Sonic hedgehog shedding results in functional activation of the solubilized protein

All Hedgehog (Hh) proteins are released from producing cells despite being synthesized as N- and C-terminally lipidated, membrane-tethered molecules. Thus, a cellular mechanism is needed for Hh solubilization. We previously suggested that a disintegrin and metalloprotease (ADAM)-mediated shedding of Sonic hedgehog (ShhNp) from its lipidated N and C termini results in protein solubilization. This finding, however, seemed at odds with the established role of N-terminal palmitoylation for ShhNp signaling activity. We now resolve this paradox by showing that N-palmitoylation of ShhNp N-terminal peptides is required for their proteolytic removal during solubilization. These peptides otherwise block ShhNp zinc coordination sites required for ShhNp binding to its receptor Patched (Ptc), explaining the essential yet indirect role of N-palmitoylation for ShhNp function. We suggest a functional model in which membrane-tethered multimeric ShhNp is at least partially autoinhibited in trans but is processed into fully active, soluble multimers upon palmitoylation-dependent cleavage of inhibitory N-terminal peptides.     

Über den Einfluß von Tryptophan und analogen Verbindungen auf die Biosynthese von Clavinalkaloiden in saprophytischer Kultur

Zusammenfassung Tryptophan steigert die Alkaloidbildung des Claviceps-Stammes SD 58, wenn es den Kulturen vor der Beimpfung oder in einem frühen Stadium zugesetzt wird, nicht jedoch bei späterem Zusatz. Den gleichen Effekt zeigen verschiedene Tryptophan-Analoga. Es handelt sich nicht um einen Nährstoff-Effekt, sondern wahrscheinlich um eine Beeinflussung von Enzymen des Alkaloidstoffwechsels. Obwohl man eine Hemmung des Alkaloidabbaus nicht ausschließen kann, vermuten wir eher, daß das Tryptophan als Induktor für Enzyme der Alkaloidsynthese wirkt. Als Arbeitshypothese wird angenommen, daß die Alkaloidbildung durch ein Nachlassen der Eiweißsynthese und eine damit verbundene Vergrößerung des Pools freier Aminosäuren ausgelöst wird, was dann zur induzeirten Synthese von Enzymen führt, die für die Alkaloidbildung erforderlich sind.     

Single nucleotide polymorphism (SNP) genotyping as basis for developing a PCR-based marker highly diagnostic for potato varieties with high resistance to Globodera pallida pathotype Pa2/3

Globodera pallida is a parasitic root cyst nematode of potato, which causes reduction of crop yield and quality in infested fields. Field populations of G. pallida containing mixtures of pathotypes Pa2 and Pa3 (Pa2/3) are currently most relevant for potato cultivation in middle Europe. Genes for resistance to G. pallida have been introgressed into the cultivated potato gene pool from the wild, tuber bearing Solanum species S. spegazzinii and S. vernei. Selection of resistant genotypes in breeding programs is hampered by the fact that the phenotypic evaluation of resistance to G. pallida is time consuming, costly and often ambiguous. DNA-based markers diagnostic for resistance to G. pallida would facilitate the development of resistant varieties. A tetraploid F₁ hybrid family SR-Gpa segregating for quantitative resistance to G.␣pallida was developed and evaluated for resistance to G. pallida population ‘Chavornay’. Two subpopulations of 30 highly resistant and 30 susceptible individuals were selected and genotyped for 96 single nucleotide polymorphism (SNP) markers tagging 12 genomic regions on 10 potato chromosomes. Seven SNPs were found significantly linked to the nematode resistance, which were all located within a resistance ‘hotspot’ on potato chromosome V. A haplotype model for these seven SNPs was deduced from the SNP patterns observed in the SR-Gpa family. A PCR assay ‘HC’ was developed, which specifically detected the SNP haplotype c that was linked with high levels of nematode resistance. The HC marker was only found in accessions of S.␣vernei. Screening with the HC marker 34 potato varieties resistant to G. pallida pathotypes Pa2 and/or Pa3 and 22 susceptible varieties demonstrated that the HC marker was highly diagnostic for presence of high levels of resistance to G. pallida pathotype Pa2/Pa3.Amirali Sattarzadeh and Ute Achenbach contributed equally to the work     

Monitoring of integrin-mediated adhesion of human ovarian cancer cells to model protein surfaces by quartz crystal resonators: evaluation in the impedance analysis mode

The quartz crystal microbalance (QCM) was used to monitor specific, integrin-mediated adhesion of human ovarian cancer cells to distinct extracellular matrix (ECM) proteins immobilized on gold-coated quartz crystal resonators. The QCM was operated in the impedance analysis mode, where frequency shift as well as bandwidth are accessible on a broad range of overtones. The increase in bandwidth caused by covering the quartz resonator with cells was reproducible and largely independent of overtone order, whereas the frequency shift displayed some variability. Thus the bandwidth proved to be the more robust parameter for sensing cell adhesive events. The bandwidth increased in proportion to the number of seeded cells to the quartz crystal as long as the number was below 150,000 cells/ml. Comparing the resonance parameters on different harmonics, one finds that viscoelastic modeling with homogeneous layer systems cannot reproduce the results: lateral heterogeneity has to be taken into account. The differences in adhesive strength of human ovarian cancer cells towards selected ECM proteins monitored by QCM was in good agreement with data obtained by conventional cell adhesion assays. Strong cell adhesion was observed to the ECM proteins vitronectin (VN) and fibronectin (FN), while only weak attachment occurred on laminin. In order to prove specific, integrin alphavbeta3-mediated cell adhesion to its ligands FN and VN, the cyclic integrin alphavbeta3-directed peptide c(RGDfV) was used as competitor and significantly reversed cell adhesion. Since integrin interaction with ECM proteins is dependent on the presence of bivalent cations, cell detachment was also seen after treatment of cell monolayers with the chelator ethylene-dinitro-tetra-acetic acid (EDTA). The QCM technique is a reliable method to monitor cell adsorption to ECM-pretreated surfaces in real time. It may be an alternative tool for screening specific and selective antagonists of integrin/ECM interaction.     

Arabidopsis CURVATURE THYLAKOID1 Proteins Modify Thylakoid Architecture by Inducing Membrane Curvature

Chloroplasts of land plants characteristically contain grana, cylindrical stacks of thylakoid membranes. A granum consists of a core of appressed membranes, two stroma-exposed end membranes, and margins, which connect pairs of grana membranes at their lumenal sides. Multiple forces contribute to grana stacking, but it is not known how the extreme curvature at margins is generated and maintained. We report the identification of the CURVATURE THYLAKOID1 (CURT1) protein family, conserved in plants and cyanobacteria. The four Arabidopsis thaliana CURT1 proteins (CURT1A, B, C, and D) oligomerize and are highly enriched at grana margins. Grana architecture is correlated with the CURT1 protein level, ranging from flat lobe-like thylakoids with considerably fewer grana margins in plants without CURT1 proteins to an increased number of membrane layers (and margins) in grana at the expense of grana diameter in overexpressors of CURT1A. The endogenous CURT1 protein in the cyanobacterium Synechocystis sp PCC6803 can be partially replaced by its Arabidopsis counterpart, indicating that the function of CURT1 proteins is evolutionary conserved. In vitro, Arabidopsis CURT1A proteins oligomerize and induce tubulation of liposomes, implying that CURT1 proteins suffice to induce membrane curvature. We therefore propose that CURT1 proteins modify thylakoid architecture by inducing membrane curvature at grana margins.     

Cloning of a Nitrilase Gene from the Cyanobacterium Synechocystis sp. Strain PCC6803 and Heterologous Expression and Characterization of the Encoded Protein

The gene encoding a putative nitrilase was identified in the genome sequence of the photosynthetic cyanobacterium Synechocystis sp. strain PCC6803. The gene was amplified by PCR and cloned into an expression vector. The encoded protein was heterologously expressed in the native form and as a His-tagged protein in Escherichia coli, and the recombinant strains were shown to convert benzonitrile to benzoate. The active enzyme was purified to homogeneity and shown by gel filtration to consist probably of 10 subunits. The purified nitrilase converted various aromatic and aliphatic nitriles. The highest enzyme activity was observed with fumarodinitrile, but also some rather hydrophobic aromatic (e.g., naphthalenecarbonitrile), heterocyclic (e.g., indole-3-acetonitrile), or long-chain aliphatic (di-)nitriles (e.g., octanoic acid dinitrile) were converted with higher specific activities than benzonitrile. From aliphatic dinitriles with less than six carbon atoms only 1 mol of ammonia was released per mol of dinitrile, and thus presumably the corresponding cyanocarboxylic acids formed. The purified enzyme was active in the presence of a wide range of organic solvents and the turnover rates of dodecanoic acid nitrile and naphthalenecarbonitrile were increased in the presence of water-soluble and water-immiscible organic solvents.     

Backbone resonance assignments for a homolog of the essential ribosome biogenesis factor Fap7 from P. horikoshii in its nucleotide-free and -bound forms

The protein “factor activating Pos9 (Skn7)”, Fap7, is an essential protein in yeast and plays an important role in the biogenesis of the small ribosomal subunit. In eukaryotes, the final processing step of the small ribosomal subunit RNA is the endonucleolytic cleavage of 20S pre-rRNA at cleavage site D yielding mature 18S rRNA. Depletion of Fap7 in yeast leads to a dramatic accumulation of 20S pre-rRNA and a concomitant decrease in 18S rRNA in the cytoplasm. In addition, these cells contain higher levels of 60S, but decreased numbers of 40S ribosomal subunits. Fap7 contains a P-loop like motif placing it in a class with NTPases and kinases and a role for it as an adenylate kinase has been suggested. Up to now both the structure of Fap7 and its detailed function during ribosome biogenesis remain elusive. Here, we present the backbone NMR assignments of a Fap7 homolog from the thermophilic archaeon Pyrococcus horikoshii in its nucleotide free form and bound to the adenylate kinase inhibitor AP₅A.     

The legumin gene family: a reconstructed Vicia faba legumin gene encoding a high-molecular-weight subunit is related to type B genes

Nucleotide sequence information from a partial genomic clone, a cDNA clone, a RACE clone and a PCR fragment was combined to reconstruct the first reported complete gene sequence encoding a large legumin subunit, designated LelB3. The length difference to the well-characterized major legumin subunits is caused by an extended glutamin/glutamic acid-rich region encoded by the C-terminal part of the chain. Amino acid sequence comparisons reveal that gene LelB3 is more closely related to B-type than to A-type legumin genes of Vicia faba. Gene LelB3 is a member of a small gene family as indicated by published (Pich and Schubert, Biol Zbl 112 (1993); 342–350) and limited own data.     

Rapid and dissimilar response of ammonia oxidizing archaea and bacteria to nitrogen and water amendment in two temperate forest soils

Biochemical processes relevant to soil nitrogen (N) cycling are performed by soil microorganisms affiliated with diverse phylogenetic groups. For example, the oxidation of ammonia, representing the first step of nitrification, can be performed by ammonia oxidizing bacteria (AOB) and, as recently reported, also by ammonia oxidizing archaea (AOA). However, the contribution to ammonia oxidation of the phylogenetically separated AOA versus AOB and their respective responsiveness to environmental factors are still poorly understood. The present study aims at comparing the capacity of AOA and AOB to momentarily respond to N input and increased soil moisture in two contrasting forest soils. Soils from the pristine Rothwald forest and the managed Schottenwald forest were amended with either NH(4)(+)-N or NO(3)(-)-N and were incubated at 40% and 70% water-filled pore space (WFPS) for four days. Nitrification rates were measured and AOA and AOB abundance and community composition were determined via quantitative PCR (qPCR) and terminal restriction length fragment polymorphism (T-RFLP) analysis of bacterial and archaeal amoA genes. Our study reports rapid and distinct changes in AOA and AOB abundances in the two forest soils in response to N input and increased soil moisture but no significant effects on net nitrification rates. Functional microbial communities differed significantly in the two soils and responded specifically to the treatments during the short-term incubation. In the Rothwald soil the abundance and community composition of AOA were affected by the water content, whereas AOB communities responded to N amendment. In the Schottenwald soil, by contrast, AOA responded to N addition. These results suggest that AOA and AOB may be selectively influenced by soil and management factors.