Molecular recognition of human eosinophil-derived neurotoxin (RNase 2) by placental ribonuclease inhibitor

Placental ribonuclease inhibitor (RI) binds diverse mammalian RNases with dissociation constants that are in the femtomolar range. Previous studies on the complexes of RI with RNase A and angiogenin revealed that RI utilises largely distinctive interactions to achieve high affinity for these two ligands. Here we report a 2.0 angstroms resolution crystal structure of RI in complex with a third ligand, eosinophil-derived neurotoxin (EDN), and a mutational analysis based on this structure. The RI-EDN interface is more extensive than those of the other two complexes and contains a considerably larger set of interactions. Few of the contacts present in the RI-angiogenin complex are replicated; the correspondence to the RI-RNase A complex is somewhat greater, but still modest. The energetic contributions of various interface regions differ strikingly from those in the earlier complexes. These findings provide insight into the structural basis for the unusual combination of high avidity and relaxed stringency that RI displays.     

Polypyrimidine tract binding protein blocks the 5' splice site-dependent assembly of U2AF and the prespliceosomal E complex

Polypyrimidine tract binding protein (PTB) represses some alternatively spliced exons by direct occlusion of splice sites. In repressing the splicing of the c-src N1 exon, we find that PTB acts by a different mechanism. PTB does not interfere with U1 snRNP binding to the N1 5' splice site. Instead, PTB prevents formation of the prespliceosomal early (E) complex across the intervening intron by preventing the assembly of the splicing factor U2AF on the 3' splice site of exon 4. When the unregulated 5' splice site of the upstream exon 3 is present, U2AF binding is restored and splicing between exons 3 and 4 proceeds in spite of the N1 exon bound PTB. Thus, rather than directly blocking the N1 splice sites, PTB prevents the 5' splice site-dependent assembly of U2AF into the E complex. This mechanism likely occurs in many other alternative exons.     

De novo alanine synthesis by bacteroids of Mesorhizobium loti is not required for nitrogen transfer in the determinate nodules of Lotus corniculatus

Deletion of both alanine dehydrogenase genes (aldA) in Mesorhizobium loti resulted in the loss of AldA enzyme activity from cultured bacteria and bacteroids but had no effect on the symbiotic performance of Lotus corniculatus plants. Thus, neither indeterminate pea nodules nor determinate L. corniculatus nodules export alanine as the sole nitrogen secretion product.     

An unexpected role for caspase-2 in neuroblastoma

Caspase-2 has been implicated in various cellular functions, including cell death by apoptosis, oxidative stress response, maintenance of genomic stability and tumor suppression. The loss of the caspase-2 gene (Casp2) enhances oncogene-mediated tumorigenesis induced by E1A/Ras in athymic nude mice, and also in the Eμ-Myc lymphoma and MMTV/c-neu mammary tumor mouse models. To further investigate the function of caspase-2 in oncogene-mediated tumorigenesis, we extended our studies in the TH-MYCN transgenic mouse model of neuroblastoma. Surprisingly, we found that loss of caspase-2 delayed tumorigenesis in the TH-MYCN neuroblastoma model. In addition, tumors from TH-MYCN/Casp2^−/− mice were predominantly thoracic paraspinal tumors and were less vascularized compared with tumors from their TH-MYCN/Casp2^+/+ counterparts. We did not detect any differences in the expression of neuroblastoma-associated genes in TH-MYCN/Casp2^−/− tumors, or in the activation of Ras/MAPK signaling pathway that is involved in neuroblastoma progression. Analysis of expression array data from human neuroblastoma samples showed a correlation between low caspase-2 levels and increased survival. However, caspase-2 levels correlated with clinical outcome only in the subset of MYCN-non-amplified human neuroblastoma. These observations indicate that caspase-2 is not a suppressor in MYCN-induced neuroblastoma and suggest a tissue and context-specific role for caspase-2 in tumorigenesis.The caspase family of cysteine proteases are highly conserved regulators of cell death by apoptosis.¹ In addition to their pro-apoptotic function, many caspases also have non-apoptotic roles in other physiological processes, such as inflammation, necrosis and tumor suppression.^{2, 3, 4} The most highly conserved caspase, caspase-2, has recently been demonstrated to function in the cellular stress response, protection against ageing, maintenance of genome stability and in tumor suppression.^{2, 5, 6, 7, 8}The tumor suppressor function of caspase-2 was first demonstrated using E1A/Ras-transformed caspase-2-deficient mouse embryonic fibroblasts (MEFs), which showed an increased tumorigenic potential in athymic nude mice.⁷ Further supporting evidence came from experiments demonstrating that caspase-2 deficiency enhances B-cell lymphoma development in Eμ-Myc transgenic mice⁷ and mammary carcinomas in MMTV/c-neu mice,⁹ suggesting that caspase-2 prevents oncogene-induced lymphomas and epithelial tumors. Importantly, tumor suppression by caspase-2 is also evident in the non-oncogene-driven Atm^−/− thymoma mouse model.¹⁰Given its role in apoptosis, the tumor suppression function of caspase-2 was thought to be associated with this role, via the elimination of mutagenic or potentially tumorigenic cells. Recent studies have now indicated that the role of caspase-2 may extend beyond apoptosis and that its tumor suppression function may, in part, be mediated by maintaining genomic stability and/or the oxidative stress response. Caspase-2-deficient MEFs and tumor cells from Eμ-Myc/Casp2^−/−, MMTV/c-neu/Casp2^−/− and Atm^−/−;Casp2^−/− mice all display aberrant proliferation, and increased genomic instability^{6, 9, 10} and indicate that caspase-2 is important for the maintenance of genome stability. Importantly, the role of caspase-2 in maintaining genomic stability in primary cells appears to be required for its tumor suppressor function.¹⁰Genomic instability is a hallmark of cancer¹¹ and the overexpression of Myc family oncoproteins is commonly associated with genomic instability and a wide spectrum of human cancers.^{12, 13, 14} Interestingly, a common feature of the oncogene-induced tumor models used in the study of caspase-2 tumor suppressor function is the overexpression of c-Myc¹⁵ or aberrant c-Myc signaling.^{16, 17, 18} Given the role of Myc proteins as key mediators of genomic instability as well as cell proliferation, cell growth and DNA damage, we were interested in further assessing whether caspase-2 can promote tumor suppression in other MYC-dependent mouse tumor models. We used the MYCN mouse model of neuroblastoma (TH-MYCN mouse), in which MYCN is constitutively expressed under the control of the rat tyrosine hydroxylase (TH) promoter leading to neural crest cell-specific expression and early-onset neuroblastoma.¹⁹ Amplification of MYCN occurs in ∼20% of human neuroblastomas and high MYCN protein levels are strongly associated with tumor progression and poor clinical outcome.^{20, 21} Thus, the TH-MYCN transgenic mouse model recapitulates many clinical features of aggressive neuroblastomas in humans and provides a powerful model of preclinical neuroblastoma.^{19, 22}MYCN-mediated neuroblastoma onset and progression is commonly associated with additional genetic events, including the expression of the key genes including Odc1, Mrp1, SirT1 and Ras.^{23, 24, 25} A recent study has found that caspase-8 is in fact a potent suppressor of neuroblastoma, with the loss of caspase-8 expression occurring in ∼70% of neuroblastoma patients.^{26, 27} Interestingly, the loss of caspase-8 also promotes bone marrow metastasis in the TH-MYCN neuroblastoma mouse model.^{26, 27} The role of other caspases in neuroblastoma has not previously been examined, and given the function of caspase-2 in tumor suppression, provided additional relevance in assessing its role in this model.This study shows that caspase-2 is not able to suppress neuroblastoma development in TH-MYCN mice. In contrast to a role for caspase-2 as a tumor suppressor, our findings demonstrate that loss of caspase-2 somewhat delays neuroblastoma onset in mice. Interestingly, expression array data from human neuroblastoma show a strong correlation between low caspase-2 levels and improved outcome. Our data demonstrate that the tumor suppressor function of caspase-2 is not specific to Myc-mediated oncogenesis and that its role is likely to be tissue- and/or context-specific.     

QSAR studies on the activation of the human carbonic anhydrase cytosolic isoforms I and II and secretory isozyme VI with amino acids and amines

The first QSAR study on the activation of the human secretory isoform of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1), CA VI, with a series of amines and amino acids is reported. A large set of topological indices have been used to obtain several tri-/tetra-parametric models. We compared the CA VI activating QSAR models with those calculated for activation of the cytosolic human isozymes hCA I and hCA II. In addition, the effect of D- and L-amino acids as activators of hCA I, hCA II and of hCA VI as compared to those of structurally related biogenic amines was investigated for obtaining statistically significant and predictive QSAR equations. The obtained models are discussed using a variety of statistical parameters. The best models were obtained for hCA II activation, followed by hCA I, whereas the QSAR models for the activation of hCA VI were statistically weaker.     

Fibronectin and laminin enhance engraftibility of cultured hematopoietic stem cells

To test the hypothesis that extracellular matrix (ECM) components maintain stem cell property, murine bone marrow (BM) cells were expanded in fibronectin and laminin coated plate in the presence of cytokines. We observed significant phenotypic and functional improvement of expanded cells. In 10 days, 800-fold expansion of colony-forming unit-granulocyte erythrocyte monocyte megakaryocyte (CFU-GEMM) was observed in the cultured cells. No apparent activation of cell cycle was observed, but CD29 and very late antigen-4 (VLA-4) expression was increased, as compared to the normal BM cells. A fraction of the expanded cells became verapamil sensitive, suggesting upregulation of multi-drug resistant gene(s), as found in the primitive hematopoietic stem cells (HSCs). Competitive repopulation assay confirmed that HSCs compartment was amplified during culture. Overall, our study clearly demonstrated that ex vivo culture of murine HSCs in the presence of fibronectin and laminin resulted in expansion of primitive stem cells and improvement in the marrow engraftibility.     

The protein kinase C delta catalytic fragment targets Mcl-1 for degradation to trigger apoptosis

Proteolytic cleavage and subsequent activation of protein kinase C (PKC) delta is required for apoptosis induced by a variety of genotoxic agent, including UV radiation. In addition, overexpression of the constitutively active PKCdelta catalytic fragment (PKCdelta-cat) is sufficient to trigger Bax activation, cytochrome c release, and apoptosis. While PKCdelta is a key apoptotic effector, the downstream target(s) responsible for the mitochondrial apoptotic cascade are not known. We found that expression of the active PKCdelta-cat in HaCaT cells triggers a reduction in the anti-apoptotic protein Mcl-1, similar to UV radiation. The down-regulation of Mcl-1 induced by PKCdelta-cat was not at the mRNA level but was due to decreased protein half-life. Overexpression of Mcl-1 protected HaCaT cells from both UV and PKCdelta-cat-induced apoptosis and blocked the release of cytochrome c from the mitochondria, indicating that Mcl-1 down-regulation was required for apoptosis signaling. Indeed, down-regulation of Mcl-1 with siRNA slightly increased the basal apoptotic rate of HaCaT cells and dramatically sensitized them to UV or PKCdelta-cat-induced apoptosis. HaCaT cells with down-regulated Mcl-1 had higher activated Bax protein, as measured by Bax cross-linking, indicating that Mcl-1 down-regulation is sufficient for Bax activation. Finally, recombinant PKCdelta could phosphorylate Mcl-1 in vitro, identifying Mcl-1 as a direct target for PKCdelta. Overall our results identify Mcl-1 as an important target for PKCdelta-cat that can mediate its pro-apoptotic effects on mitochondria to amplify the apoptotic signaling induced by a wide range of apoptotic stimuli.     

Phosphorylation on Ser-279 and Ser-282 of connexin43 regulates endocytosis and gap junction assembly in pancreatic cancer cells

The molecular mechanisms regulating the assembly of connexins (Cxs) into gap junctions are poorly understood. Using human pancreatic tumor cell lines BxPC3 and Capan-1, which express Cx26 and Cx43, we show that, upon arrival at the cell surface, the assembly of Cx43 is impaired. Connexin43 fails to assemble, because it is internalized by clathrin-mediated endocytosis. Assembly is restored upon expressing a sorting-motif mutant of Cx43, which does not interact with the AP2 complex, and by expressing mutants that cannot be phosphorylated on Ser-279 and Ser-282. The mutants restore assembly by preventing clathrin-mediated endocytosis of Cx43. Our results also document that the sorting-motif mutant is assembled into gap junctions in cells in which the expression of endogenous Cx43 has been knocked down. Remarkably, Cx43 mutants that cannot be phosphorylated on Ser-279 or Ser-282 are assembled into gap junctions only when connexons are composed of Cx43 forms that can be phosphorylated on these serines and forms in which phosphorylation on these serines is abolished. Based on the subcellular fate of Cx43 in single and contacting cells, our results document that the endocytic itinerary of Cx43 is altered upon cell–cell contact, which causes Cx43 to traffic by EEA1-negative endosomes en route to lysosomes. Our results further show that gap-junctional plaques formed of a sorting motif–deficient mutant of Cx43, which is unable to be internalized by the clathrin-mediated pathway, are predominantly endocytosed in the form of annular junctions. Thus the differential phosphorylation of Cx43 on Ser-279 and Ser-282 is fine-tuned to control Cx43’s endocytosis and assembly into gap junctions.