Nuclear Export Signal Sequence Research Articles

Enhanced C-To-T and A-To-G Base Editing in Mitochondrial DNA with Engineered DdCBE and TALED.

Mitochondrial base editing with DddA-derived cytosine base editor (DdCBE) is limited in the accessible target sequences and modest activity. Here, the optimized DdCBE tools is presented with improved editing activity and expanded C-to-T targeting scope by fusing DddA11 variant with different cytosine deaminases with single-strand DNA activity. Compared to previous DdCBE based on DddA11 variant alone, fusion of the activation-induced cytidine deaminase (AID) from Xenopus laevis not only permits cytosine editing of 5'-GC-3' sequence, but also elevates editing efficiency at 5'-TC-3', 5'-CC-3', and 5'-GC-3' targets by up to 25-, 10-, and 6-fold, respectively. Furthermore, the A-to-G editing efficiency is significantly improved by fusing the evolved DddA6 variant with TALE-linked deoxyadenosine deaminase (TALED). Notably, the authors introduce the reported high-fidelity mutations in DddA and add nuclear export signal (NES) sequences in DdCBE and TALED to reduce off-target editing in the nuclear and mitochondrial genome while improving on-target editing efficiency in mitochondrial DNA (mtDNA). Finally, these engineered mitochondrial base editors are shown to be efficient in installing mtDNA mutations in human cells or mouse embryos for disease modeling. Collectively, the study shows broad implications for the basic study and therapeutic applications of optimized DdCBE and TALED.

Experience-dependent Tip60 nucleocytoplasmic transport is regulated by its NLS/NES sequences for neuroplasticity gene control

Nucleocytoplasmic transport (NCT) in neurons is critical for enabling proteins to enter the nucleus and regulate plasticity genes in response to environmental cues. Such experience-dependent (ED) neural plasticity is central for establishing memory formation and cognitive function and can influence the severity of neurodegenerative disorders like Alzheimer's disease (AD). ED neural plasticity is driven by histone acetylation (HA) mediated epigenetic mechanisms that regulate dynamic activity-dependent gene transcription profiles in response to neuronal stimulation. Yet, how histone acetyltransferases (HATs) respond to extracellular cues in the in vivo brain to drive HA-mediated activity-dependent gene control remains unclear. We previously demonstrated that extracellular stimulation of rat hippocampal neurons in vitro triggers Tip60 HAT nuclear import with concomitant synaptic gene induction. Here, we focus on investigating Tip60 HAT subcellular localization and NCT specifically in neuronal activity-dependent gene control by using the learning and memory mushroom body (MB) region of the Drosophila brain as a powerful in vivo cognitive model system. We used immunohistochemistry (IHC) to compare the subcellular localization of Tip60 HAT in the Drosophila brain under normal conditions and in response to stimulation of fly brain neurons in vivo either by genetically inducing potassium channels activation or by exposure to natural positive ED conditions. Furthermore, we found that both inducible and ED condition-mediated neural induction triggered Tip60 nuclear import with concomitant induction of previously identified Tip60 target genes and that Tip60 levels in both the nucleus and cytoplasm were significantly decreased in our well-characterized Drosophila AD model. Mutagenesis of a putative nuclear localization signal (NLS) sequence and nuclear export signal (NES) sequence that we identified in the Drosophila Tip60 protein revealed that both are functionally required for appropriate Tip60 subcellular localization. Our results support a model by which neuronal stimulation triggers Tip60 NCT via its NLS and NES sequences to promote induction of activity-dependent neuroplasticity gene transcription and that this process may be disrupted in AD.

Nuclear translocation of MLKL enhances necroptosis by a RIP1/RIP3-independent mechanism in H9c2 cardiomyoblasts

Accumulating evidence suggests that necroptosis of cardiomyocytes contributes to cardiovascular diseases. Lethal disruption of the plasma membrane in necroptosis is induced by oligomers of mixed lineage kinase domain-like (MLKL) that is translocated to the membrane from the cytosol. However, the role played by cytoplasmic-nuclear shuttling of MLKL is unclear. Here, we tested the hypothesis that translocation of MLKL to the nucleus promotes the necroptosis of cardiomyocytes. Activation of the canonical necroptotic signaling pathway by a combination of TNF-α and zVAD (TNF/zVAD) increased nuclear MLKL levels in a RIP1-activity-dependent manner in H9c2 cells, a rat cardiomyoblast cell line. By use of site-directed mutagenesis, we found a nuclear export signal sequence in MLKL and prepared its mutant (MLKL-L280/283/284A), though a search for a nuclear import signal was unsuccessful. MLKL-L280/283/284A localized to both the cytosol and the nucleus. Expression of MLKL-L280/283/284A induced necroptotic cell death, which was attenuated by GppNHp, an inhibitor of Ran-mediated nuclear import, but not by inhibition of RIP1 activity or knockdown of RIP3 expression. GppNHp partly suppressed H9c2 cell death induced by TNF/zVAD treatment. These results suggest that MLKL that is translocated to the nucleus via RIP1-mediated necroptotic signaling enhances the necroptosis of cardiomyocytes through a RIP1-/RIP3-independent mechanism.

C-terminal deletion-induced condensation sequesters AID from IgH targets in immunodeficiency.

In activated B cells, activation‐induced cytidine deaminase (AID) generates programmed DNA lesions required for antibody class switch recombination (CSR), which may also threaten genome integrity. AID dynamically shuttles between cytoplasm and nucleus, and the majority stays in the cytoplasm due to active nuclear export mediated by its C‐terminal peptide. In immunodeficient‐patient cells expressing mutant AID lacking its C‐terminus, a catalytically active AID‐delC protein accumulates in the nucleus but nevertheless fails to support CSR. To resolve this apparent paradox, we dissected the function of AID‐delC proteins in the CSR process and found that they cannot efficiently target antibody genes. We demonstrate that AID‐delC proteins form condensates both in vivo and in vitro, dependent on its N‐terminus and on a surface arginine‐rich patch. Co‐expression of AID‐delC and wild‐type AID leads to an unbalanced nuclear AID‐delC/AID ratio, with AID‐delC proteins able to trap wild‐type AID in condensates, resulting in a dominant‐negative phenotype that could contribute to immunodeficiency. The co‐condensation model of mutant and wild‐type proteins could be an alternative explanation for the dominant‐negative effect in genetic disorders.

O-GlcNAc transferase promotes the nuclear localization of the focal adhesion–associated protein Zyxin to regulate UV-induced cell death

Zyxin is a zinc-binding phosphoprotein known to regulate cell migration, adhesion, and cell survival. Zyxin also plays a role in signal transduction between focal adhesions and the nuclear compartment. However, the mechanism of Zyxin shuttling to nucleus is still unclear. Here, we identify that the GlcNAc transferase (O-linked GlcNAc [O-GlcNAc] transferase) can O-GlcNAcylate Zyxin and regulate its nuclear localization. We show that O-GlcNAc transferase O-GlcNAcylates Zyxin at two residues, serine 169 (Ser-169) and Ser-246. In addition, O-GlcNAcylation of Ser-169, but not Ser-246, enhances its interaction with 14-3-3γ, which is a phosphoserine/threonine-binding protein and is reported to bind with phosphorylated Zyxin. Furthermore, we found that 14-3-3γ could promote the nuclear localization of Zyxin after Ser-169 O-GlcNAcylation by affecting the function of the N-terminal nuclear export signal sequence; functionally, UV treatment increases the O-GlcNAcylation of Zyxin, which may enhance the nuclear location of Zyxin. Finally, Zyxin in the nucleus maintains homeodomain-interacting protein kinase 2 stability and promotes UV-induced cell death. In conclusion, we uncover that the nuclear localization of Zyxin can be regulated by its O-GlcNAcylation, and that this protein may regulate UV-induced cell death.

Keratin 17 Promotes T Cell Response in Allergic Contact Dermatitis by Upregulating C-C Motif Chemokine Ligand 20.

Allergic contact dermatitis (ACD) is a delayed-type hypersensitivity response to skin contact allergens in which keratinocytes are critical in the initiation of early responses. Keratin 17 (K17) is a cytoskeletal protein inducible under stressful conditions and regulates multiple cellular processes, especially in skin inflammatory diseases; however, knowledge regarding its contribution to ACD pathogenesis remains ill defined. In the present study, we clarified the proinflammatory role of K17 in an oxazolone (OXA)-induced contact hypersensitivity (CHS) murine model and identified the underlying molecular mechanisms. Our results showed that K17 was highly expressed in the lesional skin of ACD patients and OXA-induced CHS mice. Mice lacking K17 exhibited alleviated OXA-induced skin inflammation, including milder ear swelling, a reduced frequency of T cell infiltration, and decreased inflammatory cytokine levels. In vitro, K17 stimulated and activated human keratinocytes to produce plenty of proinflammatory mediators, especially the chemokine CCL20, and promoted keratinocyte-mediated T cell trafficking. The neutralization of CCL20 with a CCL20-neutralizing monoclonal antibody significantly alleviated OXA-induced skin inflammation in vivo. Moreover, K17 could translocate into the nucleus of activated keratinocytes through a process dependent on the nuclear-localization signal (NLS) and nuclear-export signal (NES) sequences, thus facilitating the activation and nuclear translocation of signal transducer and activator of transcription 3 (STAT3), further promoting the production of CCL20 and T cell trafficking to the lesional skin. Taken together, these results highlight the novel roles of K17 in driving allergen-induced skin inflammation and suggest targeting K17 as a potential strategy for ACD.

Phosphorylation in the accessory domain of yeast histone chaperone protein 1 exposes the nuclear export signal sequence.

Histone chaperone proteins assist in the formation of the histone octamers, the scaffold proteins that facilitate the packing of DNA into nucleosomes in the cell nucleus. One such histone chaperone protein is yeast nucleosome assembly protein 1 (yNap1), the crystal structure of which has been determined and found to have a nuclear export signal (NES) sequence within its long α-helix. Experimental evidence obtained from mutagenesis studies of the budding yeast suggests that the NES is necessary for the transport of yNap1 from the cell nucleus to the cytosol. However, the NES sequence is masked by an accessory domain, the exact role of which has not yet been elucidated, especially in nucleocytoplasmic transport. To clarify the role of the accessory domain, we focused on its phosphorylation, because proteomic experiments have identified multiple phosphorylation sites on yNap1. To study this phenomenon computationally, all-atom molecular dynamics simulations of the non-phosphorylated yNap1 (Nap1-nonP) and phosphorylated yNap1 (Nap1-P) systems were performed. Specifically, we addressed how the NES sequence is exposed to the protein surface by measuring its solvent-accessible surface area (SASA). It was found that the median of the SASA distribution of Nap1-P was greater than that of Nap1-nonP, indicating that phosphorylation in the accessory domain exposes the NES, resulting in its increased accessibility. In conclusion, yNap1 might modulate the accessibility of the NES by dislocating the accessory domain through its phosphorylation.

Importin-\u03b1s are required for the nuclear localization and function of the Plasmopara viticola effector PvAVH53

Plant pathogenic oomycetes deliver a troop of effector proteins into the nucleus of host cells to manipulate plant cellular immunity and promote colonization. Recently, researchers have focused on identifying how effectors are transferred into the host cell nucleus, as well as the identity of the nuclear targets. In this study, we found that the RxLR effector PvAVH53 from the grapevine (Vitis vinifera) oomycete pathogen Plasmopara viticola physically interacts with grapevine nuclear import factor importin alphas (VvImpα and VvImpα4), localizes to the nucleus and triggers cell death when transiently expressed in tobacco (Nicotiana benthamiana) cells. Deletion of a nuclear localization signal (NLS) sequence from PvAVH53 or addition of a nuclear export signal (NES) sequence disrupted the nuclear localization of PvAVH53 and attenuated its ability to trigger cell death. Suppression of two tobacco importin-α genes, namely, NbImp-α1 and NbImp-α2, by virus-induced gene silencing (VIGS) also disrupted the nuclear localization and ability of PvAVH53 to induce cell death. Likewise, we transiently silenced the expression of VvImpα/α4 in grape through CRISPR/Cas13a, which has been reported to target RNA in vivo. Finally, we found that attenuating the expression of the Importin-αs genes resulted in increased susceptibility to the oomycete pathogen Phytophthora capsici in N. benthamiana and P. viticola in V. vinifera. Our results demonstrate that importin-αs are required for the nuclear localization and function of PvAVH53 and are essential for host innate immunity. The findings provide insight into the functions of importin-αs in grapevine against downy mildew.

Identification of nuclear export signal in KLLN suggests potential role in proteasomal degradation in cancer cells.

Germline and somatic promoter hypermethylation of KLLN has been found in diverse heritable and sporadic cancers, respectively. KLLN has many identified tumor suppressor functions, and when first reported, was thought to be exclusively nuclear. Here, we report on KLLN localization in both the nucleus and cytoplasm and the identification of a putative nuclear export signal (NES) sequence. KLLN overexpression in colon and breast cancer cells showed both nuclear and cytoplasmic presence. Inhibition of the CRM1 export pathway increased nuclear sequestration of KLLN, confirming the prediction of an NES sequence. Point mutations introduced in the predicted NES sequence decreased the strength of the NES and increased the nuclear sequestration of KLLN. Contrary to expectations, the transcription regulation and cellular proliferation functions of KLLN were unaffected by increased KLLN nuclear sequestration. Instead, increased nuclear KLLN correlated with increased nuclear sequestration of TRIM25 and decreased inhibitory phosphorylation of MDM2. Computational analysis of The Cancer Genome Atlas (TCGA) dataset showed positive correlation among KLLN, TRIM25 and MDM2 expression; pathway analysis of the common genes downstream of these three genes revealed protein degradation as one of the top canonical pathways. Together, our observations suggest that CRM1 pathway-based nuclear export of KLLN may impact proteasomal degradation.

A newly identified NES sequence present in spastin regulates its subcellular localization and microtubule severing activity

Spastin, a microtubule-severing AAA ATPase, regulates microtubule dynamics and plays important roles in cell division and neurogenesis. Mutations in the spastin-coding gene SPAST lead to neurodegenerative disorders and cause spastic paraplegia type 4. Spastin has two main isoforms, M1 and M87, that differ only in the presence or absence of 86 N-terminal amino acids and have alternative splicing variants that lack exon4. The N-terminal region of M1 contains a hydrophobic domain, nuclear localization signal (NLS), and nuclear export signal (NES), which partly explains the differences in the two isoforms' localization. However, the mechanisms involved in regulating isoform localization, and the effects of localization on spastin functions are not fully understood. We found endogenous M1 and M87 shuttled between the nucleus and cytoplasm during the cell cycle. We identified a NES (amino acids 195–204) that spans the microtubule-interacting and endosomal-trafficking domain and exon4 region. Furthermore, the NES sequence contains both the coiled-coil and exon4 region of spastin isoforms. Highly conserved leucine 195 in exon3 and the two residues in exon4 are crucial for predicted coiled-coil formation. Mutations in NES or leptomycin B treatment reduced cytoplasmic localization and microtubule fragmentation in M87 rather than in M1. Phosphomimetic mutation of threonine 306 adjacent to the NLS (amino acids 309–312) inhibited nuclear transport of M87. Our results indicate that the newly identified NES in the spastin isoforms containing exon4 regulates the subcellular localization of spastin in coordination with NLS controlled by the phosphorylation state of spastin, and is involved in microtubule severing.

Pro-515 of the dynamin-like GTPase MxB contributes to HIV-1 inhibition by regulating MxB oligomerization and binding to HIV-1 capsid

Interferon-regulated myxovirus resistance protein B (MxB) is an interferon-induced GTPase belonging to the dynamin superfamily. It inhibits infection with a wide range of different viruses, including HIV-1, by impairing viral DNA entry into the nucleus. Unlike the related antiviral GTPase MxA, MxB possesses an N-terminal region that contains a nuclear localization signal and is crucial for inhibiting HIV-1. Because MxB previously has been shown to reside in both the nuclear envelope and the cytoplasm, here we used bioinformatics and biochemical approaches to identify a nuclear export signal (NES) responsible for MxB's cytoplasmic location. Using the online computational tool LocNES (Locating Nuclear Export Signals or NESs), we identified five putative NES candidates in MxB and investigated whether their deletion caused nuclear localization of MxB. Our results revealed that none of the five deletion variants relocates to the nucleus, suggesting that these five predicted NES sequences do not confer NES activity. Interestingly, deletion of one sequence, encompassing amino acids 505-527, abrogated the anti-HIV-1 activity of MxB. Further mutation experiments disclosed that amino acids 515-519, and Pro-515 in particular, regulate MxB oligomerization and its binding to HIV-1 capsid, thereby playing an important role in MxB-mediated restriction of HIV-1 infection. In summary, our results indicate that none of the five predicted NES sequences in MxB appears to be required for its nuclear export. Our findings also reveal several residues in MxB, including Pro-515, critical for its oligomerization and anti-HIV-1 function.

Sj\xf6gren syndrome/scleroderma autoantigen 1 is a direct Tankyrase binding partner in cancer cells

Sjögren syndrome/scleroderma autoantigen 1 (SSSCA1) was first described as an auto-antigen over-expressed in Sjögren’s syndrome and in scleroderma patients. SSSCA1 has been linked to mitosis and centromere association and as a potential marker candidate in diverse solid cancers. Here we characterize SSSCA1 for the first time, to our knowledge, at the molecular, structural and subcellular level. We have determined the crystal structure of a zinc finger fold, a zinc ribbon domain type 2 (ZNRD2), at 2.3 Å resolution. We show that the C-terminal domain serves a dual function as it both behaves as the interaction site to Tankyrase 1 (TNKS1) and as a nuclear export signal. We identify TNKS1 as a direct binding partner of SSSCA1, map the binding site to TNKS1 ankyrin repeat cluster 2 (ARC2) and thus define a new binding sequence. We experimentally verify and map a new nuclear export signal sequence in SSSCA1.

Structural prerequisites for CRM1-dependent nuclear export signaling peptides: accessibility, adapting conformation, and the stability at the binding site

Nuclear export signal (NES) motifs function as essential regulators of the subcellular location of proteins by interacting with the major nuclear exporter protein, CRM1. Prediction of NES is of great interest in many aspects of research including cancer, but currently available methods, which are mostly based on the sequence-based approaches, have been suffered from high false positive rates since the NES consensus patterns are quite commonly observed in protein sequences. Therefore, finding a feature that can distinguish real NES motifs from false positives is desired to improve the prediction power, but it is quite challenging when only using the sequence. Here, we provide a comprehensive table for the validated cargo proteins, containing the location of the NES consensus patterns with the disordered propensity plots, known protein domain information, and the predicted secondary structures. It could be useful for determining the most plausible NES region in the context of the whole protein sequence and suggests possibilities for some non-binders of the annotated regions. In addition, using the currently available crystal structures of CRM1 bound to various classes of NES peptides, we adopted, for the first time, the structure-based prediction of the NES motifs bound to the CRM1’s binding groove. Combining sequence-based and structure-based predictions, we suggest a novel and more straight-forward approach to identify CRM1-binding NES sequences by analysis of their structural prerequisites and energetic evaluation of the stability at the CRM1’s binding site.

Blocking nuclear export of HSPA8 after heat shock stress severely alters cell survival

The nuclear translocation of endogenous heat shock cognate protein HSPA8 is a requisite for cell survival during oxidative and heat shock stress. Upon these events, cytoplasmic HSPA8 is thought to concentrate within the nucleus and nucleolus. When the situation returns to normal, HSPA8 is released from its nuclear/nucleolar anchors and redistributes into the cytoplasm. By using different stress conditions and a 21-mer phosphopeptide tool called P140, which binds HSPA8 and hampers its chaperone properties, we deciphered the cellular and molecular effects arising during this vital cytoplasmic-nuclear-cytoplasmic shuttling process. Using the non-metastatic fibroblastoid cell line MRL/N-1 derived from a MRL/MpTn-gld/gld lupus-prone mouse, we discovered that P140 treatment neutralized the egress of HSPA8 from nucleus to cytoplasm in the cell recovery phase. This lack of relocation of HSPA8 into the cytoplasm of heat-shocked MRL/N-1 cells altered the ability of these cells to survive when a second mild oxidative stress mimicking inflammatory conditions was applied. Crosslinking experiments followed by proteomics studies showed that P140 binds regions close to nuclear import and export signal sequences encompassed within the HSPA8 structure. These data are consistent with HSPA8 having a crucial cell protective role against reactive oxygen species (ROS) production by mitochondria during inflammatory conditions.

Abstract 4018: Mutations of HER2 at L755 residue results in HER2 nuclear accumulation and enhances breast cancer stem cell activity

Abstract Background: HER2 plays a critical role in tumor progression and cancer stemness in breast cancer cells. Somatic mutations in HER2 gene have been observed in low HER2-expressing cancer cells and are correlated with insensitization to tyrosine kinase inhibitors. One of the most frequently altered residue is L755, which is located at kinase domain and near a putative nuclear export signal sequence, increasing kinase activity and is associated with lapatinib resistance. Moreover, the correlation between HER2 nuclear localization and drug-resistance and poor clinical outcome has been demonstrated in cancer patients. However, the roles of HER2 mutations in its nuclear distribution and its ability to enhance cancer stemness remain unknown. Methods: We hypothesized that activation of HER2 mutations on amino acid L755 might affect HER2 cellular localization. To investigate the cellular localization of HER2 and the role of L755 mutations in expansion of cancer stem cells, T47D cell line clones expressing wile-type (WT) and mutant HER2 (L755P and L755S) were established. The cellular fractionation, immunofluorescence staining, and western blot analysis were employed to verify the cellular distribution of HER2 L755 mutations. Biochemical strategies were further performed to examine the interaction of HER2 L755 mutant and cellular transporters nuclear importers or exporters. Results: Our results showed that ectopically expressed HER2 L755P and L755S mutants exhibited a higher nuclear retention via interrupting nuclear exporting complex formation. HER2 L755P and HER2 L755S showed higher abilities to associate with CRM1 and RanBP3 but reduced the binding capacity with Ran. The increases in tyrosine phosphorylation of CRM1 and RanBP3 were found by HER2 mutants and cause the defect in the formation of nuclear exporting complex. Additionally, the HER2 L755P and HER2 L755S-expressing cell lines showed higher levels of cancer stem cell markers, CD44 and ALDH1 and increased mammosphere formation. Conclusion: HER2 L755P and L755S mutations enhance the HER2 nuclear localization and breast cancer stemness. This study provided new insight into the role of HER2 mutation in cancer stemness, and was helpful to develop novel prognostic markers for the therapeutic efficacy in breast cancer patients. Citation Format: Wen-Ling Wang, Lei Nie, Kieu-Thanh Huynh, Jhen-Yu Chen, Jing-Han Yao, Mien-Chie Hung, Wei-Chien Huang. Mutations of HER2 at L755 residue results in HER2 nuclear accumulation and enhances breast cancer stem cell activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4018.

Mapping of domains of heat stress transcription factor OsHsfA6a responsible for its transactivation activity

Elevated temperatures affect the growth and reproduction of crop plants and thus have become concern worldwide. Hsp101/ClpB protein is a major molecular chaperone, performing dis-aggregation of protein aggregates formed during heat stress. In rice, OsHsfA6a binds to the promoter of OsHsp101/ClpB-C and regulates its expression. In this study, analysis of C-terminal domains of ClassA OsHsfs revealed the presence of aromatic, hydrophobic, acidic (AHA) and nuclear export signal (NES) motifs in all the members. Using deletion constructs, we show that the activation potential of OsHsfA6a is confined in the C-terminal activation domain comprising of AHA and NES sequences. The results obtained in yeast were complemented with transient expression of reporter in protoplast (TERP) based assay. Detailed analysis of OsHsfA6a splice variants shows the presence of one full version and a DBD truncated smaller version whose existence needs experimental evidences. Phylogeny analysis revealed that OsHsfA6a has diverged from A6a/A6b forms of Arabidopsis and tomato and has no expressologs. OsHsfA6a in-silico network was enriched in MAP kinases along with Hsp70 and Hsp90 proteins. Thus, it appears that regulation of OsClpB-C by HsfA6a is unique in rice and activation potential of OsHsfA6a resides in the single AHA motif located in the C-terminal domain.

Structural basis for the regulatory interactions of proapoptotic Par-4

Par-4 is a unique proapoptotic protein with the ability to induce apoptosis selectively in cancer cells. The X-ray crystal structure of the C-terminal domain of Par-4 (Par-4CC), which regulates its apoptotic function, was obtained by MAD phasing. Par-4 homodimerizes by forming a parallel coiled-coil structure. The N-terminal half of Par-4CC contains the homodimerization subdomain. This structure includes a nuclear export signal (Par-4NES) sequence, which is masked upon dimerization indicating a potential mechanism for nuclear localization. The heteromeric-interaction models specifically showed that charge interaction is an important factor in the stability of heteromers of the C-terminal leucine zipper subdomain of Par-4 (Par-4LZ). These heteromer models also displayed NES masking capacity and therefore the ability to influence intracellular localization.

DDX17 nucleocytoplasmic shuttling promotes acquired gefitinib resistance in non-small cell lung cancer cells via activation of β-catenin

Although epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are effective for non-small cell lung cancer (NSCLC) patients with EGFR mutations, almost all these patients will eventually develop acquired resistance to EGFR-TKI. However, the molecular mechanisms responsible for gefitinib resistance remain still not fully understood. Here, we report that elevated DDX17 levels are observed in gefitinib-resistant NSCLC cells than gefitinib-sensitive cells. Upregulation of DDX17 enhances the gefitinib resistance, whereas DDX17-silenced cells partially restore gefitinib sensitivity. Mechanistically, we demonstrate that DDX17 disassociates the E-cadherin/β-catenin complex, resulting in β-catenin nuclear translocation and subsequently augmenting the transcription of β-catenin target genes. Moreover, we identify two nuclear localization signal (NLS) and four nuclear export signal (NES) sequences mediated DDX17 nucleocytoplasmic shuttling via an exportin/importin-dependent pathways. Interruption of dynamic nucleocytoplasmic shuttling of DDX17 impairs DDX17-mediating the activation of β-catenin and acquired resistance in NSCLC cells. In conclusion, our findings reveal a novel and important mechanism by which DDX17 contributes to acquired gefitinib resistance through exportin/importin-dependent cytoplasmic shuttling and followed by activation of β-catenin, and DDX17 inhibition may be a promising strategy to overcome acquired resistance of gefitinib in NSCLC patients.

The Silencing of a 14-3-3ɛ Homolog in Tenebrio molitor Leads to Increased Antimicrobial Activity in Hemocyte and Reduces Larval Survivability.

The 14-3-3 family of phosphorylated serine-binding proteins acts as signaling molecules in biological processes such as metabolism, division, differentiation, autophagy, and apoptosis. Herein, we report the requirement of 14-3-3ɛ isoform from Tenebrio molitor (Tm14-3-3ɛ) in the hemocyte antimicrobial activity. The Tm14-3-3ɛ transcript is 771 nucleotides in length and encodes a polypeptide of 256 amino acid residues. The protein has the typical 14-3-3 domain, the nuclear export signal (NES) sequence, and the peptide binding residues. The Tm14-3-3ɛ transcript shows a significant three-fold expression in the hemocyte of T. molitor larvae when infected with Escherichia coli Tm14-3-3ɛ silenced larvae show significantly lower survival rates when infected with E. coli. Under Tm14-3-3ɛ silenced condition, a strong antimicrobial activity is elicited in the hemocyte of the host inoculated with E. coli. This suggests impaired secretion of antimicrobial peptides (AMP) into the hemolymph. Furthermore, a reduction in AMP secretion under Tm14-3-3ɛ silenced condition would be responsible for loss in the capacity to kill bacteria and might explain the reduced survivability of the larvae upon E. coli challenge. This shows that Tm14-3-3ɛ is required to maintain innate immunity in T. molitor by enabling antimicrobial secretion into the hemolymph and explains the functional specialization of the isoform.

A cellular reporter to evaluate CRM1 nuclear export activity: functional analysis of the cancer-related mutant E571K.

The exportin CRM1 binds nuclear export signals (NESs), and mediates active transport of NES-bearing proteins from the nucleus to the cytoplasm. Structural and biochemical analyses have uncovered the molecular mechanisms underlying CRM1/NES interaction. CRM1 binds NESs through a hydrophobic cleft, whose open or closed conformation facilitates NES binding and release. Several cofactors allosterically modulate the conformation of the NES-binding cleft through intramolecular interactions involving an acidic loop and a C-terminal helix in CRM1. This current model of CRM1-mediated nuclear export has not yet been evaluated in a cellular setting. Here, we describe SRV100, a cellular reporter to interrogate CRM1 nuclear export activity. Using this novel tool, we provide evidence further validating the model of NES binding and release by CRM1. Furthermore, using both SRV100-based cellular assays and in vitro biochemical analyses, we investigate the functional consequences of a recurrent cancer-related mutation, which targets a residue near CRM1 NES-binding cleft. Our data indicate that this mutation does not necessarily abrogate the nuclear export activity of CRM1, but may increase its affinity for NES sequences bearing a more negatively charged C-terminal end.