Expression of microbial virulence proteins in Saccharomyces cerevisiae models mammalian infection

Bacterial virulence proteins that are translocated into eukaryotic cells were expressed in Saccharomyces cerevisiae to model human infection. The subcellular localization patterns of these proteins in yeast paralleled those previously observed during mammalian infection, including localization to the nucleus and plasma membrane. Localization of Salmonella SspA in yeast provided the first evidence that SspA interacts with actin in living cells. In many cases, expression of the bacterial virulence proteins conferred genetically exploitable growth phenotypes. In this way, Yersinia YopE toxicity was demonstrated to be linked to its Rho GTPase activating protein activity. YopE blocked polarization of the yeast cytoskeleton and cell cycle progression, while SspA altered polarity and inhibited depolymerization of the actin cytoskeleton. These activities are consistent with previously proposed or demonstrated effects on higher eukaryotes and provide new insights into the roles of these proteins in pathogenesis: SspA in directing formation of membrane ruffles and YopE in arresting cell division. Thus, study of bacterial virulence proteins in yeast is a powerful system to determine functions of these proteins, probe eukaryotic cellular processes and model mammalian infection.     

Expression of NLRP3 inflammasome proteins in ExpiCHO-S mammalian cells reveals oligomerization properties that are highly sensitive to solution conditions

The NLRP3 inflammasome is a key intracellular component of the innate immune response. It is a three-protein complex essential for the production of mature interleukin 1-β. The complex, which is comprised of three proteins, NLRP3, ASC, and pro-caspase-1, has been implicated in the physiological response to pathogenic elements of cardiovascular disease and Alzheimer's disease. Investigations into the properties of the three proteins can be aided by larger-scale recombinant expression to produce adequate amounts. In the current study, a variety of NLRP3 inflammasome proteins were expressed in the ExpiCHO-S mammalian cell system with a particular focus on ASC. ASC fusion proteins with glutathione-S transferase, maltose-binding protein, and SUMO increased solubility and aided in determining the stability and oligomerization propensity of individual ASC domains and full-length ASC. ASC oligomerization was highly sensitive to protein concentration, ionic strength, and mutation. These observations provided strategic ways to enhance protein purification and characterize ASC oligomerization. The ExpiCHO-S expression system consistently produced high-yield recombinant NLRP3 inflammasome proteins which led to a further understanding of ASC oligomerization.     

Continuous flow electrophoretic separation of proteins and cells from mammalian tissues

A new continuous flow electrophoretic separator for cells and macromolecules was built and tested in laboratory experiments and in the microgravity environment of space flight. Buffer flows upward in a 120-cm long flow chamber, which is 6 cm wide X 1.5 mm thick in the laboratory version and 16 cm wide X 3.0 mm thick in the microgravity version. Electrophoretic subpopulations are collected in 197 fractions spanning 16 cm at the upper end of the chamber. The electrode buffer is recirculated through front and back cooling chambers, which are also electrode chambers. Ovalbumin and rat serum albumin were used as test proteins in resolution and throughout tests; resolution of these two proteins at 25% total w/v concentration in microgravity was the same as that found at 0.2% w/v concentration in the laboratory. Band spreading caused by Poiseuille flow and conductance gaps was evaluated using polystyrene microspheres in microgravity, and these phenomena were quantitatively the same in microgravity as in the laboratory. Rat anterior pituitary cells were separated into subpopulations enriched with cells that secrete specific hormones; growth-hormone-secreting cells were found to have high electrophoretic mobility, whereas prolactin-secreting cells were found to have low electrophoretic mobility. Cultured human embryonic kidney cells were separated into several electrophoretic subfractions that produced different plasminogen activators; a medium-high-mobility subpopulation and a medium-low-mobility subpopulation each produced a different molecular form of urokinase, whereas a high- and an intermediate-mobility subpopulation produced tissue plasminogen activator. Canine pancreatic islets of Langerhans cells were separated into subpopulations, which, after reaggregation into pseudoislets, were found to be enriched with cells that secrete specific hormones; insulin-secreting beta cells were found in lowest mobility fractions, whereas glucagon-secreting alpha cells were found in the highest mobility fractions. Results of particle electrophoresis experiments were comparable in microgravity and in the laboratory, since cell densities that overloaded the carrier buffer (resulting in zone sedimentation) were avoided, and a 500-fold increase in protein throughput was achieved without compromising resolution in microgravity.     

Binding proteins for cyclic AMP in mammalian tissues

• 1.1. The distribution and physicochemical properties of proteins known to bind cyclic AMP in vitro and methodological aspects of their interaction with ligands is reviewed.
• 2.2. The interaction between such proteins and cyclic AMP is discussed, the allosteric binding of the nucleotide to cyclic AMP dependent protein kinase type I being considered in detail.
• 3.3. The use of naturally occurring binding proteins in assays for cyclic AMP is briefly reviewed.
• 4.4. Finally, some aspects of the control of cyclic AMP binding in the intact cell are considered.

     

Newly found selenium-containing proteins in the tissues of the rat

The Se-containing proteins in 27 tissues of the rat were investigated by in vivo labeling with⁷⁵Se-selenite, separation of the tissue homogenate proteins by SDS-polyacrylamide gel electrophoresis, and determination of the labeled proteins by autoradiography. By using Se-depleted rats and a⁷⁵Se-tracer with a high specific activity, Se compounds present at only very low concentrations could be detected. Besides the 13 Se-containing proteins previously described, for which apparent molecular masses of 12, 15, 18, 20, 22, 25, 28, 34, 56, 60, 65, 70, and 75 kD have been found here, a further 15⁷⁵Se-labeled bands, with apparent molecular masses of 8, 10, 15.5, 16.5, 24, 32, 34.5, 38, 40, 41, 44, 45, 46.5, 53 and 116 kD could be distinguished. Two-dimensional separation of the kidney homogenate proteins showed that some of the Se-containing bands could be resolved into several labeled spots. Most of the newly found compounds were present in various tissues, but with some the enrichment in certain tissues suggested specific sites of action.     

Fluorescent Citrine-IgG fusion proteins produced in mammalian cells

Genetically encoded fluorescent antibodies are desirable for many applications in biotechnology, proteomics, microscopy, cell biology and molecular diagnostics, although efficient production of fluorescent IgGs in mammalian cells has been hampered by different and mutually incompatible secretion- and folding-requirements of antibodies and green fluorescent protein-derived fluorescent entities. Here, we show that this hurdle can be overcome by generating whole antibody fusions with Citrine, a modified yellow fluorescent protein that folds properly in the endoplasmic reticulum of mammalian cells. Applying optimized connector sequences, one or more Citrine molecules can be fused to different positions of IgGs without interfering with folding, secretion or function of the fusion proteins. These proteins can be transiently expressed and purified to similar yields as unmodified antibodies using standard technologies. IgG-Citrine fusions fully retain binding specificity and affinity, and can be applied to assays that require labeled IgG. A particularly interesting feature is the pH-dependency of Citrine fluorescence. This makes IgG-Citrine fusion proteins a valuable tool to track antibody target binding, internalization and subsequent intracellular trafficking to acidic compartments.     

Stimulation by phspholipid of calcium-dependent phosphorylation of endogenous proteins from mammalian tissues

The occurrence of endogenous substrate proteins for calcium-dependent protein kinase, augmented by either phospholipid or calmodulin, was examined in extracts of several tissues. Pancreas, vas deferens, adrenal and liver were found to contain substrate proteins for phospholipid-sensitive protein kinase. Phosphorylation of pancreatic substrate protein for phospholipid-senstivie protein kinase was rapid and highly sensitive to Ca²⁺, being detectable within 15 s a following exposure to Ca²⁺ and phosphatidylserine and at concentrations of Ca²⁺ as low as 0.5 μM. These findings suggest that the phospholipid-sensitive protien kinase system may serve to mediate some effects of Ca²⁺ in a variety of mammalian cell types.     

Expression of fusion proteins of the nicotinic acetylcholine receptor from mammalian muscle identifies the membrane-spanning regions in the alpha and delta subunits

We have investigated the topology of the alpha and delta subunits of the nicotinic acetylcholine receptor (AChR) from mammalian muscle synthesized in an in vitro translation system supplemented with dog pancreatic microsomes. Fusion proteins were expressed in which a carboxy-terminal fragment of bovine prolactin was attached downstream of each of the major putative transmembrane domains, M1-M4 and MA, in the AChR subunits. The orientation of the prolactin domain relative to the microsomal membrane was then determined for each protein by a proteolysis protection assay. Since the prolactin domain contains no information which either directs or prevents its translocation, its transmembrane orientation depends solely on sequences within the AChR subunit portion of the fusion protein. When subunit-prolactin fusion proteins with the prolactin domain fused after either M2 or M4 were tested, prolactin-immunoreactive peptides that were larger than the prolactin domain itself were recovered. No prolactin-immunoreactive peptides were recovered after proteolysis of fusion proteins containing prolactin fused after M1, M3, or MA. These results support a model of AChR subunit topology in which M1-M4, but not MA, are transmembrane domains and the carboxy terminus is extracellular.     

Isoparorchis hypselobagri: ultrastructure of the tegument and associated tissues in a digenean inhabiting a gaseous aerobic environment

Adult I. hypselobagri live in the swim bladder of the Indian catfish Wallago attu, a gaseous environment with a relatively high oxygen content. The ventral tegument, which in life is applied close to the swim bladder wall, is relatively unspecialized, showing typical ultrastructural features of the digenean surface. The dorsal tegument, which is exposed to the oxygen-rich surroundings, has numerous pyriform extensions of superficial parenchymal cells closely applied to the base of the surface syncytium. These extensions bear numerous mitochondria and send finger-like processes deep into the basal cytoplasm of the syncytium where they interdigitate with corresponding infolds of the basal tegument membrane. The pyriform parenchymal extensions are connected with underlying nucleated cell bodies via irregular glycogen-filled tubular processes, many of which end blindly in the interstitial tissue or expand into glycogen-filled bulbs beneath the cell bodies. These superficial parenchymal processes associate at gap junctions with ramifications of a distinct deeper parenchymal tissue which contains lipid, residual bodies and glycogen. The dorsal tegument and associated structures may constitute a respiratory organ, taking advantage of molecular oxygen diffusing across the surface syncytium to carry out aerobic energy transduction in the superficial parenchymal extensions. ATP so generated may diffuse inwards for distribution throughout the body in the deep parenchymal tissue. The extensive network of ramifying cytoplasmic tubules is supported by a fibrous matrix of interstitial tissue.     

Pulmonary and bronchial blood circulation under conditions of changed gaseous environment

By means of ultrasonic method used in acute experiments on cats with closed chest under normal respiration the authors studied the blood flow in left low-lobar pulmonary artery and vein and in bronchial artery, as well as the blood pressure in pulmonary and femoral arteries in inhalation of next gaseous mixtures: 7.5% O2 in nitrogen; 30% O2; 3% CO2; 21% O2+ +79% He; 30% O2 + 67% He + 3% CO2. It was shown, that inhalation of the normoxic gaseous mixture, in which nitrogen is replaced by helium, did not call significant changes in pulmonary and systemic circulation. However, the presence of the helium in complicated gaseous mixture can change the reactivity of pulmonary and bronchial vessels to influence the components participating in these complicated gaseous mixtures.     

Microtubule-associated proteins characteristic of embryonic brain are found in the adult mammalian retina

We have used monoclonal antibodies against each of the major mammalian brain microtubule-associated proteins (MAPs), MAP1, MAP2, MAP3, MAP5, and tau, to study the timing of appearance and the cytological distribution of these proteins during the development of the rat retina. Western blots of adult rat retina reveal MAPs that are characteristic of embryonic brain, i.e., MAP5 and the low-molecular-weight forms of MAP2 (MAP2c) and tau (juvenile tau). At the onset of neuronal differentiation within the embryonic retina, MAP5, MAP3, MAP2c, and tau are found in the perikarya or extending axons of ganglion cells. High-molecular-weight MAP2, a dendrite marker, does not appear in the retina until the second day of postnatal development, when ganglion cell dendrites ramify within the inner plexiform layer. MAP1, which is characteristic of adult brain, does not appear in the retina until 1 week after birth, and is limited to ganglion cells and their processes. In the adult retina, MAP5 and MAP2c are concentrated within the inner segments and cell bodies of photosensitive cells, whereas tau is found in horizontal cells and more internal cell layers. Since photosensitive cells are unique among retinal neurons in their constant regeneration of their primary processes, the photoreceptive outer segments, both MAP5 and MAP2c appear not only to be involved in events associated with the embryonic differentiation and growth of neurites, but also in process regeneration in adult neurons that maintain some embryonic characteristics.     

Selective release from cultured mammalian cells of heat-shock (stress) proteins that resemble glia-axon transfer proteins

Cultured rat embryo cells were stimulated to rapidly release a small group of proteins that included several heat-shock proteins (hsp110, hsp71, hscp73) and nonmuscle actin. The extracellular proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis. Heat-shocked cells released the same set of proteins as control cells with the addition of the stress-inducible hsp110 and hsp71. Release of these proteins was not blocked by either monensin or colchicine, inhibitors of the common secretory pathway. A small amount of the glucose-regulated protein grp78 was externalized by this pathway. The extracellular accumulation of these proteins was inhibited after they were synthesized in the presence of the lysine analogue aminoethyl cysteine. It is likely that the analogue-substituted proteins were misfolded and could not be released from cells, supporting our conclusion that a selective release mechanism is involved. Remarkably, actin and the squid heat-shock proteins homologous to rat hsp71 and hsp110 are also among a select group of proteins transferred from glial cells to the squid giant axon, where they have been implicated in neuronal stress responses (Tytell et al.: Brain Res., 363:161-164, 1986). Based in part on the similarities between these two sets of proteins, we hypothesized that these proteins were released from labile cortical regions of animal cells in response to perturbations of homeostasis in cells as evolutionarily distinct as cultured rat embryo cells and squid glial cells.     

Troponin C-like proteins (calmodulins) from mammalian smooth muscle and other tissues.

1. An acidic protein with properties similar to those of troponin C from rabbit skeletal muscle has been shown to be present in bovine and rabbit smooth muscles, chicken gizzard and rabbit liver, kidney and lung. 2. A simple new method involving the use of organic solvents is described for the purification of the troponin C-like proteins from various tissues. 3. The troponin C-like proteins can be distinguished from rabbit skeletal-muscle toponin C by their electrophoretic behaviour on polyacrylamide gels at pH 8.3 in the presence and absence of Ca2+. The troponin C-like proteins have been shown to form complexes with rabbit skeletal-muscle troponin I that migrate on electrophoresis in polyacrylamide gels. 4. Behaviour on electrophoresis, amino acid analysis and the patterns of CNBr digests on polyacrylamide gels indicate that the troponin C-like proteins from bovine uterus and aorta, rabbit uterus, and liver and chicken gizzard are very similar to, if not identical with, bovine brain modulator protein. 5. With bovine cardiac muscle the organic-solvent method yields a preparation consisting of roughly similar amounts of troponin C and troponin C-like protein. 6. By the isotope-dilution technique, troponin C-like protein has been shown to represent 0.42% of the total protein in rabbit uterus. 7. In homogenates of smooth muscle, rabbit lung, kidney and brain, the troponin C-like proteins form a complex with other protein (or proteins) that requires Ca2+ for its formation and that is not dissociated in 9M-urea.     

Distribution of the ATPase inhibitor proteins of mitochondria in mammalian tissues including fibroblasts from a patient with Luft's disease.

The mitochondrial ATPase inhibitor proteins--the Pullman-Monroy inhibitor (PMI) and the Ca(2+)-binding protein (CaBI)--have a wide distribution, both being present in mitochondria of bovine heart and kidney, rat liver and brain, two mitochondrial populations of rabbit skeletal muscle, and mitochondria from human fibroblasts and the human breast cancer cell line T-47-D. The ratio of CaBI to PMI was highest in heart and skeletal muscle mitochondria. The subsarcolemmal fraction of skeletal muscle had 2.6-times as much CaBI as did the intermyofibrillar. The ratio of CaBI to PMI in the mitochondria of the other normal tissues and fibroblasts was close to 1. In contrast, mitochondria from T-47D cells had 1.5-times as much PMI as CaBI whilst mitochondria from fibroblasts from a patient with Luft's disease showed a virtual lack of PMI. The specific ATPase, ATP-synthetase and succinate dehydrogenase activities of the Luft's mitochondria were, however, in the normal range. The specific ATP synthetase activity of the T-47D cells was significantly higher than normal. We conclude that tissues like heart and skeletal muscle which experience wide fluctuations in intracellular Ca2+ have a greater need for CaBI. Why lack of PMI could lead to 'loose' coupling of oxidative phosphorylation in skeletal muscle of Luft's patients, but not in fibroblasts is discussed.     

Membrane fusion activity of purified SipB, a Salmonella surface protein essential for mammalian cell invasion

An early event in Salmonella infection is the invasion of non-phagocytic intestinal epithelial cells. The pathogen is taken up by macropinocytosis, induced by contact-dependent delivery of bacterial proteins that subvert signalling pathways and promote cytoskeletal rearrangement. SipB, a Salmonella protein required for delivery and invasion, was shown to localize to the cell surface of bacteria invading mammalian target cells and to fractionate with outer membrane proteins. To investigate the properties of SipB, we purified the native full-length protein following expression in recombinant Escherichia coli. Purified SipB assembled into hexamers via an N-terminal protease-resistant domain predicted to form a trimeric coiled coil, reminiscent of viral envelope proteins that direct homotypic membrane fusion. The SipB protein integrated into both mammalian cell membranes and phospholipid vesicles without disturbing bilayer integrity, and it induced liposomal fusion that was optimal at neutral pH and influenced by membrane lipid composition. SipB directed heterotypic fusion, allowing delivery of contents from E. coli-derived liposomes into the cytosol of living mammalian cells.