Amyloids are a class of disease-related peptides and proteins observed to undergo aggregation in vivo, resulting in the formation of insoluble plaques.217 Plaques are 10 µm to 200 µm in size, Fig. 4.6 (c), comprising an amyloid fibril core surrounded by other biological entities such as nerve terminals. Cytotoxicity of amyloid plaques is attributed to the involvement of nerve terminals and to plaque structure and insolubility, rather than to the sequence or structure of peptide monomers. Amyloid plaque formation is a hallmark of many degenerative diseases, particularly those associated with the brain or peripheral tissues, such as Alzhiemer’s disease.
Other diseases attributed to amyloid plaque formation include type 2 diabetes, cystic fibrosis, some forms of emphysema and Parkinson’s disease.218
Figure 4.6 (a) The proposed mechanism of amyloid fibril formation, resulting in amyloid plaques linked to degenerative disease induction; (b) Graph showing rate of amyloid assembly;
(c) Electron microscopy of typical amyloid fibrils.219
Successful treatment of amyloid related diseases requires mechanistic understanding of plaque formation.9,219 Fig. 4.6 (a) and (b) summarise three key steps involved in plaque formation:
1. Rate-limiting lag phase: monomers undergo folding, unfolding, dimerisation and denat-uration, eventually nucleating into small globular amyloidogenic oligomers.
90 4.3. Amyloid Intrinsic Lipidation
2. Fibril growth phase: amyloidogenic oligomers above a concentration threshold aggregate into an amyloid seed, which then elongates into rod-like filaments. Filaments bundle and intertwine forming first protofibrils then filaments.
3. Saturation: filament elongation ceases and β-sheet filaments form the dense central core of amyloid plaques.
Despite considerable study of the amyloid aggregation process, the trigger for initial amyloid monomer nucleation remains unclear. One theory suggests that protein misfolding exposes novel protein regions normally shielded, inducing aggregation through hydrophobic inter-actions.9 Alternative theories include membrane induced proximity facilitating monomer aggregation, or excess peptide accumulation resulting from increased production or reduced clearance.9 Peptide intrinsic lipidation provides a further possible mechanism for induction of amyloid plaque formation. Synthetic addition of a hydrophobic N -terminal membrane anchor analogous to intrinsic lipidation acylation, has been observed to double the rate of amyloid aggregation, shielding the hydrophobic chain from aqueous surroundings.220,221 Furthermore, intrinsic lipidation induced nucleation is consistent with both the observed lag phase, and the necessity for a catalytic lipid membrane in vitro in order to accurately mimic aggrega-tion rates in vivo.219,220 Finally, fluorescent amyloid plaques are formed in the presence of phospholipids comprised of fluorescently labelled acyl chains, suggesting incorporation during fibril formation.222
Investigation into the relationship between peptide intrinsic lipidation and amyloid aggregation was probed using three amyloid peptides, Fig. 4.7. Forty amino acid amyloid-β, is formed upon cleavage of APP precursor protein by membrane bound protease β-secretase, and the γ-secretase complex.9,223,224Implicated in Alzheimer’s disease, the 11-28 residue active region of amyloid-β includes two lysine, one serine and two histidine moieties, primed to undergo intrinsic lipidation. Sixty-seven residue hormone amylin is naturally produced by signal sequence cleavage from proamylin, and subsequence post-translational modification.225 Implicated in the loss of islet β-cells associated with type 2 diabetes, amylin contains three serine residues capable of intrinsic lipidation within its active region of residues 20-29. β-2 microglobulin, a one hundred nineteen residue component of the major histocompatibility complex, is known to aggregate in joint spaces causing dialysis related amyloidosis.226 Agregation prone regions of β-2 microglobulin, residues 22–31 and 60–70, contain three lysine residues and one serine prone to intrinsic lipidation reactivity.227
Chapter 4. Peptide Intrinsic Lipidation 91
D A E F R H D S G Y E V H H Q K L V F F A E D V G S N K G A I I G L M V G G V V
S N N F G A I L S S
M S R S V A L A V L A L L S L S G L E A I Q R T P K I Q V Y S R H P A E N G K S N F L N C Y V S G F H P S D I E V D L L K N G E R I E K V E H S D L S F S K D W S F Y L L Y Y T E F T P T E K D E Y A C R V N H V T L S Q P K I V K W D R D M
(a)
(b)
(c)
Figure 4.7 (a) Amino acid sequence of amyloid-β; (b) Amino acid sequence of amylin; (c) Amino acid sequence of β-2 microglobulin. Active regions are highlighted in green.
Amyloid stock solutions were prepared at 5 µg mL−1in hexafluoro-2-propanol (HFIP) to prevent aggregation during storage.228 Stock solution portions were incubated under physiological conditions for 72 hours in 1:10 molar ratio with POPC:SLPS (4:1) liposomes. Membrane model selection mimics plaque forming conditions in vivo, and facilitates origin of transfer determination through acyl chain diversity. Sample analysis was conducted by MALDI MS on an Autoflex II ToF/ToF (Bruker Daltonics Ltd., UK), rather than standard LCMS with ESI ionisation optimised for study of peptide intrinsic lipidation.39,40 MALDI instrumentation provides a robust analytical alternative to ESI, compatible with study of amyloid aggregates and increased solvent diversity, which can prove problematic for LCMS.109,229,230 Neither modified nor unmodified amyloid monomers were observed for amylin, amyloid-β, or β-2 microglobulin following 72 hour incubation with liposomes. Lack of visualisation was considered likely due to amyloid aggregation, preventing sample solubilisation and detection by MALDI MS.
Confirmation of amyloid aggregation under experimental conditions was determined by em-ploying two biophysical techniques, a Thioflavin T fluorescence assay, and electron microscopy (EM).231 Thioflavin T emission at 485 nm is dependent upon polarity of the dye’s external environment.232–234 Fluorescence is quenched in aqueous solution, therefore increased fluo-rescence is observed upon amyloid fibril formation providing a hydrophobic environment for Thioflavin T intercalation. Fig. 4.8 shows normalised Thioflavin T emission at 485 nm for amyloids amylin, amyloid-β, and β-2 microglobulin, in solution and in the presence of a lipid membrane. Consistent with monomer detection by MALDI MS, solution phase amyloids do not exhibit the increased Thioflavin T fluorescence indicative of plaque formation, over the time period of study. In contrast, amyloid species in the presence of a lipid membrane
92 4.3. Amyloid Intrinsic Lipidation
exhibit increased Thioflavin T fluorescence, reaching emission maxima within approximately 15 minutes of incubation. Emission plateaus, indicative of complete amyloid aggregation and dye intercalation, then decreases due to solvent evaporation.
0 100 200 300 400 500
0 2000 4000 6000 8000
Time (min)
Intensity(arb.units)
Figure 4.8 Thioflavin T fluorescence at 485 nm under physiological conditions for amylin (solid red), amyloid-β (dashed green), or β-2 microglobulin (dotted and dashed blue) in solution (lighter colours) and in the presence of a POPC:SLPS (4:1) liposomal membrane (darker colours).
Transmission electron microscopy (TEM) provides confirmation of amyloid aggregation in the presence of a lipid membrane through direct visualisation.235–237 Fig. 4.9 presents one example of plaque formation following 72 hour incubation of amyloid-β with POPC:SLPS (4:1). Fibril length is limited in vitro by the initial concentration of amyloid peptide available for aggregation. Combined TEM, Thioflavin T fluorescence, and MALDI MS data support the theory of amyloid aggregation occurring under experimental conditions. Observed aggregation under known peptide intrinsic lipidation conditions serves to support acyl transfer as a possible mechanism for the induction of amyloid nucleation.
Figure 4.9 TEM of amyloid plaques formed upon 72 hour incubation of amyloid-β with a POPC:SLPS (4:1) lipid membrane under physiological conditions.
Chapter 4. Peptide Intrinsic Lipidation 93
Modification to environmental parameters is predicted to aid in the study of amyloid intrinsic lipidation by diminishing amyloid aggregation, and facilitating product visualisation. Lit-erature reports suggest amyloid aggregation can be prevented by employing reduced NaCl concentrations, low temperatures, and by sonicating samples prior to incubation.228,238 In-fluence of temperature reduction upon aggregation cannot be tested using a Thioflavin T assay, due to instrument limitations. However, Thioflavin T emission was studied for amyloids in the presence of a lipid membrane using low salt buffer (10 mM NaHCO3 pH 7.4), and following sonication. Amyloid aggregation, indicated by increased Thioflavin T emission, was not observed over 6 hours of study for modified reaction conditions. Aggregation is therefore reduced compared to initial experimental conditions, where emission increase was observed within 15 minutes. Modified experimental conditions were taken forward for 72 hour incubation and MALDI MS analysis, hoping to detect amyloid intrinsic lipidation. However, monomer visualisation was not improved under modified experimental conditions, suggesting amyloid aggregation is slowed by not completely prevented.
Considering the predicted link between intrinsic lipidation and amyloid nucleation, preventing aggregation is likely to simultaneously prevent acyl transfer. Aggregate depolymerisation, and subsequent composition determination, provides an alternative mechanism for the study of amyloid intrinsic lipidation. Aggregates were isolated by centrifugation following 72 hour incubation of amyloids with POPC:SLPS (4:1) liposomes under standard intrinsic lipidation conditions. Amyloid plaques were treated with HFIP, a known depolymerising agent, and analysed directly by MALDI MS.228 Unmodified monomer and amyloid aggregates were observed for all three amyloid species, suggesting incomplete depolymerisation. Acylated amyloids were not observed, however since this be attributed to either inclusion within remaining aggregates, or to ion suppression, intrinsic lipidation cannot be ruled out.
Literature precedent suggests an alternative method of amyloid depolymerisation is overnight incubation of aggregates at pH 3 in formic acid.223,224 Isolated amyloid plaques were there-fore treated with formic acid and analysed by MALDI MS. Incomplete depolymerisation was observed for all amyloids, characterised by detection of small multi amyloid aggregates.
Insufficient depolymerisation occurred to facilitate visualisation of either unmodified or mod-ified amyloid monomers. Determination of intrinsic lipidation reactivity therefore proved inconclusive following formic acid depolymerisation.
Enzymatic digestion provides a third potential mechanism for depolymerisation of amyloid aggregates.40,223,224,239 Digestion conditions were optimised using monomer β2-microglobulin,
94 4.3. Amyloid Intrinsic Lipidation
due to its superior size and complexity compared to amylin and amyloid-β. Initial digestion was conducted with trypsin, trypsin:lys C, and pepsin enzymes, in order to determine peptide suitability. Trypsin:lys C was selected for further study, due to increased cleavage at lysine and arginine residues facilitating superior sequence coverage. Cleavage conditions were further modulated as shown in Table 4.4, in order to maximise observed sequence coverage based upon predicted digestion fragments shown in Fig. 4.10. Optimised conditions achieved 96 % β2-microglobulin sequence coverage, as shown in Fig. 4.11, within a 3 hour digestion time.
Dithiothreitol (DTT) addition improves sequence coverage from 71 % to 96 % by facilitating amyloid denaturation, whereas iodoacetamide (IAA) addition reduced coverage to 90 %, suggesting cysteine capping is unnecessary. Optimised digestion conditions were applied to monomer amyloid-β, resulting in 100 % sequence coverage as shown in Fig. 4.11. Amylin digestion was not attempted due to lack of relevant lysine or arginine cleavage sites.
Trypsin/LysC:Protein Time NH4HCO3 Buffer DTT IAA % Coverage
1:40 24 hours 25 mM Yes No 20 %
1:40 24 hours 25 mM Yes Yes 20 %
1:40 3 hours 50 mM No No 71 %
1:40 5 hours 50 mM No No 50 %
1:40 3 hours 50 mM Yes No 96 %
1:40 3 hours 50 mM Yes Yes 90 %
1:40 3 hours 100 mM No No 70 %
1:20 3 hours 50 mM Yes No 66 %
Table 4.4 Enzymatic digestion conditions employed for study of β2-microglobulin, and resulting sequence coverage determined by LCMS.
Optimised digestion conditions were applied to isolated amyloid aggregates, and analysis conducted by both MALDI MS and ESI LCMS.240 However, amyloid digestion was not observed in the presence of amyloid plaques, attributed to the insoluble and impenetrable nature of aggregates. Increased digestion times, up to the point of trypsin autolysis, were applied in the hope of facilitating digestion, however this too proved unsuccessful. Enzymatic digestion, along with HFIP and formic acid, was therefore considered unsuitable for aggregate depolymerisation. Inability to disrupt aggregates prevented further study of amyloid intrinsic lipidation, therefore a definitive link between amyloid nucleation and innate acyl transfer remains to be established.
Chapter 4. Peptide Intrinsic Lipidation 95
I Q R T P K I Q V Y S R H P A E N G K S N F L N C Y V
S G F H P S D I E V D L L K N G E R I E K V E H S D L
S F S K D W S F Y L L Y Y T E F T P T E K D E Y A C R
V N H V T L S Q P K I V K W D R D M
1-3 4-6 7-12 13-19 20-41
42-45 46-48 49-58
Figure 4.10 Sequence and predicted trypsin:lys C digestion fragments of: (a) β2-microglobulin; (b) amyloid-β.
Figure 4.11 TIC of amyloids digested under optimised conditions: (a) β2-microglobulin;
(b) amyloid-β.
4.4 Conclusions
Insight into the activities and physical properties of intrinsically lipidated peptides is vital to understanding the biological relevance of innate peptide reactivity. Biophysical techniques have been employed to probe the properties of acylated model peptide melittin, facilitating this understanding. CD and intrinsic tryptophan fluorescence confirm that unlike its unmodified counterpart, acylated melittin exhibits solution phase structure. Predicted to be α-helical with a possible central kink at secondary amino acid proline, structural formation is driven by favourably shielding the hydrophobic acyl moiety from external aqueous environment.
96 4.4. Conclusions
However, it remains unclear as to whether this structure is attributed to monomers or to bulk peptide structure, such as the tetramer adopted by unmodified melittin at high concentrations.
Bulk structure was probed by employing fluorescent Rhodamine 6G dye to examine possible micelle formation. Palmitoylated melittin derivatives underwent micellisation, with calculated CMCs of 1 µm and 5 µm for N -terminal and K23 modifications respectively. Oleoylated derivatives did not exhibit spontaneous aggregation into micelles, attributed to reduced packing of unsaturated acyl chains. The antimicrobial activity of acylated melittin derivatives was investigated, given the potency of unmodified melittin and similar lipidated peptides.
However, only minimal bacterial growth inhibition of E. coli and S. aureus was observed at 32 µm for acylated melittin. Preferential antimicrobial activity was attributed to palmitoylated and N -terminally modified derivatives.
The biological relevance of innate acyl transfer was further probed by investigating links between intrinsic lipidation and disease related peptides. Literature studies suggested intrinsic lipidation could induce the process of amyloid nucleation, ultimately resulting in disease related plaque formation. Significant support for this prediction is attributed to the doubled rate of plaque formation observed for amyloid-β modified with a hydrophobic N -terminal anchor.
Three amyloids, amylin, amyloid-β, and β2-microglobulin, subjected to peptide intrinsic lipidation conditions, underwent aggregation confirmed by Thioflavin T fluorescence and EM. This observation supports a link between amyloid nucleation and aggregation, however confirmation requires visualisation of acylated amyloid. In an attempt to aid visualisation of acylated amyloid, modified experimental conditions were employed, including reduced salt content, temperature, and sonication. Slowed aggregation was observed under these modified experimental conditions, but not sufficiently eliminated to study acyl transfer. Aggregate depolymerisation and subsequent content analysis was considered a preferable method to facilitate acylated amyloid visualisation. HFIP, formic acid and optimised trypsin:lys C enzy-matic digestion were studied, but ultimately proved unsuccessful in complete depolymerisation.
Therefore at present a link between intrinsic lipidation and amyloid nucleation can be neither definitively proved nor disregarded.
5 | Small Molecule Intrinsic Lipida-tion
5.1 Introduction
Small molecule intrinsic lipidation is a non-enzymatic acyl transfer reaction between membrane phospholipids and a small organic molecule.76,77 The major reaction product from small molecule intrinsic lipidation is the small molecule modified with a hydrophobic acyl chain derived from the membrane. The increased hydrophobicity of the modified small molecule dramatically changes its structure, properties and action. Such modification is particularly important if the small molecule substrate is a pharmaceutical, given the likely resulting changes to activity and side effect induction.
Past research into small molecule intrinsic lipidation has utilised lysolipids, the reaction by-product, as means of studying the reaction.76,77 Since alternative mechanisms of lysolipid production exist, notably enzymatically in vivo and by solution phase hydrolysis, their presence is not diagnostic of small molecule intrinsic lipidation. To definitively prove the existence of small molecule intrinsic lipidation at the membrane interface, direct observation of modified acyl small molecules is necessary. LCMS is the optimum technique for direct observation, due to its informative nature, sensitivity, and robustness towards complex samples. Additional benefits of pursuing LCMS for the study of small molecule intrinsic lipidation include the observed success of the technique for the analysis of peptide intrinsic lipidation, and the ability to tune instrumentation for optimum results.
97