The First Step of Neurospora crassa Molybdenum Cofactor Biosynthesis: Regulatory Aspects under N-Derepressing and Nitrate-Inducing Conditions.

Molybdenum cofactor (Moco) is the active site prosthetic group found in all Moco dependent enzymes, except for nitrogenase. Mo-enzymes are crucial for viability throughout all kingdoms of life as they catalyze a diverse set of two electron transfer reactions. The highly conserved Moco biosynthesis pathway consists of four different steps in which guanosine triphosphate is converted into cyclic pyranopterin monophosphate, molybdopterin (MPT), and subsequently adenylated MPT and Moco. Although the enzymes and mechanisms involved in these steps are well characterized, the regulation of eukaryotic Moco biosynthesis is not. Within this work, we described the regulation of Moco biosynthesis in the filamentous fungus Neurospora crassa, which revealed the first step of the multi-step pathway to be under transcriptional control. We found, that upon the induction of high cellular Moco demand a single transcript variant of the nit-7 gene is increasingly formed pointing towards, that essentially the encoded enzyme NIT7-A is the key player for Moco biosynthesis activity in Neurospora.

Generation of Recombinant Antibodies against the beta-(1,6)-Branched beta-(1,3)-D-Glucan Schizophyllan from Immunized Mice via Phage Display.

beta-(1,6)-Branched beta-(1,3)-D-glucans like schizophyllan from the basidiomycete Schizophyllum commune excite various immunostimulatory effects and have been clinically tested as adjuvants. Some of the glucans are also applicable in food or petrol industry due to their viscosity and temperature stability in aqueous solution. Antibodies against these glucans could be used as tool for analysis of glucan preparations or for further research of its bioactivity. Therefore, an immune phage display library was constructed from mice immunized with schizophyllan. Three recombinant monoclonal antibodies were isolated from this library by affinity selection (panning) on schizophyllan. The half-maximal effective concentration (EC50) values for those antibodies varied between 16.4 ng mL-1 and 21.3 ng mL-1. The clones showed binding specificity not only for schizophyllan but also for other beta-(1,6)-branched beta-(1,3)-D-glucans of similar macromolecular structure. Denaturation of the secondary structure led to a reduced antibody binding, indicating an epitope requiring the correct conformation of the triple helical structure of the glucans.

Guide-independent DNA cleavage by archaeal Argonaute from Methanocaldococcus jannaschii.

Prokaryotic Argonaute proteins acquire guide strands derived from invading or mobile genetic elements, via an unknown pathway, to direct guide-dependent cleavage of foreign DNA. Here, we report that Argonaute from the archaeal organism Methanocaldococcus jannaschii (MjAgo) possesses two modes of action: the canonical guide-dependent endonuclease activity and a non-guided DNA endonuclease activity. The latter allows MjAgo to process long double-stranded DNAs, including circular plasmid DNAs and genomic DNAs. Degradation of substrates in a guide-independent fashion primes MjAgo for subsequent rounds of DNA cleavage. Chromatinized genomic DNA is resistant to MjAgo degradation, and recombinant histones protect DNA from cleavage in vitro. Mutational analysis shows that key residues important for guide-dependent target processing are also involved in guide-independent MjAgo function. This is the first characterization of guide-independent cleavage activity for an Argonaute protein potentially serving as a guide biogenesis pathway in a prokaryotic system.

Enzymatic characterization of recombinant nitrate reductase expressed and purified from Neurospora crassa.

We established an expression and purification procedure for recombinant protein production in Neurospora crassa (N. crassa). This Strep-tag® based system was successfully used for purifying recombinant N. crassa nitrate reductase (NR), whose enzymatic activity was compared to recombinant N. crassa NR purified from Escherichia coli. The purity of the two different NR preparations was similar but NR purified from N. crassa showed a significantly higher nitrate turnover rate. Two phosphorylation sites were identified for NR purified from the endogenous expression system. We conclude that homologous expression of N. crassa NR yields a higher active enzyme and propose that NR phosphorylation causes enhanced enzymatic activity.

Thymosin beta4 inhibits ADF/cofilin stimulated F-actin cycling and hela cell migration: reversal by active Arp2/3 complex.

F-actin treadmilling plays a key part in cell locomotion. Because immunofluorescence showed colocalisation of thymosin beta4 (Tß4) with cofilin-1 and Arp2/3 complex in lamellipodia, we analyzed combinations of these proteins on F-actin-adenosine triphosphate (ATP)-hydrolysis, which provides a measure of actin treadmilling. Actin depolymerising factor (ADF)/cofilin stimulated treadmilling, while Tß4 decreased treadmilling, presumably by sequestering monomers. Tß4 added together with ADF/cofilin also inhibited the treadmilling, relative to cofilin alone, but both the rate and extent of depolymerization were markedly enhanced in the presence of both these proteins. Arp2/3 complex reversed the sequestering activity of Tß4 when equimolar to actin, but not in the additional presence of cofilin-1 or ADF. Transfection experiments to explore the effects of changing the intracellular concentration of Tß4 in HeLa cells showed that an increase in Tß4 resulted in reduced actin filaments bundles and narrower lamellipodia, and a conspicuous decrease of cell migration as seen by two different assays. In contrast, cells transfected with a vector leading to Tß4 knockdown by small interfering RNA (siRNA) displayed prominent actin filament networks within the lamellipodia and the leading lamella and enhanced migration. The experiments reported here demonstrate the importance of the interplay of these different classes of actin-binding proteins on cell behaviour.

Maturation of the cytochrome cd1 nitrite reductase NirS from Pseudomonas aeruginosa requires transient interactions between the three proteins NirS, NirN and NirF.

The periplasmic cytochrome cd1 nitrite reductase NirS occurring in denitrifying bacteria such as the human pathogen Pseudomonas aeruginosa contains the essential tetrapyrrole cofactors haem c and haem d1. Whereas the haem c is incorporated into NirS by the cytochrome c maturation system I, nothing is known about the insertion of the haem d1 into NirS. Here, we show by co-immunoprecipitation that NirS interacts with the potential haem d1 insertion protein NirN in vivo. This NirS-NirN interaction is dependent on the presence of the putative haem d1 biosynthesis enzyme NirF. Further, we show by affinity co-purification that NirS also directly interacts with NirF. Additionally, NirF is shown to be a membrane anchored lipoprotein in P. aeruginosa. Finally, the analysis by UV-visible absorption spectroscopy of the periplasmic protein fractions prepared from the P. aeruginosa WT (wild-type) and a P. aeruginosa ΔnirN mutant shows that the cofactor content of NirS is altered in the absence of NirN. Based on our results, we propose a potential model for the maturation of NirS in which the three proteins NirS, NirN and NirF form a transient, membrane-associated complex in order to achieve the last step of haem d1 biosynthesis and insertion of the cofactor into NirS.

In birds, profilin-2a is ubiquitously expressed and contributes to actin-based motility.

Profilins are small actin-binding proteins expressed in all eukaryotes. They are involved in the regulation of actin filament dynamics and various signalling pathways. The identification of a variety of profilin isoforms led to the assumption that there may be isoform-specific functions. In mammals, profilin-1 (PFN1) is ubiquitously expressed and engaged in the regulation of various motility processes in all cell types. By contrast, profilin-2a (PFN2a) is mainly restricted to neuronal cells and there is evidence that it is involved in neuronal plasticity and membrane trafficking. However, the PFN2a sequence is much better conserved than PFN1 throughout different phyla, indicating that its restricted expression and specialized function in mammals might be unique. Using isoform-specific antibodies, we show that the situation is different in birds. PFN2a is ubiquitously expressed in embryonic and adult chicken tissues at equal and frequently higher amounts than in mammals. Together with PFN1, it is present in cultivated chicken fibroblasts, but differentially localized. Knockdown experiments with miRNA reveal that PFN2a is involved in cell adhesion, spreading and locomotion, and silencing this isoform has pronounced consequences on these processes. Our results indicate profilin isoform expression is differentially regulated among vertebrates.

Control of c-myc mRNA stability by IGF2BP1-associated cytoplasmic RNPs.

The RNA-binding protein IGF2BP1 (IGF-II mRNA binding protein 1) stabilizes the c-myc RNA by associating with the Coding Region instability Determinant (CRD). If and how other proteins cooperate with IGF2BP1 in promoting stabilization of the c-myc mRNA via the CRD remained elusive. Here, we identify various RNA-binding proteins that associate with IGF2BP1 in an RNA-dependent fashion. Four of these proteins (HNRNPU, SYNCRIP, YBX1, and DHX9) were essential to ensure stabilization of the c-myc mRNA via the CRD. These factors associate with IGF2BP1 in a CRD-dependent manner, co-distribute with IGF2BP1 in non-polysomal fractions comprising c-myc mRNA, and colocalize with IGF2BP1 in the cytoplasm. A selective shift of relative c-myc mRNA levels to the polysomal fraction is observed upon IGF2BP1 knockdown. These findings suggest that IGF2BP1 in complex with at least four proteins promotes CRD-mediated mRNA stabilization. Complex formation at the CRD presumably limits the transfer of c-myc mRNA to the polysomal fraction and subsequent translation-coupled decay.

Peptide nanoparticles serve as a powerful platform for the immunogenic display of poorly antigenic actin determinants.

The role of actin in transcription and RNA processing is now widely accepted but the form of nuclear actin remains enigmatic. Monomeric, oligomeric or polymeric forms of actin seem to be involved in nuclear functions. Moreover, uncommon forms of actin such as the "lower dimer" have been observed in vitro. Antibodies have been pivotal in revealing the presence and distribution of different forms of actin in different cellular locations. Because of its high degree of conservation, actin is a poor immunogen and only few specific actin antibodies are available. To unravel the mystery of less common forms of actin, in particular those in the nucleus, we chose to tailor monoclonal antibodies to recognize distinct forms of actin. To increase the immune response, we used a new approach based on peptide nanoparticles, which are designed to mimic an icosahedral virus capsid and allow the repetitive, ordered display of a specific epitope on their surface. Actin sequences representing the highly conserved "hydrophobic loop," which is buried in the filamentous actin filament, were grafted onto the surface of nanoparticles by genetic engineering. After immunization with "loop nanoparticles," a number of monoclonal antibodies were established that bind to the hydrophobic loop both in vitro and in situ. Immunofluorescence studies on cells revealed that filamentous actin filaments were only labeled once the epitope had been exposed. Our studies indicate that self-assembling peptide nanoparticles represent a versatile platform that can easily be customized to present antigenic determinants in repetitive, ordered arrays and elicit an immune response against poor antigens.

Splice-specific functions of gephyrin in molybdenum cofactor biosynthesis.

Gephyrin is a multifunctional protein involved in the clustering of inhibitory neuroreceptors. In addition, gephyrin catalyzes the last step in molybdenum cofactor (Moco) biosynthesis essential for the activities of Mo-dependent enzymes such as sulfite oxidase and xanthine oxidoreductase. Functional complexity and diversity of gephyrin is believed to be regulated by alternative splicing in a tissue-specific manner. Here, we investigated eight gephyrin variants with combinations of seven alternatively spliced exons located in the N-terminal G domain, the central domain, and the C-terminal E domain. Their activity in Moco synthesis was analyzed in vivo by reconstitution of gephyrin-deficient L929 cells, which were found to be defective in the G domain of gephyrin. Individual domain functions were assayed in addition and confirmed that variants containing either an additional C5 cassette or missing the C6 cassette are inactive in Moco synthesis. In contrast, different alterations within the central domain retained the Moco synthetic activity of gephyrin. The recombinant gephyrin G domain containing the C5 cassette forms dimers in solution, binds molybdopterin, but is unable to catalyze molybdopterin (MPT) adenylylation. Determination of Moco and MPT content in different tissues showed that besides liver and kidney, brain was capable of synthesizing Moco most efficiently. Subsequent analysis of cultured neurons and glia cells demonstrated glial Moco synthesis due to the expression of gephyrins containing the cassettes C2 and C6 with and without C3.1.

Raver1 is an integral component of muscle contractile elements.

Raver1, a ubiquitously expressed protein, was originally identified as a ligand for metavinculin, the muscle-specific isoform of the microfilament-associated protein vinculin. The protein resides primarily in the nucleus, where it colocalises and may interact with polypyrimidine-tract-binding protein, which is involved in alternative splicing processes. During skeletal muscle differentiation, raver1 translocates to the cytoplasm and eventually targets the Z-line of sarcomeres. Here, it colocalises with metavinculin, vinculin and alpha-actinin, all of which have biochemically been identified as raver1 ligands. To obtain more information about the potential role of raver1 in muscle structure and function, we have investigated its distribution and fine localisation in mouse striated and smooth muscle, by using three monoclonal antibodies that recognise epitopes in different regions of the raver1 protein. Our immunofluorescence and immunoelectron-microscopic results indicate that the cytoplasmic accumulation of raver1 is not confined to skeletal muscle but also occurs in heart and smooth muscle. Unlike vinculin and metavinculin, cytoplasmic raver1 is not restricted to costameres but additionally represents an integral part of the sarcomere. In isolated myofibrils and in ultrathin sections of skeletal muscle, raver1 has been found concentrated at the I-Z-I band. A minor fraction of raver1 is present in the nuclei of all three types of muscle. These data indicate that, during muscle differentiation, raver1 might link gene expression with structural functions of the contractile machinery of muscle.