Properties of the recombinant glucose/galactose dehydrogenase from the extreme thermoacidophile, Picrophilus torridus

In Picrophilus torridus, a euryarchaeon that grows optimally at 60 degrees C and pH 0.7 and thus represents the most acidophilic thermophile known, glucose oxidation is the first proposed step of glucose catabolism via a nonphosphorylated variant of the Entner-Doudoroff pathway, as deduced from the recently completed genome sequence of this organism. The P. torridus gene for a glucose dehydrogenase was cloned and expressed in Escherichia coli, and the recombinant enzyme, GdhA, was purified and characterized. Based on its substrate and coenzyme specificity, physicochemical characteristics, and mobility during native PAGE, GdhA apparently resembles the main glucose dehydrogenase activity present in the crude extract of P. torridus DSM 9790 cells. The glucose dehydrogenase was partially purified from P. torridus cells and identified by MS to be identical with the recombinant GdhA. P. torridus GdhA preferred NADP+ over NAD+ as the coenzyme, but was nonspecific for the configuration at C-4 of the sugar substrate, oxidizing both glucose and its epimer galactose (Km values 10.0 and 4.5 mM, respectively). Detection of a dual-specific glucose/galactose dehydrogenase points to the possibility that a 'promiscuous' Entner-Doudoroff pathway may operate in P. torridus, similar to the one recently postulated for the crenarchaeon Sulfolobus solfataricus. Based on Zn2+ supplementation and chelation experiments, the P. torridus GdhA appears to contain structurally important zinc, and conserved metal-binding residues suggest that the enzyme also contains a zinc ion near the catalytic site, similar to the glucose dehydrogenase enzymes from yeast and Thermoplasma acidophilum. Strikingly, NADPH, one of the products of the GdhA reaction, is unstable under the conditions thought to prevail in Picrophilus cells, which have been reported to maintain the lowest cytoplasmic pH known (pH 4.6). At the optimum growth temperature for P. torridus, 60 degrees C, the half-life of NADPH at pH 4.6 was merely 2.4 min, and only 1.7 min at 65 degrees C (maximum growth temperature). This finding suggests a rapid turnover of NADPH in Picrophilus.     

Structures of the iron-sulfur flavoproteins from Methanosarcina thermophila and Archaeoglobus fulgidus

Iron-sulfur flavoproteins (ISF) constitute a widespread family of redox-active proteins in anaerobic prokaryotes. Based on sequence homologies, their overall structure is expected to be similar to that of flavodoxins, but in addition to a flavin mononucleotide cofactor they also contain a cubane-type [4Fe:4S] cluster. In order to gain further insight into the function and properties of ISF, the three-dimensional structures of two ISF homologs, one from the thermophilic methanogen Methanosarcina thermophila and one from the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus, were determined. The structures indicate that ISF assembles to form a tetramer and that electron transfer between the two types of redox cofactors requires oligomerization to juxtapose the flavin mononucleotide and [4Fe:4S] cluster bound to different subunits. This is only possible between different monomers upon oligomerization. Fundamental differences in the surface properties of the two ISF homologs underscore the diversity encountered within this protein family.     

Quantitative calcium measurements in subcellular compartments of Plasmodium falciparum-infected erythrocytes

The acidic food vacuole exerts several important functions during intraerythrocytic development of the human malarial parasite Plasmodium falciparum. Hemoglobin taken up from the host erythrocyte is degraded in the food vacuole, and the heme liberated during this process is crystallized to inert hemozoin. Several anti-malarial drugs target food vacuolar pathways, such as hemoglobin degradation and heme crystallization. Resistance and sensitization to some antimalarials is associated with mutations in food vacuolar membrane proteins. Other studies suggest a role of the food vacuole in ion homeostasis, and release of Ca2+ from the food vacuole may mediate adopted physiological responses. To investigate whether the food vacuole is an intracellular Ca2+ store, which in turn may affect other physiological functions in which this organelle partakes, we have investigated total and exchangeable Ca2+ within the parasite's food vacuole using x-ray microanalysis and quantitative confocal live cell Ca2+ imaging. Apparent free Ca2+ concentrations of approximately 90, approximately 350, and approximately 400 nM were found in the host erythrocyte cytosol, the parasite cytoplasm, and the food vacuole, respectively. In our efforts to determine free intracellular Ca2+ concentrations, we evaluated several Ca2+-sensitive fluorochromes in a live cell confocal setting. We found that the ratiometric Ca2+ indicator Fura-Red provides reliable determinations, whereas measurements using the frequently used Fluo-4 are compromised due to problems arising from phototoxicity, photobleaching, and the strong pH dependence of the dye. Our data suggest that the food vacuole contains only moderate amounts of Ca2+, disfavoring a role as a major intracellular Ca2+ store.     

Trimeric membrane-anchored gp41 inhibits HIV membrane fusion

The HIV-1 envelope glycoprotein is composed of a receptor binding subunit, gp120 that is non-covalently linked to the membrane-anchored fusion protein, gp41. Triggered by cellular receptor binding, the trimeric envelope complex mediates the fusion of viral and cellular membranes through the rearrangement of the fusion protein subunit into a six-helical bundle core structure. Here we describe the biophysical and functional properties of a membrane-anchored fragment of gp41 (gp41ctm) that includes the complete C-terminal heptad repeat region 2, the connecting part, and the transmembrane region. We show that the transmembrane domain of the envelope glycoprotein is sufficient for trimerization in vitro, contributing most of the alpha-helical content of gp41ctm. Trimeric gp41ctm is protease-resistant and recognizes neutralizing antibodies 2F5 and 4E10. However, gp41ctm and gp41ctm proteoliposomes elicit no clear neutralizing immune responses in preliminary mouse studies. We further show that gp41ctm and surprisingly also gp41ctm proteoliposomes have potent anti-viral activity. Our data suggest that liposome-anchored gp41ctm exerts its inhibitory action outside of the initial fusion contact site, and its implications for the fusion reaction are discussed.     

Regulation of monocyte apoptosis by the protein kinase Cdelta-dependent phosphorylation of caspase-3

Monocytes are central components of the innate immune response and normally circulate for a short period of time before undergoing spontaneous apoptosis. During inflammation, differentiation, or oncogenic transformation, the life span of monocytes is prolonged by preventing the activation of the apoptotic program. Here we showed that caspase-3, a cysteine protease required for monocyte apoptosis, is a phosphoprotein. We identified protein kinase Cdelta (PKCdelta) as a member of the protein kinase C family that associates with and phosphorylates caspase-3. The PKCdelta-dependent phosphorylation of caspase-3 resulted in an increase in the activity of caspase-3. This effect of PKCdelta is specific to caspase-3, as evidenced by the absence of similar effects on caspase-9. The activity of PKCdelta precedes the activation of caspase-3 during spontaneous monocyte apoptosis and in monocyte-induced apoptosis. We found that the overexpression of PKCdelta resulted in an increase of apoptosis, whereas its inhibition blocked caspase-3 activity and decreased apoptosis. Our results provided evidence that the PKCdelta-dependent phosphorylation of caspase-3 provided a novel pro-apoptotic mechanism involved in the regulation of monocyte life span.     

Lipid droplets gain PAT family proteins by interaction with specialized plasma membrane domains

Proteins of the PAT family, named after perilipin, adipophilin, and TIP47 (tail-interacting protein of 47 kDa), are associated with lipid droplets and have previously been localized by immunofluorescence microscopy exclusively to the droplet surface. These proteins are considered not to be present in any other subcellular compartment. By applying the high resolution technique of freeze-fracture electron microscopy combined with immunogold labeling, we now demonstrate that in macrophages and adipocytes PAT family proteins are, first, distributed not only in the surface but also throughout the lipid droplet core and, second, are integral components of the plasma membrane. Under normal culture conditions these proteins are dispersed in the cytoplasmic leaflet of the plasma membrane. Stimulation of lipid droplet formation by incubation of the cells with acetylated low density lipoprotein leads to clustering of the PAT family proteins in raised plasma membrane domains. Fractures penetrating beneath the plasma membrane demonstrate that lipid droplets are closely apposed to these domains. A similar distribution pattern of labeling in the form of linear aggregates within the clusters is apparent in the cytoplasmic monolayer of the plasma membrane and the immediately adjacent outer monolayer of the lipid droplet. The aggregation of the PAT family proteins into such assemblies may facilitate carrier-mediated lipid influx from the extracellular environment into the lipid droplet. Lipid droplets appear to acquire their PAT proteins by interaction with plasma membrane domains enriched in these proteins.     

Depth-dependent strain of patellofemoral articular cartilage in unconfined compression

This biomechanical study reports strain gradients in patellofemoral joint cross-sections of seven porcine specimens in response to 1% unconfined axial compression subsequent to specific amounts of off-set strain. Strain distributions were quantified with a customized laser-based electronic speckle pattern interferometry (ESPI) system in a non-contact manner, delivering high-resolution, high-sensitivity strain maps over entire patellofemoral cartilage cross-sections. Strain reports were evaluated to determine differences in strain magnitudes between the superficial, middle, and deep cartilage layers in femoral and patellar cartilage. In addition, the effect of 5%, 10%, 15%, and 20% off-set strain on depth-dependent strain gradients was quantified. Regardless of the amount of off-set strain, the superficial layer of femoral cartilage absorbed the most strain, and the deep layer absorbed the least strain. These depth-dependent strain gradients were most pronounced for 5% off-set strain, at which the superficial layer absorbed on average 5.7 and 23.7 times more strain as compared to the middle and deep layers, respectively. For increased off-set strain levels, strain gradients became less pronounced. At 20% off-set strain, differences in layer-specific strain were not statistically significant, with the superficial layer showing a 1.4 fold higher strain as the deep layer. Patellar cartilage exhibited similar strain gradients and effects of off-set strain, although the patellar strain was on average 19% larger as compared to corresponding femoral strain reports. This study quantified for the first time continuous strain gradients over patellofemoral cartilage cross-sections. Next to provision of a detailed functional characterization of normal diarthrodial joints, this novel experimental approach holds considerable attraction to investigate joint degenerative processes.     

Inhibitors of actin polymerisation stimulate arachidonic acid release and 5-lipoxygenase activation by upregulation of Ca2+ mobilisation in polymorphonuclear leukocytes involving Src family kinases

Here, we show that actin polymerisation inhibitors such as latrunculin B (LB), and to a minor extent also cytochalasin D (Cyt D), enhance the release of arachidonic acid (AA) as well as nuclear translocation of 5-lipoxygenase (5-LO) and 5-LO product synthesis in human polymorphonuclear leukocytes (PMNL), challenged with thapsigargin (TG) or N-formyl-methionyl-leucyl-phenylalanine. The concentration-dependent effects of LB (EC50 approximately 200 nM) declined with prolonged preincubation (>3 min) prior TG and were barely detectable when PMNL were stimulated with Ca2+-ionophores. Investigation of the stimulatory mechanisms revealed that LB (or Cyt D) elicits Ca2+ mobilisation and potentiates stimulus-induced elevation of intracellular Ca2+, regardless of the nature of the stimulus. LB caused rapid but only moderate activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK)2. The selective Src family kinase inhibitors PP2 and SU6656 blocked LB- or Cyt D-mediated Ca2+ mobilisation and suppressed the upregulatory effects on AA release and 5-LO product synthesis, without affecting AA metabolism evoked by ionophore alone. We conclude that in PMNL, inhibitors of actin polymerisation cause enhancement of intracellular Ca2+ levels through Src family kinase signaling, thereby facilitating stimulus-induced release of AA and 5-LO product formation.