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1.
Science ; 382(6676): 1270-1276, 2023 12 15.
Article in English | MEDLINE | ID: mdl-38096385

ABSTRACT

Current HIV vaccines designed to stimulate CD8+ T cells have failed to induce immunologic control upon infection. The functions of vaccine-induced HIV-specific CD8+ T cells were investigated here in detail. Cytotoxic capacity was significantly lower than in HIV controllers and was not a consequence of low frequency or unaccumulated functional cytotoxic proteins. Low cytotoxic capacity was attributable to impaired degranulation in response to the low antigen levels present on HIV-infected targets. The vaccine-induced T cell receptor (TCR) repertoire was polyclonal and transduction of these TCRs conferred the same reduced functions. These results define a mechanism accounting for poor antiviral activity induced by these vaccines and suggest that an effective CD8+ T cell response may require a vaccination strategy that drives further TCR clonal selection.


Subject(s)
AIDS Vaccines , Cell Degranulation , Cytotoxicity, Immunologic , HIV Infections , T-Lymphocytes, Cytotoxic , Humans , AIDS Vaccines/immunology , Clone Cells , HIV Infections/prevention & control , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes, Cytotoxic/immunology , Cell Degranulation/immunology
2.
Cell Death Discov ; 5: 101, 2019.
Article in English | MEDLINE | ID: mdl-31231549

ABSTRACT

The SARS (severe acute respiratory syndrome) outbreak was caused by a coronavirus (CoV) named the SARS-CoV. SARS pathology is propagated both by direct cytotoxic effects of the virus and aberrant activation of the innate immune response. Here, we identify several mechanisms by which a SARS-CoV open reading frame (ORF) activates intracellular stress pathways and targets the innate immune response. We show that ORF8b forms insoluble intracellular aggregates dependent on a valine at residue 77. Aggregated ORF8b induces endoplasmic reticulum (ER) stress, lysosomal damage, and subsequent activation of the master regulator of the autophagy and lysosome machinery, Transcription factor EB (TFEB). ORF8b causes cell death in epithelial cells, which is partially rescued by reducing its ability to aggregate. In macrophages, ORF8b robustly activates the NLRP3 inflammasome by providing a potent signal 2 required for activation. Mechanistically, ORF8b interacts directly with the Leucine Rich Repeat domain of NLRP3 and localizes with NLRP3 and ASC in cytosolic dot-like structures. ORF8b triggers cell death consistent with pyroptotic cell death in macrophages. While in those cells lacking NLRP3 accumulating ORF8b cytosolic aggregates cause ER stress, mitochondrial dysfunction, and caspase-independent cell death.

3.
Mol Cell Biol ; 34(22): 4186-99, 2014 Nov 15.
Article in English | MEDLINE | ID: mdl-25225330

ABSTRACT

Both chemotaxis and phagocytosis depend upon actin-driven cell protrusions and cell membrane remodeling. While chemoattractant receptors rely upon canonical G-protein signaling to activate downstream effectors, whether such signaling pathways affect phagocytosis is contentious. Here, we report that Gαi nucleotide exchange and signaling helps macrophages coordinate the recognition, capture, and engulfment of zymosan bioparticles. We show that zymosan exposure recruits F-actin, Gαi proteins, and Elmo1 to phagocytic cups and early phagosomes. Zymosan triggered an increase in intracellular Ca(2+) that was partially sensitive to Gαi nucleotide exchange inhibition and expression of GTP-bound Gαi recruited Elmo1 to the plasma membrane. Reducing GDP-Gαi nucleotide exchange, decreasing Gαi expression, pharmacologically interrupting Gßγ signaling, or reducing Elmo1 expression all impaired phagocytosis, while favoring the duration that Gαi remained GTP bound promoted it. Our studies demonstrate that targeting heterotrimeric G-protein signaling offers opportunities to enhance or retard macrophage engulfment of phagocytic targets such as zymosan.


Subject(s)
GTP-Binding Protein alpha Subunit, Gi2/immunology , Macrophages/cytology , Phagocytosis , Signal Transduction , Zymosan/immunology , Actins/analysis , Actins/immunology , Adaptor Proteins, Signal Transducing/analysis , Adaptor Proteins, Signal Transducing/immunology , Animals , Calcium/analysis , Calcium/immunology , Cell Line , GTP-Binding Protein alpha Subunit, Gi2/analysis , GTP-Binding Protein alpha Subunit, Gi2/genetics , Gene Deletion , Macrophages/immunology , Macrophages/metabolism , Macrophages/microbiology , Mice , Mice, Inbred C57BL , Phagosomes/genetics , Phagosomes/immunology , Phagosomes/microbiology , Phagosomes/ultrastructure , Saccharomyces cerevisiae/immunology
4.
J Immunol ; 193(6): 3080-9, 2014 Sep 15.
Article in English | MEDLINE | ID: mdl-25135833

ABSTRACT

Coronaviruses (CoV) have recently emerged as potentially serious pathogens that can cause significant human morbidity and death. The severe acute respiratory syndrome (SARS)-CoV was identified as the etiologic agent of the 2002-2003 international SARS outbreak. Yet, how SARS evades innate immune responses to cause human disease remains poorly understood. In this study, we show that a protein encoded by SARS-CoV designated as open reading frame-9b (ORF-9b) localizes to mitochondria and causes mitochondrial elongation by triggering ubiquitination and proteasomal degradation of dynamin-like protein 1, a host protein involved in mitochondrial fission. Also, acting on mitochondria, ORF-9b targets the mitochondrial-associated adaptor molecule MAVS signalosome by usurping PCBP2 and the HECT domain E3 ligase AIP4 to trigger the degradation of MAVS, TRAF3, and TRAF 6. This severely limits host cell IFN responses. Reducing either PCBP2 or AIP4 expression substantially reversed the ORF-9b-mediated reduction of MAVS and the suppression of antiviral transcriptional responses. Finally, transient ORF-9b expression led to a strong induction of autophagy in cells. The induction of autophagy depended upon ATG5, a critical autophagy regulator, but the inhibition of MAVS signaling did not. These results indicate that SARS-CoV ORF-9b manipulates host cell mitochondria and mitochondrial function to help evade host innate immunity. This study has uncovered an important clue to the pathogenesis of SARS-CoV infection and illustrates the havoc that a small ORF can cause in cells.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Immunity, Innate/genetics , Mitochondria/immunology , Severe acute respiratory syndrome-related coronavirus/immunology , Viral Proteins/immunology , Autophagy/genetics , Autophagy-Related Protein 5 , Cell Line , Dynamins , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , Green Fluorescent Proteins , HEK293 Cells , Humans , Immune Evasion , Microtubule-Associated Proteins/biosynthesis , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Mitochondria/genetics , Mitochondria/virology , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Open Reading Frames/genetics , Open Reading Frames/immunology , RNA Interference , RNA, Small Interfering , RNA-Binding Proteins/biosynthesis , RNA-Binding Proteins/genetics , Repressor Proteins/biosynthesis , Repressor Proteins/genetics , Severe acute respiratory syndrome-related coronavirus/genetics , Severe Acute Respiratory Syndrome/immunology , Severe Acute Respiratory Syndrome/virology , TNF Receptor-Associated Factor 3/metabolism , TNF Receptor-Associated Factor 6/metabolism , Ubiquitin-Protein Ligases/biosynthesis , Ubiquitin-Protein Ligases/genetics , Ubiquitination , Viral Proteins/genetics
5.
PLoS One ; 9(6): e97957, 2014.
Article in English | MEDLINE | ID: mdl-24911523

ABSTRACT

The omega-3 (ω3) fatty acid docosahexaenoic acid (DHA) can suppress inflammation, specifically IL-1ß production through poorly understood molecular mechanisms. Here, we show that DHA reduces macrophage IL-1ß production by limiting inflammasome activation. Exposure to DHA reduced IL-1ß production by ligands that stimulate the NLRP3, AIM2, and NAIP5/NLRC4 inflammasomes. The inhibition required Free Fatty Acid Receptor (FFAR) 4 (also known as GPR120), a G-protein coupled receptor (GPR) known to bind DHA. The exposure of cells to DHA recruited the adapter protein ß-arrestin1/2 to FFAR4, but not to a related lipid receptor. DHA treatment reduced the initial inflammasome priming step by suppressing the nuclear translocation of NF-κB. DHA also reduced IL-1ß levels by enhancing autophagy in the cells. As a consequence macrophages derived from mice lacking the essential autophagy protein ATG7 were partially resistant to suppressive effects of DHA. Thus, DHA suppresses inflammasome activation by two distinct mechanisms, inhibiting the initial priming step and by augmenting autophagy, which limits inflammasome activity.


Subject(s)
Autophagy/drug effects , Docosahexaenoic Acids/pharmacology , Inflammasomes/metabolism , Macrophage Activation/drug effects , Macrophages/drug effects , NF-kappa B/metabolism , Active Transport, Cell Nucleus/drug effects , Animals , Arrestins/metabolism , Bone Marrow Cells/cytology , Calcium/metabolism , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Gene Expression Regulation/drug effects , HeLa Cells , Humans , Interleukin-1beta/biosynthesis , Interleukin-1beta/metabolism , Intracellular Space/drug effects , Intracellular Space/metabolism , Lipopolysaccharides/pharmacology , Macrophages/cytology , Macrophages/immunology , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Receptors, G-Protein-Coupled/genetics , beta-Arrestins
6.
J Biol Chem ; 287(40): 33480-7, 2012 Sep 28.
Article in English | MEDLINE | ID: mdl-22859293

ABSTRACT

The Wnt ß-catenin pathway controls numerous cellular processes including cell differentiation and cell-fate decisions. Wnt ligands engage Frizzled receptors and the low-density-lipoprotein-related protein 5/6 (LRP5/6) receptor complex leading to the recruitment of Dishevelled (Dvl) and Axin1 to the plasma membrane. Axin1 has a regulator of G-protein signaling (RGS) domain that binds adenomatous polyposis coli and Gα subunits, thereby providing a mechanism by which Gα subunits can affect ß-catenin levels. Here we show that Wnt signaling enhances the expression of another RGS domain-containing protein, PDZ-RGS3. Reducing PDZ-RGS3 levels impaired Wnt3a-induced activation of the canonical pathway. PDZ-RGS3 bound GSK3ß and decreased its catalytic activity toward ß-catenin. PDZ-RGS3 overexpression enhanced Snail1 and led to morphological and biochemical changes reminiscent of epithelial mesenchymal transition (EMT). These results indicate that PDZ-RGS3 can enhance signals generated by the Wnt canonical pathway and that plays a pivotal role in EMT.


Subject(s)
Epithelial-Mesenchymal Transition , GTP-Binding Proteins/metabolism , GTPase-Activating Proteins/metabolism , Gene Expression Regulation , Wnt Proteins/metabolism , Actins/chemistry , Animals , Catalytic Domain , Cell Line , Dogs , HEK293 Cells , Humans , Models, Biological , Protein Structure, Tertiary , Proto-Oncogene Proteins c-myc/metabolism , RGS Proteins , RNA, Small Interfering/metabolism , Signal Transduction , Snail Family Transcription Factors , Transcription Factors/metabolism , beta Catenin/metabolism
7.
Nat Immunol ; 13(3): 255-63, 2012 Jan 29.
Article in English | MEDLINE | ID: mdl-22286270

ABSTRACT

Autophagosomes delivers cytoplasmic constituents to lysosomes for degradation, whereas inflammasomes are molecular platforms activated by infection or stress that regulate the activity of caspase-1 and the maturation of interleukin 1ß (IL-1ß) and IL-18. Here we show that the induction of AIM2 or NLRP3 inflammasomes in macrophages triggered activation of the G protein RalB and autophagosome formation. The induction of autophagy did not depend on the adaptor ASC or capase-1 but was dependent on the presence of the inflammasome sensor. Blocking autophagy potentiated inflammasome activity, whereas stimulating autophagy limited it. Assembled inflammasomes underwent ubiquitination and recruited the autophagic adaptor p62, which assisted their delivery to autophagosomes. Our data indicate that autophagy accompanies inflammasome activation to temper inflammation by eliminating active inflammasomes.


Subject(s)
Autophagy , Inflammasomes/immunology , Interleukin-1beta/biosynthesis , Signal Transduction , Ubiquitination , Animals , Carrier Proteins/immunology , Cell Line , DNA-Binding Proteins , Humans , Inflammasomes/metabolism , Inflammation/immunology , Inflammation/metabolism , Interleukin-1beta/immunology , Mice , NLR Family, Pyrin Domain-Containing 3 Protein , Nuclear Proteins/immunology , ral GTP-Binding Proteins/immunology
8.
Mol Cell Biol ; 30(14): 3519-30, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20479129

ABSTRACT

In model organisms, resistance to inhibitors of cholinesterase 8 (Ric-8), a G protein alpha (G alpha) subunit guanine nucleotide exchange factor (GEF), functions to orient mitotic spindles during asymmetric cell divisions; however, whether Ric-8A has any role in mammalian cell division is unknown. We show here that Ric-8A and G alpha(i) function to orient the metaphase mitotic spindle of mammalian adherent cells. During mitosis, Ric-8A localized at the cell cortex, spindle poles, centromeres, central spindle, and midbody. Pertussis toxin proved to be a useful tool in these studies since it blocked the binding of Ric-8A to G alpha(i), thus preventing its GEF activity for G alpha(i). Linking Ric-8A signaling to mammalian cell division, treatment of cells with pertussis toxin, reduction of Ric-8A expression, or decreased G alpha(i) expression similarly affected metaphase cells. Each treatment impaired the localization of LGN (GSPM2), NuMA (microtubule binding nuclear mitotic apparatus protein), and dynein at the metaphase cell cortex and disturbed integrin-dependent mitotic spindle orientation. Live cell imaging of HeLa cells expressing green fluorescent protein-tubulin also revealed that reduced Ric-8A expression prolonged mitosis, caused occasional mitotic arrest, and decreased mitotic spindle movements. These data indicate that Ric-8A signaling leads to assembly of a cortical signaling complex that functions to orient the mitotic spindle.


Subject(s)
Antigens, Nuclear/metabolism , Dyneins/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Nuclear Matrix-Associated Proteins/metabolism , Spindle Apparatus/metabolism , Animals , Base Sequence , Cell Cycle Proteins , Cell Division/physiology , Cell Line , Dogs , GTP-Binding Protein alpha Subunits, Gi-Go/antagonists & inhibitors , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , Guanine Nucleotide Exchange Factors/antagonists & inhibitors , Guanine Nucleotide Exchange Factors/genetics , HeLa Cells , Humans , Interphase/physiology , Metaphase/physiology , Pertussis Toxin/pharmacology , Protein Binding/drug effects , RNA, Small Interfering/genetics , Signal Transduction , Spindle Apparatus/drug effects
9.
Mol Cell Biol ; 26(17): 6511-21, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16914735

ABSTRACT

Wnt ligands bind receptors of the Frizzled (Fz) family to control cell fate, proliferation, and polarity. Canonical Wnt/Fz signaling stabilizes beta-catenin by inactivating GSK3beta, leading to the translocation of beta-catenin to the nucleus and the activation of Wnt target genes. Noncanonical Wnt/Fz signaling activates RhoA and Rac, and the latter triggers the activation of c-Jun N-terminal kinase (JNK). Here, we show that exposure of B-lymphocytes to Wnt3a-conditioned media activates JNK and raises cytosolic beta-catenin levels. Both the Rac guanine nucleotide exchange factor Asef and the mitogen-activated protein kinase kinase kinase kinase germinal center kinase-related enzyme (GCKR) are required for Wnt-mediated JNK activation in B cells. In addition, we show that GCKR positively affects the beta-catenin pathway in B cells. Reduction of GCKR expression inhibits Wnt3a-induced phosphorylation of GSK3beta at serine 9 and decreases the accumulation of cytosolic beta-catenin. Furthermore, Wnt signaling induces an interaction between GCKR and GSK3beta. Our findings demonstrate that GCKR facilitates both canonical and noncanonical Wnt signaling in B lymphocytes.


Subject(s)
B-Lymphocytes/enzymology , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , Up-Regulation/genetics , Wnt Proteins/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Adenomatous Polyposis Coli Protein/metabolism , Animals , B-Lymphocytes/cytology , Cells, Cultured , Culture Media, Conditioned , Cytosol/metabolism , Dishevelled Proteins , Enzyme Activation , Germinal Center Kinases , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Guanine Nucleotide Exchange Factors/metabolism , Humans , JNK Mitogen-Activated Protein Kinases/metabolism , Mice , Mice, Inbred C57BL , Phosphoproteins/metabolism , Phosphoserine/metabolism , Protein Binding , Wnt3 Protein , Wnt3A Protein , beta Catenin/metabolism , rac GTP-Binding Proteins/metabolism
10.
J Leukoc Biol ; 79(6): 1357-68, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16565322

ABSTRACT

Chemokines bind receptors that are members of the G-protein-coupled receptor family. Chemokine receptors transduce intracellular signals by activating heterotrimeric G-proteins. Acting to limit and modulate heterotrimeric G-protein signaling is a family of proteins, termed regulator of G-protein signaling (RGS). Two of these proteins, RGS1 and RGS13, are well-expressed in germinal center B cells and many Burkitt's lymphoma cell lines. Reducing RGS13 and to a lesser extent RGS1 expression in a Burkitt's lymphoma cell line enhances responsiveness to two chemokines, CXC chemokine ligand 12 (CXCL12) and CXCL13, and reducing both mRNAs augments the responses more dramatically. The double knock-down (KD) cells respond better to restimulation with CXCL12 or CXCL13 after a primary stimulation with CXCL12 than do the control cells. The double-KD cells also exhibit a greater propensity to polarize and to develop multiple small lamellipodia. These results indicate that RGS1 and RGS13 act together to regulate chemokine receptor signaling in human germinal center B lymphocytes and provide evidence that they contribute significantly to the rapid desensitization of the signaling pathway.


Subject(s)
B-Lymphocytes/drug effects , Chemotaxis/drug effects , Germinal Center/cytology , RGS Proteins/physiology , RNA Interference , RNA, Messenger/antagonists & inhibitors , B-Lymphocytes/physiology , Burkitt Lymphoma/pathology , Cell Line, Tumor/drug effects , Chemokine CXCL12 , Chemokines, CXC/pharmacology , Genetic Vectors/genetics , Humans , Lentivirus/genetics , Platelet Activating Factor/pharmacology , RGS Proteins/genetics , RNA, Messenger/genetics , Recombinant Fusion Proteins/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Signal Transduction/physiology
11.
J Immunol ; 175(11): 7125-34, 2005 Dec 01.
Article in English | MEDLINE | ID: mdl-16301615

ABSTRACT

Interactions between B lymphocytes and Ag-bearing dendritic cells (DC) likely occur at inflammatory sites and within lymphoid organs. To better understand these interactions we imaged B cells (TgB) from hen egg lysozyme (HEL) transgenic mice and DC pulsed with HEL (DC-HEL) in collagen matrices. Analysis of live-cell dynamics revealed autonomous movements and repeated encounters between TgB cells and DC-HEL that are best described by a "kiss-run and engage" model, whereas control B cells had only short-lived interactions. Ag localized at contact sites between TgB cells and DC-HEL, and both cell types rearranged their actin cytoskeletons toward the contact zone. The interaction of a TgB cell with a HEL-bearing DC triggered strong Ca2+ transients in the B cells. Thus, B cells can productively interact with DC displaying their cognate Ag.


Subject(s)
B-Lymphocytes/immunology , Cell Communication/immunology , Dendritic Cells/immunology , Lymphocyte Activation/immunology , Muramidase/immunology , Animals , Antigen Presentation/immunology , Cell Culture Techniques , Collagen , Cytoskeleton/physiology , Mice , Mice, Transgenic , Microscopy, Confocal
12.
Immunity ; 22(3): 343-54, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15780991

ABSTRACT

Signaling by G protein-coupled receptors coupled to Galpha(i) assists in triggering lymphocyte movement into and out of lymph nodes. Here, we show that modulating the signaling output from these receptors dramatically alters B cell trafficking. Intravital microscopy of adoptively transferred B cells from wild-type and Rgs1-/- mice revealed that Rgs1-/- B cells stick better to lymph node high endothelial venules, home better to lymph nodes, and move more rapidly within lymph node follicles than do wild-type B cells. In contrast, B cells from Gnai2-/- mice enter lymph nodes poorly and move more slowly than do wild-type B cells. The Gnai2-/- mice often lack multiple peripheral lymph nodes, and their B cells respond poorly to chemokines, indicating that Galpha(i1) and Galpha(i3) poorly compensate for the loss of Galpha(i2). These results demonstrate opposing roles for Rgs1 and Gnai2 in B cell trafficking into and within lymph nodes.


Subject(s)
B-Lymphocytes/immunology , Chemotaxis, Leukocyte/immunology , GTP-Binding Protein alpha Subunits, Gi-Go/immunology , Lymph Nodes/immunology , Proto-Oncogene Proteins/immunology , RGS Proteins/immunology , Animals , Female , Flow Cytometry , GTP-Binding Protein alpha Subunit, Gi2 , Image Processing, Computer-Assisted , Immunoblotting , Mice
13.
J Biol Chem ; 277(23): 21095-102, 2002 Jun 07.
Article in English | MEDLINE | ID: mdl-11923310

ABSTRACT

Human mitochondrial ClpP (hClpP) and ClpX (hClpX) were separately cloned, and the expressed proteins were purified. Electron microscopy confirmed that hClpP forms heptameric rings and that hClpX forms a hexameric ring. Complexes of a double heptameric ring of hClpP with hexameric hClpX rings bound on each side are stable in the presence of ATP or adenosine 5'-(3-thiotriphosphate) (ATPgammaS), indicating that a symmetry mismatch is a universal feature of Clp proteases. hClpXP displays both ATP-dependent proteolytic activity and ATP- or ATPgammaS-dependent peptidase activity. hClpXP cannot degrade lambdaO protein or GFP-SsrA, specific protein substrates recognized by Escherichia coli (e) ClpXP. However, eClpX interacts with hClpP, and, when examined by electron microscopy, the resulting heterologous complexes are indistinguishable from homologous eClpXP complexes. The hybrid eClpX-hClpP complexes degrade eClpX-specific protein substrates. In contrast, eClpA can neither associate with nor activate hClpP. hClpP has an extra C-terminal extension of 28 amino acids. A mutant lacking this C-terminal extension interacts more tightly with both hClpX and eClpX and shows enhanced enzymatic activities but still does not interact with eClpA. Our results establish that human ClpX and ClpP constitute a bone fide ATP-dependent protease and confirm that substrate selection, which differs between human and E. coli ClpX, is dependent solely on the Clp ATPase. Our data also indicate that human ClpP has conserved sites required for interaction with eClpX but not eClpA, implying that the modes of interaction with ClpP may not be identical for ClpA and ClpX.


Subject(s)
Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Escherichia coli Proteins , Mitochondria/enzymology , Serine Endopeptidases/metabolism , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/isolation & purification , Amino Acid Sequence , Base Sequence , Binding Sites , Catalysis , Cloning, Molecular , DNA Primers , DNA, Complementary , Endopeptidase Clp , Enzyme Activation , Humans , Hydrolysis , Microscopy, Electron , Molecular Sequence Data , Sequence Homology, Amino Acid , Serine Endopeptidases/chemistry , Serine Endopeptidases/genetics , Serine Endopeptidases/isolation & purification , Substrate Specificity
14.
Exp Cell Res ; 273(1): 73-84, 2002 Feb 01.
Article in English | MEDLINE | ID: mdl-11795948

ABSTRACT

Essential genes which are required for normal nuclear migration and play a role in developmental processes have been isolated from model genetic organisms. One such gene is nudC (nuclear distribution C), which is required for positioning nuclei in the cytoplasm of the filamentous fungus Aspergillus nidulans and for normal colony growth. This gene is highly conserved, structurally and functionally, throughout evolution and the human homolog, HnudC, has been cloned. To study the function of nudC in higher eukaryotic cells, HnudC was downregulated by developing triple ribozyme constructs, consisting of two cis-acting ribozymes which liberate an internal trans-acting ribozyme targeted to HnudC. Efficient cleavage sites in HnudC mRNA were identified using a library selection technique and HnudC-targeted internal ribozymes were cloned into a triple ribozyme cassette. Triple ribozyme constructs were subcloned into an ecdysone-inducible expression vector and stably transfected into human embryonic 293 cells. Muristerone A induced expression of the HnudC ribozyme and produced specific reduction of HnudC mRNA. Downregulation of HnudC mRNA resulted in significant inhibition of cell proliferation in clones expressing the HnudC-targeted triple ribozyme, which was not observed in uninduced cells or cells transfected with vector alone. In induced cultures, many mitotic cells demonstrated defects in spindle architecture during mitosis. The most common defect observed was multiple mitotic spindle poles rather than the expected bipolar structure. These data demonstrate the fundamental importance of HnudC in eukaryotic cell proliferation and a functional role for HnudC in spindle formation at mitosis.


Subject(s)
Cell Division/physiology , Proteins/physiology , Spindle Apparatus/physiology , Cell Cycle Proteins , Cells, Cultured , Down-Regulation , Gene Expression , Genetic Therapy , Humans , Kidney/cytology , Kidney/metabolism , Nuclear Proteins , Nucleic Acid Conformation , RNA, Antisense/metabolism , RNA, Antisense/pharmacology , RNA, Catalytic/metabolism , RNA, Messenger/metabolism , Transfection
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