Calcitonin Receptor-Zonula Occludens-1 Interaction Is Critical for Calcitonin-Stimulated Prostate Cancer Metastasis

The role of neuroendocrine peptide calcitonin (CT) and its receptor (CTR) in epithelial cancer progression is an emerging concept with great clinical potential. Expression of CT and CTR is frequently elevated in prostate cancers (PCs) and activation of CT–CTR axis in non-invasive PC cells induces an invasive phenotype. Here we show by yeast-two hybrid screens that CTR associates with the tight junction protein Zonula Occludens-1 (ZO-1) via the interaction between the type 1 PDZ motif at the carboxy-terminus of CTR and the PDZ3 domain of ZO-1. Mutation of either the CTR C-PDZ-binding motif or the ZO-1-PDZ3 domain did not affect binding of CTR with its ligand or G-protein-mediated signaling but abrogated destabilizing actions of CT on tight junctions and formation of distant metastases by orthotopically implanted PC cells in nude mice, indicating that these PDZ domain interactions were pathologically relevant. Further, we observed CTR-ZO-1 interactions in PC specimens by proximity ligation immunohistochemistry, and identified that the number of interactions in metastatic PC specimens was several-fold larger than in non-metastatic PC. Our results for the first time demonstrate a mechanism by which PDZ-mediated interaction between CTR and ZO1 is required for CT-stimulated metastasis of prostate cancer. Since many receptors contain PDZ-binding motifs, this would suggest that PDZ-binding motif-adaptor protein interactions constitute a common mechanism for cancer metastasis.     

Cell death and mitochondrial dysfunction induced by the dietary non-proteinogenic amino acid l-azetidine-2-carboxylic acid (Aze)

In addition to the 20 protein amino acids that are vital to human health, hundreds of naturally occurring amino acids, known as non-proteinogenic amino acids (NPAAs), exist and can enter the human food chain. Some NPAAs are toxic through their ability to mimic protein amino acids and this property is utilised by NPAA-containing plants to inhibit the growth of other plants or kill herbivores. The NPAA l-azetidine-2-carboxylic acid (Aze) enters the food chain through the use of sugar beet (Beta vulgaris) by-products as feed in the livestock industry and may also be found in sugar beet by-product fibre supplements. Aze mimics the protein amino acid l-proline and readily misincorporates into proteins. In light of this, we examined the toxicity of Aze to mammalian cells in vitro. We showed decreased viability in Aze-exposed cells with both apoptotic and necrotic cell death. This was accompanied by alterations in endosomal–lysosomal activity, changes to mitochondrial morphology and a significant decline in mitochondrial function. In summary, the results show that Aze exposure can lead to deleterious effects on human neuron-like cells and highlight the importance of monitoring human Aze consumption via the food chain.

     

Repulsive guidance molecule RGMa alters utilization of bone morphogenetic protein (BMP) type II receptors by BMP2 and BMP4

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta superfamily of multifunctional ligands that transduce their signals through type I and II serine/threonine kinase receptors and intracellular Smad proteins. Recently, we identified the glycosylphosphatidylinositol-anchored repulsive guidance molecules RGMa, DRAGON (RGMb), and hemojuvelin (RGMc) as coreceptors for BMP signaling (Babbit, J. L., Huang, F. W., Wrighting, D. W., Xia, Y., Sidis, Y., Samad, T. A., Campagna, J. A., Chung, R., Schneyer, A., Woolf, C. J., Andrews, N. C., and Lin, H. Y. (2006) Nat. Genet. 38, 531-539; Babbit, J. L., Zhang, Y., Samad, T. A., Xia, Y., Tang, J., Schneyer, A., Woolf, C. J., and Lin, H. Y. (2005) J. Biol. Chem. 280, 29820-29827; Samad, T. A., Rebbapragada, A., Bell, E., Zhang, Y., Sidis, Y., Jeong, S. J., Campagna, J. A., Perusini, S., Fabrizio, D. A., Schneyer, A. L., Lin, H. Y., Brivanlou, A. H., Attisano, L., and Woolf, C. J. (2005) J. Biol. Chem. 280, 14122-14129). However, the mechanism by which RGM family members enhance BMP signaling remains unknown. Here, we report that RGMa bound to radiolabeled BMP2 and BMP4 with Kd values of 2.4+/-0.2 and 1.4+/-0.1 nm, respectively. In KGN human ovarian granulosa cells and mouse pulmonary artery smooth muscle cells, BMP2 and BMP4 signaling required BMP receptor type II (BMPRII), but not activin receptor type IIA (ActRIIA) or ActRIIB, based on changes in BMP signaling by small interfering RNA inhibition of receptor expression. In contrast, cells transfected with RGMa utilized both BMPRII and ActRIIA for BMP2 or BMP4 signaling. Furthermore, in BmpRII-null pulmonary artery smooth muscle cells, BMP2 and BMP4 signaling was reduced by inhibition of endogenous RGMa expression, and RGMa-mediated BMP signaling required ActRIIA expression. These findings suggest that RGMa facilitates the use of ActRIIA by endogenous BMP2 and BMP4 ligands that otherwise prefer signaling via BMPRII and that increased utilization of ActRIIA leads to generation of an enhanced BMP signal.     

Molecular variation at a candidate gene implicated in the regulation of fire ant social behavior

The fire ant Solenopsis invicta and its close relatives display an important social polymorphism involving differences in colony queen number. Colonies are headed by either a single reproductive queen (monogyne form) or multiple queens (polygyne form). This variation in social organization is associated with variation at the gene Gp-9, with monogyne colonies harboring only B-like allelic variants and polygyne colonies always containing b-like variants as well. We describe naturally occurring variation at Gp-9 in fire ants based on 185 full-length sequences, 136 of which were obtained from S. invicta collected over much of its native range. While there is little overall differentiation between most of the numerous alleles observed, a surprising amount is found in the coding regions of the gene, with such substitutions usually causing amino acid replacements. This elevated coding-region variation may result from a lack of negative selection acting to constrain amino acid replacements over much of the protein, different mutation rates or biases in coding and non-coding sequences, negative selection acting with greater strength on non-coding than coding regions, and/or positive selection acting on the protein. Formal selection analyses provide evidence that the latter force played an important role in the basal b-like lineages coincident with the emergence of polygyny. While our data set reveals considerable paraphyly and polyphyly of S. invicta sequences with respect to those of other fire ant species, the b-like alleles of the socially polymorphic species are monophyletic. An expanded analysis of colonies containing alleles of this clade confirmed the invariant link between their presence and expression of polygyny. Finally, our discovery of several unique alleles bearing various combinations of b-like and B-like codons allows us to conclude that no single b-like residue is completely predictive of polygyne behavior and, thus, potentially causally involved in its expression. Rather, all three typical b-like residues appear to be necessary.     

Community response grids: using information technology to help communities respond to bioterror emergencies

Access to accurate and trusted information is vital in preparing for, responding to, and recovering from an emergency. To facilitate response in large-scale emergency situations, Community Response Grids (CRGs) integrate Internet and mobile technologies to enable residents to report information, professional emergency responders to disseminate instructions, and residents to assist one another. CRGs use technology to help residents and professional emergency responders to work together in community response to emergencies, including bioterrorism events. In a time of increased danger from bioterrorist threats, the application of advanced information and communication technologies to community response is vital in confronting such threats. This article describes CRGs, their underlying concepts, development efforts, their relevance to biosecurity and bioterrorism, and future research issues in the use of technology to facilitate community response.     

Protein oxidation implicated as the primary determinant of bacterial radioresistance

In the hierarchy of cellular targets damaged by ionizing radiation (IR), classical models of radiation toxicity place DNA at the top. Yet, many prokaryotes are killed by doses of IR that cause little DNA damage. Here we have probed the nature of Mn-facilitated IR resistance in Deinococcus radiodurans, which together with other extremely IR-resistant bacteria have high intracellular Mn/Fe concentration ratios compared to IR-sensitive bacteria. For in vitro and in vivo irradiation, we demonstrate a mechanistic link between Mn(II) ions and protection of proteins from oxidative modifications that introduce carbonyl groups. Conditions that inhibited Mn accumulation or Mn redox cycling rendered D. radiodurans radiation sensitive and highly susceptible to protein oxidation. X-ray fluorescence microprobe analysis showed that Mn is globally distributed in D. radiodurans, but Fe is sequestered in a region between dividing cells. For a group of phylogenetically diverse IR-resistant and IR-sensitive wild-type bacteria, our findings support the idea that the degree of resistance is determined by the level of oxidative protein damage caused during irradiation. We present the case that protein, rather than DNA, is the principal target of the biological action of IR in sensitive bacteria, and extreme resistance in Mn-accumulating bacteria is based on protein protection.