Modulation of an early step in the secretory machinery in hippocampal nerve terminals

In hippocampal neurons, neurotransmitter release can be regulated by protein kinase A (PKA) through a direct action on the secretory machinery. To identify the site of PKA modulation, we have taken advantage of the ability of the neurotoxin Botulinum A to cleave the synaptic protein SNAP-25. Cleavage of this protein decreases the Ca2+ responsiveness of the secretory machinery by partially uncoupling Ca2+-sensing from fusion per se. This is expressed as a shift toward higher Ca2+ levels of the Ca2+ to neurotransmitter release relationship and as a perturbation of synaptic delay under conditions where secretion induced by the Ca2+-independent secretagogue ruthenium red is unimpaired. We find that SNAP-25 cleavage also perturbs PKA-dependent modulation of secretion; facilitation of ruthenium red-evoked neurotransmitter release by the adenylyl cyclase activator forskolin is blocked completely after Botulinum toxin A action. Together with our observation that forskolin modifies the Ca2+ to neurotransmitter release relationship, our results suggest that SNAP-25 acts as a functional linker between Ca2+ detection and fusion and that PKA modulates an early step in the secretory machinery related to calcium sensing to facilitate synaptic transmission.

Governing step of metastasis visualized in vitro

Metastasis is the ultimate life-threatening stage of cancer. The lack of accurate model systems thwarted studies of the metastatic cell’s basic biology. To follow continuously the succeeding stages of metastatic colony growth, we heritably labeled cells from the human lung adenocarcinoma cell line ANIP 973 with green fluorescent protein (GFP) by transfection with GFP cDNA. Labeled cells were then injected intravenously into nude mice, where, by 7 days, they formed brilliantly fluorescing metastatic colonies on mouse lung [Chishima, T., Miyagi, Y., Wang, X., Yang, M., Tan, Y., Shimada, H., Moossa, A. R. & Hoffman, R. M. (1997) Clin. Exp. Metastasis 15, 547–552]. The seeded lung tissue was then excised and incubated in the three-dimensional sponge-gel-matrix-supported histoculture that maintained the critical features of progressive in vivo tumor colonization while allowing continuous access for measurement and manipulation. Tumor progression was continuously visualized by GFP fluorescence in the same individual cultures over a 52-day period, during which the tumors spread throughout the lung. Histoculture tumor colonization was selective for lung cancer cells to grow on lung tissue, because no growth occurred on histocultured mouse liver tissue, which was also observed in vivo. The ability to support selective organ colonization in histoculture and visualize tumor progression by GFP fluorescence allows the in vitro study of the governing processes of metastasis [Kuo, T.-H., Kubota, T., Watanbe, M., Furukawa, T., Teramoto, T., Ishibiki, K., Kitajima, M., Moossa, A. R., Penman, S. & Hoffman, R. M. (1995) Proc. Natl. Acad. Sci. USA 92, 12085–12089]. The results presented here provide significant, new opportunities to understand and to develop treatments that prevent and possibly reverse metastasis.

Use of intrinsic modes in biology: Examples of indicial response of pulmonary blood pressure to ± step hypoxia

Recently, a new method to analyze biological nonstationary stochastic variables has been presented. The method is especially suitable to analyze the variation of one biological variable with respect to changes of another variable. Here, it is illustrated by the change of the pulmonary blood pressure in response to a step change of oxygen concentration in the gas that an animal breathes. The pressure signal is resolved into the sum of a set of oscillatory intrinsic mode functions, which have zero “local mean,” and a final nonoscillatory mode. With this device, we obtain a set of “mean trends,” each of which represents a “mean” in a definitive sense, and together they represent the mean trend systematically with different degrees of oscillatory content. Correspondingly, the oscillatory content of the signal about any mean trend can be represented by a set of partial sums of intrinsic mode functions. When the concept of “indicial response function” is used to describe the change of one variable in response to a step change of another variable, we now have a set of indicial response functions of the mean trends and another set of indicial response functions to describe the energy or intensity of oscillations about each mean trend. Each of these can be represented by an analytic function whose coefficients can be determined by a least-squares curve-fitting procedure. In this way, experimental results are stated sharply by analytic functions.

The first step of aminoacylation at the atomic level in histidyl-tRNA synthetase

The crystal structure of an enzyme–substrate complex with histidyl-tRNA synthetase from Escherichia coli, ATP, and the amino acid analog histidinol is described and compared with the previously obtained enzyme–product complex with histidyl-adenylate. An active site arginine, Arg-259, unique to all histidyl-tRNA synthetases, plays the role of the catalytic magnesium ion seen in seryl-tRNA synthetase. When Arg-259 is substituted with histidine, the apparent second order rate constant (kcat/Km) for the pyrophosphate exchange reaction and the aminoacylation reaction decreases 1,000-fold and 500-fold, respectively. Crystals soaked with MnCl2 reveal the existence of two metal binding sites between β- and γ-phosphates; these sites appear to stabilize the conformation of the pyrophosphate. The use of both conserved metal ions and arginine in phosphoryl transfer provides evidence of significant early functional divergence of class II aminoacyl-tRNA synthetases.

Isopentenyl diphosphate biosynthesis via a mevalonate-independent pathway: Isopentenyl monophosphate kinase catalyzes the terminal enzymatic step

In plants, the biosynthesis of isopentenyl diphosphate, the central precursor of all isoprenoids, proceeds via two separate pathways. The cytosolic compartment harbors the mevalonate pathway, whereas the newly discovered deoxyxylulose 5-phosphate pathway, which also operates in certain eubacteria, including Escherichia coli, is localized to plastids. Only the first two steps of the plastidial pathway, which involve the condensation of pyruvate and glyceraldehyde 3-phosphate to deoxyxylulose 5-phosphate followed by intramolecular rearrangement and reduction to 2-C-methylerythritol 4-phosphate, have been established. Here we report the cloning from peppermint (Mentha × piperita) and E. coli, and expression, of a kinase that catalyzes the phosphorylation of isopentenyl monophosphate as the last step of this biosynthetic sequence to isopentenyl diphosphate. The plant gene defines an ORF of 1,218 bp that, when the proposed plastidial targeting sequence is excluded, corresponds to ≈308 aa with a mature size of ≈33 kDa. The E. coli gene (ychB), which is located at 27.2 min of the chromosomal map, consists of 852 nt, encoding a deduced enzyme of 283 aa with a size of 31 kDa. These enzymes represent a conserved class of the GHMP family of kinases, which includes galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase, with homologues in plants and several eubacteria. Besides the preferred substrate isopentenyl monophosphate, the recombinant peppermint and E. coli kinases also phosphorylate isopentenol, and, much less efficiently, dimethylallyl alcohol, but dimethylallyl monophosphate does not serve as a substrate. Incubation of secretory cells isolated from peppermint glandular trichomes with isopentenyl monophosphate resulted in the rapid production of monoterpenes and sesquiterpenes, confirming that isopentenyl monophosphate is the physiologically relevant, terminal intermediate of the deoxyxylulose 5-phosphate pathway.

Isolation and one-step preparation of A2E and iso-A2E, fluorophores from human retinal pigment epithelium

Age-related macular degeneration, a major cause of blindness for which no satisfactory treatments exist, leads to a gradual decrease in central high acuity vision. The accumulation of fluorescent materials, called lipofuscin, in retinal pigment epithelial cells of the aging retina is most pronounced in the macula. One of the fluorophores of retinal pigment epithelial lipofuscin has been characterized as A2E, a pyridinium bis-retinoid, which is derived from two molecules of vitamin A aldehyde and one molecule of ethanolamine. An investigation aimed at optimizing the in vitro synthesis of A2E has resulted in the one-step biomimetic preparation of this pigment in 49% yield, readily producing more than 50 mg in one step. These results have allowed for the optimization of HPLC conditions so that nanogram quantities of A2E can be detected from extracts of tissue samples. By using 5% of the extract from individual aged human eyes, this protocol has led to the quantification of A2E and the characterization of iso-A2E, a new A2E double bond isomer; all-trans-retinol and 13-cis-retinol also have been identified in these HPLC chromatograms. Exposure of either A2E or iso-A2E to light gives rise to 4:1 A2E:iso-A2E equilibrium mixtures, similar to the composition of these two pigments in eye extracts. A2E and iso-A2E may exhibit surfactant properties arising from their unique wedge-shaped structures.

From snapshot to movie: φ analysis of protein folding transition states taken one step further

Kinetic anomalies in protein folding can result from changes of the kinetic ground states (D, I, and N), changes of the protein folding transition state, or both. The 102-residue protein U1A has a symmetrically curved chevron plot which seems to result mainly from changes of the transition state. At low concentrations of denaturant the transition state occurs early in the folding reaction, whereas at high denaturant concentration it moves close to the native structure. In this study we use this movement to follow continuously the formation and growth of U1A's folding nucleus by φ analysis. Although U1A's transition state structure is generally delocalized and displays a typical nucleation–condensation pattern, we can still resolve a sequence of folding events. However, these events are sufficiently coupled to start almost simultaneously throughout the transition state structure.

The 9-cis-epoxycarotenoid cleavage reaction is the key regulatory step of abscisic acid biosynthesis in water-stressed bean

Abscisic acid (ABA), a cleavage product of carotenoids, is involved in stress responses in plants. A well known response of plants to water stress is accumulation of ABA, which is caused by de novo synthesis. The limiting step of ABA biosynthesis in plants is presumably the cleavage of 9-cis-epoxycarotenoids, the first committed step of ABA biosynthesis. This step generates the C15 intermediate xanthoxin and C25-apocarotenoids. A cDNA, PvNCED1, was cloned from wilted bean (Phaseolus vulgaris L.) leaves. The 2,398-bp full-length PvNCED1 has an ORF of 615 aa and encodes a 68-kDa protein. The PvNCED1 protein is imported into chloroplasts, where it is associated with the thylakoids. The recombinant protein PvNCED1 catalyzes the cleavage of 9-cis-violaxanthin and 9′-cis-neoxanthin, so that the enzyme is referred to as 9-cis-epoxycarotenoid dioxygenase. When detached bean leaves were water stressed, ABA accumulation was preceded by large increases in PvNCED1 mRNA and protein levels. Conversely, rehydration of stressed leaves caused a rapid decrease in PvNCED1 mRNA, protein, and ABA levels. In bean roots, a similar correlation among PvNCED1 mRNA, protein, and ABA levels was observed. However, the ABA content was much less than in leaves, presumably because of the much smaller carotenoid precursor pool in roots than in leaves. At 7°C, PvNCED1 mRNA and ABA were slowly induced by water stress, but, at 2°C, neither accumulated. The results provide evidence that drought-induced ABA biosynthesis is regulated by the 9-cis-epoxycarotenoid cleavage reaction and that this reaction takes place in the thylakoids, where the carotenoid substrate is located.

Limited internal friction in the rate-limiting step of a two-state protein folding reaction

Small, single-domain proteins typically fold via a compact transition-state ensemble in a process well fitted by a simple, two-state model. To characterize the rate-limiting conformational changes that underlie two-state folding, we have investigated experimentally the effects of changing solvent viscosity on the refolding of the IgG binding domain of protein L. In conjunction with numerical simulations, our results indicate that the rate-limiting conformational changes of the folding of this domain are strongly coupled to solvent viscosity and lack any significant “internal friction” arising from intrachain collisions. When compared with the previously determined solvent viscosity dependencies of other, more restricted conformational changes, our results suggest that the rate-limiting folding transition involves conformational fluctuations that displace considerable amounts of solvent. Reconciling evidence that the folding transition state ensemble is comprised of highly collapsed species with these and similar, previously reported results should provide a significant constraint for theoretical models of the folding process.

Specific Sterols Required for the Internalization Step of Endocytosis in Yeast

Sterols are major components of the plasma membrane, but their functions in this membrane are not well understood. We isolated a mutant defective in the internalization step of endocytosis in a gene (ERG2) encoding a C-8 sterol isomerase that acts in the late part of the ergosterol biosynthetic pathway. In the absence of Erg2p, yeast cells accumulate sterols structurally different from ergosterol, which is the major sterol in wild-type yeast. To investigate the structural requirements of ergosterol for endocytosis in more detail, several erg mutants (erg2Δ, erg6Δ, and erg2Δerg6Δ) were made. Analysis of fluid phase and receptor-mediated endocytosis indicates that changes in the sterol composition lead to a defect in the internalization step. Vesicle formation and fusion along the secretory pathway were not strongly affected in the ergΔ mutants. The severity of the endocytic defect correlates with changes in sterol structure and with the abundance of specific sterols in the ergΔ mutants. Desaturation of the B ring of the sterol molecules is important for the internalization step. A single desaturation at C-8,9 was not sufficient to support internalization at 37°C whereas two double bonds, either at C-5,6 and C-7,8 or at C-5,6 and C-8,9, allowed internalization.

A Novel RING Finger Protein Complex Essential for a Late Step in Protein Transport to the Yeast Vacuole

Protein transport to the lysosome-like vacuole in yeast is mediated by multiple pathways, including the biosynthetic routes for vacuolar hydrolases, the endocytic pathway, and autophagy. Among the more than 40 genes required for vacuolar protein sorting (VPS) in Saccharomyces cerevisiae, mutations in the four class C VPS genes result in the most severe vacuolar protein sorting and morphology defects. Herein, we provide complementary genetic and biochemical evidence that the class C VPS gene products (Vps18p, Vps11p, Vps16p, and Vps33p) physically and functionally interact to mediate a late step in protein transport to the vacuole. Chemical cross-linking experiments demonstrated that Vps11p and Vps18p, which both contain RING finger zinc-binding domains, are components of a hetero-oligomeric protein complex that includes Vps16p and the Sec1p homologue Vps33p. The class C Vps protein complex colocalized with vacuolar membranes and a distinct dense membrane fraction. Analysis of cells harboring a temperature-conditional vps18 allele (vps18tsf) indicated that Vps18p function is required for the biosynthetic, endocytic, and autophagic protein transport pathways to the vacuole. In addition, vps18tsf cells accumulated multivesicular bodies, autophagosomes, and other membrane compartments that appear to represent blocked transport intermediates. Overproduction of either Vps16p or the vacuolar syntaxin homologue Vam3p suppressed defects associated with vps18tsf mutant cells, indicating that the class C Vps proteins and Vam3p may functionally interact. Thus we propose that the class C Vps proteins are components of a hetero-oligomeric protein complex that mediates the delivery of multiple transport intermediates to the vacuole.

Role of adenine functional groups in the recognition of the 3′-splice-site AG during the second step of pre-mRNA splicing

The AG dinucleotide at the 3′ splice sites of metazoan nuclear pre-mRNAs plays a critical role in catalytic step II of the splicing reaction. Previous studies have shown that replacement of the guanine by adenine in the AG (AG → GG) inhibits this step. We find that the second step was even more severely inhibited by cytosine (AG → CG) or uracil (AG → UG) substitutions at this position. By contrast, a relatively moderate inhibition was observed with a hypoxanthine substitution (AG → HG). When adenine was replaced by a purine base (AG → PG) or by 7-deazaadenine (AG → c7AG), little effect on the second step was observed, suggesting that the 6-NH2 and N7 groups do not play a critical role in adenine recognition. Finally, replacement of adenine by 2-aminopurine (AG → 2-APG) had no effect on the second step. Taken together, our results suggest that the N1 group of adenine functions as an essential determinant in adenine recognition during the second step of pre-mRNA splicing.

Guanidine hydrochloride blocks a critical step in the propagation of the prion-like determinant [PSI+] of Saccharomyces cerevisiae

The cytoplasmic heritable determinant [PSI+] of the yeast Saccharomyces cerevisiae reflects the prion-like properties of the chromosome-encoded protein Sup35p. This protein is known to be an essential eukaryote polypeptide release factor, namely eRF3. In a [PSI+] background, the prion conformer of Sup35p forms large oligomers, which results in the intracellular depletion of functional release factor and hence inefficient translation termination. We have investigated the process by which the [PSI+] determinant can be efficiently eliminated from strains, by growth in the presence of the protein denaturant guanidine hydrochloride (GuHCl). Strains are “cured” of [PSI+] by millimolar concentrations of GuHCl, well below that normally required for protein denaturation. Here we provide evidence indicating that the elimination of the [PSI+] determinant is not derived from the direct dissolution of self-replicating [PSI+] seeds by GuHCl. Although GuHCl does elicit a moderate stress response, the elimination of [PSI+] is not enhanced by stress, and furthermore, exhibits an absolute requirement for continued cell division. We propose that GuHCl inhibits a critical event in the propagation of the prion conformer and demonstrate that the kinetics of curing by GuHCl fit a random segregation model whereby the heritable [PSI+] element is diluted from a culture, after the total inhibition of prion replication by GuHCl.

Nonlinear indicial response of complex nonstationary oscillations as pulmonary hypertension responding to step hypoxia

This paper is devoted to the quantization of the degree of nonlinearity of the relationship between two biological variables when one of the variables is a complex nonstationary oscillatory signal. An example of the situation is the indicial responses of pulmonary blood pressure (P) to step changes of oxygen tension (ΔpO2) in the breathing gas. For a step change of ΔpO2 beginning at time t1, the pulmonary blood pressure is a nonlinear function of time and ΔpO2, which can be written as P(t-t1 | ΔpO2). An effective method does not exist to examine the nonlinear function P(t-t1 | ΔpO2). A systematic approach is proposed here. The definitions of mean trends and oscillations about the means are the keys. With these keys a practical method of calculation is devised. We fit the mean trends of blood pressure with analytic functions of time, whose nonlinearity with respect to the oxygen level is clarified here. The associated oscillations about the mean can be transformed into Hilbert spectrum. An integration of the square of the Hilbert spectrum over frequency yields a measure of oscillatory energy, which is also a function of time, whose mean trends can be expressed by analytic functions. The degree of nonlinearity of the oscillatory energy with respect to the oxygen level also is clarified here. Theoretical extension of the experimental nonlinear indicial functions to arbitrary history of hypoxia is proposed. Application of the results to tissue remodeling and tissue engineering of blood vessels is discussed.

Mg-chelatase of tobacco: The role of the subunit CHL D in the chelation step of protoporphyrin IX

The Mg-chelation is found to be a prerequisite to direct protoporphyrin IX into the chlorophyll (Chl)-synthesizing branch of the tetrapyrrol pathway. The ATP-dependent insertion of magnesium into protoporphyrin IX is catalyzed by the enzyme Mg-chelatase, which consists of three protein subunits (CHL D, CHL I, and CHL H). We have chosen the Mg-chelatase from tobacco to obtain more information about the mode of molecular action of this complex enzyme by elucidating the interactions in vitro and in vivo between the central subunit CHL D and subunits CHL I and CHL H. We dissected CHL D in defined peptide fragments and assayed for the essential part of CHL D for protein–protein interaction and enzyme activity. Surprisingly, only a small part of CHL D, i.e., 110 aa, was required for interaction with the partner subunits and maintenance of the enzyme activity. In addition, it could be demonstrated that CHL D is capable of forming homodimers. Moreover, it interacted with both CHL I and CHL H. Our data led to the outline of a two-step model based on the cooperation of the subunits for the chelation process.