Hélène Hietter

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.

Antagonistic action of cholesterol towards the toxicity of hydroxysterols on cultured hepatoma cells

The cytostatic and cytolytic action of 22R - hydroxydesmosterol on hepatoma cells cultured in a medium containing 10% newborn-calf serum can be reversed within certain concentration limits by adding cholesterol to the culture medium. In contrast, under the same conditions, the cytotoxicity of 7 beta -hydroxycholesterol could not be reversed, whatever the concentrations of cholesterol added. However, in a lipoprotein-poor and in a chemically defined medium, the cytolytic action of both hydroxysterols can be reversed by adding cholesterol, but growth inhibition cannot be suppressed. This demonstrates the importance of serum lipids and lipoproteins for the toxicity of the hydroxysterols and for the antagonistic effect of cholesterol. Our results suggest that the action mechanisms of 7 beta-hydroxycholesterol and 22R - hydroxydesmosterol on HTC hepatoma cells are not fully identical.

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.

Electrophysiological activity of the C-peptide of the Locusta insulin-related peptide Effect on the membrane conductance of Locusta neurones in vitro

The C‐peptide of Locusta insulin‐related peptide, which is a 50 residue peptide originally isolated from the corpora cardiaca of the insect Locusta migratoria and to which we refer as 5‐kDa peptide, has been synthesised chemically by the solid‐phase metho, using a BOC strategy. Since this peptide contains in its sequence a potential monobasic cleavage site, we also synthesised its 1–38 residue‐related fragment, named 4‐kDa peptide, although we have no hints of its natural occurrence in the corpora cardiaca. Electrophysiological studies have shown that both the 5‐kDa and 4‐kDa peptides depolarise the membrane and increase the membrane conductance of neurones freshly isolated from the thoracic ganglia of Locusta. Under voltage‐clamp conditions, the current underlying these effects was inwardly directed and could be resolved into 2 components. One component, I(5‐kDa)1, activated at potentials more hyperpolarised than −50 mV, peaked at about −75 mV and was blocked by the potassium channel blockers cesium and rubidium. The second component, I(5‐kDa)2 was activated at potentials more depolarised than −50 mV, increased with depolarisation and was not blocked by cesium and rubidium. The effects of the 5‐kDa and 4‐kDa peptides on the membrane potential and membrane conductance of Locusta neurones suggest that these peptides may have a physiological role in the central nervous system of insects.

Metabolism of cytotoxic hydroxysterols in cultured cells. Chemical characterization of metabolites

The metabolism of labelled 7 alpha- and 7 beta-hydroxycholesterol was investigated in two lymphoma cell lines (YAC-1, RDM-4), in murine splenocytes and in HTC hepatoma cells. The structures of the metabolites in lymphoma cells were determined as 3 beta-esters of C14-C20 fatty acids by nuclear magnetic resonance and mass spectrometric studies. In hepatoma cells, more polar metabolites of 7 alpha- and 7 beta-hydroxycholesterol were detected whereas, in non-dividing lymphocyte cells, no metabolic transformation occurs. Therefore, metabolic transformation of the hydroxycholesterol is not required for the expression of their activity and the question of the physiological role of the metabolic products is raised.

Inhibition of high voltage-activated Ca2+ currents from cultured sensory neurones by a novel insect peptide

PMP-D2, a novel 35 amino acid peptide isolated from the brain of the locust Locusta migratoria, is localised specifically in neurosecretory cells and nerve tracts of the Pars intercerebralis. When PMP-D2 is applied onto rat sensory neurones it blocks high voltage-activated inward Ca2+ currents at concentrations ranging from 0.1 mu M to 10 mu M. The inhibitory effect of PMP-D2 is more marked on the sustained inward Ca2+ current measured at the end of 100 ms voltage step commands than on the maximum inward Ca2+ current. These results suggests that PMP-D2 may differentially inhibit the two components of the high voltage-activated inward Ca2+ currents of rat sensory neurones.