Unprecedented access of phenolic substrates to the heme active site of a catalase: Substrate binding and peroxidase‐like reactivity of Bacillus pumilus catalase monitored by X‐ray crystallography and EPR spectroscopy

Heme‐containing catalases and catalase‐peroxidases catalyze the dismutation of hydrogen peroxide as their predominant catalytic activity, but in addition, individual enzymes support low levels of peroxidase and oxidase activities, produce superoxide, and activate isoniazid as an antitubercular drug. The recent report of a heme enzyme with catalase, peroxidase and penicillin oxidase activities in Bacillus pumilus and its categorization as an unusual catalase‐peroxidase led us to investigate the enzyme for comparison with other catalase‐peroxidases, catalases, and peroxidases. Characterization revealed a typical homotetrameric catalase with one pentacoordinated heme b per subunit (Tyr340 being the axial ligand), albeit in two orientations, and a very fast catalatic turnover rate (k_cat = 339,000 s^?1). In addition, the enzyme supported a much slower (k_cat = 20 s^?1) peroxidatic activity utilizing substrates as diverse as ABTS and polyphenols, but no oxidase activity. Two binding sites, one in the main access channel and the other on the protein surface, accommodating pyrogallol, catechol, resorcinol, guaiacol, hydroquinone, and 2‐chlorophenol were identified in crystal structures at 1.65–1.95 Å. A third site, in the heme distal side, accommodating only pyrogallol and catechol, interacting with the heme iron and the catalytic His and Arg residues, was also identified. This site was confirmed in solution by EPR spectroscopy characterization, which also showed that the phenolic oxygen was not directly coordinated to the heme iron (no low‐spin conversion of the Fe^III high‐spin EPR signal upon substrate binding). This is the first demonstration of phenolic substrates directly accessing the heme distal side of a catalase. Proteins 2015; 83:853–866. © 2015 Wiley Periodicals, Inc.     

Enzyme–substrate complexes of allosteric citrate synthase: Evidence for a novel intermediate in substrate binding

The citrate synthase (CS) of Escherichia coli is an allosteric hexameric enzyme specifically inhibited by NADH. The crystal structure of wild type (WT) E. coli CS, determined by us previously, has no substrates bound, and part of the active site is in a highly mobile region that is shifted from the position needed for catalysis. The CS of Acetobacter aceti has a similar structure, but has been successfully crystallized with bound substrates: both oxaloacetic acid (OAA) and an analog of acetyl coenzyme A (AcCoA). We engineered a variant of E. coli CS wherein five amino acids in the mobile region have been replaced by those in the A. aceti sequence. The purified enzyme shows unusual kinetics with a low affinity for both substrates. Although the crystal structure without ligands is very similar to that of the WT enzyme (except in the mutated region), complexes are formed with both substrates and the allosteric inhibitor NADH. The complex with OAA in the active site identifies a novel OAA-binding residue, Arg306, which has no functional counterpart in other known CS-OAA complexes. This structure may represent an intermediate in a multi-step substrate binding process where Arg306 changes roles from OAA binding to AcCoA binding. The second complex has the substrate analog, S-carboxymethyl-coenzyme A, in the allosteric NADH-binding site and the AcCoA site is not formed. Additional CS variants unable to bind adenylates at the allosteric site show that this second complex is not a factor in positive allosteric activation of AcCoA binding.     

A physical profile of elite female ice hockey players from the USA

Despite impressive numbers of hockey participants, there is little research examining elite female ice hockey players. Therefore, the purpose of this study was to describe the physical characteristics of elite female ice hockey players who were trying out for the 2010 US Women's Ice Hockey team. Twenty-three women participated in the study and were evaluated for body mass (kilograms), height (centimeters), age (years) vertical jump (centimeters), standing long jump (centimeters), 1RM front squat (kilograms), front squat relative to body mass (percent), 1RM bench press (kilograms), bench press relative to body mass (percent), pull-ups, and body composition (percent body fat). The athletes in this sample were 24.7 years of age (SD = 3.1) and 169.7 cm tall (SD = 6.9); on average, they weighed 70.4 kg (SD = 7.1) and reported 15.8% body fat (SD = 1.9). Mean vertical jump height was 50.3 cm (SD = 5.7) and standing long jump was 214.8 cm (SD = 10.9). Mean 1RM for the upper body strength (bench press) was 65.3 kg (SD = 12.2) (95.1 ± 15.5% of body mass), and 1RM for lower body (front squat) was 88.6 kg (SD = 11.2) (127.7 ± 16.3% of body mass). This study is the first to report the physical characteristics of elite female ice hockey players from the USA. Data should assist strength and conditioning coaches in identifying talent, testing for strengths and weaknesses, comparing future teams to these indicators, and designing programs that will enhance the performance capabilities of female ice hockey athletes.     

Evidence for osteocyte regulation of bone homeostasis through RANKL expression

Osteocytes embedded in bone have been postulated to orchestrate bone homeostasis by regulating both bone-forming osteoblasts and bone-resorbing osteoclasts. We find here that purified osteocytes express a much higher amount of receptor activator of nuclear factor-κB ligand (RANKL) and have a greater capacity to support osteoclastogenesis in vitro than osteoblasts and bone marrow stromal cells. Furthermore, the severe osteopetrotic phenotype that we observe in mice lacking RANKL specifically in osteocytes indicates that osteocytes are the major source of RANKL in bone remodeling in vivo.     

Loss of the α2β1 integrin alters human papilloma virus-induced squamous carcinoma progression in vivo and in vitro

Expression of the α2β1 integrin, a receptor for collagens and laminin, is altered during tumor progression. Recent studies have linked polymorphisms in the α2 integrin gene with oral, squamous cell carcinoma (SCC). To determine the α2β1 integrin's role in SCC progression, we crossed α2-null mice with K14-HPV16 transgenic animals. Pathological progression to invasive carcinoma was evaluated in HPV-positive, α2-null (HPV/KO) and HPV-positive, wild-type (HPV/WT) animals. α2β1 integrin expression stimulated progression from hyperplasia and papillomatosis to dysplasia with concomitant dermal mast cell infiltration. Moreover, lymph node metastasis was decreased by 31.3% in HPV/KO, compared to HPV/WT, animals. To evaluate the integrin-specific impact on the malignant epithelium versus the microenvironment, we developed primary tumor cell lines. Although transition from dysplasia to carcinoma was unaltered during spontaneous tumor development, isolated primary HPV/KO SCC cell lines demonstrated decreased migration and invasion, compared to HPV/WT cells. When HPV/WT and HPV/KO SCC cells were orthotopically injected into WT or KO hosts, tumor α2β1 integrin expression resulted in decreased tumor latency, regardless of host integrin status. HPV/WT SCC lines failed to demonstrate a proliferative advantage in vitro, however, the HPV/WT tumors demonstrated increased growth compared to HPV/KO SCC lines in vivo. Although contributions of the integrin to the microenvironment cannot be excluded, our studies indicate that α2β1 integrin expression by HPV-transformed keratinocytes modulates SCC growth and progression.     

Dectin-1 Activation Controls Maturation of β-1,3-Glucan-containing Phagosomes

Elimination of fungal pathogens by phagocytes requires phagosome maturation, a process that involves the recruitment and fusion of intracellular proteins. The role of Dectin-1, a β-1,3-glucan receptor, critical for fungal recognition and triggering of Th17 responses, to phagosomal maturation has not been defined. We show that GFP-Dectin-1 translocates to the fungal phagosome, but its signal decays after 2 h. Inhibition of acidification results in retention of GFP-Dectin-1 to phagosome membranes highlighting the requirement for an acidic pH. Following β-1,3-glucan recognition, GFP-Dectin-1 undergoes tyrosine phosphorylation by Src kinases with subsequent Syk activation. Our results demonstrate that Syk is activated independently of intraphagosomal pH. Inhibition of Src or Syk results in prolonged retention of GFP-Dectin-1 to the phagosome signifying a link between Syk and intraphagosomal pH. β-1,3-glucan phagosomes expressing a signaling incompetent Dectin-1 failed to mature as demonstrated by prolonged Dectin-1 retention, presence of Rab5B, failure to acquire LAMP-1 and inability to acidify. Phagosomes containing Candida albicans also require Dectin-1-dependent Syk activation for phagosomal maturation. Taken together, these results support a model where Dectin-1 not only controls internalization of β-1,3-glucan containing cargo and triggers proinflammatory cytokines, but also acts as a master regulator for subsequent phagolysosomal maturation through Syk activation.     

Exploring Synchronicity in the Heart Rates of Familiar and Unfamiliar Pairs of Horses and Humans Undertaking an In-Hand Task

ABSTRACT

Physiological responses that occur in horses and humans during their interactions, on the ground and during ridden work, have been investigated in a number of studies with some conflicting results. These suggest that in some situations emotional state may be transferred from humans to horses and that there is the potential for the heart rates of horse–human pairs to become synchronized during ridden work. Here we explore the effect of familiarity on the physiological responses of horse–human pairs completing a task in-hand, using heart rate as an indicator for emotional state. We investigated differences in heart rate response between familiar and unfamiliar pairings and the possibility of heart rate synchronization within each pair. Complete sets of horse and human heart rate data were available for 17 horses. We found a significant order affect, with higher horse heart rates seen the first time around the course regardless of whether a familiar or unfamiliar handler was leading (Wilcoxon test: Z=–2.67, p<0.05). However, despite this, the horses’ mean heart rates for each course were significantly higher with the unfamiliar handler than with the familiar handler (Wilcoxon test: Z=–4.46, p<0.001). In contrast, human heart rates were higher when paired with a familiar horse compared with an unfamiliar horse (Mann-Whitney U test: Z=–5.08, p<0.001). Significant correlations between horse and human heart rates were seen in three familiar pairings and two unfamiliar pairings. Our findings indicate that the relationship between horse and human heart rates during interactions is not straightforward or consistent between horses and humans, and is likely to depend on a number of factors such as experience of the test situation. Although the lower heart rates seen in horses being led by their familiar handler suggest that they are more relaxed with someone they know, this could not be said for the human partner.     

Climate,not Aboriginal landscape burning,controlled the historical demography and distribution of fire-sensitive conifer populations across Australia

Climate and fire are the key environmental factors that shape the distribution and demography of plant populations in Australia. Because of limited palaeoecological records in this arid continent, however, it is unclear as to which factor impacted vegetation more strongly, and what were the roles of fire regime changes owing to human activity and megafaunal extinction (since ca 50 kya). To address these questions, we analysed historical genetic, demographic and distributional changes in a widespread conifer species complex that paradoxically grows in fire-prone regions, yet is very sensitive to fire. Genetic demographic analysis showed that the arid populations experienced strong bottlenecks, consistent with range contractions during the Last Glacial Maximum (ca 20 kya) predicted by species distribution models. In southern temperate regions, the population sizes were estimated to have been mostly stable, followed by some expansion coinciding with climate amelioration at the end of the last glacial period. By contrast, in the flammable tropical savannahs, where fire risk is the highest, demographic analysis failed to detect significant population bottlenecks. Collectively, these results suggest that the impact of climate change overwhelmed any modifications to fire regimes by Aboriginal landscape burning and megafaunal extinction, a finding that probably also applies to other fire-prone vegetation across Australia.     

RhoGTPases — NODes for effector-triggered immunity in animals

A recent study published in Nature by Keestra and colleagues addresses how the immune system detects the pathogenic potential of microbes and provides evidence that one strategy involves NOD1, which monitors the activation state of the RhoGTPases that are targeted by virulence effectors produced by pathogenic microbes. Interestingly, their findings reveal striking similarities with previous observations made in flies and plants, establishing the evolutionary conservation of this detection system in the innate immune arsenal in many taxa.The discovery that Drosophila Toll, and the homologous Toll-like receptors (TLRs) in animals, are pattern recognition receptors (PRRs) that act as cellular sensors of microbes has attracted considerable attention during the last two decades¹. The PRR system is based on the detection of conserved molecular motifs, microbe-associated molecular patterns (MAMPS), that are shared by most microbes, virulent or not. Given the conserved nature of MAMPS, this model does not explain how the host discriminates between harmful pathogens and beneficial commensal microbes. An elegant hypothesis is that, in addition to the PRR system, the host is able to monitor the pathogen-induced disruption of cellular homeostasis. This type of immune surveillance system has been demonstrated in plants and termed “effector-triggered immunity” (ETI)². Recently, the concept of ETI has been extended to metazoans and proof of its importance as an innate immune mechanism has now been provided in Drosophila melanogaster, Caenorabditis elegans and mammals³.The ETI model is of particular relevance when considering that most major pathogenic bacteria have evolved many protein effectors commonly referred to as virulence factors. These effectors are either directly injected into host cells by cell-bound bacteria, or are secreted toxins endowed with the ability to bind to and translocate into the host cell cytosol. Once within the host cell, these bacterial effectors perturb cellular homeostasis by modifying the activity of critical regulators. Common amongst the arsenal of numerous pathogenic bacteria are virulence factors that target the small RhoGTPases of the host⁴. This predilection for targeting RhoGTPases is probably because it allows bacteria to hijack the many cellular functions that contribute to immunity including phagocytosis, apoptosis, as well as production of reactive oxygen species (ROS) and inflammatory mediators⁵. In this regard, the RhoGTPases represent a common target and vulnerability in the host cell, which explains why their aberrant activity can often indicate pathogen invasion.Providing the foundation for the work of Keestra et al.⁶, previous studies have shown that effectors that activate RhoGTPase can induce unusual immune responses in the host. Both Salmonella typhimurium and Shigella spp. are enteric pathogens that invade host cells using a type III secretion system that is able to inject effectors in the cytosol of host cells. Earlier work by the group of Jorge Galan showed that Salmonella effectors could activate epithelial cells through a PRR-independent mechanism that was dependent on the GEF activity of certain effectors and involving target GTPases in the host⁷. Similarly, the Shigella effector, GEF-H1 was shown to augment NF-κB-dependent immune responses in a NOD1-dependent manner also after modifying RhoGTPases⁸. Finally, the activation of RacGTPase by the CNF1 toxin of uropathogenic Escherichia coli also triggers an immune response⁹. In this case the response is via an innate immune signaling pathway conserved in Drosophila and mammals involving IMD in flies and the related Rip proteins, RIP1 and RIP2, in mammalian cells⁹. Moreover, this response can be beneficial for the host and help clear the bacteria as shown in an in vivo fly model. Now, using a mouse model of Salmonella typhimurium infection, Keestra et al.⁶ further define the mechanism of detection of effectors that target RhoGTPase in vertebrates. They show that mammals detect the activity of the injected effector SopE once it is active in the host cytosol. Using cell-based assays, they demonstrate that this detection mechanism is through NOD1, a NOD-Like Receptor (NLR) protein, in a molecular complex containing HSP90. Together this complex detects the activation of the RhoGTPases Rac1 and Cdc42 and transduces a danger signal though RIP2 kinase. Furthermore, using NOD1-deficient mice, they show that SopE-triggered inflammation is markedly reduced.Together this emerging body of data support the idea that the activity of GTPases is monitored by NLR and related pathways, and used as a cue to augment ongoing immune responses during pathogen invasion. Interestingly, Kawano and colleagues have shown that resistance to the rice blast fungus also involves activation of Rac (OsRac1) downstream of Pit, a plant nucleotide-binding site-leucine-rich repeat (NBS-LRR) receptor¹⁰. OsRac1 contributes to NBS-LRR-mediated production of ROS and induction of a hypersensitive response for the purpose of destroying infected tissue and preventing dissemination. Although the link between RhoGTPases and NLRs in mammals and NBS-LRR in plants is likely a consequence of convergent evolution, these data highlight some striking similarities between the two systems (Figure 1). Together these papers suggest that we should now consider the NOD proteins not only as intracellular PRR but also akin to plant NBS-LRRs that are able to sense the direct and indirect perturbations of host cell homeostasis. Moreover, these data place RhoGTPases as central players in the molecular cascades of ETI in many species, explaining why monitoring the activation state of RhoGTPases as a surrogate for the presence of virulent pathogens is an evolutionarily conserved strategy. Our current challenge will now be to determine how PRR- and effector-triggered immunity collaborate to confer optimal protection during infections with virulent pathogens.Open in a separate windowFigure 1RhoGTPases are components of effector-triggered immune responses in different species. The role of the RhoGTPases OsRac1 in plants (left), Rac2 in flies and mammals (middle), and Rac1 and CDC42 in mammals (right) in ETI responses. Activation of Rac2 by CNF1 (middle), and Rac1 and CDC42 by SopE engages RIP kinase-dependent signaling pathways. In plants OsRac1 is activated downstream of the NBS-LRR, Pit, and the response to SopE (right panel) requires the NLR, NOD1. In contrast, flies lack NLRs and the ETI response occurs independently of NLRs but does require the RhoGTPase, Rac2. Middle and right panels — inactive GTPase bound to GDP is shown in grey (black square) and the active GTPase bound to GTP in blue (blue circle).