Structure, dynamics, lipid binding, and physiological relevance of the putative GTPase-binding domain of Dictyostelium formin C

Dictyostelium Formin C (ForC) is involved in the regulation of local actin cytoskeleton reorganization (e.g. during cellular adhesion or migration). ForC contains formin homology 2 and 3 (FH2 and -3) domains and an N-terminal putative GTPase-binding domain (GBD) but lacks a canonical FH1 region. To better understand the role of the GBD, its structure, dynamics, lipid-binding properties, and cellular functions were analyzed by NMR and CD spectroscopy and by in vivo fluorescence microscopy. Moreover, the program CS-Rosetta was tested for the structure prediction based on chemical shift data only. The ForC GBD adopts an ubiquitin-like α/β-roll fold with an unusually long loop between β-strands 1 and 2. Based on the lipid-binding data, the presence of DPC micelles induces the formation of α-helical secondary structure and a rearrangement of the tertiary structure. Lipid-binding studies with a mutant protein and a peptide suggest that the β1-β2 loop is not relevant for these conformational changes. Whereas small amounts of negatively charged phosphoinositides (1,2-dioctanoyl-sn-glycero-3-(phosphoinositol 4,5-bisphosphate) and 1,2-dihexanoyl-sn-glycero-3-(phosphoinositol 3,4,5-trisphosphate)) lower the micelle concentration necessary to induce the observed spectral changes, other negatively charged phospholipids (1,2-dihexanoyl-sn-glycero-3-(phospho-L-serine) and 1,2-dihexanoyl-sn-glycero-3-phospho-(1'-rac-glycerol)) had no such effect. Interestingly, bicelles and micelles composed of diacylphosphocholines had no effect on the GBD structure. Our data suggest a model in which part of the large positively charged surface area of the GBD mediates localization to specific membrane patches, thereby regulating interactions with signaling proteins. Our cellular localization studies show that both the GBD and the FH3 domain are required for ForC targeting to cell-cell contacts and early phagocytic cups and macropinosomes.     

Impairment of protein trafficking by direct interaction of gliadin peptides with actin

Intestinal celiac disease (CD) is triggered by peptic-tryptic digest of gluten, known as Frazer's Fraction (FF), in genetically predisposed individuals. Here, we investigate the immediate effects of FF on the actin cytoskeleton and the subsequent trafficking of actin-dependent and actin-independent proteins in COS-1 cells. Morphological alterations in the actin filaments were revealed concomitant with a drastic reduction in immunoprecipitated actin from cells incubated with FF. These alterations elicit impaired protein trafficking of intestinal sucrase–isomaltase, a glycoprotein that follows an actin-dependent vesicular transport to the cell surface. However, the actin-independent transport of intestinal lactase phlorizin hydrolase remains unaffected. Moreover, the morphological alteration in actin is induced by direct interaction of this protein with gliadin peptides carrying the QQQPFP epitope revealed by co-immunoprecipitation utilizing a monoclonal anti-gliadin antibody. Finally, stimulation of cells with FF directly influences the binding of actin to Arp2. Altogether, our data demonstrate that FF directly interacts with actin and alters the integrity of the actin cytoskeleton thus leading to an impaired trafficking of intestinal proteins that depend on an intact actin network. This direct interaction could be related to the endocytic segregation of gliadin peptides as well as the delayed endocytic vesicle trafficking and maturation in gliadin-positive intestinal epithelial cells and opens new insights into the pathogenesis of CD.     

Excited-state dynamics of protochlorophyllide revealed by subpicosecond infrared spectroscopy

To gain a better understanding of the light-induced reduction of protochlorophyllide (PChlide) to chlorophyllide as a key regulatory step in chlorophyll synthesis, we performed transient infrared absorption measurements on PChlide in d4-methanol. Excitation in the Q-band at 630 nm initiates dynamics characterized by three time constants: τ₁ = 3.6 ± 0.2, τ₂ = 38 ± 2, and τ₃ = 215 ± 8 ps. As indicated by the C13′=O carbonyl stretching mode in the electronic ground state at 1686 cm⁻¹, showing partial ground-state recovery, and in the excited electronic state at 1625 cm⁻¹, showing excited-state decay, τ₂ describes the formation of a state with a strong change in electronic structure, and τ₃ represents the partial recovery of the PChlide electronic ground state. Furthermore, τ₁ corresponds with vibrational energy relaxation. The observed kinetics strongly suggest a branched reaction scheme with a branching ratio of 0.5 for the path leading to the PChlide ground state on the 200 ps timescale and the path leading to a long-lived state (>>700 ps). The results clearly support a branched reaction scheme, as proposed previously, featuring the formation of an intramolecular charge transfer state with ∼25 ps, its decay into the PChlide ground state with 200 ps, and a parallel reaction path to the long-lived PChlide triplet state.     

Mx is dispensable for interferon-mediated resistance of chicken cells against influenza A virus

The type I interferon (IFN) system plays an important role in antiviral defense against influenza A viruses (FLUAV), which are natural chicken pathogens. Studies of mice identified the Mx1 protein as a key effector molecule of the IFN-induced antiviral state against FLUAV. Chicken Mx genes are highly polymorphic, and recent studies suggested that an Asn/Ser polymorphism at amino acid position 631 determines the antiviral activity of the chicken Mx protein. By employing chicken embryo fibroblasts with defined Mx-631 polymorphisms and retroviral vectors for the expression of Mx isoforms in chicken cells and embryonated eggs, we show here that neither the 631Asn nor the 631Ser variant of chicken Mx was able to confer antiviral protection against several lowly and highly pathogenic FLUAV strains. Using a short interfering RNA (siRNA)-mediated knockdown approach, we noted that the antiviral effect of type I IFN in chicken cells was not dependent on Mx, suggesting that some other IFN-induced factors must contribute to the inhibition of FLUAV in chicken cells. Finally, we found that both isoforms of chicken Mx protein appear to lack GTPase activity, which might explain the observed lack of antiviral activity.     

Synthesis, characterization and antitumor activity of polymeric copper(II) complexes with thiosemicarbazones of 3-methyl-5-oxo-1-phenyl-3-pyrazolin-4-carboxaldehyde and 5-oxo-3-phenyl-3-pyrazolin-4-carboxaldehyde

New polymeric copper(II) complexes with two tridentate ONS thiosemicarbazone ligands containing substituted pyrazolone moiety were synthesized and characterized by means of spectroscopic, electrochemical and crystallographic techniques. While both ligands exist as different tautomers in the solid state and DMSO-d₆ solution, Cu(II) ion coordinates the ligands from the same tautomeric form with square-pyramidal geometry around each Cu atom. In the crystal structures, the copper(II) complex cation forms polymeric chains {[Cu(L)Cl]⁺}_n with a bridging chlorine atom. One of the complexes was found to have a significantly higher cytotoxic potential in comparison with cisplatin in inhibition of several cell lines (HL60, REH, C6, L929 and B16). The results obtained on the basis of flow cytometry indicated that apoptosis could be possible mechanism of cell death.     

Targeting FcαRI on polymorphonuclear cells induces tumor cell killing through autophagy

Neutrophils are the most abundant circulating FcR-expressing WBCs with potent cytotoxic ability. Currently, they are recognized as promising effector cells for Ab-mediated immunotherapy of cancer, because their capacity to kill tumor cells is greatly enhanced by tumor Ag-specific mAbs. The FcαRI represents the most potent FcR on neutrophils for induction of Ab-mediated tumor cell killing. However, the mechanisms of cell death that are induced are poorly understood. Because these mechanisms can be used for modulation of anticancer treatment, we investigated the tumor cell death induced by neutrophil-mediated Ab-dependent killing via FcαRI. Human mammary carcinoma cells were efficiently killed when incubated with human neutrophils and tumor-specific FcαRI bispecific or IgA Abs. Interestingly, we observed characteristics of autophagy such as autophagic structures by electron microscopy and LC3B(+) autophagosomes in different human epithelial carcinoma cells, which resulted in tumor cell death. To a lesser extent, necrotic features, such as cellular membrane breakdown and spillage of intracellular content, were found. By contrast, apoptotic features including fragmented nuclei, Annexin V-positivity, and presence of cleaved caspase-3 were not observed. These findings indicate that neutrophils mainly facilitate autophagy to induce tumor cell death rather than the more commonly recognized apoptotic cell death mechanisms induced by NK cells or cytotoxic T cells. This knowledge not only reveals the type of tumor cell death induced in neutrophil-mediated, Ab-dependent cellular cytotoxicity, but importantly opens up additional perspectives for modulation of anticancer therapy in, for example, apoptosis-resistant tumor cells.     

Adaptive potential of northernmost tree populations to climate change, with emphasis on Scots pine (Pinus sylvestris L.)

The adaptive potential of the northernmost Pinus sylvestris L. (and other northern tree) populations is considered by examining first the current patterns of quantitative genetic adaptive traits, which show high population differentiation and clines. We then consider the postglacial history of the populations using both paleobiological and genetic data. The current patterns of diversity at nuclear genes suggest that the traces of admixture are mostly visible in mitochondrial DNA variation patterns. There is little evidence of increased diversity due to admixture between an eastern and western colonization lineage, but no signal of reduced diversity (due to sequential bottlenecks) either. Quantitative trait variation in the north is not associated with the colonizing lineages. The current clines arose rapidly and may be based on standing genetic variation. The initial phenotypic response of Scots pine in the north is predicted to be increased survival and growth. The genetic responses are examined based on quantitative genetic predictions of sustained selection response and compared with earlier simulation results that have aimed at more ecological realism. The phenotypic responses of increased growth and survival reduce the opportunity for selection and delay the evolutionary responses. The lengthening of the thermal growing period also causes selection on the critical photoperiod in the different populations. Future studies should aim at including multiple ecological and genetic factors in evaluating potential responses.     

The dual role of annexin II in targeting of brush border proteins and in intestinal cell polarity

Functional intestinal epithelium relies on complete polarization of enterocytes marked by the formation of microvilli and the accurate trafficking of glycoproteins to relevant membrane domains. Numerous transport pathways warrant the unique structural identity and protein/lipid composition of the brush border membrane. Annexin II (Ca(2+)-dependent lipid-binding protein) is an important component of one of the apical protein transport machineries, which involves detergent-resistant membranes and the actin cytoskeleton. Here, we investigate in intestinal Caco-2 cells the contribution of annexin II to the sorting and transport of brush border hydrolases and role in intestinal cell polarity. Downregulation of annexin II in Caco-2-A4 cell line results in a severe reduction of the levels of the brush border membrane resident enzyme sucrase isomaltase (SI) as well as structural components such as ezrin. This reduction is accompanied by a redistribution of these proteins to intracellular compartments and a striking morphological transition of Caco-2 cells to rudimentary epithelial cells that are characterized by an almost flat apical membrane with sparse and short microvilli. Concomitant with this alteration is the redistribution of the intermediate filament protein keratin 19 to the intracellular membranes in Caco-2-A4 cells. Interestingly, keratin 19 interacts with annexin II in wild type Caco-2 cells and this interaction occurs exclusively in lipid rafts. Our findings suggest a role for annexin II and K19 in differentiation and polarization of intestinal cells.     

Mice with human immune system components as in vivo models for infections with human pathogens

Many pathogens relevant to human disease do not infect other animal species. Therefore, animal models that reconstitute or harbor human tissues are explored as hosts for these. In this review, we will summarize recent advances to utilize mice with human immune system components, reconstituted from hematopoietic progenitor cells in vivo. Such mice can be used to study human pathogens that replicate in leukocytes. In addition to studying the replication of these pathogens, the reconstituted human immune system components can also be analyzed for initiating immune responses and control against these infections. Moreover, these new animal models of human infectious disease should replicate the reactivity of the human immune system to vaccine candidates and, especially, the adjuvants contained in them, more faithfully.     

Discovery and optimization of 2-phenyloxazole derivatives as diacylglycerol acyltransferase-1 inhibitors

In a discovery effort to find safe and effective DGAT-1 inhibitors, we have identified 2-phenyloxazole 4-carboxamide 1 as a conformationally constrained analog of a hydrazide hit, which was previously identified from high-throughput screening. Further optimization of this series has led to chemically more stable 2-phenyloxazole-based DGAT-1 inhibitor 25 with improved solubility, cell-based activity, and pharmacokinetic properties. Compound 25 also demonstrated in vivo efficacy in a diet-induced obesity (DIO) rat model.