Effect of sulfasalazine on human neuroblastoma: analysis of sepiapterin reductase (SPR) as a new therapeutic target.

BACKGROUND: Neuroblastoma (NB) is an aggressive childhood malignancy in children up to 5 years of age. High-stage tumors frequently relapse even after aggressive multimodal treatment, and then show therapy resistance, typically resulting in patient death. New molecular-targeted compounds that effectively suppress tumor growth and prevent relapse with more efficacy are urgently needed. We and others previously showed that polyamines (PA) like spermidine and spermine are essential for NB tumorigenesis and that DFMO, an inhibitor of the key PA synthesis gene product ODC, is effective both in vitro and in vivo, securing its evaluation in NB clinical trials. To find additional compounds interfering with PA biosynthesis, we tested sulfasalazine (SSZ), an FDA-approved salicylate-based anti-inflammatory and immune-modulatory drug, recently identified to inhibit sepiapterin reductase (SPR). We earlier presented evidence for a physical interaction between ODC and SPR and we showed that RNAi-mediated knockdown of SPR expression significantly reduced native ODC enzyme activity and impeded NB cell proliferation. METHODS: Human NB mRNA expression datasets in the public domain were analyzed using the R2 platform. Cell viability, isobologram, and combination index analyses as a result of SSZ treatment with our without DFMO were carried out in NB cell cultures. Molecular protein-ligand docking was achieved using the GRAMM algorithm. Statistical analyses were performed with the Kruskal-Wallis test, 2log Pearson test, and Student's t test. RESULTS: In this study, we show the clinical relevance of SPR in human NB tumors. We found that high SPR expression is significantly correlated to unfavorable NB characteristics like high age at diagnosis, MYCN amplification, and high INSS stage. SSZ inhibits the growth of NB cells in vitro, presumably due to the inhibition of SPR as predicted by computational docking of SSZ into SPR. Importantly, the combination of SSZ with DFMO produces synergistic antiproliferative effects in vitro. CONCLUSIONS: The results suggest the use of SSZ in combination with DFMO for further experiments, and possible prioritization as a novel therapy for the treatment of NB patients.

Molecular phylogeny of a newfound hantavirus in the Japanese shrew mole (Urotrichus talpoides).

Recent molecular evidence of genetically distinct hantaviruses in shrews, captured in widely separated geographical regions, corroborates decades-old reports of hantavirus antigens in shrew tissues. Apart from challenging the conventional view that rodents are the principal reservoir hosts, the recently identified soricid-borne hantaviruses raise the possibility that other soricomorphs, notably talpids, similarly harbor hantaviruses. In analyzing RNA extracts from lung tissues of the Japanese shrew mole (Urotrichus talpoides), captured in Japan between February and April 2008, a hantavirus genome, designated Asama virus (ASAV), was detected by RT-PCR. Pairwise alignment and comparison of the S-, M-, and L-segment nucleotide and amino acid sequences indicated that ASAV was genetically more similar to hantaviruses harbored by shrews than by rodents. However, the predicted secondary structure of the ASAV nucleocapsid protein was similar to that of rodent- and shrew-borne hantaviruses, exhibiting the same coiled-coil helix at the amino terminus. Phylogenetic analyses, using the maximum-likelihood method and other algorithms, consistently placed ASAV with recently identified soricine shrew-borne hantaviruses, suggesting a possible host-switching event in the distant past. The discovery of a mole-borne hantavirus enlarges our concepts about the complex evolutionary history of hantaviruses.

Altered functional properties of a TRPM2 variant in Guamanian ALS and PD.

Two related neurodegenerative disorders, Western Pacific amyotrophic lateral sclerosis (ALS) and parkinsonism-dementia (PD), originally occurred at a high incidence on Guam, in the Kii peninsula of Japan, and in southern West New Guinea more than 50 years ago. These three foci shared a unique mineral environment characterized by the presence of severely low levels of Ca(2+) and Mg(2+), coupled with high levels of bioavailable transition metals in the soil and drinking water. Epidemiological studies suggest that genetic factors also contribute to the etiology of these disorders. Here, we report that a variant of the transient receptor potential melastatin 2 (TRPM2) gene may confer susceptibility to these diseases. TRPM2 encodes a calcium-permeable cation channel highly expressed in the brain that has been implicated in mediating cell death induced by oxidants. We found a heterozygous variant of TRPM2 in a subset of Guamanian ALS (ALS-G) and PD (PD-G) cases. This variant, TRPM2(P1018L), produces a missense change in the channel protein whereby proline 1018 (Pro(1018)) is replaced by leucine (Leu(1018)). Functional studies revealed that, unlike WT TRPM2, P1018L channels inactivate. Our results suggest that the ability of TRPM2 to maintain sustained ion influx is a physiologically important function and that its disruption may, under certain conditions, contribute to disease states.

Fluorescent proteins and their use in marine biosciences, biotechnology, and proteomics.

This review explores the field of fluorescent proteins (FPs) from the perspective of their marine origins and their applications in marine biotechnology and proteomics. FPs occur in hydrozoan, anthozoan, and copepodan species, and possibly in other metazoan niches as well. Many FPs exhibit unique photophysical and photochemical properties that are the source of exciting research opportunities and technological development. Wild-type FPs can be enhanced by mutagenetic modifications leading to variants with optimized fluorescence and new functionalities. Paradoxically, the benefits from ocean-derived FPs have been realized, first and foremost, for terrestrial organisms. In recent years, however, FPs have also made inroads into aquatic biosciences, primarily as genetically encoded fluorescent fusion tags for optical marking and tracking of proteins, organelles, and cells. Examples of FPs and applications summarized here testify to growing utilization of FP-based platform technologies in basic and applied biology of aquatic organisms. Hydra, sea squirt, zebrafish, striped bass, rainbow trout, salmonids, and various mussels are only a few of numerous instances where FPs have been used to address questions relevant to evolutionary and developmental research and aquaculture.

Genomic organization, characterization, and molecular 3D model of GDE1, a novel mammalian glycerophosphoinositol phosphodiesterase.

Glycerophosphodiester phosphodiesterase (GDPD) catalyzes the hydrolysis of deacylated glycerophospholipids to glycerol phosphate and alcohol. A mammalian glycerophosphoinositol phosphodiesterase, GDE1/MIR16, was recently identified as an interacting protein of the regulator of G protein signaling 16 (RGS16) providing a link between phosphoinositide metabolism and G protein signal transduction. To further understand the function and properties of human GDE1, we determined its genomic organization and its biochemical and structural characteristics. GDE1 encodes a 331-residue protein with two hydrophobic domains and contains a GDE domain that shares strong homologies with GDE1-related proteins as well as bacterial GDPDs. The human GDE1 gene is located on chromosome 16p12-p11.2 and contains six exons and five introns. A molecular 3D model, which was built based on the crystal structure of Escherichia coli GDPD (1YDY), provides the first structural information of human GDE1 and suggests a TIM barrel core as typically found in bacterial GDPDs. Furthermore, a model of the putative catalytic motif within the GDE domain was nearly identical to the corresponding domain of GDPD and highlights the individual core residues Glu97, Asp99, and His112, which are crucial to maintaining GDE1 catalytic activity. These studies provide important new insights into understanding the function of GDE1 and GDE1-related proteins.

Fluorescence polarization/anisotropy approaches to study protein-ligand interactions: effects of errors and uncertainties.

Fluorescence techniques are widely used in the study of protein-ligand interactions because of their inherent sensitivity, and the fact that they can be implemented at true equilibrium conditions. Fluorescence polarization/anisotropy methodologies, in particular, are now extensively utilized in biotechnology and clinical chemistry. In this chapter, we shall discuss both theoretical and practical aspects of polarization/anisotropy methods. We shall also focus attention on considerations of errors and uncertainties in such measurements, and how these uncertainties affect the ultimate estimation of ligand-protein dissociation constants.

Withaferin A Inhibits STAT3 and Induces Tumor Cell Death in Neuroblastoma and Multiple Myeloma.

Signal transducer and activator of transcription 3 (STAT3) is an oncogenic transcription factor that has been implicated in many human cancers and has emerged as an ideal target for cancer therapy. Withaferin A (WFA) is a natural product with promising antiproliferative properties through its association with a number of molecular targets including STAT3. However, the effect of WFA in pediatric neuroblastoma (NB) and its interaction with STAT3 have not been reported. In this study, we found that WFA effectively induces dose-dependent cell death in high-risk and drug-resistant NB as well as multiple myeloma (MM) tumor cells, prevented interleukin-6 (IL-6)-mediated and persistently activated STAT3 phosphorylation at Y705, and blocked the transcriptional activity of STAT3. We further provide computational models that show that WFA binds STAT3 near the Y705 phospho-tyrosine residue of the STAT3 Src homology 2 (SH2) domain, suggesting that WFA prevents STAT3 dimer formation similar to BP-1-102, a well-established STAT3 inhibitor. Our findings propose that the antitumor activity of WFA is mediated at least in part through inhibition of STAT3 and provide a rationale for further drug development and clinical use in NB and MM.

Novel interaction of ornithine decarboxylase with sepiapterin reductase regulates neuroblastoma cell proliferation.

Ornithine decarboxylase (ODC) is the sentinel enzyme in polyamine biosynthesis. Both ODC and polyamines regulate cell division, proliferation, and apoptosis. Sepiapterin reductase (SPR) catalyzes the last step in the biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor of nitric oxide synthase, and has been implicated in neurological diseases but not yet in cancer. In this study, we present compelling evidence that native ODC and SPR physically interact, and we defined the individual amino acid residues involved in both enzymes using in silico protein-protein docking simulations. The resulting heterocomplex is a surprisingly compact structure, featuring two energetically and structurally equivalent binding modes both in monomer and in dimer conformations. The novel interaction between ODC and SPR proteins was confirmed under physiological conditions by co-immunoprecipitation and co-localization in neuroblastoma (NB) cells. Importantly, we showed that siRNA (small interfering RNA)-mediated knockdown of SPR expression significantly reduced endogenous ODC enzyme activity in NB cells, thus demonstrating the biological relevance of the ODC-SPR interaction. Finally, in a cohort of 88 human NB tumors, we found that high SPR mRNA expression correlated significantly with poor survival prognosis using a Kaplan-Meier analysis (log-rank test, P=5 × 10(-4)), suggesting an oncogenic role for SPR in NB tumorigenesis. In conclusion, we showed that ODC binds SPR and thus propose a new concept in which two well-characterized biochemical pathways converge via the interaction of two enzymes. We identified SPR as a novel regulator of ODC enzyme activity and, based on clinical evidence, present a model in which SPR drives ODC-mediated malignant progression in NB.

Fluorescence techniques in analysis of protein-ligand interactions.

Fluorescence spectroscopy may serve as a universal tool for the study of protein-ligand interactions. Applications of fluorometry have made use of various aspects of fluorescence such as intensity, emission and excitation spectra, lifetime, quantum yield, polarization state, and anisotropy, as well as energy transfer and other electronic phenomena. An experimentalist has to consider each of these characteristics carefully, frequently in combination with each other, for the analysis of protein-ligand complexes and for the determination of binding constants. Most of the available techniques are of a rather general nature and a wealth of possibilities exists for their utilization. In this chapter we will provide a short survey of selected techniques that can be used for measuring binding constants and probing protein-ligand interactions. Basic principles and phenomena are discussed followed by experimental considerations and examples of binding constant determination. Emphasis is placed on steady-state techniques that employ the use of intrinsic protein fluorescence, labeled ligands, as well as anisotropy and resonance energy transfer.

Evolutionary insights from a genetically divergent hantavirus harbored by the European common mole (Talpa europaea).

BACKGROUND: The discovery of genetically distinct hantaviruses in shrews (Order Soricomorpha, Family Soricidae) from widely separated geographic regions challenges the hypothesis that rodents (Order Rodentia, Family Muridae and Cricetidae) are the primordial reservoir hosts of hantaviruses and also predicts that other soricomorphs harbor hantaviruses. Recently, novel hantavirus genomes have been detected in moles of the Family Talpidae, including the Japanese shrew mole (Urotrichus talpoides) and American shrew mole (Neurotrichus gibbsii). We present new insights into the evolutionary history of hantaviruses gained from a highly divergent hantavirus, designated Nova virus (NVAV), identified in the European common mole (Talpa europaea) captured in Hungary. METHODOLOGY/PRINCIPAL FINDINGS: Pair-wise alignment and comparison of the full-length S- and L-genomic segments indicated moderately low sequence similarity of 54-65% and 46-63% at the nucleotide and amino acid levels, respectively, between NVAV and representative rodent- and soricid-borne hantaviruses. Despite the high degree of sequence divergence, the predicted secondary structure of the NVAV nucleocapsid protein exhibited the characteristic coiled-coil domains at the amino-terminal end, and the L-segment motifs, typically found in hantaviruses, were well conserved. Phylogenetic analyses, using maximum-likelihood and Bayesian methods, showed that NVAV formed a distinct clade that was evolutionarily distant from all other hantaviruses. CONCLUSIONS: Newly identified hantaviruses harbored by shrews and moles support long-standing virus-host relationships and suggest that ancestral soricomorphs, rather than rodents, may have been the early or original mammalian hosts.

Information content of fluorescence polarization and anisotropy.

The equality of information content in fluorescence polarization and emission anisotropy is a common assumption and the two quantities are used according to practical considerations. However, an information-theoretic analysis presented here reveals that their information content is substantially different. A scaling relation exists between polarization and anisotropy, and normalization allows their direct comparison. Various measures of information such as the absolute, relative, differential, and potential entropies all appear larger for anisotropy over part or all of its normalized overlap with the polarization function. The larger information content coincides with the signal range where the emitted light is polarized mostly in the parallel direction. Polarization takes on larger absolute entropy only when the emission is about perpendicular to the incident light and when the differential entropy is considered over the entire physical domain. The additional information locally afforded by polarization appears to be related to its larger signal range whereas the extra information in anisotropy may be attributed to a second perpendicular emission plane in its definition, which is oriented along the axis of propagation of light and takes the contribution of all degrees of rotational freedom into account. Thus anisotropy may be considered as a more accurate and more informative representation of the underlying physical phenomena. Some practical aspects relevant to studies of protein-ligand interactions are also discussed.