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  • 1
    Keywords: CELLS ; CELL ; MICROSCOPY ; TOOL ; NEW-YORK ; desmosome ; TISSUE ; TISSUES ; BIOLOGY ; MOLECULAR-BIOLOGY ; WATER ; PARTICLES ; REQUIRES ; ELECTRON ; tomography ; ARTIFACTS ; RECONSTRUCTION ; ARCHITECTURE ; CRYOELECTRON MICROSCOPY ; SECTIONS ; PROTEOMICS ; intermediate filament ; bacterial envelope ; BIOLOGICAL SPECIMENS ; CHROMOSOMES ; CRYO-ELECTRON MICROSCOPY ; high-pressure freezing ; INSITU ; ULTRATHIN SECTIONS ; vitrification
    Abstract: Since the beginning of the 1980s, cryo-electron microscopy of a thin film of vitrified aqueous suspension has made it possible to observe biological particles in their native state, in the absence of the usual artefacts of dehydration and staining. Combined with 3-d reconstruction, it has become an important tool for structural molecular biology. Larger objects such as cells and tissues cannot generally be squeezed in a thin enough film. Cryo-electron microscopy of vitreous sections (CEMOVIS) provides then a solution. It requires vitrification of a sizable piece of biological material and cutting it into ultrathin sections, which are observed in the vitrified state. Each of these operations raises serious difficulties that have now been overcome. In general, the native state seen with CEMOVIS is very different from what has been seen before and it is seen in more detail. CEMOVIS will give its full potential when combined with computerized electron tomography for 3-d reconstruction
    Type of Publication: Journal article published
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  • 2
    Keywords: RECEPTOR ; APOPTOSIS ; CELLS ; tumor ; Germany ; KINASE ; GENERATION ; DEATH ; PROTEIN ; MICE ; NF-KAPPA-B ; ACTIVATION ; COMPLEX ; COMPLEXES ; MECHANISM ; mechanisms ; T-CELL ; T-CELLS ; BINDING ; PHOSPHORYLATION ; SUPPRESSION ; ALPHA ; CLEAVAGE ; TRANSGENIC MICE ; activation-induced cell death ; CELL-DEATH ; INDUCED APOPTOSIS ; LYMPHOCYTES ; BETA ; T-LYMPHOCYTES ; sensitization ; TCR ; KAPPA-B ; sensitivity ; SIGNALING COMPLEX ; IMMUNOLOGICAL SYNAPSE ; T lymphocytes ; CD95 ; signaling ; PROGRAM ; RE ; INCREASE ; IMMUNE-SYSTEM ; cell death ; ANTIGEN RECEPTORS ; HPK1 ; progenitor ; INDUCE ; NEGATIVE REGULATION ; SWITCH ; AICD ; CD28 COSTIMULATION ; HEMATOPOIETIC PROGENITOR KINASE-1 ; IKK ; KINASE-C-THETA
    Abstract: Restimulation of the T-cell receptor (TCR) in activated T cells induces CD95 (Fas/Apo-1)-mediated activation-induced cell death (AICD). The TCR-proximal mechanisms leading to AICD are elusive. Here we characterize hematopoietic progenitor kinase 1 (HPK1) as a differentially regulated TCR-proximal signaling protein involved in AICD of primary T cells. We show that HPK1 is a functional component of the endogenous I kappa B kinase (IKK) complex and is crucial for TCR-mediated NF kappa B activation. While full-length HPK1 enhances IKK beta phosphorylation, siRNA-mediated knockdown of HPK1 blunts TCR-mediated NF kappa B activation and increases cell death. We also demonstrate proteolytic processing of HPK1 into HPK1-C, specifically in AICD-sensitive primary T cells. The cleavage product HPK1-C sequesters the inactive IKK complex and suppresses NF kappa B upon TCR restimulation by binding to IKK alpha and IKK beta. T cells of HPK1-C transgenic mice are sensitized towards TCR-mediated AICD. Consequently, preventing HPK1-C generation in primary T cells by siRNA-mediated knockdown results in decreased AICD. Thus, these results show a novel mechanism of sensitization of T lymphocytes towards AICD by suppression of NF kappa B, and propose that HPK1 is a life/death switch in T lymphocytes
    Type of Publication: Journal article published
    PubMed ID: 16341093
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  • 3
    Keywords: CELLS ; Germany ; human ; INHIBITION ; KINASE ; PATHWAY ; DISEASE ; DISEASES ; PROTEIN ; PROTEINS ; kidney ; MECHANISM ; MARKER ; mechanisms ; BINDING ; PHOSPHORYLATION ; ACID ; TRANSPORT ; NUMBER ; MUTATION ; genetics ; MARKERS ; TRAFFICKING ; MUTATIONS ; EPITHELIAL-CELLS ; INTERACTS ; AMINO-ACIDS ; CLUSTER ; targeting ; DISORDERS ; CHILDHOOD ; RESIDUES ; TRANSITION ; AMINO-ACID ; interaction ; INDUCE ; LOSSES ; INTERACT ; cilia ; cystic kidney disease ; INTRAFLAGELLAR TRANSPORT ; KIDNEY-DISEASE ; nephrocystin ; NEPHRONOPHTHISIS ; NPHP1 ; PACS-1 ; TRANS-GOLGI NETWORK
    Abstract: Mutations in proteins localized to cilia and basal bodies have been implicated in a growing number of human diseases. Access of these proteins to the ciliary compartment requires targeting to the base of the cilia. However, the mechanisms involved in transport of cilia proteins to this transitional zone are elusive. Here we show that nephrocystin, a ciliary protein mutated in the most prevalent form of cystic kidney disease in childhood, is expressed in respiratory epithelial cells and accumulates at the base of cilia, overlapping with markers of the basal body area and the transition zone. Nephrocystin interacts with the phosphofurin acidic cluster sorting protein (PACS)-1. Casein kinase 2 (CK2)-mediated phosphorylation of three critical serine residues within a cluster of acidic amino acids in nephrocystin mediates PACS-I binding, and is essential for colocalization of nephrocystin with PACS-1 at the base of cilia. Inhibition of CK2 activity abrogates this interaction and results in the loss of correct nephrocystin targeting. These data suggest that CK2-dependent transport processes represent a novel pathway of targeting proteins to the cilia
    Type of Publication: Journal article published
    PubMed ID: 16308564
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  • 4
    Keywords: APOPTOSIS ; CELLS ; EXPRESSION ; CELL ; IN-VIVO ; MODEL ; PATHWAY ; PATHWAYS ; GENE ; GENES ; PROTEIN ; PROTEINS ; TISSUE ; MICE ; ACTIVATION ; INDUCTION ; TISSUES ; TARGET ; TRANSGENIC MICE ; SIGNAL-TRANSDUCTION ; p53 ; EPITHELIAL-CELLS ; PHENOTYPE ; C-MYC ; REGULATOR ; CYTOKINE ; RE ; INCREASE ; GLAND ; LEVEL ; TARGET GENES ; PREGNANCY ; mammary gland ; LACTATION ; PROLACTIN ; mammary ; GP130 ; Stat3 ; mammary epithelial cells ; ONCOSTATIN-M ; CYTOKINE SIGNALING-3 ; GENE DELETION ; Socs3
    Abstract: Suppressor of cytokine signalling (SOCS) proteins are critical attenuators of cytokine-mediated signalling in diverse tissues. To determine the importance of Socs3 in mammary development, we generated mice in which Socs3 was deleted in mammary epithelial cells. No overt phenotype was evident during pregnancy and lactation, indicating that Socs3 is not a key physiological regulator of prolactin signalling. However, Socs3-deficient mammary glands exhibited a profound increase in epithelial apoptosis and tissue remodelling, resulting in precocious involution. This phenotype was accompanied by augmented Stat3 activation and a marked increase in the level of c-myc. Moreover, induction of c-myc before weaning using an inducible transgenic model recapitulated the Socs3 phenotype, and elevated expression of likely c-myc target genes, E2F-1, Bax and p53, was observed. Our data establish Socs3 as a critical attenuator of pro-apoptotic pathways that act in the developing mammary gland and provide evidence that c-myc regulates apoptosis during involution
    Type of Publication: Journal article published
    PubMed ID: 17139252
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  • 5
    Keywords: SPECTRA ; human ; MODEL ; PATHWAY ; PATHWAYS ; SITE ; SITES ; CLONING ; DISTINCT ; GENOME ; PROTEIN ; RNA ; transcription ; COMPLEX ; COMPLEXES ; IDENTIFICATION ; SUBUNIT ; PROMOTER ; NUMBER ; PROMOTERS ; HUMAN GENOME ; RECRUITMENT ; RNA-POLYMERASE-I ; INITIATION ; analysis ; PROFILES ; function ; SPECTRUM ; SET ; ChIP-on-chip ; CHROMOSOME-22 ; ENHANCERS ; HISTONE ACETYLASE COMPLEX ; TBP ; transcription factor profiling
    Abstract: Our current knowledge of the general factor requirement in transcription by the three mammalian RNA polymerases is based on a small number of model promoters. Here, we present a comprehensive chromatin immunoprecipitation (ChIP)-on-chip analysis for 28 transcription factors on a large set of known and novel TATA-binding protein (TBP)-binding sites experimentally identified via ChIP cloning. A large fraction of identified TBP-binding sites is located in introns or lacks a gene/mRNA annotation and is found to direct transcription. Integrated analysis of the ChIP-on-chip data and functional studies revealed that TAF12 hitherto regarded as RNA polymerase II (RNAP II)-specific was found to be also involved in RNAP I transcription. Distinct profiles for general transcription factors and TAF-containing complexes were uncovered for RNAP II promoters located in CpG and non-CpG islands suggesting distinct transcription initiation pathways. Our study broadens the spectrum of general transcription factor function and uncovers a plethora of novel, functional TBP-binding sites in the human genome
    Type of Publication: Journal article published
    PubMed ID: 17268553
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  • 6
    Keywords: PEPTIDE ; CELL ; Germany ; IN-VIVO ; SYSTEM ; DISEASE ; RISK ; SITE ; PROTEIN ; MECHANISM ; IMPACT ; BIOLOGY ; SEQUENCE ; CLEAVAGE ; MUTATION ; etiology ; MUTATIONS ; BETA ; PEPTIDES ; FUNCTIONAL DOMAINS ; AD ; ECTODOMAIN ; DIMERIZATION ; CYTOTOXICITY ; SENILE PLAQUES ; E-cadherin ; MOLECULAR-MECHANISM ; RESIDUES ; INCREASE ; LEADS ; SUBSTRATE ; LEVEL ; RISK-FACTOR ; PRECURSOR ; amyloid A beta ; amyloid precursor protein (APP) ; BETA-PROTEIN ; DIMERIZATION MOTIF ; FAMILIAL ALZHEIMERS-DISEASE ; GAMMA-SECRETASE ACTIVITY ; GxxxG ; HELIX ASSOCIATION ; INTRAMEMBRANE CLEAVAGE ; secretase
    Abstract: Processing of the amyloid precursor protein (APP) by beta- and gamma-secretases leads to the generation of amyloid-beta (A beta) peptides with varying lengths. Particularly A beta 42 contributes to cytotoxicity and amyloid accumulation in Alzheimer's disease ( AD). However, the precise molecular mechanism of A beta 42 generation has remained unclear. Here, we show that an amino-acid motif GxxxG within the APP transmembrane sequence (TMS) has regulatory impact on the Ab species produced. In a neuronal cell system, mutations of glycine residues G29 and G33 of the GxxxG motif gradually attenuate the TMS dimerization strength, specifically reduce the formation of A beta 42, leave the level of A beta 40 unaffected, but increase A beta 38 and shorter Ab species. We show that glycine residues G29 and G33 are part of a dimerization site within the TMS, but do not impair oligomerization of the APP ectodomain. We conclude that gamma-secretase cleavages of APP are intimately linked to the dimerization strength of the substrate TMS. The results demonstrate that dimerization of APP TMS is a risk factor for AD due to facilitating A beta 42 production
    Type of Publication: Journal article published
    PubMed ID: 17332749
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  • 7
    Keywords: CELL ; Germany ; SYSTEM ; SYSTEMS ; GENE ; PROTEIN ; PROTEINS ; COMPLEX ; DOMAIN ; MEMBERS ; cell culture ; FORM ; ESCHERICHIA-COLI ; DEGRADATION ; TRANSLOCATION ; specificity ; SUBSTRATE ; VIRULENCE ; CHAPERONE ; AAA plus protein ; CENTRAL PORE ; EDWARDSIELLA-TARDA ; ESCRT-III ; III SECRETION ; PATHOGENICITY ISLAND ; protein secretion
    Abstract: The recently identified type VI secretion systems (T6SS) have a crucial function in the virulence of various proteo-bacteria, including the human pathogen Vibrio cholerae. T6SS are encoded by a conserved gene cluster comprising approximately 15 open reading frames, mediating the appearance of Hcp and VgrG proteins in cell culture supernatants. Here, we analysed the function of the V. cholerae T6SS member ClpV, a specialized AAA+ protein. ClpV is crucial for a functional T6SS and interacts through its N-terminal domain with the VipA/VipB complex that is composed of two conserved and essential members of T6SS. Transferring ClpV substrate specificity to a distinct AAA+ protein involved in proteolysis caused degradation of VipA but not Hcp or VgrG2, suggesting that VipA rather than Hcp/VgrG2 functions as a primary ClpV substrate. Strikingly, VipA/VipB form tubular, cogwheel-like structures that are converted by a threading activity of ClpV into small complexes. ClpV-mediated remodelling of VipA/VipB tubules represents a crucial step in T6S, illuminating an unexpected role of an ATPase component in protein secretion
    Type of Publication: Journal article published
    PubMed ID: 19131969
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  • 8
    Keywords: CELLS ; INHIBITOR ; CELL ; MODEL ; SUPPORT ; PROTEIN ; RNA ; transcription ; NF-KAPPA-B ; ACTIVATION ; COMPLEX ; COMPLEXES ; BIOLOGY ; FORM ; PROMOTER ; POSTTRANSLATIONAL MODIFICATIONS ; RNA-POLYMERASE-II ; INHIBITORS ; molecular biology ; HIV ; LONG ; HIV-1 TAT ; p300 ; USA ; P-TEFB ; BINDING DOMAIN ; POLYMERASE ; 7SK SNRNP ; BROMODOMAIN PROTEIN BRD4 ; DEPENDENT TRANSCRIPTION ; HEXIM1 ; transcription elongation ; TRANSCRIPTIONAL ELONGATION
    Abstract: The elongation competence of the RNA polymerase II complex is critically dependent on the positive transcription elongation factor b (P-TEFb). P-TEFb exists in two forms in cells, an active form composed of cyclin T1 and CDK9 and an inactive form, in which cyclin T1/CDK9 is sequestered by Hexim1 and 7SK snRNA. Here, we report that partitioning of active and inactive P-TEFb is regulated by acetylation of cyclin T1. Cyclin T1 acetylation triggers dissociation of Hexim1 and 7SK snRNA from cyclin T1/CDK9 and activates the transcriptional activity of P-TEFb. This activation is lost in P-TEFb complexes containing cyclin T1 that can no longer be acetylated. An acetylation-deficient cyclin T1 mutant dominantly suppresses NF-kappa B-mediated activation of the interleukin-8 promoter but continues to synergize normally with the HIV Tat protein to transactivate the HIV long terminal repeat. These findings support the model that acetylation of cyclin T1 serves as a physiological switch that liberates P-TEFb from its endogenous inhibitors Hexim1 and 7SK snRNA, but is not required for the cooperative action with HIV Tat. The EMBO Journal (2009) 28, 1407-1417. doi:10.1038/emboj.2009.99; Published online 23 April 2009
    Type of Publication: Journal article published
    PubMed ID: 19387490
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  • 9
    Keywords: AFFINITY, ARCHITECTURE, ASSOCIATION, BINDING, BIOLOGY, CELL, CELLS, CHROMATIN, DIFFUSION, DNA, DYNAM
    Abstract: The nucleus of eukaryotes is organized into functional compartments, the two most prominent being heterochromatin and nucleoli. These structures are highly enriched in DNA, proteins or RNA, and thus thought to be crowded. In vitro, molecular crowding induces volume exclusion, hinders diffusion and enhances association, but whether these effects are relevant in vivo remains unclear. Here, we establish that volume exclusion and diffusive hindrance occur in dense nuclear compartments by probing the diffusive behaviour of inert fluorescent tracers in living cells. We also demonstrate that chromatin-interacting proteins remain transiently trapped in heterochromatin due to crowding induced enhanced affinity. The kinetic signatures of these crowding consequences allow us to derive a fractal model of chromatin organization, which explains why the dynamics of soluble nuclear proteins are affected independently of their size. This model further shows that the fractal architecture differs between heterochromatin and euchromatin, and predicts that chromatin proteins use different target-search strategies in the two compartments. We propose that fractal crowding is a fundamental principle of nuclear organization, particularly of heterochromatin maintenance.
    Type of Publication: Journal article published
    PubMed ID: 19927119
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  • 10
    Abstract: The human immunodeficiency virus (HIV) Tat protein plays an essential role in promoting efficient transcriptional elongation of viral transcripts. We report that the transcriptional co-activator PCAF and Tat interact and synergize to activate the HIV promoter. The binding of Tat and PCAF in vitro and in vivo is dependent on the acetylated state of Lys50 of Tat and on the PCAF bromodomain. Structural analysis of the acetylated Tat peptide bound to the PCAF bromodomain defined amino acids Y47 and R53 in Tat and V763, Y802, and Y809 in PCAF as critical interaction points between the two proteins. Mutation of each of these residues in either Tat or PCAF inhibited in a cumulative manner the Tat-PCAF interaction in vitro and in vivo, and abrogated the synergistic activation of the HIV promoter by both proteins. These observations demonstrate that acetylation of Tat establishes a novel protein-protein interaction domain at the surface of Tat that is necessary for the transcriptional activation of the HIV promoter.
    Type of Publication: Journal article published
    PubMed ID: 12032084
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