scholarly journals Effect of Ivermectin and Atorvastatin on Nuclear Localization of Importin Alpha and Drug Target Expression Profiling in Host Cells from Nasopharyngeal Swabs of SARS-CoV-2- Positive Patients

Viruses ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 2084
Author(s):  
Valeria Inés Segatori ◽  
Juan Garona ◽  
Lorena Grisel Caligiuri ◽  
Juan Bizzotto ◽  
Rosario Lavignolle ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nuclear Localization ◽  
Rho Gtpases ◽  
Nuclear Transport ◽  
Host Cells ◽  
Poly I:C ◽  
Nuclear Accumulation ◽  
Synthetic Analog ◽  
Importin Α

Nuclear transport and vesicle trafficking are key cellular functions involved in the pathogenesis of RNA viruses. Among other pleiotropic effects on virus-infected host cells, ivermectin (IVM) inhibits nuclear transport mechanisms mediated by importins and atorvastatin (ATV) affects actin cytoskeleton-dependent trafficking controlled by Rho GTPases signaling. In this work, we first analyzed the response to infection in nasopharyngeal swabs from SARS-CoV-2-positive and -negative patients by assessing the gene expression of the respective host cell drug targets importins and Rho GTPases. COVID-19 patients showed alterations in KPNA3, KPNA5, KPNA7, KPNB1, RHOA, and CDC42 expression compared with non-COVID-19 patients. An in vitro model of infection with Poly(I:C), a synthetic analog of viral double-stranded RNA, triggered NF-κB activation, an effect that was halted by IVM and ATV treatment. Importin and Rho GTPases gene expression was also impaired by these drugs. Furthermore, through confocal microscopy, we analyzed the effects of IVM and ATV on nuclear to cytoplasmic importin α distribution, alone or in combination. Results showed a significant inhibition of importin α nuclear accumulation under IVM and ATV treatments. These findings confirm transcriptional alterations in importins and Rho GTPases upon SARS-CoV-2 infection and point to IVM and ATV as valid drugs to impair nuclear localization of importin α when used at clinically-relevant concentrations.

scholarly journals Differential Importin-α Recognition and Nuclear Transport by Nuclear Localization Signals within the High-Mobility-Group DNA Binding Domains of Lymphoid Enhancer Factor 1 and T-Cell Factor 1

1998 ◽  
Vol 18 (8) ◽  
pp. 4819-4832 ◽  
Author(s):  
Mary G. Prieve ◽  
Katherine L. Guttridge ◽  
Jesus Munguia ◽  
Marian L. Waterman
Keyword(s):  
Dna Binding ◽  
Nuclear Localization ◽  
Nuclear Transport ◽  
High Mobility ◽  
Dna Binding Domain ◽  
T Cell Factor ◽  
Importin Α ◽  
Armadillo Repeat ◽  
Enhancer Factor

ABSTRACT The transcription factor lymphoid enhancer factor 1 (LEF-1) is directed to the nucleus by a nine-amino-acid nuclear localization signal (NLS; KKKKRKREK) located in the high-mobility-group DNA binding domain. This NLS is recognized by two armadillo repeat proteins (pendulin/Rch1/α-P1/hSrp1α and Srp1/karyopherin-α/α-S1/NPI-1) which function in nuclear transport as the importin-α subunit of NLS receptors. T-cell factor 1 (TCF-1), a related transcription factor, contains a similar sequence (KKKRRSREK) in the identical position within its HMG DNA binding domain. We show that this sequence functions as an NLS in vivo but is not recognized by these two importin-α subtypes in a yeast two-hybrid assay and only weakly recognized in an in vitro binding assay. Transfer of the LEF-1 NLS to TCF-1 can confer pendulin/Rch1 binding, demonstrating that the NLS is the primary determinant for recognition. We have constructed a set of deletion mutations in pendulin/Rch1 to examine the differential NLS recognition more closely. We find that the entire armadillo repeat array of pendulin/Rch1 is necessary to maintain high affinity and specificity for the LEF-1 NLS versus the TCF-1 NLS. Importin-β, the second subunit of the NLS receptor complex, does not influence in vitro NLS binding affinity or specificity. To test whether this differential recognition is indicative of distinct mechanisms of nuclear transport, the subcellular localization of LEF-1 and TCF-1 fused to green fluorescent protein (GFP)) was examined in an in vitro nuclear transport assay. GFP–LEF-1 readily localizes to the nucleus, whereas GFP–TCF-1 remains in the cytoplasm. Thus, LEF-1 and TCF-1 differ in several aspects of nuclear localization.

005.Efficiency of nuclear import of the chromatin-remodelling factor SRY is critical for sex determination

2005 ◽  
Vol 17 (9) ◽  
pp. 64
Author(s):  
D. A. Jans ◽  
G. Kaur ◽  
I. K. H. Poon ◽  
A. Delluc-Clavieries ◽  
K. M. Wagstaff
Keyword(s):  
Sex Determination ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Sex Reversal ◽  
Nuclear Accumulation ◽  
Missense Mutations ◽  
Nuclear Localization Signals ◽  
Testicular Cells ◽  
Xy Sex Reversal

15% of cases of human XY sex reversal are due to mutations in SRY (sex determining region on the Y chromosome), many of which map to one of SRY’s two independently acting nuclear localization signals (NLSs) flanking its DNA binding domain. The C-terminal NLS (C-NLS) targets SRY to the nucleus through a ‘conventional’ pathway dependent on the nuclear import receptor importin-β (Imp-β). No importin has been shown to bind the N-terminal NLS (N-NLS), but it is known to interact with the Ca2+-binding protein calmodulin (CaM). We examined seven distinct missense mutations in the SRY NLSs from XY sex-reversed human females for effects on nuclear import and ability to interact with CaM/Imp-β1. All mutations were found to result in reduced nuclear localization in transfected testicular cells compared to wild type. The CaM antagonist, calmidazolium chloride (CDZ), was found to significantly reduce SRY nuclear accumulation, indicating a dependence of SRY nuclear import on CaM. Intriguingly, N-NLS mutants were resistant to CDZ’s effects, implying a loss of interaction with CaM; this was confirmed directly by in vitro binding experiments using recombinantly expressed protein. Either impaired CaM or Imp-β1 binding can thus be the basis of sex-reversal in human patients. Our results implicate a CaM-dependent nuclear import pathway for SRY mediated by the N-NLS that, together with the C-NLS, is required to achieve threshold levels of SRY in the nucleus for male sex determination.

scholarly journals Inhibition of Transcription Induces Phosphorylation of YB-1 at Ser102 and Its Accumulation in the Nucleus

Cells ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 104 ◽  
Author(s):  
Dmitry A. Kretov ◽  
Daria A. Mordovkina ◽  
Irina A. Eliseeva ◽  
Dmitry N. Lyabin ◽  
Dmitry N. Polyakov ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase Ii ◽  
Nuclear Localization ◽  
Kinase Activity ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Regulatory Mechanisms ◽  
Nuclear Accumulation ◽  
Nuclear Retention ◽  
Post Translational Modifications

The Y-box binding protein 1 (YB-1) is an RNA/DNA-binding protein regulating gene expression in the cytoplasm and the nucleus. Although mostly cytoplasmic, YB-1 accumulates in the nucleus under stress conditions. Its nuclear localization is associated with aggressiveness and multidrug resistance of cancer cells, which makes the understanding of the regulatory mechanisms of YB-1 subcellular distribution essential. Here, we report that inhibition of RNA polymerase II (RNAPII) activity results in the nuclear accumulation of YB-1 accompanied by its phosphorylation at Ser102. The inhibition of kinase activity reduces YB-1 phosphorylation and its accumulation in the nucleus. The presence of RNA in the nucleus is shown to be required for the nuclear retention of YB-1. Thus, the subcellular localization of YB-1 depends on its post-translational modifications (PTMs) and intracellular RNA distribution.

scholarly journals In vitro integration of an ichnovirus genome segment into the genomic DNA of lepidopteran cells

2007 ◽  
Vol 88 (1) ◽  
pp. 105-113 ◽  
Author(s):  
Daniel Doucet ◽  
Anic Levasseur ◽  
Catherine Béliveau ◽  
Renée Lapointe ◽  
Don Stoltz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Virus Replication ◽  
Genomic Dna ◽  
Cultured Cells ◽  
Germ Line ◽  
Choristoneura Fumiferana ◽  
Genome Segment ◽  
Host Cells ◽  
Dsdna Viruses

Polydnaviruses (PDVs) are dsDNA viruses transmitted by ichneumonid and braconid endoparasitoids to their lepidopteran hosts during oviposition. Wasp carriers are asymptomatic and transmit the virus to their progeny through the germ line; replication is confined to the calyx region of the wasp ovary, where the virus accumulates in the fluid bathing the eggs. In the lepidopteran host, however, no virus replication takes place, but PDV gene expression is essential for successful parasitism. Sustained gene expression in the absence of virus replication thus requires that the circular PDV genome segments persist for days within host cells. Available evidence suggests that most genome segments persist as episomes, but recent studies have indicated that some genome segments may undergo integration within lepidopteran genomic DNA, at least in vitro. In the present study, an integrated form of a Tranosema rostrale ichnovirus (TrIV) genome segment was cloned from genomic DNA extracted from infected Choristoneura fumiferana CF-124T cells and junction regions on either side of the viral DNA sequence were sequenced. This is the first proven example of integration of an ichnovirus genome segment in infected lepidopteran cells. Interestingly, circular forms of this genome segment do not appear to persist in these cells; none the less, a gene (TrFrep1) carried by this genome segment displays long-term transcription in infected cultured cells.

scholarly journals Nuclear Localization of PTEN by a Ran-dependent Mechanism Enhances Apoptosis: Involvement of an N-Terminal Nuclear Localization Domain and Multiple Nuclear Exclusion Motifs

2006 ◽  
Vol 17 (9) ◽  
pp. 4002-4013 ◽  
Author(s):  
Anabel Gil ◽  
Amparo Andrés-Pons ◽  
Elena Fernández ◽  
Miguel Valiente ◽  
Josema Torres ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Effector Proteins ◽  
Dominant Negative ◽  
Nuclear Accumulation ◽  
Localization Domain ◽  
Nuclear Exclusion ◽  
Catalytically Active ◽  
Importin Α ◽  
Functional Consequences ◽  
Dependent Mechanism

The targeting of the tumor suppressor PTEN protein to distinct subcellular compartments is a major regulatory mechanism of PTEN function, by controlling its access to substrates and effector proteins. Here, we investigated the molecular basis and functional consequences of PTEN nuclear/cytoplasmic distribution. PTEN accumulated in the nucleus of cells treated with apoptotic stimuli. Nuclear accumulation of PTEN was enhanced by mutations targeting motifs in distinct PTEN domains, and it was dependent on an N-terminal nuclear localization domain. Coexpression of a dominant negative Ran GTPase protein blocked PTEN accumulation in the nucleus, which was also affected by coexpression of importin α proteins. The lipid- and protein-phosphatase activity of PTEN differentially modulated PTEN nuclear accumulation. Furthermore, catalytically active nuclear PTEN enhanced cell apoptotic responses. Our findings indicate that multiple nuclear exclusion motifs and a nuclear localization domain control PTEN nuclear localization by a Ran-dependent mechanism and suggest a proapoptotic role for PTEN in the cell nucleus.

scholarly journals The Arginine-Rich Domains Present in Human Immunodeficiency Virus Type 1 Tat and Rev Function as Direct Importin β-Dependent Nuclear Localization Signals

1999 ◽  
Vol 19 (2) ◽  
pp. 1210-1217 ◽  
Author(s):  
Ray Truant ◽  
Bryan R. Cullen
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signals ◽  
Immunodeficiency Virus ◽  
Importin Α

ABSTRACT Protein nuclear import is generally mediated by basic nuclear localization signals (NLSs) that serve as targets for the importin α (Imp α) NLS receptor. Imp α is in turn bound by importin β (Imp β), which targets the resultant protein complex to the nucleus. Here, we report that the arginine-rich NLS sequences present in the human immunodeficiency virus type 1 regulatory proteins Tat and Rev fail to interact with Imp α and instead bind directly to Imp β. Using in vitro nuclear import assays, we demonstrate that Imp α is entirely dispensable for Tat and Rev nuclear import. In contrast, Imp β proved both sufficient and necessary, in that other β-like import factors, such as transportin, were unable to support Tat or Rev nuclear import. Using in vitro competition assays, it was demonstrated that the target sites on Imp β for Imp α, Tat, and Rev binding either are identical or at least overlap. The interaction of Tat and Rev with Imp β is also similar to Imp α binding in that it is inhibited by RanGTP but not RanGDP, a finding that may in part explain why the interaction of the Rev nuclear RNA export factor with target RNA species is efficient in the cell nucleus yet is released in the cytoplasm. Together, these studies define a novel class of arginine-rich NLS sequences that are direct targets for Imp β and that therefore function independently of Imp α.

scholarly journals Characterization of the Human Herpesvirus 6 U69 Gene Product and Identification of Its Nuclear Localization Signal

2007 ◽  
Vol 82 (2) ◽  
pp. 710-718 ◽  
Author(s):  
Yuji Isegawa ◽  
Yoichi Miyamoto ◽  
Yoshinari Yasuda ◽  
Katsunori Semi ◽  
Kenji Tsujimura ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Nuclear Localization ◽  
Nuclear Transport ◽  
Human Herpesvirus ◽  
Small Gtpase ◽  
Terminal Region ◽  
Mutant Form ◽  
Human Herpesvirus 6 ◽  
Importin Α ◽  
Herpesvirus 6

ABSTRACT To elucidate the function of the U69 protein kinase of human herpesvirus 6 (HHV-6) in vivo, we first analyzed its subcellular localization in HHV-6-infected Molt 3 cells by using polyclonal antibodies against the U69 protein. Immunofluorescence studies showed that the U69 signal localized to the nucleus in a mesh-like pattern in both HHV-6-infected and HHV6-transfected cells. A computer program predicted two overlapping classic nuclear localization signals (NLSs) in the N-terminal region of the protein; this NLS motif is highly conserved in the N-terminal region of most of the herpesvirus protein kinases examined to date. An N-terminal deletion mutant form of the protein failed to enter the nucleus, whereas a fusion protein of green fluorescent protein (GFP) and/or glutathione S-transferase (GST) and the U69 N-terminal region was transported into the nucleus, demonstrating that the predicted N-terminal NLSs of the protein actually function as NLSs. The nuclear transport of the GST-GFP fusion protein containing the N-terminal NLS of U69 was inhibited by wheat germ agglutinin and by the Q69L Ran-GTP mutant, indicating that the U69 protein is transported into the nucleus from the cytoplasm via classic nuclear transport machinery. A cell-free import assay showed that the nuclear transport of the U69 protein was mediated by importin α/β in conjunction with the small GTPase Ran. When the import assay was performed with a low concentration of each importin-α subtype, NPI2/importin-α7 elicited more efficient transport activity than did Rch1/importin-α1 or Qip1/importin-α3. These results suggest a relationship between the localization of NPI2/importin-α7 and the cell tropism of HHV-6.

scholarly journals Evidence for Separable Functions of Srp1p, the Yeast Homolog of Importin α (Karyopherin α): Role for Srp1p and Sts1p in Protein Degradation

2000 ◽  
Vol 20 (16) ◽  
pp. 6062-6073 ◽  
Author(s):  
Michelle M. Tabb ◽  
Prasad Tongaonkar ◽  
Loan Vu ◽  
Masayasu Nomura
Keyword(s):  
Protein Degradation ◽  
Nuclear Localization ◽  
Ubiquitin Proteasome System ◽  
26S Proteasome ◽  
Localization Function ◽  
Importin Α ◽  
Yeast Homolog ◽  
Two Hybrid

ABSTRACT Srp1p (importin α) functions as the nuclear localization signal (NLS) receptor in Saccharomyces cerevisiae. Thesrp1-31 mutant is defective in this nuclear localization function, whereas an srp1-49 mutant exhibits defects that are unrelated to this localization function, as was confirmed by intragenic complementation between the two mutants. RPN11and STS1 (DBF8) were identified as high-dosage suppressors of the srp1-49 mutation but not of thesrp1-31 mutation. We found that Sts1p interacts directly with Srp1p in vitro and also in vivo, as judged by coimmunoprecipitation and two-hybrid analyses. Mutants of Sts1p that cannot interact with Srp1p are incapable of suppressingsrp1-49 defects, strongly suggesting that Sts1p functions in a complex with Srp1p. STS1 also interacted with the second suppressor, RPN11, a subunit of the 26S proteasome, in the two-hybrid system. Further, degradation of Ub-Pro-β-galactosidase, a test substrate for the ubiquitin-proteasome system, was defective in srp1-49 but not insrp1-31. This defect in protein degradation was alleviated by overexpression of either RPN11 or STS1 insrp1-49. These results suggest a role for Srp1p in regulation of protein degradation separate from its well-established role as the NLS receptor.

scholarly journals GTP-dependent Binding and Nuclear Transport of RNA Polymerase II by Npa3 Protein

2011 ◽  
Vol 286 (41) ◽  
pp. 35553-35561 ◽  
Author(s):  
Lidija Staresincic ◽  
Jane Walker ◽  
A. Barbara Dirac-Svejstrup ◽  
Richard Mitter ◽  
Jesper Q. Svejstrup
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Nuclear Import ◽  
Chromatin Immunoprecipitation ◽  
Nuclear Transport ◽  
Proteomic Screen ◽  
Genome Wide ◽  
Importin Α

We identified XAB1 in a proteomic screen for factors that interact with human RNA polymerase II (RNAPII). Because XAB1 has a conserved Saccharomyces cerevisiae homologue called Npa3, yeast genetics and biochemical analysis were used to dissect the significance of the interaction. Degron-dependent Npa3 depletion resulted in genome-wide transcription decreases, correlating with a loss of RNAPII from genes as measured by chromatin immunoprecipitation. Surprisingly, however, transcription in vitro was unaffected by Npa3, suggesting that it affects a process that is not required for transcription in yeast extracts. Indeed, Npa3 depletion in vivo affects nuclear localization of RNAPII; the polymerase accumulates in the cytoplasm. Npa3 is a member of the GPN-LOOP family of GTPases. Npa3 mutants that either cannot bind GTP or that bind but cannot hydrolyze it are inviable and unable to support nuclear transport of RNAPII. Surprisingly, we were unable to detect interactions between Npa3 and proteins in the classical importin α/β pathway for nuclear import. Interestingly, Npa3-RNAPII binding is significantly increased by the addition of GTP or its slowly hydrolyzable analogue guanosine 5′-3-O-(thio)triphosphate (GTPγS). Moreover, the Npa3 mutant that binds GTP, but cannot hydrolyze it, binds RNAPII even in the absence of added GTP, whereas the mutant that cannot bind GTP is unable to bind the polymerase. Together, our data suggest that Npa3 defines an unconventional pathway for nuclear import of RNAPII, which involves GTP-dependent binding of Npa3 to the polymerase.

Key role of endothelial importin-α in VEGF expression and gastric angiogenesis: novel insight into aging gastropathy

2014 ◽  
Vol 306 (4) ◽  
pp. G338-G345 ◽  
Author(s):  
Amrita Ahluwalia ◽  
Michael K. Jones ◽  
Andrzej S. Tarnawski
Keyword(s):  
Nuclear Transport ◽  
Gene Activation ◽  
In Vivo Studies ◽  
Vegf Gene ◽  
Vegf Expression ◽  
Importin Α ◽  
In Vitro Angiogenesis ◽  
Impaired Angiogenesis

Recent in vivo studies demonstrated that aging gastric mucosa has impaired angiogenesis and reduced expression of vascular endothelial growth factor (VEGF). Angiogenesis is triggered by hypoxia and VEGF gene activation, and the latter requires transport of transcription factor(s) into endothelial cell nuclei. We focused on gastric mucosal endothelial cells (GMEC), which are key targets and effectors of gastric angiogenesis, and determined whether and to what extent importin-α, a nuclear transport protein, regulates VEGF gene activation and gastric angiogenesis and the possible role of importin-α in aging gastropathy. GMEC were isolated from rats 3 and 24 mo of age, young (YGEC) and aging (AGEC), respectively. We examined in these cells 1) in vitro angiogenesis, 2) expression of VEGF and importin-α, 3) nuclear transport of hypoxia-inducible factor (HIF)-1α by importin-α, 4) binding of HIF-1α to the VEGF gene promoter, and 5) effects of importin-α silencing in YGEC and its upregulation in AGEC on angiogenesis and VEGF expression. AGEC exhibited significantly impaired in vitro angiogenesis by fourfold and decreased expression of VEGF, importin-α, and nuclear HIF-1α by 1.4-fold, 1.6-fold, and 2.9-fold, respectively, vs. YGEC. Upregulation of importin-α in AGEC significantly reversed all these abnormalities. In YGEC, knockdown of importins-α1 and -α3 significantly reduced in vitro angiogenesis by 93% and 73% and VEGF expression by 48% and 52%, respectively. The above findings demonstrate that importin-α is a novel and critical regulator of gastric angiogenesis. Its reduced expression in AGEC is the key mechanism for impaired angiogenesis and reduced VEGF.

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