The Transmembrane Segment of a Tail-anchored Protein Determines Its Degradative Fate through Dislocation from the Endoplasmic Reticulum

Terminally misfolded proteins that accumulate in the endoplasmic reticulum (ER) are dislocated and targeted for ubiquitin-dependent destruction by the proteasome. UBC6e is a tail-anchored E2 ubiquitin-conjugating enzyme that is part of a dislocation complex nucleated by the ER-resident protein SEL1L. Little is known about the turnover of tail-anchored ER proteins. We constructed a set of UBC6e transmembrane domain replacement mutants and found that the tail anchor of UBC6e is vital for its function, its stability, and its mode of membrane integration, the last step dependent on the ASNA1/TRC40 chaperone. We constructed a tail-anchored UBC6e variant that requires for its removal from the ER membrane not only YOD1 and p97, two cytosolic proteins involved in the extraction of ER transmembrane or luminal proteins, but also UBXD8, AUP1 and members of the Derlin family. Degradation of tail-anchored proteins thus relies on components that are also used in other aspects of protein quality control in the ER.     

Macrophage phagocytosis of apoptotic neutrophils is compromised by matrix proteins modified by cigarette smoke and lipid peroxidation products

Clearance of apoptotic cells by phagocytosis plays an important role in the resolution of an inflammatory response. Macrophages interacting with extracellular matrix (ECM) proteins upregulate their phagocytic capacity. Cigarette smoke contains highly reactive carbonyls that modify proteins which directly/indirectly affects cellular function. We observed, in vitro, that human macrophages interacting with carbonyl or cigarette smoke modified ECM proteins dramatically down regulated their ability to phagocytose apoptotic neutrophils. We also show that this interaction with carbonyl-adduct modified ECM proteins led to increased macrophage adhesion in vitro. We hypothesise that changes in the ECM environment as a result of cigarette smoking affect the ability of macrophages to remove apoptotic cells. Moreover, we postulate that this decreased phagocytic activity was as a result of sequestration of receptors involved in the uptake of apoptotic cells towards that of recognition of carbonyl adducts on the modified ECM proteins leading to increased macrophage adhesion.     

Structural descriptions of ligands in their binding site of integral membrane proteins at near physiological conditions using solid-state NMR

Using solid-state NMR approaches, it is now possible to define the structure and dynamics of binding for a small, isotopically (²H, ¹³C, ¹⁹F, ¹⁵N) labelled ligand, prosthetic group or solute in its binding site of a membrane-bound protein at near physiological conditions in natural membrane fragments or in reconstituted complexes. Studies of oriented membranes permit the orientational bond vectors of labelled groups to be determined to good precision, as shown for retinal in bacteriorhodopsin and bovine rhodopsin. Using novel magic angle spinning NMR methods on membrane dispersions, high-resolution NMR spectra can be obtained. Dipolar couplings can be reintroduced into the spectrum of labelled ligands in their binding sites of membrane-bound proteins to give interatomic distances to high precision (±0.5 Å). Relaxation and cross-polarization data give estimates for the kinetics for on-off rates for binding. In addition, chemical shifts can be measured directly to help provide details of the binding environment for a bound ligand, as shown for analogues of drugs used in peptic ulcer treatment in the gastric ATPase, and for acetylcholine in the acetylcholine receptor.     

Growth and morphogenesis of embryonic mouse organs on non-coated and extracellular matrix-coated Biopore membrane

Embryonic mouse salivary glands, pancreata, and kidneys were isolated from embryos of appropriate gestational age by microdissection, and were cultured on Biopore membrane either non-coated or coated with type I collagen or Matrigel. As expected, use of Biopore membrane allowed high quality photomicroscopy of the living organs. In all organs extensive mesenchymal spreading was observed in the presence of type I collagen or Matrigel. However, differences were noted in the effects of extracellular matrix (ECM) coatings on epithelial growth and morphogenesis: salivary glands were minimally affected, pancreas morphogenesis was adversely affected, and kidney growth and branching apparently was enhanced. It is suggested that these differences in behaviour reflect differences in the strength of interactions between the mesenchymal cells and their surrounding endogenous matrix, compared to the exogenous ECM macromolecules. This method will be useful for culture of these and other embryonic organs. In particular, culture of kidney rudiments on ECM-coated Biopore offers a great improvement over previously used methods which do not allow morphogenesis to be followed in vitro.     

Embryonic lung morphogenesis in organ culture: experimental evidence for a proteoglycan function in the extracellular matrix

The lung rudiment, isolated from mid-gestation (11 day) mouse embryos, can undergo morphogenesis in organ culture. Observation of living rudiments, in culture, reveals both growth and ongoing bronchiolar branching activity. To detect proteoglycan (PG) biosynthesis, and deposition in the extracellular matrix, rudiments were metabolically labeled with radioactive sulfate, then fixed, embedded, sectioned and processed for autoradiography. The sulfated glycosaminoglycan (GAG) types, composing the carbohydrate component of the proteoglycans, were evaluated by selective GAG degradative approaches that showed chondroitin sulfate PG principally associated with the interstitial matrix, and heparan sulfate PG principally associated with the basement membrane. Experiments using the proteoglycan biosynthesis disrupter, beta-xyloside, suggest that when chondroitin sulfate PG deposition into the ECM is perturbed, branching morphogenesis is compromised.     

Effects of aldosterone on Na transport in the toad bladder I. Glycolysis and lactate production under aerobic conditions

Previous studies indicated that aldosterone enhances active Na⁺ transport, glycolysis, lactate production and respiration of the toad bladder. Evidence was also presented that the changes in glycolysis and lactate production were secondary to the changes in active Na⁺ transport. Further analysis of the relationships between metabolism and Na⁺ transport was undertaken with the aid of two inhibitors of pyruvate metabolism, oxythiamine and phenylpyruvate. These inhibitors prevented the aldosterone-induced increase in oxidation of [6-¹⁴C]glucose but had little effect on the increase in lactate production. In contrast, the effect on Na⁺ transport (i.e., I_sc) was completely inhibited by oxythiamine plus phenylpyruvate with glucose as substrate. The effect on Na⁺ transport, however, was obtained wth the by-pass substrates, oxaloacetate plus ß-hydroxybutyrate, in the presence of these inhibitors. These results implied that steroidal enhancement of lactate production and Na⁺ transport were independent effects. To evaluate whether an increase in Na⁺ transport, per se would augment lactate production, the responses were evaluated under conditions of an imposed Na⁺ gradient (mucosal Na⁺ = 5 mM; serosal Na⁺ = 110 mM). Addition of NaCl to the mucosal media evoked the same increase in I_sc as the addition of aldosterone; both additions increased I_sc more than two-fold. Aldosterone reduced lactate production under these conditions while the re-addition of NaCl had no effect on lactate formation. These results are consistent with an action of aldosterone on pathways involved in oxidative energy metabolism, and suggest that the activation of glycolysis may be a function of the net balance between energy production and utilization.     

Endochondral bone formation in embryonic mouse pre-metatarsals

Long term exposure to a reduced gravitational environment has a deleterious effect on bone. The developmental events which occur prior to initial bone deposition will provide insight into the regulation of mature bone physiology. We have characterized a system in which the events preceding bone formation take place in an isolated in vitro organ culture environment. We show that cultured pre-metatarsal tissue parallels development of pre-metatarsal tissue in the embryo. Both undergo mesenchyme differentiation and morphogenesis to form a cartilage rod, which resembles the future bone, followed by terminal chondrocyte differentiation in a definite morphogenetic pattern. These sequential steps occur prior to osteoblast maturation and bone matrix deposition in the developing organism. Alkaline phosphatase (ALP) activity is a distinctive enzymatic marker for mineralizing tissues. We have measured this activity throughout pre-metatarsal development and show (a) where in the tissue it is predominantly found, and (b) that this is indeed the mineralizing isoform of the enzyme.     

Gravitational studies in cellular and developmental biology

The paucity of data on the role of gravity in cellular and developmental biology has been examined, and a hypothesis has been generated that unifies potential gravity sensitivity in both plant and animal systems. This hypothesis considers the macromolecular order and functional importance of the extracellular matrix compartment, the intracellular cytoskeleton compartment, and the connecting plasma membrane-signal transduction compartment of plant and animal systems as potentially sensitive to alterations in the unit gravity environment in which they evolved.     

Immunofluorescence comparisons of anti-actin specificity

The abilities of antibody populations against brain actin and two immunogenic forms of cardiac actin to react with sarcomeric muscle actin and cytoplasmic non-muscle actin were tested by indirect immunofluorescence, by using isolated skeletal muscle myofibrils and cultured non-neuronal dorsal root ganglion cells as the test systems. All three antibody preparations stained the I-bands of myofibrils, a result that demonstrated the presence of antigenic determinants shared among skeletal, cardiac, and brain actins. However, although antibodies against cytoplasmic brain actin stained the stress fibers of cultured cells, those against glutaraldehyde cross-linked cardiac actin did not, a result that implies that cardiac actin possesses determinants common to sarcomeric actins but not present on cytoplasmic actin. Finally, antibodies against SDS-treated cardiac actin readily stained the stress fibers of cultured cells, in contrast to those against glutaraldehyde cross-linked cardiac actin, a result that suggests that the state of the original immunogen can affect the actin type specificity of the resulting antibody population.     

Synthesis and biological properties of novel glucocorticoid androstene C-17 furoate esters

A series of novel corticosteroid derivatives featuring C-17 furoate ester functionality have been synthesised. Profiling in vitro and in vivo has resulted in the identification of a compound with a longer duration of action and a lower oral side effect profile in rodents compared to budesonide.