sequence-independent control of peptide conformation in liposomal vaccines for targeting protein...
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Sequence-independent Control of Peptide Conformation in Liposomal
Vaccines for Targeting Protein Misfolding Diseases
D. Hickman, M. Deber, D. Ndao, A. Silva, D. Nand, M. Pihlgren, V. Giriens, R. Madani, A. St-Pierre, H.
Karastaneva, L. Steger, D. Willbold, D. Riesner, C. Nicolau, M. Baldus, A. Pfeifer, A. Muhs
Ben KremkowNovember 9th, 2011CHEM 645 – Group 1
Motivation
Protein misfolding diseases• Neurodegenerative:
– Parkinsons’– Alzheimers’– Creutzfeldt-Jakob– Huntington– Amyotrophic lateral sclerosis
• Non-neurodegenerative:– Inherited cataracts– Type II diabetes mellitus
2
• Population affected (US)– 1.5 million– 4 million– ~200– 8,000– 15,000
– ?– 23.2 millionwww.ninds.nih.gov
Motivation• Yearly cost (US $)
– Alzheimers’ – 100+ billion– Diabetes – 156 billion
• US population– 1900:
• Life expectancy = 47 years• 3 million Americans
– 2000: • Life expectancy = 77 years• 35 million Americans
3
Worldclimatereport.comThrall, 2005.www.ninds.nih.gov
Introduction – Protein Misfolding Defenses
• Cellular defenses:
– Chaperones
– Polyubiquitin attachment
– Proteasome targeting
– Aggresome
5Bronstein, 2004.Ross, et al. 2004.
Introduction – Therapeutic Defenses
• Enhance cellular defenses:
– Geldanamycin – Modulate/enhance chaperone levels
• Reduce abnormal protein level in cell
– RNA interference – Delivery is an issue
• Small molecules
– Target protein misfolding pathway – Congo red
– Inhibit aggregation
• ID specific pathogenic mechanisms
– Proteolytic cleavage – small molecule inhibitors 6
Introduction – Therapeutic Defenses
• Issues
– Inhibition of 1 step may cause toxic accumulation
– Unknown protein misfolding pathway
– Diseases seem to have common pathways
7Ross, et al. 2004.
Objective
• Goals:
– Increased understanding of the β-sheet conformation
– Determine what variables affect liposomal protein conformation
– Establishing a structure-conformation relationship for liposomal Palm1-15 (ACI-24)
– Evaluate the constructs for the generation of antibodies
8
Methods
• CD Spectroscopy• Thioflavin T Fluorescence• Magic Angle Spinning-NMR Spectroscopy• Size Exclusion Chromatography
• Biological Methods– Vaccine Preparation– Conformational Antibody Specificity– Tissue Preparation– Immunohistochemistry
9
Common Methods
• Thioflavin T (ThT)– Benzothiazole dye, exhibits red shift upon
binding to aggregated β-sheet peptides– Measures β-sheet aggregation
• CD Spectroscopy– Measures peptide secondary structure– Minima at 220 nm is characteristic of β-sheet
conformation
10Khurana, et. al, 2005.
Magic Angle Spinning (MAS)-NMR Spectroscopy
• Sample is spun at a magic angle, θm
– cos2 θm = 1/3
– Resolution is increased as the broad lines become narrower
• Triple resonance (1H, 13C, 15N) MAS probe
• 16.4 T static magnetic field
– Palm1-15 amino acids Ala-2, Ser-8, and Gly-9 were labeled with 13C and 15N
– Incorporated into DMPC, DMPG, DMTAP, cholesterol, and MPLA liposomes
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Liposome Lipids and Molecules
• Lipids
– DMPC
– DMPG
– DMTAP
• MPLA
• Cholesterol
12Avantilipids.com
ACI-24
• Liposomal vaccine
– Tetrapalmitoylated β-amyloid 1-15 peptide
– Elicits immune response to restore cognitive impairment of amyloid precursor protein pathway
• Link between peptide immunogen conformation and in vivo efficacy
13
Conformational Analysis
• ThT Fluorescence
– 485 nm
– No liposome interference
• Kd = 2.4 μm
15Hickman, et. al, 2011.
CD Spectra• Lipidated Palm1-15
– Solid line• Acetylated native
Acetyl 1-15– Dotted line
• Liposomal Palm1-15 adopts a B-sheet secondary structure
• Acetyl1-15 is unstructured
• B-sheet aggregates similar to B-amyloid
16Hickman, et. al, 2011.
Metal Ion Effects
• B-sheet aggregates are dissociated by Cu(II)
– Similar to AB(1-42)
• Metal chelator DPTA
– Similar to before metal ion addition
• Other metals have reduced or no effect
17Hickman, et. al, 2011.
Sequence Order and Length
• Palm15-1, 1-9, and scPalm1-15– β-sheet
conformation• Palm1-5
– Mixed β-sheet and random coil
• Peptide sequence has minor influence on β-sheet aggregation– Not unique to Aβ1-
15 sequence
18
Palm15-1 (___)scPalm15(------)Palm1-9(xxxx)Palm1-5(…….)
Hickman, et. al, 2011.
Effect of Peptide Charge
• β-sheet have no dependence upon peptide net charge
– Range: 5.2-10.0
– Minima at 220 nm
• ThT findings support
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Palm1-15(D7K) (___)Palm1-15(E3A,D7K) (----)Palm1-15(E3K,D7K) (xxx)Palm1-15(E3K,D7K,E11K)
(…)
Hickman, et. al, 2011.
Liposome Surface Charge
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• Similar apparent Kd values for liposome formulations from the same peptide
– Charge does not weaken ThT binding
• ThT signal due to differences in peptide structure and aggregation
• β-strand favored in anionic, rather than cationic liposomes
– Red decrease in CB-Ala
– Red decrease in CA-Ser
• Increase in mobility
Anionic – blueCationic - red
Anionic – dark squareCationic – white
triangleEmpty – dark circle
Hickman, et. al, 2011.
Palmitoyl Chain Effects
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• Vary number and position of lipid anchors– C-terminal tetrapalmitoylated peptide forms more β-sheets
• Apparent Kd values similar, so not due to ThT binding affinity (Supp)
– Number of lipid chains is an indicative variable
Palm(4C) (___)Palm1-15(2C) (xxx)Palm1-15(1N1C)
(----)Palm1-15(1C)
(…….)
Hickman, et. al, 2011.
Lipid Chain Length Effects
• Lipid chain length is an indicative variable– Longer chains improve β-sheet conformation
and extent of aggregation 23Hickman, et. al, 2011.
Antibody Binding
• Recognition of oligomer, not monomer– Oligomer fraction – red– Monomer fraction – blue
24Hickman, et. al, 2011.
Summary
• Peptide N- or C-terminal lipidation is common to embed peptides into liposome bilayers
• Palm1-15– Adopts a β-sheet conformation, not random coil– Similar ThT fluorescence to Aβ
• Responsible variables– Net surface potential– Lipidation pattern– Lipid anchor chain length
• Induced IgG antibodies to recognize β-sheet multimers
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