Bang Luu

SERINE PROTEASE INHIBITION BY INSECT PEPTIDES CONTAINING A CYSTEINE KNOT AND A TRIPLE-STRANDED BETA -SHEET

Three insect peptides showing high sequence similarity and belonging to the same structural family incorporating a cysteine knot and a short three-stranded antiparallel β-sheet were studied. Their inhibitory effect on two serine proteases (bovine α-chymotrypsin and human leukocyte elastase) is reported. One of them, PMP-C, is a strong α-chymotrypsin inhibitor (Ki = 0.2 nM) and interacts with leukocyte elastase with a Ki of 0.12 μM. The other two peptides, PMP-D2 and HI, interact only weakly with α-chymotrypsin and do not inhibit leukocyte elastase. Synthetic variants of these peptides were prepared by solid-phase synthesis, and their action toward serine proteases was evaluated. This enabled us to locate the P1 residues within the reactive sites (Leu-30 for PMP-C and Arg-29 for PMP-D2 and HI), and, interestingly, variants of PMP-D2 and HI were converted into powerful inhibitors of both α-chymotrypsin and leukocyte elastase, the most potent elastase inhibitor obtained in this study having a Ki of 3 nM.

Neurotrophic effect of naturally occurring long-chain fatty alcohols on cultured CNS neurons

A long‐chain fatty alcochol,n‐hexacosanol, that we have isolated from the Far‐Eastern traditional medicinal plant, Hygrophila erecta, Hochr., is shown to promote the maturation of central neurons. Added at 500 nM to fetal rat brain neurons in culture, it increased both neurite outgrowth by a factor of 4–6 and the number of collaterals, especially in multipolar neurons. The biochemical differentiation of cultured neurons was also strikingly enhanced by this compound: it increased the protein content and almost doubled the activities of two neuron‐specific enzymes, phosphate‐activated glutaminase and neuron‐specific enolase, by 92 and 78%, respectively. Extensive studies with several synthetic long‐chain fatty alcohols showed that the neurotrophic activity was maximal for n‐hexacosanol. It is suggested that some long‐chain fatty alcohols with an appropriate length of hydrocarbon chain might play an important role in central neuron development.

Complexation of Two Proteic Insect Inhibitors to the Active Site of Chymotrypsin Suggests Decoupled Roles for Binding and Selectivity

The crystal structures of two homologous inhibitors (PMP-C and PMP-D2v) from the insect Locusta migratoria have been determined in complex with bovine α-chymotrypsin at 2.1- and 3.0-Å resolution, respectively. PMP-C is a potent bovine α-chymotrypsin inhibitor whereas native PMP-D2 is a weak inhibitor of bovine trypsin. One unique mutation at the P1 position converts PMP-D2 into a potent bovine α-chymotrypsin inhibitor. The two peptides have a similar overall conformation, which consists of a triple-stranded antiparallel β-sheet connected by three disulfide bridges, thus defining a novel family of serine protease inhibitors. They have in common the protease interaction site, which is composed of the classical protease binding loop (position P5 to P′4, corresponding to residues 26–34) and of an internal segment (residues 15–18), held together by two disulfide bridges. Structural divergences between the two inhibitors result in an additional interaction site between PMP-D2v (position P10 to P6, residues 21–25) and the residues 172–175 of α-chymotrypsin. This unusual interaction may be responsible for species selectivity. A careful comparison of data on bound and free inhibitors (from this study and previous NMR studies, respectively) suggests that complexation to the protease stabilizes the flexible binding loop (from P5 to P′4).

Conversion of a radiolabelled ecdysone precursor, 2,22,25-trideoxyecdysone, by embryonic and larval tissues of Locusta migratoria

A high specific activity tritiated ecdysone precursor, 2,22,25-trideoxyecdysone, was used to probe the capacity of various embryonic and larval tissues to perform the last 3 hydroxylation steps in ecdysone biosynthesis. Embryos at early stages of development, prior to the differentiation of their endocrine glands and embryonic heads, thoraces and abdomens of later stages, were found to have the capacity to hydroxylate the precursor to ecdysone. Larval epidermis and fat body are also able to transform 2,22,25-trideoxyecdysone into ecdysone; Malpighian tubules and midgut hydroxylate the precursor at C-2 but are apparently unable to hydroxylate both at C-22 and C-25. Larval prothoracic glands convert the precursor to ecdysone at a very efficient rate, which is 1-2 magnitudes higher than that of the other tissues investigated; several data argue for the existence of a privileged sequence of hydroxylations, C-25, C-22, C-2, in the larval prothoracic glands.

Developmental Study of Proteolipids in Bovine Brain: A Novel Proteolipid and DM-20 Appear Before Proteolipid Protein (PLP) During Myelination

Abstract: In a developmental study, we have shown that DM‐20 is present before proteolipid protein (PLP) in the fetal bovine cerebral hemispheres. When the white matter appears (27–30 weeks of gestation), the amount of DM‐20 drastically increases. DM‐20 remains the major proteolipid until birth. PLP is detected only 2–4 weeks after the appearance of white matter, that is, more than 4 weeks after the appearance of DM‐20. The early appearance of DM‐20 at the beginning of myelination raises the question of its particular function. In the adult bovine cerebral hemispheres, PLP is the major proteolipid but DM‐20 remains quantitatively important because the PLP/DM‐20 ratio ranges from 1.5 to 1.7. In the same developmental study we have, in the fetal cerebral hemispheres, isolated and characterized a novel proteolipid (apparent Mr 20,000), which appears even before DM‐20 and is not detected in the adult brain. It is structurally related to PLP and DM‐20 because the first 31 N‐terminal amino acid residues are the same. However, in immunoblot. it did not react either with the antitridecapeptide 117–129 antiserum of PLP or with the anti‐C‐terminal hexapeptide antiserum of PLP.

Cyclohexenonic Long-Chain Fatty Alcohols as Neuronal Growth Stimulators

Neurotrophic factors play an important role in the development and maintenance of neurons, thus providing a suitable therapeutic approach for the treatment of neurodegenerative diseases. However, their clinical use has revealed problematic because of a number of technical and biological disadvantages. Among the different strategies proposed to overcome such difficulties, the search for non-peptide substances with neurotrophic potential is giving promising results. Here we will expose major findings in this field, drawing special attention to cyclohexenonic long-chain fatty alcohols, a novel family of compounds that promote neuronal survival and neurite outgrowth.

Synthesis of a labelled putative precursor of ecdysone—II: [3H4]3β-hydroxy-5β-cholest-7en-6-one: Critical re-evaluation of its role in Locusta migratoria

Abstract We have synthesized a tritiated form of 2,14,22,25-tetradeoxyecdysone (5β-ketol) of high specific activity (115 Ci/mmol). We have examined the capacity of various tissues of Locusta migratoria to use this 5β-ketol, a putative precursor of ecdysone, in ecdysteroid biosynthesis. While larval prothoracic glands convert the radiotracer to labelled 14-deoxyecdysone they fail to hydroxylate the molecule to ecdysone itself. Other larval tissues, embryonic tissues or vitellogenic female ovaries are unable to convert the radiotracer to ecdysone, 20-hydroxyecdysone or 2-deoxyecdysone, the terminal products of biosynthesis in different developmental stages. Using subcellular preparations of prothoracic glands or follicle cells we have been unable to show a biological C-14 hydroxylation of 5β-ketol. It thus appears that the step of C-14 hydroxylation in the biosynthesis of ecdysteroids requires a substrate other than 5β-ketol.

Effect of cyclohexenonic long chain fatty alcohols on neurite outgrowth. Study on structure-activity relationship

Abstract Four series of long chain fatty alcohols bearing a cyclohexenone moiety in addition to a ω-alkanol side chain were synthesized using “Umpolung” reactivity strategy. Their effect on neurite outgrowth was evaluated by means of fetal rat neurons in culture. The length of the ω-hydroxy side chain is a crucial factor for biological activity.

Acetylenic cholesteryl derivatives as irreversible inhibitors of ecdysone biosynthesis

Abstract Two series of acetylenic derivatives of cholesterol were synthesized from stigmasterol and pregnenolone. These compounds carry an acetylenic function at C-22 and were devised with the aim to inhibit the C-22 hydroxylation of ecdysone biosynthesis by a suicide-substrate mechanism. Two of these compounds ( 15a , 15f ) inhibit the synthesis of ecdysone in follicular cells under in vitro conditions. The inhibition is selective of the C-22 hydroxylase system.

Characterization of three structurally-related 8–9 kDa monomeric peptides present in the Corpora cardiaca of Locusta: A revised structure for the neuroparsins

Abstract Recent developments in automated peptide microsequencing, liquid secondary-ion and electrospray mass spectrometry enable unambiguous primary structure determinations of minute amounts of biological material. We have used these methods in combination to characterize the predominant peptides from HPLC eluates of aqueous extracts of corpora cardiaca from adults of Locusta migratoria . Among the molecules or families of molecules clearly predominating in the extracts, we had previously characterized novel peptides (Hietter et al. , 1989, 1990), and we recently identified three structurally-related, cysteine-rich, 8–9 kDa peptides. We present in this paper their complete structure determination. The amino acid sequence of these peptides is superimposable to that of neuroparsins isolated as dimers by Girardie et al. , 1989. However, our experimental data lead us to propose that these molecules are monomers containing six intramolecular disulfide bridges.