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Sequence-independent Control of Peptide Conformation in Liposomal Vaccines for Targeting Protein Misfolding Diseases. - PowerPoint PPT Presentation
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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
MotivationProtein 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
3Worldclimatereport.comThrall, 2005.www.ninds.nih.gov
Motivation
4Nationmaster.com
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
11
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
Peptides Created
14Avantilipids.comHickman, et. al, 2011.
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.
Acetyl CD Spec
• Lack of minima around 220 nm – Lacks β-sheets
19Hickman, 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
20
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
22
• 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.
25Hickman, 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
26
Thanks for your attention
27
Questions?
Supplemental Figures/Info
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