Christoph Gelhaus

In an early branching metazoan, bacterial colonization of the embryo is controlled by maternal antimicrobial peptides

Early embryos of many organisms develop outside the mother and are immediately confronted with myriads of potential colonizers. How these naive developmental stages control and shape the bacterial colonization is largely unknown. Here we show that early embryonic stages of the basal metazoan Hydra are able to control bacterial colonization by using maternal antimicrobial peptides. Antimicrobial peptides of the periculin family selecting for a specific bacterial colonization during embryogenesis are produced in the oocyte and in early embryos. If overexpressed in hydra ectodermal epithelial cells, periculin1a drastically reduces the bacterial load, indicating potent antimicrobial activity. Unexpectedly, transgenic polyps also revealed that periculin, in addition to bactericidal activity, changes the structure of the bacterial community. These findings delineate a role for antimicrobial peptides both in selecting particular bacterial partners during development and as important components of a “be prepared” strategy providing transgenerational protection.

Hydramacin-1, Structure and Antibacterial Activity of a Protein from the Basal Metazoan Hydra

Hydramacin-1 is a novel antimicrobial protein recently discovered during investigations of the epithelial defense of the ancient metazoan Hydra. The amino acid sequence of hydramacin-1 shows no sequence homology to any known antimicrobial proteins. Determination of the solution structure revealed that hydramacin-1 possesses a disulfide bridge-stabilized αβ motif. This motif is the common scaffold of the knottin protein fold. The structurally closest relatives are the scorpion oxin-like superfamily. Within this superfamily hydramacin-1 establishes a new family of proteins that all share antimicrobial activity. Hydramacin-1 is potently active against Gram-positive and Gram-negative bacteria including multi-resistant human pathogenic strains. It leads to aggregation of bacteria as an initial step of its bactericidal mechanism. Aggregated cells are connected via electron-dense contacts and adopt a thorn apple-like morphology. Analysis of the hydramacin-1 structure revealed an unusual distribution of amino acid side chains on the surface. A belt of positively charged residues is sandwiched by two hydrophobic areas. Based on this characteristic surface feature and on biophysical analysis of protein-membrane interactions, we propose a model that describes the aggregation effect exhibited by hydramacin-1.

The natural anticancer compounds rocaglamides inhibit the Raf-MEK-ERK pathway by targeting prohibitin 1 and 2.

Rocaglamides are potent natural anticancer products that inhibit proliferation of various cancer cells at nanomolar concentrations. We have recently shown that these compounds prevent tumor growth and sensitize resistant cancer cells to apoptosis by blocking the MEK-ERK-eIF4 pathway. However, their direct molecular target(s) remain(s) unknown. In this study, using an affinity chromatography approach we discovered that prohibitin (PHB) 1 and 2 are the direct targets of rocaglamides. Binding of rocaglamides to PHB prevents interaction between PHB and CRaf and, thereby, inhibits CRaf activation and subsequently CRaf-MEK-ERK signaling. Moreover, knockdown of PHB mimicked the effects of rocaglamides on the CRaf-MEK-ERK pathway and cell cycle progression. Thus, our finding suggests that rocaglamides are a new type of anticancer agent and that they may serve as a small-molecular tool for studying PHB-mediated cellular processes.

Novel Peptidomimetics Containing a Vinyl Ester Moiety as Highly Potent and Selective Falcipain-2 Inhibitors

This paper describes the synthesis and biological evaluation of a new class of peptidomimetic falcipain-2 inhibitors based on a 1,4-benzodiazepine scaffold combined with various alpha,beta-unsaturated electrophilic functions such as vinyl-ketone, -amide, -ester, and -nitrile. The profile of reactivity of this class of derivatives has been evaluated and 4c, containing a vinyl ester warhead, proved to be a highly potent and selective falcipain-2 inhibitor.

Caenopores are antimicrobial peptides in the nematode Caenorhabditis elegans instrumental in nutrition and immunity.

For the soil nematode Caenorhabditis elegans, microbes are both food source and potential pathogens. Intrinsic antibiotic agents such as antimicrobial peptides (AMP) are important to protect the worm against infection. Here, we show that among potential antimicrobial peptides of C. elegans, with respect to gene number, the majority belongs to the SPP-protein family which we named caenopores as they resemble structurally and functionally amoebapores. SPP-5 kills bacteria by permeabilizing their cytoplasmic membrane and displays pore-forming activity as judged by liposome depolarization. The antimicrobial polypeptide is required to cope with Escherichia coli, the regular food source of C. elegans in the laboratory, as worms devoid of this weapon develop poorly, permitting a substantial number of bacteria to survive in the intestine. As numerous caenopores exert their activity in the intestinal lumen, an environment mimicking with its acidic pH and the presence of hydrolytic enzymes, the interior of phagolysosomes, individual members may be operative in eliminating distinct groups of microorganisms that enter this tract by food consumption. Individual spp genes are induced upon contact with particular bacteria, whereas others are expressed regardless of the bacteria they live on. The multifarious caenopore family of antimicrobial peptides may have been a key event that enables C. elegans to live and survive in its natural habitat, on rotting organic material.

Nuclear Death Receptor TRAIL-R2 Inhibits Maturation of Let-7 and Promotes Proliferation of Pancreatic and Other Tumor Cells

BACKGROUND & AIMS Tumor necrosis factor-related apoptosis inducing ligand (TRAIL-R1) (TNFRSF10A) and TRAIL-R2 (TNFRSF10B) on the plasma membrane bind ligands that activate apoptotic and other signaling pathways. Cancer cells also might have TRAIL-R2 in the cytoplasm or nucleus, although little is known about its activities in these locations. We investigated the functions of nuclear TRAIL-R2 in cancer cell lines. METHODS Proteins that interact with TRAIL-R2 initially were identified in pancreatic cancer cells by immunoprecipitation, mass spectrometry, and immunofluorescence analyses. Findings were validated in colon, renal, lung, and breast cancer cells. Functions of TRAIL-R2 were determined from small interfering RNA knockdown, real-time polymerase chain reaction, Drosha-activity, microRNA array, proliferation, differentiation, and immunoblot experiments. We assessed the effects of TRAIL-R2 overexpression or knockdown in human pancreatic ductal adenocarcinoma (PDAC) cells and their ability to form tumors in mice. We also analyzed levels of TRAIL-R2 in sections of PDACs and non-neoplastic peritumoral ducts from patients. RESULTS TRAIL-R2 was found to interact with the core microprocessor components Drosha and DGCR8 and the associated regulatory proteins p68, hnRNPA1, NF45, and NF90 in nuclei of PDAC and other tumor cells. Knockdown of TRAIL-R2 increased Drosha-mediated processing of the let-7 microRNA precursor primary let-7 (resulting in increased levels of mature let-7), reduced levels of the let-7 targets (LIN28B and HMGA2), and inhibited cell proliferation. PDAC tissues from patients had higher levels of nuclear TRAIL-R2 than non-neoplastic pancreatic tissue, which correlated with increased nuclear levels of HMGA2 and poor outcomes. Knockdown of TRAIL-R2 in PDAC cells slowed their growth as orthotopic tumors in mice. Reduced nuclear levels of TRAIL-R2 in cultured pancreatic epithelial cells promoted their differentiation. CONCLUSIONS Nuclear TRAIL-R2 inhibits maturation of the microRNA let-7 in pancreatic cancer cell lines and increases their proliferation. Pancreatic tumor samples have increased levels of nuclear TRAIL-R2, which correlate with poor outcome of patients. These findings indicate that in the nucleus, death receptors can function as tumor promoters and might be therapeutic targets.

2-D DIGE analysis of the proteome of extracts from peanut variants reveals striking differences in major allergen contents

Over the last decade, an increasing prevalence of peanut allergies was observed worldwide. Peanuts are meanwhile categorized among the most dangerous food allergens. This is particularly relevant since peanut‐derived ingredients are widely used in industrial food production. To minimize the problem of hidden food allergens causing severe anaphylactic reactions, pre‐packaged food containing peanut components needs to be classified according to European ruling since 2005. Food companies search for strategies to reduce the allergenicity of peanut‐derived food additives either by genetically altering the allergen content or by identifying peanut varieties with low levels of major allergens. In our study, we focused on peanut extracts from Indonesia that apparently contain lower levels of the major Arachis hypogaea allergen 1 (Ara h 1). Basic extracts of Virginia‐type and Indonesian peanuts were compared by 1‐ and 2‐DE. We identified more than hundred individual components in these extracts by MS and provide a high‐resolution allergen map that also includes so far unknown fragments of major peanut allergens. The reduced level of Ara h 1 associated with a significantly lower abundance of the most potent peanut allergen Ara h 2 in various Indonesian peanuts was also confirmed by Western blotting with monoclonal antibodies and sera of allergic patients.

Allicin and derivates are cysteine protease inhibitors with antiparasitic activity.

Allicin and derivatives thereof inhibit the CAC1 cysteine proteases falcipain 2, rhodesain, cathepsin B and L in the low micromolar range. The structure-activity relationship revealed that only derivatives with primary carbon atom in vicinity to the thiosulfinate sulfur atom attacked by the active-site Cys residue are active against the target enzymes. Some compounds also show potent antiparasitic activity against Plasmodium falciparum and Trypanosoma brucei brucei.

The human antimicrobial protein psoriasin acts by permeabilization of bacterial membranes.

Psoriasin, a member of the S100 family of calcium-binding proteins (S100A7) is highly upregulated in the skin of psoriasis patients. As it has recently been found to exhibit antimicrobial activity, an important role of psoriasin in surface defence has been suggested. The similarity of the three-dimensional structures of psoriasin and amoebapore A, an ancient antimicrobial, pore-forming peptide from Entamoeba histolytica, intrigued us to investigate whether the human psoriasin is also able to permeabilize bacterial membranes. Here, we demonstrate that psoriasin exerts pore-forming activity at pH values below 6 demonstrating that disruption of microbial membranes is the basis of its antimicrobial activity at low pH. Furthermore, the killing activity of psoriasin shows pH-dependent target specificity. At neutral pH, the Gram-negative bacterium E. coli is killed apparently without compromising its membrane, whereas at low pH exclusively the Gram-positive bacterium B. megaterium is killed by permeabilization of its cytoplasmic membrane.

The Antimicrobial Peptide NK-2, the Core Region of Mammalian NK-Lysin, Kills Intraerythrocytic Plasmodium falciparum

ABSTRACT In a time of dramatically increasing resistance of microbes to all kinds of antibiotics, natural antimicrobial peptides and synthetic analogs thereof have emerged as compounds with potentially significant therapeutical applications against human pathogens. Only very few of these peptide antibiotics have been tested against protozoan pathogens that are a major cause of morbidity and mortality in large parts of the world. Here, we studied the effect of NK-2, a peptide representing the cationic core region of the lymphocytic effector protein NK-lysin, on the malaria parasite Plasmodium falciparum. Whereas noninfected red blood cells were hardly affected, human erythrocytes infected with the parasite were rapidly permeabilized by NK-2 in the micromolar range. Loss of plasma membrane asymmetry and concomitant exposure of phosphatidylserine upon infection appears to be the molecular basis for the observed target preference of NK-2, as can be demonstrated by annexin V binding. The peptide also affects the viability of the intracellular parasite, as evidenced by the drop in DNA content of cultured parasites. Accumulated evidence derived from permeabilization assays using parasites and liposomes as targets and from fluorescence microscopy of infected erythrocytes treated with fluorescently labeled NK-2 indicates that the positively charged peptide electrostatically interacts with the altered and negatively charged plasma membrane of the infected host cell and traverses this membrane as well as the parasitophorous vacuole membrane to reach its final target, the intracellular parasite. The apparent affinity for foreign membranes that resulted in the death of a eukaryotic parasite residing in human host cells makes NK-2 a promising template for novel anti-infectives.