Lactoferrin

The first function attributed to lactoferrin (Lf), an iron binding protein belonging to the non-immune natural defences, was antimicrobial activity that depended on its capacity to sequester iron. Iron-independent microbicidal activities, requiring direct interaction between this cationic protein and microbial surface components, were later demonstrated. Many other anti-microbial and anti-viral functions have since been ascribed to Lf. In mucosal secretions, iron and Lf modulate the motility and aggregation of pathogenic bacteria. Lf inhibits bacterial adhesion on abiotic surfaces through ionic binding to biomaterials, or specific binding to bacterial structures or both. Lf inhibition of bacterial adhesion to host cells requires Lf binding to bacteria and/or host cells. Lf hinders microbial internalization by binding to both glycosaminoglycans and bacterial proteins which can be degraded by Lf-mediated proteolysis. Moreover, Lf internalisation and localisation to the host cell nuclei could modulate bacterial entry into cells through gene regulation. Finally, the capability of Lf to exert antiviral activity, through its binding to host cells and/or viral particles, strengthens the idea that it is an important brick in the mucosal wall, effective against both microbial and viral attacks.     

Bacterial signal peptide recognizes HeLa cell mitochondrial import receptors and functions as a mitochondrial leader sequence

Phage display was used to identify new components of the mammalian mitochondrial receptor complex using Tom20 as a binding partner. Two peptides were identified. One had partial identity (SMLTVMA) with a bacterial signal peptide from Toho-1, a periplasmic protein. The other had partial identity with a mitochondrial inner membrane glutamate carrier. The bacterial signal peptide could carry a protein into mitochondria both in vivo and in vitro. The first six residues of the sequence, SMLTVM, were necessary for import but the two adjacent arginine residues in the 30-amino-acid leader were not critical for import. The signal peptides of Escherichia coli β-lactamase and Bacillsus subtilis lipase could not carry proteins into mitochondria. Presumably, the Toho-1 leader can adopt a structure compatible for recognition by the import apparatus.Received 29 April 2005; received after revision 8 June 2005; accepted 17 June 2005     

Lytic transglycosylases in macromolecular transport systems of Gram-negative bacteria

The cell wall of Gram-negative bacteria is essential for the integrity of the bacterial cell but also imposes a physical barrier to trans-envelope transport processes in which DNA and/or proteins are taken up or secreted by complex protein assemblies. The presence of genes encoding lytic transglycosylases in macromolecular transport systems (bacteriophage entry, type II secretion and type IV pilus synthesis, type III secretion, type IV secretion) suggests an important role for these specialised cell-wall-degrading enzymes. Such enzymes are capable of locally enlarging gaps in the peptidoglycan meshwork to allow the efficient assembly and anchoring of supramolecular transport complexes in the cell envelope. In this review, current knowledge on the role and distribution of these specialised murein-degrading enzymes in diverse macromolecular transport systems is summarised and discussed.Received 13 February 2003; received after revision 25 April 2003; accepted 12 May 2003     

Bacterial resistance mechanisms against host defense peptides

Host defense peptides and proteins are important components of the innate host defense against pathogenic microorganisms. They target negatively charged bacterial surfaces and disrupt microbial cytoplasmic membranes, which ultimately leads to bacterial destruction. Throughout evolution, pathogens devised several mechanisms to protect themselves from deleterious damage of host defense peptides. These strategies include (a) inactivation and cleavage of host defense peptides by production of host defense binding proteins and proteases, (b) repulsion of the peptides by alteration of pathogen’s surface charge employing modifications by amino acids or amino sugars of anionic molecules (e.g., teichoic acids, lipid A and phospholipids), (c) alteration of bacterial membrane fluidity, and (d) expulsion of the peptides using multi drug pumps. Together with bacterial regulatory network(s) that regulate expression and activity of these mechanisms, they represent attractive targets for development of novel antibacterials.     

Sex-peptides: seminal peptides of the <Emphasis Type="Italic">Drosophila</Emphasis> male

Mating affects the reproductive behaviour of insect females: the egg-laying rate increases and courting males are rejected. These post-mating responses are induced mainly by seminal fluid. In Drosophila melanogaster, males transfer two peptides (sex-peptides, = Sps) that reduce receptivity and elicit increased egg laying in their mating partners. Similarities in the open reading frames of the genes suggest that they have arisen by gene duplication. In females, Sps bind to specific sites in the central and peripheral nervous system, and to the genital tract. The binding proteins of the nervous system and genital tract are membrane proteins, but they differ molecularly. The former protein is proposed to be a receptor located at the top of a signalling cascade leading to the two post-mating responses, whereas the latter is a carrier protein moving Sps from the genital tract into the haemolymph. Sps bind to sperm. Together with sperm they are responsible for the persistence of the two post-mating responses. But Sps are the molecular basis of the sperm effect; sperm is merely the carrier.Received 10 February 2003; received after revision 25 April 2003; accepted 1 May 2003This article is dedicated to the 85th birthday of the discover of the sex-peptide, Prof. Dr. Pei Shen Chen, Zoological Institute, University of Zürich, Switzerland. P. S. Chen has served on the Editorial Board of Experientia (now CMLS) from 1974 to 1988.     

Implication of phosphoinositide phosphatases in genetic diseases: the case of myotubularin

Phosphoinositides play a central role in the control of major eukaryotic cell signaling mechanisms. Accordingly, the list of phosphoinositide-metabolizing enzymes implicated in human diseases has considerably increased these last years. Here we will focus on myotubularin, the protein mutated in the X-linked myotubular myopathy (XLMTM) and the founding member of a family of 13 related proteins. Recent data demonstrate that myotubularin and several other members of the family are potent lipid phosphatases showing a marked specificity for phosphatidylinositol 3-phosphate [PtdIns(3)P]. This finding has raised considerable interest as PtdIns(3)P is implicated in vesicular trafficking and sorting through its binding to specific protein domains. The structure of myotubularin, the molecular mechanisms of its function and its implication in the etiology of XLMTM will be discussed, as well as the potential function and role of the other members of the family.Received 14 February 2003; received after revision 10 April 2003; accepted 14 April 2003     

Structure and function of eukaryotic peptide transporters

The cotransport of protons and peptides is now recognised as a major route by which dietary nitrogen is absorbed from the intestine, and filtered protein reabsorbed in the kidney. Recently, molecular biology has had a very substantial impact on the study of peptide transport, and here we review the molecular and functional information available within the framework of physiology. To this end we consider not only the mammalian peptide transporters and their tissue distribution and regulation but also those from other species (including Caenorhabditis elegans) which make up the proton-dependent oligopeptide transport superfamily. In addition, understanding the binding requirements for transported substrates may allow future design and targeted tissue delivery of peptide and peptidomimetic drugs. Finally, we aim to highlight some of the less well understood areas of peptide transport, in the hope that it will stimulate further research into this challenging yet exciting topic.     

Yeast as a sensor of factors affecting the accuracy of protein synthesis

The cell monitors and maintains the fidelity of translation during the three stages of protein synthesis: initiation, elongation and termination. Errors can arise by multiple mechanisms, such as altered start site selection, reading frame shifts, misincorporation or nonsense codon suppression. All of these events produce incorrect protein products. Translational accuracy is affected by both cis- and trans-acting elements that insure the proper peptide is synthesized by the protein synthetic machinery. Many cellular components are involved in the accuracy of translation, including RNAs (transfer RNAs, messenger RNAs and ribosomal RNAs) and proteins (ribosomal proteins and translation factors). The yeast Saccharomyces cerevisiae has proven an ideal system to study translational fidelity by integrating genetic approaches with biochemical analysis. This review focuses on the ways studies in yeast have contributed to our understanding of the roles translation factors and the ribosome play in assuring the accuracy of protein synthesis.Received 27 November 2002; received after revision 16 April 2003; accepted 25 April 2003     

Structure and function of desmosomal proteins and their role in development and disease

Desmosomes represent major intercellular adhesive junctions at basolateral membranes of epithelial cells and in other tissues. They mediate direct cell-cell contacts and provide anchorage sites for intermediate filaments important for the maintenance of tissue architecture. There is increasing evidence now that desmosomes in addition to a simple structural function have new roles in tissue morphogenesis and differentiation. Transmembrane glycoproteins of the cadherin superfamily of Ca²⁺-dependent cell-cell adhesion molecules which mediate direct intercellular interactions in desmosomes appear to be of central importance in this respect. The complex network of proteins forming the desmosomal plaque associated with the cytoplasmic domain of the desmosomal cadherins, however, is also involved in junction assembly and regulation of adhesive strength. This re-view summarizes the structural features of these desmosomal proteins, their function during desmosome assembly and maintenance, and their role in development and disease.Received 5 February 2003; received after revision 14 March 2003; accepted 1 April 2003     

Proline-rich antimicrobial peptides: converging to a non-lytic mechanism of action

Proline-rich antimicrobial peptides are a group of cationic host defense peptides of vertebrates and invertebrates characterized by a high content of proline residues, often associated with arginine residues in repeated motifs. Those isolated from some mammalian and insect species, although not evolutionarily related, use a similar mechanism to selectively kill Gram-negative bacteria, with a low toxicity to animals. Unlike other types of antimicrobial peptides, their mode of action does not involve the lysis of bacterial membranes but entails penetration into susceptible cells, where they then act intracellularly. Some aspects of the transport system and cytoplasmic targets have been elucidated. These features make them attractive both as anti-infective lead compounds and as a new class of potential cell-penetrating peptides capable of internalising membrane-impermeant drugs into both bacterial and eukaryotic cells     

The vault complex

Vaults are large ribonucleoprotein particles found in eukaryotic cells. They are composed of multiple copies of a M _r 100,000 major vault protein and two minor vault proteins of M _r 193,000 and 240,000, as well as small untranslated RNAs of 86–141 bases. The vault components are arranged into a highly characteristic hollow barrel-like structure of 35 × 65 nm in size. Vaults are predominantly localized in the cytoplasm where they may associate with cytoskeletal elements. A small fraction of vaults are found to be associated with the nucleus. As of yet, the precise cellular function of the vault complex is unknown. However, their distinct morphology and intracellular distribution suggest a role in intracellular transport processes. Here we review the current knowledge on the vault complex, its structure, components and possible functions.Received 23 January 2003; received after revision 13 March 2003; accepted 26 March 2003     

Soluble proteins of chemical communication in the social wasp <Emphasis Type="Italic">Polistes dominulus</Emphasis>

Members of the odorant-binding protein (OBP) and chemosensory protein (CSP) families were identified and characterised in the sensory tissues of the social wasp Polistes dominulus (Hymenoptera: Vespidae). Unlike most insects so far investigated, OBPs were detected in antennae, legs and wings, while CSPs appeared to be preferentially expressed in the antennae. The OBP is very different from the homologous proteins of other Hymenopteran species, with around 20% of identical residues, while the CSP appears to be much better conserved. Both OBP and CSP, not showing other post-translational modifications apart from disulphide bridges, were expressed with high yields in a bacterial system. Cysteine pairing in the recombinant and native proteins follows the classical arrangements described for other members of these classes of proteins. OBPs isolated from the wings were found to be associated with a number of long-chain aliphatic amides and other small organic molecules. Binding of these ligands and other related compounds was measured for both recombinant OBP and CSP.Received 14 May 2003; received after revision 8 June 2003; accepted 12 June 2003     

Macro domains as metabolite sensors on chromatin

How metabolism and epigenetics are molecularly linked and regulate each other is poorly understood. In this review, we will discuss the role of direct metabolite-binding to chromatin components and modifiers as a possible regulatory mechanism. We will focus on globular macro domains, which are evolutionarily highly conserved protein folds that can recognize NAD⁺-derived metabolites. Macro domains are found in histone variants, histone modifiers, and a chromatin remodeler among other proteins. Here we summarize the macro domain-containing chromatin proteins and the enzymes that generate relevant metabolites. Focusing on the histone variant macroH2A, we further discuss possible implications of metabolite binding for chromatin function.     

The bacterial translocase: a dynamic protein channel complex

The major route of protein translocation in bacteria is the so-called general secretion pathway (Sec-pathway). This route has been extensively studied in Escherichia coli and other bacteria. The movement of preproteins across the cytoplasmic membrane is mediated by a multimeric membrane protein complex called translocase. The core of the translocase consists of a proteinaceous channel formed by an oligomeric assembly of the heterotrimeric membrane protein complex SecYEG and the peripheral adenosine triphosphatase (ATPase) SecA as molecular motor. Many secretory proteins utilize the molecular chaperone SecB for targeting and stabilization of the unfolded state prior to translocation, while most nascent inner membrane proteins are targeted to the translocase by the signal recognition particle and its membrane receptor. Translocation is driven by ATP hydrolysis and the proton motive force. In the last decade, genetic and biochemical studies have provided detailed insights into the mechanism of preprotein translocation. Recent crystallographic studies on SecA, SecB and the SecYEG complex now provide knowledge about the structural features of the translocation process. Here, we will discuss the mechanistic and structural basis of the translocation of proteins across and the integration of membrane proteins into the cytoplasmic membrane.Received 10 January 2003; received after revision 2 April 2003; accepted 4 April 2003     

Multiple flavonoid-binding sites within multidrug resistance protein MRP1

Recombinant nucleotide-binding domains (NBDs) from human multidrug resistance protein MRP1 were overexpressed in bacteria and purified to measure their direct interaction with high-affinity flavonoids, and to evaluate a potential correlation with inhibition of MRP1-mediated transport activity and reversion of cellular multidrug resistance. Among different classes of flavonoids, dehydrosilybin exhibited the highest affinity for both NBDs, the binding to N-terminal NBD1 being prevented by ATP. Dehydrosilybin increased vanadate-induced 8-N₃-[-³²P]ADP trapping, indicating stimulation of ATPase activity. In contrast, dehydrosilybin strongly inhibited leukotriene C₄ (LTC₄) transport by membrane vesicles from MRP1-transfected cells, independently of reduced glutathione, and chemosensitized cell growth to vincristine. Hydrophobic C-isoprenylation of dehydrosilybin increased the binding affinity for NBD1, but outsite the ATP site, lowered the increase in vanadate-induced 8-N₃-[-³²P]ADP trapping, weakened inhibition of LTC₄ transport which became glutathione dependent, and induced some cross-resistance. The overall results indicate multiple binding sites for dehydrosilybin and its derivatives, on both cytosolic and transmembrane domains of MRP1.Received 1 May 2003; received after revision 18 June 2003; accepted 24 June 2003     

Massive glycosaminoglycan-dependent entry of Trp-containing cell-penetrating peptides induced by exogenous sphingomyelinase or cholesterol depletion

Among non-invasive cell delivery strategies, cell-penetrating peptide (CPP) vectors represent interesting new tools. To get fundamental knowledge about the still debated internalisation mechanisms of these peptides, we modified the membrane content of cells, typically by hydrolysis of sphingomyelin or depletion of cholesterol from the membrane outer leaflet. We quantified and visualised the effect of these viable cell surface treatments on the internalisation efficiency of different CPPs, among which the most studied Tat, R₉, penetratin and analogues, that all carry the N-terminal biotin-Gly₄ tag cargo. Under these cell membrane treatments, only penetratin and R₆W₃ underwent a massive glycosaminoglycan (GAG)-dependent entry in cells. Internalisation of the other peptides was only slightly increased, similarly in the absence or the presence of GAGs for R₉, and only in the presence of GAGs for Tat and R₆L₃. Ceramide formation (or cholesterol depletion) is known to lead to the reorganisation of membrane lipid domains into larger platforms, which can serve as a trap and cluster receptors. These results show that GAG clustering, enhanced by formation of ceramide, is efficiently exploited by penetratin and R₆W₃, which contains Trp residues in their sequence but not Tat, R₉ and R₆L₃. Hence, these data shed new lights on the differences in the internalisation mechanism and pathway of these peptides that are widely used in delivery of cargo molecules.     

Differential protein expression in pancreatic islets after treatment with an imidazoline compound

The effects of an imidazoline compound (BL11282) on protein expression in rat pancreatic islets were investigated with a proteomic approach. The compound increases insulin release selectively at high glucose concentrations and is therefore of interest in type 2 diabetes. Whole cell extracts from isolated drug-treated and native pancreatic rat islets were compared after separation by 2-D gel electrophoresis. Differentially expressed proteins were identified by mass spectrometry; 15 proteins were selectively up-regulated and 7 selectively down-regulated in drug-treated islets. Of special interest among the differentially expressed proteins are those involved in protein folding (Hsp60, protein disulfide isomerase, calreticulin), Ca²⁺ binding (calgizzarin, calcyclin and annexin I) and metabolism or signalling (pyruvate kinase, alpha enolase and protein kinase C inhibitor 1). Received 19 March 2007; received after revision 11 April 2007; accepted 11 April 2007     

Receptor binding, internalization, and retrograde transport of neurotrophic factors

This review deals with the receptor interactions of neurotrophic factors, focusing on the neurotrophins of the nerve growth factor (NGF) family, the glial cell derived neurotrophic factor (GDNF) family, and the ciliary neurotrophic factor (CNTF) family. The finding that two proteins, p75^NTR and Trk, act as receptors for NGF in neurons generated the discovery of other neurotrophic factors/receptor families and has enhanced our understanding of the development, survival, regeneration, and degeneration of the nervous system. The kinetics of binding, the structure of the ligand-receptor complex, and the mechanism of retrograde transport of the neurotrophins are discussed in detail and compared to information available on the GDNF and CNTF families. Each neurotrophic factor family, i.e., NGF, GDNF, and CNTF, has a set of receptors with specificity for individual members of the family and a common receptor without member specificity that, in some families, generates the cellular signal and retrograde transport.     

The invasion-associated type III secretion system of Salmonella typhimurium: common and unique features

Several bacterial pathogens make use of a specialized protein secretion system to inject effector proteins into host cells. This system, commonly referred to as type III secretion, is always associated with phenotypes related to intimate interactions between the pathogen and its respective host cells. The enteric pathogen Salmonella typhimurium utilizes a type III secretion system to invade nonphagocytic intestinal epithelial cells. Whereas the invasion-associated type III system of S. typhimurium has evolved to perform a specific function, many of the components of this system are conserved among the type III systems of other bacterial pathogens. This review will discuss the common and unique features of the S. typhimurium system in relation to the type III systems of other human pathogens. Topics discussed include the phenotypes associated with various type III systems, the genetic loci encoding these systems, the components of the type III secretion apparatus, the effector proteins and the mechanisms by which they enter host cells as well as the mechanisms used to regulate the expression of type III systems.