Sox11 Reduces Caspase-6 Cleavage and Activity.

The apoptotic cascade is an orchestrated event, whose final stages are mediated by effector caspases. Regulatory binding proteins have been identified for caspases such as caspase-3, -7, -8, and -9. Many of these proteins belong to the inhibitor of apoptosis (IAP) family. By contrast, caspase-6 is not believed to be influenced by IAPs, and little is known about its regulation. We therefore performed a yeast-two-hybrid screen using a constitutively inactive form of caspase-6 for bait in order to identify novel regulators of caspase-6 activity. Sox11 was identified as a potential caspase-6 interacting protein. Sox11 was capable of dramatically reducing caspase-6 activity, as well as preventing caspase-6 self- cleavage. Several regions, including amino acids 117-214 and 362-395 within sox11 as well as a nuclear localization signal (NLS) all contributed to the reduction in caspase-6 activity. Furthermore, sox11 was also capable of decreasing other effector caspase activity but not initiator caspases -8 and -9. The ability of sox11 to reduce effector caspase activity was also reflected in its capacity to reduce cell death following toxic insult. Interestingly, other sox proteins also had the ability to reduce caspase-6 activity but to a lesser extent than sox11.

NLS-RARα promotes proliferation and inhibits differentiation in HL-60 cells.

A unique mRNA produced in leukemic cells from a t(15;17) acute promyelocytic leukemia (APL) patient encodes a fusion protein between the retinoic acid receptor α (RARα) and a myeloid gene product called PML. Studies have reported that neutrophil elastase (NE) cleaves bcr-1-derived PML-RARα in early myeloid cells, leaving only the nuclear localization signal (NLS) of PML attached to RARα. The resultant NLS-RARα fusion protein mainly localizes to, and functions within, the cell nucleus. It is speculated that NLS-RARα may act in different ways from the wild-type RARα, but its biological characteristics have not been reported. This study takes two approaches. Firstly, the NLS-RARα was silenced with pNLS-RARα-shRNA. The mRNA and protein expression of NLS-RARα were detected by RT-PCR and Western blot respectively. Cell proliferation in vitro was assessed by MTT assay. Flow cytometry (FCM) was used to detect the differentiation of cells. Secondly, the NLS-RARα was over-expressed by preparation of recombinant adenovirus HL-60/pAd-NLS-RARα. The assays of mRNA and protein expression of NLS-RARα, and cell proliferation, were as above. By contrast, cell differentiation was stimulated by all trans retinoic acid (ATRA) (2.5µmol/L) at 24h after virus infection of pAd-NLS-RARα, and then detected by CD11b labeling two days later. The transcription and translation of C-MYC was detected in HL-60/pAd-NLS-RARα cells which treated by ATRA. Our results showed that compared to the control groups, the expression of NLS-RARα was significantly reduced in the HL-60/pNLS-RARα-shRNA cells, and increased dramatically in the HL-60/pAd-NLS-RARα cells. The proliferation was remarkably inhibited in the HL-60/pNLS-RARα-shRNA cells in a time-dependent manner, but markedly promoted in the HL-60/pAd-NLS-RARα cells. FCM outcome revealed the differentiation increased in HL-60/pNLS-RARα-shRNA cells, and decreased in the HL-60/pAd-NLS-RARα cells treated with 2.5µmol/L ATRA. The expression of C-MYC increased strikingly in HL-60/pAd-NLS-RARα cells treated with 2.5µmol/L ATRA. Down-regulation of NLS-RARα expression inhibited the proliferation and induced the differentiation of HL-60 cells. On the contrary, over-expression of NLS-RARα promoted proliferation and reduced the ATRA-induced differentiation of HL-60 cells.

Nordihydroguaiaretic acid induces Nrf2 nuclear translocation in vivo and attenuates renal damage and apoptosis in the ischemia and reperfusion model.

It has been shown that the pretreatment with nordihydroguaiaretic acid (NDGA), a lignan with direct and indirect antioxidant properties, protects against the ischemia-reperfusion (I/R)-induced renal oxidant damage. Although it has been shown that NDGA induces Nrf2 nuclear translocation in renal epithelial LLC-PK1 cells in culture, it is unknown if NDGA may induce Nrf2 translocation in vivo. In this work was explored if NDGA is able to induce in vivo Nrf2 nuclear translocation in kidneys of rats submitted to uni-nephrectomy (U-NX) or I/R injury. Four groups of male Wistar rats were used: U-NX, NDGA, I/R, and I/R+NDGA. NDGA was injected i.p. (10mg/kg/day) starting 48 h before I/R. Kidney samples were obtained at 3 h of reperfusion after to measure Nrf2 translocation. Additional groups of rats were studied at 24 h of reperfusion to measure histological damage and apoptosis. NDGA was able to induce Nrf2 translocation in vivo in kidneys of rats submitted to both U-NX and I/R injury and to protect against renal histological damage and apoptosis. It is concluded that the pretreatment of NDGA is able to induce in vivo nuclear Nrf2 translocation in kidney of rats suggesting that this may be involved in the renoprotection against I/R.

Generation of new cytotoxic human ribonuclease variants directed to the nucleus.

Ribonucleases are promising agents for use in anticancer therapy. Engineering a nuclear localization signal into the sequence of the human pancreatic ribonuclease has been revealed as a new strategy to endow this enzyme with cytotoxic activity against tumor cells. We previously described a cytotoxic human pancreatic ribonuclease variant, named PE5, which is able to cleave nuclear RNA, inducing the apoptosis of cancer cells and reducing the amount of P-glycoprotein in different multidrug-resistant cell lines. These results open the opportunity to use this ribonuclease in combination with other chemotherapeutics. In this work, we have investigated how to improve the properties of PE5 as an antitumor drug candidate. When attempting to develop a recombinant protein as a drug, two of the main desirable attributes are minimum immunogenicity and maximum potency. The improvements of PE5 have been designed in both senses. First, in order to reduce the potential immunogenicity of the protein, we have studied which residues mutated on PE5 can be reverted to those of the wild-type human pancreatic ribonuclease sequence without affecting its cytotoxicity. Second, we have investigated the effect of introducing an additional nuclear localization signal at different sites of PE5 in an effort to obtain a more cytotoxic enzyme. We show that the nuclear localization signal location is critical for the cytotoxicity. One of these variants, named NLSPE5, presents about a 10-fold increase in cytotoxicity respective to PE5. This variant induces apoptosis and kills the cells using the same mechanism as PE5.

Nuclear import and export signals of human cohesins SA1/STAG1 and SA2/STAG2 expressed in Saccharomyces cerevisiae.

BACKGROUND: Human SA/STAG proteins, homologues of the yeast Irr1/Scc3 cohesin, are the least studied constituents of the sister chromatid cohesion complex crucial for proper chromosome segregation. The two SA paralogues, SA1 and SA2, show some specificity towards the chromosome region they stabilize, and SA2, but not SA1, has been shown to participate in transcriptional regulation as well. The molecular basis of this functional divergence is unknown. METHODOLOGY/PRINCIPAL FINDINGS: In silico analysis indicates numerous putative nuclear localization (NLS) and export (NES) signals in the SA proteins, suggesting the possibility of their nucleocytoplasmic shuttling. We studied the functionality of those putative signals by expressing fluorescently tagged SA1 and SA2 in the yeast Saccharomyces cerevisiae. Only the N-terminal NLS turned out to be functional in SA1. In contrast, the SA2 protein has at least two functional NLS and also two functional NES. Depending on the balance between these opposing signals, SA2 resides in the nucleus or is distributed throughout the cell. Validation of the above conclusions in HeLa cells confirmed that the same N-terminal NLS of SA1 is functional in those cells. In contrast, in SA2 the principal NLS functioning in HeLa cells is different from that identified in yeast and is localized to the C-terminus. CONCLUSIONS/SIGNIFICANCE: This is the first demonstration of the possibility of non-nuclear localization of an SA protein. The reported difference in the organization between the two SA homologues may also be relevant to their partially divergent functions. The mechanisms determining subcellular localization of cohesins are only partially conserved between yeast and human cells.

Construction of ordered protein arrays.

Artificially ordered protein arrays provide a facile approach to a variety of problems in biology and nanoscience. Current demonstration systems use either nucleic acid tethers or methyltransferase fusions in order to target proteins or peptides of interest to nucleic acid scaffolds. These demonstrations point to the large number of useful devices and assemblies that can be envisioned using this approach, including smart biological probes and drug delivery systems. In principle, these systems are now capable of imitating the earliest forms of prebiotic organisms and can be expected to reach the complexity of a small virus in the near future. Third-generation methyltransferase inhibitors provide an example of a smart chemotherapeutics that can be constructed with this approach. We describe the use of mechanistic enzymology, computer-aided design, and microfluidic chip-based capillary electrophoresis in assessing the final assembly and testing of designs of this type.

A nuclear localization sequence endows human pancreatic ribonuclease with cytotoxic activity.

Some members of the ribonuclease superfamily, such as Onconase, are cytotoxic to cancer cells. This is not the case for human pancreatic ribonuclease. This lack of cytotoxicity is probably a result of the inhibition exerted by the cytosolic ribonuclease inhibitor once the protein has reached the cytosol. Until now, all cytotoxic human pancreatic ribonuclease variants have been described as being resistant to the inhibitor. Here, we report on the characterization of a cytotoxic variant of human pancreatic ribonuclease which has an Arg triplet introduced onto one of its surface-exposed loops. Despite its sensitivity to the inhibitor, this variant, called PE5, was only 5-15 times less cytotoxic than Onconase. When it was taken up by cells, it was only observed within late compartments of the endocytic pathway, probably because the number of molecules transported to the cytosol was too small to allow their visualization. Nuclear import assays showed that the Arg triplet endows PE5 with a nuclear localization signal. In these experiments, PE5 was efficiently transported to the nucleus where it was initially localized in the nucleolus. Although the Arg introduction modified the net charge of the protein and somehow impaired recognition by the cytosolic inhibitor, control variants, which had the same number of charges or were not recognized by the inhibitor, were not toxic. We concluded that targeting a ribonuclease to the nucleus results in cytotoxicity. This effect is probably due to ribonuclease interference with rRNA processing and ribosome assembly within the nucleolus.

Divergent roles of hepatitis B virus X-associated protein 2 (XAP2) in human versus mouse Ah receptor complexes.

The aryl hydrocarbon receptor (AhR) mediates the toxicologic and carcinogenic properties of 2,3,7,8-tetrachlorodibenzo-p-dioxin. In the cytoplasm, the AhR is complexed with a dimer of hsp90, and the hepatitis B virus X-associated protein 2 (XAP2). Most studies that have examined the ability of XAP2 to modulate the AhR have characterized the mouse receptor (mAhR). However, the amino acid sequence of mAhR is significantly different from human AhR (hAhR) in the carboxy terminal half of the protein, and this could lead to differences in the behavior of the two receptors. mAhR-yellow fluorescent protein (YFP) and hAhR-YFP were used to compare nucleocytoplasmic shuttling properties and the ability of XAP2 to modulate their activity. As reported previously, mAhR localized predominantly in the nucleus and was redistributed to the cytoplasm by coexpression of XAP2 in COS-1 cells. Leptomycin B treatment revealed that XAP2 blocked mAhR-YFP translocation to the nucleus in the absence of ligand. In contrast, hAhR-YFP localized predominantly in the cytoplasm, and coexpression of XAP2 did not affect this localization, and did not block nuclear accumulation in the presence of leptomycin B. An XAP2 fusion protein with a nuclear localization signal fused to the carboxy terminus (XAP2-NLS) was utilized to test whether this protein could drag the AhR into the nucleus. Coexpression of mAhR-YFP and XAP2-NLS caused cytoplasmic localization of the mAhR, while hAhR-YFP was partially localized in the nucleus, suggesting that XAP2 remains bound to the hAhR during nucleocytoplasmic shuttling. The presence of XAP2 in the ligand-bound hAhR complex enhanced the rate of nuclear translocation but repressed transcriptional activity. Together, these results suggest that the hAhR differs biochemically from the mAhR.

Nuclear localization of basic fibroblast growth factor is mediated by heparan sulfate proteoglycans through protein kinase C signaling.

Understanding the process of wound healing will provide valuable insight for the development of new strategies to treat diseases associated with improper regeneration, such as blindness induced by corneal scarring. Heparan sulfate proteoglycans (HSPG) are not normally expressed in the corneal stroma, but their presence at sites of injury suggests their involvement in the wound healing response. Primary cultured corneal stromal fibroblasts constitutively express HSPG and represent an injured phenotype. Recently, nuclear localization of HSPG was shown to increase in corneal stromal fibroblasts plated on fibronectin (FN), an extracellular matrix protein whose appearance in the corneal stroma correlates with injury. One possible role for the nuclear localization of HSPG is to function as a shuttle for the nuclear transport of heparin-binding growth factors, such as basic fibroblast growth factor (FGF-2). Once in the nucleus, these growth factors might directly modulate cellular activities. To investigate this hypothesis, cells were treated with (125)I-labelled FGF-2 under various conditions and fractionated. Our results show that nuclear localization of FGF-2 was increased in cells plated on FN compared to those on collagen type I (CO). Interestingly, FGF-2-stimulated proliferation was increased in cells plated on FN compared to CO and this effect was absent in the presence of heparinase III. Furthermore, pre-treatment with heparinase III decreased nuclear FGF-2, and CHO cells defective in the ability to properly synthesize heparan sulfate chains showed reduced nuclear FGF-2 indicating that the heparan sulfate chains of HSPG are critical for this process. HSPG signaling, particularly through the cytoplasmic tails of syndecans, was investigated as a potential mechanism for the nuclear localization of FGF-2. Treatment with phorbol 12-myristate-13-acetate (PMA), under conditions that caused downregulation of protein kinase Calpha (PKCalpha), decreased nuclear FGF-2. Using pharmacological inhibitors of specific PKC isozymes, we elucidated a potential mode of regulation whereby PKCalpha mediates the nuclear localization of FGF-2 and PKCdelta inhibits it. Our studies suggest a novel mechanism in which FGF-2 translocates to the nucleus in response to injury.

Identification of the nuclear localization signal in Xenopus cyclin E and analysis of its role in replication and mitosis.

Cyclin-dependent kinase (Cdk)2/cyclin E is imported into nuclei assembled in Xenopus egg extracts by a pathway that requires importin-alpha and -beta. Here, we identify a basic nuclear localization sequence (NLS) in the N-terminus of Xenopus cyclin E. Mutation of the NLS eliminated nuclear accumulation of both cyclin E and Cdk2, and such versions of cyclin E were unable to trigger DNA replication. Addition of a heterologous NLS from SV40 large T antigen restored both nuclear targeting of Cdk2/cyclin E and DNA replication. We present evidence indicating that Cdk2/cyclin E complexes must become highly concentrated within nuclei to support replication and find that cyclin A can trigger replication at much lower intranuclear concentrations. We confirmed that depletion of endogenous cyclin E increases the concentration of cyclin B necessary to promote entry into mitosis. In contrast to its inability to promote DNA replication, cyclin E lacking its NLS was able to cooperate with cyclin B in promoting mitotic entry.

Identification of a short amino acid sequence essential for efficient nuclear targeting of the Kaposi's sarcoma-associated herpesvirus/human herpesvirus-8 K8 protein.

The K8 protein of Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus-8 is a member of the bZIP family of transcription factors, which has homology with the Epstein-Barr virus transcription and replication factor, EB1. In this report, we have analysed the subcellular localization of the K8 protein and characterized a 12 amino acid sequence rich in basic residues which is responsible for targeting the protein to the cell nucleus. Furthermore, we show that a K8 mutant lacking the nuclear localization sequence can be directed to the nucleus by co-expression with an intact K8 protein, suggesting that K8 homodimerizes in the cytoplasm of the cell in vivo.