Role of the 14-3-3 C-terminal loop in ligand interaction

14-3-3 proteins are a family of conserved dimeric molecules that interact with a broad range of target proteins, most of which contain phosphoserine/threonine. The amphipathic groove of 14-3-3 is the main structural feature involved in mediating its associations. We have studied another domain of 14-3-3, the C-terminal loop, to determine what role it plays in ligand interaction. A truncated form of 14-3-3zeta lacking this C-terminal loop was generated and found to bind with higher affinity than the wild-type 14-3-3zeta protein to the ligands Raf-1 and Bad. Interestingly, the truncated 14-3-3zeta also showed increased association with the 14-3-3 binding-deficient Bad/S136A mutant. Taken together, these data support a role for the C-terminal loop as a general inhibitor of 14-3-3/ligand interactions. This may provide a mechanism by which inappropriate associations with 14-3-3 are prevented.     

BDNF VAL66MET polymorphism and memory decline across the spectrum of Alzheimer's disease

The brain-derived neurotrophic factor (BDNF) Val66Met (rs6265) polymorphism has been shown to moderate the extent to which memory decline manifests in preclinical Alzheimer's disease (AD). To date, no study has examined the relationship between BDNF and memory in individuals across biologically confirmed AD clinical stages (i.e., Aβ+). We aimed to understand the effect of BDNF on episodic memory decline and clinical disease progression over 126 months in individuals with preclinical, prodromal and clinical AD. Participants enrolled in the Australian Imaging, Biomarkers and Lifestyle (AIBL) study who were Aβ + (according to positron emission tomography), and cognitively normal (CN; n = 238), classified as having mild cognitive impairment (MCI; n = 80), or AD (n = 66) were included in this study. Cognition was evaluated at 18 month intervals using an established episodic memory composite score over 126 months. We observed that in Aβ + CNs, Met66 was associated with greater memory decline with increasing age and were 1.5 times more likely to progress to MCI/AD over 126 months. In Aβ + MCIs, there was no effect of Met66 on memory decline or on disease progression to AD over 126 months. In Aβ + AD, Val66 homozygotes showed greater memory decline, while Met66 carriers performed at a constant and very impaired level. Our current results illustrate the importance of time and disease severity to clinicopathological models of the role of BDNF Val66Met in memory decline and AD clinical progression. Specifically, the effect of BDNF on memory decline is greatest in preclinical AD and reduces as AD clinical disease severity increases.     

The Restoration of Phytophagous Beetles in Species‐Rich Chalk Grasslands

This study focuses on the restoration of chalk grasslands over a 6‐year period and tests the efficacy of two management practices, hay spreading and soil disturbance, in promoting this process for phytophagous beetles. Restoration success for the beetles, measured as similarity to target species–rich chalk grassland, was not found to be influenced by either management practice. In contrast, restoration success for the plants did increase in response to hay spreading management. Although the presence of suitable host plants was considered to dictate the earliest point at which phytophagous beetles could successfully colonized, few beetle species colonized as soon as their host plants became established. Morphological characteristics and feeding habits of 27 phytophagous beetle species were therefore tested to identify factors that limited their colonization and persistence. The lag time between host plant establishment and colonization was greatest for flightless beetles. Beetles with foliage‐feeding larvae both colonized at slower rates than seed‐, stem‐, or root‐feeding species and persisted within the swards for shorter periods. Although the use of hay spreading may benefit plant communities during chalk grassland restoration, it did not directly benefit phytophagous beetles. Without techniques for overcoming colonization limitation for invertebrate taxa, short‐term success of restoration may be limited to the plants only.     

The beta-amyloid domain is essential for axonal sorting of amyloid precursor protein.

We have analysed the axonal sorting signals of amyloid precursor protein (APP). Wild-type and mutant versions of human APP were expressed in hippocampal neurons using the Semliki forest virus system. We show that wild-type APP and mutations implicated in Alzheimer's disease and another brain beta-amyloidosis are sorted to the axon. By analysis of deletion mutants we found that the membrane-inserted APP ectodomain but not the cytoplasmic tail is required for axonal sorting. Systematic deletions of the APP ectodomain identified two regions required for axonal delivery: one encoded by exons 11-15 in the carbohydrate domain, the other encoded by exons 16-17 in the juxtamembraneous beta-amyloid domain. Treatment of the cells with the N-glycosylation inhibitor tunicamycin induced missorting of wild-type APP, supporting the importance of glycosylation in axonal sorting of APP. The data revealed a hierarchy of sorting signals on APP: the beta-amyloid-dependent membrane proximal signal was the major contributor to axonal sorting, while N-glycosylation had a weaker effect. Furthermore, recessive somatodendritic signals, most likely in the cytoplasmic tail, directed the protein to the dendrites when the ectodomain was deleted. Analysis of detergent solubility of APP and another axonally delivered protein, hemagglutinin, demonstrated that only hemagglutinin formed CHAPS-insoluble complexes, suggesting distinct mechanisms of axonal sorting for these two proteins. This study is the first delineation of sorting requirements of an axonally targeted protein in polarized neurons and indicates that the beta-amyloid domain plays a major role in axonal delivery of APP.     

The cytokineplast: purified, stable, and functional motile machinery from human blood polymorphonuclear leukocytes

We examined the formation of motile, chemotactically active, anucleate fragments from human blood polymorphonuclear leukocytes (PMN, granulocytes), induced by the brief application of heat. These granule-poor fragments are former protopods (leading fronts, lamellipodia) that become uncoupled from the main body of the cell and leave it, at first with a connecting filament that breaks and seals itself. The usual random orientation of such filaments can be controlled by preorientation of cells in a gradient of the chemotactic peptide, N-formylmethionylleucylphenylalanine (F-Met-Leu-Phe) (2x10(-9) M- 1x10(-8)). Cytochalsin B, 2.5-5 μg/ml, prevents fragment formation; colchicine, 10(-5) M, does not. In scanning electron micrographs, fragments are ruffled and the cell body rounded up and rather smooth. In transmission electron micrographs, fragments contain microfilaments but lack centrioles and microtubules. Like intact cells, both bound and free fragments can respond chemotactically to an erythrocyte destroyed by laser microirradiation (necrotaxis); the free, anucleate fragments may do so repeatedly, even after having been held overnight at ambient temperatures. We propse the name cytokineplast for the result of this self-purification of motile apparatus. The exodus of the motile machinery from the granulocyte requires anchoring of the bulk of the cell to glass and uncoupling, which may involve heat-induced dysfunction of the centrosome. In ultrastructural studies of the centrosomal region after heat, centriolar structure remains intact, but pericentriolar osmiophilic material appears condensed, and microtubules are sparse. These changes are found in all three blood cell types examined: PMN, eosinophil, and monocyte. Of these, the first two make fragments under our conditions; the more sluggish monocyte does not. Uncoupling is further linked to centrosomal dysfunction by the observation that colchicines-treated granulocytes (10(-5)M, to destroy the centrosome’s efferent arm) make fragments after less heat than controls. If motive force and orientation are specified mainly from the organelle-excluding leading front, then endoplasmic streaming in PMN is a catch-up phenomenon, and microtubules do not provide the vector of locomotion but rather stabilize and orient the “baggage” (nucleus, granuloplasm)—i.e., they prevent fishtailing. Moreover, constraints emanating from the centrosome may now be extended to include, maintenance of the motile machinery as an integral part of the cell.     

Cooperative interaction of reduced pyridine nucleotides with estrogen synthetase of human placental microsomes

The estrogen synthetase present in human placental microsomes appears to be dependent on the cooperative interaction of the reduced cofactors NADPH and NADH for optimal activity. Using steady-state concentrations of either cofactor, it was found that while the estrogen synthetase activity followed hyperbolic saturation kinetics with NADPH (K_mapp = 14 μM), the enzyme followed sigmoidal saturation kinetics when the cofactor was NADH, with the half-maximum velocity attained at a cofactor concentration of 1.1 mm. The maximum velocity obtained with NADPH as the cofactor was greater than with corresponding concentrations of NADH. Estrogen synthetase activity in the presence of NADH was not due to NADPH contamination. NADH, in the presence of small concentrations of NADPH (0.5 to 5 μm), stimulated significantly the rate of estrogen formation from androstenedione by placental microsomes and, in addition, the enzyme saturation kinetics changed from sigmoidal to hyperbolic, thus mimicking the effect of NADPH. Estrogen synthetase activity, measured in the presence of 1 mm NADH, was stimulated in a dose-dependent manner by NADPH (K_mapp = 0.4 μM NADPH) and, when the enzyme was measured in the presence of 5 μm NADPH, the activity was stimulated in a dose-dependent manner by NADH (K_mapp = 45 μM NADH). Estrogen synthetase activity measured in the presence of NADH, without and with NADPH (1 μm) remained linear both with time of incubation for approximately 15 min and with microsomal protein concentration up to 3 mg/ml. The apparent K_m of estrogen synthetase for androstenedione, when measured in the presence of NADH, was 1 μm. The synergistic interaction between NADH and NADPH in stimulating placental estrogen synthetase activity observed in vitro may, conceivably, take place in vivo in the intact placenta.     

On the differential release of glycolytic enzymes from cellular structure

In an endeavour to extend the available information on the biological significance of the interactions between glycolytic enzymes and cellular ultrastructure, the role of release of enzymes from digitonized fibroblasts has been studied. Lactate dehydrogenase and phosphofructokinase were rapidly and quantitatively eluted under the experimental conditions, while glyceraldehyde-3-phosphate dehydrogenase and aldolase were retained to an appreciably greater extent by the cells. This differential release of glycolytic enzymes has been related to the known binding propensities between those enzymes and subcellular structures, and are interpreted as providing additional confirmatory evidence of the importance of aldolase and glyceraldehyde-3-phosphate dehydrogenase, in particular, to these associations. The data also shed light on the order of binding of these glycolytic components - phosphofructokinase being indicated as binding subsequently (and probably separately) to aldolase and glyceraldehyde-3-phosphate dehydrogenase. These results have been discussed in relation to the available data on the associations between glycolytic enzymes and cellular structure, the possible physiological significance of this phenomenon, and the access to these problems provided by the present technique.     

Metal ions,pH, and cholesterol regulate the interactions of Alzheimer's disease amyloid-beta peptide with membrane lipid

The interaction of A beta peptides with the lipid matrix of neuronal cell membranes plays an important role in the pathogenesis of Alzheimer's disease. By using EPR and CD spectroscopy, we found that in the presence of Cu(2+) or Zn(2+), pH, cholesterol, and the length of the peptide chain influenced the interaction of these peptides with lipid bilayers. In the presence of Zn(2+), A beta 40 and A beta 42 both inserted into the bilayer over the pH range 5.5-7.5, as did A beta 42 in the presence of Cu(2+). However, A beta 40 only penetrated the lipid bilayer in the presence of Cu(2+) at pH 5.5-6.5; at higher pH there was a change in the Cu(2+) coordination sphere that inhibited membrane insertion. In the absence of the metals, insertion of both peptides only occurred at pH < 5.5. Raising cholesterol to 0.2 mol fraction of the total lipid inhibited insertion of both peptides under all conditions investigated. Membrane insertion was accompanied by the formation of alpha-helical structures. The nature of these structures was the same irrespective of the conditions used, indicating a single low energy structure for A beta in membranes. Peptides that did not insert into the membrane formed beta-sheet structures on the surface of the lipid.     

The journey from NADPH-cytochrome P450 oxidoreductase to nitric oxide synthases

This mini-review will reflect the perspective of its author on two fields of research, which have merged as the result of the insights of investigators whose work has influenced both areas immeasurably. It cannot be overlooked, however, that the research activities of many during a period of over five decades have produced the chemical and biological bases for the exciting discoveries now encompassing the cytochromes P450 and their redox partners, and the three isoforms of nitric oxide synthase as they function in their respective biological milieux. Following the remarkable discovery that, indeed, molecular oxygen can be adducted to organic molecules by enzymatic systems and that such processes require a supply of reducing equivalents, it is the purpose of this review to provide a chart, with some of its detours, of the road that followed in the pursuit of interesting biological phenomena involving these two major oxygenation systems. It is not intended to be a balanced review and apologies must be offered in advance to those whose contributions may be overlooked or simply were not directly germane to the development of the author's journey.     

Copper Modulation of Ion Channels of PrP[106–126] Mutant Prion Peptide Fragments

We have shown previously that the protease-resistant and neurotoxic prion peptide fragment PrP[106-126] of human PrP incorporates into lipid bilayer membranes to form heterogeneous ion channels, one of which is a Cu(2+)-sensitive fast cation channel. To investigate the role of PrP[106-126]'s hydrophobic core, AGAAAAGA, on its ability to form ion channels and their regulation with Cu(2+), we used the lipid-bilayer technique to examine membrane currents induced as a result of PrP[106-126] (AA/SS) and PrP[106-126] (VVAA/SSSS) interaction with lipid membranes and channel formation. Channel analysis of the mutant (VVAAA/SSS), which has a reduced hydrophobicity due to substitution of hydrophobic residues with the hydrophilic serine residue, showed a significant change in channel activity, which reflects a decrease in the beta-sheet structure, as shown by CD spectroscopy. One of the channels formed by the PrP[106-126] mutant has fast kinetics with three modes: burst, open and spike. The biophysical properties of this channel are similar to those of channels formed with other aggregation-prone amyloids, indicating their ability to form the common beta sheet-based channel structure. The current-voltage (I-V) relationship of the fast cation channel, which had a reversal potential, E(rev), between -40 and -10 mV, close to the equilibrium potential for K(+) ( E(K) = -35 mV), exhibited a sigmoidal shape. The value of the maximal slope conductance (g(max)) was 58 pS at positive potentials between 0 and 140 mV. Cu(2+) shifted the kinetics of the channel from being in the open and "burst" states to the spike mode. Cu(2+) reduced the probability of the channel being open (P(o)) and the mean open time (T(o)) and increased the channel's opening frequency (F(o)) and the mean closed time (T(c)) at a membrane potential ( V(m)) between +20 and + 140 mV. The fact that Cu(2+) induced changes in the kinetics of this channel with no changes in its conductance, indicates that Cu(2+) binds at the mouth of the channel via a fast channel block mechanism. The Cu(2+)-induced changes in the kinetic parameters of this channel suggest that the hydrophobic core is not a ligand Cu(2+) site, and they are in agreement with the suggestion that the Cu(2+)-binding site is located at M(109) and H(111) of this prion fragment. Although the data indicate that the hydrophobic core sequence plays a role in PrP[106-126] channel formation, it is not a binding site for Cu(2+). We suggest that the role of the hydrophobic region in modulating PrP toxicity is to influence PrP assembly into neurotoxic channel conformations. Such conformations may underlie toxicity observed in prion diseases. We further suggest that the conversions of the normal cellular isoform of prion protein (PrP(c)) to abnormal scrapie isoform (PrP(Sc)) and intermediates represent conversions to protease-resistant neurotoxic channel conformations.