Definition
Fluorescence resonance energy transfer (FRET) is a distance-dependent interaction between the electronic excited states of two dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon1. Following are some of the standard dye combinations used: a)Fluorescein and Dabcyl or Dabsyl b) Fluorescein and Tamra c) Methoxy-coumarin-acetic-acid(MCA) and 2,4-Dinitrophenyl(DNP).
Discovery
The first internally quenched fluorescent peptide reported was a substrate for angiotensin I-converting enzyme (ACE), namely Abz-Gly-Phe(NO2)-Pro where the fluorescence of the N-terminal ortho-amino benzoic acid (Abz) was quenched by the p-nitro-phenylalanine [Phe(NO2)] group (Carmel and Yaron 1978) 2. A new generation of fluorescence-quenched substrates was developed by Chagas et al., (1991), using the FRET peptide concept described substrates for tissue and plasma kallikrein containing Abz as the fluorescent group and EDDnp (2,4-dinitrophenyl ethylenediamine) as the quencher group. The FRET peptides introduced by Chagas et al., (1991) was a breakthrough in the study of proteases’ specificity, and the synthesis of different Abzpeptidyl-EDDnp sequences provided the opportunity for us to study the activity of various endopeptidases such as human rennin3.
Structural Characteristics
FRET peptides are labelled with two dye molecules. These can be identical, but in most applications, different dyes are commonly used. FRET describes the transfer of energy from an initially excited donor (dye 1) to an acceptor (dye 2). Typically, this donor emits light at a wavelength λd that overlaps with the absorption wavelength λa of the acceptor. If donor and acceptor dye are in close proximity (10 – 100 Å),
this energy transfer happens in one of two ways, depending on the chemical structure of the acceptor:
a) the transferred energy is converted to molecular vibrations (acceptor is dark quencher)
b) the transferred energy is emitted as light with a longer wavelength (acceptor is fluorescent).
Mode of Action
In a FRET peptide fluorescent donor group attached to one of the amino acid residues of the peptide transfers energy to a quenching acceptor attached to another residue in the sequence following the resonance mechanism. This process occurs whenever the emission spectrum of the fluorophore overlaps with the absorption spectrum of the acceptor (Sapsford et al., 2006)4. The FRET peptides exhibit internal fluorescence quenching when intact, but cleavage of any peptide bond between the donor/acceptor pair liberates fluorescence that can be detected continuously, allowing a quantitative measurement of the enzyme activity.
Functions
Fluorescence Energy Resonance Transfer (FRET) peptides are an excellent alternative for enzyme kinetic studies and for analysis of the enzymatic activity in biological fluids, crude tissue extracts or on the surface of cells in culture.
FRET peptides are used as suitable substrates in enzyme studies, such as:
1. Functional characterization of peptidases / proteases / kinases / phosphatases.
2. Kinetic characterization of peptidases / proteases / kinases / phosphatases.
3. Screening and detection of new proteolytic enzymes.
4. They can be used for conformational investigation of peptide folding.
References
1.Hossain MA, Mihara H, Ueno A (2003). Fluorescence resonance energy transfer in a novel cyclodextrin-peptide conjugate for detecting steroid molecules. Bioorg Med Chem Lett., 13(24):4305-4308.
2.Carmel A, Yaron A (1978). An intramolecularly quenched fluorescent tripeptide as a fluorogenic substrate of angiotensin-I-converting enzyme and of bacterial dipeptidyl carboxypeptidase. Eur J Biochem., 87(2):265-273.
3.Chagas JR, Juliano L, Prado ES (1991). Intramolecularly quenched fluorogenic tetrapeptide substrates for tissue and plasma kallikreins. Anal Biochem., 192(2):419-425.
4.Sapsford KE, Berti L, Medintz IL (2006). Materials for fluorescence resonance energy transfer analysis: beyond traditional donor-acceptor combinations. Angew Chem Int Ed Engl., 45(28):4562-4589.
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Monday, February 22, 2010
Tuesday, February 9, 2010
Secretase Inhibitors and Substrates
Definition
ß- and g-secretases generate the amyloid ß-protein (Aß) 1 from amyloid ß-protein precursor (APP) that is the hallmark of Alzheimer disease. Gamma (g)?-secretase has also been implicated in the cleavage of other substrates, the most notable one being the Notch receptor. Inhibition of Notch processing is the key factor of mechanism-based side effects associated with g-secretase inhibitors.
Discovery
Vassar R in 2000 discussed about a beta generating enzyme in Alzheimer disease (AD) 2. Esler in 2002 carried out activity-dependent isolation of the presenilin- g-secretase complex. The functional g-secretase complex was isolated by using an immobilized active site-directed inhibitor of the protease. Presenilin heterodimers and nicastrin bound specifically to this inhibitor under conditions tightly correlating with protease activity, whereas several other presenilin-interacting proteins (beta-catenin, calsenilin, and presenilin-associated protein) did not bind. Moreover, anti-nicastrin antibodies immunoprecipitated g-secretase activity from detergent-solubilized microsomes. Unexpectedly, C83, the major endogenous amyloid-beta precursor protein substrate of g-secretase, was also quantitatively associated with the complex 3.
Structural Characteristics
AD is characterized by the progressive accumulation of Aß in brain regions subserving memory and cognition. ß-Secretase is a single membrane-spanning aspartyl pro-tease expressed at high levels in neurons 2. ?-Secretase is also an aspartyl protease but with an unprecedented intramembranous catalytic site that is required for the cleavage of a wide range of type I membrane proteins that include APP and the Notch receptors3. g-secretase is a multi-subunit protease complex, which is an integral membrane protein that cleaves single-pass transmembrane proteins. g???secretase complex consists of four individual proteins: presenilin, nicastrin, APH-1 (anterior pharynx-defective 1), and PEN-2 (presenilin enhancer 2). PEN-2 associates with the complex via binding of a transmembrane domain of presenilin. APH-1, which is required for proteolytic activity, binds to the complex via a conserved alpha helix interaction motif and aids in initiating assembly of premature components4,5. Substrate of g?? secretase is amyloid precursor protein, a large integral membrane protein that, which is cleaved by both g and b secretase, into a short 39-42 amino acid peptide known as amyloid beta whose abnormally folded fibrillar form is the primary component of amyloid plaques found in the brains of AD patients. Substrate recognition occurs via nicastrin ectodomain binding to the N-terminus of the target, which is then passed via a poorly understood process between the two presenilin fragments to a water-containing active site at which the catalytic aspartate residue resides. Structure of novel g-secretase inhibitor is [1S-benzyl-4R-[1-(5-cyclohexyl-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3(R,S)-ylcarbamoyl)-S-ethylcarbamoyl]-2R-hydroxy-5-phenyl-pentyl]-carbamic acid tert-butyl ester (CBAP), which not only physically interacts with presenilin 1 (PS1), but upon chronic treatment produces a "pharmacological knock-down" of PS1 fragments 6.
Mode of Action
Development of AD pathology appears to be causally related to age-dependent changes in the metabolism of the Aß, leading to its enhanced aggregation and deposition. g-Secretase is a crucial enzyme for the generation of antibody from the APP and thus represents a valid potential therapeutic target for the treatment or prevention of AD. Enzyme activity has been shown to be dependent on the expression of presenilins and the identification of inhibitors containing transition-state analogue mimics, together with mutagenesis and knockout studies, confirms that presenilins may provide at least a component of the catalytic site for this putative aspartyl protease. Considerable effort has been expended to identify compounds which specifically reduce g-secretase activity in the central nervous system, and those with the appropriate properties are being utilized in on-going proof-of-concept studies in animals and humans, to determine the extent and duration of g-secretase inhibition required to elicit therapeutic benefits. g-secretase-mediated substrate cleavage fall into the category of 'regulated intramembrane proteolysis'. g -secretase itself displays characteristics of an aspartyl protease as it is inhibited by structurally diverse transition-state mimetics for this class of protease. These include compounds with moderate affinities such as substrate-based difluoroketones or an inhibitor with potency in the low-nM range that contains a hydroxyethylene dipeptide isostere 7,8.
Functions
Cause of Alzheimer's disease, sequential action of b-secretase and g-secretase enzymes leads to releases the APP. This custom peptide entity is thought to be the cause of AD. Therefore, inhibiting either of the two critical enzymes involved in antibody peptide generation provides opportunities to develop drugs which could have the potential to slow down the progression and delay the onset of AD 9.
Gamma-secretase inhibitors may have the ability to interfere with both intermolecular and intramolecular cleavage reactions, which require presenilins. Thus, from a drug-discovery point of view it is important that g-secretase inhibitors interfere with presenilin functions 10.
Tunicamycin-induced expression of the chaperone BiP, an indicator of the unfolded protein response, was not changed in the presence of functional g-secretase inhibitors. These findings suggest that presenilins are multi-functional proteins and that gamma-secretase inhibitors are valuable tools to discriminate presenilin-associated protease functions from unrelated functions.
References
1. Selkoe DJ (2001). Alzheimer's disease: genes, proteins, and therapy. Physiol Rev., 81:741–766.Vassar R, Citron M. (2000). A beta-generating enzymes: recent advances in beta- and gamma-secretase research. Neuron, 27:419–422.
2. Esler WP, Kimberly WT, Ostaszewski BL, Ye W, Diehl TS, Selkoe DJ, Wolfe MS (2002). Activity-dependent isolation of the presenilin- gamma -secretase complex reveals nicastrin and a gamma substrate. PNAS., 99:2720–2725.
3. Kaether C, Haass C, Steiner H. (2006). Assembly, trafficking and function of gamma-secretase. Neurodegener Dis., 3(4-5):275-283.
4. Lee SF, Shah S, Yu C, Wigley WC, Li H, Lim M, Pedersen K, Han W, Thomas P, Lundkvist J, Hao YH, Yu G (2004). A conserved GXXXG motif in APH-1 is critical for assembly and activity of the gamma-secretase complex. J Biol Chem., 279(6):4144-4152.
5. Beher D, Wrigley JD, Nadin A, Evin G, Masters CL, Harrison T, Castro JL, Shearman MS (2001). Pharmacological knock-down of the presenilin 1 heterodimer by a novel gamma -secretase inhibitor: implications for presenilin biology. J. Biol. Chem., 276:45394–45402.
6. Shearman MS, Beher D, Clarke EE, Lewis HD, Harrison T, Hunt P, Nadin A, Smith AL, Stevenson G, Castro JL (2000.) L-685,458, an aspartyl protease transition state mimic, is a potent inhibitor of amyloid beta-protein precursor gamma-secretase activity. Biochemistry, 38:8698–8704.
7. Brown MS, Ye J, Rawson RB, Goldstein JL (2000). Regulated intramembrane proteolysis: a control mechanism conserved from bacteria to humans. Cell, 100:391–398.
8. Hardy J, Selkoe DJ (2002). The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science, 297(5580):353-356.
9. Mumm JS, Kopan R (2000) Notch signaling: from the outside in. Dev. Biol., 228:151-165
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ß- and g-secretases generate the amyloid ß-protein (Aß) 1 from amyloid ß-protein precursor (APP) that is the hallmark of Alzheimer disease. Gamma (g)?-secretase has also been implicated in the cleavage of other substrates, the most notable one being the Notch receptor. Inhibition of Notch processing is the key factor of mechanism-based side effects associated with g-secretase inhibitors.
Discovery
Vassar R in 2000 discussed about a beta generating enzyme in Alzheimer disease (AD) 2. Esler in 2002 carried out activity-dependent isolation of the presenilin- g-secretase complex. The functional g-secretase complex was isolated by using an immobilized active site-directed inhibitor of the protease. Presenilin heterodimers and nicastrin bound specifically to this inhibitor under conditions tightly correlating with protease activity, whereas several other presenilin-interacting proteins (beta-catenin, calsenilin, and presenilin-associated protein) did not bind. Moreover, anti-nicastrin antibodies immunoprecipitated g-secretase activity from detergent-solubilized microsomes. Unexpectedly, C83, the major endogenous amyloid-beta precursor protein substrate of g-secretase, was also quantitatively associated with the complex 3.
Structural Characteristics
AD is characterized by the progressive accumulation of Aß in brain regions subserving memory and cognition. ß-Secretase is a single membrane-spanning aspartyl pro-tease expressed at high levels in neurons 2. ?-Secretase is also an aspartyl protease but with an unprecedented intramembranous catalytic site that is required for the cleavage of a wide range of type I membrane proteins that include APP and the Notch receptors3. g-secretase is a multi-subunit protease complex, which is an integral membrane protein that cleaves single-pass transmembrane proteins. g???secretase complex consists of four individual proteins: presenilin, nicastrin, APH-1 (anterior pharynx-defective 1), and PEN-2 (presenilin enhancer 2). PEN-2 associates with the complex via binding of a transmembrane domain of presenilin. APH-1, which is required for proteolytic activity, binds to the complex via a conserved alpha helix interaction motif and aids in initiating assembly of premature components4,5. Substrate of g?? secretase is amyloid precursor protein, a large integral membrane protein that, which is cleaved by both g and b secretase, into a short 39-42 amino acid peptide known as amyloid beta whose abnormally folded fibrillar form is the primary component of amyloid plaques found in the brains of AD patients. Substrate recognition occurs via nicastrin ectodomain binding to the N-terminus of the target, which is then passed via a poorly understood process between the two presenilin fragments to a water-containing active site at which the catalytic aspartate residue resides. Structure of novel g-secretase inhibitor is [1S-benzyl-4R-[1-(5-cyclohexyl-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3(R,S)-ylcarbamoyl)-S-ethylcarbamoyl]-2R-hydroxy-5-phenyl-pentyl]-carbamic acid tert-butyl ester (CBAP), which not only physically interacts with presenilin 1 (PS1), but upon chronic treatment produces a "pharmacological knock-down" of PS1 fragments 6.
Mode of Action
Development of AD pathology appears to be causally related to age-dependent changes in the metabolism of the Aß, leading to its enhanced aggregation and deposition. g-Secretase is a crucial enzyme for the generation of antibody from the APP and thus represents a valid potential therapeutic target for the treatment or prevention of AD. Enzyme activity has been shown to be dependent on the expression of presenilins and the identification of inhibitors containing transition-state analogue mimics, together with mutagenesis and knockout studies, confirms that presenilins may provide at least a component of the catalytic site for this putative aspartyl protease. Considerable effort has been expended to identify compounds which specifically reduce g-secretase activity in the central nervous system, and those with the appropriate properties are being utilized in on-going proof-of-concept studies in animals and humans, to determine the extent and duration of g-secretase inhibition required to elicit therapeutic benefits. g-secretase-mediated substrate cleavage fall into the category of 'regulated intramembrane proteolysis'. g -secretase itself displays characteristics of an aspartyl protease as it is inhibited by structurally diverse transition-state mimetics for this class of protease. These include compounds with moderate affinities such as substrate-based difluoroketones or an inhibitor with potency in the low-nM range that contains a hydroxyethylene dipeptide isostere 7,8.
Functions
Cause of Alzheimer's disease, sequential action of b-secretase and g-secretase enzymes leads to releases the APP. This custom peptide entity is thought to be the cause of AD. Therefore, inhibiting either of the two critical enzymes involved in antibody peptide generation provides opportunities to develop drugs which could have the potential to slow down the progression and delay the onset of AD 9.
Gamma-secretase inhibitors may have the ability to interfere with both intermolecular and intramolecular cleavage reactions, which require presenilins. Thus, from a drug-discovery point of view it is important that g-secretase inhibitors interfere with presenilin functions 10.
Tunicamycin-induced expression of the chaperone BiP, an indicator of the unfolded protein response, was not changed in the presence of functional g-secretase inhibitors. These findings suggest that presenilins are multi-functional proteins and that gamma-secretase inhibitors are valuable tools to discriminate presenilin-associated protease functions from unrelated functions.
References
1. Selkoe DJ (2001). Alzheimer's disease: genes, proteins, and therapy. Physiol Rev., 81:741–766.Vassar R, Citron M. (2000). A beta-generating enzymes: recent advances in beta- and gamma-secretase research. Neuron, 27:419–422.
2. Esler WP, Kimberly WT, Ostaszewski BL, Ye W, Diehl TS, Selkoe DJ, Wolfe MS (2002). Activity-dependent isolation of the presenilin- gamma -secretase complex reveals nicastrin and a gamma substrate. PNAS., 99:2720–2725.
3. Kaether C, Haass C, Steiner H. (2006). Assembly, trafficking and function of gamma-secretase. Neurodegener Dis., 3(4-5):275-283.
4. Lee SF, Shah S, Yu C, Wigley WC, Li H, Lim M, Pedersen K, Han W, Thomas P, Lundkvist J, Hao YH, Yu G (2004). A conserved GXXXG motif in APH-1 is critical for assembly and activity of the gamma-secretase complex. J Biol Chem., 279(6):4144-4152.
5. Beher D, Wrigley JD, Nadin A, Evin G, Masters CL, Harrison T, Castro JL, Shearman MS (2001). Pharmacological knock-down of the presenilin 1 heterodimer by a novel gamma -secretase inhibitor: implications for presenilin biology. J. Biol. Chem., 276:45394–45402.
6. Shearman MS, Beher D, Clarke EE, Lewis HD, Harrison T, Hunt P, Nadin A, Smith AL, Stevenson G, Castro JL (2000.) L-685,458, an aspartyl protease transition state mimic, is a potent inhibitor of amyloid beta-protein precursor gamma-secretase activity. Biochemistry, 38:8698–8704.
7. Brown MS, Ye J, Rawson RB, Goldstein JL (2000). Regulated intramembrane proteolysis: a control mechanism conserved from bacteria to humans. Cell, 100:391–398.
8. Hardy J, Selkoe DJ (2002). The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science, 297(5580):353-356.
9. Mumm JS, Kopan R (2000) Notch signaling: from the outside in. Dev. Biol., 228:151-165
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Malaria Peptides
Definition
Malaria is spread by Anopheles mosquitoes, the protozoan parasite grows and multiplies within erythrocytes in its asexual cycle. Different malaria peptides were isolated by human antibodies in plasma samples of individuals exposed to chronic malaria.
Discovery
The first approach of a malaria peptide vaccine was a polymeric chimeric molecule named SPf66, which conferred limited protective efficacy in monkey and human trial 1. Apical membrane antigen-1 is a protein found in the merozoite stage of the malaria parasite Plasmodium falciparum and has been shown to be critical in the invasion of host erythrocytes. Using a random peptide library displayed on the surface of phage, several peptides were identified which specifically recognized and bound to P. falciparum antigen 2. Different malaria peptide specific antibodies were obtained both by affinity-purification from malaria immune sera and by immunization of mice and these peptide candidates are used as vaccine candidates for malaria. Malaria peptides that are used as therapeutic purpose are Malaria CSP (334 - 342), Malaria aspartyl proteinase FRET substrate I, MSP - 1 P2, Malaria merozoite surface peptide – 1, peptides from circumsporozoite (CS) Protein, hemoglobin, 3037a etc. 3,4,5,6.
Structural Characteristics
These are smaller peptides of different sizes. The sequence of Malaria CSP (334 - 342) is H - Tyr - Leu - Lys - Lys - Ile - Lys - Asn - Ser - Leu – OH. MSP - 1 P3, Malaria Merozoite Surface Peptide – 1 sequence is H - Lys - Leu - Asn - Ser - Leu - Asn - Asn - Pro - His - Asn - Val - Leu - Gln - Asn - Phe - Ser - Val - Phe - Phe - Asn - Lys – OH. The amino acid sequence of Hemoglobin, 3037a, Malaria FRET Substrate II—is DABCYL - GABA - Glu - Arg - Met - Phe - Leu - Ser - Phe - Pro – EDANS. The immunodominant T cell epitope of the merozoite antigen ring-infected erythrocyte surface antigen sequence is H-Leu-Gly-Arg-Ser-Gly-Gly-Asp-Ile-Ile-Lys-Lys-Met-Gln-Thr-Leu-OH 3,4,5,6.
Mode of Action
Malaria peptides has been shown to accelerate the humoral antibody immune responses and as a free peptide increase the protective efficacy of cellular immune response and sustain the immunity for a longer time against malaria A. Peptides copying malaria protein sequences often stimulate human CD4+ T cells and it was thought that they represented T cell epitopes present in the parasite and may thus have particular relevance to malaria vaccine development. A 15 residue peptide (PP1) inhibited falcipain-2 enzyme of Plasmodium falciparum activity in vitro. PP1 fused with Antennapedia homeoprotein internalization domain blocked hemoglobin hydrolysis, merozoite release and markedly inhibited Plasmodium falciparum growth and maturation 7. A peptide derived from the proregion of falcipain-2 that blocks late-stage malaria parasite development in RBCs, suggesting the development of peptide and peptidometric drugs against the human malaria parasite 2.
Functions
Malaria CSP (334 - 342) - This is amino acids 334 to 342 fragment of malaria CSP derived from malaria circumsporozoite protein. It is commonly used as a control peptide for melanoma vaccine studies 8.
Malaria Aspartyl Proteinase FRET Substrate I- A useful fluorogenic substrate for the continuous assay of malaria aspartyl proteinase.
MSP - 1 P2, Malaria Merozoite Surface Peptide - 1- This peptide is a fragment of malaria Merozoite Protein 1, MSP-1 (codons 250–271), a malaria vaccine candidate designated P2. It is used to assess T cell response to N-terminal region of MSP-1 4.
MSP - 1 P3, Malaria Merozoite Surface Peptide - 1-This N-terminal region fragment of Malaria, Merozoite Surface Protein (codons 1101–1121), is a malaria vaccine candidate designated as P3. It is used to assess T cell response to the N-terminal region of major merozoite surface protein 1 (MSP-1).
Hemoglobin, 3037a, Malaria FRET Substrate II- The sequence of this peptide is based on the primary site of cleavage within hemoglobin (Hb). This peptide was used to characterize the molecular mechanism underlying Hb degradation by plasmepsin II (PM II). N-terminal (GABA) extension results in higher maximal velocity and dramatic concentration-dependent substrate inhibition 5.
Circumsporozoite (CS) Protein Sequence-This cell-adhesive motif in region II of malarial circumsporozoite protein has also been found in thrombospondin, properdin, and in a blood-stage antigen of Plasmodium falciparum. It was shown to be the critical sequence for the observed cell-adhesive properties 9.
RESA Peptides-This peptide is identical to the immunodominant T cell epitope of the merozoite antigen ring-infected erythrocyte surface antigen Pf155/RESA in its nonrepeated amino-terminal region (residues 181-195) 10.
Malaria Aspartyl Proteinase Substrate- Useful peptide substrate for a continuous fluorescence-based assay of the malaria aspartyl proteinase. The peptide sequence is derived from the cleavage site present in haemoglobin 11.
References
1.Moorthy VS, Good MF, Hill AV (2004). Malaria vaccine developments. Lancet., 363(9403):150-156.
2.Scanlon D, Harris KS, Coley AM, Karas JA, Casey JL, Hughes AB, Foley M (2008). Comprehensive N-Methyl Scanning of a Potent Peptide Inhibitor of Malaria Invasion into Erythrocytes Leads to Pharmacokinetic Optimization of the Molecule. International Journal of Peptide Research and Therapeutics, 14(4):381-386.
3.Yamshchikov GV, Mullins DW, Chang CC, Ogino T, Thompson L, Presley J, Galavotti H, Aquila W, Deacon D, Ross W, Patterson JW, Engelhard VH, Ferrone S, Slingluff CL Jr. (2005). Sequential immune escape and shifting of T cell responses in a long-term survivor of melanoma. J Immunol., 174(11):6863-6871.
4.King CL, Malhotra I, Wamachi A, Kioko J, Mungai P, Wahab SA, Koech D, Zimmerman P, Ouma J, Kazura JW.(2002). Acquired immune responses to Plasmodium falciparum merozoite surface protein-1 in the human fetus. J. Immunol., 168(1):356-64.
5.Istvan E, Goldberet D (2005). Distal Substrate Interactions Enhance Plasmepsin Activity. The Journal of Biological Chemistry, 280:6890-6896.
6.Savi P, Bernat A, Lalé A, Roque C, Zamboni G, Herbert JM (2000). Effect of aspirin on platelet desaggregation induced by SR121566, a potent GP-IIb/IIIa antagonist. Platelets, 11(1):43-48
7.Korde R, Bhardwaj A, Singh R, Srivastava A, Chauhan VS, Bhatnagar RK, Malhotra P (2008). A prodomain peptide of plasmodium falciparum cysteine protease(falcipain-2) inhibits malaria parasite development. J. Med. Chem., 51(11): 3116–3123.
8.Blum-Tirouvanziam U, Servis C, Habluetzel A, Valmori D, Men Y, Esposito F, Del Nero L, Holmes N, Fasel N, Corradin G (1995). Localization of HLA-A2.1-restricted T cell epitopes in the circumsporozoite protein of Plasmodium falciparum. J. Immunol., 154(8):3922-3931.
9.Flotow H, Leong CY, Buss AD (2002). Development of a plasmepsin II fluorescence polarization assay suitable for high throughput antimalarial drug discovery. J Biomol Screen., 7(4):367-371.
10. Chougnet C, Troye-Blomberg M, Deloron P, Kabilan L, Lepers JP, Savel J, Perlmann P (1991). Human immune response in Plasmodium falciparum malaria. Synthetic peptides corresponding to known epitopes of the Pf155/RESA antigen induce production of parasite-specific antibodies in vitro. J. Immunol., 147(7):2295-2301.
11. Jiang S, Prigge ST, Wei L, Gao Ye, Hudson TH, Gerena L, Dame JB, Kyle DE (2001). New class of small nonpeptidyl compounds blocks Plasmodium falciparum development in vitro by inhibiting plasmepsins. Antimicrob Agents Chemother., 45(9):2577-2584.
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Malaria is spread by Anopheles mosquitoes, the protozoan parasite grows and multiplies within erythrocytes in its asexual cycle. Different malaria peptides were isolated by human antibodies in plasma samples of individuals exposed to chronic malaria.
Discovery
The first approach of a malaria peptide vaccine was a polymeric chimeric molecule named SPf66, which conferred limited protective efficacy in monkey and human trial 1. Apical membrane antigen-1 is a protein found in the merozoite stage of the malaria parasite Plasmodium falciparum and has been shown to be critical in the invasion of host erythrocytes. Using a random peptide library displayed on the surface of phage, several peptides were identified which specifically recognized and bound to P. falciparum antigen 2. Different malaria peptide specific antibodies were obtained both by affinity-purification from malaria immune sera and by immunization of mice and these peptide candidates are used as vaccine candidates for malaria. Malaria peptides that are used as therapeutic purpose are Malaria CSP (334 - 342), Malaria aspartyl proteinase FRET substrate I, MSP - 1 P2, Malaria merozoite surface peptide – 1, peptides from circumsporozoite (CS) Protein, hemoglobin, 3037a etc. 3,4,5,6.
Structural Characteristics
These are smaller peptides of different sizes. The sequence of Malaria CSP (334 - 342) is H - Tyr - Leu - Lys - Lys - Ile - Lys - Asn - Ser - Leu – OH. MSP - 1 P3, Malaria Merozoite Surface Peptide – 1 sequence is H - Lys - Leu - Asn - Ser - Leu - Asn - Asn - Pro - His - Asn - Val - Leu - Gln - Asn - Phe - Ser - Val - Phe - Phe - Asn - Lys – OH. The amino acid sequence of Hemoglobin, 3037a, Malaria FRET Substrate II—is DABCYL - GABA - Glu - Arg - Met - Phe - Leu - Ser - Phe - Pro – EDANS. The immunodominant T cell epitope of the merozoite antigen ring-infected erythrocyte surface antigen sequence is H-Leu-Gly-Arg-Ser-Gly-Gly-Asp-Ile-Ile-Lys-Lys-Met-Gln-Thr-Leu-OH 3,4,5,6.
Mode of Action
Malaria peptides has been shown to accelerate the humoral antibody immune responses and as a free peptide increase the protective efficacy of cellular immune response and sustain the immunity for a longer time against malaria A. Peptides copying malaria protein sequences often stimulate human CD4+ T cells and it was thought that they represented T cell epitopes present in the parasite and may thus have particular relevance to malaria vaccine development. A 15 residue peptide (PP1) inhibited falcipain-2 enzyme of Plasmodium falciparum activity in vitro. PP1 fused with Antennapedia homeoprotein internalization domain blocked hemoglobin hydrolysis, merozoite release and markedly inhibited Plasmodium falciparum growth and maturation 7. A peptide derived from the proregion of falcipain-2 that blocks late-stage malaria parasite development in RBCs, suggesting the development of peptide and peptidometric drugs against the human malaria parasite 2.
Functions
Malaria CSP (334 - 342) - This is amino acids 334 to 342 fragment of malaria CSP derived from malaria circumsporozoite protein. It is commonly used as a control peptide for melanoma vaccine studies 8.
Malaria Aspartyl Proteinase FRET Substrate I- A useful fluorogenic substrate for the continuous assay of malaria aspartyl proteinase.
MSP - 1 P2, Malaria Merozoite Surface Peptide - 1- This peptide is a fragment of malaria Merozoite Protein 1, MSP-1 (codons 250–271), a malaria vaccine candidate designated P2. It is used to assess T cell response to N-terminal region of MSP-1 4.
MSP - 1 P3, Malaria Merozoite Surface Peptide - 1-This N-terminal region fragment of Malaria, Merozoite Surface Protein (codons 1101–1121), is a malaria vaccine candidate designated as P3. It is used to assess T cell response to the N-terminal region of major merozoite surface protein 1 (MSP-1).
Hemoglobin, 3037a, Malaria FRET Substrate II- The sequence of this peptide is based on the primary site of cleavage within hemoglobin (Hb). This peptide was used to characterize the molecular mechanism underlying Hb degradation by plasmepsin II (PM II). N-terminal (GABA) extension results in higher maximal velocity and dramatic concentration-dependent substrate inhibition 5.
Circumsporozoite (CS) Protein Sequence-This cell-adhesive motif in region II of malarial circumsporozoite protein has also been found in thrombospondin, properdin, and in a blood-stage antigen of Plasmodium falciparum. It was shown to be the critical sequence for the observed cell-adhesive properties 9.
RESA Peptides-This peptide is identical to the immunodominant T cell epitope of the merozoite antigen ring-infected erythrocyte surface antigen Pf155/RESA in its nonrepeated amino-terminal region (residues 181-195) 10.
Malaria Aspartyl Proteinase Substrate- Useful peptide substrate for a continuous fluorescence-based assay of the malaria aspartyl proteinase. The peptide sequence is derived from the cleavage site present in haemoglobin 11.
References
1.Moorthy VS, Good MF, Hill AV (2004). Malaria vaccine developments. Lancet., 363(9403):150-156.
2.Scanlon D, Harris KS, Coley AM, Karas JA, Casey JL, Hughes AB, Foley M (2008). Comprehensive N-Methyl Scanning of a Potent Peptide Inhibitor of Malaria Invasion into Erythrocytes Leads to Pharmacokinetic Optimization of the Molecule. International Journal of Peptide Research and Therapeutics, 14(4):381-386.
3.Yamshchikov GV, Mullins DW, Chang CC, Ogino T, Thompson L, Presley J, Galavotti H, Aquila W, Deacon D, Ross W, Patterson JW, Engelhard VH, Ferrone S, Slingluff CL Jr. (2005). Sequential immune escape and shifting of T cell responses in a long-term survivor of melanoma. J Immunol., 174(11):6863-6871.
4.King CL, Malhotra I, Wamachi A, Kioko J, Mungai P, Wahab SA, Koech D, Zimmerman P, Ouma J, Kazura JW.(2002). Acquired immune responses to Plasmodium falciparum merozoite surface protein-1 in the human fetus. J. Immunol., 168(1):356-64.
5.Istvan E, Goldberet D (2005). Distal Substrate Interactions Enhance Plasmepsin Activity. The Journal of Biological Chemistry, 280:6890-6896.
6.Savi P, Bernat A, Lalé A, Roque C, Zamboni G, Herbert JM (2000). Effect of aspirin on platelet desaggregation induced by SR121566, a potent GP-IIb/IIIa antagonist. Platelets, 11(1):43-48
7.Korde R, Bhardwaj A, Singh R, Srivastava A, Chauhan VS, Bhatnagar RK, Malhotra P (2008). A prodomain peptide of plasmodium falciparum cysteine protease(falcipain-2) inhibits malaria parasite development. J. Med. Chem., 51(11): 3116–3123.
8.Blum-Tirouvanziam U, Servis C, Habluetzel A, Valmori D, Men Y, Esposito F, Del Nero L, Holmes N, Fasel N, Corradin G (1995). Localization of HLA-A2.1-restricted T cell epitopes in the circumsporozoite protein of Plasmodium falciparum. J. Immunol., 154(8):3922-3931.
9.Flotow H, Leong CY, Buss AD (2002). Development of a plasmepsin II fluorescence polarization assay suitable for high throughput antimalarial drug discovery. J Biomol Screen., 7(4):367-371.
10. Chougnet C, Troye-Blomberg M, Deloron P, Kabilan L, Lepers JP, Savel J, Perlmann P (1991). Human immune response in Plasmodium falciparum malaria. Synthetic peptides corresponding to known epitopes of the Pf155/RESA antigen induce production of parasite-specific antibodies in vitro. J. Immunol., 147(7):2295-2301.
11. Jiang S, Prigge ST, Wei L, Gao Ye, Hudson TH, Gerena L, Dame JB, Kyle DE (2001). New class of small nonpeptidyl compounds blocks Plasmodium falciparum development in vitro by inhibiting plasmepsins. Antimicrob Agents Chemother., 45(9):2577-2584.
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Monday, February 1, 2010
Human Rhinovirus-14 (HRV14) 3C Protease Substrates
Definition
The human rhinoviruses (HRVs), implicated as the major causative agents of upper respiratory tract infections collectively known as the common cold, belong to the largest genus of the picornavirus family. Small plus-strand Human rhinoviruses (HRVs), encode a single open reading frame which is translated into a single large polyprotein with a size of 220 kDa. Maturation cleavage of the polyprotein to generate functional viral proteins is mainly performed by two virally encoded proteases, designated 2A and 3C.
Discovery
The first cleavage is catalyzed by the 2A protease, which takes place at the junction of capsid protein VP1 and the N terminus of the 2A protease itself, separates the viral capsid from the nonstructural proteins. Most of the remaining cleavages are further processed by either the 3C protease or its precursor 3CD enzyme 1,2.
Structural Characteristics
From a structural point of view, rhinovirus 3C proteins display a strong similarity to trypsin-like serine proteases, 3C contains a cysteine as the active site nucleophile. The X-ray crystal structures of 3C proteases from both hepatitis A virus and HRV14 have been solved, revealing their structural similarity to the typical serine proteases 3. Colorimetric assay for 3C using peptides suggests amino acids downstream from the original cleavage site have all been replaced with a chromophoric p-nitroaniline moiety which is directly linked to the bond undergoing enzymatic cleavage, thereby generating a new cleavage site Gln-pNA for the enzyme 4.Cleavage by the 3C proteinase are predicted to occur at a Gln-Gly junction. The hydrolysis was shown (by reverse phase fast protein liquid chromatography and amino acid analysis) to occur specifically at the Gln-Gly bond in each of the peptides. The ready availability of such convenient substrates facilitated the further characterization of the 3C proteinase 5.
Mode of Action
The ability of the HRV-14 3C proteinase to hydrolyse the synthetic peptides was inhibited if a Cys~Ser(146) mutation was introduced into the protein. Studies with known proteinase inhibitors substantiated the conclusion that the HRV- 14 3C protein appears to be a cysteine proteinase and that the Cys residue at position 146 may be the active site nucleophile 5. Kinetic parameters of 3C protease toward p-nitroanilides (pNA) peptides have been measured and analyzed. The pNA peptides have been modeled within the active site of the 3C protease to investigate the ability of the pNA group to act as a replacement for Gly-Pro in the prime side. Hydrolysis of these pNA peptides by 3C at the newly formed scissile bond releases free p-nitroaniline which is yellow-colored and can be continuously monitored at a visible wavelength 3. Compound LY343814, one of the most potent inhibitors against HRV14 3C protease, had an antiviral 50% inhibitory concentration of 4.2 µM in the cell-based assay. Results suggest that the antiviral activity associated with these compounds might result from inactivation of both 2A and 3C proteases in vivo. Since the processing of the viral polyprotein is hierarchical, dual inhibition of the two enzymes may result in cooperative inhibition of viral replication 4.
Functions
Drug development, the 3C proteases encoded by HRV are attractive targets for antiviral drug development due to their important roles in viral replication. The HRV-14 3C proteinase probably plays animportant role, analogous to that implied for the poliovirus 3C proteinase, in the replication of the virus and thus represents a potential target for antiviral chemotherapy 6.
RNA binding, in addition to its proteolytic activity, viral 3C protease has been shown to be a RNA-binding protein and may be involved in formation of the viral replication complex .
Serine protease, as illustrated by its crystal structure, HRV 3C protease represents a novel class of cysteine protease that contains a cysteine as the active site nucleophile but is structurally like a serine protease 7.
References
1.Palmenberg AC (1990). Proteolytic procession of picornaviral polyprotein. Annu. Rev. Microbiol., 44:603–623.
2.Porter AG (1993). Picornavirus nonstructural proteins: emerging roles in virus replication and inhibition of host cell functions. J. Virol., 67:6917–6921.
3.Malcolm BA (1995). The picornaviral 3C proteinases: cysteine nucleophiles in serine proteinase folds. Protein Sci., 4:1439–1445.
4.Wang QM, Johnson RB, Cox GA, Villarreal EC, Loncharich RJ (1997). A continuous colorimetric assay for rhinovirus-14 3C protease using peptide
5.David C, LONG AC, Kay J, Dunn BM, Cameron JM (1989). Hydrolysis of a Series S of synthetic Peptide
6.Leong LE, Walker PA, Porter AG (1993). Human rhinovirus-14 protease 3C (3Cpro) binds specifically to the 5'-noncoding region of the viral RNA. Evidence that 3Cpro has different domains for the RNA binding and proteolytic activities. J. Biol. Chem., 268: 25735–25739.
7.Bazan JF, Fletterick RJ (1988). Viral cysteine proteases are homologous to the trypsin-like family of serine proteases: structural and functional implications.PNAS., 85:7872–7876.
p-nitroanilides (pNA) as substrates p-nitroanilides as substrates. Anal Biochem., 252(2):238-245. Substrates by the Human Rhinovirus 14 3C Proteinase, Cloned and Expressed in E coli. J. gen. Virol., 70: 2931-2942.
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The human rhinoviruses (HRVs), implicated as the major causative agents of upper respiratory tract infections collectively known as the common cold, belong to the largest genus of the picornavirus family. Small plus-strand Human rhinoviruses (HRVs), encode a single open reading frame which is translated into a single large polyprotein with a size of 220 kDa. Maturation cleavage of the polyprotein to generate functional viral proteins is mainly performed by two virally encoded proteases, designated 2A and 3C.
Discovery
The first cleavage is catalyzed by the 2A protease, which takes place at the junction of capsid protein VP1 and the N terminus of the 2A protease itself, separates the viral capsid from the nonstructural proteins. Most of the remaining cleavages are further processed by either the 3C protease or its precursor 3CD enzyme 1,2.
Structural Characteristics
From a structural point of view, rhinovirus 3C proteins display a strong similarity to trypsin-like serine proteases, 3C contains a cysteine as the active site nucleophile. The X-ray crystal structures of 3C proteases from both hepatitis A virus and HRV14 have been solved, revealing their structural similarity to the typical serine proteases 3. Colorimetric assay for 3C using peptides suggests amino acids downstream from the original cleavage site have all been replaced with a chromophoric p-nitroaniline moiety which is directly linked to the bond undergoing enzymatic cleavage, thereby generating a new cleavage site Gln-pNA for the enzyme 4.Cleavage by the 3C proteinase are predicted to occur at a Gln-Gly junction. The hydrolysis was shown (by reverse phase fast protein liquid chromatography and amino acid analysis) to occur specifically at the Gln-Gly bond in each of the peptides. The ready availability of such convenient substrates facilitated the further characterization of the 3C proteinase 5.
Mode of Action
The ability of the HRV-14 3C proteinase to hydrolyse the synthetic peptides was inhibited if a Cys~Ser(146) mutation was introduced into the protein. Studies with known proteinase inhibitors substantiated the conclusion that the HRV- 14 3C protein appears to be a cysteine proteinase and that the Cys residue at position 146 may be the active site nucleophile 5. Kinetic parameters of 3C protease toward p-nitroanilides (pNA) peptides have been measured and analyzed. The pNA peptides have been modeled within the active site of the 3C protease to investigate the ability of the pNA group to act as a replacement for Gly-Pro in the prime side. Hydrolysis of these pNA peptides by 3C at the newly formed scissile bond releases free p-nitroaniline which is yellow-colored and can be continuously monitored at a visible wavelength 3. Compound LY343814, one of the most potent inhibitors against HRV14 3C protease, had an antiviral 50% inhibitory concentration of 4.2 µM in the cell-based assay. Results suggest that the antiviral activity associated with these compounds might result from inactivation of both 2A and 3C proteases in vivo. Since the processing of the viral polyprotein is hierarchical, dual inhibition of the two enzymes may result in cooperative inhibition of viral replication 4.
Functions
Drug development, the 3C proteases encoded by HRV are attractive targets for antiviral drug development due to their important roles in viral replication. The HRV-14 3C proteinase probably plays animportant role, analogous to that implied for the poliovirus 3C proteinase, in the replication of the virus and thus represents a potential target for antiviral chemotherapy 6.
RNA binding, in addition to its proteolytic activity, viral 3C protease has been shown to be a RNA-binding protein and may be involved in formation of the viral replication complex .
Serine protease, as illustrated by its crystal structure, HRV 3C protease represents a novel class of cysteine protease that contains a cysteine as the active site nucleophile but is structurally like a serine protease 7.
References
1.Palmenberg AC (1990). Proteolytic procession of picornaviral polyprotein. Annu. Rev. Microbiol., 44:603–623.
2.Porter AG (1993). Picornavirus nonstructural proteins: emerging roles in virus replication and inhibition of host cell functions. J. Virol., 67:6917–6921.
3.Malcolm BA (1995). The picornaviral 3C proteinases: cysteine nucleophiles in serine proteinase folds. Protein Sci., 4:1439–1445.
4.Wang QM, Johnson RB, Cox GA, Villarreal EC, Loncharich RJ (1997). A continuous colorimetric assay for rhinovirus-14 3C protease using peptide
5.David C, LONG AC, Kay J, Dunn BM, Cameron JM (1989). Hydrolysis of a Series S of synthetic Peptide
6.Leong LE, Walker PA, Porter AG (1993). Human rhinovirus-14 protease 3C (3Cpro) binds specifically to the 5'-noncoding region of the viral RNA. Evidence that 3Cpro has different domains for the RNA binding and proteolytic activities. J. Biol. Chem., 268: 25735–25739.
7.Bazan JF, Fletterick RJ (1988). Viral cysteine proteases are homologous to the trypsin-like family of serine proteases: structural and functional implications.PNAS., 85:7872–7876.
p-nitroanilides (pNA) as substrates p-nitroanilides as substrates. Anal Biochem., 252(2):238-245. Substrates by the Human Rhinovirus 14 3C Proteinase, Cloned and Expressed in E coli. J. gen. Virol., 70: 2931-2942.
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Herpes Simplex Virus (HSV) Peptides
Definition
Herpes simplex virus (HSV) is a lytic virus. Infection of cells with the HSV leads to a gradual inhibition of cell-specific protein synthesis and a concomitant increase in the rate of synthesis of virus-specific proteins and peptides. Several interactions between HSV proteins have been proposed as attractive targets for antiviral drug discovery.
Discovery
JP Vasilakos in 1993 used a short synthetic peptide representing amino acid residues 497-507 from HSV-1 glycoprotein B to induce Cytotoxic T cells. C57BL/6 mice were immunized with a single dose of a short synthetic peptide representing amino acid residues 497-507 from HSV-1 glycoprotein B. The CTL were CD8+ and H-2b restricted. They were capable of lysing target cells exogenously sensitized with peptide or endogenously processed glycoprotein B 1. Paradis H in 1991 identified a nonapeptide, HSV R2-(329-337), corresponding to the subunit 2 (R2) carboxyl terminus of HSV ribonucleotide reductases, specifically inhibits this enzyme activity 2. HSV envelope glycoproteins are the prime targets of adaptive antiviral immunity. Three overlapping peptides showed ability to stimulate immunity which cross-reacts with HSV-1 3.
Structural Characteristics
Sequence of HSV-1 Glycoprotein (gB) (497-507) is H-Thr-Ser-Ser-Ile-Glu-Phe-Ala-Arg-Leu-Gln-Phe-OH. This peptide represents a H-2Kb T cell epitope of HSV-1 gB. It activates CD8+ cytotoxic T lymphocytes in vivo in a manner independent of CD4+ T cells. HSV R2-(329-337) is a nine amino acid peptide, this peptide was rapidly degraded by proteases present in the partially purified enzyme extract. The main process of proteolysis involves the successive removal of Tyr329 and Ala330, which corresponds to an aminopeptidase activity 2.
Mode of action
CTL primed with custom peptide sequence 497-507 from HSV-1 glycoprotein B produce an anamnestic response in vivo upon peptide challenge. The requirement of CD4+ T cells for CD8+ CTL activation was shown by depleting CD4+ cells in vivo with GK1.5 mAb. CD4+ T cell depletion did not abrogate CTL generation. These results suggest that glycoprotein B peptide 497-507 activates CD8+ CTL in vivo in a manner independent of CD4+ T cells 1. Nx-acetylation, a modification known to protect nonapeptide, HSV R2-(329-337), against aminopeptidase attacks, greatly improved the proteolytic resistance of HSVs 2.
Two peptides, the gB 18-mer and 20.1-mer, were recognized by MAb B6 and HSV-immune antibody but were unable to stimulate virus-neutralizing antibody or serum able to protect against zosteriform spread in vivo. The 20.2-mer peptide, however, which was not recognized by MAb B6 or HSV-generated immune antibody, stimulated the production of neutralizing antibody and serum able to protect against zosteriform spread 3.
A synthetic peptide derived from the secreted portion of HSV type 2 glycoprotein G, denoted gG-2p20, which has proinflammatory properties in vitro. The gG-2p20 peptide, corresponding to aa 190–205 of glycoprotein G-2, was a chemoattractant for both monocytes and neutrophils in a dose-dependent fashion, and also induced the release of reactive oxygen from these cells. The receptor mediating the responses was identified as the formyl peptide receptor. The gG-2p20-induced activation of phagocytes had a profound impact on NK cell functions. The reactive oxygen species produced by gG-2p20-activated phagocytes both inhibited NK cell cytotoxicity and accelerated the apoptotic cell death in NK cell-enriched lymphocyte populations 4.
Functions
Cellular and humoral immunity, investigation of the immune response to herpes simplex virus (HSV) has indicated that certain viral envelope glycoproteins, especially gB, gC, and gD, generate both cellular and humoral immunity 5,6.
Host cell entry, glycoproteins B and D, essential for virus entry into host cells, act as protective immunogens in animals when introduced in recombinant virus vectors, as purified proteins, or in transfected L cells 7.
Neutralizing antibody, three overlapping peptides, corresponding to the wild-type gB-1 sequence amino acids 63 to 110 have the ability to stimulate immunity in mice which cross-reacts with HSV-1. One peptide was able to generate HSV-1 neutralizing antibody which protected in the murine zosteriform spread model.
CCAP, the complex of native cytokines and antimicrobial peptides (CCAP or Superlymph) proved to inhibit the HSV reproduction in vitro. Protegrines, as a CCAP component, were active against the virus 8.
References
1.Vasilakos JP, Michael JG (1993). Herpes simplex virus class I-restricted peptide induces cytotoxic T lymphocytes in vivo independent of CD4+ T cells. J. Immunol., 150(6):2346-2355.
2.Paradis H, Langelier Y, Michaud J, Brazeau P, Gaudreau P (1991). Studies on in vitro proteolytic sensitivity of peptides inhibiting herpes simplex virus ribonucleotide reductases lead to discovery of a stable and potent inhibitor. Int J Pept Protein Res., 37(1):72-79.
3.Mester JC, Highlander SL, Osmand AP, Glorioso JC, Rouse BT (1990). Herpes Simplex Virus Type 1-Specific Immunity Induced by Peptides Corresponding to an Antigenic Site of Glycoprotein B. J. Virology., 64(11):5277-5283.
4.Bellner L (2005). A Proinflammatory Peptide from Herpes Simplex Virus Type 2 Glycoprotein G Affects Neutrophil, Monocyte, and NK Cell Functions. The Journal of Immunology, 174:2235-2241.
5.Vestergaard BF (1980). Herpes simplex virus antigens and antibodies: a survey of studies based on quantitative immunoelectrophoresis. Rev. Infect. Dis., 2:899-913.
6.Zarling JM (1986). T cell-mediated immunity to herpes simplex viruses, p. 103-114. In C. Lopez and B. Roizman (ed.), Human herpesvirus infections. Raven Press, New York.
7.Corey L, Spear PG (1986). Infections with herpes simplex viruses. N. Engl. J. Med., 314: 686-691.
8.Kovalchuk LV, Gankovskaya LV, Gankovskaya OA, Lavrov VF (2007). Herpes simplex virus: treatment with antimicrobial peptides. Adv Exp Med Biol., 601:369-376.
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Herpes simplex virus (HSV) is a lytic virus. Infection of cells with the HSV leads to a gradual inhibition of cell-specific protein synthesis and a concomitant increase in the rate of synthesis of virus-specific proteins and peptides. Several interactions between HSV proteins have been proposed as attractive targets for antiviral drug discovery.
Discovery
JP Vasilakos in 1993 used a short synthetic peptide representing amino acid residues 497-507 from HSV-1 glycoprotein B to induce Cytotoxic T cells. C57BL/6 mice were immunized with a single dose of a short synthetic peptide representing amino acid residues 497-507 from HSV-1 glycoprotein B. The CTL were CD8+ and H-2b restricted. They were capable of lysing target cells exogenously sensitized with peptide or endogenously processed glycoprotein B 1. Paradis H in 1991 identified a nonapeptide, HSV R2-(329-337), corresponding to the subunit 2 (R2) carboxyl terminus of HSV ribonucleotide reductases, specifically inhibits this enzyme activity 2. HSV envelope glycoproteins are the prime targets of adaptive antiviral immunity. Three overlapping peptides showed ability to stimulate immunity which cross-reacts with HSV-1 3.
Structural Characteristics
Sequence of HSV-1 Glycoprotein (gB) (497-507) is H-Thr-Ser-Ser-Ile-Glu-Phe-Ala-Arg-Leu-Gln-Phe-OH. This peptide represents a H-2Kb T cell epitope of HSV-1 gB. It activates CD8+ cytotoxic T lymphocytes in vivo in a manner independent of CD4+ T cells. HSV R2-(329-337) is a nine amino acid peptide, this peptide was rapidly degraded by proteases present in the partially purified enzyme extract. The main process of proteolysis involves the successive removal of Tyr329 and Ala330, which corresponds to an aminopeptidase activity 2.
Mode of action
CTL primed with custom peptide sequence 497-507 from HSV-1 glycoprotein B produce an anamnestic response in vivo upon peptide challenge. The requirement of CD4+ T cells for CD8+ CTL activation was shown by depleting CD4+ cells in vivo with GK1.5 mAb. CD4+ T cell depletion did not abrogate CTL generation. These results suggest that glycoprotein B peptide 497-507 activates CD8+ CTL in vivo in a manner independent of CD4+ T cells 1. Nx-acetylation, a modification known to protect nonapeptide, HSV R2-(329-337), against aminopeptidase attacks, greatly improved the proteolytic resistance of HSVs 2.
Two peptides, the gB 18-mer and 20.1-mer, were recognized by MAb B6 and HSV-immune antibody but were unable to stimulate virus-neutralizing antibody or serum able to protect against zosteriform spread in vivo. The 20.2-mer peptide, however, which was not recognized by MAb B6 or HSV-generated immune antibody, stimulated the production of neutralizing antibody and serum able to protect against zosteriform spread 3.
A synthetic peptide derived from the secreted portion of HSV type 2 glycoprotein G, denoted gG-2p20, which has proinflammatory properties in vitro. The gG-2p20 peptide, corresponding to aa 190–205 of glycoprotein G-2, was a chemoattractant for both monocytes and neutrophils in a dose-dependent fashion, and also induced the release of reactive oxygen from these cells. The receptor mediating the responses was identified as the formyl peptide receptor. The gG-2p20-induced activation of phagocytes had a profound impact on NK cell functions. The reactive oxygen species produced by gG-2p20-activated phagocytes both inhibited NK cell cytotoxicity and accelerated the apoptotic cell death in NK cell-enriched lymphocyte populations 4.
Functions
Cellular and humoral immunity, investigation of the immune response to herpes simplex virus (HSV) has indicated that certain viral envelope glycoproteins, especially gB, gC, and gD, generate both cellular and humoral immunity 5,6.
Host cell entry, glycoproteins B and D, essential for virus entry into host cells, act as protective immunogens in animals when introduced in recombinant virus vectors, as purified proteins, or in transfected L cells 7.
Neutralizing antibody, three overlapping peptides, corresponding to the wild-type gB-1 sequence amino acids 63 to 110 have the ability to stimulate immunity in mice which cross-reacts with HSV-1. One peptide was able to generate HSV-1 neutralizing antibody which protected in the murine zosteriform spread model.
CCAP, the complex of native cytokines and antimicrobial peptides (CCAP or Superlymph) proved to inhibit the HSV reproduction in vitro. Protegrines, as a CCAP component, were active against the virus 8.
References
1.Vasilakos JP, Michael JG (1993). Herpes simplex virus class I-restricted peptide induces cytotoxic T lymphocytes in vivo independent of CD4+ T cells. J. Immunol., 150(6):2346-2355.
2.Paradis H, Langelier Y, Michaud J, Brazeau P, Gaudreau P (1991). Studies on in vitro proteolytic sensitivity of peptides inhibiting herpes simplex virus ribonucleotide reductases lead to discovery of a stable and potent inhibitor. Int J Pept Protein Res., 37(1):72-79.
3.Mester JC, Highlander SL, Osmand AP, Glorioso JC, Rouse BT (1990). Herpes Simplex Virus Type 1-Specific Immunity Induced by Peptides Corresponding to an Antigenic Site of Glycoprotein B. J. Virology., 64(11):5277-5283.
4.Bellner L (2005). A Proinflammatory Peptide from Herpes Simplex Virus Type 2 Glycoprotein G Affects Neutrophil, Monocyte, and NK Cell Functions. The Journal of Immunology, 174:2235-2241.
5.Vestergaard BF (1980). Herpes simplex virus antigens and antibodies: a survey of studies based on quantitative immunoelectrophoresis. Rev. Infect. Dis., 2:899-913.
6.Zarling JM (1986). T cell-mediated immunity to herpes simplex viruses, p. 103-114. In C. Lopez and B. Roizman (ed.), Human herpesvirus infections. Raven Press, New York.
7.Corey L, Spear PG (1986). Infections with herpes simplex viruses. N. Engl. J. Med., 314: 686-691.
8.Kovalchuk LV, Gankovskaya LV, Gankovskaya OA, Lavrov VF (2007). Herpes simplex virus: treatment with antimicrobial peptides. Adv Exp Med Biol., 601:369-376.
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