Structural and functional properties of isocitrate dehydrogenase from the psychrophilic bacterium Desulfotalea psychrophila reveal a cold-active enzyme with an unusual high thermal stability

Isocitrate dehydrogenase (IDH) has been studied extensively due to its central role in the Krebs cycle, catalyzing the oxidative NAD(P)(+)-dependent decarboxylation of isocitrate to alpha-ketoglutarate and CO(2). Here, we present the first crystal structure of IDH from a psychrophilic bacterium, Desulfotalea psychrophila (DpIDH). The structural information is combined with a detailed biochemical characterization and a comparative study with IDHs from the mesophilic bacterium Desulfitobacterium hafniense (DhIDH), porcine (PcIDH), human cytosolic (HcIDH) and the hyperthermophilic Thermotoga maritima (TmIDH). DpIDH was found to have a higher melting temperature (T(m)=66.9 degrees C) than its mesophilic homologues and a suboptimal catalytic efficiency at low temperatures. The thermodynamic activation parameters indicated a disordered active site, as seen also for the drastic increase in K(m) for isocitrate at elevated temperatures. A methionine cluster situated at the dimeric interface between the two active sites and a cluster of destabilizing charged amino acids in a region close to the active site might explain the poor isocitrate affinity. On the other hand, DpIDH was optimized for interacting with NADP(+) and the crystal structure revealed unique interactions with the cofactor. The highly acidic surface, destabilizing charged residues, fewer ion pairs and reduced size of ionic networks in DpIDH suggest a flexible global structure. However, strategic placement of ionic interactions stabilizing the N and C termini, and additional ionic interactions in the clasp domain as well as two enlarged aromatic clusters might counteract the destabilizing interactions and promote the increased thermal stability. The structure analysis of DpIDH illustrates how psychrophilic enzymes can adjust their flexibility in dynamic regions during their catalytic cycle without compromising the global stability of the protein.     

Protein interactions between surface annexin A2 and S100A10 mediate adhesion of breast cancer cells to microvascular endothelial cells

Annexin A2 (AnxA2) and S100A10 are known to form a molecular complex. Using fluorescence-based binding assays, we show that both proteins are localised on the cell surface, in a molecular form that allows mutual interaction. We hypothesized that binding between these proteins could facilitate cell–cell interactions. For cells that express surface S100A10 and surface annexin A2, cell–cell interactions can be blocked by competing with the interaction between these proteins. Thus an annexin A2-S100A10 molecular bridge participates in cell–cell interactions, revealing a hitherto unexplored function of this protein interaction.     

SmeA, a small membrane protein with multiple functions in Streptomyces sporulation including targeting of a SpoIIIE/FtsK-like protein to cell division septa

Sporulation in aerial hyphae of Streptomyces coelicolor involves profound changes in regulation of fundamental morphogenetic and cell cycle processes to convert the filamentous and multinucleoid cells to small unigenomic spores. Here, a novel sporulation locus consisting of smeA (encoding a small putative membrane protein) and sffA (encoding a SpoIIIE/FtsK-family protein) is characterized. Deletion of smeA-sffA gave rise to pleiotropic effects on spore maturation, and influenced the segregation of chromosomes and placement of septa during sporulation. Both smeA and sffA were expressed specifically in apical cells of sporogenic aerial hyphae simultaneously with or slightly after Z-ring assembly. The presence of smeA-like genes in streptomycete chromosomes, plasmids and transposons, often paired with a gene for a SpoIIIE/FtsK- or Tra-like protein, indicates that SmeA and SffA functions might be related to DNA transfer. During spore development SffA accumulated specifically at sporulation septa where it colocalized with FtsK. However, sffA did not show redundancy with ftsK, and SffA function appeared distinct from the DNA translocase activity displayed by FtsK during closure of sporulation septa. The septal localization of SffA was dependent on SmeA, suggesting that SmeA may act as an assembly factor for SffA and possibly other proteins required during spore maturation.     

Diving behaviour of dugongs, Dugong dugon

The diving behaviour of 15 dugongs (Dugong dugon) was documented using time-depth recorders (TDRs), which logged a total of 39,507 dives. The TDRs were deployed on dugongs caught at three study sites in northern Australia: Shark Bay, the Gulf of Carpentaria and Shoalwater Bay. The average time for which the dive data were collected per dugong was 10.4±1.1 (S.E.) days. Overall, these dugongs spent 47% of their daily activities within 1.5 m of the sea surface and 72% less than 3 m from the sea surface. Their mean maximum dive depth was 4.8±0.4 m (S.E.), mean dive duration was 2.7±0.17 min and the number of dives per hour averaged 11.8±1.2. The maximum dive depth recorded was 20.5 m; the maximum dive time in water >1.5 m deep was 12.3 min. The effects of dugong sex, location (study site), time of day and tidal cycle on diving rates (dives per hour), mean maximum dive depths, durations of dives, and time spent ≤1.5 m from the surface were investigated using weighted split-plot analysis of variance. The dugongs exhibited substantial interindividual variation in all dive parameters. The interaction between location and time of day was significant for diving rates, mean maximum dive depths and time spent within 1.5 m of the surface. In all these cases, there was substantial variation among individuals within locations among times of day. Thus, it was the variation among individuals that dominated all other effects. Dives were categorised into five types based on the shape of the time-depth profile. Of these, 67% of dives were interpreted as feeding dives (square and U-shaped), 8% as exploratory dives (V-shaped), 22% as travelling dives (shallow-erratic) and 3% as shallow resting dives. There was systematic variation in the distribution of dive types among the factors examined. Most of this variation was among individuals, but this differed across both time of day and tidal state. Not surprisingly, there was a positive relationship between dive duration and depth and a negative relationship between the number of dives per hour and the time spent within 1.5 m of the surface after a dive.     

Symbiotic leghemoglobins are crucial for nitrogen fixation in legume root nodules but not for general plant growth and development

Hemoglobins are ubiquitous in nature and among the best-characterized proteins. Genetics has revealed crucial roles for human hemoglobins, but similar data are lacking for plants. Plants contain symbiotic and nonsymbiotic hemoglobins; the former are thought to be important for symbiotic nitrogen fixation (SNF). In legumes, SNF occurs in specialized organs, called nodules, which contain millions of nitrogen-fixing rhizobia, called bacteroids. The induction of nodule-specific plant genes, including those encoding symbiotic leghemoglobins (Lb), accompanies nodule development. Leghemoglobins accumulate to millimolar concentrations in the cytoplasm of infected plant cells prior to nitrogen fixation and are thought to buffer free oxygen in the nanomolar range, avoiding inactivation of oxygen-labile nitrogenase while maintaining high oxygen flux for respiration. Although widely accepted, this hypothesis has never been tested in planta. Using RNAi, we abolished symbiotic leghemoglobin synthesis in nodules of the model legume Lotus japonicus. This caused an increase in nodule free oxygen, a decrease in the ATP/ADP ratio, loss of bacterial nitrogenase protein, and absence of SNF. However, LbRNAi plants grew normally when fertilized with mineral nitrogen. These data indicate roles for leghemoglobins in oxygen transport and buffering and prove for the first time that plant hemoglobins are crucial for symbiotic nitrogen fixation.     

Switches, excitable responses and oscillations in the Ring1B/Bmi1 ubiquitination system

In an active, self-ubiquitinated state, the Ring1B ligase monoubiquitinates histone H2A playing a critical role in Polycomb-mediated gene silencing. Following ubiquitination by external ligases, Ring1B is targeted for proteosomal degradation. Using biochemical data and computational modeling, we show that the Ring1B ligase can exhibit abrupt switches, overshoot transitions and self-perpetuating oscillations between its distinct ubiquitination and activity states. These different Ring1B states display canonical or multiply branched, atypical polyubiquitin chains and involve association with the Polycomb-group protein Bmi1. Bistable switches and oscillations may lead to all-or-none histone H2A monoubiquitination rates and result in discrete periods of gene (in)activity. Switches, overshoots and oscillations in Ring1B catalytic activity and proteosomal degradation are controlled by the abundances of Bmi1 and Ring1B, and the activities and abundances of external ligases and deubiquitinases, such as E6-AP and USP7.     

RAB23 mutations in Carpenter syndrome imply an unexpected role for hedgehog signaling in cranial-suture development and obesity

Carpenter syndrome is a pleiotropic disorder with autosomal recessive inheritance, the cardinal features of which include craniosynostosis, polysyndactyly, obesity, and cardiac defects. Using homozygosity mapping, we found linkage to chromosome 6p12.1-q12 and, in 15 independent families, identified five different mutations (four truncating and one missense) in RAB23, which encodes a member of the RAB guanosine triphosphatase (GTPase) family of vesicle transport proteins and acts as a negative regulator of hedgehog (HH) signaling. In 10 patients, the disease was caused by homozygosity for the same nonsense mutation, L145X, that resides on a common haplotype, indicative of a founder effect in patients of northern European descent. Surprisingly, nonsense mutations of Rab23 in open brain mice cause recessive embryonic lethality with neural-tube defects, suggesting a species difference in the requirement for RAB23 during early development. The discovery of RAB23 mutations in patients with Carpenter syndrome implicates HH signaling in cranial-suture biogenesis--an unexpected finding, given that craniosynostosis is not usually associated with mutations of other HH-pathway components--and provides a new molecular target for studies of obesity.