Crystal structure of NblA from Anabaena sp. PCC 7120, a small protein playing a key role in phycobilisome degradation

Cyanobacterial light-harvesting complexes, the phycobilisomes, are proteolytically degraded when the organisms are starved for combined nitrogen, a process referred to as chlorosis or bleaching. Gene nblA, present in all phycobilisome-containing organisms, encodes a protein of about 7 kDa that plays a key role in phycobilisome degradation. The mode of action of NblA in this degradation process is poorly understood. Here we presented the 1.8-A crystal structure of NblA from Anabaena sp. PCC 7120. In the crystal, NblA is present as a four-helix bundle formed by dimers, the basic structural units. By using pull-down assays with immobilized NblA and peptide scanning, we showed that NblA specifically binds to the alpha-subunits of phycocyanin and phycoerythrocyanin, the main building blocks of the phycobilisome rod structure. By site-directed mutagenesis, we identified amino acid residues in NblA that are involved in phycobilisome binding. The results provided evidence that NblA is directly involved in phycobilisome degradation, and the results allowed us to present a model that gives insight into the interaction of this small protein with the phycobilisomes.     

Bivalent monoclonal IgY antibody formats by conversion of recombinant antibody fragments

Monoclonal IgY have the potential to become unique tools for diagnostic research and therapeutic purposes since avian antibodies provide several advantages due to their phylogenetic difference when compared to mammalian antibodies. The mechanism of avian immunoglobulin gene diversification renders chicken an excellent source for the generation of recombinant scFv as well as Fab antibody libraries of high diversity. One major limitation of these antibody fragments, however, is their monovalent format, impairing the functional affinity of the molecules and, thereby, their applicability in prevalent laboratory methods. In this study, we generated vectors for conversion of avian recombinant antibody fragments into different types of bivalent IgY antibody formats. To combine the properties of established mammalian monoclonal antibodies with those of IgY constant domains, we additionally generated bivalent murine/avian chimeric antibody constructs. When expressed in HEK-293 cells, all constructs yielded bivalent disulfide-linked antibodies, which exhibit a glycosylation pattern similar to that of native IgY as assessed by lectin blot analysis. After purification by one step procedures, the chimeric and the entire avian bivalent antibody formats were analyzed for antigen binding and interaction with secondary reagents. The data demonstrate that all antibody formats provide comparable antigen binding characteristics and the well established properties of avian constant domains.     

Genome-wide CRISPR Screens in T Helper Cells Reveal Pervasive Crosstalk between Activation and Differentiation

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Mechanism of cell killing after ionizing radiation by a dominant negative DNA polymerase beta

Several types of DNA lesion are induced after ionizing irradiation (IR) of which double strand breaks (DSBs) are expected to be the most lethal, although single strand breaks (SSBs) and DNA base damages are quantitatively in the majority. Proteins of the base excision repair (BER) pathway repair these numerous lesions. DNA polymerase beta has been identified as a crucial enzyme in BER and SSB repair (SSBR). We showed previously that inhibition of BER/SSBR by expressing a dominant negative DNA polymerase beta (polβDN) resulted in radiosensitization. We hypothesized increased kill to result from DSBs arising from unrepaired SSBs and BER intermediates. We find here higher numbers of IR-induced chromosome aberrations in polβDN expressing cells, confirming increased DSB formation. These aberrations did not result from changes in DSB induction or repair of the majority of lesions. SSB conversion to DSBs has been shown to occur during replication. We observed an increased induction of chromatid aberrations in polβDN expressing cells after IR, suggesting such a replication-dependence of secondary DSB formation. We also observed a pronounced increase of chromosomal deletions, the most likely cause of the increased kill. After H₂O₂ treatment, polβDN expression only resulted in increased chromatid (not chromosome) aberrations. Together with the lack of sensitization to H₂O₂, these data further suggest that the additional secondarily induced lethal DSBs resulted from repair attempts at complex clustered damage sites, unique to IR. Surprisingly, the polβDN induced increase in residual γH2AX foci number was unexpectedly low compared with the radiosensitization or induction of aberrations. Our data thus demonstrate the formation of secondary DSBs that are reflected by increased kill but not by residual γH2AX foci, indicating an escape from γH2AX-mediated DSB repair. In addition, we show that in the polβDN expressing cells secondary DSBs arise in a radiation-specific and partly replication-dependent manner.     

Mutations of the mitochondrial holocytochrome c-type synthase in X-linked dominant microphthalmia with linear skin defects syndrome

The microphthalmia with linear skin defects syndrome (MLS, or MIDAS) is an X-linked dominant male-lethal disorder almost invariably associated with segmental monosomy of the Xp22 region. In two female patients, from two families, with MLS and a normal karyotype, we identified heterozygous de novo point mutations--a missense mutation (p.R217C) and a nonsense mutation (p.R197X)--in the HCCS gene. HCCS encodes the mitochondrial holocytochrome c-type synthase that functions as heme lyase by covalently adding the prosthetic heme group to both apocytochrome c and c(1). We investigated a third family, displaying phenotypic variability, in which the mother and two of her daughters carry an 8.6-kb submicroscopic deletion encompassing part of the HCCS gene. Functional analysis demonstrates that both mutant proteins (R217C and Delta 197-268) were unable to complement a Saccharomyces cerevisiae mutant deficient for the HCCS orthologue Cyc3p, in contrast to wild-type HCCS. Moreover, ectopically expressed HCCS wild-type and the R217C mutant protein are targeted to mitochondria in CHO-K1 cells, whereas the C-terminal-truncated Delta 197-268 mutant failed to be sorted to mitochondria. Cytochrome c, the final product of holocytochrome c-type synthase activity, is implicated in both oxidative phosphorylation (OXPHOS) and apoptosis. We hypothesize that the inability of HCCS-deficient cells to undergo cytochrome c-mediated apoptosis may push cell death toward necrosis that gives rise to severe deterioration of the affected tissues. In summary, we suggest that disturbance of both OXPHOS and the balance between apoptosis and necrosis, as well as the X-inactivation pattern, may contribute to the variable phenotype observed in patients with MLS.     

Homo- and heterotypic fibrillin-1 and -2 interactions constitute the basis for the assembly of microfibrils

Fibrillin-1 and fibrillin-2 constitute the backbone of extracellular filaments, called microfibrils. Fibrillin assembly involves complex multistep mechanisms to result in a periodical head-to-tail alignment in microfibrils. Impaired assembly potentially plays a role in the molecular pathogenesis of genetic disorders caused by mutations in fibrillin-1 (Marfan syndrome) and fibrillin-2 (congenital contractural arachnodactyly). Presently, the basic molecular interactions involved in fibrillin assembly are obscure. Here, we have generated recombinant full-length human fibrillin-1, and two overlapping recombinant polypeptides spanning the entire human fibrillin-2 in a mammalian expression system. Characterization by gel electrophoresis, electron microscopy after rotary shadowing, and reactivity with antibodies demonstrated correct folding of these recombinant polypeptides. Analyses of homotypic and heterotypic interaction repertoires showed N- to C-terminal binding of fibrillin-1, and of fibrillin-1 with fibrillin-2. The interactions were of high affinity with dissociation constants in the low nanomolar range. However, the N- and C-terminal fibrillin-2 polypeptides did not interact with each other. These results demonstrate that fibrillins can directly interact in an N- to C-terminal fashion to form homotypic fibrillin-1 or heterotypic fibrillin-1/fibrillin-2 microfibrils. This conclusion was further strengthened by double immunofluorescence labeling of microfibrils. In addition, the binding epitopes as well as the entire fibrillin molecules displayed very stable properties.     

Three-dimensional modelling of honeybee venom allergenic proteases: relation to allergenicity

Api SI and Api SII are serine proteases of the honeybee venom containing allergenic determinants. Each protease consists of two structural modules: an N-terminal CUB (Api SI) or a clip domain (Api SII) and a C-terminal serine protease-like (SPL) domain. Both domains are connected with a linker peptide. The knowledge about the structure and function of Api SI and Api SII is limited mainly to their amino acid sequences. We constructed 3-D models of the two proteases using their amino acid sequences and crystallographic coordinates of related proteins. The models of the SPL domains were built using the structure of the prophenoloxidase-activating factor (PPAF)-II as a template. For modelling of the Api SI CUB domain the coordinates of porcine spermadhesin PSP-I were used. The models revealed the catalytic and substrate-binding sites and the negatively charged residue responsible for the trypsin-like activity. IgE-binding and antigenic sites in the two allergens were predicted using the models and programs based on the structure of known epitopes. Api SI and Api SII show structural and functional similarity to the members of the PPAF-II family. Most probably, they are part of the defence system of Apis mellifera.     

Root–root interactions: extending our perspective to be more inclusive of the range of theories in ecology and agriculture using in-vivo analyses

Background

There is a large body of literature on competitive interactions among plants, but many studies have only focused on above-ground interactions and little is known about root–root dynamics between interacting plants. The perspective on possible mechanisms that explain the outcome of root–root interactions has recently been extended to include non-resource-driven mechanisms (as well as resource-driven mechanisms) of root competition and positive interactions such as facilitation. These approaches have often suffered from being static, partly due to the lack of appropriate methodologies for in-situ non-destructive root characterization.

Scope

Recent studies show that interactive effects of plant neighbourhood interactions follow non-linear and non-additive paths that are hard to explain. Common outcomes such as accumulation of roots mainly in the topsoil cannot be explained solely by competition theory but require a more inclusive theoretical, as well as an improved methodological framework. This will include the question of whether we can apply the same conceptual framework to crop versus natural species.

Conclusions

The development of non-invasive methods to dynamically study root–root interactions in vivo will provide the necessary tools to study a more inclusive conceptual framework for root–root interactions. By following the dynamics of root–root interactions through time in a whole range of scenarios and systems, using a wide variety of non-invasive methods, (such as fluorescent protein which now allows us to separately identify the roots of several individuals within soil), we will be much better equipped to answer some of the key questions in root physiology, ecology and agronomy.     

Single-cell electroporation

Electroporation is a widely used method for the introduction of polar and charged agents such as dyes, drugs, DNA, RNA, proteins, peptides, and amino acids into cells. Traditionally, electroporation is performed with large electrodes in a batch mode for treatment of a large number of cells in suspension. Recently, microelectrodes that can produce extremely localized electric fields, such as solid carbon fiber microelectrodes, electrolyte-filled capillaries and micropipettes as well as chip-based microfabricated electrode arrays, have proven useful to electroporate single cells and subcellular structures. Single-cell electroporation opens up a new window of opportunities in manipulating the genetic, metabolic, and synthetic contents of single targeted cells in tissue slices, cell cultures, in microfluidic channels or at specific loci on a chip-based device.