Matthias Michalek

Membrane interactions of the amphipathic amino terminus of huntingtin.

The amino-terminal domain of huntingtin (Htt17), located immediately upstream of the decisive polyglutamine tract, strongly influences important properties of this large protein and thereby the development of Huntingtons disease. Htt17 markedly increases polyglutamine aggregation rates and the level of huntingtins interactions with biological membranes. Htt17 adopts a largely helical conformation in the presence of membranes, and this structural transition was used to quantitatively analyze membrane association as a function of lipid composition. The apparent membrane partitioning constants increased in the presence of anionic lipids but decreased with increasing amounts of cholesterol. When membrane permeabilization was tested, a pronounced dye release was observed from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles and 75:25 (molar ratio) POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine vesicles but not across bilayers that better mimic cellular membranes. Solid-state nuclear magnetic resonance structural investigations indicated that the Htt17 α-helix adopts an alignment parallel to the membrane surface, and that the tilt angle (∼75°) was nearly constant in all of the membranes that were investigated. Furthermore, the addition of Htt17 resulted in a decrease in the lipid order parameter in all of the membranes that were investigated. The lipid interactions of Htt17 have pivotal implications for membrane anchoring and functional properties of huntingtin and concomitantly the development of the disease.

Structure and Topology of the Huntingtin 1-17 Membrane Anchor by a Combined Solution and Solid-State NMR Approach.

The very amino-terminal domain of the huntingtin protein is directly located upstream of the proteins polyglutamine tract, plays a decisive role in several important properties of this large protein and in the development of Huntingtons disease. This huntingtin 1-17 domain is on the one hand known to markedly increase polyglutamine aggregation rates and on the other hand has been shown to be involved in cellular membrane interactions. Here, we determined the high-resolution structure of huntingtin 1-17 in dodecyl phosphocholine micelles and the topology of its helical domain in oriented phosphatidylcholine bilayers. Using two-dimensional solution NMR spectroscopy the low-energy conformations of the polypeptide were identified in the presence of dodecyl phosphocholine detergent micelles. In a next step a set of four solid-state NMR angular restraints was obtained from huntingtin 1-17 labeled with (15)N and (2)H at selected sites. Of the micellar ensemble of helical conformations only a limited set agrees in quantitative detail with the solid-state angular restraints of huntingtin 1-17 obtained in supported planar lipid bilayers. Thereby, the solid-state NMR data were used to further refine the domain structure in phospholipid bilayers. At the same time its membrane topology was determined and different motional regimes of this membrane-associated domain were explored. The pronounced structural transitions of huntingtin 1-17 upon membrane-association result in a α-helical conformation from K6 to F17, i.e., up to the very start of the polyglutamine tract. This amphipathic helix is aligned nearly parallel to the membrane surface (tilt angle ∼77°) and is characterized by a hydrophobic ridge on one side and an alternation of cationic and anionic residues that run along the hydrophilic face of the helix. This arrangement facilitates electrostatic interactions between huntingtin 1-17 domains and possibly with the proximal polyglutamine tract.

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.

An exceptional salt-tolerant antimicrobial peptide derived from a novel gene family of haemocytes of the marine invertebrate Ciona intestinalis

A novel gene family coding for putative antimicrobial peptides was identified in the EST (expressed sequence tag) database of the sea squirt Ciona intestinalis, and one of these genes was molecularly cloned from the Northern European Ciona subspecies. In situ hybridization and immunocytochemical analysis revealed that the natural peptide is synthesized and stored in a distinct haemocyte type, the univacuolar non-refractile granulocytes. By semiquantitative RT-PCR (reverse transcription-PCR) analysis, it was shown that the expression of the gene is markedly up-regulated in haemocytes after immune challenge. To evaluate the antimicrobial potency of the putative defence protein, we synthesized a peptide corresponding to its cationic core region. The peptide was highly effective against Gram-negative and Gram-positive bacteria including several human and marine pathogens as well as the yeast Candida albicans. Notably, the antibacterial activity of the peptide was retained at salt concentrations of up to 450 mM NaCl. Using two different methods we demonstrated that the peptide kills Gram-negative and Gram-positive bacteria by permeabilizing their cytoplasmic membranes. CD spectroscopy revealed that, in the presence of liposomes composed of negatively charged phospholipids, the peptide undergoes a conformational change and adopts an alpha-helical structure. Moreover, the peptide was virtually non-cytolytic for mammalian erythrocytes. Hence, the designed salt-tolerant antimicrobial peptide may represent a valuable template for the development of novel antibiotics.

Shedding of Endogenous Interleukin-6 Receptor (IL-6R) Is Governed by A Disintegrin and Metalloproteinase (ADAM) Proteases while a Full-length IL-6R Isoform Localizes to Circulating Microvesicles.

Background: A soluble form of IL-6 receptor mediates pathogenic IL-6 trans-signaling. Results: ADAM10 and ADAM17 release IL-6 receptor from both human and murine monocytes/macrophages, whereas in the blood IL-6 receptor is also present on microvesicles. Conclusion: Shedding of endogenous IL-6 receptor is similar in humans and mice. Significance: Microvesicle release represents a novel mode of soluble IL-6 receptor generation with potential clinical implications. Generation of the soluble interleukin-6 receptor (sIL-6R) is a prerequisite for pathogenic IL-6 trans-signaling, which constitutes a distinct signaling pathway of the pleiotropic cytokine interleukin-6 (IL-6). Although in vitro experiments using ectopically overexpressed IL-6R and candidate proteases revealed major roles for the metalloproteinases ADAM10 and ADAM17 in IL-6R shedding, the identity of the protease(s) cleaving IL-6R in more physiological settings, or even in vivo, remains unknown. By taking advantage of specific pharmacological inhibitors and primary cells from ADAM-deficient mice we established that endogenous IL-6R of both human and murine origin is shed by ADAM17 in an induced manner, whereas constitutive release of endogenous IL-6R is largely mediated by ADAM10. Although circulating IL-6R levels are altered in various diseases, the origin of blood-borne IL-6R is still poorly understood. It has been shown previously that ADAM17 hypomorphic mice exhibit unaltered levels of serum sIL-6R. Here, by quantification of serum sIL-6R in protease-deficient mice as well as human patients we also excluded ADAM10, ADAM8, neutrophil elastase, cathepsin G, and proteinase 3 from contributing to circulating sIL-6R. Furthermore, we ruled out alternative splicing of the IL-6R mRNA as a potential source of circulating sIL-6R in the mouse. Instead, we found full-length IL-6R on circulating microvesicles, establishing microvesicle release as a novel mechanism for sIL-6R generation.

Congenital Lipoid Adrenal Hyperplasia: Functional Characterization of Three Novel Mutations in the STAR Gene

CONTEXT The steroidogenic acute regulatory protein (StAR) has been shown to be essential for steroidogenesis by mediating cholesterol transfer into mitochondria. Inactivating StAR mutations cause the typical clinical picture of congenital lipoid adrenal hyperplasia. OBJECTIVE The objective of the investigation was to study the functional and structural consequences of three novel StAR mutations (p.N148K in an Italian patient; p.P129fs and p.Q128R in a Turkish patient). METHODS AND RESULTS Transient in vitro expression of the mutant proteins together with P450 side-chain cleavage enzyme, adrenodoxin, and adrenodoxin reductase yielded severely diminished cholesterol conversion of the p.N148K mutant, the combined p.P129fs and p.Q128R mutant, and the p.P129fs mutant by itself. The p.Q128R mutant led to a higher cholesterol conversion than the wild-type StAR protein. As derived from three-dimensional protein modeling, the residue N148 is lining the ligand cavity of StAR. A positively charged lysine residue at position 148 disturbs the hydrophobic cluster formed by the alpha4-helix and the sterol binding pocket. The frame shift mutation p.P129fs truncates the StAR protein. Residue p.Q128 is situated at the surface of the molecule and is not part of any functionally characterized region of the protein. CONCLUSION The mutations p.N148K and p.P129fs cause adrenal insufficiency in both cases and lead to a disorder of sex development with complete sex reversal in the 46, XY case. The mutation p.Q128R, which is not relevant for the patients phenotype, is the first reported variant showing a gain of function. We speculate that the substitution of hydrophilic glutamine with basic arginine at the surface of the molecule may accelerate cholesterol transfer.

A novel horse α-defensin: gene transcription, recombinant expression and characterization of the structure and function

Defensins are a predominant class of antimicrobial peptides, which act as endogenous antibiotics. Defensins are classified into three distinct sub-families: theta-, beta-, and alpha-defensins. Synthesis of alpha-defensin has been confirmed only in primates and glires to date and is presumably unique for a few tissues, including neutrophils and Paneth cells of the small intestine. Antimicrobial activities of these peptides were shown against a wide variety of microbes including bacteria, fungi, viruses and protozoan parasites. In the present study, we report the characterization of the equine alpha-defensin DEFA (defensin alpha) 1. Transcription analysis revealed that the transcript of the gene is present in the small intestine only. An alignment with known alpha-defensins from primates and glires displayed a homology with Paneth-cell-specific alpha-defensins. DEFA1 was recombinantly expressed in Escherichia coli and subsequently analysed structurally by CD and molecular modelling. To examine the antimicrobial properties, a radial diffusion assay was performed with 12 different micro-organisms and the LD90 (lethal dose killing > or =90% of target organism) and MBC (minimal bactericidal concentration) values were examined. DEFA1 showed an antimicrobial activity against different Gram-positive and Gram-negative bacteria and against the yeast Candida albicans. Using viable bacteria in combination with a membrane-impermeable fluorescent dye, as well as depolarization of liposomes as a minimalistic system, it became evident that membrane permeabilization is at least an essential part of the peptides mode of action.

Solid-state NMR approaches to study protein structure and protein-lipid interactions.

Solid-state NMR spectroscopy has been developed for the investigation of membrane-associated polypeptides and remains one of the few techniques to reveal high-resolution structural information in liquid-disordered phospholipid bilayers. In particular, oriented samples have been used to investigate the structure, dynamics, and topology of membrane polypeptides. Much of the previous solid-state NMR work has been developed and performed on peptides, but the technique is constantly expanding towards larger membrane proteins. Here, a number of protocols are presented describing among other the reconstitution of membrane proteins into oriented membranes, monitoring membrane alignment by (31)P solid-state NMR spectroscopy; investigations of the protein by one- and two-dimensional (15)N solid-state NMR; and measurements of the lipid order parameters using (2)H solid-state NMR spectroscopy. Using such methods solid-state NMR spectroscopy has revealed a detailed picture of the ensemble of both lipids and proteins and their mutual interdependence in the bilayer environment.

Structure and function of a unique pore-forming protein from a pathogenic acanthamoeba.

Human pathogens often produce soluble protein toxins that generate pores inside membranes, resulting in the death of target cells and tissue damage. In pathogenic amoebae, this has been exemplified with amoebapores of the enteric protozoan parasite Entamoeba histolytica. Here we characterize acanthaporin, to our knowledge the first pore-forming toxin to be described from acanthamoebae, which are free-living, bacteria-feeding, unicellular organisms that are opportunistic pathogens of increasing importance and cause severe and often fatal diseases. We isolated acanthaporin from extracts of virulent Acanthamoeba culbertsoni by tracking its pore-forming activity, molecularly cloned the gene of its precursor and recombinantly expressed the mature protein in bacteria. Acanthaporin was cytotoxic for human neuronal cells and exerted antimicrobial activity against a variety of bacterial strains by permeabilizing their membranes. The tertiary structures of acanthaporins active monomeric form and inactive dimeric form, both solved by NMR spectroscopy, revealed a currently unknown protein fold and a pH-dependent trigger mechanism of activation.

Expression and Function of Psoriasin (S100A7) and Koebnerisin (S100A15) in the Brain

ABSTRACT The expression and function of psoriasin in the brain have been insufficiently characterized. Here, we show the induction of psoriasin expression in the central nervous system (CNS) after bacterial and viral stimulation. We used a pneumococcal meningitis in vivo model that revealed S100A15 expression in astrocytes and meningeal cells. These results were confirmed by a cell-based in vivo assay using primary rat glial and meningeal cell cultures. We investigated psoriasin expression in glial and meningeal cells using polyinosinic-polycytidylic acid, a synthetic analog of double-stranded RNA that mimics viral infection. Furthermore, previous results showed that antimicrobial peptides have not only bactericidal but also immunomodulatory functions. To test this statement, we used recombinant psoriasin as a stimulus. Glial and meningeal cells were treated with recombinant psoriasin at concentrations from 25 to 500 ng/ml. Treated microglia and meningeal cells showed phosphorylation of the extracellular signal-regulated kinase 1 (ERK1)/ERK2 (ERK1/2) signal transduction pathway. We demonstrated that this activation of ERK depends on RAGE, the receptor for advanced glycation end products. Furthermore, microglia cells treated with recombinant psoriasin change their phenotype to an enlarged shape. In conclusion, our results indicate an occurrence of psoriasin in the brain. An involvement of psoriasin as an antimicrobial protein that modulates the innate immune system after bacterial or viral stimulation is possible.