Anti herpes simplex virus activity of lactoferrin/lactoferricin – an example of antiviral activity of antimicrobial protein/peptide

One of the most common viral infections in humans is caused by the herpes simplex virus (HSV). It was first effectively treated in the 1970s with the introduction of acyclovir, which is still the most commonly used treatment. Naturally occurring antimicrobial proteins and peptides have also been shown to possess antiviral activity against HSV. This review will focus on the anti-HSV activity of one such protein, lactoferrin, and a small peptide fragment from its N-terminal domain, lactoferricin. Both components have been shown to effectively block entry of HSV into the host cell. In addition to blocking HSV entry, the peptides appear to have immune stimulatory activity, although this is still somewhat controversial. Mode of action studies and knowledge about the anti-HSV activity of lactoferricin have also been successfully employed in the design of new, more specific HSV blockers. Received 25 May 2005; received after revision 24 August 2005; accepted 6 September 2005     

Heat shock proteins in immune response to cancer: The Fourth Paradigm

The involvement of heat shock proteins in immune response is categorized into four distinct paradigms. In the First Paradigm, HSP derived from foreign organisms act as classical foreign antigens, and they elicit immune response to the non-conserved HSP epitopes. The Second Paradigm refers to instances where the host responds to self HSP to which there is no central or peripheral tolerance. The Third Paradigm involves molecular mimicry, where cross-reactivity between an HSP and another protein leads to an immune response to the latter under conditions which elicit an immune response to the former, such as infection with a bacterium whose immunodominant antigen is an HSP. The Fourth Paradigm refers to situations where an HSP-antigen complex elicits an effective response to the antigen andnot to the HSP. Thus the HSP acts as a carrier for the antigenic peptide. The role of HSP in recognition by γδ T cells may also fall into this paradigm. In this article, the Fourth Paradigm is considered as a crucial element in the development of vaccines against cancers and infectious diseases, and is analyzed through the prism of the observed association of hsp70 species with antigenic peptides.     

Cell penetrating peptides: overview and applications to the delivery of oligonucleotides

Crossing biological barriers represents a major limitation for clinical applications of biomolecules such as nucleic acids, peptides or proteins. Cell penetrating peptides (CPP), also named protein transduction domains, comprise short and usually basic amino acids-rich peptides originating from proteins able to cross biological barriers, such as the viral Tat protein, or are rationally designed. They have emerged as a new class of non-viral vectors allowing the delivery of various biomolecules across biological barriers from low molecular weight drugs to nanosized particles. Encouraging data with CPP-conjugated oligonucleotides have been obtained both in vitro and in vivo in animal models of diseases such as Duchenne muscular dystrophy. Whether CPP-cargo conjugates enter cells by direct translocation across the plasma membrane or by endocytosis remains controversial. In many instances, however, endosomal escape appears as a major limitation of this new delivery strategy.     

CD4<Superscript>+</Superscript> T cell-mediated immunity against prion proteins

The prion protein (PrP(C)) is essential for susceptibility to transmissible spongiform encephalopathies. A specific conformer of this protein (PrP(Sc)) is, according to the 'protein only' hypothesis, the principal or only component of the infectious agent, designated prion. Transmission of prions between species is often inefficient, resulting in low attack rates and/or prolonged incubation times and is ascribed to a 'species barrier' caused by differences in the amino acid sequence of PrP between recipient and donor. In this report, we demonstrate that these differences in amino acid sequence result in presentation of distinct peptides on major histocompatibility complex class II molecules. These peptides result in activation of specific CD4+ T cells which leads to the induction of an effective immune response against foreign PrP as demonstrated by antibody production. Therefore, CD4+ T cells represent a crucial component of the immune system to distinguish between foreign and self PrP.     

Phylogenetic distribution, functional epitopes and evolution of the CSαβ superfamily

A superfamily of proteins often conserves a common structural scaffold but develops diverse biochemical and biological functions during evolution. The understanding of evolutionary mechanisms responsible for this diversity is of fundamental importance not only in structural genomics but also in nature-guided drug design. A superfamily of peptides with a conserved CSalphabeta structural motif provides a considerably intriguing example to approach such an issue. The peptides from this superfamily have wide origins, ranging from plants to animals, and exhibit diverse biological activities, varying from a sweet-tasting protein to antibacterial defensins and animal toxins targeting ion channels. This review describes the phylogenetic distribution and structural classifi cation of this unique scaffold and provides new insights into its functional diversity from the perspective of sequence, structure and evolution.     

Casein, a prohormone with an immunomodulating role for the newborn?

Maternal colostrum and milk, the earliest food of the newborn, should not only be considered as supplying nutrients, but also as agents providing protection against aggressions from the new environment. Indeed by enzymatic digestion of the main milk proteins, the caseins, biologically active peptides are released; they may be implicated in the stimulation of the newborn's immune system. From this point of view a 'strategic active zone' has been characterized in beta-casein. A possible role of casein as a 'prohormone' for the newborn is suggested.     

The role of glycosylation in protein antigenic properties

Glycosylation of proteins is a common event and contributes to protein antigenic properties. Most data have been obtained from model studies on glycoprotens with well-defined structure or synthetic glycopeptides and their respective monoclonal antibodies. Antibodies raised against glycoprotein antigens may be specific for their carbohydrate units which are recognized irrespective of the protein carrier (carbohydrate epitopes), or in the context of the adjacent amino acid residues (glycopeptidic epitopes). Conformation or proper exposure of peptidic epitopes of glycoproteins is also frequently modulated by glycosylation due to intramolecular carbohydrate-protein interactions. The effects of glycosylation are broad: glycosylation may 'inactivate' the peptidic epitope or may be required for its reactivity with the antibody, depending on the structure of the antigenic site and antibody fine specificity. Evidence is increasing that similar effects of glycosylation pertain to T cell-dependent cellular immune responses. Glycosylated peptides can be bound and presented by MHC class I or II molecules and elicit glycopeptide-specific T cell clones. Received 5 July 2001; received after revision 9 October 2001; accepted 11 October 2001     

Evolutionary and functional perspectives of the major histocompatibility complex class I antigen-processing machinery

Major histocompatibility complex (MHC) class I molecules present antigenic peptides to CD8+ T cells, providing the basis for immune recognition of pathogen-infected cells. Peptides generated mainly by proteasomes in the cytosol are transported into the lumen of the endoplasmic reticulum by transporters associated with antigen processing (TAP). The maturation of MHC class I molecules is controlled by a number of accessory proteins and chaperones that are to a varying degree dedicated to the assembly of MHC class I. Several newly characterised proteins have been demonstrated to play important roles in this process. This review focuses on the functional relationship and evolutionary history of the antigen-processing machinery (APM) components and MHC class I itself. These are of great interest for further elucidating the origin of the immune system and understanding the mechanisms of antigen presentation and immunology in general.     

Molecular mimicry and the role of B lymphocytes in the processing of autoantigens

The immune system has evolved several mechanisms that provide lymphocytes with the intelligence to ignore self proteins while attacking foreign pathogenic agents. Notably, B and T lymphocytes that encounter self antigen at either the inappropriate levels or affinity are usually instructed to perish or become anergized. However, the presence of autoimmune disease suggests that the induction of self tolerance is not foolproof. In fact, autoreactive cells are now found to be normal inhabitants of the B and T lymphocyte repertoire. This review examines how foreign peptides which resemble self proteins can elicit autoimmunity that is amplified to many sites on a target autoantigen. In particular, B lymphocytes initiated by foreign molecular mimics can process and present self peptides in the shaping of autoimmune T cell responses.     

The macromolecular peptide-loading complex in MHC class I-dependent antigen presentation

A challenging task for the adaptive immune system of vertebrates is to identify and eliminate intracellular antigens. Therefore a highly specialized antigen presentation machinery has evolved to display fragments of newly synthesized proteins to effector cells of the immune system at the cell surface. After proteasomal degradation of unwanted proteins or defective ribosome products, resulting peptides are translocated into the endoplasmic reticulum by the transporter associated with antigen processing and loaded onto major histocompatibility complex (MHC) class I molecules. Peptide-MHC I complexes are transported via the secretory pathway to the cell surface where they are then inspected by cytotoxic T lymphocytes, which can trigger an immune response. This review summarizes the current view of the intracellular machinery of antigen processing and of viral immune escape mechanisms to circumvent destruction by the host. Received 4 October 2005; received after revision 19 November 2005; accepted 24 November 2005     

Intestinal epithelial barrier and mucosal immunity

The innate immune system plays a crucial role in maintaining the integrity of the intestine and protecting the host against a vast number of potential microbial pathogens from resident and transient gut microflora. Mucosal epithelial cells and Paneth cells produce a variety of antimicrobial peptides (defensins, cathelicidins, crytdinrelated sequence peptides, bactericidal/permeabilityincreasing protein, chemokine CCL20) and bacteriolytic enzymes (lysozyme, group IIA phospholipase A2) that protect mucosal surfaces and crypts containing intestinal stem cells against invading microbes. Many of the intestinal antimicrobial molecules have additional roles of attracting leukocytes, alarming the adaptive immune system or neutralizing proinflammatory bacterial molecules. Dysfunction of components of the innate immune system has recently been implicated in chronic inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, illustrating the pivotal role of innate immunity in maintaining the delicate balance between immune tolerance and immune response in the gut.     

Helix insertion into bilayers and the evolution of membrane proteins

Polytopic α-helical membrane proteins cannot spontaneously insert into lipid bilayers without assistance from polytopic α-helical membrane proteins that already reside in the membrane. This raises the question of how these proteins evolved. Our current knowledge of the insertion of α-helices into natural and model membranes is reviewed with the goal of gaining insight into the evolution of membrane proteins. Topics include: translocon-dependent membrane protein insertion, antibiotic peptides and proteins, in vitro insertion of membrane proteins, chaperone-mediated insertion of transmembrane helices, and C-terminal tail-anchored (TA) proteins. Analysis of the E. coli genome reveals several predicted C-terminal TA proteins that may be descendents of proteins involved in pre-cellular membrane protein insertion. Mechanisms of pre-translocon polytopic α-helical membrane protein insertion are discussed.     

A dynamic view of peptides and proteins in membranes

Biological membranes are highly dynamic supramolecular arrangements of lipids and proteins, which fulfill key cellular functions. Relatively few high-resolution membrane protein structures are known to date, although during recent years the structural databases have expanded at an accelerated pace. In some instances the structures of reaction intermediates provide a stroboscopic view on the conformational changes involved in protein function. Other biophysical approaches add dynamic aspects and allow one to investigate the interactions with the lipid bilayers. Membrane-active peptides fulfill many important functions in nature as they act as antimicrobials, channels, transporters or hormones, and their studies have much increased our understanding of polypeptide-membrane interactions. Interestingly several proteins have been identified that interact with the membrane as loose arrays of domains. Such conformations easily escape classical high-resolution structural analysis and the lessons learned from peptides may therefore be instructive for our understanding of the functioning of such membrane proteins. Received 11 March 2008; received after revision 2 May 2008; accepted 5 May 2008     

The translocation motif of hepatitis B virus improves protein vaccination

Cell-penetrating peptides (CPPs) have been shown to improve antigen loading of dendritic cell vaccines. Here we asked whether fusion of a CPP to a protein improves its immunogenicity when this fusion protein is directly applied as vaccine. We used the cell-penetrating translocation motif (TLM) derived from the hepatitis B virus, because no size limitation of cargos has been observed. Increased immunogenicity was observed when TLM was fused to ovalbumin (TLM-ova). TLM-ova was found to be superior to ova in inducing proliferation and cytotoxicity of ova-specific CD8+ T cells in vitro and in vivo. Using ovalbumin-expressing thymoma cells (EG7-ova), an improved anti-tumor immune response was observed for TLM-ova vaccination versus vaccination with ova. Moreover, TLM-ova vaccination induced a higher titer of anti-ovalbumin IgG2a antibodies compared to ova. These data demonstrate that CPP-protein vaccines can improve cellular as well as humoral immune responses. Received 16 November 2005; received after revision 12 December 2005; accepted 10 January 2006 †These authors contributed equally to this work     

Inhibitors of protein translocation across membranes of the secretory pathway: novel antimicrobial and anticancer agents

Proteins routed to the secretory pathway start their journey by being transported across biological membranes, such as the endoplasmic reticulum. The essential nature of this protein translocation process has led to the evolution of several factors that specifically target the translocon and block translocation. In this review, various translocation pathways are discussed together with known inhibitors of translocation. Properties of signal peptide-specific systems are highlighted for the development of new therapeutic and antimicrobial applications, as compounds can target signal peptides from either host cells or pathogens and thereby selectively prevent translocation of those specific proteins. Broad inhibition of translocation is also an interesting target for the development of new anticancer drugs because cancer cells heavily depend on efficient protein translocation into the endoplasmic reticulum to support their fast growth.     

Generation of MHC class I ligands in the secretory and vesicular pathways

CD8⁺ T lymphocytes screen the surface of all cells in the body to detect pathogen infection or oncogenic transformation. They recognize peptides derived from cellular proteins displayed at the plasma membrane by major histocompatibility complex (MHC) class I molecules. Peptides are mostly by-products of cytosolic proteolytic enzymes. Peptidic ligands of MHC class I molecules are also generated in the secretory and vesicular pathways. Features of protein substrates, of proteases and of available MHC class I molecules for loading peptides in these compartments shape a singular collection of ligands that also contain different, longer, and lower affinity peptides than ligands produced in the cytosol. Especially in individuals who lack the transporters associated with antigen processing, TAP, and in infected and tumor cells where TAP is blocked, which thus have no supply of peptides derived from the cytosol, MHC class I ligands generated in the secretory and vesicular pathways contribute to shaping the CD8⁺ T lymphocyte response.     

G proteins as drug targets

The structure and function of heterotrimeric G protein subunits is known in considerable detail. Upon stimulation of a heptahelical receptor by the appropriate agonists, the cognate G proteins undergo a cycle of activation and deactivation; the α-subunits and the βγ-dimers interact sequentially with several reaction partners (receptor, guanine nucleotides and effectors as well as regulatory proteins) by exposing appropriate binding sites. For most of these domains, low molecular weight ligands have been identified that either activate or inhibit signal transduction. These ligands include short peptides derived from receptors, G protein subunits and effectors, mastoparan and related insect venoms, modified guanine nucleotides, suramin analogues and amphiphilic cations. Because compounds that act on G proteins may be endowed with new forms of selectivity, we propose that G protein subunits may therefore be considered as potential drug targets. Received 18 September 1998; received after revision 6 November 1998; accepted 11 November 1998     

Antimicrobial skin peptides and proteins

Human skin is permanently exposed to microorganisms, but rarely infected. One reason for this natural resistance might be the existence of a ‘chemical barrier’ consisting in constitutively and inducibly produced antimicrobial peptides and proteins (AMPs). Many of these AMPs can be induced in vitro by proinflammatory cytokines or bacteria. Apart from being expressed in vivo in inflammatory lesions, some AMPs are also focally expressed in skin in the absence of inflammation. This suggests that non-inflammatory stimuli of endogenous and/or exogenous origin can also stimulate AMP synthesis without inflammation. Such mediators might be ideal ‘immune stimulants’ to induce only the innate antimicrobial skin effector molecules without causing inflammation. Received 9 August 2005; received after revision 21 October 2005; accepted 16 November 2005     

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