The Cyst Nematode SPRYSEC Protein RBP-1 Elicits Gpa2- and RanGAP2-Dependent Plant Cell Death

Plant NB-LRR proteins confer robust protection against microbes and metazoan parasites by recognizing pathogen-derived avirulence (Avr) proteins that are delivered to the host cytoplasm. Microbial Avr proteins usually function as virulence factors in compatible interactions; however, little is known about the types of metazoan proteins recognized by NB-LRR proteins and their relationship with virulence. In this report, we demonstrate that the secreted protein RBP-1 from the potato cyst nematode Globodera pallida elicits defense responses, including cell death typical of a hypersensitive response (HR), through the NB-LRR protein Gpa2. Gp-Rbp-1 variants from G. pallida populations both virulent and avirulent to Gpa2 demonstrated a high degree of polymorphism, with positive selection detected at numerous sites. All Gp-RBP-1 protein variants from an avirulent population were recognized by Gpa2, whereas virulent populations possessed Gp-RBP-1 protein variants both recognized and non-recognized by Gpa2. Recognition of Gp-RBP-1 by Gpa2 correlated to a single amino acid polymorphism at position 187 in the Gp-RBP-1 SPRY domain. Gp-RBP-1 expressed from Potato virus X elicited Gpa2-mediated defenses that required Ran GTPase-activating protein 2 (RanGAP2), a protein known to interact with the Gpa2 N terminus. Tethering RanGAP2 and Gp-RBP-1 variants via fusion proteins resulted in an enhancement of Gpa2-mediated responses. However, activation of Gpa2 was still dependent on the recognition specificity conferred by amino acid 187 and the Gpa2 LRR domain. These results suggest a two-tiered process wherein RanGAP2 mediates an initial interaction with pathogen-delivered Gp-RBP-1 proteins but where the Gpa2 LRR determines which of these interactions will be productive.     

Haplotyping barley <Emphasis Type="Italic">bmy1</Emphasis> using the SNaPshot assay

The allelic status at bmy1, which encodes the enzyme β-amylase 1 in the barley grain, has an important influence over a cultivar’s malting quality. Changes in the malting process have been responsible for the need to improve the thermostability of this enzyme. We have compared a published bmy1 haplotyping assay based on TDI-FRET (template-directed dye-terminator incorporation fluorescence resonance energy transfer) with a SNaPshot protocol by jointly analysing a set of 21 cultivars of known haplotype. The two methods gave the same result, but the SNaPshot assay was easier to interpret. The SNaPshot assay was therefore used to haplotype the Czech malting barley core collection with respect to bmy1. The old Czech cultivar Kasticky was the only entry identified as carrying the high thermostability haplotype, with the remainder carrying either the intermediate or the low thermostability haplotypes. Older materials were the most variable in terms of bmy1 haplotype, but the majority carried the intermediate type. Most of the descendants of cv. Diamant carried the low thermostability haplotype. The most recently released cultivars recommended for the brewing of Czech beer tend to carry the intermediate allele.     

Cloning and functional expression of a nitrile hydratase (NHase) from Rhodococcus equi TG328-2 in Escherichia coli, its purification and biochemical characterisation

The nitrile hydratase (NHase, EC 4.2.1.84) genes (α and β subunit) and the corresponding activator gene from Rhodococcus equi TG328-2 were cloned and sequenced. This Fe-type NHase consists of 209 amino acids (α subunit, M_r 23 kDa) and 218 amino acids (β subunit, M_r 24 kDa) and the NHase activator of 413 amino acids (M_r 46 kDa). Various combinations of promoter, NHase and activator genes were constructed to produce active NHase enzyme recombinantly in E. coli. The maximum enzyme activity (844 U/mg crude cell extract towards methacrylonitrile) was achieved when the NHase activator gene was separately co-expressed with the NHase subunit genes in E. coli BL21 (DE3). The overproduced enzyme was purified with 61% yield after French press, His-tag affinity chromatography, ultrafiltration and lyophilization and showed typical Fe-type NHase characteristics: besides aromatic and heterocyclic nitriles, aliphatic ones were hydrated preferentially. The purified enzyme had a specific activity of 6,290 U/mg towards methacrylonitrile. Enantioselectivity was observed for aromatic compounds only with E values ranging 5–17. The enzyme displayed a broad pH optimum from 6 to 8.5, was most active at 30°C and showed the highest stability at 4°C in thermal inactivation studies between 4°C and 50°C.     

BAG1 restores formation of functional DJ-1 L166P dimers and DJ-1 chaperone activity

Mutations in the gene coding for DJ-1 protein lead to early-onset recessive forms of Parkinson’s disease. It is believed that loss of DJ-1 function is causative for disease, although the function of DJ-1 still remains a matter of controversy. We show that DJ-1 is localized in the cytosol and is associated with membranes and organelles in the form of homodimers. The disease-related mutation L166P shifts its subcellular distribution to the nucleus and decreases its ability to dimerize, impairing cell survival. Using an intracellular foldase biosensor, we found that wild-type DJ-1 possesses chaperone activity, which is abolished by the L166P mutation. We observed that this aberrant phenotype can be reversed by the expression of the cochaperone BAG1 (Bcl-2–associated athanogene 1), restoring DJ-1 subcellular distribution, dimer formation, and chaperone activity and ameliorating cell survival.     

Tyrosine Phosphorylation of Integrin β3 Regulates Kindlin-2 Binding and Integrin Activation

Kindlins are essential for integrin activation in cell systems and do so by working in a cooperative fashion with talin via their direct interaction with integrin β cytoplasmic tails (CTs). Kindlins interact with the membrane-distal NxxY motif, which is distinct from the talin-binding site within the membrane-proximal NxxY motif. The Tyr residues in both motifs can be phosphorylated, and it has been suggested that this modification of the membrane-proximal NxxY motif negatively regulates interaction with the talin head domain. However, the influence of Tyr phosphorylation of the membrane-distal NxxY motif on kindlin binding is unknown. Using mutational analyses and phosphorylated peptides, we show that phosphorylation of the membrane-distal NITY⁷⁵⁹ motif in the β₃ CT disrupts kindlin-2 recognition. Phosphorylation of this membrane-distal Tyr also disables the ability of kindlin-2 to coactivate the integrin. In direct binding studies, peptides corresponding to the non-phosphorylated β₃ CT interacted well with kindlin-2, whereas the Tyr⁷⁵⁹-phosphorylated peptide failed to bind kindlin-2 with measurable affinity. These observations indicate that transitions between the phosphorylated and non-phosphorylated states of the integrin β₃ CT determine reactivity with kindlin-2 and govern the role of kindlin-2 in regulating integrin activation.     

Effect of saliva processing on bacterial DNA extraction

More than 700 bacterial species inhabit oral cavity of humans. Various oral diseases are related to changes in the structure of this complex community. Their pathogenesis can, thus, be better understood by study of oral microbial flora. As many bacteria are refractory to cultivation, molecular approaches based on PCR followed by downstream analysis are more suitable for community analysis than culture dependent methods. Effective DNA extraction from the sample matrix is a fundamental part of the pre-analytical phase but it can be influenced by processing of the starting material. The aim of this study was to analyze the effects of saliva processing on DNA extraction using several non-commercial isolation procedures. Bacterial chromosomal DNA was extracted from three different sample matrices: fresh saliva, diluted saliva and pelleted saliva using four different extraction methods: phenol chloroform protocol, benzyl-chloride protocol, extraction with Chelex-100 and extraction with Triton X. Extraction from different saliva samples and the use of different extraction methods significantly affected the effectiveness of DNA extraction. The most suitable material for bacterial DNA extraction for molecular analysis is a fresh saliva sample. The most effective methods for isolating salivary DNA are the benzyl-chloride protocol and Chelex-100 extraction. Our results have implications for studies concentrating on salivary microbiome and its role in the pathogenesis of oral diseases.     

Epimorphic regeneration in mice is p53-independent

The process of regeneration is most readily studied in species of sponge, hydra, planarian and salamander (i.e., newt and axolotl). The closure of MRL mouse ear pinna through-and-through holes provides a mammalian model of unusual wound healing/regeneration in which a blastema-like structure closes the ear hole and cartilage and hair follicles are replaced. Recent studies, based on a broad level of DNA damage and a cell cycle pattern of G₂/M “arrest,” showed that p21^Cip1/Waf1 was missing from the MRL mouse ear and that a p21-null mouse could close its ear holes. Given the p53/p21 axis of control of DNA damage, cell cycle arrest, apoptosis and senescence, we tested the role of p53 in the ear hole regenerative response. Using backcross mice, we found that loss of p53 in MRL mice did not show reduced healing. Furthermore, cross sections of MRL. p53^−/− mouse ears at 6 weeks post-injury showed an increased level of adipocytes and chondrocytes in the region of healing whereas MRL or p21^−/− mice showed chondrogenesis alone in this same region, though at later time points. In addition, we also investigated other cell cyclerelated mutant mice to determine how p21 was being regulated. We demonstrate that p16 and Gadd45 null mice show little healing capacity. Interestingly, a partial healing phenotype in mice with a dual Tgfβ/Rag2 knockout mutation was seen. These data demonstrate an independence of p53 signaling for mouse appendage regeneration and suggest that the role of p21 in this process is possibly through the abrogation of the Tgfβ/Smad pathway.Key words: mouse, regeneration, p53, p21, MRL, ear-hole, Tgfβ     

Long-residency hydration, cation binding, and dynamics of loop E/helix IV rRNA-L25 protein complex

Molecular dynamics simulations of RNA-protein complex between Escherichia coli loop E/helix IV (LE/HeIV) rRNA and L25 protein reveal a qualitative agreement between the experimental and simulated structures. The major groove of LE is a prominent rRNA cation-binding site. Divalent cations rigidify the LE major groove geometry whereas in the absence of divalent cations LE extensively interacts with monovalent cations via inner-shell binding. The HeIV region shows bistability of its major groove explaining the observed differences between x-ray and NMR structures. In agreement with the experiments, the simulations suggest that helix-alpha1 of L25 is the least stable part of the protein. Inclusion of Mg2+ cations into the simulations causes perturbation of basepairing at the LE/HeIV junction, which does not, however, affect the protein binding. The rRNA-protein complex is mediated by a number of highly specific hydration sites with long-residing water molecules and two of them are bound throughout the entire 24-ns simulation. Long-residing water molecules are seen also outside the RNA-protein contact areas with water-binding times substantially enhanced compared to simulations of free RNA. Long-residency hydration sites thus represent important elements of the three-dimensional structure of rRNA.     

Non-Watson-Crick basepairing and hydration in RNA motifs: molecular dynamics of 5S rRNA loop E

Explicit solvent and counterion molecular dynamics simulations have been carried out for a total of >80 ns on the bacterial and spinach chloroplast 5S rRNA Loop E motifs. The Loop E sequences form unique duplex architectures composed of seven consecutive non-Watson-Crick basepairs. The starting structure of spinach chloroplast Loop E was modeled using isostericity principles, and the simulations refined the geometries of the three non-Watson-Crick basepairs that differ from the consensus bacterial sequence. The deep groove of Loop E motifs provides unique sites for cation binding. Binding of Mg(2+) rigidifies Loop E and stabilizes its major groove at an intermediate width. In the absence of Mg(2+), the Loop E motifs show an unprecedented degree of inner-shell binding of monovalent cations that, in contrast to Mg(2+), penetrate into the most negative regions inside the deep groove. The spinach chloroplast Loop E shows a marked tendency to compress its deep groove compared with the bacterial consensus. Structures with a narrow deep groove essentially collapse around a string of Na(+) cations with long coordination times. The Loop E non-Watson-Crick basepairing is complemented by highly specific hydration sites ranging from water bridges to hydration pockets hosting 2 to 3 long-residing waters. The ordered hydration is intimately connected with RNA local conformational variations.     

Molecular dynamics simulations of RNA kissing-loop motifs reveal structural dynamics and formation of cation-binding pockets

Explicit solvent molecular dynamics (MD) simulations were carried out for three RNA kissing–loop complexes. The theoretical structure of two base pairs (2 bp) complex of H3 stem–loop of Moloney murine leukemia virus agrees with the NMR structure with modest violations of few NMR restraints comparable to violations present in the NMR structure. In contrast to the NMR structure, however, MD shows relaxed intermolecular G-C base pairs. The core region of the kissing complex forms a cation-binding pocket with highly negative electrostatic potential. The pocket shows nanosecond-scale breathing motions coupled with oscillations of the whole molecule. Additional simulations were carried out for 6 bp kissing complexes of the DIS HIV-1 subtypes A and B. The simulated structures agree well with the X-ray data. The subtype B forms a novel four-base stack of bulged-out adenines. Both 6 bp kissing complexes have extended cation-binding pockets in their central parts. While the pocket of subtype A interacts with two hexacoordinated Mg²⁺ ions and one sodium ion, pocket of subtype B is filled with a string of three delocalized Na⁺ ions with residency times of individual cations 1–2 ns. The 6 bp complexes show breathing motions of the cation-binding pockets and loop major grooves.