The compromised recognition of turnip crinkle virus1 subfamily of microrchidia ATPases regulates disease resistance in barley to biotrophic and necrotrophic pathogens.

MORC1 and MORC2, two of the seven members of the Arabidopsis (Arabidopsis thaliana) Compromised Recognition of Turnip Crinkle Virus1 subfamily of microrchidia Gyrase, Heat Shock Protein90, Histidine Kinase, MutL (GHKL) ATPases, were previously shown to be required in multiple layers of plant immunity. Here, we show that the barley (Hordeum vulgare) MORCs also are involved in disease resistance. Genome-wide analyses identified five MORCs that are 37% to 48% identical on the protein level to AtMORC1. Unexpectedly, and in clear contrast to Arabidopsis, RNA interference-mediated knockdown of MORC in barley resulted in enhanced basal resistance and effector-triggered, powdery mildew resistance locus A12-mediated resistance against the biotrophic powdery mildew fungus (Blumeria graminis f. sp. hordei), while MORC overexpression decreased resistance. Moreover, barley knockdown mutants also showed higher resistance to Fusarium graminearum. Barley MORCs, like their Arabidopsis homologs, contain the highly conserved GHKL ATPase and S5 domains, which identify them as members of the MORC superfamily. Like AtMORC1, barley MORC1 (HvMORC1) binds DNA and has Mn2+-dependent endonuclease activities, suggesting that the contrasting function of MORC1 homologs in barley versus Arabidopsis is not due to differences in their enzyme activities. In contrast to AtMORCs, which are involved in silencing of transposons that are largely restricted to pericentromeric regions, barley MORC mutants did not show a loss-of-transposon silencing regardless of their genomic location. Reciprocal overexpression of MORC1 homologs in barley and Arabidopsis showed that AtMORC1 and HvMORC1 could not restore each other's function. Together, these results suggest that MORC proteins function as modulators of immunity, which can act negatively (barley) or positively (Arabidopsis) dependent on the species.

CRT1 is a nuclear-translocated MORC endonuclease that participates in multiple levels of plant immunity.

Arabidopsis thaliana CRT1 (compromised for recognition of Turnip Crinkle Virus) was previously shown to be required for effector-triggered immunity. Sequence analyses previously revealed that CRT1 contains the ATPase and S5 domains characteristic of Microchidia (MORC) proteins; these proteins are associated with DNA modification and repair. Here we show that CRT1 and its closest homologue, CRH1, are also required for pathogen-associated molecular pattern (PAMP)-triggered immunity, basal resistance, non-host resistance and systemic acquired resistance. Consistent with its role in PAMP-triggered immunity, CRT1 interacted with the PAMP recognition receptor FLS2. Subcellular fractionation and transmission electron microscopy detected a subpopulation of CRT1 in the nucleus, whose levels increased following PAMP treatment or infection with an avirulent pathogen. These results, combined with the demonstration that CRT1 binds DNA, exhibits endonuclease activity, and affects tolerance to the DNA-damaging agent mitomycin C, argue that this prototypic eukaryotic member of the MORC superfamily has important nuclear functions during immune response activation.

The pro-form of BMP-2 exhibits a delayed and reduced activity when compared to mature BMP-2.

Pro-forms of growth factors receive increasing attention since it was shown that they can affect the function(s) of the mature proteins. Here, we extend our previous investigations on the biological function of the pro-form of bone morphogenetic protein-2 (BMP-2). We demonstrate that proBMP-2, upon prolonged incubation with C2C12 cells, induces alkaline phosphatase, a marker enzyme for osteoblastic differentiation. Expression studies with three different bone marker transcripts reveal that proBMP-2 induces bone-specific transcripts, however, to a smaller extent than the mature growth factor. To resolve this finding at the protein level, the fate of proBMP-2 and BMP-2 was studied in cell culture. We demonstrate that both proteins become internalized, and proBMP-2 is processed to mature BMP-2 within the cells. The data presented here suggest that proBMP-2 elicits biological functions as mature BMP-2 at a delayed and reduced level, which might depend on intracellular cleavage and subsequent secretion as mature BMP-2.

The influence of covalently linked and free polyethylene glycol on the structural and release properties of rhBMP-2 loaded microspheres.

The current clinical success of therapies with recombinant human Bone Morphogenetic Protein 2 (rhBMP-2) is limited due to inefficient delivery. The high doses applied have frequently been related to severe adverse effects such as tissue swelling, seroma, inflammatory effects and heterotopic ossification. The controlled delivery of lower doses is supposed to reduce adverse effect incidence as well as costs. In this study, novel polyethylene glycol-poly(lactic-co-glycolic acid) (PEG-PLGA) diblock copolymers were used to produce low dose controlled delivery vehicles for rhBMP-2. A method to fabricate a variety of microsphere formulations with a high encapsulation efficiency in high yields was developed. The influence of PEG as an inner phase cosolvent and linked PLGA as copolymer was investigated. Six different microsphere systems with varying PEG amounts in both core and shell were characterised thoroughly with respect to the specific properties of rhBMP-2. The particle size of the microspheres was investigated with both laser diffraction and environmental scanning electron microscopy. Higher PEG/PLGA ratios showed a tendency to increase in size and a wider distribution. Due to the low rhBMP-2 doses, a profound characterisation was very challenging. The growth factor was covalently attached to rhodamine B for the first time. Studies on drug distribution in the microspheres were performed by means of confocal laser scanning microscopy. The addition of PEG to the inner phase was found to impair the formation of spherical microdomains with localized higher growth factor concentrations. Release profiles, determined with ELISA, were linked to the structural changes that were monitored. Distinct, controlled release profiles were achieved in all formulations and showed that PEG is a versatile tool in the effective control of release rates from microspheres. Higher PEG/PLGA ratios in the polymer were shown to increase the release rate from the microspheres. In contrast, PEG administered to the inner phase decreased the release rate. The biological activity of released protein was shown in vitro in an alkaline phosphatase assay. It was demonstrated that PEG-PLGA microspheres are a promising sustained delivery system which allows a reduction of the required rhBMP-2 dose to limit both adverse effects and costs. Furthermore, the data indicated that the use of PEG as an inner phase cosolvent is not suitable for rhBMP-2 in contrast the reported beneficial effects for other growth factors.

The pro-form of BMP-2 interferes with BMP-2 signalling by competing with BMP-2 for IA receptor binding.

Pro-forms of growth factors have received increasing attention since it was shown that they can affect both the maturation and functions of mature growth factors. Here, we assessed the biological function of the pro-form of bone morphogenetic protein-2 (BMP-2), a member of the transforming growth factor beta (TGFbeta)/BMP superfamily. The role of the 263 amino acids of the pro-peptide is currently unclear. In order to obtain an insight into the function of the pro-form (proBMP-2), the ability of proBMP-2 to induce alkaline phosphatase (AP), a marker enzyme for cells differentiating into osteoblasts, was tested. Interestingly, in contrast to mature BMP-2, proBMP-2 did not lead to induction of AP. Instead, proBMP-2 inhibited the induction of AP by BMP-2. This result raised the question of whether proBMP-2 may compete with mature BMP-2 for receptor binding. ProBMP-2 was found to bind to the purified extracellular ligand binding domain (ECD) of BMPR-IA, a high-affinity receptor for mature BMP-2, with a similar affinity as mature BMP-2. Binding of proBMP-2 to BMPR-IA was confirmed in cell culture by cross-linking proBMP-2 to BMPR-IA presented on the cell surface. In contrast to this finding, proBMP-2 did not bind to the ECD of BMPR-II. ProBMP-2 also differed from BMP-2 in its capacity to induce p38 and Smad phosphorylation. The data presented here suggest that the pro-domain of BMP-2 can alter the signalling properties of the growth factor by modulating the ability of the mature part to interact with the receptors.

A folded and functional protein domain in an amyloid-like fibril.

The effect of the polypeptide environment on polyalanine-induced fibril formation was investigated with amyloidogenic fragments from PAPBN1, a nuclear protein controlling polyadenylation. Mutation-caused extensions of the natural 10 alanine sequence up to maximally 17 alanines result in fibril formation of PABPN1 and the development of the disease oculopharyngeal muscular dystrophy (OPMD). We explored the influence of fibril formation on the structure and function of a one-domain protein linked to the fibril-forming part of PABPN1. The well-characterized, stably folded, one-domain protein, cold-shock protein CspB from Bacillus subtilis, was fused either to the C terminus of the entire N-terminal domain of PABPN1 or directly to peptides consisting of 10 or 17 alanine residues. The fusion protein between the N-terminal domain of PABPN1 and CspB formed fibrils in which the structure and activity of CspB were retained. In the fibrils formed by fusions in which the polyalanine sequence was directly linked to CspB, CspB was unfolded. These results indicate that the folded conformation and the function of a protein domain can be maintained in amyloid-like fibrils, and that the distance between this domain and the fibril plays an important role.