Shuttling Protein Research Articles

Substrate mechanics controls adipogenesis through YAP phosphorylation by dictating cell spreading

The mechanoregulated proteins YAP/TAZ are involved in the adipogenic/osteogenic switch of mesenchymal stem cells (MSCs).MSC fate decision can be unbalanced by controlling substrate mechanics, in turn altering the transmission of tension through cell cytoskeleton. MSCs have been proposed for orthopedic and reconstructive surgery applications. Thus, a tight control of their adipogenic potential is required in order to avoid their drifting towards fat tissue. Substrate mechanics has been shown to drive MSC commitment and to regulate YAP/TAZ protein shuttling and turnover. The mechanism by which YAP/TAZ co-transcriptional activity is mechanically regulated during MSC fate acquisition is still debated.Here, we design few bioengineering tools suited to disentangle the contribution of mechanical from biological stimuli to MSC adipogenesis. We demonstrate that the mechanical repression of YAP happens through its phosphorylation, is purely mediated by cell spreading downstream of substrate mechanics as dictated by dimensionality. YAP repression is sufficient to prompt MSC adipogenesis, regardless of a permissive biological environment, TEAD nuclear presence or focal adhesion stabilization.Finally, by harnessing the potential of YAP mechanical regulation, we propose a practical example of the exploitation of adipogenic transdifferentiation in tumors.

ABA inhibits myristoylation and induces shuttling of the RGLG1 E3 ligase to promote nuclear degradation of PP2CA.

Hormone- and stress-induced shuttling of signaling or regulatory proteins is an important cellular mechanism to modulate hormone signaling and cope with abiotic stress. Hormone-induced ubiquitination plays a crucial role to determine the half-life of key negative regulators of hormone signaling. For ABA signaling, the degradation of clade-A PP2Cs, such as PP2CA or ABI1, is a complementary mechanism to PYR/PYL/RCAR-mediated inhibition of PP2C activity. ABA promotes the degradation of PP2CA through the RGLG1 E3 ligase, although it is not known how ABA enhances the interaction of RGLG1 with PP2CA given that they are predominantly found in the plasma membrane and the nucleus, respectively. We demonstrate that ABA modifies the subcellular localization of RGLG1 and promotes nuclear interaction with PP2CA. We found RGLG1 is myristoylated invivo, which facilitates its attachment to the plasma membrane. ABA inhibits the myristoylation of RGLG1 through the downregulation of N-myristoyltransferase1 (NMT1) and promotes nuclear translocation of RGLG1 in a cycloheximide-insensitive manner. Enhanced nuclear recruitment of the E3 ligase was also promoted by increasing PP2CA protein levels and the formation of RGLG1-receptor-phosphatase complexes. We show that RGLG1Gly2Ala mutated at the N-terminal myristoylation site shows constitutive nuclear localization and causes an enhanced response to ABA and salt or osmotic stress. RGLG1/5 can interact with certain monomeric ABA receptors, which facilitates the formation of nuclear complexes such as RGLG1-PP2CA-PYL8. In summary, we provide evidence that an E3 ligase can dynamically relocalize in response to both ABA and increased levels of its target, which reveals a mechanism to explain how ABA enhances RGLG1-PP2CA interaction and hence PP2CA degradation.

Nucleocytoplasmic shuttling of SAMHD1 is important for LINE-1 suppression

Currently, SAMHD1 is the only known dNTPase in human cells. It also suppresses the replication of both retroviruses and retroelements. SAMHD1 contains a classic nuclear localization sequence (NLS) and resides in the nucleus in live or fixed cells. It has been reported that alteration or removal of NLS does not affect the dNTPase or the antiviral activity of SAMHD1. However, it was unclear whether the nuclear localization was involved in SAMHD1-mediated suppression against retroelements such as long interspersed element type 1 (LINE-1 or L1). In this study, we reported that SAMHD1 is a nucleocytoplasmic shuttling protein. Digitonin-based cytoplasm/nucleus fractionation tests suggested that SAMHD1 is capable of being exported from the nucleus, which was confirmed by introducing exogenous exportin Xpo1 in live cells. Interestingly, altering the protein's subcellular localization by mutating or removing NLS significantly enhances SAMHD1's potency in L1 suppression. Further tests with SAMHD1 mutants indicated that nucleocytoplasmic shuttling is important for SAMHD1-mediated L1 suppression. Finally, we demonstrated that the cytoplasmic distribution of SAMHD1 leads to an enhanced depletion of L1 ORF2p. Taken together, our data have revealed SAMHD1 as a nucleocytoplasmic shuttling protein, and associated such a new feature of SAMHD1 with its potency against L1 retrotransposition, which provides more insights to the understanding of SAMHD1 and its role in L1 suppression.

The catalytic subunit of DNA polymerase δ is a nucleocytoplasmic shuttling protein

The DNA polymerase δ catalytic subunit (PolD1) is a highly conserved protein with established functions in both the nucleus and the cytoplasm: whereas PolD1 participates in the replication and repair of nuclear DNA, it plays a role in the control of cytoplasmic microtubule growth by directly acting on microtubule-nucleator γ-tubulin ring complexes. Here, we show that PolD1 shuttles between the nucleus and the cytoplasm. PolD1 harbors two nuclear localization signals that mediate the active transport of PolD1 to the nucleus; conversely, PolD1 is exported from the nucleus by the exportin CRM1-dependent mechanism, a major nuclear-export pathway that mediates the export of various cargos. These findings suggest that the nucleocytoplasmic distribution of PolD1 is influenced by both the nuclear import and export activities of the protein.

Nuclear/Cytoplasmic Fractionation to Study Hippo Effectors.

The translocation or shuttling of Hippo proteins between the nucleus and cytoplasm is a rapid event following cytoskeletal or mechanical cues as well as stimulation with extracellular growth factors. Here we describe an experimental procedure for a simple and fast separation of nuclear and cytoplasmic fractions which maintains protein integrity and integrity of protein-protein complexes, indicating that it should be applicable to many experimental questions.

Membrane topologies of PEX13 and PEX14 provide new insights on the mechanism of protein import into peroxisomes.

PEX13 and PEX14 are two core components of the so-called peroxisomal docking/translocation module, the transmembrane hydrophilic channel through which newly synthesized peroxisomal proteins are translocated into the organelle matrix. The two proteins interact with each other and with PEX5, the peroxisomal matrix protein shuttling receptor, through relatively well characterized domains. However, the topologies of these membrane proteins are still poorly defined. Here, we subjected proteoliposomes containing PEX13 or PEX14 and purified rat liver peroxisomes to protease-protection assays and analyzed the protected protein fragments by mass spectrometry, Edman degradation and western blotting using antibodies directed to specific domains of the proteins. Our results indicate that PEX14 is a bona fide intrinsic membrane protein with a Nin -Cout topology, and that PEX13 adopts a Nout -Cin topology, thus exposing its carboxy-terminal Src homology 3 [SH3] domain into the organelle matrix. These results reconcile several enigmatic findings previously reported on PEX13 and PEX14 and provide new insights into the organization of the peroxisomal protein import machinery. ENZYMES: Trypsin, EC3.4.21.4; Proteinase K, EC3.4.21.64; Tobacco etch virus protease, EC3.4.22.44.

DNA Methylation Predicts the Response of Triple-Negative Breast Cancers to All-Trans Retinoic Acid.

All-trans retinoic acid (atRA) regulates gene expression and is used to treat acute promyelocytic leukemia. Attempts to use atRA in breast cancer without a stratification strategy have resulted in limited overall effectiveness. To identify biomarkers for the treatment of triple-negative breast cancer (TNBC) with atRA, we characterized the effects of atRA on the tumor growth of 13 TNBC cell lines. This resulted in a range of effects that was not predictable based on previously hypothesized predictors of response, such as the levels of atRA nuclear shuttling proteins fatty acid binding protein 5 (FABP5) and cellular retinoic acid binding protein 2 (CRABP2). Transcriptional profiling revealed that atRA induced distinct gene expression changes in the sensitive versus resistant cell lines that were mostly independent of the presence of retinoic acid response elements (RAREs) or peroxisome proliferator response elements (PPREs). Given the importance of DNA methylation in regulating gene expression, we hypothesized that differential DNA methylation could predict the response of TNBCs to atRA. We identified over 1400 sites that were differentially methylated between atRA resistant and sensitive cell lines. These CpG sites predicted the response of four TNBC patient-derived xenografts to atRA, and we utilized these xenografts to refine the profile and identified that as many as 17% of TNBC patients could benefit from atRA treatment. These data illustrate that differential methylation of specific CpGs may be useful biomarkers for predicting the response of patient tumors to atRA treatment.

Changes in mRNA abundance drive shuttling of RNA binding proteins, linking cytoplasmic RNA degradation to transcription.

Alterations in global mRNA decay broadly impact multiple stages of gene expression, although signals that connect these processes are incompletely defined. Here, we used tandem mass tag labeling coupled with mass spectrometry to reveal that changing the mRNA decay landscape, as frequently occurs during viral infection, results in subcellular redistribution of RNA binding proteins (RBPs) in human cells. Accelerating Xrn1-dependent mRNA decay through expression of a gammaherpesviral endonuclease drove nuclear translocation of many RBPs, including poly(A) tail-associated proteins. Conversely, cells lacking Xrn1 exhibited changes in the localization or abundance of numerous factors linked to mRNA turnover. Using these data, we uncovered a new role for relocalized cytoplasmic poly(A) binding protein in repressing recruitment of TATA binding protein and RNA polymerase II to promoters. Collectively, our results show that changes in cytoplasmic mRNA decay can directly impact protein localization, providing a mechanism to connect seemingly distal stages of gene expression.

Phenotype of vigilin expressing breast cancer cells binding to the 69 nt 3′UTR element in CSF-1R mRNA

Vigilin, a nucleocytoplasmic shuttling protein, post-transcriptionally suppresses proto-oncogene c-fms expression (encoding CSF-1R) in breast cancer by binding to a 69 nt cis-acting 3-UTR element in CSF-1R mRNA. CSF-1R is an important mediator of breast cancer development, metastasis, and survival. We confirm that vigilin decreases in vitro reporter luciferase activity as well as the translation rate of target mRNAs. We further explore the mechanism of suppression of CSF-1R. We show that the 69 nt binding element has profound effects on translation efficiency of CSF-1R mRNA, not seen in the presence of mutation of the element. Also, mutation of the 69 nt element in the CSF-1R mRNA 3′UTR both interferes with direct vigilin binding and obviates effect of vigilin overexpression on translational repression of CSF-1R. We show that stable vigilin binding requires the full length 69 nt CSF-1R element, including the 26 nt pyrimidine-rich core. Furthermore, titration of endogenous vigilin and other proteins which bind the 69 nt element, by exogenously introduced CSF-1R mRNA 3′UTR containing the pyrimidine-rich sequence, increases the adhesion, motility, and invasion of breast cancer cells. This phenotypic effect is not seen when the 69 nt element is deleted. Lastly, we are the first to show that human breast tissues exhibit strong vigilin expression in normal breast epithelium. Our pilot data suggest decreased vigilin protein expression, along with shift from the nucleus to the cytoplasmic location, in the transition to ductal carcinoma in situ.

Nucleocytoplasmic shuttling: The ins and outs of quantitative imaging.

Nucleocytoplasmic protein shuttling is integral to the transmission of signals between the nucleus and the cytoplasm. The nuclear/cytoplasmic distribution of proteins of interest can be determined via fluorescence microscopy, following labelling of the target protein with fluorophore-conjugated antibodies (immunofluorescence) or by tagging the target protein with an autofluorescent protein, such as green fluorescent protein (GFP). The latter enables live cell imaging, a powerful approach that precludes many of the artefacts associated with indirect immunofluorescence in fixed cells. In this review, we discuss important considerations for the design and implementation of fluorescence microscopy experiments to quantify the nuclear/cytoplasmic distribution of a protein of interest. We summarise the pros and cons of detecting endogenous proteins in fixed cells by immunofluorescence and ectopically-expressed fluorescent fusion proteins in living cells. We discuss the suitability of widefield fluorescence microscopy and of 2D, 3D and 4D imaging by confocal microscopy for different applications, and describe two different methods for quantifying the nuclear/cytoplasmic distribution of a protein of interest from the fluorescent signal. Finally, we discuss the importance of eliminating sources of bias and subjectivity during image acquisition and post-imaging analyses. This is critical for the accurate and reliable quantification of nucleocytoplasmic shuttling.

Adapt your shuttling proteins for virulence: a lesson from the corn smut fungus Ustilago maydis.

This article is a Commentary on Krombach et al., 220: 553–566.

Modeling the crosstalk between the circadian clock and ROS in Neurospora crassa

The circadian clock regulates the expression of clock-controlled genes and adapts to environmental changes. Reactive oxygen species (ROS) also undergo circadian rhythms and have a role in circadian timekeeping. To elucidate crosstalk between ROS and the circadian clock in Neurospora crassa, we build an integrative network model, characterizing the circadian oscillator, ROS system and their interactions. Notably, the (de)phosphorylation and nuclear-cytoplasmic shuttling of clock proteins are modeled in detail. Simulation results quantitatively reproduce the essential features of circadian rhythm (both in constant darkness and under light/dark cycles) and the changes in period length and phase when ROS levels are altered under diverse conditions. This work clarifies the effects of three interactions between ROS and the clock on the circadian rhythm, suggesting that the regulation of WCC activity by protein phosphatase 2A in an O2−-dependent manner plays a predominant role. The functional significance of such modulations is also discussed.

Silencing of the nucleocytoplasmic shuttling protein karyopherin a2 promotes cell-cycle arrest and apoptosis in glioblastoma multiforme.

We have previously shown that the nucleocytoplasmic carrier karyopherin a2 (KPNA2) is overexpressed in glioblastoma multiforme (GBM) whereas its expression is inversely associated with patient prognosis. However, the promoting role of KPNA2 in gliomagenesis is still poorly understood. This study aims to further elucidate this role of KPNA2 in in vitro GBM models.From four different tested GBM cell lines, the U87MG showed the highest proliferation, low adherence and outgrowth in 3D clusters as well as the highest expression of KPNA2, all features conferring greater malignant behaviour. Silencing of KPNA2 via siRNA interference in those cells significantly decreased their proliferative capacity (p = 0.001). We further observed both a significant cell cycle phase arrest (p = 0.040) and the promoting of cellular apoptosis (p = 0.016) as well as a strong trend (p = 0.062) for an inhibition of nuclear import of c-Myc.This study confirms that a higher expression of KPNA2 in GBM is associated with a more malignant phenotype also in in vitro models. While increased expression of KPNA2 promotes proliferation and survival of GBM tumour cells, silencing of KPNA2 conferred a less malignant behaviour. Our results strongly suggest that silencing of KPNA2 may play an important role in modulation of malignant features of GBM cells.

14-3-3γ binds regulator of G protein signaling 14 (RGS14) at distinct sites to inhibit the RGS14:Gαi–AlF4− signaling complex and RGS14 nuclear localization

Regulator of G protein signaling 14 (RGS14) is a multifunctional brain scaffolding protein that integrates G protein and Ras/ERK signaling pathways. It is also a nucleocytoplasmic shuttling protein. RGS14 binds active Gαi/o via its RGS domain, Raf and active H-Ras-GTP via its R1 Ras-binding domain (RBD), and inactive Gαi1/3 via its G protein regulatory (GPR) domain. RGS14 suppresses long-term potentiation (LTP) in the CA2 region of the hippocampus, thereby regulating hippocampally based learning and memory. The 14-3-3 family of proteins is necessary for hippocampal LTP and associative learning and memory. Here, we show direct interaction between RGS14 and 14-3-3γ at two distinct sties, one phosphorylation-independent and the other phosphorylation-dependent at Ser-218 that is markedly potentiated by signaling downstream of active H-Ras. Using bioluminescence resonance energy transfer (BRET), we show that the pSer-218-dependent RGS14/14-3-3γ interaction inhibits active Gαi1-AlF4- binding to the RGS domain of RGS14 but has no effect on active H-Ras and inactive Gαi1-GDP binding to RGS14. By contrast, the phosphorylation-independent binding of 14-3-3 has no effect on RGS14/Gαi interactions but, instead, inhibits (directly or indirectly) RGS14 nuclear import and nucleocytoplasmic shuttling. Together, our findings describe a novel mechanism of negative regulation of RGS14 functions, specifically interactions with active Gαi and nuclear import, while leaving the function of other RGS14 domains intact. Ongoing studies will further elucidate the physiological function of this interaction between RGS14 and 14-3-3γ, providing insight into the functions of both RGS14 and 14-3-3 in their roles in modulating synaptic plasticity in the hippocampus.

Mechanistic insights into avian reovirus p17-modulated suppression of cell cycle CDK–cyclin complexes and enhancement of p53 and cyclin H interaction

The avian reovirus p17 protein is a nucleocytoplasmic shuttling protein. Although we have demonstrated that p17 causes cell growth retardation via activation of p53, the precise mechanisms remain unclear. This is the first report that avian reovirus p17 possesses broad inhibitory effects on cell cycle CDKs, cyclins, CDK-cyclin complexes, and CDK-activating kinase activity in various mammalian, avian, and cancer cell lines. Suppression of CDK activity by p17 occurs by direct binding to CDKs, cyclins, and CDK-cyclin complexes; transcriptional down-regulation of CDKs; cytoplasmic retention of CDKs and cyclins; and inhibition of CDK-activating kinase activity by promoting p53-cyclin H interaction. p17 binds to CDK-cyclin except for CDK1-cyclin B1 and CDK7-cyclin H complexes. We have determined that the negatively charged 151LAVXDVDA(E/D)DGADPN165 motif in cyclin B1 interacts with a positively charged region of CDK1. p17 mimics the cyclin B1 sequence to compete for CDK1 binding. The PSTAIRE motif is not required for interaction of CDK1-cyclin B1, but it is required for other CDK-cyclin complexes. p17 interacts with cyclins by its cyclin-binding motif, 125RXL127 Sequence and mutagenic analyses of p17 indicated that a 140WXFD143 motif and residues Asp-113 and Lys-122 in p17 are critical for CDK2 and CDK6 binding, leading to their sequestration in the cytoplasm. Exogenous expression of p17 significantly enhanced virus replication, whereas p17 mutants with low binding ability to cell cycle CDKs had no effect on virus yield, suggesting that p17 inhibits cell growth and the cell cycle, benefiting virus replication. An in vivo tumorigenesis assay also showed a significant reduction in tumor size.

Cellular Mechanotransduction: From Tension to Function.

Living cells are constantly exposed to mechanical stimuli arising from the surrounding extracellular matrix (ECM) or from neighboring cells. The intracellular molecular processes through which such physical cues are transformed into a biological response are collectively dubbed as mechanotransduction and are of fundamental importance to help the cell timely adapt to the continuous dynamic modifications of the microenvironment. Local changes in ECM composition and mechanics are driven by a feed forward interplay between the cell and the matrix itself, with the first depositing ECM proteins that in turn will impact on the surrounding cells. As such, these changes occur regularly during tissue development and are a hallmark of the pathologies of aging. Only lately, though, the importance of mechanical cues in controlling cell function (e.g., proliferation, differentiation, migration) has been acknowledged. Here we provide a critical review of the recent insights into the molecular basis of cellular mechanotransduction, by analyzing how mechanical stimuli get transformed into a given biological response through the activation of a peculiar genetic program. Specifically, by recapitulating the processes involved in the interpretation of ECM remodeling by Focal Adhesions at cell-matrix interphase, we revise the role of cytoskeleton tension as the second messenger of the mechanotransduction process and the action of mechano-responsive shuttling proteins converging on stage and cell-specific transcription factors. Finally, we give few paradigmatic examples highlighting the emerging role of malfunctions in cell mechanosensing apparatus in the onset and progression of pathologies.

Abstract 293: Heterogeneous nucleocytoplasmic regulation of an incompletely penetrant ErbB2 onco-phenotype

Abstract Perturbation of cancer cells often leads to heterogeneous outcomes, in that most cells exhibit a dominant phenotype, but the rest appear resistant or hypersensitive to the perturbation. If the penetrance of such a phenotype is heritably incomplete, then it becomes extremely difficult to decipher the upstream molecular events that heterogenize the population and cause response variability. By combining quantitative measurements with dynamical models, systems approaches should be useful if provided with a core network of important biomolecules. The daunting hurdle lies in identifying phenotype-relevant regulatory heterogeneities that define the network for penetrance at the single-cell level. Here, we exploit a new approach, called stochastic frequency matching (SFM), which elaborates the molecular networks upstream of incompletely penetrant phenotypes. SFM identifies and parameterizes single-cell heterogeneities—which emerge after a uniform perturbation but before the appearance of a variable phenotype—to hone in on regulatory states corresponding to future penetrance. For a multi-acinar onco-phenotype incompletely triggered by ErbB receptor tyrosine kinase signaling in 3D cultured breast epithelia, we implemented SFM using microarrays to uncover a network of critical nucleocytoplasmic regulators, including the inner-ring nucleoporin NUP37 and the exportin CSE1L. Gain- and loss-of-function perturbations of NUP37 and CSE1L alter the frequency of ErbB2-triggered multiacinus formation, supporting that SFM can identify mechanisms of incompletely penetrant phenotypes. In addition, population-level transcriptomics revealed other ErbB2-induced changes in nucleocytoplasmic shuttling proteins: KPNA2, RANBP1, and XPO1. The net result of these alterations on shuttling is complicated, and the specific cargo relocalized by ErbB2 signaling is not known. We hypothesize that ErbB signaling heterogeneously reconfigures the nucleocytoplasmic shuttling state of cells to determine incomplete penetrance of the onco-phenotype. Using proximity labeling, we seek to identify novel ErbB2-induced NUP37 and CSE1L interactors that may serve as effectors for the multiacinar phenotype. Systems models of the nucleocytoplasmic shuttling network will evaluate the impact of observed abundance changes and identify characteristics of cargo predicted to be most sensitive to network reconfiguration. The importance of key effectors will ultimately be tested in vivo with murine cells engineered for spontaneous Erbb2 amplification and in patient samples with HER2 amplification. Citation Format: Lixin Wang, Kevin A. Janes. Heterogeneous nucleocytoplasmic regulation of an incompletely penetrant ErbB2 onco-phenotype [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 293.

SET1A-Mediated Mono-Methylation at K342 Regulates YAP Activation by Blocking Its Nuclear Export and Promotes Tumorigenesis

YAP, a key effector of Hippo pathway, is activated by its translocation from cytoplasm to nucleus to regulate gene expression and promote tumorigenesis. Although the mechanism by which YAP is suppressed in cytoplasm has been well-studied, how the activated YAP is sequestered in the nucleus remains unknown. Here, we demonstrate that YAP is a nucleocytoplasmic shuttling protein and its nuclear export is controlled by SET1A-mediated mono-methylation of YAP at K342, which disrupts the binding of YAP to CRM1. YAPmimetic methylation knockin mice are more susceptible to colorectal tumorigenesis. Clinically, YAP K342 methylation is reversely correlated with cancer survival. Collectively, our study identifies SET1A-mediatedmono-methylation at K342 as an essential regulatory mechanism for regulating YAP activity and tumorigenesis.

Structure-activity relationship study of itraconazole, a broad-range inhibitor of picornavirus replication that targets oxysterol-binding protein (OSBP)

Itraconazole (ITZ) is a well-known, FDA-approved antifungal drug that is also in clinical trials for its anticancer activity. ITZ exerts its anticancer activity through several disparate targets and pathways. ITZ inhibits angiogenesis by hampering the functioning of the vascular endothelial growth receptor 2 (VEGFR2) and by indirectly inhibiting mTOR signaling. Furthermore, ITZ directly inhibits the growth of several types of tumor cells by antagonizing Hedgehog signaling. Recently, we reported that ITZ also has broad-spectrum antiviral activity against enteroviruses, cardioviruses and hepatitis C virus, independent of established ITZ-activities but instead via a novel target, oxysterol-binding protein (OSBP), a cellular lipid shuttling protein. In this study, we analyzed which structural features of ITZ are important for the OSBP-mediated antiviral activity. The backbone structure, consisting of five rings, and the sec-butyl chain are important for antiviral activity, whereas the triazole moiety, which is critical for antifungal activity, is not. The features required for OSBP-mediated antiviral activity of ITZ overlap mostly with published features required for inhibition of VEGFR2 trafficking, but not Hh signaling. Furthermore, we use in silico studies to explore how ITZ could bind to OSBP. Our data show that several pharmacological activities of ITZ can be uncoupled, which is a critical step in the development of ITZ-based antiviral compounds with greater specificity and reduced off-target effects.

A novel cell-penetrating peptide protects against neuron apoptosis after cerebral ischemia by inhibiting the nuclear translocation of annexin A1.

Nuclear translocation of annexin A1 (ANXA1) has recently been reported to participate in neuronal apoptosis after cerebral ischemia. Prevention of the nuclear translocation of ANXA1 should therefore inhibit neuronal apoptosis and protect against cerebral stroke. Here, we found that, in the repeat III domain of ANXA1, the amino-acid residues from R228 to F237 function as a unique nuclear translocation signal (NTS) and are required for nuclear translocation of ANXA1. Intriguingly, we synthesized a cell-penetrating peptide derived by conjugating the trans-activator of transcription (Tat) domain to the NTS sequence. This Tat-NTS peptide specifically blocked the interaction of ANXA1 with importin β and, consequently, the nuclear translocation of ANXA1 without affecting the nucleocytoplasmic shuttling of other proteins. The Tat-NTS peptide inhibited the transcriptional activity of p53, decreased Bid expression, suppressed activation of the caspase-3 apoptosis pathway and improved the survival of hippocampal neurons subjected to oxygen-glucose deprivation and reperfusion in vitro. Moreover, using a focal brain ischemia animal model, we showed that the Tat-NTS peptide could be efficiently infused into the ischemic hippocampus and cortex by unilateral intracerebroventricular injection. Injection of the Tat-NTS peptide alleviated neuronal apoptosis in the ischemic zone. Importantly, further work revealed that administration of the Tat-NTS peptide resulted in a dramatic reduction in infarct volume and that this was correlated with a parallel improvement in neurological function after reperfusion. Interestingly, the effects of Tat-NTS were injury specific, with little impact on neuronal apoptosis or cognitive function in sham-treated nonischemic animals. In conclusion, based on its profound neuroprotective and cognitive-preserving effects, it is suggested that the Tat-NTS peptide represents a novel and potentially promising new therapeutic candidate for the treatment of ischemic stroke.