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.

PB1792: NGS VS PCR FOR THE DETECTION OF NPM1 GENE MUTATIONS IN ACUTE MYELOID LEUKEMIA.

Background:NPM1 gene mutations are found in 30% of acute myeloid leukemia (AML) cases. It is a reliable marker for the evaluation of therapy outcome and minimal residual disease (MRD) monitoring. The most often mutations are 4bp insertions in exon 12, affecting 2 functionally important tryptophan residues (W288 and W290) in the C-terminal region of nucleophosmin and causing a frameshift. As a result, a leucine-rich motif (L-xxx-V-xx-V-x-L) is formed, which is a signal sequence for protein export from the nucleus to the cytoplasm. Commonly NPM1 gene mutations are detected by fragment analysis (FA) of PCR fragment of 12 exon for the presence of 4bp insert. Alternatively, allele-specific polymerase chain reaction (AS-PCR) to detect inserts belonging to the A, B, or D type (representing up to 90% of all inserts) is used. However, about 10% remaining cases are non-standard inserts and intron polymorphisms that require more precise evaluation. Aims: Evaluation of diverse approaches for efficient NPM1 gene mutation analysis and their robustness for the diagnostics. Methods: The study included 80 DNA samples from AML patients admitted to the National Research Center for Hematology in 2019-2021 that were positive for NPM1 gene mutations detected by FA performed with the Nanophor-5 genetic analyzer (Synthol, Russia). The type of insert was assessed by AC-PCR. Samples in doubt were evaluated by targeted next-generation sequencing (NGS) on MySeq (Illumina, USA). Results: In 62 out of 80 samples, the type of insert (A, B, or D) was reliably determined by AS-PCR. In 9 cases type of insert was not detected. In further 9 cases, amplification ΔCt between wild-type and mutant allele was out of reliable range. These 18 samples were examined by NGS, and 2 out of 18 showed an intron deletion, which, presumably, does not affect the function of the protein, but leads to the appearance of an additional peak on FA. In 16 out of 18 samples, non-standard inserts were detected (Table 1). Type A inserts were found in 48 out of 78 cases (61.5%), type B - in 2 out of 78 (4.5%), and type D - 15.4% (12 out of 78). We have detected atypical inserts in 16 out of 78 cases (20.5%), which is more than reported in the literature. 14 out of 16 atypical inserts result in a leucine-rich motif, as do standard inserts. In 4 out of 16 cases, one of the 2 functionally important tryptophan residues was retained (W280), therefore possibly conserving nuclear localization signal. The effect of partial preservation of the nucleolar localization of nucleophosmin on prognosis in AML has not been studied yet. Image:Summary/Conclusion: FA is still a golden standard for screening NPM1 gene mutations in AML. AS-PCR could be effectively used for monitoring cases with standard inserts. However, NGS greatly facilitates non-standard inserts identification, appropriate allele-specific primers design for MRD analysis, and avoiding false-positive FA results for cases with intron localization of the insert.

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.

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.

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.

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.

TMD1 domain and CRAC motif determine the association and disassociation of MxIRT1 with detergent-resistant membranes.

Iron is essential for most living organisms. The iron-regulated transporter1 (IRT1) plays a major role in iron uptake in roots, and its trafficking from endoplasmic reticulum (ER) to plasma membrane (PM) is tightly coordinated with changes in iron environment. However, studies on the IRT1 response are limited. Here, we report that Malus xiaojinesis IRT1 (MxIRT1) associates with detergent-resistant membranes (DRMs, a biochemical counterpart of PM microdomains), whereas the PM microdomains are known platforms for signal transduction in the PM. Depending on the shift of MxIRT1 from microdomains to homogeneous regions in PM, MxIRT1-mediated iron absorption is activated by the cholesterol recognition/interaction amino acid consensus (CRAC) motif of MxIRT1. MxIRT1 initially associates with DRMs in ER via its transmembrane domain 1 (TMD1), and thus begins DRMs-dependent intracellular trafficking. Subsequently, MxIRT1 is sequestered in COPII vesicles via the ER export signal sequence in MxIRT1. These studies suggest that iron homeostasis is influenced by the CRAC motif and TMD1 domain due to their determination of MxIRT1-DRMs association.

Heterologous expression of a β-D-glucosidase in Caldicellulosiruptor bescii has a surprisingly modest effect on the activity of the exoproteome and growth on crystalline cellulose.

Members of the genus Caldicellulosiruptor are the most thermophilic cellulolytic bacteria so far described and are capable of efficiently utilizing complex lignocellulosic biomass without conventional pretreatment. Previous studies have shown that accumulation of high concentrations of cellobiose and, to a lesser extent, cellotriose, inhibits cellulase activity both in vivo and in vitro and high concentrations of cellobiose are present in C. bescii fermentations after 90h of incubation. For some cellulolytic microorganisms, β-D-glucosidase is essential for the efficient utilization of cellobiose as a carbon source and is an essential enzyme in commercial preparations for efficient deconstruction of plant biomass. In spite of its ability to grow efficiently on crystalline cellulose, no extracellular β-D-glucosidase or its GH1 catalytic domain could be identified in the C. bescii genome. To investigate whether the addition of a secreted β-D-glucosidase would improve growth and cellulose utilization by C. bescii, we cloned and expressed a thermostable β-D-glucosidase from Acidothermus cellulolyticus (Acel_0133) in C. bescii using the CelA signal sequence for protein export. The effect of this addition was modest, suggesting that β-D-glucosidase is not rate limiting for cellulose deconstruction and utilization by C. bescii.

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.

Cytoplasmic, full length and novel cleaved variant, TBLR1 reduces apoptosis in prostate cancer under androgen deprivation.

TBLR1/TBL1XR1, a core component of the nuclear receptor corepressor (NCoR) complex critical for the regulation of multiple nuclear receptors, is a transcriptional coactivator of androgen receptor (AR) and functions as a tumor suppressor when expressed in the nucleus in prostate. Subcellular localization of a protein is critical for its function, and although TBLR1, as a transcriptional cofactor, has been primarily viewed as a nuclear protein, many cells also express variable levels of cytoplasmic TBLR1 and its cytoplasmic specific functions have not been studied. Prostate cancer (PCa) cells express moderately higher level of cytoplasmic TBLR1 compared to benign prostate cells. When comparing androgen-dependent (AD) to androgen-independent (AI) PCa, AI cells contain very high levels of TBLR1 cytoplasmic expression and low levels of nuclear expression. Overexpression of cytoplasmic TBLR1 in AD cells inhibits apoptosis induced by androgen deprivation therapy, either in an androgen free condition or in the presence of bicalutamide. Additionally, we identified a cytoplasmic specific isoform of TBLR1 (cvTBLR1) approximately 5 kDa lower in molecular weight, that is expressed at higher levels in AI PCa cells. By immunoprecipitation, we purified cvTBLR1 and using mass spectrometry analysis combined with N-terminal TMPP labeling and Edman degradation, we identified the cleavage site of cvTBLR1 at amino acid 89, truncating the first 88 amino acids of the N-terminus of the full length protein. Functionally, cvTBLR1 expressed in the cytoplasm reduced apoptosis in PCa cells and promoted growth, migration, and invasion. Finally, we identified a nuclear export signal sequence for TBLR1 cellular localization by deletion and site-directed mutagenesis. The roles of TBLR1 and cvTBLR1 provide novel insights into the mechanism of castration resistance and new strategies for PCa therapy.

Chromosome region maintenance 1 expression and its association with clinical pathological features in primary carcinoma of the liver.

Liver cancer is the third leading cause of cancer-associated mortality worldwide. Recurrence and metastasis are the major factors affecting the prognosis; thus, investigation of the underlying molecular mechanisms of invasion and metastasis, and detection of novel drug target may improve the mortality rate of liver cancer patients. Chromosome region maintenance 1 (CRM1) recognizes specific leucine-rich nuclear export signal sequences, and its overexpression is associated with tumor-suppressor gene inactivation, proliferation, invasion and resistance to chemotherapy. The aim of the present study was to examine the association of CRM1 expression with the clinical and pathological features of primary liver cancer. In total, 152 cases diagnosed with liver cancer were included. CRM1 expression was detected in cancer tissues and adjacent normal tissues by immunohistochemical assay. No statistically significant difference was found between the CRM1 expression levels in tumor and adjacent normal tissues (P=0.106). However, CRM1 expression in adjacent normal tissues was higher compared with that in tumor tissues in the negative hepatitis B envelope antigen (HBeAg; P=0.029) and low differentiation (P=0.004) groups. In tumor tissues, CRM1 expression was significantly correlated with differentiation (P=0.045), whereas in adjacent normal tissues, CRM1 expression was significantly correlated with the tumor diameter (P=0.004). Therefore, it can be concluded that CRM1 is highly expressed in both tumor and adjacent normal tissues. Furthermore, CRM1 expression is associated with the tumor differentiation degree and diameter. Lower differentiation and larger tumor diameter resulted in higher CRM1 expression in adjacent normal tissues, and higher tendency for invasion and metastasis. In addition, the risk of invasion and metastasis remains in chronic hepatitis B patients with negative HBeAg.

Oocyte-Specific Expression of Mouse MEX3C652AA in the Ovary and Its Potential Role in Regulating Maternal Fos mRNA.

Currently, the human MEX3C gene is known to encode an RNA-binding protein of 659 amino acid residues. Here we show that the MEX3C gene has alternative splicing forms giving rise to multiple MEX3C variants, and some cells express MEX3C transcripts coding for short MEX3C isoforms but not transcripts for MEX3C(659AA) MEX3C(659AA) functions as an adaptor protein for Exportin 1 (XPO1)-mediated nuclear export since it increases the cytoplasmic distribution of poly(A)(+) RNA and since addition of the nuclear export signal (NES) sequence to a short MEX3C isoform MEX3C(464AA) confers similar cytoplasmic poly(A)(+) RNA accumulation activity as MEX3C(659AA) FOS mRNA is a potential MEX3C target mRNA. One mechanism by which MEX3C(659AA) could regulate FOS mRNA is by promoting its nuclear export. Overexpressing MEX3C(659AA) significantly increased FOS mRNA expression, whereas mutating the NES of MEX3C(659AA) and treating cells with leptomycin B to inhibit XPO1-mediated nuclear export attenuated FOS upregulation. FOS mRNA is unstable in somatic cells but less so in oocytes; how it is stabilized in the oocytes is unknown. Transcripts for the mouse counterpart of human MEX3C(659AA) (MEX3C(652AA)) are specifically expressed in developing oocytes in the ovary, although total Mex3c transcripts are expressed in both granulosa cells and oocytes. The specific expression of this long MEX3C isoform in oocytes and its ability to enhance FOS mRNA nuclear export and stability all suggest that MEX3C(659AA) is an RNA-binding protein that preserves maternal FOS mRNA in oocytes.

Interleukin 33 as a mechanically responsive cytokine secreted by living cells

Background: Interleukin 33 (IL-33), a member of the Interleukin 1 cytokine family, is implicated in numerous human inflammatory diseases such as asthma, atherosclerosis and rheumatoid arthritis. Despite its pathophysiologic importance, fundamental questions regarding the basic biology of IL-33 remain. Nuclear localization and lack of an export signal sequence are consistent with the view of IL-33 as a nuclear factor with the ability to repress RNA transcription. However, signaling via the transmembrane receptor ST2 and documented caspase-dependent inactivation have suggested IL-33 is liberated during cellular necrosis to effect paracrine signaling.