Biomedical subjects

J A Hoffmann

Publications and source records attributed to J A Hoffmann.

At least 19 recordsLinked to original sources

PubMed1995

Metchnikowin, a novel immune-inducible proline-rich peptide from Drosophila with antibacterial and antifungal properties.

One of the characteristics of the host defense of higher insects is the rapid and transient synthesis of a variety of potent antimicrobial peptides. To date, several distinct inducible antimicrobial peptides or peptide families have been totally or partially characterized. We present here the isolation and characterization of a novel 26-residue proline-rich immune-inducible peptide from Drosophila, which exhibits both antibacterial (Gram-positive) and antifungal activities. Peptide sequencing and cDNA cloning indicate the presense of two isoforms in our Drosophila Oregon strain, which differ by one residue (His compared to Arg) as a consequence of a single nucleotide change. The gene, which maps in position 52A1-2 on the right arm of the second chromosome, is expressed in the fat body after immune challenge. The novel peptide, which we propose to name metchnikowin, is a member of a family of proline-rich peptides, and we discuss the possible evolutionary relationships within this family.

Amino Acid Sequence↗

PubMed1995

A recessive mutation, immune deficiency (imd), defines two distinct control pathways in the Drosophila host defense.

In this paper we report a recessive mutation, immune deficiency (imd), that impairs the inducibility of all genes encoding antibacterial peptides during the immune response of Drosophila. When challenged with bacteria, flies carrying this mutation show a lower survival rate than wild-type flies. We also report that, in contrast to the antibacterial peptides, the antifungal peptide drosomycin remains inducible in a homozygous imd mutant background. These results point to the existence of two different pathways leading to the expression of two types of target genes, encoding either the antibacterial peptides or the antifungal peptide drosomycin.

Animals↗

PubMed1995

Insect immunity. The inducible antibacterial peptide diptericin carries two O-glycans necessary for biological activity.

A bacterial challenge of larvae of the dipteran insect Phormia terranovae induces the rapid synthesis of diptericin, an antibacterial polypeptide, previously characterized at the amino acid level and indirectly by cDNA cloning studies. This 82-residue polypeptide consists of an N-terminal proline-rich domain and a central and C-terminal glycine-rich domain. Using liquid chromatography coupled to electrospray ionization-mass spectrometry, we demonstrate here that this molecule is more complex than anticipated and carries two O-substitutions on threonine residues, one in the proline-rich domain (residue 10) and one in the glycine-rich domain (residue 54). These substitutions consist of identical trisaccharides: glucose-->galactose-->N-acetylgalactosamine-->(threonine). Treatment of diptericin with O-glycosidase, which selectively removes the substitutions without altering the polypeptide proper, abolishes the antibacterial activity, indicating that this posttranslational modification is essential for biological activity of the polypeptide. We also show that diptericin is posttranslationally modified by a C-terminal amidation.

Amino Acid Sequence↗

PubMed1995

Refined three-dimensional solution structure of insect defensin A.

BACKGROUND: Insect defensin A is a basic 4 kDa protein secreted by Phormia terranovae larvae in response to bacterial challenges or injuries. Previous biological tests suggest that the bacterial cytoplasmic membrane is the target of defensin A. The structural study of this protein is the first step towards establishing a structure-activity relationship and forms the basis for understanding its antibiotic activity at the molecular level. RESULTS: We describe a refined model of the three-dimensional structure of defensin A derived from an extensive analysis of 786 inter-proton nuclear Overhauser effects. The backbone fold involves an N-terminal loop and an alpha-helical fragment followed by an antiparallel beta-structure. The helix and the beta-structure are connected by two of the three disulphide bridges present in defensin A, forming a so-called 'cysteine-stabilized alpha beta' (CS alpha beta) motif. The N-terminal loop, which is locally well defined, can occupy different positions with respect to the other moieties of the molecule. CONCLUSIONS: The CS alpha beta motif, which forms the core of the defensin A structure, appears to be a common organization for several families of small proteins with toxic properties. The distribution of amino acid side chains in the protein structure creates several hydrophobic or hydrophilic patches. This leads us to propose that the initial step in the action of positively charged defensin A molecules with cytoplasmic membranes may involve interactions with acidic phospholipids.

Amino Acid Sequence↗

PubMed1995

Drosophila immunity. A sequence homologous to mammalian interferon consensus response element enhances the activity of the diptericin promoter.

Bacterial challenge of larvae or adults of Drosophila induces the rapid transcription of several genes encoding antibacterial peptides with a large spectrum of activity. One of these peptides, the 82-residue anti-gram negative diptericin, is encoded by a single intronless gene and we are investigating the control of expression of this gene. Previous studies using both transgenic experiments and footprint analysis have highlighted the role in the induction of this gene of a 30 nucleotide region which contains three partially overlapping motifs with sequence homology to mammalian NF-kappa B and NF-IL6 response elements and to the GAAANN sequence present in the interferon consensus response elements of some mammalian interferon-induced genes. We now show that the latter sequence binds in immune responsive tissues (fat body, blood cells) of Drosophila a approximately 45 kDa polypeptide which cross-reacts with a polyserum directed against mammalian interferon Regulatory Factor-I. Using a transfection assay of Drosophila tumorous blood cells, we show that the GAAANN sequence positively regulates the activity of the diptericin promoter. We propose that this motif cooperatively interacts with the other response elements in the regulation of the diptericin gene expression.

Animals↗

PubMed1995

Functional analysis and regulation of nuclear import of dorsal during the immune response in Drosophila.

In addition to its function in embryonic development, the NF-kappa B/rel-related gene dorsal (dl) of Drosophila is expressed in larval and adult fat body where its RNA expression is enhanced upon injury. Injury also leads to a rapid nuclear translocation of dl from the cytoplasm in fat body cells. Here we present data which strongly suggest that the nuclear localization of dl during the immune response is controlled by the Toll signaling pathway, comprising gene products that participate in the intracellular part of the embryonic dorsoventral pathway. We also report that in mutants such as Toll or cactus, which exhibit melanotic tumor phenotypes, dl is constitutively nuclear. Together, these results point to a potential link between the Toll signaling pathway and melanotic tumor induction. Although dl has been shown previously to bind to kappa B-related motifs within the promoter of the antibacterial peptide coding gene diptericin, we find that injury-induced expression of diptericin can occur in the absence of dl. Furthermore, the melanotic tumor phenotype of Toll and cactus is not dl dependent. These data underline the complexity of the Drosophila immune response. Finally, we observed that like other rel proteins, dl can control the level of its own transcription.

Animals↗

PubMed1995

Innate immunity of insects.

Insects are particularly resistant to microorganisms. Their host-defense system relies on several innate reactions: upon injury, the immediate onset of two proteolytic cascades leading to localized blood clotting and to melanization, the latter process involving production of cytotoxic molecules (namely reactive oxygen intermediates); the phagocytosis of bacteria and the encapsulation of larger parasites by blood cells; the induced synthesis by the fat body of a battery of potent antimicrobial peptides/polypeptides which are secreted into the hemolymph where they act synergistically to kill the invading microorganisms. The insect host defence system shares many of the basic characteristics of the mammalian acute phase response, especially at the level of the coordinate control of gene expression, where similar cis-regulatory and inducible transactivators appear to play key functions. The powerful techniques developed to study the genetics of Drosophila provide a unique opportunity to dissect the development and differentiation of this primordial immune system and may contribute to our understanding of the innate immune response in higher organisms.

Animals↗

PubMed1995

Insect immunity: isolation of three novel inducible antibacterial defensins from the vector mosquito, Aedes aegypti.

The injection of Escherichia coli and Micrococcus luteus into the hemocoel of Aedes aegypti induces a potent antibacterial activity in the hemolymph. We have purified and fully characterized three 40-residue antibacterial peptides from the hemolymph of bacteria-challenged mosquitoes that are absent in naive mosquitoes. The peptides are potently active against Gram-positive bacteria and against one of the Gram-negative bacteria that were tested. The amino acid sequences clearly show that the three peptides are novel isoforms of the insect defensin family of antibacterial peptides. They differ from each other by one or two amino acid residues. We present here the complete amino acid sequences of the three isoforms and the activity spectrum of the predominant Aedes defensin.

Aedes↗

PubMed1995

Structure of a protein in a kinetic trap.

We have determined the structure of a metastable disulphide isomer of human insulin. Although not observed for proinsulin folding or insulin-chain recombination, the isomer retains ordered secondary structure and a compact hydrophobic core. Comparison with native insulin reveals a global rearrangement in the orientation of A- and B-chains. One face of the protein's surface is nevertheless in common between native and non-native structures. This face contains receptor-binding determinants, rationalizing the partial biological activity of the isomer. Structures of native and non-native disulphide isomers also define alternative three-dimensional templates. Threading of insulin-like sequences provide an experimental realization of the inverse protein-folding problem.

Amino Acid Sequence↗

PubMed1994

Insect immunity. Septic injury of Drosophila induces the synthesis of a potent antifungal peptide with sequence homology to plant antifungal peptides.

In response to a septic injury (pricking with a bacteria-soaked needle) larvae and adults of Drosophila produce considerable amounts of a 44-residue peptide containing 8 cysteines engaged in intramolecular disulfide bridges. The peptide is synthesized in the fat body, a functional homologue of the mammalian liver, and secreted into the blood of the insect. It exhibits potent antifungal activity but is inactive against bacteria. This novel inducible peptide, which we propose to name drosomycin, shows a significant homology with a family of 5-kDa cysteine-rich plant antifungal peptides recently isolated from seeds of Brassicaceae. This finding underlines that plants and insects can rely on similar molecules in their innate host defense.

Amino Acid Sequence↗

PubMed1994

Insect immunity. A transgenic analysis in Drosophila defines several functional domains in the diptericin promoter.

Diptericins are antibacterial polypeptides which are strongly induced in the fat body and blood cells of dipteran insects in response to septic injury. The promoter of the single-copy, intronless diptericin gene of Drosophila contains several nucleotide sequences homologous to mammalian cis-regulatory motifs involved in the control of acute phase response genes. Extending our previous studies on the expression of the diptericin gene, we now report a quantitative analysis of the contribution of various putative regulatory elements to the bacterial inducibility of this gene, based on the generation of 60 transgenic fly lines carrying different elements fused to a reporter gene. Our data definitively identify two Kappa B-related motifs in the proximal promoter as the sites conferring inducibility and tissue-specific expression to the diptericin gene. These motifs alone, however, mediate only minimal levels of expression. Additional proximal regulatory elements are necessary to attain some 20% of the full response and we suspect a role for sequences homologous to mammalian IL6 response elements and interferon-gamma responsive sites in this up-regulation. The transgenic experiments also reveal the existence of a distal regulatory element located upstream of -0.6 kb which increases the level of expression by a factor of five.

Animals↗

PubMed1993

Insect immunity: the diptericin promoter contains multiple functional regulatory sequences homologous to mammalian acute-phase response elements.

We are using the diptericin gene as a model system to study the control of expression of the genes encoding antibacterial peptides during the Drosophila immune reaction. In order to investigate the putative regulatory regions in the diptericin promoter, we performed DNaseI footprinting experiments combined with gel-shift assays in two inducible systems: the larval fat body and a tumorous Drosophila blood cell line. Our results confirm the importance of kappa B-like elements previously described in the immune response of insects and reveal for the first time the involvement of other regions containing sequences homologous to mammalian acute-phase response elements.

Acute-Phase Proteins↗

PubMed1993

Insect defensin, an inducible antibacterial peptide, forms voltage-dependent channels in Micrococcus luteus.

Insect defensins are cationic, cysteine-rich peptides (approximately 4 kDa) that appear after bacterial challenge or injury in the hemolymph of insects belonging to a large variety of orders. These peptides possess anti-Gram-positive activity and participate in the potent antibacterial defense reactions of insects. Using recombinant insect defensin and the strain Micrococcus luteus as a test organism, we have investigated the mode of action of this peptide. We show that defensin disrupts the permeability barrier of the cytoplasmic membrane of M. luteus, resulting in a loss of cytoplasmic potassium, a partial depolarization of the inner membrane, a decrease in cytoplasmic ATP, and an inhibition of respiration. Potassium loss is inhibited below the order-disorder transition of the lipid hydrocarbon chains. It is also inhibited by divalent cations and by a decrease in the membrane potential below a threshold of 110 mV. We propose that these permeability changes reflect the formation of channels in the cytoplasmic membrane by defensin oligomers. This proposal is supported by patch-clamp experiments that show that insect defensins form channels in giant liposomes.

Adenosine Triphosphate↗

PubMed1992

A novel insect defensin mediates the inducible antibacterial activity in larvae of the dragonfly Aeschna cyanea (Paleoptera, Odonata).

The injection of low doses of bacteria into the aquatic larvae of dragonflies (Aeschna cyanea, Odonata, Paleoptera) induces the appearance in their hemolymph of a potent antibacterial activity. We have isolated a 38-residue peptide from this hemolymph which is strongly active against Gram-positive bacteria and also shows activity against one of the Gram-negative bacteria which was tested. The peptide is a novel member of the insect defensin family of inducible antibacterial peptides, which had so far only been reported from the higher insect orders believed to have evolved 100 million years after the Paleoptera. Aeschna defensin is more potent than defensin from the dipteran Phormia, from which its structure differs in several interesting aspects, which are discussed in the paper.

Amino Acid Sequence↗

PubMed1992

(A-C-B) human proinsulin, a novel insulin agonist and intermediate in the synthesis of biosynthetic human insulin.

The hormone insulin is synthesized in the beta cell of the pancreas as the precursor, proinsulin, where the carboxyl terminus of the B-chain is connected to the amino terminus of the A-chain by a connecting or C-peptide. Proinsulin is a weak insulin agonist that possesses a longer in vivo half-life than does insulin. A form of proinsulin clipped at the Arg65-Gly66 bond has been shown to be more potent than the parent molecule with protracted in vivo activity, presumably as a result of freeing the amino terminal residue of the A-chain. To generate a more active proinsulin-like molecule, we have constructed an "inverted" proinsulin molecule where the carboxyl terminus of the A-chain is connected to the amino terminus of the B-chain by the C-peptide, leaving the critical Gly1 residue free. Transformation of Escherichia coli with a plasmid coding for A-C-B human proinsulin led to the stable production of the protein. By a process of cell disruption, sulfitolysis, anion-exchange chromatography, refolding, and reversed-phase high-performance liquid chromatography, two forms of the inverted proinsulin differing at their amino termini as Gly1 and Met0-Gly1 were identified and purified to homogeneity. Both proteins were shown by a number of analytical techniques to be of the inverted sequence, with insulin-like disulfide bonding. Biological analyses by in vitro techniques revealed A-C-B human proinsulin to be intermediate in potency when compared to human insulin and proinsulin. The time to maximal lowering of blood glucose in the fasted normal rat appeared comparable to that of proinsulin. Additionally, we were able to generate fully active, native insulin from A-C-B human proinsulin by proteolytic transformation. The results of this study lend themselves to the generation of novel insulin-like peptides while providing a simplified route to the biosynthetic production of insulin.

Amino Acid Sequence↗

PubMed1992

Insect immunity: developmental and inducible activity of the Drosophila diptericin promoter.

Diptericins are 9 kDa inducible antibacterial peptides initially isolated from immune haemolymph of Phormia (Diptera). Following the isolation of a Drosophila cDNA encoding a diptericin homologue, we have now cloned a genomic fragment containing the Drosophila diptericin gene. To dissect the regulation of this gene, we have transformed flies with a fusion gene in which the reporter beta-galactosidase gene is under the control of 2.2 kb upstream sequences of the diptericin gene. We show that such a fusion gene is inducible by injection of live bacteria or complete Freund's adjuvant and respects the tissue specific expression pattern of the resident diptericin gene. Our analysis reveals at least four distinct phases in the regulation of this gene: young larvae, late third instar larvae, pupae and adults. This complexity may be related to the presence in the upstream sequences of multiple copies of response elements previously characterized in genes encoding acute phase response proteins in mammals (e.g. NK-kappa B, NF-kappa B related, NF-IL6 response elements).

Acute-Phase Proteins↗

PubMed1992

Two-dimensional 1H NMR study of recombinant insect defensin A in water: resonance assignments, secondary structure and global folding.

A 500 MHz 2D 1H NMR study of recombinant insect defensin A is reported. This defense protein of 40 residues contains 3 disulfide bridges, is positively charged and exhibits antibacterial properties. 2D NMR maps of recombinant defensin A were fully assigned and secondary structure elements were localized. The set of NOE connectivities, 3JNH-alpha H coupling constants as well as 1H/2H exchange rates and delta delta/delta T temperature coefficients of NH protons strongly support the existence of an alpha-helix (residues 14-24) and of an antiparallel beta-sheet (residues 27-40). Models of the backbone folding were generated by using the DISMAN program and energy refined by using the AMBER program. This was done on the basis of: (i) 133 selected NOEs, (ii) 21 dihedral restraints from 3JNH-alpha H coupling constants, (iii) 12 hydrogen bonds mostly deduced from 1H/2H exchange rates or temperature coefficients, in addition to 9 initial disulfide bridge covalent constraints. The two secondary structure elements and the two bends connecting them involve approximately 70% of the total number of residues, which impose some stability in the C-terminal part of the molecule. The remaining N-terminal fragment forms a less well defined loop. This spatial organization, in which a beta-sheet is linked to an alpha-helix by two disulfide bridges and to a large loop by a third disulfide bridge, is rather similar to that found in scorpion charybdotoxin and seems to be partly present in several invertebrate toxins.

Amino Acid Sequence↗

PubMed1992

Insect defensins: inducible antibacterial peptides.

In response to bacterial challenge or trauma, insects produce a battery of bactericidal or bacteriostatic molecules with a broad spectrum of activity against Gram-positive and/or Gram-negative bacteria; most are small-sized cationic peptides. This review focuses on insect defensins, a large group of inducible antibacterial peptides that are present both in ancient and recent insect orders. This immune response of insects shares many of the characteristics of the mammalian acute phase response.

Amino Acid Sequence↗