scholarly journals Antiviral activity of a purine synthesis enzyme reveals a key role of deamidation in regulating protein nuclear import

2019 ◽  
Vol 5 (10) ◽  
pp. eaaw7373 ◽  
Author(s):  
Junhua Li ◽  
Jun Zhao ◽  
Simin Xu ◽  
Shu Zhang ◽  
Junjie Zhang ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Transcriptional Activation ◽  
Nuclear Import ◽  
Nuclear Translocation ◽  
Lytic Replication ◽  
Metabolic Enzyme ◽  
Purine Synthesis ◽  
Localization Signal ◽  
Positively Charged

Protein nuclear translocation is highly regulated and crucial for diverse biological processes. However, our understanding concerning protein nuclear import is incomplete. Here we report that a cellular purine synthesis enzyme inhibits protein nuclear import via deamidation. Employing human Kaposi’s sarcoma-associated herpesvirus (KSHV) to probe the role of protein deamidation, we identified a purine synthesis enzyme, phosphoribosylformylglycinamidine synthetase (PFAS) that inhibits KSHV transcriptional activation. PFAS deamidates the replication transactivator (RTA), a transcription factor crucial for KSHV lytic replication. Mechanistically, deamidation of two asparagines flanking a positively charged nuclear localization signal impaired the binding of RTA to an importin β subunit, thus diminishing RTA nuclear localization and transcriptional activation. Finally, RTA proteins of all gamma herpesviruses appear to be regulated by PFAS-mediated deamidation. These findings uncover an unexpected function of a metabolic enzyme in restricting viral replication and a key role of deamidation in regulating protein nuclear import.

scholarly journals Identification of a putative nuclear localization signal in maspin protein shed light into its nuclear import regulation

2018 ◽  
Author(s):  
Jeffrey Reina ◽  
Lixin Zhou ◽  
Marcos R.M. Fontes ◽  
Nelly Panté ◽  
Nathalie Cella
Keyword(s):  
Subcellular Localization ◽  
Nuclear Localization ◽  
Hela Cells ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Biological Activities ◽  
Dependent Manner ◽  
Localization Signal

AbstractMaspin (SERPINB5) is a potential tumor suppressor gene with pleiotropic biological activities, including regulation of cell proliferation, death, adhesion, migration and gene expression. Several studies suggest that subcellular localization plays an essential role on maspin tumor suppression activity. In this study we investigated the molecular mechanisms underlying maspin nucleocytoplasmic shuttling. Anin vitronuclear-import assay using digitonin-permeabilized HeLa cells demonstrated that maspin enters the nucleus by an energy-and carrier-independent mechanism. However, previous studies indicated that maspin subcellular localization is regulated in the cell. Using a nuclear localization signal (NLS) prediction software, we identified a putative NLS in the maspin amino acid sequence. To distinguish between passive and regulated nuclear translocation, maspinNLS or the full-length protein (MaspinFL) were fused to 5GFP, rendering the construct too large to enter the nucleus passively. Unexpectedly, 5GFP-maspinNLS, but not maspinFL-5GFP, entered the nucleus of HeLa cells. Dominant-negative Ran-GTPase mutants RanQ69L or RanT24N, suppressed 5GFP-maspinNLS nuclear localization. In summary, we provide evidence that maspin translocates to the nucleus passively. In addition, we identified a peptide in the maspin protein sequence, which is able to drive a 5GFP construct to the nucleus in an energy-dependent manner.

scholarly journals Pol α-primase dependent nuclear localization of the mammalian CST complex

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Joseph M. Kelich ◽  
Harry Papaioannou ◽  
Emmanuel Skordalakes
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Intracellular Localization ◽  
Telomere Maintenance ◽  
Dna Polymerase Alpha ◽  
Localization Signal ◽  
Initiation Of Dna Replication ◽  
Cycle Dependent ◽  
Lagging Strand

AbstractThe human CST complex composed of CTC1, STN1, and TEN1 is critically involved in telomere maintenance and homeostasis. Specifically, CST terminates telomere extension by inhibiting telomerase access to the telomeric overhang and facilitates lagging strand fill in by recruiting DNA Polymerase alpha primase (Pol α-primase) to the telomeric C-strand. Here we reveal that CST has a dynamic intracellular localization that is cell cycle dependent. We report an increase in nuclear CST several hours after the initiation of DNA replication, followed by exit from the nucleus prior to mitosis. We identify amino acids of CTC1 involved in Pol α-primase binding and nuclear localization. We conclude, the CST complex does not contain a nuclear localization signal (NLS) and suggest that its nuclear localization is reliant on Pol α-primase. Hypomorphic mutations affecting CST nuclear import are associated with telomere syndromes and cancer, emphasizing the important role of this process in health.

scholarly journals YB-1 Phosphorylation at Serine 209 Inhibits Its Nuclear Translocation

2021 ◽  
Vol 23 (1) ◽  
pp. 428
Author(s):  
Ekaterina M. Sogorina ◽  
Ekaterina R. Kim ◽  
Alexey V. Sorokin ◽  
Dmitry N. Lyabin ◽  
Lev P. Ovchinnikov ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Posttranslational Modifications ◽  
Nuclear Import ◽  
Rna Binding ◽  
Nuclear Translocation ◽  
Dna And Rna ◽  
Localization Signal ◽  
And Migration ◽  
Dna And Rna Binding

YB-1 is a multifunctional DNA- and RNA-binding protein involved in cell proliferation, differentiation, and migration. YB-1 is a predominantly cytoplasmic protein that is transported to the nucleus in certain conditions, including DNA-damaging stress, transcription inhibition, and viral infection. In tumors, YB-1 nuclear localization correlates with high aggressiveness, multidrug resistance, and a poor prognosis. It is known that posttranslational modifications can regulate the nuclear translocation of YB-1. In particular, well-studied phosphorylation at serine 102 (S102) activates YB-1 nuclear import. Here, we report that Akt kinase phosphorylates YB-1 in vitro at serine 209 (S209), which is located in the vicinity of the YB-1 nuclear localization signal. Using phosphomimetic substitutions, we showed that S209 phosphorylation inhibits YB-1 nuclear translocation and prevents p-S102-mediated YB-1 nuclear import.

scholarly journals Specific Nucleoporin Requirement for Smad Nuclear Translocation

2010 ◽  
Vol 30 (16) ◽  
pp. 4022-4034 ◽  
Author(s):  
Xiaochu Chen ◽  
Lan Xu
Keyword(s):  
Nuclear Localization ◽  
Nuclear Pore Complex ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Transforming Growth Factor ◽  
Nuclear Translocation ◽  
Nuclear Periphery ◽  
Transforming Growth Factor Β ◽  
Nuclear Pore ◽  
Localization Signal

ABSTRACT Cytoplasm-to-nucleus translocation of Smad is a fundamental step in transforming growth factor β (TGF-β) signal transduction. Here we identify a subset of nucleoporins that, in conjunction with Msk (Drosophila Imp7/8), specifically mediate activation-induced nuclear translocation of MAD (Drosophila Smad1) but not the constitutive import of proteins harboring a classic nuclear localization signal (cNLS) or the spontaneous nuclear import of Medea (Drosophila Smad4). Surprisingly, many of these nucleoporins, including Sec13, Nup75, Nup93, and Nup205, are scaffold nucleoporins considered important for the overall integrity of the nuclear pore complex (NPC) but not known to have cargo-specific functions. We demonstrate that the roles of these nucleoporins in supporting Smad nuclear import are separate from their previously assigned functions in NPC assembly. Furthermore, we uncovered novel pathway-specific functions of Sec13 and Nup93; both Sec13 and Nup93 are able to preferentially interact with the phosphorylated/activated form of MAD, and Nup93 acts to recruit the importin Msk to the nuclear periphery. These findings, together with the observation that Sec13 and Nup93 could interact directly with Msk, suggest their direct involvement in the nuclear import of MAD. Thus, we have delineated the nucleoporin requirement of MAD nuclear import, reflecting a unique trans-NPC mechanism.

Nuclear import of glycoconjugates is distinct from the classical NLS pathway

1995 ◽  
Vol 108 (4) ◽  
pp. 1325-1332 ◽  
Author(s):  
E. Duverger ◽  
C. Pellerin-Mendes ◽  
R. Mayer ◽  
A.C. Roche ◽  
M. Monsigny
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Peptide Sequence ◽  
Nuclear Pore ◽  
Dependent Manner ◽  
Permeabilized Cells ◽  
Localization Signal ◽  
Energy Dependent ◽  
Cytosolic Factors

The nuclear import of many proteins depends on a short peptide sequence called the nuclear localization signal. However, glycosylated proteins, which lack such a nuclear localization signal, upon their injection into the cytosol by electroporation, enter the nucleus in a sugar-dependent manner. This paper brings new insights on the mechanism of this process, based on a study of neoglycoprotein nuclear uptake by digitonin-permeabilized cells. The nuclear import of neoglycoproteins is energy dependent: it does not occur when cells are maintained at 4 degrees C or when cells are ATP-depleted by treatment with apyrase. The nuclear import of neoglycoproteins occurs through the nuclear pore: it is inhibited by preincubation of cells with wheat germ agglutinin, a lectin which binds the nuclear pore glycoproteins and blocks the translocation step of nuclear localization signal bearing proteins through the nuclear pore. Furthermore, the nuclear import of neoglycoproteins does not use the pathway of nuclear localization signal bearing proteins: nuclear import of nuclear localization signal bearing proteins depends on cytosolic factors and is inhibited by treatment of cells with N-ethylmaleimide, while the nuclear import of neoglycoproteins neither requires added cytosolic factors nor is sensitive to alkylation by N-ethylmaleimide. In addition, upon incubation in the presence of a large excess of nuclear localization signal bearing protein, the nuclear import of neoglycoproteins is not inhibited.

Nuclear transport of the U2 snRNP-specific U2B'' protein is mediated by both direct and indirect signalling mechanisms

1994 ◽  
Vol 107 (7) ◽  
pp. 1807-1816 ◽  
Author(s):  
C. Kambach ◽  
I.W. Mattaj
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Nuclear Transport ◽  
U2 Snrna ◽  
Central Segment ◽  
U2 Snrnp ◽  
U1 Snrnp ◽  
Localization Signal ◽  
Protein Amino Acids

Experiments investigating the nuclear import of the U2 snRNP-specific B'' protein (U2B'') are presented. U2B'' nuclear transport is shown to be able to occur independently of binding to U2 snRNA. The central segment of the protein (amino acids 90–146) encodes an unusual nuclear localization signal (NLS) that is related to that of the U1 snRNP-specific A protein. However, nuclear import of U2B'' does not depend on this NLS. Sequences in the N-terminal RNP motif of the protein are sufficient to direct nuclear transport, and evidence is presented that the interaction of U2B'' with the U2A' protein mediates this effect. This suggests that U2B'' can ‘piggy-back’ to the nucleus in association with U2A’, and thus be imported to the nucleus by two different mechanisms. U2A' nuclear transport, on the other hand, can occur independently of both U2B'' binding and of U2 snRNA.

scholarly journals OsWRKY62 and OsWRKY76 interact with Importin α1s for Negative Regulation of Defensive Responses in Nucleus

2021 ◽  
Author(s):  
Xiaohui Xu ◽  
Han Wang ◽  
Jiqin Liu ◽  
Shuying Han ◽  
Miaomiao Lin ◽  
...  
Keyword(s):  
Amino Acids ◽  
Nuclear Localization ◽  
Nuclear Export ◽  
Nuclear Translocation ◽  
Nuclear Export Signal ◽  
Defense Responses ◽  
Nuclear Localization Signals ◽  
Defensive Responses ◽  
Export Signal ◽  
Positively Charged

Abstract Background: OsWRKY62 and OsWRKY76, two close members of WRKY transcription factors, function together as transcriptional repressors. OsWRKY62 is predominantly localized in the cytosol. What are the regulatory factors for OsWRKY62 nuclear translocation?Results: In this study, we characterized they interacted with rice importin, OsIMα1a and OsIMα1b, for nuclear translocation. Chimeric OsWRKY62.1-GFP, which is predominantly localized in the cytoplasm, was translocated to the nucleus of Nicotiana benthamiana leaf cells in the presence of OsIMα1a or OsIMαDIBB1a lacking the auto-inhibitory importin β-binding domain. OsIMαDIBB1a interacted with the WRKY domain of OsWRKY62.1, which has specific bipartite positively charged concatenated amino acids functioning as a nuclear localization signal. Similarly, we found that OsIMαDIBB1a interacted with the AvrPib effector of rice blast fungus Magnaporthe oryzae, which contains a scattered distribution of positively charged amino acids. Furthermore, we identified a nuclear export signal in OsWRKY62.1 that inhibited nuclear transportation. Overexpression of OsIMα1a or OsIMα1b enhanced resistance to M. oryzae, whereas knockout mutants decreased resistance to the pathogen. However, overexpressing both OsIMα1a and OsWRKY62.1 were slightly more susceptible to M. oryzae than OsWRKY62.1 alone. Ectopic overexpression of OsWRKY62.1 with an extra nuclear export signal compromised the enhanced susceptibility of OsWRKY62.1 to M. oryzae.Conclusion: These results indicated that OsWRKY62 localization is a consequence of competition binding between rice importins and exportins. OsWRKY62, OsWRKY76, and AvrPib effector translocate to nucleus in association with importin α1s through new types of nuclear localization signals for negatively regulating defense responses.

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