Extraction and identification by high resolution mass spectrometry of bioactive substances in different extracts obtained from pomegranate peel.

This work proposes a method to separate proteins and polyphenols in a food byproduct with high bioactive properties and demonstrate by high performance liquid chromatography tandem mass spectrometry its efficiency. Bioactive substances were extracted using high intensity focused ultrasounds. Resulting extract (SR) was submitted to a step for the purification of proteins and to obtain a protein isolate (PI). Both extracts (SR and PI) were digested using two different enzymes (alcalase and thermolysin). Antioxidant, hypocholesterolemic, and antihypertensive properties of hydrolysates were explored. High antioxidant capacity, mainly due to the presence of polyphenols, was observed in SR and its hydrolysates. Hydrolysates obtained from PI using the alcalase enzyme showed simultaneously a high capacity to inhibit cholesterol esterase and to reduce micellar cholesterol solubility. Hydrolysate obtained from PI using the thermolysin enzyme showed a high antihypertensive capacity. Peptides and polyphenols in hydrolysates were identified by RP-HPLC-ESI-Q-TOF. Hydrolysates obtained from PI showed a high amount of peptides and a negligible amount of polyphenols while polyphenols were mainly present in hydrolysates from SR.

Natural Compounds and Their Analogues as Potent Antidotes against the Most Poisonous Bacterial Toxin.

Botulinum neurotoxins (BoNTs), the most poisonous proteins known to humankind, are a family of seven (serotype A to G) immunologically distinct proteins synthesized primarily by different strains of the anaerobic bacterium Clostridium botulinum Being the causative agents of botulism, the toxins block neurotransmitter release by specifically cleaving one of the three soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins, thereby inducing flaccid paralysis. The development of countermeasures and therapeutics against BoNTs is a high-priority research area for public health because of their extreme toxicity and potential for use as biowarfare agents. Extensive research has focused on designing antagonists that block the catalytic activity of BoNTs. In this study, we screened 300 small natural compounds and their analogues extracted from Indian plants for their activity against BoNT serotype A (BoNT/A) as well as its light chain (LCA) using biochemical and cellular assays. One natural compound, a nitrophenyl psoralen (NPP), was identified to be a specific inhibitor of LCA with an in vitro 50% inhibitory concentration (IC50) value of 4.74 ± 0.03 µM. NPP was able to rescue endogenous synaptosome-associated protein 25 (SNAP-25) from cleavage by BoNT/A in human neuroblastoma cells with an IC50 of 12.2 ± 1.7 µM, as well as to prolong the time to the blocking of neutrally elicited twitch tensions in isolated mouse phrenic nerve-hemidiaphragm preparations.IMPORTANCE The long-lasting endopeptidase activity of BoNT is a critical biological activity inside the nerve cell, as it prompts proteolysis of the SNARE proteins, involved in the exocytosis of the neurotransmitter acetylcholine. Thus, the BoNT endopeptidase activity is an appropriate clinical target for designing new small-molecule antidotes against BoNT with the potential to reverse the paralysis syndrome of botulism. In principle, small-molecule inhibitors (SMIs) can gain entry into BoNT-intoxicated cells if they have a suitable octanol-water partition coefficient (log P) value and other favorable characteristics (P. Leeson, Nature 481:455-456, 2012, https://doi.org/10.1038/481455a). Several efforts have been made in the past to develop SMIs, but inhibitors effective under in vitro conditions have not in general been effective in vivo or in cellular models (L. M. Eubanks, M. S. Hixon, W. Jin, S. Hong, et al., Proc Natl Acad Sci U S A 104:2602-2607, 2007, https://doi.org/10.1073/pnas.0611213104). The difference between the in vitro and cellular efficacy presumably results from difficulties experienced by the compounds in crossing the cell membrane, in conjunction with poor bioavailability and high cytotoxicity. The screened nitrophenyl psoralen (NPP) effectively antagonized BoNT/A in both in vitro and ex vivo assays. Importantly, NPP inhibited the BoNT/A light chain but not other general zinc endopeptidases, such as thermolysin, suggesting high selectivity for its target. Small-molecule (nonpeptidic) inhibitors have better oral bioavailability, better stability, and better tissue and cell permeation than antitoxins or peptide inhibitors.

Quantifying the Effects of Different Neutral Proteases on Human Islet Integrity.

Efficient islet release from the pancreas requires the combination of collagenase, neutral protease (cNP), or thermolysin (TL). Recently, it has been shown that clostripain (CP) may also contribute to efficient islet release from the human pancreas. The aim of this study was to evaluate the impact of these proteases on human islet integrity in a prospective approach. Islets were isolated from the pancreas of 10 brain-dead human organ donors. Purified islets were precultured for 3 to 4 d at 37 °C to ensure that preparations were cleared of predamaged islets, and only integral islets were subjected to 90 min of incubation at 37 °C in Hank's balanced salt solution supplemented with cNP, TL, or CP. The protease concentrations were calculated for a pancreas of 100 g trimmed weight utilizing 120 dimethyl-casein units of cNP, 70,000 caseinase units of TL, or 200 benzoyl-l-arginine-ethyl-ester units of CP (1×). These activities were then increased both 5× and 10×. After subsequent 24-h culture in enzyme-free culture medium, treated islets were assessed and normalized to sham-treated controls. Compared with controls and CP, islet yield was significantly reduced by using the 5× activity of cNP and TL, inducing also fragmentation and DNA release. Viability significantly decreased not until adding the 1× activity of cNP, 5× activity of TL, or 10× activity of CP. Although mitochondrial function was significantly lowered by 1× cNP and 5× TL, CP did not affect mitochondria at any concentration. cNP- and TL-incubated islets significantly lost intracellular insulin already at 1× activity, while the 10× activity of CP had to be added to observe a similar effect. cNP and TL have a similar toxic potency regarding islet integrity. CP also induces adverse effects on islets, but the toxic threshold is generally higher. We hypothesize that CP can serve as supplementary protease to minimize cNP or TL activity for efficient pancreas digestion.

Selective isolation of hydrophobin SC3 by solid-phase extraction with polytetrafluoroethylene microparticles and subsequent mass spectrometric analysis.

Hydrophobins are small proteins that play a role in a number of processes during the filamentous fungi growth and development. These proteins are characterized by the self-assembly of their molecules into an amphipathic membrane at hydrophilic-hydrophobic interfaces. Isolation and purification of hydrophobins generally present a challenge in their analysis. Hydrophobin SC3 from Schizophyllum commune was selected as a representative of class I hydrophobins in this work. A novel procedure for selective and effective isolation of hydrophobin SC3 based on solid-phase extraction with polytetrafluoroethylene microparticles loaded in a small self-made microcolumn is reported. The tailored binding of hydrophobins to polytetrafluoroethylene followed by harsh elution conditions resulted in a highly specific isolation of hydrophobin SC3 from the model mixture of ten proteins. The presented isolation protocol can have a positive impact on the analysis and utilization of these proteins including all class I hydrophobins. Hydrophobin SC3 was further subjected to reduction of its highly stable disulfide bonds and to chymotryptic digestion followed by mass spectrometric analysis. The isolation and digestion protocols presented in this work make the analysis of these highly hydrophobic and compact proteins possible.

Discovery of novel plant peptides as strong inhibitors of metalloproteinases.

Five novel metalloproteinase protein inhibitors (MPIs) with molecular mass between 5.6 and 8.9 kDa and acid/neutral pI were detected in lupin seeds and exhibited strong inhibitory activities against thermolysin and/or gelatinase B. These novel peptides constitute not only the first MPIs described in plants but also the first plant peptides with inhibitory activity against a matrixin.

Discovery of potent thermolysin inhibitors using structure based virtual screening and binding assays.

In the present work, 22 compounds of the U.S. NCI compound library (size 273K) were identified as putative thermolysin binders by structure based virtual screening with the ICM software (ICM-VLS). In vitro competitive binding assays confirmed that 12 were thermolysin binders. Thermolysin binding modes of the 12 compounds were studied by docking using ICM and Molegro Virtual Docker (MVD). The most potent inhibitor had an IC(50) value of 6.4 x 10(-8) mM (NSC250686, 1 beta-D-arabinofuranosyl-N(4)-lauroylcytosine). The structure of this compound is quite different from the other 11 compounds. Nine out of the 12 compounds contained a similar chemical skeleton (3-nitrobenzamide derivatives) and have IC(50) values ranging from 697.48 to 0.047 mM. The ICM-VLS score and the activity profiles (pIC(50) values) were compared and found to be somewhat linearly correlated (R(2) = 0.78). Kinetic studies showed that, except for NSC285166 (oxyquinoline), the compounds are competitive thermolysin inhibitors.

MedusaScore: an accurate force field-based scoring function for virtual drug screening.

Virtual screening is becoming an important tool for drug discovery. However, the application of virtual screening has been limited by the lack of accurate scoring functions. Here, we present a novel scoring function, MedusaScore, for evaluating protein-ligand binding. MedusaScore is based on models of physical interactions that include van der Waals, solvation, and hydrogen bonding energies. To ensure the best transferability of the scoring function, we do not use any protein-ligand experimental data for parameter training. We then test the MedusaScore for docking decoy recognition and binding affinity prediction and find superior performance compared to other widely used scoring functions. Statistical analysis indicates that one source of inaccuracy of MedusaScore may arise from the unaccounted entropic loss upon ligand binding, which suggests avenues of approach for further MedusaScore improvement.

Proteolytic cleavage of ostrich and turkey pancreatic lipases: production of an active N-terminal domain.

OBJECTIVES: The aim of this study was to check some biochemical and structural properties of ostrich and turkey pancreatic lipases (OPL and TPL, respectively). METHODS: Limited proteolysis of OPL and TPL was performed in conditions similar to those reported for porcine pancreatic lipase. RESULTS: In the absence of bile salts and colipase, OPL failed to catalyze the hydrolysis of pure tributyrin or efficiently hydrolyze olive oil emulsion. When bile salts and colipase were preincubated with the substrate, the OPL kinetic behavior remained linear for more than 30 minutes. The enzyme presented a penetration power value into an egg phosphatidylcholine monomolecular film that was comparable to that of HPL and lower than that of TPL. Chymotrypsin, trypsin, and thermolysin were able to hydrolyze OPL and TPL in different ways. In both cases, only N-terminal fragments accumulated during the hydrolysis, whereas no C-terminal fragment was obtained in either case. Tryptic cleavage of OPL and TPL completely degraded the enzymes. Nevertheless, chymotryptic attack generated 35-kd and 43-kd forms for TPL and OPL, respectively. Interestingly, the OPL 43-kd form was inactive, whereas the TPL 35-kd protein conserved its lipolytic activity. CONCLUSIONS: OPL, TPL, and mammal pancreatic lipases share a high amino acid sequence homology. Further investigations are, however, needed to identify key residues involved in substrate recognition responsible for biochemical differences between the 2 classes of lipases.

Use of thermolysin in the diagnosis of prion diseases.

The molecular diagnosis of prion diseases almost always involves the use of a protease to distinguish PrPC from PrPSc and invariably the protease of choice is proteinase K. Here, we have applied the protease thermolysin to the diagnosis of animal prion diseases. This thermostable protease cleaves at the hydrophobic residues Leu, Ile, Phe, Val, Ala, and Met, residues that are absent from the protease accessible aminoterminal region of PrPSc. Therefore, although thermolysin readily digests PrPC into small protein fragments, full-length PrPSc is resistant to such proteolysis. This contrasts with proteinase K digestion where an aminoterminally truncated PrPSc species is produced, PrP27-30. Thermolysin was used in the diagnosis of ovine scrapie and bovine spongiform encephalopathy and produced comparable assay sensitivity to assays using proteinase K digestion. Furthermore, we demonstrated the concentration of thermolysin-resistant PrPSc using immobilized metal-affinity chromatography. The use of thermolysin to reveal a full-length PrPSc has application for the development of novel immunodiagnostics by exploiting the wide range of commercially available immunoreagents and metal affinity matrices that bind the amino-terminal region of PrP. In addition, thermolysin provides a complementary tool to proteinase K to allow the study of the contribution of the amino-terminal domain of PrPSc to disease pathogenesis.

Conformational properties of the SDS-bound state of alpha-synuclein probed by limited proteolysis: unexpected rigidity of the acidic C-terminal tail.

Alpha-synuclein (alpha-syn) is a "natively unfolded" protein constituting the major component of intracellular inclusions in several neurodegenerative disorders. Here, we describe proteolysis experiments conducted on human alpha-syn in the presence of SDS micelles. Our aim was to unravel molecular features of micelle-bound alpha-syn using the limited proteolysis approach. The nonspecific proteases thermolysin and proteinase K, as well as the Glu-specific V8-protease, were used as proteolytic probes. While alpha-syn at neutral pH is easily degraded to a variety of relatively small fragments, in the presence of 10 mM SDS the proteolysis of the protein is rather selective. Complementary fragments 1-111 and 112-140, 1-113 and 114-140, and 1-123 and 124-140 are obtained when thermolysin, proteinase K, and V8 protease, respectively, are used. These results are in line with a conformational model of alpha-syn in which it acquires a folded helical structure in the N-terminal region in its membrane-bound state. At the same time, they indicate that the C-terminal portion of the molecule is rather rigid, as seen in its relative resistance to extensive proteolytic degradation. It is likely that, under the specific experimental conditions of proteolysis in the presence of SDS, the negatively charged C-terminal region can be rigidified by binding a calcium ion, as shown before with intact alpha-syn. In this study, some evidence of calcium binding properties of isolated C-terminal fragments 112-140, 114-140, and 124-140 was obtained by mass spectrometry measurements, since molecular masses for calcium-loaded fragments were obtained. Our results indicate that the C-terminal portion of the membrane-bound alpha-syn is quite rigid and structured, at variance from current models of the membrane-bound protein deduced mostly from NMR. Considering that the aggregation process of alpha-syn is modulated by its C-terminal tail, the results of this study may provide useful insights into the behavior of alpha-syn in a membrane-mimetic environment.

A virtual active compound produced from the negative image of a ligand-binding pocket, and its application to in-silico drug screening.

We developed a new structure-based in-silico screening method using a negative image of a ligand-binding pocket and a multi-protein-compound interaction matrix. Based on the structure of the ligand pocket of the target protein, we designed a negative image, which consists of virtual atoms whose radii are close to those of carbon atoms. The virtual atoms fit the pocket ideally and achieve an optimal Coulomb interaction. A protein-compound docking program calculates the protein-compound interaction matrix for many proteins and many compounds including the negative image, which can be treated as a virtual compound. With specific attention to a vector of docking scores for a single compound with many proteins, we selected a compound whose score vector was similar to that of the negative image as a candidate hit compound. This method was applied to representative target proteins and showed high database enrichment with a relatively quick procedure.

A novel suite of cyclotides from Viola odorata: sequence variation and the implications for structure, function and stability.

Cyclotides are a fascinating family of plant-derived peptides characterized by their head-to-tail cyclized backbone and knotted arrangement of three disulfide bonds. This conserved structural architecture, termed the CCK (cyclic cystine knot), is responsible for their exceptional resistance to thermal, chemical and enzymatic degradation. Cyclotides have a variety of biological activities, but their insecticidal activities suggest that their primary function is in plant defence. In the present study, we determined the cyclotide content of the sweet violet Viola odorata, a member of the Violaceae family. We identified 30 cyclotides from the aerial parts and roots of this plant, 13 of which are novel sequences. The new sequences provide information about the natural diversity of cyclotides and the role of particular residues in defining structure and function. As many of the biological activities of cyclotides appear to be associated with membrane interactions, we used haemolytic activity as a marker of bioactivity for a selection of the new cyclotides. The new cyclotides were tested for their ability to resist proteolysis by a range of enzymes and, in common with other cyclotides, were completely resistant to trypsin, pepsin and thermolysin. The results show that while biological activity varies with the sequence, the proteolytic stability of the framework does not, and appears to be an inherent feature of the cyclotide framework. The structure of one of the new cyclotides, cycloviolacin O14, was determined and shown to contain the CCK motif. This study confirms that cyclotides may be regarded as a natural combinatorial template that displays a variety of peptide epitopes most likely targeted to a range of plant pests and pathogens.

Molecular characterization of the human Calpha-formylglycine-generating enzyme.

Calpha-formylglycine (FGly) is the catalytic residue in the active site of sulfatases. In eukaryotes, it is generated in the endoplasmic reticulum by post-translational modification of a conserved cysteine residue. The FGly-generating enzyme (FGE), performing this modification, is an endoplasmic reticulum-resident enzyme that upon overexpression is secreted. Recombinant FGE was purified from cells and secretions to homogeneity. Intracellular FGE contains a high mannose type N-glycan, which is processed to the complex type in secreted FGE. Secreted FGE shows partial N-terminal trimming up to residue 73 without loosing catalytic activity. FGE is a calcium-binding protein containing an N-terminal (residues 86-168) and a C-terminal (residues 178-374) protease-resistant domain. The latter is stabilized by three disulfide bridges arranged in a clamp-like manner, which links the third to the eighth, the fourth to the seventh, and the fifth to the sixth cysteine residue. The innermost cysteine pair is partially reduced. The first two cysteine residues are located in the sequence preceding the N-terminal protease-resistant domain. They can form intramolecular or intermolecular disulfide bonds, the latter stabilizing homodimers. The C-terminal domain comprises the substrate binding site, as evidenced by yeast two-hybrid interaction assays and photocross-linking of a substrate peptide to proline 182. Peptides derived from all known human sulfatases served as substrates for purified FGE indicating that FGE is sufficient to modify all sulfatases of the same species.

Flexible docking under pharmacophore type constraints.

FLEXX-PHARM, an extended version of the flexible docking tool FLEXX, allows the incorporation of information about important characteristics of protein-ligand binding modes into a docking calculation. This information is introduced as a simple set of constraints derived from receptor-based type pharmacophore features. The constraints are determined by selected FLEXX interactions and inclusion volumes in the receptor active site. They guide the docking process to produce a set of docking solutions with particular properties. By applying a series of look-ahead checks during the flexible construction of ligand fragments within the active site, FLEXX-PHARM determines which partially built docking solutions can potentially obey the constraints. Solutions that will not obey the constraints are deleted as early as possible, often decreasing the calculation time and enabling new docking solutions to emerge. FLEXX-PHARM was evaluated on various individual protein-ligand complexes where the top docking solutions generated by FLEXX had high root mean square deviations (RMSD) from the experimentally observed binding modes. FLEXX-PHARM showed an improvement in the RMSD of the top solutions in most cases, along with a reduction in run time. We also tested FLEXX-PHARM as a database screening tool on a small dataset of molecules for three target proteins. In two cases, FLEXX-PHARM missed one or two of the active molecules due to the constraints selected. However, in general FLEXX-PHARM maintained or improved the enrichment shown with FLEXX, while completing the screen in considerably less run time.

Identification of a domain conferring nucleotide binding to the N-acetyl-d-glucosamine 2-epimerase (Renin binding protein).

Renin binding protein (RnBP), a cellular renin inhibitor, has been identified as the enzyme N-acetyl-D-glucosamine (GlcNAc) 2-epimerase. Our recent studies demonstrated that rat GlcNAc 2-epimerase has a ten-times higher affinity for ATP, dATP, and ddATP than the human enzyme [Takahashi, S. et al. (2001) J. Biochem. 130, 815-821]. To identify the domain conferring nucleotide binding to GlcNAc 2-epimerase, we constructed a series of chimeric enzymes successively replacing the three domains of the human enzyme (N-terminal, middle, and C-terminal domains) with the corresponding domains of the rat enzyme. Chimeras were expressed in Escherichia coli JM109 cells under the control of the Taq promoter. The purified chimeric enzymes had GlcNAc 2-epimerase activity and inhibited renin activity in a dose-dependent manner. The recombinant human and rat enzymes required catalytic amounts of ATP with apparent K(m) values of 73 and 5.5 microM, respectively. Chimeric enzymes of HHR, RHH, and RHR (H, human type domain; R, rat type domain) had nearly the same nucleotide specificity as the human GlcNAc 2-epimerase. On the other hand, HRR, HRH, and RRH chimeras had the same nucleotide specificity as the rat enzyme. These results indicate that the middle domain of the GlcNAc 2-epimerase molecule participates in the specificity for and binding of nucleotides, and that nucleotides are essential to form the catalytic domain of the enzyme.

Characterization of five new low-molecular-mass trypsin inhibitors from white mustard (Sinapis alba L.) seed.

Five new low-molecular-mass trypsin inhibitors belonging to the RTI/MTI-2 family were identified from white mustard (Sinapis alba L. ; MTI-2) seed. Purified MTI-2 consisted of a peptide mixture, displaying Ile or Arg at position 43, Trp or kynurenine (Kyn) at position 44, and C-terminal ragged ends. The occurrence of Ile or Arg at position 43 suggested that MTI-2 inhibitors originated from different genes. The presence of 5-oxo-proline (pyroglutamic acid; 5-oxoPro1) and Kyn44 reflected post-translational processing of the serine proteinase inhibitor. MTI-2 showed approximately 70% amino-acid identity with low-molecular-mass trypsin inhibitors isolated from oil rape (Brassica napus var. oleifera; RTI-III) seed and with serine proteinase inhibitors mapped in Arabidopsis thaliana chromosome II (ATTI). Furthermore, MTI-2 was homologous to brazzein, the sweet-tasting protein from Pentadiplandra brazzeana Baillon fruit ( approximately 30% amino-acid identity). Although snake-venom toxins showed a low amino-acid identity (< 20%) with MTI-2, RTI-III, and ATTI, some structurally relevant residues were conserved. The disulfide bridge pattern of MTI-2 (Cys5-Cys27, Cys18-Cys31, Cys42-Cys52, and Cys54-Cys57) corresponded to that of RTI-III and of snake-venom toxins, being different from that of brazzein. Therefore, protein similarity might be attributable to the three-dimensional arrangement rather than to the amino-acid sequence. Values of Ka for MTI-2 binding to bovine beta-trypsin (trypsin) and bovine alpha-chymotrypsin were 6.3 x 109 M-1 and 2.0 x 106 M-1, respectively, at pH 8.0 and 21.0 degrees C. Moreover, values of kon for MTI-2 binding to trypsin and of koff for the dissociation of the serine proteinase:inhibitor complex were 5.6 x 105 M-1.s-1 and 8.9 x 10-5 M-1.s-1, respectively, at pH 8.0 and 21.0 degrees C. Despite the heterogeneity of the purified inhibitor peptide mixture, the inhibition properties of the different MTI-2 inhibitors were indistinguishable.

Aminopeptidase A, which generates one of the main effector peptides of the brain renin-angiotensin system, angiotensin III, has a key role in central control of arterial blood pressure.

Overactivity of the brain renin-angiotensin system (RAS) has been implicated in the development and maintenance of hypertension in several experimental animal models. We have recently reported that, in the murine brain RAS, angiotensin II (AngII) is converted by aminopeptidase A (APA) into angiotensin III (AngIII),which is itself degraded by aminopeptidase N (APN), both peptides being equipotent to increase vasopressin release and arterial blood pressure when injected by the intracerebroventricular (i.c.v.) route. Because AngII is converted in vivo into AngIII, the exact nature of the active peptide is not precisely known. To delineate their respective roles in the central control of cardiovascular functions, specific and selective APA and APN inhibitors are needed to block the metabolic pathways of AngII and AngIII respectively. In the absence of such compounds for APA, we first explored the organization of the APA active site by site-directed mutagenesis. This led us to propose a molecular mechanism of action for APA similar to that proposed for the bacterial enzyme thermolysin deduced from X-ray diffraction studies. Secondly, we developed a specific and selective APA inhibitor, compound EC33 [(S)-3-amino-4-mercaptobutylsulphonic acid], as well as a potent and selective APN inhibitor, PC18 (2-amino-4-methylsulphonylbutane thiol). With these new tools we examined the respective roles of AngII and AngIII in the central control of arterial blood pressure. A central blockade of APA with the APA inhibitor EC33 suppressed the pressor effect of exogenous AngII, suggesting that brain AngII must be converted into AngIII to increase arterial blood pressure. Furthermore, EC33, injected alone i.c.v. but not intravenously, caused a dose-dependent decrease in arterial blood pressure by blocking the formation of brain AngIII but not systemic AngIII. This is corroborated by the fact that the selective APN inhibitor PC18 administered alone via the i.c.v. route increased arterial blood pressure. This pressor response was blocked by prior treatment with the angiotensin type 1 receptor antagonist losartan, showing that blocking the action of APN on AngIII metabolism leads to an increase in endogenous AngIII levels, resulting in arterial blood pressure increase through an interaction with angiotensin type 1 receptors. These results demonstrate that AngIII is a major effector peptide of the brain RAS, exerting a tonic stimulatory control over arterial blood pressure. Thus APA, the enzyme responsible for the formation of brain AngIII, represents a potential central therapeutic target that justifies the development of APA inhibitors, crossing the blood-brain barrier, as central anti-hypertensive agents.

Lipid phosphorylation in chloroplast envelopes. Evidence for galactolipid CTP-dependent kinase activities.

Lipid phosphorylation takes place within the chloroplast envelope. In addition to phosphatidic acid, phosphatidylinositol phosphate, and their corresponding lyso-derivatives, we found that two novel lipids underwent phosphorylation in envelopes, particularly in the presence of carrier-free [gamma-(32)P]ATP. These two lipids incorporated radioactive phosphate in chloroplasts in the presence of [gamma-(32)P]ATP or [(32)P]P(i) and light. Interestingly, these two lipids were preferentially phosphorylated in envelope membranes in the presence [gamma-(32)P]CTP, as the phosphoryl donor, or [gamma-(32)P]ATP, when supplemented with CDP and nucleoside diphosphate kinase II. The lipid kinase activity involved in this reaction was specifically inhibited in the presence of cytosine 5'-O-(thiotriphosphate) (CTPgammaS) and sensitive to CTP chase, thereby showing that both lipids are phosphorylated by an envelope CTP-dependent lipid kinase. The lipids were identified as phosphorylated galactolipids by using an acid hydrolysis procedure that generated galactose 6-phosphate. CTPgammaS did not affect the import of the small ribulose-bisphosphate carboxylase/oxygenase subunit into chloroplasts, the possible physiological role of this novel CTP-dependent galactolipid kinase activity in the chloroplast envelope is discussed.

Assignment of the disulfide bonds of Ole e 1, a major allergen of olive tree pollen involved in fertilization.

The most prevalent allergen from olive tree pollen, Ole e 1, consists of a single polymorphic polypeptide chain of 145 amino acids which includes six cysteine residues at positions 19, 22, 43, 78, 90 and 131. By using an homogeneous form of the allergen expressed in Pichia pastoris, the array of the disulfide bridges has been elucidated. Specific proteolysis with thermolysin and reverse-phase HPLC separation of the peptides allowed the determination of the disulfide bond between Cys43 and Cys78. Another thermolytic product, which contained three peptides linked by the remaining four cysteines, was digested with Glu-specific staphylococcal V8 protease and the products isolated by reverse-phase HPLC. Amino acid compositions and Edman degradation of the peptide products indicated the presence of the disulfide bonds at Cys19-Cys90 and Cys22-Cys131. These data can help in the analysis of the three-dimensional structure of the protein as well as in studies of its allergenic determinants.

Elucidation of the mode of interaction of thermolysin with a proteinaceous metalloproteinase inhibitor, SMPI, based on a model complex structure and a structural dynamics analysis.

SMPI is a proteinaceous microbial metalloproteinase inhibitor that was isolated from Streptomyces nigrescens TK-23 in 1979. SMPI is known to selectively inhibit the metalloproteinases in the gluzincin family, according to the Rawling and Barrett classification. There has been no report on the interaction of a metalloproteinase in the family of gluzincins with its specific proteinaceous inhibitor. We have solved the solution structure of SMPI by NMR. Here, we report the binding mode of SMPI to thermolysin, based on the model complex structure generated using our high-resolution NMR structure of SMPI and the crystal structure of thermolysin. The obtained complex model shows that the extruded loop of SMPI, with the scissile bond Cys64-Val65, is complementary in shape to the active cleft of thermolysin. In the complex, the Cys64 (P1) carbonyl oxygen atom can form a tetrahedral coordination to the active zinc in thermolysin, and simultaneously, the methyl groups of Val65 (P1') are closely located in the hydrophobic S1' pocket in thermolysin. From the electrostatic potential surface calculation, the active loop of SMPI and the active cleft in thermolysin have been shown to be complementary in the surface charge distribution, resulting in the stabilization of the complex. The apparently large active loop is less flexible, but maintains a conformation in the nano- to picosecond time-scale, as elucidated from the 15N spin relaxation analysis. This is a quite different structural feature of SMPI from the flexible binding loop generally found in the serine proteinase inhibitors, such as SSI and eglin c, and can be related to the narrow specificity of SMPI. The present study provides the first insight into the interaction between a proteinaceous inhibitor and a gluzincin metalloproteinase.