Browse
Submit Data
Databases
API
Help

Dataset Information

17 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Human Enzyme PADI4 Binds to the Nuclear Carrier Importin α3

ABSTRACT: PADI4 is a peptidyl-arginine deiminase (PADI) involved in the conversion of arginine to citrulline. PADI4 is present in macrophages, monocytes, granulocytes, and several cancer cells. It is the only PADI family member observed within both the nucleus and the cytoplasm. PADI4 has a predicted nuclear localization sequence (NLS) comprising residues Pro56 to Ser83, to allow for nuclear translocation. Recent predictors also suggest that the region Arg495 to Ile526 is a possible NLS. To understand how PADI4 is involved in cancer, we studied the ability of intact PADI4 to bind importin α3 (Impα3), a nuclear transport factor that plays tumor-promoting roles in several cancers, and its truncated species (ΔImpα3) without the importin-binding domain (IBB), by using fluorescence, circular dichroism (CD), and isothermal titration calorimetry (ITC). Furthermore, the binding of two peptides, encompassing the first and the second NLS regions, was also studied using the same methods and molecular docking simulations. PADI4 interacted with both importin species, with affinity constants of ~1–5 µM. The isolated peptides also interacted with both importins. The molecular simulations predict that the anchoring of both peptides takes place in the major binding site of Impα3 for the NLS of cargo proteins. These findings suggest that both NLS regions were essentially responsible for the binding of PADI4 to the two importin species. Our data are discussed within the framework of a cell mechanism of nuclear transport that is crucial in cancer.

SUBMITTER: Neira J

PROVIDER: S-EPMC9319256 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

The Paralogue of the Intrinsically Disordered Nuclear Protein 1 Has a Nuclear Localization Sequence that Binds to Human Importin α3.

Project description:Numerous carrier proteins intervene in protein transport from the cytoplasm to the nucleus in eukaryotic cells. One of those is importin α, with several human isoforms; among them, importin α3 (Impα3) features a particularly high flexibility. The protein NUPR1L is an intrinsically disordered protein (IDP), evolved as a paralogue of nuclear protein 1 (NUPR1), which is involved in chromatin remodeling and DNA repair. It is predicted that NUPR1L has a nuclear localization sequence (NLS) from residues Arg51 to Gln74, in order to allow for nuclear translocation. We studied in this work the ability of intact NUPR1L to bind Impα3 and its depleted species, ∆Impα3, without the importin binding domain (IBB), using fluorescence, isothermal titration calorimetry (ITC), circular dichroism (CD), nuclear magnetic resonance (NMR), and molecular docking techniques. Furthermore, the binding of the peptide matching the isolated NLS region of NUPR1L (NLS-NUPR1L) was also studied using the same methods. Our results show that NUPR1L was bound to Imp α3 with a low micromolar affinity (~5 μM). Furthermore, a similar affinity value was observed for the binding of NLS-NUPR1L. These findings indicate that the NLS region, which was unfolded in isolation in solution, was essentially responsible for the binding of NUPR1L to both importin species. This result was also confirmed by our in silico modeling. The binding reaction of NLS-NUPR1L to ∆Impα3 showed a larger affinity (i.e., lower dissociation constant) compared with that of Impα3, confirming that the IBB could act as an auto-inhibition region of Impα3. Taken together, our findings pinpoint the theoretical predictions of the NLS region in NUPR1L and, more importantly, suggest that this IDP relies on an importin for its nuclear translocation.

| S-EPMC7583046 | biostudies-literature

Calcitriol decreases expression of importin α3 and attenuates RelA translocation in human bronchial smooth muscle cells.

Project description:PurposeA potent immunomodulatory role of Vitamin D in both innate and adaptive immunity has recently been appreciated. In allergic asthma, activation of NF-кB induces transcription of various cytokines and chemokines involved in allergic airway inflammation. The nuclear import of activated NF-кB p50/RelA subunit is dependent on importin α3 (KPNA4) and importin α4 (KPNA3). In this study, we examined the role of importin α3 in immunomodulatory effect of calcitriol in human bronchial smooth muscle cells (HBSMCs).MethodsCultured HBSMCs were stimulated with calcitriol in the presence and absence of cytokines, TNF-α, IL-1β, and IL-10. The mRNA transcripts of importin α3 and α4 were analyzed using qPCR while protein expression of importin α3, α4 and nuclear RelA was analyzed by immunoblotting.ResultsCalcitriol significantly decreased mRNA and protein expression of importin α3 as well as nuclear protein expression of NF-кB p65 (RelA). The decreased activation of RelA by calcitriol was confirmed by decreased release of RelA-inducible molecules, including IL-5, IL-6 and IL-8, by HBSMCs upon calcitriol treatment. Calcitriol attenuated the effect of TNF-α and IL-1β to upregulate mRNA and protein expression of importin α3. IL-10 significantly decreased the TNF-α induced expression of importin α3 and this effect was further potentiated by calcitriol.ConclusionsThese data suggest that under inflammatory conditions, calcitriol decreases the expression of importin α3 resulting in decreased nuclear import of activated RelA. This could be a novel mechanism by which calcitriol could exert its immunomodulatory effects to reduce allergic airway inflammation and thus may alleviate the symptoms in allergic asthma.

| S-EPMC3444658 | biostudies-literature

Importin α3 (KPNA3) Deficiency Augments Effortful Reward-Seeking Behavior in Mice.

Project description:Importin α3 (Gene: Kpna3, the ortholog of human Importin α4) is a member of the importin α family and participates in nucleocytoplasmic transport by forming trimeric complexes between cargo proteins and importin β1. Evidence from human studies has indicated that single nucleotide polymorphisms (SNP) in the KPNA3 gene are associated with the occurrence of several psychiatric disorders accompanied by abnormal reward-related behavior, including schizophrenia, major depression, and substance addiction. However, the precise roles of importin α3 in controlling reward processing and motivation are still unclear. In this study, we evaluated the behavioral effects of Kpna3 knockout (KO) in mice on performance in touchscreen operant chamber-based tasks evaluating simple (fixed-ratio) and effortful (progressive-ratio) reward-seeking behaviors. While Kpna3 KO mice showed no significant differences in operant reward learning on a fixed-ratio schedule, they demonstrated significantly increased motivation (increased break point) to instrumentally respond for sucrose on a progressive-ratio schedule. We additionally measured the number of c-Fos-positive cells, a marker of neural activity, in 20 regions of the brain and identified a network of brain regions based on their interregional correlation coefficients. Network and graph-theoretic analyses suggested that Kpna3 deficiency enhanced overall interregional functional connectivity. These findings suggest the importance of Kpna3 in motivational control and indicate that Kpna3 KO mice may be an attractive line for modeling motivational abnormalities associated with several psychiatric disorders.

| S-EPMC9279672 | biostudies-literature

Crystallographic data of an importin-α3 dimer in which the two protomers are bridged by a bipartite nuclear localization signal.

Project description:53BP1 (TP53-binding protein 1), a key player in DNA double-strand break repair, has a classical bipartite nuclear localization signal (NLS) of sequence 1666-GKRKLITSEEERSPAKRGRKS-1686 that binds to importin-α, a nuclear import adaptor protein. Nucleoporin Nup153 is involved in nuclear import of 53BP1, and the binding of Nup153 to importin-α has been proposed to promote efficient import of classical NLS-containing proteins. Here, the ARM-repeat domain of human importin-α3 bound to 53BP1 NLS was crystallized in the presence of a synthetic peptide corresponding to the extreme C-terminus of Nup153 (sequence: 1459-GTSFSGRKIKTAVRRRK-1475). The crystal belonged to space group I2, with unit-cell parameters a = 95.70, b = 79.60, c = 117.44 Å, β = 95.57°. The crystal diffracted X-rays to 1.9 Å resolution, and the structure was solved by molecular replacement. The asymmetric unit contained two molecules of importin-α3 and two molecules of 53BP1 NLS. Although no convincing density was observed for the Nup153 peptide, the electron density corresponding to 53BP1 NLS was unambiguous and continuous along the entire length of the bipartite NLS. The structure revealed a novel dimer of importin-α3, in which two protomers of importin-α3 are bridged by the bipartite NLS of 53BP1. In this structure, the upstream basic cluster of the NLS is bound to the minor NLS-binding site of one protomer of importin-α3, whereas the downstream basic cluster of the same chain of NLS is bound to the major NLS-binding site of another protomer of importin-α3. This quaternary structure is distinctly different from the previously determined crystal structure of mouse importin-α1 bound to the 53BP1 NLS. The atomic coordinates and structure factors have been deposited in the Protein Data Bank (accession code 8HKW).

| S-EPMC9975678 | biostudies-literature

Importin-11, a nuclear import receptor for the ubiquitin-conjugating enzyme, UbcM2.

Project description:Importins are members of a family of transport receptors (karyopherins) that mediate the nucleocytoplasmic transport of protein and RNA cargoes. We identified importin-11 as a potential new human member of this family, on the basis of limited similarity to the Saccharomyces cerevisiae protein, Lph2p, and cloned the complete open reading frame. Importin-11 interacts with the Ran GTPase, and constitutively shuttles between the nuclear and cytoplasmic compartments. A yeast dihybrid screen identified UbcM2, an E2-type ubiquitin-conjugating enzyme, as a binding partner and potential transport cargo for importin-11. Importin-11 and UbcM2 interact directly, and the complex is disassembled by Ran:GTP but not by Ran:GDP. UbcM2 is constitutively nuclear and shuttles between the nuclear and cytoplasmic compartments. Nuclear import of UbcM2 requires Ran and importin-11, and is inhibited by wheatgerm agglutinin, energy depletion or dominant interfering mutants of Ran and importin-beta. These data establish importin-11 as a new member of the karyopherin family of transport receptors, and identify UbcM2 as a nuclear member of the E2 ubiquitin-conjugating enzyme family.

| S-EPMC314000 | biostudies-literature

A minimal nuclear localization signal (NLS) in human phospholipid scramblase 4 that binds only the minor NLS-binding site of importin alpha1.

Project description:Importin ?1 can bind classical nuclear localization signals (NLSs) in two NLS-binding sites, known as "major" and "minor." The major site is located between ARM repeats 2-4, whereas the minor site spans ARM 7-8. In this study, we have characterized the cellular localization of human phospholipid scramblase 4 (hPLSCR4), a member of the phospholipid scramblase protein family. We identified a minimal NLS in hPLSCR4 ((273)GSIIRKWN(280)) that contains only two basic amino acids. This NLS is both necessary for nuclear localization of hPLSCR4 in transfected HeLa cells and sufficient for nuclear import of a non-diffusible cargo in permeabilized cells. Mutation of only one of the two basic residues, Arg(277), correlates with loss of nuclear localization, suggesting this amino acid plays a key role in nuclear transport. Crystallographic analysis of mammalian importin ?1 in complex with the hPLSCR4-NLS reveals this minimal NLS binds specifically and exclusively to the minor binding site of importin ?. These data provide the first structural and functional evidence of a novel NLS-binding mode in importin ?1 that uses only the minor groove as the exclusive site for nuclear import of nonclassical cargos.

| S-EPMC3151061 | biostudies-literature

Importin ?/? mediates nuclear import of individual SUMO E1 subunits and of the holo-enzyme.

Project description:SUMOylation, reversible attachment of small ubiquitin-related modifier (SUMO), serves to regulate hundreds of proteins. Consistent with predominantly nuclear targets, enzymes required for attachment and removal of SUMO are highly enriched in this compartment. This is true also for the first enzyme of the SUMOylation cascade, the SUMO E1 enzyme heterodimer, Aos1/Uba2 (SAE1/SAE2). This essential enzyme serves to activate SUMO and to transfer it to the E2-conjugating enzyme Ubc9. Although the last 40 amino acids in yeast Uba2 have been implicated in its nuclear localization, little was known about the import pathways of Aos1, Uba2, and/or of the assembled E1 heterodimer. Here we show that the mammalian E1 subunits can be imported separately, identify nuclear localization signals (NLSs) in Aos1 and in Uba2, and demonstrate that their import is mediated by importin ?/? in vitro and in intact cells. Once assembled into a stable heterodimer, the E1 enzyme can still be efficiently imported by importin ?/?, due to the Uba2 NLS that is still accessible. These pathways may serve distinct purposes: import of nascent subunits prior to assembly and reimport of stable E1 enzyme complex after mitosis.

| S-EPMC3046061 | biostudies-literature

Cytosolic calcium regulates cytoplasmic accumulation of TDP-43 through Calpain-A and Importin α3.

Project description:Cytoplasmic accumulation of TDP-43 in motor neurons is the most prominent pathological feature in amyotrophic lateral sclerosis (ALS). A feedback cycle between nucleocytoplasmic transport (NCT) defect and TDP-43 aggregation was shown to contribute to accumulation of TDP-43 in the cytoplasm. However, little is known about cellular factors that can control the activity of NCT, thereby affecting TDP-43 accumulation in the cytoplasm. Here, we identified via FRAP and optogenetics cytosolic calcium as a key cellular factor controlling NCT of TDP-43. Dynamic and reversible changes in TDP-43 localization were observed in Drosophila sensory neurons during development. Genetic and immunohistochemical analyses identified the cytosolic calcium-Calpain-A-Importin α3 pathway as a regulatory mechanism underlying NCT of TDP-43. In C9orf72 ALS fly models, upregulation of the pathway activity by increasing cytosolic calcium reduced cytoplasmic accumulation of TDP-43 and mitigated behavioral defects. Together, these results suggest the calcium-Calpain-A-Importin α3 pathway as a potential therapeutic target of ALS.

| S-EPMC7748415 | biostudies-literature

The Drosophila importin-α3 is required for nuclear import of notch in vivo and it displays synergistic effects with notch receptor on cell proliferation.

Project description:The Notch signaling pathway controls diverse cell-fate specification events throughout development. The versatility of this pathway to influence different aspects of development comes from its multiple levels of regulation. Upon ligand-induced Notch activation, the Notch intracellular domain (Notch-ICD) is released from the membrane and translocates to the nucleus, where it transduces Notch signals by regulating the transcription of downstream target genes. But the exact mechanism of translocation of Notch-ICD into the nucleus is not clear. Here, we implicate Importin-α3 (also known as karyopherin-α3) in the nuclear translocation of Notch-ICD in Drosophila. Our present analyses reveal that Importin-α3 can directly bind to Notch-ICD and loss of Importin-α3 function results in cytoplasmic accumulation of the Notch receptor. Using MARCM (Mosaic Analysis with a Repressible Cell Marker) technique, we demonstrate that Importin-α3 is required for nuclear localization of Notch-ICD. These results reveal that the nuclear transport of Notch-ICD is mediated by the canonical Importin-α3/Importin-β transport pathway. In addition, co-expression of both Notch-ICD and Importin-α3 displays synergistic effects on cell proliferation. Taken together, our results suggest that Importin-α3 mediated nuclear import of Notch-ICD may play important role in regulation of Notch signaling.

| S-EPMC3698139 | biostudies-literature

Importin-beta-like nuclear transport receptors.

Project description:SummaryIn recent years, our understanding of macromolecular transport processes across the nuclear envelope has grown dramatically, and a large number of soluble transport receptors mediating either nuclear import or nuclear export have been identified. Most of these receptors belong to one large family of proteins, all of which share homology with the protein import receptor importin beta (also named karyopherin beta). Members of this family have been classified as importins or exportins on the basis of the direction they carry their cargo. To date, the family includes 14 members in the yeast Saccharomyces cerevisiae and at least 22 members in humans. Importins and exportins are regulated by the small GTPase Ran, which is thought to be highly enriched in the nucleus in its GTP-bound form. Importins recognize their substrates in the cytoplasm and transport them through nuclear pores into the nucleus. In the nucleoplasm, RanGTP binds to importins, inducing the release of import cargoes. In contrast, exportins interact with their substrates only in the nucleus in the presence of RanGTP and release them after GTP hydrolysis in the cytoplasm, causing disassembly of the export complex. Thus, common features of all importin-beta-like transport factors are their ability to shuttle between the nucleus and the cytoplasm, their interaction with RanGTP as well as their ability to recognize specific transport substrates.

| S-EPMC138946 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data