Piperonylic Acid, a Selective, Mechanism-Based Inactivator of the trans-Cinnamate 4-Hydroxylase: A New Tool to Control the Flux of Metabolites in the Phenylpropanoid Pathway1

Piperonylic acid (PA) is a natural molecule bearing a methylenedioxy function that closely mimics the structure of trans-cinnamic acid. The CYP73A subfamily of plant P450s catalyzes trans-cinnamic acid 4-hydroxylation, the second step of the general phenylpropanoid pathway. We show that when incubated in vitro with yeast-expressed CYP73A1, PA behaves as a potent mechanism-based and quasi-irreversible inactivator of trans-cinnamate 4-hydroxylase. Inactivation requires NADPH, is time dependent and saturable (KI = 17 μm, kinact = 0.064 min−1), and results from the formation of a stable metabolite-P450 complex absorbing at 427 nm. The formation of this complex is reversible with substrate or other strong ligands of the enzyme. In plant microsomes PA seems to selectively inactivate the CYP73A P450 subpopulation. It does not form detectable complexes with other recombinant plant P450 enzymes. In vivo PA induces a sharp decrease in 4-coumaric acid concomitant to cinnamic acid accumulation in an elicited tobacco (Nicotiana tabacum) cell suspension. It also strongly decreases the formation of scopoletin in tobacco leaves infected with tobacco mosaic virus.

Potent Inhibition of Ribulose-Bisphosphate Carboxylase by an Oxidized Impurity in Ribulose-1,5-Bisphosphate1

Oxidation of d-ribulose-1,5-bisphosphate (ribulose-P2) during synthesis and/or storage produces d-glycero-2,3-pentodiulose-1,5-bisphosphate (pentodiulose-P2), a potent slow, tight-binding inhibitor of spinach (Spinacia oleracea L.) ribulose-P2 carboxylase/oxygenase (Rubisco). Differing degrees of contamination with pentodiulose-P2 caused the decline in Rubisco activity seen during Rubisco assay time courses to vary between different preparations of ribulose-P2. With some ribulose-P2 preparations, this compound can be the dominant cause of the decline, far exceeding the significance of the catalytic by-product, d-xylulose-1,5-bisphosphate. Unlike xylulose-1,5-bisphosphate, pentodiulose-P2 did not appear to be a significant by-product of catalysis by wild-type Rubisco at saturating CO2 concentration. It was produced slowly during frozen storage of ribulose-P2, even at low pH, more rapidly in Rubisco assay buffers at room temperature, and particularly rapidly on deliberate oxidation of ribulose-P2 with Cu2+. Its formation was prevented by the exclusion of transition metals and O2. Pentodiulose-P2 was unstable and decayed to a variety of other less-inhibitory compounds, particularly in the presence of some buffers. However, it formed a tight, stable complex with carbamylated spinach Rubisco, which could be isolated by gel filtration, presumably because its structure mimics that of the enediol intermediate of Rubisco catalysis. Rubisco catalyzes the cleavage of pentodiulose-P2 by H2O2, producing P-glycolate.

Auxin Deprivation Induces Synchronous Golgi Differentiation in Suspension-Cultured Tobacco BY-2 Cells1

To date, the lack of a method for inducing plant cells and their Golgi stacks to differentiate in a synchronous manner has made it difficult to characterize the nature and extent of Golgi retailoring in biochemical terms. Here we report that auxin deprivation can be used to induce a uniform population of suspension-cultured tobacco (Nicotiana tabacum cv BY-2) cells to differentiate synchronously during a 4-d period. Upon removal of auxin, the cells stop dividing, undergo elongation, and differentiate in a manner that mimics the formation of slime-secreting epidermal and peripheral root-cap cells. The morphological changes to the Golgi apparatus include a proportional increase in the number of trans-Golgi cisternae, a switch to larger-sized secretory vesicles that bud from the trans-Golgi cisternae, and an increase in osmium staining of the secretory products. Biochemical alterations include an increase in large, fucosylated, mucin-type glycoproteins, changes in the types of secreted arabinogalactan proteins, and an increase in the amounts and types of molecules containing the peripheral root-cap-cell-specific epitope JIM 13. Taken together, these findings support the hypothesis that auxin deprivation can be used to induce tobacco BY-2 cells to differentiate synchronously into mucilage-secreting cells.

Differential Expression of a Novel Gene in Response to Coronatine, Methyl Jasmonate, and Wounding in the Coi1 Mutant of Arabidopsis1

Coronatine is a phytotoxin produced by some plant-pathogenic bacteria. It has been shown that coronatine mimics the action of methyl jasmonate (MeJA) in plants. MeJA is a plant-signaling molecule involved in stress responses such as wounding and pathogen attack. In Arabidopsis thaliana, MeJA is essential for pollen grain development. The coi1 (for coronatine-insensitive) mutant of Arabidopsis, which is insensitive to coronatine and MeJA, produces sterile male flowers and shows an altered response to wounding. When the differential display technique was used, a message that was rapidly induced by coronatine in wild-type plants but not in coi1 was identified and the corresponding cDNA was cloned. The coronatine-induced gene ATHCOR1 (for A. thaliana coronatine-induced) is expressed in seedlings, mature leaves, flowers, and siliques but was not detected in roots. The expression of this gene was dramatically reduced in coi1 plants, indicating that COI1 affects its expression. ATHCOR1 was rapidly induced by MeJA and wounding in wild-type plants. The sequence of ATHCOR1 shows no strong homology to known proteins. However, the predicted polypeptide contains a conserved amino acid sequence present in several bacterial, animal, and plant hydrolases and includes a potential ATP/GTP-binding-site motif (P-loop).

Expression and functional significance of VE-cadherin in aggressive human melanoma cells: Role in vasculogenic mimicry

We recently have introduced the term vasculogenic mimicry to describe the unique ability of aggressive melanoma tumor cells to form tubular structures and patterned networks in three-dimensional culture, which “mimics” embryonic vasculogenic networks formed by differentiating endothelial cells. In the current study, we address the biological significance of several endothelial-associated molecules (revealed by microarray analysis) with respect to expression and function in highly aggressive and poorly aggressive human cutaneous melanoma cell lines (established from the same patient). In a comparative analysis, CD31 was not expressed by any of the melanoma cell lines, whereas TIE-1 (tyrosine kinase with Ig and epidermal growth factor homology domains-1) was strongly expressed in the highly aggressive tumor cells with a low level of expression in one of the poorly aggressive cell lines. Vascular endothelial (VE)-cadherin was exclusively expressed by highly aggressive melanoma cells and was undetectable in the poorly aggressive tumor cells, suggesting the possibility of a vasculogenic switch. Down-regulation of VE-cadherin expression in the aggressive melanoma cells abrogated their ability to form vasculogenic networks and directly tested the hypothesis that VE-cadherin is critical in melanoma vasculogenic mimicry. These results highlight the plasticity of aggressive melanoma cells and call into question their possible genetic reversion to an embryonic phenotype. This finding could pose a significant clinical challenge in targeting tumor cells that may masquerade as circulating endothelial cells or other embryonic-like stem cells.

Induction of pheromone production in a moth by topical application of a pseudopeptide mimic of a pheromonotropic neuropeptide.

An amphiphilic analog of Locusta myotropin II (Lom-MT-II), Glu-Gly-Asp-Phe-Thr-Pro-Arg-Leu-amide, was synthesized by addition of 6-phenylhexanoic acid (6-Pha) linked through alanine to the amino terminus. This pseudopeptide, [6-Pha-Ala0]Lom-MT-II, was found to have pheromonotropic activity equivalent to pheromone biosynthesis activating neuropeptide when injected into females of Heliothis virescens. Topical application of [6-Pha-Ala0]Lom-MT-II or Helicoverpa zea-pheromone biosynthesis activating neuropeptide (PBAN), dissolved in dimethyl sulfoxide, to the descaled abdomen of females induced production of pheromone, although more Hez-PBAN than [6-Pha-Ala0]Lom-MT-II was required to obtain significant production of pheromone. Application of [6-Pha-Ala0]Lom-MT-II, dissolved in water, to the abdomen induced production of pheromone, but neither Hez-PBAN nor Lom-MT-II dissolved in water stimulated production of significant amounts of pheromone. Dose- and time-response studies indicated that application of the amphiphilic mimetic in water induced pheromone production in as little as 15 min after application and that the effects were maintained for prolonged periods. These findings show that amphiphilic pseudopeptide mimics of insect neuropeptides will penetrate the insect cuticle when applied topically in water and induce an endogenous response.

Minimizing a binding domain from protein A.

We present a systematic approach to minimizing the Z-domain of protein A, a three-helix bundle (59 residues total) that binds tightly (Kd = 10 nM) to the Fc portion of an immunoglobin IgG1. Despite the fact that all the contacts seen in the x-ray structure of the complex with the IgG are derived from residues in the first two helices, when helix 3 is deleted, binding affinity is reduced > 10(5)-fold (Kd > 1 mM). By using structure-based design and phage display methods, we have iteratively improved the stability and binding affinity for a two-helix derivative, 33 residues in length, such that it binds IgG1, with a Kd of 43 nM. This was accomplished by stepwise selection of random mutations from three regions of the truncated Z-peptide: the 4 hydrophobic residues from helix 1 and helix 2 that contacted helix 3 (the exoface), followed by 5 residues between helix 1 and helix 2 (the intraface), and lastly by 19 residues at or near the interface that interacts with Fc (the interface). As selected mutations from each region were compiled (12 in total), they led to progressive increases in affinity for IgG, and concomitant increases in alpha-helical content reflecting increased stabilization of the two-helix scaffold. Thus, by sequential increases in the stability of the structure and improvements in the quality of the intermolecular contacts, one can reduce larger binding domains to smaller ones. Such mini-protein binding domains are more amenable to synthetic chemistry and thus may be useful starting points for the design of smaller organic mimics. Smaller binding motifs also provide simplified and more tractable models for understanding determinants of protein function and stability.

Traumatic brain damage prevented by the non-N-methyl-D-aspartate antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[f] quinoxaline.

The mechanisms of neuronal degeneration following traumatic head injury are not well understood and no adequate treatment is currently available for the prevention of traumatic brain damage in humans. Traumatic head injury leads to primary (at impact) and secondary (distant) damage to the brain. Mechanical percussion of the rat cortex mimics primary damage seen after traumatic head injury in humans; no animal model mimicking the secondary damage following traumatic head injury has yet been established. Rats subjected to percussion trauma of the cortex showed primary damage in the cortex and secondary damage in the hippocampus. Morphometric analysis demonstrated that both cortical and hippocampal damage was mitigated by pretreatment with either the N-methyl-D-aspartate (NMDA) antagonist 3-((+/-)- 2-carboxypiperazin-4-yl)-propyl-1-phosphonate (CPP) or the non-NMDA antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline (NBQX). Neither treatment prevented primary damage in the cortex when therapy was started after trauma. Surprisingly, delayed treatment of rats with NBQX, but not with CPP, beginning between 1 and 7 hr after trauma prevented hippocampal damage. No protection was seen when therapy with NBQX was started 10 hr after trauma. These data indicate that both NMDA- and non-NMDA-dependent mechanisms contribute to the development of primary damage in the cortex, whereas non-NMDA mechanisms are involved in the evolution of secondary damage in the hippocampus in rats subjected to traumatic head injury. The wide therapeutic time-window documented for NBQX suggests that antagonism at non-NMDA receptors may offer a novel therapeutic approach for preventing deterioration of the brain after head injury.

Mechanism of photoreceptor cGMP phosphodiesterase inhibition by its gamma-subunits.

cGMP phosphodiesterase (PDE) is the key effector enzyme of vertebrate photoreceptor cells that regulates the level of the second messenger, cGMP. PDE consists of catalytic alpha and beta subunits (Palpha and Pbeta) and two inhibitory gamma subunits (Pgamma) that block PDE activity in the dark. The major inhibitory region has been localized to the C terminus of Pgamma. The last C-terminal residues -IleIle form an important hydrophobic domain critical for the inhibition of PDE activity. In this study, mutants of Pgamma were designed for cross-linking experiments to identify regions on Palpha and Pbeta subunits that bind to the Pgamma C terminus. In one of the mutants, the cysteine at position 68 was substituted with serine, and the last four C-terminal residues of Pgamma were replaced with a single cysteine. This mutant, Pgamma83Cys, was labeled with photoprobe 4-(N-maleimido) benzophenone (MBP) at the cysteine residue. The labeled Pgamma83CysMBP mutant was a more potent inhibitor of PDE activity than the unlabeled mutant, indicating that the hydrophobic MBP probe mimics the Pgamma hydrophobic C terminus. A specific, high-yield cross-linking of up to 70% was achieved between the Pgamma83CysMBP and PDE catalytic subunits. Palpha and the N-terminally truncated Pbeta (lacking 147 aa residues) cross-linked to Pgamma83CysMBP with the same efficiency. Using mass spectrometric analysis of tryptic fragments from the cross-linked PDE, we identified the site of cross-linking to aa residues 751-763 of Palpha. The corresponding region of Pbeta, Pbeta-749-761, also may bind to the Pgamma C terminus. Our data suggest that Pgamma blocks PDE activity through the binding to the catalytic site of PDE, near the NKXD motif, a consensus sequence for interaction with the guanine ring of cGMP.

The contrasting roles of ICE family proteases and interleukin-1beta in apoptosis induced by trophic factor withdrawal and by copper/zinc superoxide dismutase down-regulation.

We compare here the mechanisms of apoptotic death of PC12 cells induced by down-regulation of Cu2+,Zn2+ superoxide dismutase (SOD1) and withdrawal of trophic support (serum/nerve growth factor). Our previous results indicated that the initiating causes of death are different in each paradigm. However, bcl-2 rescues cells in either paradigm, suggesting common downstream elements to the cell death pathway. To determine whether the ICE [interleukin 1beta converting enzyme] family of proteases, which is required for apoptosis on trophic factor withdrawal, is also required for apoptosis induced by oxidative stress, we have developed a novel peptide inhibitor that mimics the common catalytic site of these enzymes and thereby blocks their access to substrates. This differs from the more usual pseudosubstrate approach to enzyme inhibition. Blockade of ICE family proteases by either this inhibitor or by a permeant competitive ICE family antagonist rescues PC12 cells from apoptotic death following apoptosis induced by down-regulation of SOD1, as well as from trophic factor/nerve growth factor deprivation. SOD1 down-regulation results in an increase in interleukin 1beta (IL- 1beta) production by the cells, and cell death under these conditions can be prevented by either blocking antibodies against IL-1beta or the IL-1 receptor antagonist (IL-1Ralpha). In contrast, trophic factor withdrawal does not increase IL-1beta secretion, and the blocking antibody failed to protect PC12 cells from trophic factor withdrawal, whereas the receptor antagonist was only partially protective at very high concentrations. There were substantial differences in the concentrations of pseudosubstrate inhibitors which rescued cells from SOD1 down-regulation and trophic factor deprivation. These results suggest the involvement of different members of the ICE family, different substrates, or both in the two different initiating causes of cell death.

DNA damage enhances melanogenesis.

Although the ability of UV irradiation to induce pigmentation in vivo and in vitro is well documented, the intracellular signals that trigger this response are poorly understood. We have recently shown that increasing DNA repair after irradiation enhances UV-induced melanization. Moreover, addition of small DNA fragments, particularly thymine dinucleotides (pTpT), selected to mimic sequences excised during the repair of UV-induced DNA photoproducts, to unirradiated pigment cells in vitro or to guinea pig skin in vivo induces a pigment response indistinguishable from UV-induced tanning. Here we present further evidence that DNA damage and/or the repair of this damage increases melanization. (i) Treatment with the restriction enzyme Pvu II or the DNA-damaging chemical agents methyl methanesulfonate (MMS) or 4-nitroquinoline 1-oxide (4-NQO) produces a 4- to 10-fold increase in melanin content in Cloudman S91 murine melanoma cells and an up to 70% increase in normal human melanocytes, (ii) UV irradiation, MMS, and pTpT all upregulate the mRNA level for tyrosinase, the rate-limiting enzyme in melanin biosynthesis. (iii) Treatment with pTpT or MMS increases the response of S91 cells to melanocyte-stimulating hormone (MSH) and increases the binding of MSH to its cell surface receptor, as has been reported for UV irradiation. Together, these data suggest that UV-induced DNA damage and/or the repair of this damage is an important signal in the pigmentation response to UV irradiation. Because Pvu II acts exclusively on DNA and because MMS and 4-NQO, at the concentrations used, primarily interact with DNA, such a stimulus alone appears sufficient to induce melanogenesis. Of possible practical importance, the dinucleotide pTpT mimics most, if not all, of the effects of UV irradiation on pigmentation, tyrosinase mRNA regulation, and response to MSH without the requirement for antecedent DNA damage.

A histologically distinctive interstitial pneumonia induced by overexpression of the interleukin 6, transforming growth factor beta 1, or platelet-derived growth factor B gene.

Interstitial pneumonia is characterized by alveolitis with resulting fibrosis of the interstitium. To determine the relevance of humoral factors in the pathogenesis of interstitial pneumonia, we introduced expression vectors into Wistar rats via the trachea to locally overexpress humoral factors in the lungs. Human interleukin (IL) 6 and IL-6 receptor genes induced lymphocytic alveolitis without marked fibroblast proliferation. In contrast, overexpression of human transforming growth factor beta 1 or human platelet-derived growth factor B gene induced only mild or apparent cellular infiltration in the alveoli, respectively. However, both factors induced significant proliferation of fibroblasts and deposition of collagen fibrils. These histopathologic changes induced by the transforming growth factor beta 1 and platelet-derived growth factor B gene are partly akin to those changes seen in lung tissues from patients with pulmonary fibrosis and markedly contrast with the changes induced by overexpression of the IL-6 and IL-6 receptor genes that mimics lymphocytic interstitial pneumonia.

Enhanced conformational sampling in Monte Carlo simulations of proteins: application to a constrained peptide.

A Monte Carlo simulation method for globular proteins, called extended-scaled-collective-variable (ESCV) Monte Carlo, is proposed. This method combines two Monte Carlo algorithms known as entropy-sampling and scaled-collective-variable algorithms. Entropy-sampling Monte Carlo is able to sample a large configurational space even in a disordered system that has a large number of potential barriers. In contrast, scaled-collective-variable Monte Carlo provides an efficient sampling for a system whose dynamics is highly cooperative. Because a globular protein is a disordered system whose dynamics is characterized by collective motions, a combination of these two algorithms could provide an optimal Monte Carlo simulation for a globular protein. As a test case, we have carried out an ESCV Monte Carlo simulation for a cell adhesive Arg-Gly-Asp-containing peptide, Lys-Arg-Cys-Arg-Gly-Asp-Cys-Met-Asp, and determined the conformational distribution at 300 K. The peptide contains a disulfide bridge between the two cysteine residues. This bond mimics the strong geometrical constraints that result from a protein's globular nature and give rise to highly cooperative dynamics. Computation results show that the ESCV Monte Carlo was not trapped at any local minimum and that the canonical distribution was correctly determined.

A nonpeptide agonist of the invertebrate receptor for SchistoFLRFamide (PDVDHVFLRFamide), a member of a subfamily of insect FMRFamide-related peptides.

We describe a nonpeptide mimetic analog of an invertebrate peptide receptor. Benzethonium chloride (Bztc) is an agonist of the SchistoFLRFamide (PDVDHVFLRFamide) receptors found on locust oviducts. Bztc competitively displaces [125I-labeled Y1]SchistoFLRFamide binding to both high- and low-affinity receptors of membrane preparations. Bztc mimics the physiological effects of SchistoFLRFamide on locust oviduct, by inhibiting myogenic and induced contractions in a dose-dependent manner. Bztc is therefore recognized by the binding and activation regions of the SchistoFLRFamide receptors. This discovery provides a unique opportunity within insects to finally target a peptide receptor for the development of future pest management strategies.

A channeled tRNA cycle during mammalian protein synthesis.

In earlier studies it was shown that the mammalian translation system is highly organized in vivo and that the intermediates in the process, aminoacyl-tRNAs, are channeled--i.e., they are directly transferred from the aminoacyl-tRNA synthetases to the elongation factor to the ribosomes without dissociating into the cellular fluid. Here, we examine whether spent tRNAs leaving the ribosome enter the fluid phase or are transferred directly to their cognate aminoacyl-tRNA synthetases to complete a channeled tRNA cycle. Using a permeabilized CHO cell system that closely mimics living cells, we find that there is no leakage of endogenous tRNA during many cycles of translation, and protein synthesis remains linear during this period, even though free aminoacyl-tRNA is known to rapidly equilibrate between the inside and outside of these cells. We also find that exogenous tRNA and periodate-oxidized tRNA have no effect on protein synthesis in this system, indicating that they do not enter the translation machinery, despite the fact that exogenous tRNA rapidly distributes throughout the cells. Furthermore, most of the cellular aminoacyl-tRNA synthetases function only with endogenous tRNAs, although a portion can use exogenous tRNA molecules. However, aminoacylation of these exogenous tRNAs is strongly inhibited by oxidized tRNA; this inhibitor has no effect on endogenous aminoacylation. On the basis of these and the earlier observations, we conclude that endogenous tRNA is never free of the protein synthetic machinery at any stage of the translation process and, consequently, that there is a channeled tRNA cycle during protein synthesis in mammalian cells.