Nucleus and Mitochondria Targeting Theranostic Plasmonic Surface-Enhanced Raman Spectroscopy Nanoprobes as a Means for Revealing Molecular Stress Response Differences in Hyperthermia Cell Death between Cancerous and Normal Cells.

Metallic plasmonic nanoparticles have been intensively exploited as theranostic nanoprobes for plasmonic photothermal therapy (PPT) and surface-enhanced Raman spectroscopy (SERS) applications. But the underlying molecular mechanisms associated with PPT-induced apoptosis between cancerous and normal cells have remained largely unknown or disputed. In this study, we designed an organelle-targeting theranostic plasmonic SERS nanoprobe (CDs-Ag/Au NS) composed of porous Ag/Au nanoshell (p-Ag/Au NSs) and carbon dots (CDs) for nucleus and mitochondria targeted PPT of cells. The differences in molecular stress response in the PPT-induced hyperthermia cell death between cancerous HeLa and normal L929 and H8 cells have been revealed by site-specific single-cell SERS detection. The contents of tryptophan (Trp), phenylalanine (Phe), and tyrosine (Tyr) in HeLa cells were found more evidently increased than L929 and H8 cells during the PPT-induced cell-death process. And from the mitochondria point of view, we found that the PPT-induced cell apoptosis for HeLa cells mainly stems from (or is regulated through) cellular thermal stress-responsive proteins, while for L929 and H8 cells it seems more related to DNA. Understanding molecular stress response difference of the PPT-induced cell apoptosis between cancerous and normal cells is helpful for diagnosis and treatment of cancer, and the method will open an avenue for single-cell studies.

Synthesis and in vitro evaluation of a PDT active BODIPY-NLS conjugate.

Two new photosensitizers based on the BODIPY scaffold have been synthesized, of which one bears an NLS peptide, which is linked to the BODIPY's core using the copper catalysed azide-alkyne click reaction. The phototoxicities of these BODIPY based photosensitizers have been determined, as well as their dark toxicities. Although the conjugation of a single NLS peptide to the BODIPY did not lead to any observable nuclear localization, the photosensitizer did exhibit a superior photoxicity. Cellular co-localization experiments revealed a localization of both dyes in the lysosomes, as well as a partial localization within the ER (for the peptide-bearing BODIPY).

Complex assembly mechanism and an RNA-binding mode of the human p14-SF3b155 spliceosomal protein complex identified by NMR solution structure and functional analyses.

The spliceosomal protein p14, a component of the SF3b complex in the U2 small nuclear ribonucleoprotein (snRNP), is essential for the U2 snRNP to recognize the branch site adenosine. The elucidation of the dynamic process of the splicing machinery rearrangement awaited the solution structural information. We identified a suitable complex of human p14 and the SF3b155 fragment for the determination of its solution structure by NMR. In addition to the overall structure of the complex, which was recently reported in a crystallographic study (typical RNA recognition motif fold beta1-alpha1-beta2-beta3-alpha2-beta4 of p14, and alphaA-betaA fold of the SF3b155 fragment), we identified three important features revealed by the NMR solution structure. First, the C-terminal extension and the nuclear localization signal of p14 (alpha3 and alpha4 in the crystal structure, respectively) were dispensable for the complex formation. Second, the proline-rich segment of SF3b155, following betaA, closely approaches p14. Third, interestingly, the beta1-alpha1 loop and the alpha2-beta4 beta-hairpin form a positively charged groove. Extensive mutagenesis analyses revealed the functional relevance of the residues involved in the protein-protein interactions: two aromatic residues of SF3b155 (Phe408 and Tyr412) play crucial roles in the complex formation, and two hydrophobic residues (Val414 and Leu415) in SF3b 155 serve as an anchor for the complex formation, by cooperating with the aromatic residues. These findings clearly led to the conclusion that SFb155 binds to p14 with three contact points, involving Phe408, Tyr412, and Val414/Leu415. Furthermore, to dissect the interactions between p14 and the branch site RNA, we performed chemical-shift-perturbation experiments, not only for the main-chain but also for the side-chain resonances, for several p14-SF3b155 complex constructs upon binding to RNA. These analyses identified a positively charged groove and the C-terminal extension of p14 as RNA-binding sites. Strikingly, an aromatic residue in the beta1-alpha1 loop, Tyr28, and a positively charged residue in the alpha2-beta4 beta-hairpin, Agr85, are critical for the RNA-binding activity of the positively charged groove. The Tyr28Ala and Arg85Ala point mutants and a deletion mutant of the C-terminal extension clearly revealed that their RNA binding activities were independent of each other. Collectively, this study provides details for the protein-recognition mode of p14 and insight into the branch site recognition.

Cloning and expression analysis of a protein kinase C gene, PKCmu, and its regulation of the promoter region in zebrafish.

The cDNA and genomic DNA of zebrafish (Danio rerio) protein kinase Cmu (PKCmu), with its promoter region, were obtained. The 508-amino acid zebrafish PKCmu has 86.17% similarity to human PKCmu. Real-time reverse-transcription polymerase chain reaction analysis with starvation and hormonal treatment found significant differences between the control group and the experimental group after 14 days of starvation. After injecting insulin-like growth factor II (IGF-II), growth hormone (GH), insulin, or human chorionic gonadotropin, significant differences were observed between the control and experimental groups 24 h after treatment. After injecting the gonadotropin-releasing hormone or luteotropin-releasing hormone, significant differences were seen between the control and experimental groups 15 h after treatment. These results suggest that in vivo PKCmu expression is regulated by the insulin family or by the GH, but other sex hormones produced a significant expression level more quickly than the insulin family and GH. The zebrafish PKCmu gene is located on zebrafish chromosome 17 and consists of 16 exons. A 2.6 kilobase pair on the 5' flanking region displayed maximal promoter activity in the zebrafish liver (ZFL) cell line after treatment with IGF-I, IGF-II, and GH. However, a 1.6 kilobase pair on the 5' flanking region displayed maximal promoter activity in the HeLa cell line after treatment with IGF-I, IGF-II, and GH. Finally, PKCmu may have important nuclear effects on cell growth and may involve nuclear localization. By transiently transfecting ZFL cells with various zebrafish PKCmu segments, we identified a nuclear localization signal: the amino acid sequence between amino acids 206 and 209 was able to predominantly direct enhanced green fluorescence protein (EGFP) into the nucleus, whereas a deletion of this motif abrogated the nuclear localization property.

Influence of serine O-glycosylation or O-phosphorylation close to the vJun nuclear localisation sequence on nuclear import.

Nuclear import triggered by the nuclear-localisation sequence (NLS) of the viral Jun (vJun) protein is mediated by phosphorylation of a serine close to the NLS. Since phosphorylation and glycosylation of serine residues are often in a reciprocal "yin-yang" relationship, we investigated whether glycosylation of this serine with O-linked N-acetylglucosamine (O-GlcNAc) would also regulate nuclear import via the vJun NLS. Peptides containing the vJun NLS with an adjacent O-phosphorylated, O-GlcNAc-functionalised or unmodified serine, and equipped with an N-terminal biotin or a 7-nitrobenz-2-oxa-1,3-diazolyl (NBD) fluorescent label, were synthesised on the solid phase by means of an Fmoc/Boc strategy and a Pd0-sensitive HYCRON linker. Fluorescence-polarisation measurements on the NBD-labelled peptides indicated that modification with phosphate or O-GlcNAc leads to a decrease in affinity to the import-mediating adapter protein, importin alpha, of about one order of magnitude compared to the unmodified NLS. Microinjection of biotinylated NLS peptide conjugated with fluorescently labelled avidin into NIH/3T3 and MDCK cells, revealed that avidin-unmodified-NLS peptide was rapidly imported into the nucleus. However, either phosphate or O-GlcNAc next to the NLS caused almost complete exclusion of the protein conjugate from nuclear import. These findings indicate that nuclear import by the vJun NLS might not be regulated by a "yin-yang" modification of an adjacent serine with phosphate or O-GlcNAc. Rather, negative regulation of binding between the polybasic NLS and importin by a negatively charged or a bulky, uncharged residue appears likely.

Identification of novel nuclear localization signal within the ErbB-2 protein.

ErbB2, a member of the receptor tyrosine kinase family, is frequently over-expressed in breast cancer. Proteolysis of the extracellular domain of ErbB2 results in constitutive activation of ErbB2 kinase. Recent study reported that ErbB2 is found in the nucleus. Here, we showed that ErbB2 is imported into the nucleus through a nuclear localization signal (NLS)-mediated mechanism. The NLS sequence KRRQQKIRKYTMRR (aa655-668) contains three clusters of basic amino acids and it is sufficient to target GFP into the nucleus. However, mutation in any basic amino acid cluster of this NLS sequence significantly affects its nuclear localization. Furthermore, it was found that this NLS is essential for the nuclear localization of ErbB2 since the intracellular domain of Erb2 lacking NLS completely abrogates its nuclear translocation. Taken together, our study identified a novel nuclear localization signal and reveals a novel mechanism underlying ErbB2 nuclear trafficking and localization.

Modular domain structure of Arabidopsis COP1. Reconstitution of activity by fragment complementation and mutational analysis of a nuclear localization signal in planta.

The Arabidopsis COP1 protein functions as a developmental regulator, in part by repressing photomorphogenesis in darkness. Using complementation of a cop1 loss-of-function allele with transgenes expressing fusions of cop1 mutant proteins and beta-glucuronidase, it was confirmed that COP1 consists of two modules, an amino terminal module conferring a basal function during development and a carboxyl terminal module conferring repression of photomorphogenesis. The amino-terminal zinc-binding domain of COP1 was indispensable for COP1 function. In contrast, the debilitating effects of site-directed mutations in the single nuclear localization signal of COP1 were partially compensated by high-level transgene expression. The carboxyl-terminal module of COP1, though unable to substantially ameliorate a cop1 loss-of-function allele on its own, was sufficient for conferring a light-quality-dependent hyperetiolation phenotype in the presence of wild-type COP1. Moreover, partial COP1 activity could be reconstituted in vivo from two non-covalently linked, complementary polypeptides that represent the two functional modules of COP1. Evidence is presented for efficient association of the two sub-fragments of the split COP1 protein in Arabidopsis and in a yeast two-hybrid assay.

Zn finger and nuclear localization of transcription factor Sp1.

Transcription factor Sp1 is located in the nucleus of a mammalian cell and importantly related to expression of many cellular genes. In order to elucidate the nuclear localization mechanism of Sp1, various truncated fragments of Sp1 were fused to green fluorescent protein (GFP) and expressed in HeLa cell. The results show significance of the DNA binding region, especially, zinc finger (Zn finger) domain for nuclear localization of Sp1 in HeLa cell.