Enzymatic actions of Pasteurella multocida toxin detected by monoclonal antibodies recognizing the deamidated α subunit of the heterotrimeric GTPase Gq

Pasteurella multocida toxin (PMT) is a virulence factor responsible for the pathogenesis of some Pasteurellosis. PMT exerts its toxic effects through the activation of heterotrimeric GTPase (G(q), G(12/13) and G(i))-dependent pathways, by deamidating a glutamine residue in the α subunit of these GTPases. However, the enzymatic characteristics of PMT are yet to be analyzed in detail because the deamidation has only been observed in cell-based assays. In the present study, we developed rat monoclonal antibodies, specifically recognizing the deamidated Gα(q), to detect the actions of PMT by immunological techniques such as western blotting. Using the monoclonal antibodies, we found that the toxin deamidated Gα(q) only under reducing conditions. The C-terminal region of PMT, C-PMT, was more active than the full-length PMT. The C3 domain possessing the enzyme core catalyzed the deamidation in vitro without any other domains. These results not only support previous observations on toxicity, but also provide insights into the enzymatic nature of PMT. In addition, we present several lines of evidence that Gα(11), as well as Gα(q), could be a substrate for PMT.     

Chemical Characterization of Ceramide and Cerebroside in Soybean Leaves

During experiments on protoplast fusion of complementary auxotrophic mutants (194 and 11M-21) of Streptomyces antibioticus for strain improvement, the clones (typified by F-40) regenerated on minimal regeneration medium (MRM) were found to be prototrophs, and to produce an antibiotic different from those produced by the parent strain. The protoplast regeneration of each parent was examined as a negative control experiment.

In the regenerated clones of 194, half of them produced actinomycins similar to those produced by the original mutant 194, but others (typified by R-20) seemed to produce antibiotics similar to those produced by F-40. In the taxonomic characterization of morphological, cultural, and physiological properties of each strain, F-40, R-20, and the parent mutant 194 had no significant differences with a few exceptions. The problem here is whether the antibiotic of R-20 is the same as that of F-40, which was first isolated and found to be a peptide antibiotic different from actinomycins, with activity against Gram-negative and Gram-positive bacteria.     

A neuronal transmembrane protein LRFN4 induces monocyte/macrophage migration via actin cytoskeleton reorganization

Leucine-rich repeat and fibronectin type III domain-containing (LRFN) family proteins are thought to be neuronal-specific proteins that play essential roles in neurite outgrowth and synapse formation. Here, we focused on expression and function of LRFN4, the fourth member of the LRFN family, in non-neural tissues. We found that LRFN4 was expressed in a wide variety of cancer and leukemia cell lines. We also found that expression of LRFN4 in the monocytic cell line THP-1 and in primary monocytes was upregulated following macrophage differentiation. Furthermore, we demonstrated that LRFN4 signaling regulated both the transendothelial migration of THP-1 cells and the elongation of THP-1 cells via actin cytoskeleton reorganization. Our data indicate that LRFN4 signaling plays an important role in the migration of monocytes/macrophages.     

Bordetella dermonecrotic toxin exerting toxicity through activation of the small GTPase Rho

Bordetella dermonecrotic toxin (DNT) is a virulence factor produced by bacteria belonging to the genus Bordetella. The toxin possesses novel transglutaminase activity that catalyzes polyamination or deamidation of the small GTPases of the Rho family. The modified GTPases loose their GTP hydrolyzing activity, function as a constitutive active molecule, and continuously transduce signals to downstream effectors, which mediate the consequent phenotypes of cells intoxicated by DNT. A dynamin-dependent endocytosis is required for the toxin to be internalized into cells although it is unlikely transported to deep organelles such as the Golgi apparatus or the ER. Several lines of evidence show that the toxin undergoes proteolytic cleavage by furin or furin-like protease probably in the early endosome, and then escapes into the cytoplasm to reach the GTPase.     

Holliday Junction Resolvase MOC1 Maintains Plastid and Mitochondrial Genome Integrity in Algae and Bryophytes

When DNA double-strand breaks occur, four-stranded DNA structures called Holliday junctions (HJs) form during homologous recombination. Because HJs connect homologous DNA by a covalent link, resolution of HJ is crucial to terminate homologous recombination and segregate the pair of DNA molecules faithfully. We recently identified Monokaryotic Chloroplast1 (MOC1) as a plastid DNA HJ resolvase in algae and plants. Although Cruciform cutting endonuclease1 (CCE1) was identified as a mitochondrial DNA HJ resolvase in yeasts, homologs or other mitochondrial HJ resolvases have not been identified in other eukaryotes. Here, we demonstrate that MOC1 depletion in the green alga Chlamydomonas reinhardtii and the moss Physcomitrella patens induced ectopic recombination between short dispersed repeats in ptDNA. In addition, MOC1 depletion disorganized thylakoid membranes in plastids. In some land plant lineages, such as the moss P. patens, a liverwort and a fern, MOC1 dually targeted to plastids and mitochondria. Moreover, mitochondrial targeting of MOC1 was also predicted in charophyte algae and some land plant species. Besides causing instability of plastid DNA, MOC1 depletion in P. patens induced short dispersed repeat-mediated ectopic recombination in mitochondrial DNA and disorganized cristae in mitochondria. Similar phenotypes in plastids and mitochondria were previously observed in mutants of plastid-targeted (RECA2) and mitochondrion-targeted (RECA1) recombinases, respectively. These results suggest that MOC1 functions in the double-strand break repair in which a recombinase generates HJs and MOC1 resolves HJs in mitochondria of some lineages of algae and plants as well as in plastids in algae and plants.

Mitochondria and plastids were established in eukaryotic cells by endosymbiotic events of α-proteobacterial and cyanobacterial ancestors, respectively (Gray, 1992; Archibald, 2015). Reminiscent of their bacterial ancestors, both organelles possess their own genomes and proliferate by division of preexisting ones (Martin and Kowallik, 1999). Plastid DNA (ptDNA) and mitochondrial DNA (mtDNA) encode some components of the photosynthetic apparatus and respiratory chain, respectively (Allen, 2003). Thus, to maintain the functions of plastids and mitochondria, ptDNA and mtDNA must faithfully replicate and segregate during proliferation of the organelles.The mitochondrion and plastid possess multiple copies of DNA, which are organized with proteins into nucleoids (Kuroiwa, 1991; Pfalz and Pfannschmidt, 2013). Nucleoids, which can be visualized as dot-like or globular structures in mitochondria and plastids when stained with DNA-specific fluorochromes such as 4′, 6-diamidino-2-phenylindole (DAPI) or SYBR Green I, are ubiquitously observed in diverse lineages of algae and plants (Kuroiwa, 1991; Sato, 2001; Kobayashi et al., 2016). The morphology of nucleoids dynamically changes according to cell cycle progression and development (Powikrowska et al., 2014). For example, in the unicellular green alga Chlamydomonas reinhardtii, ∼80 copies of ptDNA are packaged into 5 to 8 globular nucleoids in a single cup-shaped plastid during the gap 1 (G1) phase (Armbrust, 1998). Prior to plastid division, during the synthesis (S) and mitosis (M) phases, plastid nucleoids change into filamentous structures and are scattered throughout the plastids. Then the nucleoids are inherited by two daughter plastids stochastically (Ehara et al., 1990; Kamimura et al., 2018). A similar morphological change of plastid nucleoids is also observed in the plant Arabidopsis (Arabidopsis thaliana; Terasawa and Sato, 2005), and thus, the mechanism of nucleoid segregation is apparently conserved in algae and plants. However, the molecular mechanisms underlying organelle DNA segregation and changes in nucleoid morphology have remained largely unknown.In a previous study using the green alga C. reinhardtii, mutants defective in nucleoid segregation were isolated (Misumi et al., 1999). One of the mutants possessed only a single enlarged nucleoid in a plastid (Fig. 1A), which was inherited by daughter plastids unevenly (Misumi et al., 1999). Later, the mutation responsible for this phenotype was identified in a previously uncharacterized gene, Monokaryotic Chloroplast1 (MOC1), which is conserved in eukaryotic algae and plants (Kobayashi et al., 2017). MOC1 exhibited endonuclease activity in vitro, where it specifically cleaved Holliday junctions (HJs), four-stranded DNA structures formed during homologous recombination (HR; Kobayashi et al., 2017). MOC1 symmetrically introduced nicks between consecutive cytosines (C↓C, where the arrow indicates the cleavage point) at the core of HJs (Kobayashi et al., 2017). Because HJs provide a covalent link between recombining DNA molecules and must be removed prior to genome segregation (Liu and West, 2004; West, 2009), it was suggested that HR affects the nucleoid structure and MOC1 segregates plastid nucleoids by cleaving HJs between ptDNA molecules prior to plastid division (Kobayashi et al., 2017).Open in a separate windowFigure 1.MOC1 depletion destabilizes ptDNA by increasing ectopic recombination between SDRs in C. reinhardtii. A, Differential interference contrast (DIC) and fluorescent images of SYBR Green I-stained wild-type (WT) and MOC1 KO cells. Green is SYBR Green I-stained DNA and magenta is plastid chlorophyll fluorescence (Chl). The arrow indicates a plastic nucleoid. N, Nucleus. Scale bars = 5 μm. B, qPCR comparing relative copy number of ptDNA between the wild type and CrMOC1 KO. The values of plastid rpl2, psbB, chlN, and psbD loci were normalized with that of the nuclear CBLP locus. For CrMOC1 KO, two independent clones (clones 1 and 2) were analyzed. The normalized value of the wild type was defined as 1.0. The error bar represents the mean ± sd (n = 3). Asterisks indicate significant difference by Student’s t test (**P < 0.01). C, Positions of SDRs CD5, CD5′, CI12, and CD15 in C. reinhardtii ptDNA (Odahara et al., 2016). Large inverted repeats (IRa and IRb) are shown by bold lines. SDRs are indicated by triangles. D to F, qPCR comparing relative copy numbers of ectopic recombinants of CD5 (D), CI12 (E), and CD15 (F). The recombinants were quantified with primers designed as shown in Supplemental Figure S2. Each value was normalized with that of the plastid psbB locus. The error bar represents the mean ± sd (n = 3). Asterisks indicate significant difference by Student’s t test (ns, P ≥ 0.05, *P < 0.05, and **P < 0.01).In general, HR follows either of two main pathways, the double-strand break repair (DSBR) or the synthesis-dependent strand annealing (SDSA) pathway (Supplemental Fig. S1; Holliday, 1964; Szostak et al., 1983; Pâques and Haber, 1999). The two pathways are similar in the initial step. After a double-strand break occurs, 5′ ends of the break are cut back to create 3′ overhangs of single-strand DNA (ssDNA). Recombinases bind the 3′ overhangs of ssDNA and search through vast quantities of DNA sequence to align and pair ssDNA with a homologous double-strand DNA template, facilitating the formation of a d-loop (Dunderdale et al., 1991; Murayama et al., 2008). In the DSBR pathway, the end of the invading 3′ strand is extended by a DNA polymerase and converted into a HJ. The other 3′ overhang strand also forms a HJ with the homologous DNA. After that, there are two pathways to convert the double HJs into recombination products (Sung and Klein, 2006). One pathway is mediated by HJ resolvases, which cleave HJs and produce either crossover or noncrossover products (Szostak et al., 1983). Various HJ resolvases have been found in archaea, bacteria, and eukaryotes (West, 2009). The eukaryotic nucleus also possesses another pathway, which is driven by the BTR complex consisting of the Bloom syndrome helicase, topoisomerase IIIα (TOP3A), and recombination-deficient Q-mediated genome instability subcomplex proteins (Wu and Hickson, 2003). The BTR complex does not cleave but dissolves the double HJs (Wu and Hickson, 2003). During the dissolution, the two HJ branches migrate toward each other until they form a hemicatenated intermediate, which is decatenated by TOP3A. Therefore, the dissolution pathway never produces crossover products (Supplemental Fig. S1). In contrast to DSBR, in the SDSA pathway, the invading 3′ ssDNA is utilized as a primer and extended along the recipient DNA duplex by a DNA polymerase without forming HJs. The newly synthesized strand dissociates from the template DNA and anneals with the other 3′ overhang strand through complementary base pairing. After the strands anneal, the remaining single-stranded gaps are filled by a DNA polymerase (Supplemental Fig. S1; Pâques and Haber, 1999).In nuclear, mitochondrial, and plastid genomes, numerous short dispersed repeats (SDRs) exist (Ottaviani et al., 2014). Thanks to the accuracy of recombinases in finding the homologous sequences despite the existence of myriad SDRs, the genomic sequence of the broken DNA is restored precisely via HR (McEntee et al., 1979; Shibata et al., 1979; Qi et al., 2015). However, in the absence of recombinases, SDRs anneal through complementary base pairing and produce recombinants between SDRs (Supplemental Fig. S1). Intramolecular recombination between SDRs results in deletion or inversion of the flanking region when they are oriented as direct or inverted repeats, respectively (Supplemental Fig. S2). This pathway is known as microhomology-mediated end joining (MMEJ; Supplemental Fig. S1; McVey and Lee, 2008).Recombinase genes, including Rad51 (eukaryotic type), radA (archaeal type), and recA (bacterial type), are believed to have evolved from a common ancestral gene (Lin et al., 2006). Two phylogenetically distinct RECA proteins are encoded in the nuclear genome of land plants, of which one is targeted to plastids (ptRECA) and the other to mitochondria (mtRECA; Lin et al., 2006). ptRECA and mtRECA are most closely related to cyanobacterial and proteobacterial counterparts, respectively, suggesting endosymbiotic origins of these proteins (Lin et al., 2006). Two observations should be made regarding the functions of these two plant recombinases: (1) Suppression or loss of function of ptRECA causes ectopic recombination between SDRs at a high frequency and destabilizes the plastid genome in the green alga C. reinhardtii and the moss Physcomitrella patens (Odahara et al., 2015a, 2016), suggesting that ptRECA maintains integrity of the plastid genome by promoting HR and thus suppressing MMEJ. Because MOC1 possesses HJ-resolving activity in vitro and is required for segregation of ptDNA in vivo (Kobayashi et al., 2017), RECA-mediated HR is likely accomplished with MOC1 through the DSBR pathway in plastids. However, how MOC1 functions in vivo has not been investigated. (2) Like ptRECA, mtRECA suppresses ectopic recombination between SDRs in the mitochondrial genome in P. patens and the seed plant Arabidopsis (Shedge et al., 2007; Odahara et al., 2009; Miller-Messmer et al., 2012). These results suggest that HJs are formed in plant mitochondria. However, except for CCE1, which is specific to yeasts (Saccharomyces cerevisiae and Schizosaccharomyces pombe; Kleff et al., 1992; Oram et al., 1998), mitochondrial HJ resolvases have not been identified in eukaryotes. Thus, it remains unclear whether HJs are formed and, if they are formed, how they are removed in mitochondria in plants.Regarding issues 1 and 2 described above, in this study, we show first that MOC1 suppresses ectopic recombination between SDRs in C. reinhardtii plastids, as does ptRECA, suggesting that MOC1 is involved in DSBR in the plastid. Next, we show that MOC1 dually targets plastids and mitochondria in the moss P. patens and maintains the integrity of ptDNA and mtDNA via suppression of ectopic recombination in both of these organelles. Putative dual-targeted transit peptides are also predicted in MOC1s of charophyte algae, a liverwort, a fern, and some seed plants, and we show that some of them are targeted to both plastids and mitochondria. Thus, it is suggested that HJs are formed during HR and removed by MOC1 in both plastids and mitochondria in some algae and plants.     

Ischemic Postconditioning Reduces NMDA Receptor Currents Through the Opening of the Mitochondrial Permeability Transition Pore and KATP Channel in Mouse Neurons

Ischemic postconditioning (PostC) is known to reduce cerebral ischemia/reperfusion (I/R) injury; however, whether the opening of mitochondrial ATP-dependent potassium (mito-K_ATP) channels and mitochondrial permeability transition pore (mPTP) cause the depolarization of the mitochondrial membrane that remains unknown. We examined the involvement of the mito-K_ATP channel and the mPTP in the PostC mechanism. Ischemic PostC consisted of three cycles of 15 s reperfusion and 15 s re-ischemia, and was started 30 s after the 7.5 min ischemic load. We recorded N-methyl-d-aspartate receptors (NMDAR)-mediated currents and measured cytosolic Ca²⁺ concentrations, and mitochondrial membrane potentials in mouse hippocampal pyramidal neurons. Both ischemic PostC and the application of a mito-K_ATP channel opener, diazoxide, reduced NMDAR-mediated currents, and suppressed cytosolic Ca²⁺ elevations during the early reperfusion period. An mPTP blocker, cyclosporine A, abolished the reducing effect of PostC on NMDAR currents. Furthermore, both ischemic PostC and the application of diazoxide potentiated the depolarization of the mitochondrial membrane potential. These results indicate that ischemic PostC suppresses Ca²⁺ influx into the cytoplasm by reducing NMDAR-mediated currents through mPTP opening. The present study suggests that depolarization of the mitochondrial membrane potential by opening of the mito-K_ATP channel is essential to the mechanism of PostC in neuroprotection against anoxic injury.