Membrane Structure in Disease and Drug Therapy

Svante Cornell

• Publisher: CRC Press
• Year: 2000
• ISBN:
  9781135549169
• Edition: 1
• Topic: Medicine
• Subtopic: Biochemistry in Medicine

This study asserts that cellular and intracellular membranes are active in every aspect of the body's physiology and pathophysiology. It compares secondary through to quaternary structures and protien sequences and guages their influence on health, disease and drug therapy. The book highlights the importance of correlations, homologies and categori

1
Antibacterial and Hemolytic
Activity of Amphipathic
Helical Peptides

Margitta Dathe
Institute of Molecular Pharmacology, Berlin, Germany

I. INTRODUCTION

Over the past decade a multitude of peptides with cell lytic activity has been discovered in almost all kinds of species (1–4). The diversity of physiological systems in which the peptides are expressed and their broad-spectrum activities have confirmed their role as offensive and defensive weapons of creatures. Many of these peptides exhibit antimicrobial specificity and have been suggested to constitute a system of innate immunity (2,5–8). These properties have made several compounds promising candidates for the development of a new class of antibiotics (6,9,10). Stimulated by the challenge of bacterial resistance to conventional antibiotics (11–13) and by evidence for anticancer activity (9), considerable effort is being made to understand the structural basis of membrane selectivity and to elucidate the molecular mechanism of action (14,15). The peptides exert their effect by permeabilization of the lipid matrix of the target cell. It is generally accepted that the peptide–membrane interaction is the consequence of several common structural features of the peptides, such as the tendency to adopt an amphipathic conformation, as well as of properties of the anisotropic cell membrane (1,16,17). However, the molecular mechanism of membrane disturbance by the diverse membrane-active peptides is still controversial.

This review covers membrane-active peptides with an amphipathic helical structure. We address the question of what distinguishes those peptides that are capable of lysing bacteria or normal eukaryotic cells and focus on advances in understanding the mode of membrane permeabilization. Finally, the potential of peptides as therapeutic agents and as tools for pharmaceutical research is outlined.

II. MEMBRANE-PERMEABILIZING PEPTIDES

A. Classification and Biological Function

On the basis of their origin, activity, or structure, peptides with cell lytic activity can be classified into several groups. The peptides have been discovered in all organisms in which they have been sought: bacteria, fungi, insects, and vertebrates such as fish, amphibians, birds, and mammals including humans (4). With respect to their activity against eukaryotic or prokaryotic cells they can be classified as either hemolytic or antimicrobial compounds. Apart from sequence homology within peptide families, the sizes and amino acid compositions vary widely. With respect to secondary structure, the peptides can be categorized as (1) linear, mostly assuming an α-helical conformation under appropriate conditions, (2) peptides with one to three disulfide bonds leading to the formation loops or of stabilized antiparallel β-sheets, and (3) linear peptides rich in specific amino acid residues with a preferred structure distinct from the α-helix or β- conformation.

Table 1 summarizes some properties of representative peptides from diverse sources. δ-Hemolysin is a peptidic toxin from Staphylococcus aureus with high hemolytic activity (18). The alamethicin-producing fungus Trichoderma virides (19) and insect venoms are other sources of hemolytic peptides. Peptidic toxins produced by microorganisms or as components of bee and wasp venoms such as melittin (20) and mastoparan (21) serve preferentially as offensive weapons. However, in addition to their hemolytic activity, the latter are potent antibiotics. Other peptides from insect species such as the cecropins from the pupae of silk moths and flies (22,23) are produced as a response to microbial infections. The peptides possess pronounced antibacterial selectivity but are only weakly hemolytic. Insects, which have no highly specific immune response (24,25), use antimicrobial peptides to combat microbial invaders.

Lytic peptides from vertebrates are often less potent against eukaryotic cells but act on a rather broad spectrum of microorganisms (Table 1). The antimicrobial peptides from amphibians comprise a major group (26). Two representatives are the magainins from a family of antibacterial peptides that are expressed in the skin and intestine of the African clawed frog (27,28) and buforin (29), which was recently purified from the stomach of an Asian toad. Magainin was discovered when Zasloff noticed that incisions in the frog skin healed without infection, inflammation, or notable scarring despite being exposed to a bacteria-filled environment. Most of the amphibian peptides are linear and assume an α-helical conformation on interaction with the target membrane.

Table 1 Origin and Properties of Representative Cytolytic

Peptide-based antimicrobial activity is also well established in mammals. The first mammalian cecropin was isolated from the porcine small intestine (30). The potentially helical peptide exhibits high activity against gram-negative bacteria but is less active against gram-positive bacteria and practically inactive against erythrocytes. The largest group of mammalian antimicrobial peptides comprise the β-structured defensins (see reviews 31,32). The peptides are found in rabbit, rat, mouse, guinea pig, cattle, and human. While the α-defensins are stored in circulating macrophages and neutrophils, β-defensins, among them the recently found β-defensin hBD-2 (33), are produced in skin, salivary glands, and airway tissues and by epithelia of the urogenital tract in response to bacterial infections. Other mammalian antibacterial peptides with remarkable structural variability are derived from cathelicidin precursor peptides. All those peptides that incorporate a high proportion of specific amino acid residues such as arginine, proline, or tryptophan belong to this group. Among them is indolicidin (34), the smallest of the known naturally occurring linear antimicrobial peptides that may assume an extended helix (35).

Antimicrobial peptides in vertebrates are expressed in the epithelia and are used to protect the epithelial surface from microbial infections or for local control of functionally important microorganisms. Furthermore, vertebrate antibiotic peptides play a decisive role in the microbicidal mechanism after delivery to phagocytic vacuoles containing ingested microorganisms. Actually, the peptides are poorly efficient compared to the more sophisticated toxins and generally function without high specificity and memory, but they allow the animal an immediate response to microbial invasion prior to mobilization of the adaptive immune system (3,8,25). They thus constitute a secondary, chemical immune system for host defense.

B. Mechanism of Action and Properties of the Target Membranes

All available evidence suggests that peptides exert their effect by disrupting the barrier function of the target cell membrane (2,14,15,17). This activity is not mediated by membrane receptors. The facts that much higher peptide concentrations (in the micromolar range) are required than are needed for specific peptide receptor binding and that l-amino acid peptides and their all-d enantiomers are equipotent show that protein chirality does not play a role. Additionally, activity against a broad spectrum of biological membranes exhibiting varying protein and enzyme patterns as well as peptide-induced permeabilization of different lipid model membranes suggest a rather nonspecific effect of the peptides on the membrane lipid matrix.

According to our current knowledge, membrane perturbation appears to be determined by a balance of electrostatic and hydrophobic interactions between the peptides and target membranes. The envelope of gram-negative bacteria consists of the outer wall and the cytoplasmic target membrane, interspaced by a peptidoglycan layer (36) (Fig. 1a). Highly negatively charged lipopolysaccharides (LPSs) are the main constituents of the outer membrane, and metal cations help stabilize the membrane by decreasing electrostatic repulsion between the LPS molecules. This wall allows the direct passage of only small hydrophobic residues by direct crossing through the bilayer or hydrophilic permeation of small molecules through the water-filled porin channels. Antimicrobial peptides have been postulated to overcome this barrier by displacement of the native cations from their binding sites. Binding of the much larger peptides disrupts the LPS arrangement. The resulting transient lesions in the outer membrane are large enough to permit the passage of peptides (self-promoted uptake) (10,35). The pronounced negative charge of the outer wall favors the accumulation of basic host defense peptides while remaining a barrier for electrically neutral and hydrophobic sequences. The inner target membrane of gram-negative bacteria is rich in phosphatidylethanolamine (PE), a zwitterionic lipid with the tendency to destabilize membranes at low bilayer–hexagonal transition temperatures. Negatively charged phosphatidyglycerol (PG) and a markedly negative inside transmembrane potential of approximately -170 mV (37) support binding and disturbance of the cytoplasmic membrane by cationic peptides. Gram-positive bacteria have no outer membrane barrier, thus leaving the cytoplasmic membrane directly exposed to the lytic peptides. Here the presence of negatively charged teichoic and teichuroic acids (38) may facilitate the peptide–membrane interaction. Taken together, electrostatic interactions should play a decisive role for the peptide-induced permeabilization of bacterial membranes.

The lipid composition of the eukaryotic membrane is highly complex (Fig. 1b) (36). The presence of sphingomyelin (SM), phosphatidylcholine (PC), and phosphatidylethanolamine (PE) in the outer leaflet renders the membrane surface of red blood cells electrically neutral at physiological pH. A high content of cholesterol stabilizes the lipid bilayer. The transmembrane potential (-9 mV) (39) is rather low, and the anionic charge of sialic acid molecules is located too far from the bilayer surface (40) to be significant. Consequently, electrostatic forces are of minor importance and hydrophobic interactions should dominate the peptide-induced damage of eukaryotic membranes.

The precise events that cause inhibition of bacterial growth and lysis of red blood cells are not clear. Howeve...

Table of contents

• COVER PAGE
• TITLE PAGE
• COPYRIGHT PAGE
• PREFACE
• CONTRIBUTORS
• INTRODUCTION
• 1. ANTIBACTERIAL AND HEMOLYTIC ACTIVITY OF AMPHIPATHIC HELICAL PEPTIDES
• 2. BACTERIAL MEMBRANE AS A TARGET FOR A NOVEL CLASS OF DIASTEREOMERS OF CYTOLYTIC PEPTIDES
• 3. BEE VENOM TOXICITY: SYNERGISTIC ACTION OF MELITTIN ON INTERFACIAL HYDROLYSIS BY PHOSPHOLIPASE A2
• 4. POLYMYXINS: PROTOTYPE FOR A NEW CLASS OF ANTIBIOTICS
• 5. ENVIRONMENTAL EFFECTS ON SKIN LIPIDS AND IMPAIRMENT OF BARRIER FUNCTION
• 6. OXIDATIVE STRESS AND LOOSE COUPLING/UNCOUPLING
• 7. UNCOUPLERS OF OXIDATIVE PHOSPHORYLATION: ACTIVITIES AND PHYSIOLOGICAL SIGNIFICANCE
• 8. TETRAETHER LIPID LIPOSOMES
• 9. TRANSFECTION OF EUKARYOTIC CELLS WITH BIPOLAR CATIONIC DERIVATIVES OF TETRAETHER LIPID
• 10. CHOLERA TOXIN CONFORMATIONAL CHANGES ASSOCIATED WITH CHANGES IN MEMBRANE STRUCTURE
• 11. PARASITE MITOCHONDRIAL MEMBRANE FUNCTIONS AS TARGETS FOR CHEMOTHERAPY
• 12. HEPATIC CELL FUNCTION IN LIVER FLUKE INFECTION
• 13. LIPOPOLYSACCHARIDE: A MEMBRANE-FORMING AND INFLAMMATION-INDUCING BACTERIAL MACROMOLECULE
• 14. EFFECT OF CHOLESTASIS ON BIOMEMBRANES
• 15. 3,5-DIIODOTHYRONINE BINDS TO SUBUNIT VA OF CYTOCHROME c OXIDASE: POSSIBLE MECHANISM OF SHORT-TERM EFFECTS OF THYROID HORMONES
• 16. REGULATION OF GLUCOSE TRANSPORT BY INSULIN IN MUSCLE AND FAT CELLS: TRANSLOCATION AND ACTIVATION OF GLUCOSE TRANSPORTERS
• 17. GLUCOSE-6-PHOSPHATASE: A MEMBER OF THE NEWLY IDENTIFIED HPP SUPERFAMILY THAT CONSISTS OF HISTIDINE PHOSPHATASES AND VANADIUM-CONTAINING PEROXIDASES AND CONSEQUENCES FOR MEMBRANE TOPOLOGY, ACTIVE SITE, AND REACTION MECHANISM
• 18. ALZHEIMER’S AMYLOID β-PEPTIDE-ASSOCIATED OXIDATIVE STRESS: BRAIN MEMBRANE LIPID PEROXIDATION AND PROTEIN OXIDATION
• 19. MEMBRANE ORIENTATION OF THE ALZHEIMER’S DISEASE–ASSOCIATED PRESENILINS
• 20. MEMBRANE STRUCTURE ANALYSIS IN APOPTOTIC PROCESSES OF LEUKEMIC BLASTS AND LEUKEMIA-DERIVED CELL LINES
• 21. USE OF MONOCLONAL ANTIBODIES IN CLONING AND IDENTIFICATION OF MEMBRANE ANTIGENS
• 22. POLYCYSTINS: MEMBRANE-ASSOCIATED PROTEINS INVOLVED IN AUTOSOMAL DOMINANT POLYCYSTIC KIDNEY DISEASE
• 23. ACTION OF β-AGONISTS COMPARED TO CROMOGLYCATE ON MONONUCLEAR CELL MEMBRANES: STABILIZING OR DESTABILIZING?
• 24. CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR: A CHLORIDE CHANNEL REGULATOR OF ION CHANNELS
• 25. ROLE OF CA2+ -INDEPENDENT LYSOSOMAL PHOSPHOLIPASE A2 IN TURNOVER OF LUNG SURFACTANT PHOSPHOLIPIDS
• 26. ROLE OF SODIUM PUMP IN DISEASE