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Neuronal cell death is a pathological hallmark of Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS) and prion protein diseases (PrP). Therapy directed at eliminating the primary neurotoxic event that leads to cell death may be very effective against neurodegenerative diseases.
Several studies in the literature have now shown that an endogenously produced peptide, Humanin, can abolish cell death in neuronal cells. Humanin is a 24 amino acid secreted peptide encoded by both nuclear and mitochondrial genomes. (PNAS, 98:6336-6341, 2001, J Neurosci, 21:9235-9245, 2001, Neuroscience Letters, 324:227-231, 2002). Humanin has been shown to rescue cortical neurons from PrP118-135 fragment induced apoptosis (Mol Cell Neurosci, 25:95-102, 2004). Humanin also protected neurons from the neurotoxic effects of beta amyloid 1-42 and beta amyloid 25-35 (J Neurosci, 21:9235-9245, 2001).
Additional studies on various derivatives of Humanin reveal that replacing serine at position 14 with a glycine residue produced a peptide that was 500 to 1000 fold more potent than Humanin. Humanin S14G prevented the beta amyloid 25-35 induced impairment of short term memory in mice (Mol Cell Neurosci, 25:95-102, 2004, J Neurosci Res, 79: 714-723, 2004). Combining a 17 amino acid derivative of Humanin with the activity-dependent neurotrophic factor (ADNF) resulted in a derivative of Humanin, named Colivelin,that was 1000 fold more potent. Both Humanin and Colivelin are effective against mouse dementia and neuronal death induced by injection of beta amyloid. Colivelin also has been shown to be as effective as VEGF and IGF-1 in prolonging survival of ALS mice. Colivelin improves both motor performance and survival of ALS mice (J Neurosci, 25:10252-10261, 2005, BBRC, 343:793-798, 2006, CNS Drug Reviews, 12:113-122, 2006, Mol Neurobiol, 35:55-84, 2007).
Humanin has been shown to suppress apoptosis by interfering with Bax activation. Humanin prevents the translocation of Bax from the cytosol to the mitochondria. Humanin also binds BimEL and Bid and inhibits their induced release of SMAC and cytochrome c from mitochondria. Addition of a membrane penetrating polyarginine sequence to Humanin resulted in suppressed caspase activation in HEK293T cells (Nature, 423:456-461, 2003, J Biol Chem, 280:15825-15835, 2005, J Biol Chem, 280:15815-15824, 2005).
Humanin is a secreted peptide and may function through a cell surface receptor. Both beta amyloid 1-42 and prion peptide PrP 106-126 interact with the formyl peptide receptor-like 1 (FPRL 1)(Cytokine and Growth Factor Reviews, 12:91-105, 2001, International Immunopharmacology, 2:1-13, 2002). Studies now show that Humanin acts as a ligand for FPRL 1 and shares the receptor with beta amyloid and PrP106-126. The blocking of beta amyloid and PrP106-126 by Humanin to the cell surface receptor FPRL 1 may be a mechanism by which Humanin suppresses neuronal death (J Immunology, 172:7078-7085, 2004). The discovery that Humanin binds to FPRL 1 suggested that the N-formyl derivative of Humanin would be more potent than Humanin. A recent study has shown that N-Formylated Humanin was more potent as a ligand for FPRL 1 than Humanin (BBRC, 324:255-261, 2004).
In summary Humanin inhibits neuronal death by competing with beta amyloid and PrP106-126 for binding to the cell surface receptor FPRL 1. Colivelin acts through the Humanin receptor and the ADNF receptor. Humanin also has antiapoptotic properties. Humanin binds to Bid and BimEL and inhibits their release of SMAC and cytochrome c from mitochondria. When a cell penetrating sequence is added to Humanin the peptide is taken up by the cell and blocks Bid induced apoptosis.
Numerous bioactive peptides are encrypted within the primary structure of milk proteins and released during gastrointestinal digestion and the processing of food (J Dairy Sci, 76: 301-310, 1993, J Dairy Sci, 88: 2348-2360, 2005). Upon release, these peptides have been shown to display antithrombotic, antihypertension, immunomodulation, antimicrobial and mineral absorption activities as well as opioid agonist and antagonist behaviors.
The majority of milk content is comprised of casein and whey proteins. Caseins are phosphoproteins that consist of four major groups: alpha s1, alpha s2, beta and kappa. The main whey proteins are lactalbumin and lactoglobulin.
Bioactive peptides from kappa casein (169 amino acids) are derived from the hydrolyzed C-terminal fragment known as caseinomacropeptide (106-169). The phosphorylated form of caseinomacropeptide, kappacin, has been identified as a potent antimicrobial (Antimicrobial Agents Chemotherapy, 45:2309-2315, 2001, Antimicrobial Agents Chemotherapy, 49: 2322-2328, 2005).
Fragments of kappacin also have shown antimicrobial activity (106-115, 138-158, pS149). Fragment 106-116, named casoplatelin, is antithrombotic with inhibition of platelet aggregation (J Dairy Sci, 76: 301-310, 1993, Biochimie, 80: 155-165, 1998).
Bioactive peptides from alpha s1 casein and beta casein are active opioid agonists, antimicrobial agents as well as ACE (Angiotensin I Converting Enzyme) inhibitors (Eur Food Res Technol, 2007). Beta casein fragments (74-76) and (84-86) are potent ACE inhibitors while fragment (60-64 (casomorphin-5) is an opioid agonist and beta casein 60-66 (casomorphin-7) is an opioid agonist and an ACE inhibitor (J Nutr 134: 980S-988S, 2004, Appl Environ Microbiol, 73:4658-4667, 2007). Alpha s1 casein 90-96 (exorphin) is an opioid agonist while alpha s1 casein fragment 23-34 is an ACE inhibitor.
Bovine lactoferricin is a peptide fragment produced by acid hydrolysis of lactoferrin from cow’s milk. Lactoferricin consist of 25 amino acids (FKCRRWQWRMKKLGAPSITCVRRAF) with the antimicrobial and cytotoxic activity residing in the N-terminus (FKCRRWQWRM, FKCRRWQWRMKK). Lactoferricin has shown to be both antibacterial and antiendotoxin (J Biol Chem, 280: 16955-16961, 2005). Additional studies have shown that lactoferricin has anticancer activity both in vitro and in vivo (J. Pept Sci. 2005; 11: 379-389, Mol. Cancer Ther. 2005; 4: 612-624, Jpn J. Cancer Res. 1997; 88: 184-190, J. Natl Cancer Inst. 2004; 96: 1015-1022).
Innate, or nonspecific, immunity refers to the basic resistance to disease that all plants and animals possess. The innate immune system is the first line of defense against microbial invasions. One component of the innate immune system is the host-defense antimicrobial peptides. These peptides are active against a broad range of pathogenic microorganisms including Gram-positive and Gram-negative bacteria, fungi, yeast and viruses. The antimicrobial peptides are a diverse group of peptides but are usually short (10-60 amino acids in length), amphipathic and can be either cationic or anionic. These antimicrobial peptides can be further classified according to their secondary structure: alpha helices, beta sheet, extended helices and loops. The toxicity of antimicrobial peptides to bacteria has not yet been fully elucidated but it is believed that these peptides act by disrupting the bacterial cell membrane.
The fact that antimicrobial peptides are active against a wide range of pathogenic microorganisms makes them attractive candidates for possible use as antibiotics (Hancock & Sahl, Nature Biotechnology, 24(12):1551, 2006; L. Saiman et al, Antimicrobial Agents and Chemotherapy, 45(10):2838, 2001), bio-preservatives (R.C. Anderson et al, Australian J. Agricultural Res., 55:69, 2004), as agents in wound healing (J.D. Heilborn et al, J Invest. Dermatol., 120:379, 2003; R.A. Dorschner et al, J Invest. Dermatol., 117:91, 2001), as anticancer agents (Mol. Cancer Ther. 2005; 4: 612-624, J. Natl Cancer Inst. 2004; 96: 1015-1022) or to enhance disease resistance in aquaculture (X. Jia et al, Applied and Environmental Microbiology, 66(5):1928, 2000).
Antimicrobial peptides are a diverse group of peptides that are subgrouped according to their amino acid composition and structure:
One subgroup contains anionic peptides and is present in pulmonary surfactant and airway epithelial cells. These peptides are short (7 amino acids) and are rich in glutamic and aspartic acid residues. They are active against Gram-negative and Gram-positive bacteria and require zinc as a cofactor. Included in this group are the surfactant associated anionic peptides (SAAP) found in sheep and cattle.
A second subgroup of peptides (<40 amino acids in length) is linear, cationic, alpha helical and lack cysteine residues. These peptides show antibacterial activity against Gram-negative and Gram-positive bacteria. This group includes LL-37, SMAP29, Cecropin A, Magainin 1, Melittin, Ovispirins (Ov-1, Ov-2, Ov-3) and Pleurocidin. LL-37 is a member of the cathelicidin family and is the only cathelicidin identified in humans. In addition to LL-37 being bactericidal against a wide variety of microorganisms, it also has been shown that LL-37 is found in inflammatory cells and is upregulated in some inflamed tissues such as the skin in psoriasis. SMAP29 (Sheep Myeloid Antimicrobial Peptide) is also a cathelicidin peptide composed of 29 amino acids. SMAP29 is a very potent, broad spectrum antimicrobial peptide. It is active against Pseudomonas aeruginosa, Staphylococcus aureus and E. coli. Pleurocidin has been found in flounder and has been shown to exert activity against Gram-positve and Gram-negative bacteria. Melittin, a main component of bee venom, is one of the most potent ant-inflammatory agents know. This antimicrobial peptides also exhibits in vitro inhibitory effects on the Lyme disease spirochete.
A third subgroup of peptides contains cationic peptides enriched for specific amino acids. This group includes the tryptophan containing peptide indolicidin, the histidine-rich peptides histatin-5 and histatin-8, the arginine-rich peptide bactenecin, the proline/arginine rich peptide apidaecin and the trytophan/arginine peptide lactoferricin. Apidaecin has been shown to be active against Gram-negative bacteria while the histatins have both bactericidal and antifungal activity. Indolicidin is active against a wide range of microorganisms including Gram-negative and Gram-positive bacteria and human immunodeficiency virus (HIV-1). Bactenecin is classified as a loop peptide with one disulfide bridge and is active against Escherichia coli and Staphylococcus aureus.
Bovine lactoferricin is a peptide fragment produced by acid hydrolysis of lactoferrin from cow’s milk. Lactoferricin consist of 25 amino acids (FKCRRWQWRMKKLGAPSITCVRRAF) with the antimicrobial and cytotoxic activity residing in the N-terminus (FKCRRWQWRM, FKCRRWQWRMKK). Studies have shown that lactoferricin has anticancer activity both in vitro and in vivo (J. Pept Sci. 2005; 11: 379-389, Mol. Cancer Ther. 2005; 4: 612-624, Jpn J. Cancer Res. 1997; 88: 184-190, J. Natl Cancer Inst. 2004; 96: 1015-1022).
Lactoferricin has activity against Gram-negative bacteria. However lysis of Gram-negative bacteria results in the release of bacterial endotoxin lipopolysaccharide (LPS) which triggers sepsis. Therefore it is important that the antimicrobial peptide possesses both antibacterial and LPS neutralizing properties. Lactoferricin has shown to be both antibacterial and antiendotoxin (J Biol Chem. 2005; 280: 16955-16961).
A recent study has shown that conjugation of long chain fatty acids to the N-terminus of antimicrobial peptides increase both the bactericidal and LPS neutralizing activity by increasing the hydrophobicity of the cationic peptides (Biochem. J. 2004; 378: 93-103). Fatty acid conjugation to lactoferricin may also enhance the antibacterial, anticancer, antiviral and antiendotoxin activity of this peptide.
A fourth subgroup contains both cationic and anionic peptides. These peptides contain cysteine residues, form multiple intermolecular disulphide bonds and are stable beta sheets. This subgroup includes brevinins, protegrin and a family of defensins (alpha-defensins, beta-defensins, theta-defensins). The defensins are cationic antimicrobial peptides that are active against Gram-negative and Gram-positive bacteria, fungi, spirochetes, mycobacteria and the envelope virus HIV-1.
Antimicrobial peptides have the potential to be used as potent antibiotics, biopreservatives and wound healing agents. However more research is needed to understand the mechanism of action of these peptides. Research is needed on how to produce more efficacious antimicrobial peptides, increase the peptides serum half-life, reduce toxicity of these antimicrobial peptides and lower the cost of production.
QCB has been manufacturing and producing antibodies to antimicrobial peptides for over 10 years. Our experience in peptide libraries has enabled us to produce derivatives of peptides in a very cost effective manner. QCB is ready to produce a large variety of peptides for screening assays, modify peptides or synthesize them with D-amino acids for serum half-life studies, produce large scale quantities for animal studies and generate specific antibodies for detection assays. We are ready to partner with you to produce antimicrobial and host-defense peptides. Contact our Technical Sales Department to see how we can support your research needs.