Folding of proteins with diverse folds

Using parallel tempering simulations with high statistics, we investigate the folding and thermodynamic properties of three small proteins with distinct native folds: the all-helical 1RIJ, the all-sheet beta3s, and BBA5, which has a mixed helix-sheet fold. In all three cases, simulations with our energy function find the native structures as global minima in free energy at experimentally relevant temperatures. However, the folding process strongly differs for the three molecules, indicating that the folding mechanism is correlated with the form of the native structure.     

Oxidation of Arg-410 promotes the elimination of human serum albumin

The effect of the oxidation of amino acid residues on albumin on its in vivo elimination was investigated using mutants and oxidized HSAs. The single-residue mutants (H146A, K199A, W214A, R218H, R410A, Y411A) and oxidized HSAs were produced by the recombinant DNA techniques and incubation with a metal ion-catalyzed oxidation (MCO) system for 12, 24, 48 or 72 h. Pharmacokinetics were evaluated in mice after labeling with 111In. Structural and functional properties were examined by several spectroscopic techniques. Time-dependent increase in carbonyl group content resulted in increase in the liver clearance of oxidized HSAs. Slight decreases in alpha-helical content as the result of oxidation was induced by the increases in accessible hydrophobic areas and the net negative charge on the HSA molecule. No significant change in the pharmacokinetics and structural properties was observed for the W214A, R218H and Y411A mutants, but the properties for the H146A, K199A and R410A mutants were affected (extent of effect: R410A > K199A > H146A). The liver clearance of these proteins is closely correlated to hydrophobicity (r = 0.929, P < 0.01) and the net charge of the proteins (r=0.930, P < 0.01). The rate of elimination of HSA is closely related to the hydrophobicity and net charge of the molecule. Further, the R410A mutants had a short half-life and structure similar to oxidized HSA after oxidation. Therefore, the modification of Arg-410 via oxidative stress may promote the elimination of HSA.     

Heterocyclic analogues of squamocin as inhibitors of mitochondrial complex I. On the role of the terminal lactone of annonaceous acetogenins

Heterocyclic analogues of squamocin have been semisynthesized by condensation reactions between squamocin-derived alpha-keto esters and heterodinucleophiles. The strong complex I inhibitory potency of squamocin-benzimidazole, a hybrid derivative, illustrates for the first time the functional analogy existing between the terminal butenolide of annonaceous acetogenins and heteroaromatic substructures of classic inhibitors of the enzyme. This finding supports the categorization of this atypical group of inhibitors as antagonists of the ubiquinone substrates. In addition, competition experiments of squamocin-benzimidazole versus squamocin and rolliniastatin-2 suggest that the binding of this hybrid inhibitor is responsible for a negative allosteric effect at the level of the first ubiquinone-binding site (A site) of mitochondrial complex I. This result supports the existence of a large cooperatively regulated inhibitor/ubiquinone-binding pocket located within the catalytic core of the enzyme, consisting of the association of the previously defined affinity sites A and B.     

Discovery of genes expressed in Hydra embryogenesis

Hydra's remarkable capacity to regenerate, to proliferate asexually by budding, and to form a pattern de novo from aggregates allows studying complex cellular and molecular processes typical for embryonic development. The underlying assumption is that patterning in adult hydra tissue relies on factors and genes which are active also during early embryogenesis. Previously, we reported that in Hydra the timing of expression of conserved regulatory genes, known to be involved in adult patterning, differs greatly in adults and embryos (Fr?bius, A.C., Genikhovich, G., Kürn, U., Anton-Erxleben, F. and Bosch, T.C.G., 2003. Expression of developmental genes during early embryogenesis of Hydra. Dev. Genes Evol. 213, 445-455). Here, we describe an unbiased screening strategy to identify genes that are relevant to Hydra vulgaris embryogenesis. The approach yielded two sets of differentially expressed genes: one set was expressed exclusively or nearly exclusively in the embryos, while the second set was upregulated in embryos in comparison to adult polyps. Many of the genes identified in hydra embryos had no matches in the database. Among the conserved genes upregulated in embryos is the Hydra orthologue of Embryonic Ectoderm Development (HyEED). The expression pattern of HyEED in developing embryos suggests that interstitial stem cells in Hydra originate in the endoderm. Importantly, the observations uncover previously unknown differences in genes expressed by embryos and polyps and indicate that not only the timing of expression of developmental genes but also the genetic context is different in Hydra embryos compared to adults.     

Chloroplast heat shock protein Cpn60 from Chlamydomonas reinhardtii exhibits a novel function as a group II intron-specific RNA-binding protein

Intron-binding proteins in eukaryotic organelles are mainly encoded by the nuclear genome and are thought to promote the maturation of precursor RNAs. Here, we present a biochemical approach that enable the isolation of a novel nuclear-encoded protein from Chlamydomonas reinhardtii showing specific binding properties to organelle group II intron RNA. Using FPLC chromatography of chloroplast protein extracts, a 61-kDa RNA-binding protein was isolated and then tentatively identified by mass spectrometry as the chloroplast heat shock protein Cpn60. Heterologous Cpn60 protein was used in RNA protein gel mobility shift assays and revealed that the ATPase domains of Cpn60 mediates the specific binding of two group II intron RNAs, derived from the homologous chloroplast psaA gene and the heterologous mitochondrial LSU rRNA gene. The function of Cpn60 as a general organelle splicing factor is discussed.     

Lipid-specific membrane activity of human beta-defensin-3

Defensins represent a major component of innate host defense against bacteria, fungi, and enveloped viruses. One potent defensin found, e.g., in epithelia, is the polycationic human beta-defensin-3 (hBD3). We investigated the role of the lipid matrix composition, and in particular the presence of negatively charged lipopolysaccharides (LPS) from sensitive (Escherichia coli, Salmonella enterica serovar Minnesota) or resistant (Proteus mirabilis) Gram-negative bacteria or of the zwitterionic phospholipids of human cells, in determining the action of polycationic hBD3 on the different membranes, and related to their biological activity. The main focus was directed on data derived from electrical measurements on a reconstitution system of the OM as a planar asymmetric bilayer composed on one side of LPS and on the other of a phospholipid mixture. Our results demonstrate that the antimicrobial activity and the absence of cytotoxicity can be explained by the lipid-specificity of the peptide. A clear correlation between these aspects of the biological activity of hBD3 and its interaction with lipid matrices could be found. In particular, hBD3 could only induce lesions in those membranes resembling the lipid composition of the OM of sensitive bacterial strains. The permeation through the membrane is a decisive first step for the biological activity of many antimicrobial peptides. Therefore, we propose that the lipid-specificity of hBD3 as well as some other membrane-active antimicrobial peptides is important for their activity against bacteria or mammalian cells.     

A biophysical approach to the optimisation of dendritic-tumour cell electrofusion

Electrofusion of tumour and dendritic cells (DCs) is a promising approach for production of DC-based anti-tumour vaccines. Although human DCs are well characterised immunologically, little is known about their biophysical properties, including dielectric and osmotic parameters, both of which are essential for the development of efficient electrofusion protocols. In the present study, human DCs from the peripheral blood along with a tumour cell line used as a model fusion partner were examined by means of time-resolved cell volumetry and electrorotation. Based on the biophysical cell data, the electrofusion protocol could be rapidly optimised with respect to the sugar composition of the fusion medium, duration of hypotonic treatment, frequency range for stable cell alignment, and field strengths of breakdown pulses triggering membrane fusion. The hypotonic electrofusion consistently gave a tumour-DC hybrid rate of up to 19%, as determined by counting dually labelled fluorescent hybrids in a microscope. This fusion rate is nearly twice as high as that usually reported in the literature for isotonic media. The experimental findings and biophysical approach presented here are generally useful for the development of efficient electrofusion protocols, especially for rare and valuable human cells.     

Strategy for the development of a matched set of transport-competent, angiotensin receptor-deficient proximal tubule cell lines

In the proximal convoluted tubule (PCT) angiotensin II (Ang II) modulates fluid and electrolyte transport through at least two pharmacologically distinct receptor subtypes: AT(1) and AT(2). Development of cell lines that lack these receptors are potentially useful models to probe the complex cellular details of Ang II regulation. To this end, angiotensin receptor- deficient mice were bred with an Immortomouse(R), which harbors a thermolabile SV40 large-T antigen (Tag). S1 PCT segments from kidneys of F(2) mice were microdissected, placed in culture, and maintained under conditions that enhanced cell growth, i.e., promoted Tag expression and thermostability. Three different types of angiotensin receptor-deficient cell lines, (AT(1A) [-/-], Tag [+/-]), (AT(1B) [-/-], Tag [+/-]), and (AT(1A) [-/-], AT(1B) [-/-], Tag [+/+]), as well as wild type cell lines were generated. Screening and characterization, which were conducted under culture conditions that promoted cellular differentiation, included: measurements of transepithelial transport, such as basal monolayer short-circuit current (Isc; -3 to 3 microA/cm2), basal monolayer conductance (G, 2 to 10 mS/cm2), Na3(+)-phosphate cotransport (DeltaIsc of 2 to 3 microA/cm(2) at 1 mM), and Na(3)(+)-succinate cotransport (DeltaIsc of 1 to 9 microA/cm(2) at 2 mM). Morphology of cell monolayers showed an extensive brush border, well-defined tight junctions, and primary cilia. Receptor functionality was assessed by Ang II-stimulated beta-arrestin 2 translocation and showed an Ang II-mediated response in wild type but not (AT(1A) [-/ -], AT(1B) [-/-]) cells. Cell lines were amplified, yielding a virtually unlimited supply of highly differentiated, transport-competent, angiotensin receptor-deficient PCT cell lines.     

REV1 protein interacts with PCNA: significance of the REV1 BRCT domain in vitro and in vivo

REV1 protein, a eukaryotic member of the Y family of DNA polymerases, is involved in the tolerance of DNA damage by translesion DNA synthesis. It is unclear how REV1 is recruited to replication foci in cells. Here, we report that mouse REV1 can bind directly to PCNA and that monoubiquitylation of PCNA enhances this interaction. The interaction between REV1 protein and PCNA requires a functional BRCT domain located near the N terminus of the former protein. Deletion or mutational inactivation of the BRCT domain abolishes the targeting of REV1 to replication foci in unirradiated cells, but not in UV-irradiated cells. In vivo studies in both chicken DT40 cells and yeast directly support the requirement of the BRCT domain of REV1 for cell survival and DNA damage-induced mutagenesis.