Food and Indoor Air Isolated Bacillus Non-Protein Toxins: : Structures, Physico-Chemical Properties and Mechanisms of Effects on Eukaryotic Cells

We report here the structures and properties of heat-stable, non-protein, and mammalian cell-toxic compounds produced by spore-forming bacilli isolated from indoor air of buildings and from food. Little information is available on the effects and occurrence of heat-stable non-protein toxins produced by bacilli in moisture-damaged buildings. Bacilli emit spores that move in the air and can serve as the carriers of toxins, in a manner similar to that of the spores of toxic fungi found in contaminated indoor air. Bacillus spores in food cause problems because they tolerate the temperatures applied in food manufacture and the spores later initiate growth when food storage conditions are more favorable. Detection of the toxic compounds in Bacillus is based on using the change in mobility of boar spermatozoa as an indicator of toxic exposure. GC, LC, MS, and nuclear magnetic resonance NMR spectroscopy were used for purification, detection, quantitation, and analysis of the properties and structures of the compounds. Toxicity and the mechanisms of toxicity of the compounds were studied using boar spermatozoa, feline lung cells, human neural cells, and mitochondria isolated from rat liver. The ionophoric properties were studied using the BLM (black-lipid membrane) method. One novel toxin, forming ion channels permeant to K+ > Na+ > Ca2+, was found and named amylosin. It is produced by B. amyloliquefaciens isolated from indoor air of moisture-damaged buildings. Amylosin was purified with an RP-HPLC and a monoisotopic mass of 1197 Da was determined with ESI-IT-MS. Furthermore, acid hydrolysis of amylosin followed by analysis of the amino acids with the GS-MS showed that it was a peptide. The presence of a chromophoric polyene group was found using a NMR spectroscopy. The quantification method developed for amylosin based on RP-HPLC-UV, using the macrolactone polyene, amphotericin B (MW 924), as a reference compound. The B. licheniformis strains isolated from a food poisoning case produced a lipopeptide, lichenysin A, that ruptured mammalian cell membranes and was purified with a LC. Lichenysin A was identified by its protonated molecules and sodium- and potassium- cationized molecules with MALDI-TOF-MS. Its protonated forms were observed at m/z 1007, 1021 and 1035. The amino acids of lichenysin A were analyzed with ESI-TQ-MS/MS and, after acid hydrolysis, the stereoisomeric forms of the amino acids with RP-HPLC. The indoor air isolates of the strain of B. amyloliquefaciens produced not only amylosin but also lipopeptides: the cell membrane-damaging surfactin and the fungicidal fengycin. They were identified with ESI-IT-MS observing their protonated molecules, the sodium- and potassium-cationized molecules and analysing the MS/MS spectra. The protonated molecules of surfactin and fengycin showed m/z values of 1009, 1023, and 1037 and 1450, 1463, 1493, and 1506, respectively. Cereulide (MW 1152) was purified with RP-HPLC from a food poisoning strain of B. cereus. Cereulide was identified with ESI-TQ-MS according to the protonated molecule observed at m/z 1154 and the ammonium-, sodium- and potassium-cationized molecules observed at m/z 1171, 1176, and 1192, respectively. The fragment ions of the MS/MS spectrum obtained from the protonated molecule of cereulide at m/z 1154 were also interpreted. We developed a quantification method for cereulide, using RP-HPLC-UV and valinomycin (MW 1110, which structurally resembles cereulide) as the reference compound. Furthermore, we showed empirically, using the BLM method, that the emetic toxin cereulide is a specific and effective potassium ionophore of whose toxicity target is especially the mitochondria.

Molecular Modelling of Estrogen-Producing Enzymes CYP450 Aromatase and 17beta-Hydroxysteroid Dehydrogenase Type 1

Breast cancer is the most common cancer in women in the western countries. Approximately two-thirds of breast cancer tumours are hormone dependent, requiring estrogens to grow. Estrogens are formed in the human body via a multistep route starting from cholesterol. The final steps in the biosynthesis include the CYP450 aromatase enzyme, converting the male hormones androgens (preferred substrate androstenedione ASD) into estrogens(estrone E1), and the 17beta-HSD1 enzyme, converting the biologically less active E1 into the active hormone 17beta-hydroxyestradiol E2. E2 is bound to the nuclear estrogen receptors causing a cascade of biochemical reactions leading to cell proliferation in normal tissue, and to tumour growth in cancer tissue. Aromatase and 17beta-HSD1 are expressed in or near the breast tumour, locally providing the tissue with estrogens. One approach in treating hormone dependent breast tumours is to block the local estrogen production by inhibiting these two enzymes. Aromatase inhibitors are already on the market in treating breast cancer, despite the lack of an experimentally solved structure. The structure of 17beta-HSD1, on the other hand, has been solved, but no commercial drugs have emerged from the drug discovery projects reported in the literature. Computer-assisted molecular modelling is an invaluable tool in modern drug design projects. Modelling techniques can be used to generate a model of the target protein and to design novel inhibitors for them even if the target protein structure is unknown. Molecular modelling has applications in predicting the activities of theoretical inhibitors and in finding possible active inhibitors from a compound database. Inhibitor binding at atomic level can also be studied with molecular modelling. To clarify the interactions between the aromatase enzyme and its substrate and inhibitors, we generated a homology model based on a mammalian CYP450 enzyme, rabbit progesterone 21-hydroxylase CYP2C5. The model was carefully validated using molecular dynamics simulations (MDS) with and without the natural substrate ASD. Binding orientation of the inhibitors was based on the hypothesis that the inhibitors coordinate to the heme iron, and were studied using MDS. The inhibitors were dietary phytoestrogens, which have been shown to reduce the risk for breast cancer. To further validate the model, the interactions of a commercial breast cancer drug were studied with MDS and ligand–protein docking. In the case of 17beta-HSD1, a 3D QSAR model was generated on the basis of MDS of an enzyme complex with active inhibitor and ligand–protein docking, employing a compound library synthesised in our laboratory. Furthermore, four pharmacophore hypotheses with and without a bound substrate or an inhibitor were developed and used in screening a commercial database of drug-like compounds. The homology model of aromatase showed stable behaviour in MDS and was capable of explaining most of the results from mutagenesis studies. We were able to identify the active site residues contributing to the inhibitor binding, and explain differences in coordination geometry corresponding to the inhibitory activity. Interactions between the inhibitors and aromatase were in agreement with the mutagenesis studies reported for aromatase. Simulations of 17beta-HSD1 with inhibitors revealed an inhibitor binding mode with hydrogen bond interactions previously not reported, and a hydrophobic pocket capable of accommodating a bulky side chain. Pharmacophore hypothesis generation, followed by virtual screening, was able to identify several compounds that can be used in lead compound generation. The visualisation of the interaction fields from the QSAR model and the pharmacophores provided us with novel ideas for inhibitor development in our drug discovery project.

Sympatric prey responses to lethal top-predator control: predator manipulation experiments

Introduction Many prey species around the world are suffering declines due to a variety of interacting causes such as land use change, climate change, invasive species and novel disease. Recent studies on the ecological roles of top-predators have suggested that lethal top-predator control by humans (typically undertaken to protect livestock or managed game from predation) is an indirect additional cause of prey declines through trophic cascade effects. Such studies have prompted calls to prohibit lethal top-predator control with the expectation that doing so will result in widespread benefits for biodiversity at all trophic levels. However, applied experiments investigating in situ responses of prey populations to contemporary top-predator management practices are few and none have previously been conducted on the eclectic suite of native and exotic mammalian, reptilian, avian and amphibian predator and prey taxa we simultaneously assess. We conducted a series of landscape-scale, multi-year, manipulative experiments at nine sites spanning five ecosystem types across the Australian continental rangelands to investigate the responses of sympatric prey populations to contemporary poison-baiting programs intended to control top-predators (dingoes) for livestock protection. Results Prey populations were almost always in similar or greater abundances in baited areas. Short-term prey responses to baiting were seldom apparent. Longer-term prey population trends fluctuated independently of baiting for every prey species at all sites, and divergence or convergence of prey population trends occurred rarely. Top-predator population trends fluctuated independently of baiting in all cases, and never did diverge or converge. Mesopredator population trends likewise fluctuated independently of baiting in almost all cases, but did diverge or converge in a few instances. Conclusions These results demonstrate that Australian populations of prey fauna at lower trophic levels are typically unaffected by top-predator control because top-predator populations are not substantially affected by contemporary control practices, thus averting a trophic cascade. We conclude that alteration of current top-predator management practices is probably unnecessary for enhancing fauna recovery in the Australian rangelands. More generally, our results suggest that theoretical and observational studies advancing the idea that lethal control of top-predators induces trophic cascades may not be as universal as previously supposed.

Increased virulence of rabbit haemorrhagic disease virus associated with genetic resistance in wild Australian rabbits (Oryctolagus cuniculus)

The release of myxoma virus (MYXV) and Rabbit Haemorrhagic Disease Virus (RHDV) in Australia with the aim of controlling overabundant rabbits has provided a unique opportunity to study the initial spread and establishment of emerging pathogens, as well as their co-evolution with their mammalian hosts. In contrast to MYXV, which attenuated shortly after its introduction, rapid attenuation of RHDV has not been observed. By studying the change in virulence of recent field isolates at a single field site we show, for the first time, that RHDV virulence has increased through time, likely because of selection to overcome developing genetic resistance in Australian wild rabbits. High virulence also appears to be favoured as rabbit carcasses, rather than diseased animals, are the likely source of mechanical insect transmission. These findings not only help elucidate the co-evolutionary interaction between rabbits and RHDV, but reveal some of the key factors shaping virulence evolution.

Eurasian Neogene large herbivorous mammals and climate

The development and changes in the distribution of herbivorous mammal communities during the Neogene is complex. The Eurasian scale environmental patterns reflect the large scale geographical and climatic patterns. The reorganization of these affect the biome distribution throughout the continent. The distribution of mammal taxa was closely associated with the distribution of biomes. In Eurasia the Neogene development of environments was twofold. The early and middle Miocene that seemed to have been advantageous for mammals was followed by drying of environments during the late Neogene. The mid-latitude drying was the main trend, and it is the combined result of the retreat of Paratethys, the uplift of Tibetan Plateau and changes in the ocean currents and temperatures. The common mammals were "driving" the evolution of mammalian communities. During the late Miocene we see the drying affecting more and more regions, and we see changes in the composition of mammalian communities.

Eurasian large mammal dynamics in response to changing environments during the Late Neogene

Nisäkkäiden levinneisyyteen, niiden morfologisiin ja ekologisiin piirteisiin vaikuttavat ympäristön sekä lyhyet että pitkäkestoiset muutokset, etenkin ilmaston ja kasvillisuuden vaihtelut. Työssä tutkittiin nisäkkäiden sopeutumista ilmastonmuutoksiin Euraasiassa viimeisen 24 miljoonan vuoden aikana. Tutkimuksessa keskityttiin varsinkin viimeiseen kahteen miljoonaan vuoteen, jonka aikana ilmasto muuttui voimakkaasti ja ihmisen toiminta alkoi tulla merkittäväksi. Tämän takia on usein vaikea erottaa, kummasta em. seikasta jonkin nisäkäslajin sukupuutto tai häviäminen alueelta johtui. Aineistona käytettiin laajaa venäjänkielistä kirjallisuutta, josta löytyvät tiedot ovat kääntämättöminä jääneet aiemmin länsimaisen tutkimuksen ulkopuolelle. Työssä käytettiin myös NOW-tietokantaa, jossa on fossiilisten nisäkkäiden löytöpaikat sekä niiden iät.

The Miocene of Western Asia; Fossil mammals at the crossroads of faunal provinces and climate regimes

Being at the crossroads of the Old World continents, Western Asia has a unique position through which the dispersal and migration of mammals and the interaction of faunal bioprovinces occurred. Despite its critical position, the record of Miocene mammals in Western Asia is sporadic and there are large spatial and temporal gaps between the known fossil localities. Although the development of the mammalian faunas in the Miocene of the Old World is well known and there is ample evidence for environmental shifts in this epoch, efforts toward quantification of habitat changes and development of chronofaunas based on faunal compositions were mostly neglected. Advancement of chronological, paleoclimatological, and paleogeographical reconstruction tools and techniques and increased numbers of new discoveries in recent decades have brought the need for updating and modification of our level of understanding. We under took fieldwork and systematic study of mammalian trace and body fossils from the northwestern parts of Iran along with analysis of large mammal data from the NOW database. The data analysis was used to study the provinciality, relative abundance, and distribution history of the closed- and open-adapted taxa and chronofaunas in the Miocene of the Old World and Western Asia. The provinciality analysis was carried out, using locality clustering, and the relative abundance of the closed- and open-adapted taxa was surveyed at the family level. The distribution history of the chronofaunas was studied, using faunal resemblance indices and new mapping techniques, together with humidity analysis based on mean ordinated hypsodonty. Paleoichnological studies revealed the abundance of mammalian footprints in several parts of the basins studied, which are normally not fossiliferous in terms of body fossils. The systematic study and biochronology of the newly discovered mammalian fossils in northwestern Iran indicates their close affinities with middle Turolian faunas. Large cranial remains of hipparionine horses, previously unknown in Iran and Western Asia, are among the material studied. The initiation of a new field project in the famous Maragheh locality also brings new opportunities to address questions regarding the chronology and paleoenvironment of this classical site. Provinciality analysis modified our previous level of understandings, indicating the interaction of four provinces in Western Asia. The development of these provinces was apparently due to the presence of high mountain ranges in the area, which affected the dispersal of mammals and also climatic patterns. Higher temperatures and possibly higher co2 levels in the Middle Miocene Climatic Optimum apparently favored the development of the closed forested environments that supported the dominance of the closed-adapted taxa. The increased seasonality and the progressive cooling and drying of the midlatitudes toward the Late Miocene maintained the dominance of open-adapted faunas. It appears that the late Middle Miocene was the time of transition from a more forested to a less forested world. The distribution history of the closed- and open-adapted chronofaunas shows the presence of cosmopolitan and endemic faunas in Western Asia. The closed-adapted faunas, such as the Arabian chronofauna of the late Early‒early Middle Miocene, demonstrated a rapid buildup and gradual decline. The open-adapted chronofaunas, such as the Late Miocene Maraghean fauna, climaxed gradually by filling the opening environments and moving in response to changes in humidity patterns. They abruptly declined due to demise of their favored environments. The Siwalikan chronofauna of the early Late Miocene remained endemic and restricted through all its history. This study highlights the importance of field investigations and indicates that new surveys in the vast areas of Western Asia, which are poorly sampled in terms of fossil mammal localities, can still be promising. Clustering of the localities supports the consistency of formerly known patterns and augments them. Although the quantitative approach to relative abundance history of the closed- and open-adapted mammals harks back to more than half a century ago, it is a novel technique providing robust results. Tracking the history of the chronofaunas in space and time by means of new computational and illustration methods is also a new practice that can be expanded to new areas and time spans.

Using UHF proximity loggers to quantify male–female interactions: A scoping study of estrous activity in cattle

Reproductive efficiency is an important determinant of profitable cattle breeding systems and the success of assisted reproductive techniques (ART) in wildlife conservation programs. Methods of estrous detection used in intensive beef and dairy cattle systems lack accuracy and remain the single biggest issue for improvement of reproductive rates and such methods are not practical for either large-scale extensive beef cattle enterprises or free-living mammalian species. Recent developments in UHF (ultra high frequency) proximity logger telemetry devices have been used to provide a continuous pair-wise measure of associations between individual animals for both livestock and wildlife. The objective of this study was to explore the potential of using UHF telemetry to identify the reproductive cycle phenotype in terms of intensity and duration of estrus. The study was conducted using Belmont Red (interbred Africander Brahman Hereford–Shorthorn) cattle grazing irrigated pasture on Belmont Research Station, northeastern Australia. The cow-bull associations from three groups of cows each with one bull were recorded over a 7-week breeding season and the stage of estrus was identified using ultrasonography. Telemetry data from bull and cows, collected over 4 8-day logger deployments, were log transformed and analyzed by ANOVA. Both the number and duration of bull-cow affiliations were significantly (P < 0.001) greater in estrous cows compared to anestrus cows. These results support the development of the UHF technology as a hands-off and noninvasive means of gathering socio-sexual information on both wildlife and livestock for reproductive management.

Strategies to limit the impact of nematode pressure on sugarcane productivity in the Isis

Two trials were done in this project. One was a continuation of work started under a previous GRDC/SRDC-funded activity, 'Strategies to improve the integration of legumes into cane based farming systems'. This trial aimed to assess the impact of trash and tillage management options and nematicide application on nematodes and crop performance. Methods and results are contained in the following publication: Halpin NV, Stirling GR, Rehbein WE, Quinn B, Jakins A, Ginns SP. The impact of trash and tillage management options and nematicide application on crop performance and plant-parasitic nematode populations in a sugarcane/peanut farming system. Proc. Aust. Soc. Sugar Cane Technol. 37, 192-203. Nematicide application in the plant crop significantly reduced total numbers of plant parasitic nematodes (PPN) but there was no impact on yield. Application of nematicide to the ratoon crop significantly reduced sugar yield. The study confirmed other work demonstrating that implementation of strategies like reduced tillage reduced populations of total PPN, suggesting that the soil was more suppressive to PPN in those treatments. The second trial, a variety trial, demonstrated the limited value of nematicide application in sugarcane farming systems. This study has highlighted that growers shouldn’t view nematicides as a ‘cure all’ for paddocks that have historically had high PPN numbers. Nematicides have high mammalian toxicity, have the potential to contaminate ground water (Kookana et al. 1995) and are costly. The cost of nematicide used in R1 was approx. $320 - $350/ha, adding $3.50/t of cane in a 100 t/ha crop. Also, our study demonstrated that a single nematicide treatment at the application rate registered for sugarcane is not very effective in reducing populations of nematode pests. There appears to be some levels of resistance to nematodes within the current suite of varieties available to the southern canelands. For example the soil in plots that were growing Q183 had 560% more root knot nematodes / 200mL soil compared to plots that grew Q245. The authors see great value in investment into a nematode screening program that could rate varieties into groups of susceptibility to both major sugarcane nematode pests. Such a rating could then be built into a decision support ‘tree’ or tool to better enable producers to select varieties on a paddock by paddock basis.

Spatiotemporal Organization of AT- and GC-rich DNA and Their Association With Transition Proteins TP1 and TP2 in Rat Condensing Spermatids

Transition protein 1 (TP1) and TP2 replace histones during midspermiogenesis (stages 12-15) and are finally replaced by protamines. TPs play a predominant role in DNA condensation and chromatin remodeling during mammalian spermiogenesis. TP2 is a zinc metalloprotein with two novel zinc finger modules that condenses DNA in vitro in a GC-preference manner. TP2 also localizes to the nucleolus in transfected HeLa and Cos-7 cells, suggesting a GC-rich preference, even in vivo. We have now studied the localization pattern of TP2 in the rat spermatid nucleus. Colocalization studies using GC-selective DNA-binding dyes chromomycin A3 and 7-amino actinomycin D and an AT-selective dye, 4',6-diamidino-2-phenylindole, indicate that TP2 is preferentially localized to GC-rich sequences. Interestingly, as spermatids mature, TP2 and GC-rich DNA moves toward the nuclear periphery, and in the late stages of spermatid maturation, TP2 is predominantly localized at the nuclear periphery. Another interesting observation is the mutually exclusive localization of GC- and AT-rich DNA in the elongating and elongated spermatids. A combined immunofluorescence experiment with anti-TP2 and anti-TP1 antibodies revealed several foci of overlapping localization, indicating that TP1 and TP2 may have concerted functional roles during chromatin remodeling in mammalian spermiogenesis.

Functional studies of purified transmembrane proteases, omptins, of Yersinia pestis and Salmonella enterica.

Surface proteolysis is important in migration of cells through tissue barriers. In the case of prokaryotes, surface proteolysis has been associated with invasiveness of pathogenic bacteria from the primary infection site into circulation and secondary infection sites in the host. This study addressed surface proteases of two important bacterial pathogens, Yersinia pestis which is the causative agent of the lethal systemic zoonosis, plague, and Salmonella enterica serovar Typhimurium which is an oral-faecal pathogen that annually causes millions of cases of gastoenteritis that may develop to septicaemia. Both bacterial species express an ortholog of the omptin family of transmembrane β-barrel, outer membrane proteases/adhesins. This thesis work addressed the functions of isolated plasminogen activator Pla of Y. pestis and the PgtE omptin of S. enterica. Pla and PgtE were isolated as His6-fusion proteins in denaturing conditions from recombinant Escherichia coli and activated by adding lipopolysaccharide (LPS). The structural features in LPS that enhance plasminogen activation by His6-Pla were determined, and it was found that the lack of O-specifi c chain, the presence of outer core oligosaccharide, the presence of phosphates in lipid A, as well as a low level of acylation in lipid A influence the enhancement of Pla activity by LPS. A conserved lipid A phosphate binding motif in Pla and PgtE was found important for the enhancement of enzymatic activity by LPS. The results help to explain the biological signifi cance of the genetic loss of the O-specifi c chain biosynthesis in Y. pestis as well as the variations in LPS structure upon entry of Y. pestis into the human host. Expression of Pla in Y. pestis is associated with adhesiveness to lamin of basement membranes. Here, isolated and LPS-activated His6-Pla was coated onto fluorescent microparticles. The coating conferred specifi c adhesiveness of the particles to laminin and reconstituted basement membrane, thus confi rming the intrinsic adhesive characteristics of the Pla protein. The adhesiveness is thought to direct plasmin proteolysis at tissue barriers, thus increasing tissue damage and bacterial spread. Gelatinase activity has not been previously reported in enteric bacteria. Expression of PgtE in S. enterica was associated with cleavage of porcine skin gelatin, denaturated human type I collagen, as well as DQ-gelatin. Purifi ed His6-PgtE also degraded porcine skin gelatin and human type I gelatin but did not react with DQ-gelatin, indicating that minor differences are seen in proteolysis by isolated and cell-bound PgtE. Pla was less effective in gelatin degradation. The novel gelatinase activity in S. enterica is likely to enhance bacterial dissemination during infection.

N-syndecan and HB-GAM in neural migration and differentiation : Modulation of growth factor activity in brain

The juvenile sea squirt wanders through the sea searching for a suitable rock or hunk of coral to cling to and make its home for life. For this task it has a rudimentary nervous system. When it finds its spot and takes root, it doesn't need its brain any more so it eats it. It's rather like getting tenure. Daniel C. Dennett (from Consciousness Explained, 1991) The little sea squirt needs its brain for a task that is very simple and short. When the task is completed, the sea squirt starts a new life in a vegetative state, after having a nourishing meal. The little brain is more tightly structured than our massive primate brains. The number of neurons is exact, no leeway in neural proliferation is tolerated. Each neuroblast migrates exactly to the correct position, and only a certain number of connections with the right companions is allowed. In comparison, growth of a mammalian brain is a merry mess. The reason is obvious: Squirt brain needs to perform only a few, predictable functions, before becoming waste. The more mobile and complex mammals engage their brains in tasks requiring quick adaptation and plasticity in a constantly changing environment. Although the regulation of nervous system development varies between species, many regulatory elements remain the same. For example, all multicellular animals possess a collection of proteoglycans (PG); proteins with attached, complex sugar chains called glycosaminoglycans (GAG). In development, PGs participate in the organization of the animal body, like in the construction of parts of the nervous system. The PGs capture water with their GAG chains, forming a biochemically active gel at the surface of the cell, and in the extracellular matrix (ECM). In the nervous system, this gel traps inside it different molecules: growth factors and ECM-associated proteins. They regulate the proliferation of neural stem cells (NSC), guide the migration of neurons, and coordinate the formation of neuronal connections. In this work I have followed the role of two molecules contributing to the complexity of mammalian brain development. N-syndecan is a transmembrane heparan sulfate proteoglycan (HSPG) with cell signaling functions. Heparin-binding growth-associated molecule (HB-GAM) is an ECM-associated protein with high expression in the perinatal nervous system, and high affinity to HS and heparin. N-syndecan is a receptor for several growth factors and for HB-GAM. HB-GAM induces specific signaling via N-syndecan, activating c-Src, calcium/calmodulin-dependent serine protein kinase (CASK) and cortactin. By studying the gene knockouts of HB-GAM and N-syndecan in mice, I have found that HB-GAM and N-syndecan are involved as a receptor-ligand-pair in neural migration and differentiation. HB-GAM competes with the growth factors fibriblast growth factor (FGF)-2 and heparin-binding epidermal growth factor (HB-EGF) in HS-binding, causing NSCs to stop proliferation and to differentiate, and affects HB-EGF-induced EGF receptor (EGFR) signaling in neural cells during migration. N-syndecan signaling affects the motility of young neurons, by boosting EGFR-mediated cell migration. In addition, these two receptors form a complex at the surface of the neurons, probably creating a motility-regulating structure.

Membrane Insertion of C-tail Anchored Proteins

The correct localization of proteins is essential for cell viability. In order to achieve correct protein localization to cellular membranes, conserved membrane targeting and translocation mechanisms have evolved. The focus of this work was membrane targeting and translocation of a group of proteins that circumvent the known targeting and translocation mechanisms, the C-tail anchored protein family. Members of this protein family carry out a wide range of functions, from protein translocation and recognition events preceding membrane fusion, to the regulation of programmed cell death. In this work, the mechanisms of membrane insertion and targeting of two C-tail anchored proteins were studied utilizing in vivo and in vitro methods, in yeast and mammalian cell systems. The proteins studied were cytochrome b(5), a well characterized C-tail anchored model protein, and N-Bak, a novel member of the Bcl-2 family of regulators of programmed cell death. Membrane insertion of cytochrome b(5) into the endoplasmic reticulum membrane was found to occur independently of the known protein conducting channels, through which signal peptide-containing polypeptides are translocated. In fact, the membrane insertion process was independent of any protein components and did not require energy. Instead membrane insertion was observed to be dependent on the lipid composition of the membrane. The targeting of N-Bak was found to depend on the cellular context. Either the mitochondrial or endoplasmic reticulum membranes were targeted, which resulted in morphological changes of the target membranes. These findings indicate the existence of a novel membrane insertion mechanism for C-tail anchored proteins, in which membrane integration of the transmembrane domain, and the translocation of C-terminal fragments, appears to be spontaneous. This mode of membrane insertion is regulated by the target membrane fluidity, which depends on the lipid composition of the bilayer, and the hydrophobicity of the transmembrane domain of the C-tail anchored protein, as well as by the availability of the C-tail for membrane integration. Together these mechanisms enable the cell to achieve spatial and temporal regulation of sub-cellular localization of C-tail anchored proteins.

Mechanisms of KCC2 upregulation during development

Gamma-aminobutyric acid (GABA) acting through ionotropic GABAA receptors plays a crucial role in the activity of the central nervous system (CNS). It triggers Ca2+ rise providing trophic support in developing neurons and conducts fast inhibitory function in mature neuronal networks. There is a developmental change in the GABAA reversal potential towards more negative levels during the first two postnatal weeks in rodent hippocampus. This change provides the basis for mature GABAergic activity and is attributable to the developmental expression of the neuron-specific potassium chloride cotransporter 2 (KCC2). In this work we have studied the mechanisms responsible for the control of KCC2 developmental expression. As a model system we used hippocampal dissociated cultures plated from embryonic day (E) 17 mice embryos before the onset of KCC2 expression. We showed that KCC2 was significantly up-regulated during the first two weeks of culture development. Interestingly, the level of KCC2 upregulation was not altered by chronic pharmacological blockage of action potentials as well as GABAergic and glutamatergic synaptic transmission. By in silico analysis of the proximal KCC2 promoter region we identified 10 candidate transcription factor binding sites that are highly conserved in mammalian KCC2 genes. One of these transcription factors, namely early growth response factor 4 (Egr4), had similar developmental profile as KCC2 and considerably increased the activity of mouse KCC2 gene in neuronal cells. Next we investigated the involvement of neurotrophic factors in regulation of Egr4 and KCC2 expression. We found that in immature hippocampal cultures Egr4 and KCC2 levels were strongly up-regulated by brain derived neurotrophic factor (BDNF)and neurturin. The effect of neurotrophic factors was dependent on the activation of a mitogen activated protein kinase (MAPK) signal transduction pathway. Intact Egr4-binding site in proximal KCC2 promoter was required for BDNF-induced KCC2 transcription. In vitro data were confirmed by several in vivo experiments where we detected an upregulation of KCC2 protein levels after intrahippocampal administration of BDNF or neurturin. Importantly, a MAPK-dependent rise in Egr4 and KCC2 expression levels was also observed after a period of kainic acid-induced seizure activity in neonatal rats suggesting that neuronal activity might be involved in Egr4-mediated regulation of KCC2 expression. Finally we demonstrated that the mammalian KCC2 gene (alias Slc12a5) generated two neuron-specific isoforms by using alternative promoters and first exons. A novel isoform of KCC2, termed KCC2a, differed from the previously known KCC2b isoform by 40 unique N-terminal amino acid residues. KCC2a expression was restricted to CNS,remained relatively constant during postnatal development, and contributed 20 50% of total KCC2 mRNA expression in the neonatal mouse brainstem and spinal cord. In summary, our data provide insight into the complex regulation of KCC2 expression during early postnatal development. Although basal KCC2 expression seems to be intrinsically regulated, it can be further augmented by neurotrophic factors or by enhanced activity triggering MAPK phosphorylation and Egr4 induction. Additional KCC2a isoform, regulated by another promoter, provides basal KCC2 level in neonatal brainstem and spinal cord required for survival of KCC2b knockout mice.

Computational analyses of the catalytic and heparin-binding sites and their interactions with glycosaminoglycans in glycoside hydrolase family 79 endo-d-glucuronidase (heparanase)

Mammalian heparanase is an endo-β-glucuronidase associated with cell invasion in cancer metastasis, angiogenesis and inflammation. Heparanase cleaves heparan sulfate proteoglycans in the extracellular matrix and basement membrane, releasing heparin/heparan sulfate oligosaccharides of appreciable size. This in turn causes the release of growth factors, which accelerate tumor growth and metastasis. Heparanase has two glycosaminoglycan-binding domains; however, no three-dimensional structure information is available for human heparanase that can provide insights into how the two domains interact to degrade heparin fragments. We have constructed a new homology model of heparanase that takes into account the most recent structural and bioinformatics data available. Heparin analogs and glycosaminoglycan mimetics were computationally docked into the active site with energetically stable ring conformations and their interaction energies were compared. The resulting docked structures were used to propose a model for substrates and conformer selectivity based on the dimensions of the active site. The docking of substrates and inhibitors indicates the existence of a large binding site extending at least two saccharide units beyond the cleavage site (toward the nonreducing end) and at least three saccharides toward the reducing end (toward heparin-binding site 2). The docking of substrates suggests that heparanase recognizes the N-sulfated and O-sulfated glucosamines at subsite +1 and glucuronic acid at the cleavage site, whereas in the absence of 6-O-sulfation in glucosamine, glucuronic acid is docked at subsite +2. These findings will help us to focus on the rational design of heparanase-inhibiting molecules for anticancer drug development by targeting the two heparin/heparan sulfate recognition domains.