Structural basis for the modulation of plant cytosolic triosephosphate isomerase activity by mimicry of redox‐based modifications

Reactive oxidative species (ROS) and S‐glutathionylation modulate the activity of plant cytosolic triosephosphate isomerases (cTPI). Arabidopsis thaliana cTPI (AtcTPI) is subject of redox regulation at two reactive cysteines that function as thiol switches. Here we investigate the role of these residues, AtcTPI‐Cys13 and At‐Cys218, by substituting them with aspartic acid that mimics the irreversible oxidation of cysteine to sulfinic acid and with amino acids that mimic thiol conjugation. Crystallographic studies show that mimicking AtcTPI‐Cys13 oxidation promotes the formation of inactive monomers by reposition residue Phe75 of the neighboring subunit, into a conformation that destabilizes the dimer interface. Mutations in residue AtcTPI‐Cys218 to Asp, Lys, or Tyr generate TPI variants with a decreased enzymatic activity by creating structural modifications in two loops (loop 7 and loop 6) whose integrity is necessary to assemble the active site. In contrast with mutations in residue AtcTPI‐Cys13, mutations in AtcTPI‐Cys218 do not alter the dimeric nature of AtcTPI. Therefore, modifications of residues AtcTPI‐Cys13 and AtcTPI‐Cys218 modulate AtcTPI activity by inducing the formation of inactive monomers and by altering the active site of the dimeric enzyme, respectively. The identity of residue AtcTPI‐Cys218 is conserved in the majority of plant cytosolic TPIs, this conservation and its solvent‐exposed localization make it the most probable target for TPI regulation upon oxidative damage by reactive oxygen species. Our data reveal the structural mechanisms by which S‐glutathionylation protects AtcTPI from irreversible chemical modifications and re‐routes carbon metabolism to the pentose phosphate pathway to decrease oxidative stress.     

Genetic and ultrastructural analysis of different mutants of Pseudomonas putida affected in the poly-3-hydroxy-n-alkanoate gene cluster

Functional analyses of the different proteins involved in the synthesis and accumulation of polyhydroxyalkanoates (PHAs) in P. putida U were performed using a mutant in which the pha locus had been deleted (PpUDeltapha). These studies showed that: (i) Pha enzymes cannot be replaced by other proteins in this bacterium, (ii) the transformation of PpDeltapha with a plasmid containing the locus pha fully restores the synthesis of bioplastics, (iii) the transformation of PpDeltapha with a plasmid harbouring the gene encoding the polymerase PhaC1 (pMCphaC1) permits the synthesis of polyesters (even in absence of phaC2ZDFI); however, in this strain (PpUDeltapha-pMCphaC1) the number of PHAs granules was higher than in the wild type, (iv) the expression of phaF in PpUDeltapha-pMCphaC1 restores the original phenotype, showing that PhaF is involved in the coalescence of the PHAs granules. Furthermore, the deletion of the phaDFI genes in P. putida U considerably decreases (> 70%) the biosynthesis of PHAs consisting of hydroxyalkanoates with aliphatic constituents, and completely prevents the synthesis of those ones containing aromatic monomers. Additional experiments revealed that the deletion of phaD in P. putida U strongly reduces the synthesis of PHA, this effect being restored by PhaF. Moreover, the overexpression of phaF in P. putida U, or in its DeltafadBA mutant, led to the collection of PHA over-producer strains.     

Real-time studies of the progression of bacterial infections and immediate tissue responses in live animals

By combining intravital multiphoton microscopy and bacterial genetics we have developed a technique enabling real-time imaging of bacterial proliferation and tissue responses in a live animal. Spatial and temporal control of the infection process was achieved by microinjecting GFP(+)-expressing uropathogenic Escherichia coli (UPEC) into tubules of exteriorized kidneys in live rats. GFP(+) was introduced in the clinical UPEC strain CFT073 as a single-copy chromosomal gene fusion. Within hours, bacterial colonization was accompanied by marked ischaemic effects, perivascular leakage, loss of tubular integrity and localized recruitment of immune cells. The pathophysiology was altered in response to an isogenic bacterial strain lacking the exotoxin haemolysin, revealing the subtle and temporal roles of bacterial virulence factors in vivo. Microdissection and RNA extraction of the injected nephron allowed molecular analysis of prokaryotic and eukaryotic gene expression. The techniques described here can be applied to study the integrated cell communication evoked by a variety of bacterial pathogens, assisting in the design of strategies to combat bacterial infections.     

The hisC1 gene, encoding aromatic amino acid aminotransferase-1 in Azospirillum brasilense Sp7, expressed in wheat

Background and aims

Production of indole-3-acetic acid (IAA) by Azospirillum brasilense is one of the most important mechanisms underlying the beneficial effects observed in plants after inoculation with this bacterium. This study determined the contribution of the hisC1 gene, which encodes aromatic amino acid aminotransferase-1 (AAT1), to IAA production, and analyzed its expression in the free-living state and in association with the roots of wheat.

Methods

We determined production of IAA and AAT activity in the mutant hisC::gusA-sm ^R . To study the expression of hisC1, a chromosomal gene fusion was analyzed by following β-glucuronidase (GUS) activity in vitro, in the presence of root exudates, and in association with roots.

Results

IAA production in the hisC mutant was not reduced significantly compared to the activity of the wild-type strain. AAT1 activity was reduced by 50% when tyrosine was used as the amino acid donor, whereas there was a 30% reduction when tryptophan was used, compared to the activity of the wild-type strain. Expression of the fusion protein was up-regulated in both logarithmic and stationary phases by several compounds, including IAA, tryptophan, tyrosine, and phenyl acetic acid. We observed the expression of hisC1 in bacteria associated with wheat roots. Root exudates of wheat and maize were able to stimulate hisC1 expression.

Conclusions

The expression data indicate that hisC1 is under a positive feedback control in the presence of root exudates and on plants, suggesting that AAT1 activity plays a role in Azospirillum–plant interactions.     

Familial hemiplegic migraine type 1 mutations W1684R and V1696I alter G protein-mediated regulation of CaV2.1 voltage-gated calcium channels

Familial hemiplegic migraine type 1 (FHM-1) is a monogenic form of migraine with aura that is characterized by recurrent attacks of a typical migraine headache with transient hemiparesis during the aura phase. In a subset of patients, additional symptoms such as epilepsy and cerebellar ataxia are part of the clinical phenotype. FHM-1 is caused by missense mutations in the CACNA1A gene that encodes the pore-forming subunit of Ca_V2.1 voltage-gated Ca^2 + channels. Although the functional effects of an increasing number of FHM-1 mutations have been characterized, knowledge on the influence of most of these mutations on G protein regulation of channel function is lacking. Here, we explored the effects of G protein-dependent modulation on mutations W1684R and V1696I which cause FHM-1 with and without cerebellar ataxia, respectively. Both mutations were introduced into the human Ca_V2.1α₁ subunit and their functional consequences investigated after heterologous expression in human embryonic kidney 293 (HEK‐293) cells using patch-clamp recordings. When co-expressed along with the human μ-opioid receptor, application of the agonist [d‐Ala2, N‐MePhe4, Gly‐ol]‐enkephalin (DAMGO) inhibited currents through both wild-type (WT) and mutant Ca_V2.1 channels, which is consistent with the known modulation of these channels by G protein-coupled receptors. Prepulse facilitation, which is a way to characterize the relief of direct voltage-dependent G protein regulation, was reduced by both FHM-1 mutations. Moreover, the kinetic analysis of the onset and decay of facilitation showed that the W1684R and V1696I mutations affect the apparent dissociation and reassociation rates of the Gβγ dimer from the channel complex, suggesting that the G protein-Ca^2 + channel affinity may be altered by the mutations. These biophysical studies may shed new light on the pathophysiology underlying FHM-1.     

Proneural gene requirement for hair cell differentiation in the zebrafish lateral line

The lateral line system comprises an array of mechanosensory organs, the neuromasts, distributed over the body surface. Each neuromast consists of a patch of mechanosensory hair cells surrounded by support cells. We show that, in the zebrafish, two proneural genes are essential for differentiation of the hair cells, neuroD (nrd) and atonal homolog 1 (ath1). Gene knockdown experiments demonstrate that loss of function of either gene, but not of the related proneural gene neurogenin1 (ngn1), abrogate the appearance of hair cell markers. This is in contrast to other sensory systems, such as the neurons of the lateral line ganglion, where nrd is regulated by ngn1 and not by ath1. Overexpression of ath1 can induce nrd, and the phenotype produced by loss of ath1 function can be partially rescued by injection of nrd mRNA. This supports the conclusion that the activation of nrd probably requires ath1 in the hair cell lineage, whereas in sensory neurons nrd activation requires ngn1. We propose that the emergence of two atonal homologs, ath1 and ngn1, allowed the cellular segregation of mechanoreception and signal transmission that were originally performed by a single cell type as found in insects.     

Jurassic biostratigraphy and paleoenvironmental evolution of the Malaguide complex from Sierra Espuña (Internal Betic Zone, SE Spain)

Jurassic studies in the Internal Zones of the Betic Cordillera are scarce since this zone is composed mainly of pre-Jurassic metamorphic rocks. Only the “Dorsal” and the Malaguide domains include fossiliferous Jurassic successions, as in Sierra Espuña (SE Spain), which is one of the bigger and well-exposed Jurassic outcrops of the Internal Zones. Collected Ammonite assemblages update and improve the precision of previous biostratigraphic data by the recognition of: the Domerian (= Upper Pliensbachian, in the Mediterranean Domain) Lavinianum (Cornacaldense Subzone), Algovianum (Ragazzoni, Bertrandi, Accuratum and Levidorsatum Subzones) and Emaciatum (Solare and Elisa Subzones) Zones; the Lower Toarcian Polymorphum and Serpentinum Zones; the Middle Toarcian, Bifrons and Gradata Zone; the Upper Toarcian Reynesi Zone; the Lower/Upper Bajocian, the Lower Callovian Bullatus and Gracilis Zones; the Middle/Upper Oxfordian Transversarium, Bifurcatus, Bimammatum and Planula Zones; and the Lower and Upper Kimmeridgian Platynota, Strombecki, Divisum and Beckeri Zones.The paleoenvironmental evolution of the Malaguide Jurassic at Sierra Espuña shows similarities with other Mediterranean Tethyan paleomargins. The biostratigraphic precision along with the litho- and biofacies analyses has enabled the interpretation that the Malaguide paleomargin evolved as a passive margin, developing shallow carbonate platforms, until the Domerian (Lavinianum Zone). Then, the platform broke up (Domerian, Lavinianum Zone-Upper Toarcian, Reynesi Zone) with the beginning of the rifting stage, beginning the development of horst-graben systems and the coeval drowning of the area. This stage ended in the upper Lower Callovian (Gracilis Zone) to the Middle Oxfordian (Transversarium Zone) interval, starting the drifting stage, which accentuated the horst-graben systems, leading to the deposition of condensed nodular limestones in the raised sea bottom.     

Differential targeting of Tetrahymena ORC to ribosomal DNA and non‐rDNA replication origins

The Tetrahymena thermophila origin recognition complex (ORC) contains an integral RNA subunit, 26T RNA, which confers specificity to the amplified ribosomal DNA (rDNA) origin by base pairing with an essential cis‐acting replication determinant—the type I element. Using a plasmid maintenance assay, we identified a 6.7 kb non‐rDNA fragment containing two closely associated replicators, ARS1‐A (0.8 kb) and ARS1‐B (1.2 kb). Both replicators lack type I elements and hence complementarity to 26T RNA, suggesting that ORC is recruited to these sites by an RNA‐independent mechanism. Consistent with this prediction, although ORC associated exclusively with origin sequences in the 21 kb rDNA minichromosome, the interaction between ORC and the non‐rDNA ARS1 chromosome changed across the cell cycle. In G₂ phase, ORC bound to all tested sequences in a 60 kb interval spanning ARS1‐A/B. Remarkably, ORC and Mcm6 associated with just the ARS1‐A replicator in G₁ phase when pre‐replicative complexes assemble. We propose that ORC is stochastically deposited onto newly replicated non‐rDNA chromosomes and subsequently targeted to preferred initiation sites prior to the next S phase.     

Dynamin Reduces Pyk2 Y402 Phosphorylation and Src Binding in Osteoclasts

Signaling via the Pyk2-Src-Cbl complex downstream of integrins contributes to the assembly, organization, and dynamics of podosomes, which are the transient adhesion complexes of highly motile cells such as osteoclasts and dendritic cells. We previously demonstrated that the GTPase dynamin is associated with podosomes, regulates actin flux in podosomes, and promotes bone resorption by osteoclasts. We report here that dynamin associates with Pyk2, independent of dynamin''s GTPase activity, and reduces Pyk2 Y402 phosphorylation in a GTPase-dependent manner, leading to decreased Src binding to Pyk2. Overexpressing dynamin decreased the macrophage colony-stimulating factor- and adhesion-induced phosphorylation of Pyk2 in osteoclastlike cells, suggesting that dynamin is likely to regulate Src-Pyk2 binding downstream of integrins and growth factor receptors with important cellular consequences. Furthermore, catalytically active Src promotes dynamin-Pyk2 association, and mutating specific Src-phosphorylated tyrosine residues in dynamin blunts the dynamin-induced decrease in Pyk2 phosphorylation. Thus, since Src binds to Pyk2 through its interaction with phospho-Y402, our results suggest that Src activates a negative-feedback loop downstream of integrin engagement and other stimuli by promoting both the binding of dynamin to Pyk2-containing complexes and the dynamin-dependent decrease in Pyk2 Y402 phosphorylation, ultimately leading to the dissociation of Src from Pyk2.Podosomes are specialized transient actin-containing adhesion structures (11, 14, 37, 60) that are found in highly motile cells, such as osteoclasts, macrophages, dendritic cells, transformed metastatic cells, and v-src-transformed cells (37, 43), where they are thought to play important roles in cellular migration and invasion (34). In resorbing osteoclasts on bone, podosomes are concentrated within the sealing zone, a beltlike actin-rich structure that is important for adhesion and which delineates the resorptive region of the cell known as the ruffled border. Unlike focal adhesions, which are relatively stable structures (11, 60), the assembly and disassembly of podosomes occurs within minutes (t_1/2 = 2 to 4 min) and involves the recruitment and activation of integrins, signaling proteins and scaffolding proteins (11, 14, 35, 47, 60). However, the mechanisms of action of key signaling proteins involved in podosome assembly and disassembly are only partially understood.The focal adhesion kinase Pyk2 has been linked to the proliferation, migration, and activity of a variety of mesenchymal, epithelial, and hematopoietic cell types. Several groups, including our own, have reported the importance of Pyk2 in podosome belt organization, cell spreading, and bone-resorbing activity in osteoclasts (18, 26, 31, 40, 65, 66). Pyk2 is recruited to activated β₂ and β₃ integrins (9, 20) at adhesion sites and is autophosphorylated at Y402 (17, 47, 50) via an intermolecular trans-acting mechanism (46). Although Pyk2 is partially activated by integrin-induced Ca²⁺ signaling (20, 50), the induction of Pyk2''s full catalytic activity requires the binding of Src via its SH2 domain to autophosphorylated Pyk2 Y402 and the subsequent phosphorylation of Pyk2 at functionally distinct sites, including Y579, Y580, and Y881 (17, 31, 46). The binding of Src to phosphorylated Pyk2, which leads to the formation of a multiprotein signaling complex at adhesion sites (17, 40, 50), is critical for Pyk2 activity, as demonstrated by the fact that Pyk2 phosphorylation and activity are significantly reduced in osteoclasts derived from Src^−/− mice (17, 40). Src^−/− osteoclasts also exhibit decreased motility (50) and decreased bone-resorbing activity (40, 54, 59), and we recently demonstrated that Src promotes both podosome formation and disassembly, as well as actin flux into existing podosomes and the organization of podosomes into a peripheral belt in osteoclasts (15).We have also demonstrated that the GTP-hydrolyzing protein dynamin-2, which is ubiquitously expressed and well known for its role in endocytosis (53), regulates actin remodeling in the podosomes of osteoclasts and Rous sarcoma virus-transformed baby hamster kidney cells (43). In addition, a dynamin-2 mutant that binds GTP with reduced affinity (dynK44A) (12) decreased the flux of actin into podosomes (43) and disrupted podosome belt formation in osteoclasts, thereby affecting osteoclast migration and bone-resorbing activity (8). The dynamin proteins, of which there are three homologous isoforms (3), contain several protein domains: a GTP-hydrolyzing domain (GTPase), a plextrin homology domain that mediates binding to phosphoinositides, a GTPase effector domain (GED), and a C-terminal proline-rich domain (PRD) (38, 45, 55) through which dynamin binds a number of functionally diverse SH3-containing molecules, such as Src, cortactin, Grb2, and N-Wasp (1, 7, 27, 39, 58). We previously reported that dynamin-2 partially colocalizes and associates with the E3-ubiquitin ligase Cbl within the podosome belt/sealing zone of osteoclasts, as well as in SYF cells, which lack the Src family kinases Src, Yes, and Fyn, and in HEK 293 cells that stably express the vitronectin receptor (293VnR) (8). Protein complexes containing dynamin-2 and Cbl, which are both substrates of Src (1, 2, 23, 50, 56), were disrupted in the presence of activated Src and stabilized in the absence of Src (8), demonstrating a key role of Src in regulating the formation of signaling complexes in osteoclasts downstream of integrins.In the present study, we sought to determine whether dynamin, which regulates podosome actin dynamics and bone resorption in osteoclasts, also associates with Pyk2 and/or regulates Pyk2''s activities in osteoclasts. We report here that dynamin associates with Pyk2 and promotes the dephosphorylation of Pyk2 Y402 and that catalytically active Src promotes both dynamin''s association with Pyk2 and the dynamin-induced dephosphorylation of Pyk2 Y402, resulting, in turn, in the decreased binding of Src to Pyk2. Thus, we propose that dynamin regulates podosome dynamics and osteoclast bone-resorbing activity by promoting the disassembly of the Pyk2-Src-Cbl complex that is formed in osteoclasts downstream of β₃ integrin activation.