Delivery of lethal dsRNAs in insect diets by branched amphiphilic peptide capsules.

Development of new and specific insect pest management methods is critical for overcoming pesticide resistance and collateral off-target killings. Gene silencing by feeding dsRNA to insects shows promise in this area. Here we described the use of a peptide nano-material, branched amphiphilic peptide capsules (BAPCs), that facilitates cellular uptake of dsRNA by insects through feeding. The insect diets included dsRNA with and without complexation with BAPCs. The selected insect species come from two different orders with different feeding mechanisms: Tribolium castaneum and Acyrthosiphon pisum. The gene transcripts tested (BiP and Armet) are part of the unfolded protein response (UPR) and suppressing their translation resulted in lethality. For Acyrthosiphon pisum, ingestion of BiP-dsRNA associated with BAPCs led to the premature death of the aphids (t1/2=4-5days) compared to ingestion of the same amounts of free BiP-dsRNA (t1/2=11-12days). Tribolium castaneum was effectively killed using a combination of BiP-dsRNA and Armet-dsRNA complexed with BAPCs; most dying as larvae or during eclosion (~75%). Feeding dsRNA alone resulted in fewer deaths (~30%). The results show that complexation of dsRNA with BAPCs enhanced the oral delivery of dsRNA over dsRNA alone.

Gene delivery and immunomodulatory effects of plasmid DNA associated with Branched Amphiphilic Peptide Capsules.

We recently reported on a new class of branched amphiphilic peptides that associate with double stranded DNA and promote in vitro transfection of eukaryotic cells. In the present study, we tested a different formulation in which plasmid DNA associates with the surface of preformed 20-30nm cationic capsules formed through the self-assembly of the two branched amphiphilic peptides. Under these conditions, the negatively charged DNA interacts with the cationic surface of the Branched Amphiphilic Peptide Capsules (BAPCs) through numerous electrostatic interactions generating peptide-DNA complexes with sizes ranging from 50 to 250nm. The BAPCs-DNA nanoparticles are capable of delivering plasmid DNA of different size into cells in culture, yielding high transfection rates and minimal cytotoxicity. Furthermore, BAPCs were tested for in vivo delivery of a DNA vaccine previously designed to activate immune responses and capable of controlling tumors induced by type 16 human papilloma virus (HPV-16). The BAPCs-DNA nanoparticles enhanced the vaccine-induced antitumor protection and promoted activation of murine dendritic cells without significant toxic effects. These results indicate that branched amphiphilic oligo-peptides nanoparticles represent a new and promising nonviral DNA/gene delivery approach endowing immunomodulatory properties for DNA vaccines.

Differential protein expression in the midgut of Culex quinquefasciatus mosquitoes induced by the insecticide temephos.

Mosquitoes are vectors for pathogens of malaria, lymphatic filariasis, dengue, chikungunya, yellow fever and Japanese encephalitis. Culex quinquefasciatus Say, 1823 (Diptera: Culicidae) is a known vector of lymphatic filariasis. Its control in Brazil has been managed using the organophosphate temephos. Studies examining the proteins of Cx. quinquefasciatus that are differentially expressed in response to temephos further understanding of the modes of action of the insecticide and may potentially identify resistance factors in the mosquito. In the present study, a comparative proteomic analysis, using 2-dimensional electrophoresis coupled with matrix-assisted laser desorption/ionization (MALDI) time of flight (TOF)/TOF mass spectrometry, and bioinformatics analyses were performed to identify midgut proteins in Cx. quinquefasciatus larvae that were differentially expressed in response to exposure to temephos relative to those in untreated controls. A total of 91 protein spots were differentially expressed; 40 were upregulated and 51 were downregulated by temephos. A total of 22 proteins, predominantly upregulated, were identified as known to play a role in the immune response, whereas the downregulated proteins were involved in energy and protein catabolism. This is the first proteome study of the midgut of Cx. quinquefasciatus and it provides insights into the molecular mechanisms of insecticide-induced responses in the mosquito.

Proteomic analysis of Frankliniella occidentalis and differentially expressed proteins in response to tomato spotted wilt virus infection.

Tomato spotted wilt virus (TSWV) is transmitted by Frankliniella occidentalis in a persistent propagative manner. Despite the extensive replication of TSWV in midgut and salivary glands, there is little to no pathogenic effect on F. occidentalis. We hypothesize that the first-instar larva (L1) of F. occidentalis mounts a response to TSWV that protects it from pathogenic effects caused by virus infection and replication in various insect tissues. A partial thrips transcriptome was generated using 454-Titanium sequencing of cDNA generated from F. occidentalis exposed to TSWV. Using these sequences, the L1 thrips proteome that resolved on a two-dimensional gel was characterized. Forty-seven percent of the resolved protein spots were identified using the thrips transcriptome. Real-time quantitative reverse transcriptase PCR (RT-PCR) analysis of virus titer in L1 thrips revealed a significant increase in the normalized abundance of TSWV nucleocapsid RNA from 2 to 21 h after a 3-h acquisition access period on virus-infected plant tissue, indicative of infection and accumulation of virus. We compared the proteomes of infected and noninfected L1s to identify proteins that display differential abundances in response to virus. Using four biological replicates, 26 spots containing 37 proteins were significantly altered in response to TSWV. Gene ontology assignments for 32 of these proteins revealed biological roles associated with the infection cycle of other plant- and animal-infecting viruses and antiviral defense responses. Our findings support the hypothesis that L1 thrips display a complex reaction to TSWV infection and provide new insights toward unraveling the molecular basis of this interaction.

Structural and biophysical properties of a synthetic channel-forming peptide: designing a clinically relevant anion selective pore.

The design, synthesis, modeling and in vitro testing of channel-forming peptides derived from the cys-loop superfamily of ligand-gated ion channels are part of an ongoing research focus. Over 300 different sequences have been prepared based on the M2 transmembrane segment of the spinal cord glycine receptor α-subunit. A number of these sequences are water-soluble monomers that readily insert into biological membranes where they undergo supramolecular assembly, yielding channels with a range of selectivities and conductances. Selection of a sequence for further modifications to yield an optimal lead compound came down to a few key biophysical properties: low solution concentrations that yield channel activity, greater ensemble conductance, and enhanced ion selectivity. The sequence NK(4)-M2GlyR T19R, S22W (KKKKPARVGLGITTVLTMRTQW) addressed these criteria. The structure of this peptide has been analyzed by solution NMR as a monomer in detergent micelles, simulated as five-helix bundles in a membrane environment, modified by cysteine-scanning and studied for insertion efficiency in liposomes of selected lipid compositions. Taken together, these results define the structural and key biophysical properties of this sequence in a membrane. This model provides an initial scaffold from which rational substitutions can be proposed and tested to modulate anion selectivity. This article is part of a Special Issue entitled: Protein Folding in Membranes.

Expression of an artificial Cl- channel in microperfused renal proximal tubules.

To better understand the process of fluid movement driven by Cl- conductance, a Cl- channel-forming peptide was delivered to the luminal membrane of microperfused rabbit renal proximal tubules. When the peptide (NK4-M2GlyR) was perfused, a significant new conductance was observed within 3 min and stabilized at 10 min. Alteration of the ion composition revealed it to be a Cl(-)-specific conductance. Reabsorption of Cl- (JCl) was increased by NK4-M2GlyR, but not by a scramble NK4-M2GlyR sequence, suggesting that the active peptide formed de novo Cl- channels in the luminal membrane of the perfused tubules. In the presence of the peptide, reabsorption of fluid (Jv) was dramatically increased and JNa and JCa were concomitantly increased. We propose that introduction of the new Cl- conductance in the luminal membrane leads to a coordinated efflux of water across the membrane and an increase in cation translocation via the paracellular pathway, resulting in an increase in Jv. This novel method could prove useful in characterizing mechanisms of fluid transport driven by Cl- gradients.

Structural implications of placing cationic residues at either the NH2- or COOH-terminus in a pore-forming synthetic peptide.

Restoration of chloride conductance via introduction of an anion-selective pore, formed by a channel-forming peptide, has been hypothesized as a novel treatment modality for patients with cystic fibrosis. Delivery of these peptides from an aqueous environment in the absence of organic solvents is paramount. M2GlyR peptides, designed based on the glycine receptor, insert into lipid bilayers and polarized epithelial cells and assemble spontaneously into chloride-conducting pores. Addition of 4 lysine residues to either terminus increases the solubility of M2GlyR peptides. Both orientations of the helix within the membrane form an anion-selective pore, however, differences in solubility, associations and channel-forming activity are observed. To determine how the positioning of the lysine residues affects these properties, structural characteristics of the lysyl-modified peptides were explored utilizing chemical cross-linking, NMR and molecular modeling. Initial model structures of the a-helical peptides predict that lysine residues at the COOH-terminus form a capping structure by folding back to form hydrogen bonds with backbone carbonyl groups and hydroxyl side chains of residues in the helical segment of the peptide. In contrast, lysine residues at the NH2-terminus form fewer H-bonds and extend away from the helical backbone. Results from NMR and chemical cross-linking support the model structures. The C-cap formed by H-bonding of lysine residues is likely to account for the different biophysical properties observed between NH2- and COOH-terminal-modified M2GlyR peptides.

Synthetic chloride channel restores glutathione secretion in cystic fibrosis airway epithelia.

Cystic fibrosis (CF), an inherited disease characterized by defective epithelial Cl- transport, damages lungs via chronic inflammation and oxidative stress. Glutathione, a major antioxidant in the epithelial lung lining fluid, is decreased in the apical fluid of CF airway epithelia due to reduced glutathione efflux (Gao L, Kim KJ, Yankaskas JR, and Forman HJ. Am J Physiol Lung Cell Mol Physiol 277: L113-L118, 1999). The present study examined the question of whether restoration of chloride transport would also restore glutathione secretion. We found that a Cl- channel-forming peptide (N-K4-M2GlyR) and a K+ channel activator (chlorzoxazone) increased Cl- secretion, measured as bumetanide-sensitive short-circuit current, and glutathione efflux, measured by high-performance liquid chromatography, in a human CF airway epithelial cell line (CFT1). Addition of the peptide alone increased glutathione secretion (181 +/- 8% of the control value), whereas chlorzoxazone alone did not significantly affect glutathione efflux; however, chlorzoxazone potentiated the effect of the peptide on glutathione release (359 +/- 16% of the control value). These studies demonstrate that glutathione efflux is associated with apical chloride secretion, not with the CF transmembrane conductance regulator per se, and the defect of glutathione efflux in CF can be overcome pharmacologically.

NH(2)-terminal modification of a channel-forming peptide increases capacity for epithelial anion secretion.

A synthetic, channel-forming peptide, derived from the alpha-subunit of the glycine receptor (M2GlyR), has been synthesized and modified by adding four lysine residues to the NH(2) terminus (N-K(4)-M2GlyR). In Ussing chamber experiments, apical N-K(4)-M2GlyR (250 microM) increased transepithelial short-circuit current (I(sc)) by 7.7 +/- 1.7 and 10.6 +/- 0.9 microA/cm(2) in Madin-Darby canine kidney and T84 cell monolayers, respectively; these values are significantly greater than those previously reported for the same peptide modified by adding the lysines at the COOH terminus (Wallace DP, Tomich JM, Iwamoto T, Henderson K, Grantham JJ, and Sullivan LP. Am J Physiol Cell Physiol 272: C1672-C1679, 1997). N-K(4)-M2GlyR caused a concentration-dependent increase in I(sc) (k([1/2]) = 190 microM) that was potentiated two- to threefold by 1-ethyl-2-benzimidazolinone. N-K(4)-M2GlyR-mediated increases in I(sc) were insensitive to changes in apical cation species. Pharmacological inhibitors of endogenous Cl(-) conductances [glibenclamide, diphenylamine-2-dicarboxylic acid, 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4,4'-dinitrostilben-2,2'-disulfonic acid, indanyloxyacetic acid, and niflumic acid] had little effect on N-K(4)-M2GlyR-mediated I(sc). Whole cell membrane patch voltage-clamp studies revealed an N-K(4)-M2GlyR-induced anion conductance that exhibited modest outward rectification and modest time- and voltage-dependent activation. Planar lipid bilayer studies yielded results indicating that N-K(4)-M2GlyR forms a 50-pS anion conductance with a k([1/2]) for Cl(-) of 290 meq. These results indicate that N-K(4)-M2GlyR forms an anion-selective channel in epithelial monolayers and shows therapeutic potential for the treatment of hyposecretory disorders such as cystic fibrosis.

A synthetic channel-forming peptide induces Cl(-) secretion: modulation by Ca(2+)-dependent K(+) channels.

A synthetic Cl(-) channel-forming peptide, C-K4-M2GlyR, applied to the apical membrane of human epithelial cell monolayers induces transepithelial Cl(-) and fluid secretion. The sequence of the core peptide, M2GlyR, corresponds to the second membrane-spanning region of the glycine receptor, a domain thought to line the pore of the ligand-gated Cl(-) channel. Using a pharmacological approach, we show that the flux of Cl(-) through the artificial Cl(-) channel can be regulated by modulating basolateral K(+) efflux through Ca(2+)-dependent K(+) channels. Application of C-K4-M2GlyR to the apical surface of monolayers composed of human colonic cells of the T84 cell line generated a sustained increase in short-circuit current (I(SC)) and caused net fluid secretion. The current was inhibited by the application of clotrimazole, a non-specific inhibitor of K(+) channels, and charybdotoxin, a potent inhibitor of Ca(2+)-dependent K(+) channels. Direct activation of these channels with 1-ethyl-2-benzimidazolinone (1-EBIO) greatly amplified the Cl(-) secretory current induced by C-K4-M2GlyR. The effect of the combination of C-K4-M2GlyR and 1-EBIO on I(SC) was significantly greater than the sum of the individual effects of the two compounds and was independent of cAMP. Treatment with 1-EBIO also increased the magnitude of fluid secretion induced by the peptide. The cooperative action of C-K4-M2GlyR and 1-EBIO on I(SC) was attenuated by Cl(-) transport inhibitors, by removing Cl(-) from the bathing solution and by basolateral treatment with K(+) channel blockers. These results indicate that apical membrane insertion of Cl(-) channel-forming peptides such as C-K4-M2GlyR and direct activation of basolateral K(+) channels with benzimidazolones may coordinate the apical Cl(-) conductance and the basolateral K(+) conductance, thereby providing a pharmacological approach to modulating Cl(-) and fluid secretion by human epithelia deficient in cystic fibrosis transmembrane conductance regulator Cl(-) channels.

Aqueous solubilization of transmembrane peptide sequences with retention of membrane insertion and function.

We recently reported that the peptide C-K4-M2GlyR mimics the action of chloride channels when incorporated into the apical membrane of cultured renal epithelial monolayers. C-K4-M2GlyR is one of a series of peptides that were prepared by the addition of lysine residues to the N- or C-terminus of the M2 transmembrane sequence of the brain glycine receptor. This study addresses how such modifications affect physical properties such as aqueous solubility, aggregation, and secondary structure, as well as the ability of the modified peptides to form channels in epithelial monolayers. A graded improvement in solubility with a concomitant decrease in aggregation in aqueous media was observed for the M2GlyR transmembrane sequences. Increases in short-circuit current (I(SC)) of epithelial monolayers were observed after treatment with some but not all of the peptides. The bioactivity was higher for the more soluble, less aggregated M2GlyR peptides. As described in our previous communication, sensitivity of channel activity to diphenylamine-2-carboxylate, a chloride channel blocker, and bumetanide, an inhibitor of the Na/K/2Cl cotransporter, was used to assess changes in chloride selectivity for the different assembled channel-forming peptides. The unmodified M2GlyR sequence and the modified peptides with less positive charge are more sensitive to these agents than are the more highly charged forms. This study shows that relatively insoluble transmembrane sequences can be modified such that they are easier to purify and deliver in the absence of organic solvents with retention of membrane association, insertion, and assembly.

A synthetic peptide derived from glycine-gated Cl- channel induces transepithelial Cl- and fluid secretion.

M2GlyR is a synthetic 23-amino acid peptide that mimics the second membrane-spanning region of the alpha-subunit of the postsynaptic glycine receptor. This peptide has been shown to form an anion-selective channel in phospholipid bilayers. We have investigated the possibility that the peptide may incorporate into the apical membrane of secretory epithelia and induce the secretion of Cl- and water. We improved the solubility of this peptide by adding four lysine residues to the carboxy terminus, C-K4-M2GlyR, and assayed its channel-forming activity using a subculture of Madin-Darby canine kidney (MDCK) cells. The addition of 100 microM C-K4-M2GlyR to the apical surface of MDCK monolayers significantly increased short-circuit current (Ise), hyperpolarized transepithelial potential difference, and induced fluid secretion. The increase in Ise was inhibited by 100 microM bumetanide and by Cl- channel inhibitors. The effectiveness of the channel blockers followed the sequence niflumic acid > or = 5-nitro-2-(3-phenylpropylamino)benzoate > diphenylamine-2-carboxylate (DPC) > glibenclamide. The effect of the peptide was not inhibited by 4.4'-diisothiocyanostilbene-2-2'-disulfonic acid. Removing Cl from the bathing solutions also inhibited the effect of the peptide. The Cl- efflux pathway induced by C-K4-M2GlyR differs from the native pathway activated by the adenosine 3',5'-cyclic monophosphate (cAMP) agonist, forskolin. First, intracellular cAMP levels were unaffected. Second, the concentration of DPC required to inhibit the effect of the peptide was much lower than that needed to block the forskolin response (100 microM vs. 3 mM). These results support the hypothesis that the synthetic peptide C-K4-M2GlyR can from Cl -selective channels in the apical membrane of secretory epithelial cells and can induce sustained transepithelial secretion of Cl- and fluid.

Physical characterization of a totally synthetic rubredoxin.

The entire polypeptide of hyperthermophilic Pyrococcus furiosus rubredoxin was synthesized in order to specifically probe structural determinants of protein thermostability. The uv-visible, circular dichroic, electron paramagnetic, and nuclear magnetic resonance spectra, and electrochemical properties, of the native and synthetic proteins were essentially identical. The synthetic protein had a half-life for denaturation of 24 hr at 80 degrees C. The synthetic protein is considerably more thermostable than nonhyperthermophilic rubredoxins, but not as stable as the native protein. Based on the spectroscopic evidence, it appears that the synthetic protein is incorporating iron properly to form holoprotein, but the peptide still may not be folded correctly.

Synthetic mutants of Clostridium pasteurianum ferredoxin: open iron sites and testing carboxylate coordination.

The entire polypeptide chains for two new Clostridium pasteurianum ferredoxin (Fd) mutants were prepared with the following site-specific substitutions: Cys11Asp and Cys11 alpha-aminobutyric acid (Cys11 alpha-Aba), the latter being a non-naturally occurring amino acid. Standard t-Boc procedures were used for the synthesis and the peptides. The two apoproteins were reconstituted to the 2[4Fe-4S] holoprotein and their spectroscopic, redox and thermal properties were compared with those of native C.pasteurianum Fds. The fully reconstituted Cys11Asp and Cys11 alpha-Aba mutants were initially found to have both clusters intact, i.e. they were 2[4Fe-4S] ferredoxins. The unconventional ligands of Asp and alpha-Aba led to holo-Fds that were not very stable and easily released an iron to form the [3Fe-4S] cluster, presumably through oxidation. The Cys11 alpha-Aba mutant was somewhat more thermally stable than Cys11Asp. In contrast, while both mutants were less stable than the native protein upon exposure to oxygen, the Cys11 alpha-Aba mutant was less stable than Cys11Asp. The Cys11Gly mutant was also prepared, but all attempts, despite repeated and varied experimental conditions, at reconstitution to the Cys11Gly holo 2[4Fe-4S] Fd were unsuccessful, probably because a Gly-Gly sequence is known to break structure. This work, when compared with molecular biological site-specific mutagenesis, shows some of the advantages of chemical/in vitro reconstitution: certain mutants which cannot be detected as holoproteins by site-specific mutagenesis can be formed after all in vitro. Nonetheless, it seems apparent that altering any of the Cys coordination sites of the Fd clusters results in fundamentally more unstable ferredoxins.

Membrane topography and near-neighbor relationships of the mitochondrial ATP synthase subunits e, f, and g.

The well characterized subunits of the bovine ATP synthase complex are the alpha, beta, gamma, delta, and epsilon subunits of the catalytic sector, F1; the ATPase inhibitor protein; and subunits a, b, c, and d, OSCP (oligomycin sensitivity-conferring protein), F6, and A6L, which are present in the membrane sector, F0, and the 45-A-long stalk that connects F1 to F0. It has been shown recently that bovine ATP synthase preparations also contain three small polypeptides, designated e, f, and g, with respective molecular masses of 8.2, 10. 2, and 11.3 kDa. To ascertain their involvement as bona fide subunits of the ATP synthase and to investigate their membrane topography and proximity to the above ATP synthase subunits, polyclonal antipeptide antibodies were raised in the rabbit to the COOH-terminal amino acid residues 57-70 of e, 75-86 of f, and 91-102 of g. It was shown that (i) e, f, and g could be immunoprecipitated with anti-OSCP IgG from a fraction of bovine submitochondrial particles enriched in oligomycin-sensitive ATPase; (ii) the NH2 termini of f and g are exposed on the matrix side of the mitochondrial inner membrane and can be curtailed by proteolysis; (iii) the COOH termini of all three polypeptides are exposed on the cytosolic side of the inner membrane; and (iv) f cross-links to A6L and to g, and e cross-links to g and appears to form an e-e dimer. Thus, the bovine ATP synthase complex appears to have 16 unlike subunits, twice as many as its counterpart in Escherichia coli.

The multiple endocrine neoplasia type 2B point mutation alters long-term regulation and enhances the transforming capacity of the epidermal growth factor receptor.

The RET proto-oncogene encodes a member of the receptor tyrosine kinase family. Multiple endocrine neoplasia type 2B (MEN 2B) is caused by the mutation of a conserved methionine to a threonine in the catalytic domain of the RET kinase. When the MEN 2B point mutation was introduced into the epidermal growth factor (EGF) receptor (M857T EGFR), the intrinsic tyrosine kinase activity of the mutant receptor was similar to that of wild-type EGF receptor and remained ligand-dependent. However, the mutant receptor showed an enhanced transforming capacity compared to the wild-type receptor as judged by its ability to mediate the growth of NIH 3T3 cells in soft agar. Using the oriented peptide library approach to examine substrate specificity, the M857T mutation was found to be associated with a decrease in the selectivity of the receptor for Phe and an increase in the selectivity for acidic residues at the P + 1 position as compared to wild-type EGF receptor. Short-term responses to EGF were similar in cells expressing wild-type and M857T EGF receptors. However, significant differences in receptor down-regulation were observed between the two receptors. These data demonstrate that the MEN 2B point mutation alters the substrate specificity of receptor tyrosine kinases and suggest that the enhanced oncogenesis associated with the MEN 2B mutation may be due in part to alterations in receptor regulation.

Identification of the neurotrophic factor sequence of prosaposin.

Prosaposin, recently identified as a neurotrophic factor (1), is the precursor of saposins A, B, C, and D. The neurotrophic activity of prosaposin resides in the saposin C domain. We have pinpointed the active sequence to a linear 12-mer located in the NH2-terminal sequence of saposin C (LIDNNKTEKEIL). Nanomolar concentrations of a 22-mer peptide encompassing this region stimulated neurite outgrowth and choline acetyltransferase activity, and prevented cell death in neuroblastoma cells. In primary cerebellar granule cells, the 22-mer also stimulated neurite outgroth. Studies of the neuroblastoma line NS20Y using a radiolabeled 18-mer from the neurotrophic region identified a high-affinity (Kd = 70 pM) binding site indicative of receptor-ligand interaction. The 22-mer stimulated protein phosphorylation of several proteins, some of which were tyrosine-phosphorylated after brief exposure similar to saposin C. Circular dichroism studies demonstrated that the 22-mer was converted from a random to a helical structure by addition of ganglioside GM1. The results are consistent with receptor-ligand binding by the peptide initiating a signal transduction cascade and resulting in neuronal differentiation.

Identification of the binding and activating sites of the sphingolipid activator protein, saposin C, with glucocerebrosidase.

Saposin C is a sphingolipid activator protein of 8.5 kDa that activates lysosomal glucocerebrosidase. Previously, we synthesized and characterized a synthetic full-length human saposin C protein that displays 85% of the activity of the native saposin C. In this study we use shorter synthetic peptides derived from the saposin C sequence to map binding and activation sites. By determining the activity and kinetic constant (Kact) values of these peptides, we have identified two functional domains, each comprising a binding site adjacent to or partially overlapping with an activation site. Domains 1 and 2 are located within amino acid positions 6-34 and 41-60, respectively. The activation sites span residues 27-34 and 41-49, whereas binding sites encompass residues 6-27 and 45-60. Peptides containing the sequences of either domain displayed 90% of the activity of the full-length synthetic saposin C. Domain 2, however, bound to glucocerebrosidase by at least an order of magnitude more strongly than domain 1. Binding sites within these domains contain sequences that are excellent candidates for forming amphipathic helical structures. Competition assays demonstrated that the binding of one domain to glucocerebrosidase prevents binding of the other domain, and that saposin A and saposin C bind to the same sites on glucocerebrosidase. A model predicting a saposin C:glucocerebrosidase complex with a stoichiometry of 4:2, respectively, is presented.

Position of the sulfhydryl group and the disulfide bonds of human glucocerebrosidase.

Purified human glucocerebrosidase isolated from placenta was modified with [14C]-iodoacetic acid without reduction and digested with both protease-V8 at pH 4.0 followed by alpha-chymotrypsin at pH 7.5. The majority of radioactivity was found in a peptide that contained the [14C]-carboxymethylated-cysteine identified as CM-Cys18. Direct sequencing of the N-terminus of the intact labeled protein confirmed the modification of Cys18. For identification of disulfide bond-containing peptides, another portion of glucocerebrosidase was alkylated with nonlabeled iodoacetic acid and then digested with protease V8 and alpha-chymotrypsin as before. Twenty-eight HPLC fragments were collected. These purified peaks were then reduced with beta-mercaptoethanol followed by S-carboxymethylation with [14C]-iodoacetic acid. Three peptides among these 28 peptides generated two radioactive daughter peptides. These peptides were sequenced and the position of the radioactive CM-cysteines identified. The locations of these disulfides are Cys4-Cys16, Cys23-Cys342, and Cys126-Cys248. Attempts to reproduce the free sulfhydryl labeling experiments using the glucocerebrosidase isolated from Ceredase proved unsuccessful. No label was incorporated by this enzyme prior to reduction. This result suggests that the form of the protein used in the clinic differs from the native protein.

Benzodiazepines and peptides stimulate pregnenolone synthesis in brain mitochondria.

Mitochondria isolated from rat brain were found to cleave cholesterol to produce pregnenolone, the precursor for hormonal steroids, at a mean rate of 21.0 pmol pregnenolone.mg protein-1.min-1. This rate-limiting step in steroidogenesis was significantly stimulated by PK 11195 (1-(2-chlorophenyl)-N-methyl-(1-methylpropyl)-3-isoquinoline carboxamide) and Ro5 4864 (4'-chlorodiazepam), ligands which bind to peripheral benzodiazepine receptors with high affinity. Low-affinity ligands for the peripheral benzodiazepine receptor such as Ro15 1788 (ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5 alpha][1,4] benzo-3-carboxylate) and clonazepam had no significant effect on the rate of pregnenolone synthesis. Furthermore, the rank order of potency of these compounds as inhibitors of [3H]Ro5 4864 binding was identical to the rank order for steroid production. Since the 86-amino acid peptide diazepam binding inhibitor is also thought to bind to the peripheral benzodiazepine receptor, four fragments of this peptide, a random sequence and steroidogenesis activator peptide were also evaluated for their ability to interact with peripheral benzodiazepine receptors and to stimulate steroidogenesis in rat brain mitochondria. Steroidogenesis activator peptide and two fragments of diazepam binding inhibitor significantly stimulated pregnenolone biosynthesis. In contrast to the peripheral benzodiazepine receptor ligands, no correlation between peptide potency in displacing [3H]Ro5 4864 binding and steroidogenesis was observed.