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‣ Biophysical Characterization of Interactions Involving Importin-α during Nuclear Import

Catimel, Bruno; Teh, Trazel; Fontes, Marcos R. M.; Jennings, Ian G.; Jans, David A.; Howlett, Geoffrey J.; Nice, Edouard C.; Kobe, Bostjan
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 34189-34198
Português
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Proteins containing the classical nuclear localization sequences (NLSs) are imported into the nucleus by the importin-α/β heterodimer. Importin-α contains the NLS binding site, whereas importin-β mediates the translocation through the nuclear pore. We characterized the interactions involving importin-α during nuclear import using a combination of biophysical techniques (biosensor, crystallography, sedimentation equilibrium, electrophoresis, and circular dichroism). Importin-α is shown to exist in a monomeric autoinhibited state (association with NLSs undetectable by biosensor). Association with importin-β (stoichiometry, 1:1; K D = 1.1 × 10 -8 M) increases the affinity for NLSs; the importin-α/β complex binds representative monopartite NLS (simian virus 40 large T-antigen) and bipartite NLS (nucleoplasmin) with affinities (K D = 3.5 × 10 -8 M and 4.8 × 10 -8 M, respectively) comparable with those of a truncated importin-α lacking the autoinhibitory domain (T-antigen NLS, K D = 1.7 × 10 -8 M; nucleoplasmin NLS, K D = 1.4 × 10 -8 M). The autoinhibitory domain (as a separate peptide) binds the truncated importin-α, and the crystal structure of the complex resembles the structure of full-length importin-α. Our results support the model of regulation of nuclear import mediated by the intrasteric autoregulatory sequence of importin-α and provide a quantitative description of the binding and regulatory steps during nuclear import.

‣ Active Nucleocytoplasmic Shuttling Required for Function and Regulation of Stress-Activated Kinase Spc1/StyI in Fission Yeast

Gaits, Frédérique; Russell, Paul
Fonte: The American Society for Cell Biology Publicador: The American Society for Cell Biology
Tipo: Artigo de Revista Científica
Publicado em /05/1999 Português
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48.126704%
Transcriptional induction of many stress-response genes is dependent on stress-induced nuclear accumulation of stress-activated protein kinases (SAPKs). In the fission yeast Schizosaccharomyces pombe, nuclear accumulation of the SAPK Spc1 (also known as StyI) requires activating phosphorylation catalyzed by the SAPK kinase Wis1; however, it is unknown whether the localization of Spc1 is regulated by nuclear transport factors. Herein are reported studies that show that Spc1 localization is regulated by active transport mechanisms during osmotic stress. Nuclear import of Spc1 requires Pim1, a homologue of the guanine nucleotide exchange factor RCC1 that is essential for nucleocytoplasmic shuttling of proteins. Nuclear export of Spc1 is regulated by the export factor Crm1. An Spc1–Crm1 complex forms as Spc1 is exported from the nucleus. Wis1 and the tyrosine phosphatases Pyp1 and Pyp2 that inactivate Spc1 are excluded from the nucleus by a Crm1-independent mechanism; hence the nuclear import of Spc1 leads to transient isolation from its regulatory proteins. Thus, active nucleocytoplasmic shuttling is required for both the function and regulation of Spc1 during the osmotic shock response.

‣ Active nuclear import of human immunodeficiency virus type 1 preintegration complexes.

Bukrinsky, M I; Sharova, N; Dempsey, M P; Stanwick, T L; Bukrinskaya, A G; Haggerty, S; Stevenson, M
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/07/1992 Português
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After cell infection by the human immunodeficiency virus type 1 (HIV-1), nascent viral DNA in the form of a high molecular weight nucleoprotein preintegration complex must be transported to the nucleus of the host cell prior to integration of viral DNA with the host genome. The mechanism used by retroviruses for nuclear targeting of preintegration complexes and dependence on cell division has not been established. Our studies show that, after infection, the preintegration complex of HIV-1 was rapidly transported to the nucleus of the host cell by a process that required ATP but was independent of cell division. Functional HIV-1 integrase, an essential component of the preintegration complex, was not required for nuclear import of these complexes. The ability of HIV-1 to use host cell active transport processes for nuclear import of the viral preintegration complex obviates the requirement for host cell division in establishment of the integrated provirus. These findings are pertinent to our understanding of early events in the life cycle of HIV-1 and to the mode of HIV-1 replication in terminally differentiated nondividing cells such as macrophages (monocytes, tissue macrophages, follicular dendritic cells, and microglial cells).

‣ Active transport of messenger ribonucleoprotein particles in a reconstituted cell-free system.

French, B T; Schumm, D E; Webb, T E
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1987 Português
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48.209766%
The ability of a reconstituted cell-free system to transport mRNA as a ribonucleoprotein particle has been examined. Poly(A) messenger ribonucleoproteins (mRNPs), UV cross-linked after release from isolated liver nuclei in a cell-free system, exhibited a buoyant density of 1.33 g/cm3 in cesium sulfate and 1.47 g/cm3 in cesium chloride, values identical to those of poly(A) mRNP isolated directly from liver polysomes. Furthermore, the in vivo and in vitro transported mRNP showed a similar degree of resistance to RNase digestion and had sedimentation coefficients approximately 2.5 times that of the isolated mRNA. Release of both total mRNA and alpha 2 mu-globulin mRNA was proportional to the concentration of a specific cytoplasmic protein. Removal of the transport proteins from the cytosol with streptomycin sulfate provided a basal system incapable of supporting the active transport of alpha 2 mu-globulin mRNA. Hybridization of released RNA with a recombinant probe specific for intron 6 of alpha 2 mu-globulin showed that intron sequences were retained within the nucleus under optimal alpha 2 mu-globulin mRNA transport conditions and that the transported alpha 2 mu-globulin mRNA was of mature size.

‣ Apoptosis leads to a degradation of vital components of active nuclear transport and a dissociation of the nuclear lamina

Kramer, A.; Liashkovich, I.; Oberleithner, H.; Ludwig, S.; Mazur, I.; Shahin, V.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
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38.149604%
Apoptosis, a physiologically critical process, is characterized by a destruction of the cell after sequential degradation of key cellular components. Here, we set out to explore the fate of the physiologically indispensable nuclear envelope (NE) in this process. The NE mediates the critical nucleocytoplasmic transport through nuclear pore complexes (NPCs). In addition, the NE is involved in gene expression and contributes significantly to the overall structure and mechanical stability of the cell nucleus through the nuclear lamina, which underlies the entire nucleoplasmic face of the NE and thereby interconnects the NPCs, the NE, and the genomic material. Using the nano-imaging and mechanical probing approach atomic force microscopy (AFM) and biochemical methods, we unveiled the fate of the NE during apoptosis. The doomed NE sustains a degradation of both the mediators of the critical selective nucleocytoplasmic transport, namely NPC cytoplasmic filaments and basket, and the nuclear lamina. These observations are paralleled by marked softening and destabilization of the NE and the detection of vesicle-like nuclear fragments. We conclude that destruction of the cell nucleus during apoptosis proceeds in a strategic fashion. Degradation of NPC cytoplasmic filaments and basket shuts down the critical selective nucleocytoplasmic cross-talk. Degradation of the nuclear lamina disrupts the pivotal connection between the NE and the chromatin...

‣ Rhinovirus 3C Protease Can Localize in the Nucleus and Alter Active and Passive Nucleocytoplasmic Transport▿

Ghildyal, Reena; Jordan, Benjamin; Li, Dongsheng; Dagher, Hayat; Bardin, Phillip G.; Gern, James E.; Jans, David A.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
Português
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38.50253%
The degradation of nuclear pore components and disruption of nucleocytoplasmic trafficking during rhinovirus infection have been attributed to viral 2A protease. Here we show for the first time that rhinovirus 3C protease may also have a role. Specifically, we show that 3C and its precursor, 3CD, can target green fluorescent protein to the nucleus of living cells, leading to degradation of nuclear pore components, and that incubation with recombinant 3C disrupts active and passive nucleocytoplasmic transport in a semi-intact cell nuclear transport system dependent on 3C protease activity. 3C may thus contribute to host cell shutoff in infected cells by localizing in the nucleus and facilitating nuclear pore breakdown.

‣ Active maintenance of nuclear actin by importin 9 supports transcription

Dopie, Joseph; Skarp, Kari-Pekka; Kaisa Rajakylä, Eeva; Tanhuanpää, Kimmo; Vartiainen, Maria K.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
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38.57757%
Besides its essential and well established role as a component of the cytoskeleton, actin is also present in the cell nucleus, where it has been linked to many processes that control gene expression. For example, nuclear actin regulates the activity of specific transcription factors, associates with all three RNA polymerases, and is a component of many chromatin remodelling complexes. Despite the fact that two export receptors, Crm1 and exportin 6, have been linked to nuclear export of actin, the mechanism by which actin enters the nucleus to elicit these essential functions has not been determined. It is also unclear whether actin is actively exchanged between the nucleus and the cytoplasm, and whether this connection has any functional significance for the cell. By applying a variety of live-cell imaging techniques we revealed that actin constantly shuttles in and out of the nucleus. The fast transport rates, which depend on the availability of actin monomers, suggest an active transport mechanism in both directions. Importantly, we identified importin 9 as the nuclear import factor for actin. Furthermore, our RNAi experiments showed that the active maintenance of nuclear actin levels by importin 9 is required for maximal transcriptional activity. Measurements of nuclear export rates and depletion studies also clarified that nuclear export of actin is mediated by exportin 6...

‣ Mechanical Activation of Cells Induces Chromatin Remodeling Preceding MKL Nuclear Transport

Iyer, K. Venkatesan; Pulford, S.; Mogilner, A.; Shivashankar, G.V.
Fonte: The Biophysical Society Publicador: The Biophysical Society
Tipo: Artigo de Revista Científica
Publicado em 03/10/2012 Português
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For cells to adapt to different tissues and changes in tissue mechanics, they must be able to respond to mechanical cues by changing their gene expression patterns. Biochemical signaling pathways for these responses have been elucidated, and recent evidence points to the involvement of force-induced deformation of the nucleus. However, it is still unclear how physical cues received at the plasma membrane (PM) spatiotemporally integrate to the functional chromatin organization of the cell nucleus. To investigate this issue, we applied mechanical forces through magnetic particles adhered to the PM of single cells and mapped the accompanying changes in actin polymerization, nuclear morphology, chromatin remodeling, and nuclear transport of soluble signaling intermediates using high-resolution fluorescence anisotropy imaging. Using this approach, we show the timescales associated with force-induced polymerization of actin and changes in the F/G actin ratio resulting in nuclear translocation of the G-actin-associated transcriptional cofactor, megakaryoblastic acute leukemia factor-1 (MKL). Further, this method of measuring nuclear organization at high spatiotemporal resolution with simultaneous force application revealed the physical propagation of forces to the nucleus...

‣ Active nuclear import and cytoplasmic retention of activation-induced deaminase

Patenaude, Anne-Marie; Orthwein, Alexandre; Hu, Yi; Campo, Vanina A.; Kavli, Bodil; Buschiazzo, Alejandro; Di Noia, Javier M.
Fonte: Université de Montréal Publicador: Université de Montréal
Tipo: Artigo de Revista Científica
Português
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The enzyme activation-induced deaminase (AID) triggers antibody diversification in B cells by catalyzing deamination and consequently mutation of immunoglobulin genes. To minimize off-target deamination, AID is restrained by several regulatory mechanisms including nuclear exclusion, thought to be mediated exclusively by active nuclear export. Here we identify two other mechanisms involved in controlling AID subcellular localization. AID is unable to passively diffuse into the nucleus, despite its small size, and its nuclear entry requires active import mediated by a conformational nuclear localization signal. We also identify in its C terminus a determinant for AID cytoplasmic retention, which hampers diffusion to the nucleus, competes with nuclear import and is crucial for maintaining the predominantly cytoplasmic localization of AID in steady-state conditions. Blocking nuclear import alters the balance between these processes in favor of cytoplasmic retention, resulting in reduced isotype class switching.; This work was supported by the Canadian Institutes of Health Research (MOP 84543) and a Canada Research Chair (to J.M.D.). A.O. was supported by a fellowship from the Canadian Institutes of Health Research Cancer Training Program at the IRCM. V.A.C. was supported in part by a Michel Saucier fellowship from the Louis-Pasteur Canadian Fund through the University of Montreal.

‣ Defining the role for XAP2 in stabilization of the dioxin receptor

Lees, M.; Peet, D.; Whitelaw, M.
Fonte: Amer Soc Biochemistry Molecular Biology Inc Publicador: Amer Soc Biochemistry Molecular Biology Inc
Tipo: Artigo de Revista Científica
Publicado em //2003 Português
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The dioxin receptor (DR) is a ligand-activated transcription factor that is activated upon binding of dioxins or structurally related forms of xenobiotics. Upon binding ligand the DR translocates from the cytoplasm to the nucleus where it complexes with the partner protein Arnt to form a DNA binding heterodimer, which activates transcription of target genes involved in xenobiotic metabolism. Latency of the DR signaling pathway is maintained by association of the DR with a number of molecular chaperones including the 90-kDa heat shock protein (hsp90), the hepatitis B virus X-associated protein (XAP2), and the 23-kDa heat shock protein (p23). Here we investigated the role of XAP2 in DR signaling and demonstrated that reduced levels of XAP2 labilize the DR, arguing for a function of XAP2 beyond its reported role as a cytoplasmic retention factor. In addition, we showed that a constitutively nuclear DR is degraded in the nucleus and does not require nuclear export for efficient degradation. We also provided evidence implicating the ubiquitin ligase protein C-terminal hsp70-interacting protein (CHIP) in the degradation of the DR, and we demonstrated that this degradation can be overcome by overexpression of XAP2. XAP2 protection of CHIP-mediated degradation is dependent on the tetratricopeptide repeat domain of XAP2 and suggests a mechanism whereby competition for the C-terminal tetratricopeptide repeat acceptor site of hsp90 guides the protein triage decision...

‣ Contribution of the Per/Arnt/Sim (PAS) domains to DNA binding by the basic helix-loop-helix PAS transcriptional regulators

Chapman-Smith, A.; Lutwyche, J.; Whitelaw, M.
Fonte: Amer Soc Biochemistry Molecular Biology Inc Publicador: Amer Soc Biochemistry Molecular Biology Inc
Tipo: Artigo de Revista Científica
Publicado em //2004 Português
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The basic helix-loop-helix (bHLH) PAS transcriptional regulators control critical developmental and metabolic processes, including transcriptional responses to stimuli such as hypoxia and environmental pollutants, mediated respectively by hypoxia inducible factors (HIF-α) and the dioxin (aryl hydrocarbon) receptor (DR). The bHLH proteins contain a basic DNA binding sequence adjacent to a helix-loop-helix dimerization domain. Dimerization among bHLH.PAS proteins is additionally regulated by the PAS region, which controls the specificity of partner choice such that HIF-α and DR must dimerize with the aryl hydrocarbon nuclear translocator (Arnt) to form functional DNA binding complexes. Here, we have analyzed purified bacterially expressed proteins encompassing the N-terminal bHLH and bHLH.PAS regions of Arnt, DR, and HIF-1α and evaluated the contribution of the PAS domains to DNA binding in vitro. Recovery of functional DNA binding proteins from bacteria was dramatically enhanced by coexpression of the bHLH.PAS regions of DR or HIF-1α with the corresponding region of Arnt. Formation of stable protein-DNA complexes by DR/Arnt and HIF-1α /Arnt heterodimers with their cognate DNA sequences required the PAS A domains and exhibited KD values of 0.4 nM and ~50 nM...

‣ A family of RS domain proteins with novel subcellular localization and trafficking

Kavanagh, S.; Schulz, T.; Davey, P.; Claudianos, C.; Russell, C.; Rathjen, P.
Fonte: Oxford Univ Press Publicador: Oxford Univ Press
Tipo: Artigo de Revista Científica
Publicado em //2005 Português
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We report the sequence, conservation and cell biology of a novel protein, Psc1, which is expressed and regulated within the embryonic pluripotent cell population of the mouse. The Psc1 sequence includes an RS domain and an RNA recognition motif (RRM), and a sequential arrangement of protein motifs that has not been demonstrated for other RS domain proteins. This arrangement was conserved in a second mouse protein (BAC34721. The identification of Psc1 and BAC34721homologues in vertebrates and related proteins, more widely throughout evolution, defines a new family of RS domain proteins termed acidic rich RS (ARRS) domain proteins. Psc1 incorporated into the nuclear speckles, but demonstrated novel aspects of subcellular distribution including localization to speckles proximal to the nuclear periphery and localization to punctate structures in the cytoplasm termed cytospeckles. Integration of Psc1 into cytospeckles was dependent on the RRM. Cytospeckles were dynamic within the cytoplasm and appeared to traffic into the nucleus. These observations suggest a novel role in RNA metabolism for ARRS proteins.

‣ Dengue virus (DV) replication in monocyte-derived macrophages is not affected by tumor necrosis factor alpha (TNF-alpha), and DV infection induces altered responsiveness to TNF-alpha stimulation

Wati, S.; Li, P.; Burrell, C.; Carr, J.
Fonte: Amer Soc Microbiology Publicador: Amer Soc Microbiology
Tipo: Artigo de Revista Científica
Publicado em //2007 Português
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Tumor necrosis factor alpha (TNF-alpha) is believed to play a significant role in the pathogenesis of dengue virus (DV) infection, with elevated levels of TNF-alpha in the sera of DV-infected patients paralleling the severity of disease and TNF-alpha release being coincident with the peak of DV production from infected monocyte-derived macrophages (MDM) in vitro. Since macrophages are a primary cell target in vivo for DV infection, we investigated the potential antiviral role of TNF-alpha in regulating DV replication in MDM. While pretreatment of MDM with TNF-alpha had a minor inhibitory effect, addition of TNF-alpha to MDM with established DV infection had no effect on DV replication as measured by DV RNA levels or progeny virus production. Blocking endogenous TNF-alpha using short interfering RNA or inhibitory TNF-alpha antibodies also had no effect on infectious DV production or viral RNA synthesis. Together, these results demonstrate that DV replication in MDM is not affected by TNF-alpha. Additionally, normal cellular TNF-alpha signaling, measured by quantitation of TNF-alpha-induced stimulation of transcription from an NF-kappaB-responsive reporter plasmid or NF-kappaB protein nuclear translocation, was blocked in DV-infected MDM and Huh7 cells. Thus...

‣ Molecular pathology of expanded polyalanine tract mutations in the Aristaless-related homeobox gene

Shoubridge, C.; Cloosterman, D.; Parkinson-Lawrence, E.; Brooks, D.; Gecz, J.
Fonte: Academic Press Inc Elsevier Science Publicador: Academic Press Inc Elsevier Science
Tipo: Artigo de Revista Científica
Publicado em //2007 Português
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The Aristaless-related homeobox gene (ARX) is one of the major genes causing X-linked mental retardation. We have been interested in the pathogenic mechanism of expanded polyalanine tract mutations in ARX. We showed that the c.304ins(GCG)7 mutation causing an increase from 16 to 23 alanines increased the propensity of ARX protein aggregation and a shift from nuclear to cytoplasmic localization. We proposed that mislocalization of ARX via cytoplasmic aggregation and subsequent degradation leads to a partial loss of function, contributing to the pathogenesis. We identified importin 13 (IPO13), a mediator of nuclear import for a variety of proteins, as a novel ARX interacting protein. We predicted that the transport of ARX by IPO13 from the cytoplasm to the nucleus might be disrupted by expanded polyalanine tract mutations, but our data showed that in both yeast and mammalian cells these mutant ARX proteins were still able to interact with IPO13. We established the nuclear localization regions of the ARX homeodomain that were required for the interaction with IPO13 and correct localization of the full-length ARX transcription factor to the nucleus.; http://www.elsevier.com/wps/find/journaldescription.cws_home/622838/description#description; Cheryl Shoubridge...

‣ Ribosomal stress induces processing of Mybbp1a and its translocation from the nucleolus to the nucleoplasm

Yamauchi, T.; Keough, R.; Gonda, T.; Ishii, S.
Fonte: Blackwell Science Ltd Publicador: Blackwell Science Ltd
Tipo: Artigo de Revista Científica
Publicado em //2008 Português
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58.10384%
Myb-binding protein 1a (Mybbp1a) was originally identified as a c-myb proto-oncogene product (c-Myb)-interacting protein, and also binds to various other transcription factors. The 160-kDa Mybbp1a protein (p160(MBP)) is ubiquitously expressed and is post-translationally processed in some types of cells to generate an amino-terminal 67 kDa fragment (p67(MBP)). Despite its interaction with various transcription factors, Mybbp1a is localized predominantly, but not exclusively, in nucleoli. Here, we have purified the two Mybbp1a-containing complexes. The smaller complex contained p67(MBP) and p140(MBP), which lacked the C-terminal region of p160(MBP) containing the nucleolar localization sequences. The larger complex contained the intact p160(MBP) and various ribosomal subunits. Treatment of cells with actinomycin D (ActD), cisplatin or UV, all of which inhibit ribosome biogenesis, induced processing of p160(MBP) into p140(MBP) and p67(MBP). ActD, cisplatin and UV also induced a translocation of Mybbp1a from the nucleolus to the nucleoplasm. Both small and large Mybbp1a complexes contained nucleophosmin and nucleolin. In contrast, nucleostemin was detected only in the large complex, while the cell cycle-regulated protein EBP1 was only in the small complex. These results suggest that Mybbp1a may connect the ribosome biogenesis and the Myb-dependent transcription...

‣ ARX homeodomain mutations abolish DNA binding and lead to a loss of transcriptional repression

Shoubridge, C.; Tan, M.; Seiboth, G.; Gecz, J.
Fonte: Oxford Univ Press Publicador: Oxford Univ Press
Tipo: Artigo de Revista Científica
Publicado em //2012 Português
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Mutations in the Aristaless-related homeobox (ARX) gene are one of the most frequent causes of X-linked intellectual disability (ID). Several missense mutations, clustered in the paired-type homeodomain of ARX, have been identified. These mutations lead to a range of phenotypes from X-linked lissencephaly with abnormal genitalia to seizure disorders without brain malformations including X-linked infantile spasms with ID (ISSX-ID) and X-linked myoclonic epilepsy with spasticity and ID (XMESID). The effect of these mutations on the DNA-binding and transcriptional activity has been evaluated. Luciferase reporter assays showed altered repression activity of ARX by all mutations, causing brain malformations and ISSX-ID phenotypes, but not by the P353L mutation implicated in a milder phenotype of XMESID. Similarly, transient overexpression of wild-type ARX repressed endogenous expression of known ARX targets, LMO1 and SHOX2, when measured by real-time quantitative polymerase chain reaction. Overall, the molecular consequence of missense mutations correlated well with the severity of the clinical phenotype. In all mutations tested, except P353L, the DNA binding was abolished. Electrophoretic mobility shift assay results were validated using chromatin immunoprecipitation following overexpression of normal and selected missense mutations. Unlike wild-type ARX and clinically less severe mutations...

‣ Labile zinc and zinc transporter ZnT4 in mast cell granules: Role in regulation of caspase activation and NF-KB translocation

Ho, L.; Ruffin, R.; Murgia, C.; Li, L.; Krilis, S.; Zalewski, P.
Fonte: Amer Assoc Immunologists Publicador: Amer Assoc Immunologists
Tipo: Artigo de Revista Científica
Publicado em //2004 Português
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The granules of mast cells and other inflammatory cells are known to be rich in zinc (Zn), a potent caspase inhibitor. The functions of granular Zn, its mechanism of uptake, and its relationship to caspase activation in apoptosis are unclear. The granules of a variety of mast cell types fluoresced intensely with the Zn-specific fluorophore Zinquin, and fluorescence was quenched by functional depletion of Zn using a membrane-permeable Zn chelator N, N, N', N'-tetrakis (2-pyridyl-methyl)ethylenediamine (TPEN). Zn levels were also depleted by various mast cell activators, including IgE/anti-IgE, and Zn was rapidly replenished during subsequent culture, suggesting an active uptake mechanism. In support of the latter, mast cells contained high levels of the vesicular Zn transporter ZnT4, especially in the more apical granules. Immunofluorescence and immunogold labeling studies revealed significant pools of procaspase-3 and -4 in mast cell granules and their release during degranulation. Functional depletion of Zn by chelation with TPEN, but not by degranulation, resulted in greatly increased susceptibility of mast cells to toxin-induced caspase activation, as detected using a fluorogenic substrate assay. Release of caspases during degranulation was accompanied by a decreased susceptibility to toxins. Zn depletion by chelation...

‣ Quality control of gene expression in the mammalian cell nucleus

Custódio, Noélia Maria Fernandes, 1971-
Fonte: Universidade de Lisboa Publicador: Universidade de Lisboa
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em //2008 Português
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38.264194%
Tese de doutoramento em Ciências Biomédicas (Ciências Morfológicas) apresentada à Universidade de Lisboa através da Faculdade de Medicina, 2008; Protein-encoding genes are transcribed in the nucleus by RNA polymerase II as precursor RNAs that undergo extensive processing before being translocated to the cytoplasm for translation by the ribosomes. This spatial and temporal separation between RNA and protein synthesis offers an immense opportunity for regulation and quality control.When this study was initiated it was known from biochemical studies that human ß-globin (HBB) transcripts with mutations that impaired splicing or 3' end formation were unable to be exported to the cytoplasm, a phenotype identical to that seen in ß-thalassemia patients harbouring similar mutations (Antoniou et al., 1998). This result was consistent with the retention in the nucleus of incorrectly processed transcripts. Based on this initial observation we hypothesised the existence of a quality control mechanism to retain the incorrectly processed transcripts in the nucleus and proposed to elucidate the mechanism responsible for the observed retention. The first goal of this work was to determine the intranuclear localisation of the retained transcripts. To address this we used as a model system murine erythroleukemia (MEL) cells stably transfected with either wild-type or processing mutant HBB genes. The experimental approach was based on the direct visualisation of both normal and defective HBB transcripts using fluorescence in situ hybridisation and confocal microscopy. Nuclear transcripts of both wild-type and mutant HBB genes were detected only as intranuclear foci co-localising with the template gene locus. To determine the kinetics of transcript release from the site of transcription the cells were treated with the transcriptional inhibitors actinomycin D...

‣ Poly(A)+ RNAs roam the cell nucleus and pass through speckle domains in transcriptionally active and inactive cells

Molenaar, Chris; Abdulle, Abadir; Gena, Aarti; Tanke, Hans J.; Dirks, Roeland W.
Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 26/04/2004 Português
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48.37949%
Many of the protein factors that play a role in nuclear export of mRNAs have been identified, but still little is known about how mRNAs are transported through the cell nucleus and which nuclear compartments are involved in mRNA transport. Using fluorescent 2'O-methyl oligoribonucleotide probes, we investigated the mobility of poly(A)+ RNA in the nucleoplasm and in nuclear speckles of U2OS cells. Quantitative analysis of diffusion using photobleaching techniques revealed that the majority of poly(A)+ RNA move throughout the nucleus, including in and out of speckles (also called SC-35 domains), which are enriched for splicing factors. Interestingly, in the presence of the transcription inhibitor 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole, the association of poly(A)+ RNA with speckles remained dynamic. Our results show that RNA movement is energy dependent and that the proportion of nuclear poly(A)+ RNA that resides in speckles is a dynamic population that transiently interacts with speckles independent of the transcriptional status of the cell. Rather than the poly(A)+ RNA within speckles serving a stable structural role, our findings support the suggestion of a more active role of these regions in nuclear RNA metabolism and/or transport.

‣ Efficient active transport of gene nanocarriers to the cell nucleus

Suh, Junghae; Wirtz, Denis; Hanes, Justin
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
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The intracellular transport of therapeutic gene carriers is poorly understood, limiting the rational design of efficient new vectors. We used live-cell real-time multiple particle tracking to quantify the intracellular transport of hundreds of individual nonviral DNA nanocarriers with 5-nm and 33-ms resolution. Unexpected parallels between several of nature's most efficient DNA viruses and nonviral polyethylenimine/DNA nanocomplexes were revealed to include motor protein-driven transport through the cytoplasm toward the nucleus on microtubules. Active gene carrier transport led to efficient perinuclear accumulation within minutes. The results provide direct evidence to dispute the common belief that the efficiency of nonviral gene carriers is dramatically reduced because of the need for their relatively slow random diffusion through the cell cytoplasm to the nucleus and, instead, focuses the attention of rational carrier design on overcoming barriers downstream of perinuclear accumulation.