The Penicillium chrysogenum antifungal protein PAF, a promising tool for the development of new antifungal therapies and fungal cell biology studies

In recent years the interest in antimicrobial proteins and peptides and their mode of action has been rapidly increasing due to their potential to prevent and combat microbial infections in all areas of life. A detailed knowledge about the function of such proteins is the most important requirement to consider them for future application. Our research in recent years has been focused on the low molecular weight, cysteine-rich and cationic antifungal protein PAF from Penicillium chrysogenum, which inhibits the growth of opportunistic zoo-pathogens including Aspergillus fumigatus, numerous plant-pathogenic fungi and the model organism Aspergillus nidulans. So far, the experimental results indicate that PAF elicits hyperpolarization of the plasma membrane and the activation of ion channels, followed by an increase in reactive oxygen species in the cell and the induction of an apoptosis-like phenotype. Detailed knowledge about the molecular mechanism of action of antifungal proteins such as PAF contributes to the development of new antimicrobial strategies that are urgently needed. Received 09 August 2007; received after revision 17 September 2007; accepted 19 September 2007     

Structure, function and evolution of antifreeze proteins

Antifreeze proteins bind to ice crystals and modify their growth. These proteins show great diversity in structure, and they have been found in a variety of organisms. The ice-binding mechanisms of antifreeze proteins are not completely understood. Recent findings on the evolution of antifreeze proteins and on their structures and mechanisms of action have provided new understanding of these proteins in different contexts. The purpose of this review is to present the developments in contrasting research areas and unite them in order to gain further insight into the structure and function of the antifreeze proteins. Received 2 September 1998; received after revision 21 October 1998; accepted 2 November 1998     

Life behind cell walls: paradigm lost, paradigm regained

This review of the living cell wall [1] and its protein components is in two parts. The first is anecdotal. A personal account spanning over 40 years research may perhaps be an antidote to one stereotypical view of scientists as detached and humorless. The second part deals with the meaning of function, particularly as it applies to hydroxyproline-rich glycoproteins. Function is a difficult word to define objectively. However, with help from such luminaries as Humpty Dumpty: "A word means what I want it to mean, neither more nor less," and Wittgenstein: "Giving examples of usage ... is the only way to talk about meaning," it is possible to construct a ziggurat representing increasingly complex levels of organization from molecular structure to ecology. Forty years ago I suggested that hydroxyproline-rich structural proteins played a key role in cell wall functioning. But because the bulk of the wall is carbohydrate, there has been an understandable resistance to paradigm change. Expansins, paradoxically, contribute greatly to this resistance because their modus operandi as cell-wall-loosening proteins is based on the idea that they break hydrogen bonds between polysaccharide chains allowing slippage. However, this view is not consistent with the recent discovery [Grobe et al. (1999) Eur. J. Biochem 263: 33-40] that β-expansins may be proteases, as it implies that the extensin network is not a straightjacket but a substrate for expansin in muro. Such a direct role for extensins in both negative and positive regulation of cell expansion and elongation may constitute a major morphogenetic mechanism operating at all levels of plant growth and development.     

Kank proteins: structure, functions and diseases

The Kank family of proteins, Kank1–Kank4, are characterized by their unique structure, coiled-coil motifs in the N-terminal region, and ankyrin-repeats in the C-terminal region, with an additional motif, the KN motif, at the N-terminus. Kank1 was obtained by positional cloning of a tumor suppressor gene in renal cell carcinoma, while the other members were found by homology search. The family is involved in the regulation of actin polymerization and cell motility through signaling pathways containing PI3K/Akt and/or unidentified modulators/effectors. Their relationship to diseases such as cancer, and to neuronal and developmental disorders, will be an important subject of future study.     

Scaffolds,levers, rods and springs: diverse cellular functions of long coiled-coil proteins

Long alpha-helical coiled-coil proteins are involved in a variety of organizational and regulatory processes in eukaryotic cells. They provide cables and networks in the cyto- and nucleoskeleton, molecular scaffolds that organize membrane systems, motors, levers, rotating arms and possibly springs. A growing number of human diseases are found to be caused by mutations in long coiled-coil proteins. This review summarizes our current understanding of the multifaceted group of long coiled-coil proteins in the cytoskeleton, nucleus, Golgi and cell division apparatus. The biophysical features of coiled-coil domains provide first clues toward their contribution to the diverse protein functions and promise potential future applications in the area of nanotechnology. Combining the power of fully sequenced genomes and structure prediction algorithms, it is now possible to comprehensively summarize and compare the complete inventory of coiled-coil proteins of different organisms.Received 27 January 2004; received after revision 23 February 2004; accepted 10 March 2004     

Autophagy: molecular mechanisms,physiological functions and relevance in human pathology

Autophagy is a degradative mechanism mainly involved in the recycling and turnover of cytoplasmic constituents from eukaryotic cells. Over the last years, yeast genetic screens have considerably increased our knowledge about the molecular mechanisms of autophagy, and a number of genes involved in fundamental steps of the autophagic pathway have been identified. Most of these autophagy genes are present in higher eukaryotes indicating that this process has been evolutionarily conserved. In yeast, autophagy is mainly involved in adaptation to starvation, but in multicellular organisms this route has emerged as a multifunctional pathway involved in a variety of additional processes such as programmed cell death, removal of damaged organelles and development of different tissue-specific functions. Furthermore, autophagy is associated with a growing number of pathological conditions, including cancer, myopathies and neurodegenerative disorders. The physiological and pathological roles of autophagy, as well as the molecular mechanisms underlying this multifunctional pathway, are discussed in this review.Received 12 January 2004; received after revision 29 January 2004; accepted 4 February 2004     

N-acetylmuramic acid 6-phosphate lyases (MurNAc etherases): role in cell wall metabolism, distribution, structure, and mechanism

MurNAc etherases cleave the uniqued-lactyl ether bond of the bacterial cell wall sugar N-acetylmuramic acid (MurNAc). Members of this newly discovered family of enzymes are widely distributed among bacteria and are required to utilize peptidoglycan fragments obtained either from the environment or from the endogenous cell wall (i.e., recycling). MurNAc etherases are strictly dependent on the substrate MurNAc possessing a free reducing end and a phosphoryl group at C6. They carry a single conserved sugar phosphate isomerase/sugar phosphate- binding (SIS) domain to which MurNAc 6-phosphate is bound. Two subunits form an enzymatically active homodimer that structurally resembles the isomerase module of the double-SIS domain protein GlmS, the glucosamine 6-phosphate synthase. Structural comparison provides insights into the two-step lyase-type reaction mechanism of MurNAc etherases: β-elimination of the D-lactic acid substituent proceeds through a 2,3-unsaturated sugar intermediate to which water is subsequently added. Received 31 August 2007; received after revision 12 October 2007; accepted 1 November 2007     

From MDR to MXR: new understanding of multidrug resistance systems, their properties and clinical significance

The ATP binding cassette (ABC) superfamily of membrane transporters is one of the largest protein classes known, and counts numerous proteins involved in the trafficking of biological molecules across cell membranes. The first known human ABC transporter was P-glycoprotein (P-gp), which confers multidrug resistance (MDR) to anticancer drugs. In recent years, we have obtained an increased understanding of the mechanism of action of P-gp as its ATPase activity, substrate specificity and pharmacokinetic interactions have been investigated. This review focuses on the functional characterization of P-gp, as well as other ABC transporters involved in MDR: the family of multidrug-resistance-associated proteins (MRP1-7), and the recently discovered ABC half-transporter MXR (also known as BCRP, ABCP and ABCG2). We describe recent progress in the analysis of protein structure-function relationships, and consider the conceptual problem of defining and identifying substrates and inhibitors of MDR. An in-depth discussion follows of how coupling of nucleotide hydrolysis to substrate transport takes place, and we propose a scheme for the mechanism of P-gp function. Finally, the clinical correlations, both for reversal of MDR in cancer and for drug delivery, are discussed.     

Scavenger receptor family proteins: roles for atherosclerosis, host defence and disorders of the central nervous system

In this review, we summarize the structure and function of the scavenger receptor family of proteins including class A (type I and II macrophage scavenger receptors, MARCO), class B (CD36, scavenger receptor class BI), mucinlike (CD68/macrosialin, dSR-CI) and endothelial (LOX-1) receptors. Two motifs have been identified as ligand-binding domains a charged collagen structure of type I and II receptors, and an immunodominant domain of CD36. These structures can recognize a wide range of negatively charged macromolecules, including oxidized low-density lipoproteins, damaged or apoptotic cells, and pathogenic microorganisms. After binding, these ligands can be either internalized by endocytosis or phagocytosis, or remain at the cell surface and mediate adhesion or lipid transfer through caveolae. Under physiological conditions, scavenger receptors serve to scavenge or clean up cellular debris and other related materials, and they play a role in host defence. In pathological states, they mediate the recruitment, activation and transformation of macrophages and other cells which may be related to the development of atherosclerosis and to disorders caused by the accumulation of denatured materials, such as Alzheimer's disease. Received 17 September 1997; received after revision 16 March 1998; accepted 17 March 1998     

Myelin sheaths: glycoproteins involved in their formation,maintenance and degeneration

Myelin sheaths are formed around axons by extending, biochemically modifying and spiraling plasma membranes of Schwann cells in the peripheral nervous system (PNS) and oligodendrocytes in the central nervous system (CNS). Because glycoproteins are prominent components of plasma membranes, it is not surprising that they have important roles in the formation, maintenance and degeneration of myelin sheaths. The emphasis in this review is on four integral membrane glycoproteins. Two of them, protein zero (P0) and peripheral myelin protein-22 (PMP-22), are components of compact PNS myelin. The other two are preferentially localized in membranes of sheaths that are distinct from compact myelin. One is the myelin-associated glycoprotein, which is localized at the inside of sheaths where it functions in glia-axon interactions in both the PNS and CNS. The other is the myelin-oligodendrocyte glycoprotein, which is preferentially localized on the outside of CNS myelin sheaths and appears to be an important target antigen in autoimmune demyelinating diseases such as multiple sclerosis. Received 8 April 2002; received after revision 13 May 2002; accepted 22 May 2002     

Prolactin inducible protein in cancer, fertility and immunoregulation: structure, function and its clinical implications

Prolactin inducible protein (PIP) is a 17- kDa single polypeptide chain, known by various names due to its versatile nature and function in human reproductive and immunological systems. It is expressed in several exocrine tissues such as the lacrimal, salivary, and sweat glands. Its expression is up regulated by prolactin and androgens, and estrogens down regulate it. Due to its over-expression in metastatic breast and prostate cancer, presently PIP is considered as a prognostic biomarker. Moreover, its aspartyl-proteinase nature suggests its role in tumor progression. PIP has unique features because it is small in size and plays multiple important functions. Its ability to bind potentially with CD4-T cell receptor, immunoglobulin G (IgG), actin, zinc α2-glycoprotein (ZAG), fibronectin and enamel pellicle, reveals its important biological functions. This is the first comprehensive review on the structure and functional analysis of PIP and its clinical applications. Received 04 August 2008; received after revision 09 September 2008; accepted 15 September 2008     

CSTX-9, a toxic peptide from the spider Cupiennius salei: amino acid sequence, disulphide bridge pattern and comparison with other spider toxins containing the cystine knot structure

CSTX-9 (68 residues, 7530.9 Da) is one of the most abundant toxic polypeptides in the venom of the wandering spider Cupiennius salei. The amino acid sequence was determined by Edman degradation using reduced and alkylated CSTX-9 and peptides generated by cleavages with endoproteinase Asp-N and trypsin, respectively. Sequence comparison with CSTX-1, the most abundant and the most toxic polypeptide in the crude spider venom, revealed a high degree of similarity (53% identity). By means of limited proteolysis with immobilised trypsin and RP-HPLC, the cystine-containing peptides of CSTX-9 were isolated and the disulphide bridges were assigned by amino acid analysis, Edman degradation and nanospray tandem mass spectrometry. The four disulphide bonds present in CSTX-9 are arranged in the following pattern: 1-4, 2-5, 3-8 and 6-7 (Cys₆-Cys₂₁, Cys₁₃-Cys₃₀, Cys₂₀-Cys₄₈, Cys₃₂-Cys₄₆). Sequence comparison of CSTX-1 with CSTX-9 clearly indicates the same disulphide bridge pattern, which is also found in other spider polypeptide toxins, e.g. agatoxins (ω-AGA-IVA, ω-AGA-IVB, μ-AGA-I and μ-AGA-VI) from Agelenopsis aperta, SNX-325 from Segestria florentina and curtatoxins (CT-I, CT-II and CT-III) from Hololena curta. CSTX-1/CSTX-9 belong to the family of ion channel toxins containing the inhibitor cystine knot structural motif. CSTX-9, lacking the lysine-rich C-terminal tail of CSTX-1, exhibits a ninefold lower toxicity to Drosophila melanogaster than CSTX-1. This is in accordance with previous observations of CSTX-2a and CSTX-2b, two truncated forms of CSTX-1 which, like CSTX-9, also lack the C-terminal lysine-rich tail. Received 23 July 2001; accepted 31 July 2001