repair protein DNA damage-binding protein 1 (DDB1). These findings indicate impaired DNA damage repair in LEM2-depleted cells. Overall, this interactome study identifies new potential interaction partners of emerin, MAN1 and particularly LEM2, and describes a novel potential involvement of LEM2 in nucleotide excision repair at the nuclear periphery.
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http://dx.doi.org/10.3390/cells9020463DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072835PMC
February 2020

Simultaneous dual-channel imaging to quantify interdependent protein recruitment to laser-induced DNA damage sites.

Nucleus 2018 ;9(1):474-491

a Department of Biochemistry, Max F. Perutz Laboratories , University of Vienna, Vienna Biocenter (VBC) , Vienna , Austria.

Fluorescence microscopy in combination with the induction of localized DNA damage using focused light beams has played a major role in the study of protein recruitment kinetics to DNA damage sites in recent years. Currently published methods are dedicated to the study of single fluorophore/single protein kinetics. However, these methods may be limited when studying the relative recruitment dynamics between two or more proteins due to cell-to-cell variability in gene expression and recruitment kinetics, and are not suitable for comparative analysis of fast-recruiting proteins. To tackle these limitations, we have established a time-lapse fluorescence microscopy method based on simultaneous dual-channel acquisition following UV-A-induced local DNA damage coupled with a standardized image and recruitment analysis workflow. Simultaneous acquisition is achieved by spectrally splitting the emitted light into two light paths, which are simultaneously imaged on two halves of the same camera chip. To validate this method, we studied the recruitment of poly(ADP-ribose) polymerase 1 (PARP1), poly (ADP-ribose) glycohydrolase (PARG), proliferating cell nuclear antigen (PCNA) and the chromatin remodeler ALC1. In accordance with the published data based on single fluorophore imaging, simultaneous dual-channel imaging revealed that PARP1 regulates fast recruitment and dissociation of PARG and that in PARP1-depleted cells PARG and PCNA are recruited with comparable kinetics. This approach is particularly advantageous for analyzing the recruitment sequence of fast-recruiting proteins such as PARP1 and ALC1, and revealed that PARP1 is recruited faster than ALC1. Split-view imaging can be incorporated into any laser microirradiation-adapted microscopy setup together with a recruitment-dedicated image analysis package.
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http://dx.doi.org/10.1080/19491034.2018.1516485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6284507PMC
September 2019

A novel non-canonical PIP-box mediates PARG interaction with PCNA.

Nucleic Acids Res 2017 Sep;45(16):9741-9759

Department of Biochemistry, Max F. Perutz Laboratories, University of Vienna, Vienna Biocenter (VBC), Dr. Bohr-Gasse 9, 1030 Vienna, Austria.

Poly(ADP-ribose) glycohydrolase (PARG) regulates cellular poly(ADP-ribose) (PAR) levels by rapidly cleaving glycosidic bonds between ADP-ribose units. PARG interacts with proliferating cell nuclear antigen (PCNA) and is strongly recruited to DNA damage sites in a PAR- and PCNA-dependent fashion. Here we identified PARG acetylation site K409 that is essential for its interaction with PCNA, its localization within replication foci and its recruitment to DNA damage sites. We found K409 to be part of a non-canonical PIP-box within the PARG disordered regulatory region. The previously identified putative N-terminal PIP-box does not bind PCNA directly but contributes to PARG localization within replication foci. X-ray structure and MD simulations reveal that the PARG non-canonical PIP-box binds PCNA in a manner similar to other canonical PIP-boxes and may represent a new type of PIP-box. While the binding of previously described PIP-boxes is based on hydrophobic interactions, PARG PIP-box binds PCNA via both stabilizing hydrophobic and fine-tuning electrostatic interactions. Our data explain the mechanism of PARG-PCNA interaction through a new PARG PIP-box that exhibits non-canonical sequence properties but a canonical mode of PCNA binding.
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http://dx.doi.org/10.1093/nar/gkx604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5766153PMC
September 2017

The endonuclease Ankle1 requires its LEM and GIY-YIG motifs for DNA cleavage in vivo.

J Cell Sci 2012 Feb 7;125(Pt 4):1048-57. Epub 2012 Mar 7.

Max F. Perutz Laboratories, Medical University of Vienna, Dr. Bohr-Gasse 9, A-1030, Vienna, Austria.

The LEM domain (for lamina-associated polypeptide, emerin, MAN1 domain) defines a group of nuclear proteins that bind chromatin through interaction of the LEM motif with the conserved DNA crosslinking protein, barrier-to-autointegration factor (BAF). Here, we describe a LEM protein annotated in databases as 'Ankyrin repeat and LEM domain-containing protein 1' (Ankle1). We show that Ankle1 is conserved in metazoans and contains a unique C-terminal GIY-YIG motif that confers endonuclease activity in vitro and in vivo. In mammals, Ankle1 is predominantly expressed in hematopoietic tissues. Although most characterized LEM proteins are components of the inner nuclear membrane, ectopic Ankle1 shuttles between cytoplasm and nucleus. Ankle1 enriched in the nucleoplasm induces DNA cleavage and DNA damage response. This activity requires both the catalytic C-terminal GIY-YIG domain and the LEM motif, which binds chromatin via BAF. Hence, Ankle1 is an unusual LEM protein with a GIY-YIG-type endonuclease activity in higher eukaryotes.
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http://dx.doi.org/10.1242/jcs.098392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4335191PMC
February 2012

Molecular mechanisms of centrosome and cytoskeleton anchorage at the nuclear envelope.

Cell Mol Life Sci 2011 May 5;68(9):1593-610. Epub 2010 Oct 5.

School of Biological and Biomedical Sciences, University of Durham, Durham DH1 3LE, UK.

Cell polarization is a fundamental process underpinning organismal development, and tissue homeostasis, which requires an orchestrated interplay of nuclear, cytoskeletal, and centrosomal structures. The underlying molecular mechanisms, however, still remain elusive. Here we report that kinesin-1/nesprin-2/SUN-domain macromolecular assemblies, spanning the entire nuclear envelope (NE), function in cell polarization by anchoring cytoskeletal structures to the nuclear lamina. Nesprin-2 forms complexes with the kinesin-1 motor protein apparatus by associating with and recruiting kinesin light chain 1 (KLC1) to the outer nuclear membrane. Similar to nesprin-2, KLC1 requires lamin A/C for proper NE localization. The depletion of nesprin-2 or KLC1, or the uncoupling of nesprin-2/SUN-domain protein associations impairs cell polarization during wounding and dislodges the centrosome from the NE. In addition nesprin-2 loss has profound effects on KLC1 levels, the cytoskeleton, and Golgi apparatus organization. Collectively these data show that NE-associated proteins are pivotal determinants of cell architecture and polarization.
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http://dx.doi.org/10.1007/s00018-010-0535-zDOI Listing
May 2011

Sun1 forms immobile macromolecular assemblies at the nuclear envelope.

Biochim Biophys Acta 2008 Dec 19;1783(12):2415-26. Epub 2008 Sep 19.

Center for Biochemistry, Medical Faculty, University of Cologne, Joseph-Stelzmann-Strasse 52, 50931 Cologne, Germany.

SUN-domain proteins form a novel and conserved family of inner nuclear membrane (INM) proteins, which establish physical connections between the nucleoplasm and the cytoskeleton. In the current study, we provide evidence that within the nuclear envelope (NE) Sun1 proteins form highly immobile oligomeric complexes in interphase cells. By performing inverse fluorescence recovery after photobleaching analysis, we demonstrate in vivo that both perinuclear and nucleoplasmic Sun1 segments are essential for maintenance of Sun1 immobility at the NE. Our data in particular underline the self-association properties of the C-terminal coiled-coil Sun1 segment, the ability of which to form dimers and tetramers is demonstrated. Furthermore, the Sun1 tertiary structure involves interchain disulfide bonds that might contribute to higher homo-oligomer formation, although the overall dynamics of the Sun1 C-terminus remains unaffected when the cysteins involved are mutated. While a major Sun1 pool colocalizes with nuclear pore complex proteins, a large fraction of the Sun1 protein assemblies colocalize with immunoreactive foci of Sun2, another SUN-domain paralogue at the NE. We demonstrate that the Sun1 coiled-coil domain permits these heterophilic associations with Sun2. Sun1 therefore provides a non-dynamic platform for the formation of different macromolecular assemblies at the INM. Our data support a model in which SUN-protein-containing multi-variate complexes may provide versatile outer nuclear membrane attachment sites for cytoskeletal filaments.
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http://dx.doi.org/10.1016/j.bbamcr.2008.09.001DOI Listing
December 2008

Nucleoplasmic lamins and their interaction partners, LAP2alpha, Rb, and BAF, in transcriptional regulation.

FEBS J 2007 Mar;274(6):1362-73

Department of Medical Biochemistry, Max F. Perutz Laboratories, Medical University of Vienna, Dr. Bohrgasse 9/3, A-1030 Vienna, Austria.

Lamins are major structural components of the nuclear envelope in multicellular eukaryotes. Particularly A-type lamins are also located in the nucleoplasm, likely involving a specific binding partner, lamina-associated polypeptide 2alpha (LAP2alpha). LAP2alpha-lamins A/C complexes in the nucleoplasm have been implicated in the regulation of gene expression by various means. They bind chromatin proteins and chromatin modifying enzymes, and can thus participate in epigenetic control pathways. Furthermore, binding of lamins A/C complexes to specific transcription factors and repressors may directly affect their transcriptional activity. LAP2alpha-lamins A/C also regulate retinoblastoma protein and influence cell cycle progression and differentiation, which could have important implications for molecular mechanisms of laminopathic diseases, linked to lamins A/C mutations.
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http://dx.doi.org/10.1111/j.1742-4658.2007.05695.xDOI Listing
March 2007

Barrier-to-autointegration factor--a BAFfling little protein.

Trends Cell Biol 2007 Apr 21;17(4):202-8. Epub 2007 Feb 21.

Department of Genetics, The Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.

Barrier-to-autointegration factor (BAF) is an abundant, highly conserved, small and essential protein that binds to dsDNA, chromatin, nuclear lamina proteins, histones and various transcription factors. It was discovered as a cellular component of retrovirus pre-integration complex that inhibits their autointegration in vitro. BAF is also required for many cellular functions, including the higher-order organization of chromatin and the transcription of specific genes. Recent findings suggest further roles for BAF, including nuclear envelope assembly, regulating specific developmental processes and regulating retrovirus infectivity. At least some of these roles are controlled by phosphorylation of the BAF N-terminus by the vaccinia-related kinase. Here, we give an overview of recent advances in the field of BAF with special emphasis on evolution, interacting partners and functions.
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http://dx.doi.org/10.1016/j.tcb.2007.02.004DOI Listing
April 2007

LAP2alpha-binding protein LINT-25 is a novel chromatin-associated protein involved in cell cycle exit.

J Cell Sci 2007 Mar 6;120(Pt 5):737-47. Epub 2007 Feb 6.

Max F. Perutz Laboratories, Department of Medical Biochemistry, Medical University of Vienna, Dr. Bohr-Gasse 9, Vienna Biocenter, Dr. Bohr-Gasse 7, A-1030 Vienna, Austria.

Lamina-associated polypeptide 2alpha (LAP2alpha) is a nuclear protein dynamically associating with chromatin during the cell cycle. In addition, LAP2alpha interacts with A-type lamins and retinoblastoma protein and regulates cell cycle progression via the E2F-Rb pathway. Using yeast two-hybrid analysis and three independent in vitro binding assays we identified a new LAP2alpha interaction partner of hitherto unknown functions, which we termed LINT-25. LINT-25 protein levels were upregulated during G1 phase in proliferating cells and upon cell cycle exit in quiescence, senescence and differentiation. Upon cell cycle exit LINT-25 accumulated in heterochromatin foci, and LAP2alpha protein levels were downregulated by proteasomal degradation. Although LAP2alpha was not required for the upregulation and reorganization of LINT-25 during cell cycle exit, transient expression of LINT-25 in proliferating cells caused loss of LAP2alpha and subsequent cell death. Our data show a role of LINT-25 and LAP2alpha during cell cycle exit, in which LINT-25 acts upstream of LAP2alpha.
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http://dx.doi.org/10.1242/jcs.03390DOI Listing
March 2007

Lamina-associated polypeptide 2alpha regulates cell cycle progression and differentiation via the retinoblastoma-E2F pathway.

J Cell Biol 2006 Apr;173(1):83-93

Max F. Perutz Laboratories, Department of Medical Biochemistry, Medical University of Vienna, A-1030 Vienna, Austria.

Lamina-associated polypeptide (LAP) 2alpha is a nonmembrane-bound LAP2 isoform that forms complexes with nucleoplasmic A-type lamins. In this study, we show that the overexpression of LAP2alpha in fibroblasts reduced proliferation and delayed entry into the cell cycle from a G0 arrest. In contrast, stable down-regulation of LAP2alpha by RNA interference accelerated proliferation and interfered with cell cycle exit upon serum starvation. The LAP2alpha-linked cell cycle phenotype is mediated by the retinoblastoma (Rb) protein because the LAP2alpha COOH terminus directly bound Rb, and overexpressed LAP2alpha inhibited E2F/Rb-dependent reporter gene activity in G1 phase in an Rb-dependent manner. Furthermore, LAP2alpha associated with promoter sequences in endogenous E2F/Rb-dependent target genes in vivo and negatively affected their expression. In addition, the expression of LAP2alpha in proliferating preadipocytes caused the accumulation of hypophosphorylated Rb, which is reminiscent of noncycling cells, and initiated partial differentiation into adipocytes. The effects of LAP2alpha on cell cycle progression and differentiation may be highly relevant for the cell- and tissue-specific phenotypes observed in laminopathic diseases.
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http://dx.doi.org/10.1083/jcb.200511149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2063793PMC
April 2006

LEM2 is a novel MAN1-related inner nuclear membrane protein associated with A-type lamins.

J Cell Sci 2005 Dec;118(Pt 24):5797-810

Department of Medical Biochemistry, Max F. Perutz Laboratories, Vienna Biocenter, Medical University of Vienna, Dr Bohrgasse 9/3, A-1030 Vienna, Austria.

The LEM (lamina-associated polypeptide-emerin-MAN1) domain is a motif shared by a group of lamin-interacting proteins in the inner nuclear membrane (INM) and in the nucleoplasm. The LEM domain mediates binding to a DNA-crosslinking protein, barrier-to-autointegration factor (BAF). We describe a novel, ubiquitously expressed LEM domain protein, LEM2, which is structurally related to MAN1. LEM2 contains an N-terminal LEM motif, two predicted transmembrane domains and a MAN1-Src1p C-terminal (MSC) domain highly homologous to MAN1, but lacks the MAN1-specific C-terminal RNA-recognition motif. Immunofluorescence microscopy of digitonin-treated cells and subcellular fractionation identified LEM2 as a lamina-associated protein residing in the INM. LEM2 binds to the lamin C tail in vitro. Targeting of LEM2 to the nuclear envelope requires A-type lamins and is mediated by the N-terminal and transmembrane domains. Highly overexpressed LEM2 accumulates in patches at the nuclear envelope and forms membrane bridges between nuclei of adjacent cells. LEM2 structures recruit A-type lamins, emerin, MAN1 and BAF, whereas lamin B and lamin B receptor are excluded. Our data identify LEM2 as a novel A-type-lamin-associated INM protein involved in nuclear structure organization.
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http://dx.doi.org/10.1242/jcs.02701DOI Listing
December 2005

Thymopoietin (lamina-associated polypeptide 2) gene mutation associated with dilated cardiomyopathy.

Hum Mutat 2005 Dec;26(6):566-74

CU-Cardiovascular Institute, University of Colorado Health Sciences Center, Denver, Colorado, USA.

Thymopoietin or TMPO (indicated by its alternative gene symbol, LAP2, in this work) has been proposed as a candidate disease gene for dilated cardiomyopathy (DCM), since a LAP2 product associates with nucleoplasmic lamins A/C, which are encoded by the DCM gene LMNA. We developed a study to screen for genetic mutations in LAP2 in a large collection of DCM patients and families. A total of 113 subjects from 88 families (56 with familial DCM (FDC) and 32 with sporadic DCM) were screened for LAP2 mutations using denaturing high-performance liquid chromatography and sequence analysis. We found a single putative mutation affecting the LAP2alpha isoform in one FDC pedigree. The mutation predicts an Arg690Cys substitution (c.2068C>T; p.R690C) located in the C-terminal domain of the LAP2alpha protein, a region that is known to interact with lamin A/C. RT-PCR, Western blot analyses, and immunolocalization revealed low-level LAP2alpha expression in adult cardiac muscle, and localization to a subset of nuclei. Mutated Arg690Cys LAP2alpha expressed in HeLa cells localized to the nucleoplasm like wild-type LAP2alpha, with no effect on peripheral and nucleoplasmic lamin A distribution. However, the in vitro interaction of mutated LAP2alpha with the pre-lamin A C-terminus was significantly compromised compared to the wild-type protein. LAP2 mutations may represent a rare cause of DCM. The Arg690Cys mutation altered the observed LAP2alpha interaction with A-type lamins. Our finding implicates a novel nuclear lamina-associated protein in the pathogenesis of genetic forms of dilated cardiomyopathy.
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http://dx.doi.org/10.1002/humu.20250DOI Listing
December 2005

A-type lamin complexes and regenerative potential: a step towards understanding laminopathic diseases?

Histochem Cell Biol 2006 Jan 2;125(1-2):33-41. Epub 2005 Sep 2.

Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, Department of Medical Biochemistry, Medical University of Vienna, Dr. Bohrgasse 9/3, 1030, Vienna, Austria.

The lamins are nuclear intermediate filament-type proteins forming the nuclear lamina meshwork at the inner nuclear membrane as well as complexes in the nucleoplasm. The recent discoveries that mutated A-type lamins and lamin-binding nuclear membrane proteins can be linked to numerous rare human diseases (laminopathies) affecting a multitude of tissues has changed the cell biologist's view of lamins as mere structural nuclear scaffold proteins. It is still unclear how mutations in these ubiquitously expressed proteins give rise to tissue-restricted pathological phenotypes. Potential disease models include mutation-caused defects in lamin structure and stability, the deregulation of gene expression, and impaired cell cycle control. This review brings together various previously proposed ideas and suggests a novel, more general, disease model based on an impairment of adult stem cell function and thus compromised tissue regeneration in laminopathic diseases.
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http://dx.doi.org/10.1007/s00418-005-0050-8DOI Listing
January 2006

The inner nuclear membrane protein Sun1 mediates the anchorage of Nesprin-2 to the nuclear envelope.

J Cell Sci 2005 Aug;118(Pt 15):3419-30

Center for Biochemistry, Medical Faculty, University of Cologne, Joseph-Stelzmann-Strasse 52, 50931 Cologne, Germany.

Nesprins form a novel class of nuclear envelope-anchored spectrin-repeat proteins. We show that a direct association of their highly conserved C-terminal luminal domain with the inner nuclear membrane protein Sun1 mediates their nuclear envelope localisation. In Nesprin-1 and Nesprin-2 the conserved C-terminal amino acids PPPX are essential for the interaction with a C-terminal region in Sun1. In fact, Sun1 is required for the proper nuclear envelope localisation of Nesprin-2 as shown using dominant-negative mutants and by knockdown of Sun1 expression. Sun1 itself does not require functional A-type lamins for its localisation at the inner nuclear membrane in mammalian cells. Our findings propose a conserved nuclear anchorage mechanism between Caenorhabditis elegans and mammals and suggest a model in which Sun1 serves as a ;structural bridge' connecting the nuclear interior with the actin cytoskeleton.
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http://dx.doi.org/10.1242/jcs.02471DOI Listing
August 2005

Integration of Ras subeffector signaling in TGF-beta mediated late stage hepatocarcinogenesis.

Carcinogenesis 2005 May 10;26(5):931-42. Epub 2005 Feb 10.

Department of Medicine I, Institute of Cancer Research, Medical University of Vienna, Borschke-Gasse 8a, A-1090 Vienna, Austria.

Immortalized p19(ARF) null hepatocytes (MIM) feature a high degree of functional differentiation and are susceptible to transforming growth factor (TGF)-beta driven growth arrest and apoptosis. In contrast, polarized MIM hepatocytes expressing hyperactive Ha-Ras continue proliferation in cooperation with TGF-beta, and adopt an invasive phenotype by executing an epithelial to mesenchymal transition (EMT). In this study, we analyzed the involvement of Ras subeffectors in TGF-beta mediated hepatocellular EMT by employing MIM hepatocytes, which express Ras mutants allowing selective activation of either mitogen-activated protein kinase (MAPK) signaling (V12-S35) or phosphoinositide 3-OH (PI3)3 kinase (PI3K) signaling (V12-C40). We found that MAPK signaling in MIM-S35 hepatocytes was necessary and sufficient to promote resistance to TGF-beta mediated inhibition of proliferation in vitro and in vivo. MIM-S35 hepatocytes showed also PI3K activation during EMT, however, MAPK signaling on its own protected hepatocytes from apoptosis. Yet, MIM-C40 hepatocytes failed to form tumors and required additional MAPK stimulation to overcome TGF-beta mediated growth arrest. In vivo, the collaboration of MAPK signaling and TGF-beta activity drastically accelerated the cell-cycle progression of the hepatocytes, leading to vast tumor formation. From these data we conclude that MAPK is crucial for the cooperation with TGF-beta to regulate the proliferation as well as the survival of hepatocytes during EMT, and causes the fatal increase in hepatocellular tumor progression.
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http://dx.doi.org/10.1093/carcin/bgi043DOI Listing
May 2005

beta-Catenin and TGFbeta signalling cooperate to maintain a mesenchymal phenotype after FosER-induced epithelial to mesenchymal transition.

Oncogene 2004 Apr;23(15):2672-2680

Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, Department of Medical Biochemistry, University of Vienna, Vienna, Austria.

Several signalling pathways contribute to the regulation of epithelial to mesenchymal transition (EMT), either during developmentally regulated processes or in cancer progression and metastasis. Induction of EMT in fully polarized mouse mammary epithelial cells (EpH4) by an inducible c-fos estrogen receptor (FosER) oncoprotein involves loss of E-cadherin expression, nuclear translocation of beta-catenin, and autocrine production of TGFbeta. Reporter assays demonstrate that both beta-catenin/LEF-TCF- and TGFbeta-Smad-dependent signalling activities are upregulated, probably coregulating mesenchymal-specific gene expression during EMT. Stable expression of E-cadherin in mesenchymal FosER cells decreased beta-catenin activity and reduced cell proliferation. However, these cells still exhibited a defect in epithelial polarization and expressed E-cadherin/beta-catenin complexes in the entire plasma membrane. On the other hand, inhibition of TGFbeta-Smad signalling in mesenchymal FosER cells induced flat, cobblestone-like clusters of cells, which relocalized beta-catenin to the plasma membrane but still lacked detectable E-cadherin. Interestingly, inhibition of TGFbeta signalling in the E-cadherin-expressing mesenchymal FosER cells caused their reversion to a polarized epithelial phenotype, in which E-cadherin, beta-catenin, and ZO-1 were localized at their correct lateral plasma membrane domains. These results demonstrate that loss of E-cadherin can contribute to increased LEF/TCF-beta-catenin signalling, which in turn cooperates with autocrine TGFbeta signalling to maintain an undifferentiated mesenchymal phenotype.
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http://dx.doi.org/10.1038/sj.onc.1207416DOI Listing
April 2004

Molecular aspects of epithelial cell plasticity: implications for local tumor invasion and metastasis.

Mutat Res 2004 Jan;566(1):9-20

Institute of Cancer Research, University of Vienna, Borschkegasse 8a, A-1090 Vienna, Austria.

Carcinomas arising from epithelial cells represent the most prevalent malignancies in humans, and metastasis is the major cause for the death of carcinoma patients. The breakdown of epithelial cell homeostasis leading to aggressive cancer progression has been correlated with the loss of epithelial characteristics and the acquisition of a migratory phenotype. This phenomenon, referred to as epithelial to mesenchymal transition (EMT), is considered as a crucial event in late stage tumorigenesis. Here we summarize the multitude of EMT models derived from different tissues, and review the diversity of molecular mechanisms contributing to the plasticity of epithelial cells. In particular, the synergism between activation of Ras, provided by the aberrant stimulation of receptor tyrosine kinases, and transforming growth factor (TGF)-beta signaling plays a pivotal role in inducing EMT of various epithelial cell types. Cytokines such as TGF-beta and extracellular matrix molecules are thought to fundamentally contribute to the microenvironmental interaction between stromal and malignant cells, and provide the basis for a broad repertoire of epithelial differentiation. Investigations of EMT tumor models, which represent in vitro correlates to local invasion and metastasis in vivo, facilitate the identification of diagnostic markers for a more accurate and faithful clinical and pathological assessment of epithelial tumors. In addition, the analysis of molecular mechanisms involved in EMT might yield novel therapeutic targets for the specific treatment of aggressive carcinomas.
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http://dx.doi.org/10.1016/s1383-5742(03)00033-4DOI Listing
January 2004

The proto-oncoprotein c-Fos negatively regulates hepatocellular tumorigenesis.

Oncogene 2003 Oct;22(43):6725-38

Institute of Cancer Research, University of Vienna, Borschke-Gasse 8a, A-1090 Vienna, Austria.

Hepatocytes adopt an invasive and metastatic phenotype caused by the cooperation of transforming growth factor (TGF)-beta and oncogenic Ha-Ras. In the initial phase of this process, c-Fos is rapidly induced by TGF-beta, but then decreases to undetectable levels. Here, we investigated the functional implications of c-Fos activation and its contribution to hepatocellular tumorigenesis. By employing conditional c-Fos expression, we observed that continuous activation of c-Fos and consequently AP-1 activity leads to depolarization of differentiated murine epithelial hepatocytes. Most remarkably, this change in morphology was associated with inhibition of proliferation and induction of cell death. Coexpression of antiapoptotic Bcl-XL or scavenging of reactive oxygen species was sufficient to prevent the c-Fos-mediated phenotype. In contrast, the cooperation of c-Fos with oncogenic Ha-Ras or a Ras mutant selectively activating the MAPK pathway even enhanced c-Fos-induced effects. Showing the negative role in hepatocellular tumorigenesis, c-Fos repressed oncogenic Ras-driven anchorage-independent growth in vitro and strongly suppressed tumour formation in vivo. Taken together, we demonstrate that c-Fos modulates plasticity of epithelial hepatocytes and acts tumour suppressive in neoplastic hepatocytes by stimulating cell cycle inhibition and cell death.
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http://dx.doi.org/10.1038/sj.onc.1206781DOI Listing
October 2003

Proto-oncoprotein TLS/FUS is associated to the nuclear matrix and complexed with splicing factors PTB, SRm160, and SR proteins.

Exp Cell Res 2003 Feb;283(2):184-95

Institute of Cancer Research, Borschkegasse 8a, University of Vienna, A-1090 Vienna, Austria.

TLS/FUS is a nucleic acid-binding protein whose N-terminal half functions as a transcriptional activator domain in fusion oncoproteins found in human leukemias and liposarcomas. Previous reports have suggested a role for TLS/FUS in transcription and splicing processes. Here we report the association of TLS/FUS with the nuclear matrix and investigate its role in splicing. Splicing of two pre-mRNAs was inhibited in a TLS/FUS-immunodepleted extract and could only be partly restored by addition of recombinant TLS/FUS or/and SR proteins, known interaction partners of TLS/FUS. The subsequent analysis of TLS/FUS immunoprecipitates revealed that, in addition to the SR proteins SC35 and SRp75, the splicing factor PTB (hnRNPI) and the splicing coactivator SRm160 are complexed with TLS/FUS, thus explaining the inability to restore splicing completely. Coimmunolocalization confirmed the nuclear matrix association and interaction of TLS/FUS with PTB, SR proteins, and SRm160. Our results suggest that the matrix protein TLS/FUS plays a role in spliceosome assembly.
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http://dx.doi.org/10.1016/s0014-4827(02)00046-0DOI Listing
February 2003

Concomitant determination of absolute values of cellular protein amounts, synthesis rates, and turnover rates by quantitative proteome profiling.

Mol Cell Proteomics 2002 Jul;1(7):528-37

Institute of Cancer Research, University of Vienna, 1090 Vienna, Austria.

Two-dimensional gel electrophoresis of protein fractions isolated from (35)S-radiolabeled cells provides qualitative information on intracellular amounts, (35)S incorporation rates, protein modifications, and subcellular localizations of up to thousands of individual proteins. In this study we extended proteome profiling to provide quantitative data on synthesis rates of individual proteins. We combined fluorescence detection of radiolabeled proteins with SYPRO ruby(TM) staining and subsequent autoradiography of the same gels, thereby quantifying protein amounts and (35)S incorporation. To calibrate calculation of absolute synthesis rates, we determined the amount and autoradiograph intensity of radiolabeled haptoglobin secreted by interleukin-6 pretreated HepG2 cells. This allowed us to obtain a standard calibration value for (35)S incorporation per autoradiograph intensity unit. This value was used to measure protein synthesis rates during time course experiments of heat-shocked U937 cells. We measured the increasing amounts of hsp70 and calculated it by integration of the determined hsp70 synthesis rates over time. Similar results were obtained by both methods, validating our standardization procedure. Based on the assumption that the synthesis rate of proteins in a steady state of cell metabolism would essentially compensate protein degradation, we calculated biological half-lives of proteins from protein amounts and synthesis rates determined from two-dimensional gels. Calculated protein half-lives were found close to those determined by pulse-chase experiments, thus validating this new method. In conclusion, we devised a method to assess quantitative proteome profiles covering determination of individual amounts, synthesis, and turnover rates of proteins.
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http://dx.doi.org/10.1074/mcp.m200026-mcp200DOI Listing
July 2002

Hepatocytes convert to a fibroblastoid phenotype through the cooperation of TGF-beta1 and Ha-Ras: steps towards invasiveness.

J Cell Sci 2002 Mar;115(Pt 6):1189-202

Institute of Cancer Research, University of Vienna, Borschke-Gasse 8a, A-1090 Vienna, Austria.

In hepatocarcinogenesis, it is an open question whether transforming growth factor (TGF)-beta1 provides a tumor-suppressive or a tumor-promoting role. To address this question, we employed immortalized murine hepatocytes, which display a high degree of differentiation and, expectedly, arrest in the G1 phase under exposure to TGF-beta1. These hepatocytes maintain epithelial polarization upon expression of oncogenic Ha-Ras. However, Ras-transformed hepatocytes rapidly convert to a spindle-shaped, fibroblastoid morphology upon treatment with TGF-beta1, which no longer inhibits proliferation. This epithelial to fibroblastoid conversion (EFC) is accompanied by disruption of intercellular contacts and remodeling of the cytoskeletal framework. Fibroblastoid derivatives form elongated branching cords in collagen gels and grow to severely vascularized tumors in vivo, indicating their increased malignancy and even invasive phenotype. Additionally, fibroblastoid cells secrete strongly enhanced levels of TGF-beta1, suggesting an autocrine regulation of TGF-beta signaling. Expression profiling further revealed that the loss of the adhesion component E-cadherin correlates with the upregulation of its transcriptional repressor Snail in fibroblastoid cells. Moreover, the phosphoinositide 3-OH (PI3) kinase pathway was required for the maintenance of EFC, as inhibition of PI3 kinase reverted fibroblastoid cells to an epithelial-like phenotype. Taken together, these data indicate a dual role of TGF-beta1 in hepatocytes: it induces proliferation arrest but provides a crucial function in promoting late malignant events in collaboration with activated Ha-Ras.
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March 2002