When protein/peptides aggregate, they usually form the amyloid state consisting of cross β-sheet ... more When protein/peptides aggregate, they usually form the amyloid state consisting of cross β-sheet structure built by repetitively stacked β-strands forming long fibrils. Amyloids are usually associated with disease including Alzheimer's. However, amyloid has many useful features. It efficiently transforms protein from the soluble to the insoluble state in an essentially two-state process, while its repetitive structure provides high stability and a robust prion-like replication mechanism. Accordingly, amyloid is used by nature in multifaceted and ingenious ways of life, ranging from bacteria and fungi to mammals. These include (1) Structure: Templating for small chemical molecules (Pmel17), biofilm formation in bacteria (curli), assisting aerial hyphae formation in streptomycetes (chaplins) or monolayer formation at a surface (hydrophobins). (2) Reservoirs: A storage state for peptide/proteins to protect them from their surroundings or vice versa (storage of peptide hormones in mammalian secretory granules or major basic protein in eosinophils). (3) Information carriers: The fungal immune system (HET-s prion in Podospora anserina, yeast prions) or long-term memory (e.g., mnemons in yeast, cytoplasmic polyadenylation element-binding protein in aplysia). Aggregation is also used to (4) "suppress" the function of the soluble protein (e.g., Cdc19 in yeast stress granules), or (5) "signaling" through formation of oligomers (e.g., HET-s prion, necroptosis-related proteins RIP1/RIP3). This review summarizes current knowledge on functional amyloids with a focus on the amyloid systems curli in bacteria, HET-s prion in P. anserina, and peptide hormone storage in mammals together with an attempt to highlight differences between functional and disease-associated amyloids.
Several human proteins cause disease by misfolding and aggregating into amyloid fibril deposits a... more Several human proteins cause disease by misfolding and aggregating into amyloid fibril deposits affecting the surrounding tissues. Multiple other proteins co-associate with the diseased deposits but little is known about how this association is influenced by the nature of the amyloid aggregate and the properties of the amyloid-forming protein. In this study, we investigated the co-aggregation of plasma and cerebrospinal proteins in the presence of pre-formed amyloid fibrils. We evaluated the fibril-associated proteome across multiple amyloid fibril types that differ in their amino acid sequences, ultrastructural morphologies, and recognition by amyloid-binding dyes. The fibril types included aggregates formed by Amyloid β, α-synuclein, and FAS4 that are associated with pathological disorders, and aggregates formed by the glucagon and C-36 peptides, currently not linked to any human disease. Our results highlighted a highly similar response to the amyloid fold within the body fluid of interest. Fibrils with diverse primary sequences and ultrastructural morphologies only differed slightly in the composition of the co-aggregated proteins but were clearly distinct from less fibrillar and amorphous aggregates. The type of body fluid greatly affected the resulting amyloid interactome, underlining the role of the in vivo environment. We conclude that protein fibrils lead to a specific response in protein co-aggregation and discuss the effects hereof in the context of amyloid deposition.
HAMLET (human α-lactalbumin made lethal to tumour cells) is a complex of α-lactalbumin (aLA) and ... more HAMLET (human α-lactalbumin made lethal to tumour cells) is a complex of α-lactalbumin (aLA) and oleic acid (OA) which kills transformed cells, while leaving fully differentiated cells largely unaffected. Other protein-lipid complexes show similar anti-cancer potential. We call such complexes liprotides. The cellular impact of liprotides, while intensely investigated, remains unresolved. To address this, we report on the cell-killing mechanisms of liprotides prepared by incubating aLA with OA for 1 h at 20 or 80 °C (lip20 and lip80, respectively). The liprotides showed similar cytotoxicity against MCF7 cells, though lip80 acts more slowly, possibly due to intermolecular disulphide bonds formed during preparation. Liprotides are known to increase the fluidity of a membrane and transfer OA to vesicles, prompting us to focus on the effect of liprotides on the cell membrane. Extracellular Cainflux is important for activation of the plasma membrane repair system, and we found that remova...
Chemistry (Weinheim an der Bergstrasse, Germany), Jun 6, 2016
Incorporation of silicon-containing amino acids in peptides is known to endow the peptide with de... more Incorporation of silicon-containing amino acids in peptides is known to endow the peptide with desirable properties such as improved proteolytic stability and increased lipophilicity. In the presented study, we demonstrate that incorporation of β-silicon-β3-amino acids into the antimicrobial peptide alamethicin provides the peptide with improved membrane permeabilizing properties. A robust synthetic procedure for the construction of β-silicon-β3-amino acids was developed and the amino acid analogues were incorporated into alamethicin at different positions of the hydrophobic face of the amphipathic helix by using SPPS. The incorporation was shown to provide up to 20-fold increase in calcein release as compared with wild-type alamethicin.
The feature article focuses on the interaction between nanoparticles and α-synuclein, whose aggre... more The feature article focuses on the interaction between nanoparticles and α-synuclein, whose aggregation is central in the development of Parkinson′s Disease.
Functional amyloid (FA) is widespread in bacteria and serves multiple purposes such as strengthen... more Functional amyloid (FA) is widespread in bacteria and serves multiple purposes such as strengthening of biofilm and contact with eukaryotic hosts. Unlike pathological amyloid, FA has been subjected to evolutionary optimization which is likely to be reflected in the aggregation mechanism. FA from different bacteria, including Escherichia coli (CsgA) and Pseudomonas (FapC), contains a number of imperfect repeats which may be key to efficient aggregation. Here we report on the aggregative behavior of FapC constructs which represent all single, double, and triple deletions of the protein's three imperfect repeats. Analysis of the fibrillation kinetics by the program Amylofit reveals that the removal of these repeats increases the tendency of the growing fibrils to fragment and also generally increases aggregation half-times. Remarkably, even the mutant lacking all three repeats was able to fibrillate, although fibrillation was much more irregular and led to significantly altered and destabilized fibrils. We conclude that imperfect repeats can promote fibrillation efficiency thanks to their modular design, though the context of the imperfect repeats also plays a significant role.
Amyloid : the international journal of experimental and clinical investigation : the official journal of the International Society of Amyloidosis, 2017
Thioflavin T (ThT) has been widely used to investigate amyloid formation since 1989. While concer... more Thioflavin T (ThT) has been widely used to investigate amyloid formation since 1989. While concerns have recently been raised about its use as a probe specific for amyloid, ThT still continues to be a very valuable tool for studying kinetic aspects of fibrillation and associated inhibition mechanisms. This review aims to provide a conceptual instruction manual, covering appropriate considerations and pitfalls related to the use of ThT. We start by giving a brief introduction to amyloid formation with focus on the morphology of different aggregate species, followed by a discussion of the quality of protein needed to obtain reliable fibrillation data. After an overview of the photochemical basis for ThT's amyloid binding properties and artifacts that may arise from this, we describe how to plan and analyze ThT assays. We conclude with recommendations for complementary techniques to address shortcomings in the ThT assay.
Traditional approaches to achieve sustained delivery of pharmaceutical peptides traditionally use... more Traditional approaches to achieve sustained delivery of pharmaceutical peptides traditionally use co-excipients (e.g., microspheres and hydrogels). Here, we investigate the release of an amyloidogenic glucagon analogue (3474) from an aggregated state and the influence of surfactants on this process. The formulation of peptide 3474 in dodecyl maltoside (DDM), rhamnolipid (RL), and sophorolipid (SL) led to faster fibrillation. When the aggregates were subjected to multiple cycles of release by repeated resuspension in fresh buffer, the kinetics of the release of soluble peptide 3474 from different surfactant aggregates all followed a simple exponential decay fit, with half-lives of 5–18 min and relatively constant levels of release in each cycle. However, different amounts of peptide are released from different aggregates, ranging from 0.015 mg/mL (3475-buffer) up to 0.03 mg/mL (3474-DDM), with 3474-buffer and 3474-RL in between. In addition to higher release levels, 3474-DDM aggregat...
A high-quality draft genome sequence of ATCC 31213 is presented here. This bacterium produces sev... more A high-quality draft genome sequence of ATCC 31213 is presented here. This bacterium produces several important bioactive compounds and may also produce functional amyloids. This is the first sequenced genome from the genus , and it will be essential in determining the nature of the potential amyloid protein.
Despite extensive studies of the effects of herbal-derived small molecules in the biopharmaceutic... more Despite extensive studies of the effects of herbal-derived small molecules in the biopharmaceutical and biomedical sciences, their low solubility and stability remain a challenge. Here we focus on baicalein, a small molecule showing potential against neurodegenerative diseases such as Parkinson's and Alzheimer's. However, therapeutic usage in vivo is challenged by low solubility and stability. To address this we have applied neutrally-charged nanoliposome (NLP) as carrier for baicalein. Baicalein was incorporated into NLP to form NLP-Ba at molar baicalain:lipid ratios of up to 1:3, giving a drug entrapment efficiency of 96.71%, slow release of approximately 22% after a week and increased baicalein stability up to 27%. Ascorbic acid increased baicalein's stability further, particularly when incorporated in NLP where baicalein stability intensified by 53% in NLP-Ba. Moreover, NLP-Ba did not show significant cytotoxic effects against neurons; rather, showed considerable pro...
Cholesterol (chol) is important in all mammalian cells as a modulator of membrane fluidity. Howev... more Cholesterol (chol) is important in all mammalian cells as a modulator of membrane fluidity. However, its low solubility is a challenge for controlled delivery to membranes. Here we introduce a new tool to deliver chol to membranes, namely, liprotides, i.e., protein-lipid complexes composed of a fatty acid core decorated with partially denatured protein. We focus on liprotides prepared by incubating Ca-depleted α-lactalbumin with oleic acid (OA) for 1 h at 20 °C (lip20) or 80 °C (lip80). The binding and membrane delivery properties of liprotides is compared to the widely chol transporter methyl-β-cyclodextrin (mBCD). Both lip20 and lip80 increase the solubility of chol ~ 50% more than mBCD and deliver chol to membranes with comparable efficiency. Although OA is cytotoxic at high concentrations, its effects are counterbalanced by chol. Further, cytotoxicity is strongly reduced when OA is replaced by cis-palmitoleic acid or cis-vaccenic acid. This makes liprotides good tools to deliver...
Previous studies on metal-ion catalyzed oxidation of α-synuclein oxidation have mostly used condi... more Previous studies on metal-ion catalyzed oxidation of α-synuclein oxidation have mostly used conditions that result in extensive modification precluding an understanding of the early events in this process. In this study, we have examined time-dependent oxidative events related to α-synuclein modification using six different molar ratios of Cu/HO/protein and Cu/HO/ascorbate/protein resulting in mild to moderate extents of oxidation. For a Cu/HO/protein molar ratio of 2.3:7.8:1 only low levels of carbonyls were detected (0.078 carbonyls per protein), whereas a molar ratio of 4.7:15.6:1 gave 0.22 carbonyls per α-synuclein within 15 min. With the latter conditions, rapid conversion of 3 out of 4 methionines (Met) to methionine sulfoxide, and 2 out of 4 tyrosines (Tyr) were converted to products including inter- and intra-molecular dityrosine cross-links and protein oligomers, as determined by SDS-PAGE and Western blot analysis. Limited histidine (His) modification was observed. The rapi...
The Parkinson's disease-associated protein α-synuclein (αSN) is natively unfolded but its str... more The Parkinson's disease-associated protein α-synuclein (αSN) is natively unfolded but its structure can be modulated by membranes and surfactants. The opportunistic pathogen Pseudomonas aeruginosa (PA) produces and secretes the biosurfactant rhamnolipid (RL) which modulates bacterial biofilm. Here, we show that monomeric RL enhances the ability of αSN to permeabilize membranes, while micellar RL rapidly induces protein β-sheet structure with a worm-like fibrillary appearance, which cannot seed RL-free fibrillation but transforms into linear fibrils faster than αSN fibrillating on its own. Exposure to αSN reduces the degree of biofilm formation by PA unless RL is present. Our data suggest that RL interactions with αSN may affect both αSN aggregation and cell toxicity, potentially implicating microbiomic metabolites in the origin and propagation of Parkinson's disease. This article is protected by copyright. All rights reserved.
Gram-negative bacteria possess specialised biogenesis machineries that facilitate the export of a... more Gram-negative bacteria possess specialised biogenesis machineries that facilitate the export of amyloid subunits for construction of a biofilm matrix. The secretion of bacterial functional amyloid requires a bespoke outer-membrane protein channel through which unfolded amyloid substrates are translocated. Here, we combine X-ray crystallography, native mass spectrometry, single-channel electrical recording, molecular simulations and circular dichroism measurements to provide high-resolution structural insight into the functional amyloid transporter from Pseudomonas, FapF. FapF forms a trimer of gated β-barrel channels in which opening is regulated by a helical plug connected to an extended coil-coiled platform spanning the bacterial periplasm. Although FapF represents a unique type of secretion system, it shares mechanistic features with a diverse range of peptide translocation systems. Our findings highlight alternative strategies for handling and export of amyloid protein sequences...
Biosurfactants are of growing interest as sustainable alternatives to fossil-fuel-derived chemica... more Biosurfactants are of growing interest as sustainable alternatives to fossil-fuel-derived chemical surfactants, particularly for the detergent industry. To realize this potential, it is necessary to understand how they affect proteins which they may encounter in their applications. However, knowledge of such interactions is limited. Here, we present a study of the interactions between the model protein apo-α-lactalbumin (apo-aLA) and the biosurfactant sophorolipid (SL) produced by the yeast Starmerella bombicola. SL occurs both as an acidic and a lactonic form; the lactonic form (lactSL) is sparingly soluble and has a lower critical micelle concentration (cmc) than the acidic form [non-acetylated acidic sophorolipid (acidSL)]. We show that acidSL affects apo-aLA in a similar way to the related glycolipid biosurfactant rhamnolipid (RL), with the important difference that RL is also active below the cmc in contrast to acidSL. Using isothermal titration calorimetry data, we show that a...
The Gal repressor regulates transport and metabolism of D-galactose in Escherichia coli and can m... more The Gal repressor regulates transport and metabolism of D-galactose in Escherichia coli and can mediate DNA loop formation by forming a bridge between adjacent or distant sites. GalR forms insoluble aggregates at lower salt concentrations in vitro, which can be solubilized at higher salt concentrations. Here, we investigate the assembly and disassembly of GalR aggregates. We find that a sharp transition from aggregates to soluble species occurs between 200 and 400 mM NaCl, incompatible with a simple salting-in effect. The aggregates are highly ordered rod-like structures, highlighting a remarkable ability for organized self-assembly. Mutant studies reveal that aggregation is dependent on two separate interfaces of GalR. The highly ordered structures dissociate to smaller aggregates in the presence of D-galactose. We propose that these self-assembled structures may constitute galactose-tolerant polymers for chromosome compaction in stationary phase cells, in effect linking self-assembly with regulatory function. GalR is the main regulator of galactose transport and metabolism in Escherichia coli. The GalR protein can be purified as a homodimer of a 37 kDa subunit at 0.6 M KCl 1. The dimeric repressor protein can bind to specific 16-bp operator sites and regulate transcription of the associated promoters. GalR represses transcription by diverse mechanisms including steric hindrance, contact inhibition, and DNA looping 2-5. GalR can also activate transcription by interacting with the C-terminal domain of the α-subunit of RNA polymerase 6. Repression of the P1 and P2 promoters of the galETKM operon by DNA looping requires assembly of a higher order nucleoprotein complex, the Gal repressosome. Repressosome formation requires (i) binding of two individual GalR dimers to two operator elements separated by 113 base pairs 2,7,8 ; (ii) negatively supercoiled DNA 9 ; (iii) optimal angular orientation of the two operator sites 10 ; (iv) direct interaction of the two DNA-bound GalR dimers, looping out the intervening DNA segment that contains the P1 and P2 promoters 11,12 ; and (v) specific binding of the HU protein to a DNA site (hbs) in the inter-operator region 13. Increased inter-operator distance or enhanced GalR tetramerization alleviates the requirement of supercoiling and HU binding 12,14. Structure-based genetic analysis has defined the GalR surfaces interacting to form a stacked, V-shaped, tetrameric structure 15-17. Finally, binding of D-galactose (D-gal) to GalR inhibits both DNA binding and tetramerization 18,19. GalR can self-associate to the level of octamers that can connect two or more segments of DNA on small synthetic plasmids 20 and to higher order insoluble structures 1. An intriguing feature of GalR self-association is its regulation by salt. At less than 0.2 M salt, GalR forms aggregates that precipitate, a process which is at least partially reversed in the presence of 0.6 M KCl 1. The observed reversibility suggests that the aggregation process could be a salt-dependent thermodynamic equilibrium between soluble and aggregated GalR. The structure of GalR in these aggregates remains unknown. The low ionic strength in the protoplasm of cells grown in typical media (~150 mM) should favor GalR aggregation 21. Indeed, a recent study where GalR was fused to the fluorescent Venus protein showed that GalR likely exists in vivo in an aggregated form when cells are in stationary phase 20. Association of the DNA-bound GalR dimers was suggested both to allow coordination of regulation at distant promoter sites and to contribute to
Proceedings of the National Academy of Sciences, 2016
Emerging evidence strongly suggests that chaperone proteins are cytoprotective in neurodegenerati... more Emerging evidence strongly suggests that chaperone proteins are cytoprotective in neurodegenerative proteinopathies involving protein aggregation; for example, in the accumulation of aggregated α-synuclein into the Lewy bodies present in Parkinson’s disease. Of the various chaperones known to be associated with neurodegenerative disease, the small secretory chaperone known as proSAAS (named after four residues in the amino terminal region) has many attractive properties. We show here that proSAAS, widely expressed in neurons throughout the brain, is associated with aggregated synuclein deposits in the substantia nigra of patients with Parkinson’s disease. Recombinant proSAAS potently inhibits the fibrillation of α-synuclein in an in vitro assay; residues 158–180, containing a largely conserved element, are critical to this bioactivity. ProSAAS also exhibits a neuroprotective function; proSAAS-encoding lentivirus blocks α-synuclein-induced cytotoxicity in primary cultures of nigral d...
When protein/peptides aggregate, they usually form the amyloid state consisting of cross β-sheet ... more When protein/peptides aggregate, they usually form the amyloid state consisting of cross β-sheet structure built by repetitively stacked β-strands forming long fibrils. Amyloids are usually associated with disease including Alzheimer's. However, amyloid has many useful features. It efficiently transforms protein from the soluble to the insoluble state in an essentially two-state process, while its repetitive structure provides high stability and a robust prion-like replication mechanism. Accordingly, amyloid is used by nature in multifaceted and ingenious ways of life, ranging from bacteria and fungi to mammals. These include (1) Structure: Templating for small chemical molecules (Pmel17), biofilm formation in bacteria (curli), assisting aerial hyphae formation in streptomycetes (chaplins) or monolayer formation at a surface (hydrophobins). (2) Reservoirs: A storage state for peptide/proteins to protect them from their surroundings or vice versa (storage of peptide hormones in mammalian secretory granules or major basic protein in eosinophils). (3) Information carriers: The fungal immune system (HET-s prion in Podospora anserina, yeast prions) or long-term memory (e.g., mnemons in yeast, cytoplasmic polyadenylation element-binding protein in aplysia). Aggregation is also used to (4) "suppress" the function of the soluble protein (e.g., Cdc19 in yeast stress granules), or (5) "signaling" through formation of oligomers (e.g., HET-s prion, necroptosis-related proteins RIP1/RIP3). This review summarizes current knowledge on functional amyloids with a focus on the amyloid systems curli in bacteria, HET-s prion in P. anserina, and peptide hormone storage in mammals together with an attempt to highlight differences between functional and disease-associated amyloids.
Several human proteins cause disease by misfolding and aggregating into amyloid fibril deposits a... more Several human proteins cause disease by misfolding and aggregating into amyloid fibril deposits affecting the surrounding tissues. Multiple other proteins co-associate with the diseased deposits but little is known about how this association is influenced by the nature of the amyloid aggregate and the properties of the amyloid-forming protein. In this study, we investigated the co-aggregation of plasma and cerebrospinal proteins in the presence of pre-formed amyloid fibrils. We evaluated the fibril-associated proteome across multiple amyloid fibril types that differ in their amino acid sequences, ultrastructural morphologies, and recognition by amyloid-binding dyes. The fibril types included aggregates formed by Amyloid β, α-synuclein, and FAS4 that are associated with pathological disorders, and aggregates formed by the glucagon and C-36 peptides, currently not linked to any human disease. Our results highlighted a highly similar response to the amyloid fold within the body fluid of interest. Fibrils with diverse primary sequences and ultrastructural morphologies only differed slightly in the composition of the co-aggregated proteins but were clearly distinct from less fibrillar and amorphous aggregates. The type of body fluid greatly affected the resulting amyloid interactome, underlining the role of the in vivo environment. We conclude that protein fibrils lead to a specific response in protein co-aggregation and discuss the effects hereof in the context of amyloid deposition.
HAMLET (human α-lactalbumin made lethal to tumour cells) is a complex of α-lactalbumin (aLA) and ... more HAMLET (human α-lactalbumin made lethal to tumour cells) is a complex of α-lactalbumin (aLA) and oleic acid (OA) which kills transformed cells, while leaving fully differentiated cells largely unaffected. Other protein-lipid complexes show similar anti-cancer potential. We call such complexes liprotides. The cellular impact of liprotides, while intensely investigated, remains unresolved. To address this, we report on the cell-killing mechanisms of liprotides prepared by incubating aLA with OA for 1 h at 20 or 80 °C (lip20 and lip80, respectively). The liprotides showed similar cytotoxicity against MCF7 cells, though lip80 acts more slowly, possibly due to intermolecular disulphide bonds formed during preparation. Liprotides are known to increase the fluidity of a membrane and transfer OA to vesicles, prompting us to focus on the effect of liprotides on the cell membrane. Extracellular Cainflux is important for activation of the plasma membrane repair system, and we found that remova...
Chemistry (Weinheim an der Bergstrasse, Germany), Jun 6, 2016
Incorporation of silicon-containing amino acids in peptides is known to endow the peptide with de... more Incorporation of silicon-containing amino acids in peptides is known to endow the peptide with desirable properties such as improved proteolytic stability and increased lipophilicity. In the presented study, we demonstrate that incorporation of β-silicon-β3-amino acids into the antimicrobial peptide alamethicin provides the peptide with improved membrane permeabilizing properties. A robust synthetic procedure for the construction of β-silicon-β3-amino acids was developed and the amino acid analogues were incorporated into alamethicin at different positions of the hydrophobic face of the amphipathic helix by using SPPS. The incorporation was shown to provide up to 20-fold increase in calcein release as compared with wild-type alamethicin.
The feature article focuses on the interaction between nanoparticles and α-synuclein, whose aggre... more The feature article focuses on the interaction between nanoparticles and α-synuclein, whose aggregation is central in the development of Parkinson′s Disease.
Functional amyloid (FA) is widespread in bacteria and serves multiple purposes such as strengthen... more Functional amyloid (FA) is widespread in bacteria and serves multiple purposes such as strengthening of biofilm and contact with eukaryotic hosts. Unlike pathological amyloid, FA has been subjected to evolutionary optimization which is likely to be reflected in the aggregation mechanism. FA from different bacteria, including Escherichia coli (CsgA) and Pseudomonas (FapC), contains a number of imperfect repeats which may be key to efficient aggregation. Here we report on the aggregative behavior of FapC constructs which represent all single, double, and triple deletions of the protein's three imperfect repeats. Analysis of the fibrillation kinetics by the program Amylofit reveals that the removal of these repeats increases the tendency of the growing fibrils to fragment and also generally increases aggregation half-times. Remarkably, even the mutant lacking all three repeats was able to fibrillate, although fibrillation was much more irregular and led to significantly altered and destabilized fibrils. We conclude that imperfect repeats can promote fibrillation efficiency thanks to their modular design, though the context of the imperfect repeats also plays a significant role.
Amyloid : the international journal of experimental and clinical investigation : the official journal of the International Society of Amyloidosis, 2017
Thioflavin T (ThT) has been widely used to investigate amyloid formation since 1989. While concer... more Thioflavin T (ThT) has been widely used to investigate amyloid formation since 1989. While concerns have recently been raised about its use as a probe specific for amyloid, ThT still continues to be a very valuable tool for studying kinetic aspects of fibrillation and associated inhibition mechanisms. This review aims to provide a conceptual instruction manual, covering appropriate considerations and pitfalls related to the use of ThT. We start by giving a brief introduction to amyloid formation with focus on the morphology of different aggregate species, followed by a discussion of the quality of protein needed to obtain reliable fibrillation data. After an overview of the photochemical basis for ThT's amyloid binding properties and artifacts that may arise from this, we describe how to plan and analyze ThT assays. We conclude with recommendations for complementary techniques to address shortcomings in the ThT assay.
Traditional approaches to achieve sustained delivery of pharmaceutical peptides traditionally use... more Traditional approaches to achieve sustained delivery of pharmaceutical peptides traditionally use co-excipients (e.g., microspheres and hydrogels). Here, we investigate the release of an amyloidogenic glucagon analogue (3474) from an aggregated state and the influence of surfactants on this process. The formulation of peptide 3474 in dodecyl maltoside (DDM), rhamnolipid (RL), and sophorolipid (SL) led to faster fibrillation. When the aggregates were subjected to multiple cycles of release by repeated resuspension in fresh buffer, the kinetics of the release of soluble peptide 3474 from different surfactant aggregates all followed a simple exponential decay fit, with half-lives of 5–18 min and relatively constant levels of release in each cycle. However, different amounts of peptide are released from different aggregates, ranging from 0.015 mg/mL (3475-buffer) up to 0.03 mg/mL (3474-DDM), with 3474-buffer and 3474-RL in between. In addition to higher release levels, 3474-DDM aggregat...
A high-quality draft genome sequence of ATCC 31213 is presented here. This bacterium produces sev... more A high-quality draft genome sequence of ATCC 31213 is presented here. This bacterium produces several important bioactive compounds and may also produce functional amyloids. This is the first sequenced genome from the genus , and it will be essential in determining the nature of the potential amyloid protein.
Despite extensive studies of the effects of herbal-derived small molecules in the biopharmaceutic... more Despite extensive studies of the effects of herbal-derived small molecules in the biopharmaceutical and biomedical sciences, their low solubility and stability remain a challenge. Here we focus on baicalein, a small molecule showing potential against neurodegenerative diseases such as Parkinson's and Alzheimer's. However, therapeutic usage in vivo is challenged by low solubility and stability. To address this we have applied neutrally-charged nanoliposome (NLP) as carrier for baicalein. Baicalein was incorporated into NLP to form NLP-Ba at molar baicalain:lipid ratios of up to 1:3, giving a drug entrapment efficiency of 96.71%, slow release of approximately 22% after a week and increased baicalein stability up to 27%. Ascorbic acid increased baicalein's stability further, particularly when incorporated in NLP where baicalein stability intensified by 53% in NLP-Ba. Moreover, NLP-Ba did not show significant cytotoxic effects against neurons; rather, showed considerable pro...
Cholesterol (chol) is important in all mammalian cells as a modulator of membrane fluidity. Howev... more Cholesterol (chol) is important in all mammalian cells as a modulator of membrane fluidity. However, its low solubility is a challenge for controlled delivery to membranes. Here we introduce a new tool to deliver chol to membranes, namely, liprotides, i.e., protein-lipid complexes composed of a fatty acid core decorated with partially denatured protein. We focus on liprotides prepared by incubating Ca-depleted α-lactalbumin with oleic acid (OA) for 1 h at 20 °C (lip20) or 80 °C (lip80). The binding and membrane delivery properties of liprotides is compared to the widely chol transporter methyl-β-cyclodextrin (mBCD). Both lip20 and lip80 increase the solubility of chol ~ 50% more than mBCD and deliver chol to membranes with comparable efficiency. Although OA is cytotoxic at high concentrations, its effects are counterbalanced by chol. Further, cytotoxicity is strongly reduced when OA is replaced by cis-palmitoleic acid or cis-vaccenic acid. This makes liprotides good tools to deliver...
Previous studies on metal-ion catalyzed oxidation of α-synuclein oxidation have mostly used condi... more Previous studies on metal-ion catalyzed oxidation of α-synuclein oxidation have mostly used conditions that result in extensive modification precluding an understanding of the early events in this process. In this study, we have examined time-dependent oxidative events related to α-synuclein modification using six different molar ratios of Cu/HO/protein and Cu/HO/ascorbate/protein resulting in mild to moderate extents of oxidation. For a Cu/HO/protein molar ratio of 2.3:7.8:1 only low levels of carbonyls were detected (0.078 carbonyls per protein), whereas a molar ratio of 4.7:15.6:1 gave 0.22 carbonyls per α-synuclein within 15 min. With the latter conditions, rapid conversion of 3 out of 4 methionines (Met) to methionine sulfoxide, and 2 out of 4 tyrosines (Tyr) were converted to products including inter- and intra-molecular dityrosine cross-links and protein oligomers, as determined by SDS-PAGE and Western blot analysis. Limited histidine (His) modification was observed. The rapi...
The Parkinson's disease-associated protein α-synuclein (αSN) is natively unfolded but its str... more The Parkinson's disease-associated protein α-synuclein (αSN) is natively unfolded but its structure can be modulated by membranes and surfactants. The opportunistic pathogen Pseudomonas aeruginosa (PA) produces and secretes the biosurfactant rhamnolipid (RL) which modulates bacterial biofilm. Here, we show that monomeric RL enhances the ability of αSN to permeabilize membranes, while micellar RL rapidly induces protein β-sheet structure with a worm-like fibrillary appearance, which cannot seed RL-free fibrillation but transforms into linear fibrils faster than αSN fibrillating on its own. Exposure to αSN reduces the degree of biofilm formation by PA unless RL is present. Our data suggest that RL interactions with αSN may affect both αSN aggregation and cell toxicity, potentially implicating microbiomic metabolites in the origin and propagation of Parkinson's disease. This article is protected by copyright. All rights reserved.
Gram-negative bacteria possess specialised biogenesis machineries that facilitate the export of a... more Gram-negative bacteria possess specialised biogenesis machineries that facilitate the export of amyloid subunits for construction of a biofilm matrix. The secretion of bacterial functional amyloid requires a bespoke outer-membrane protein channel through which unfolded amyloid substrates are translocated. Here, we combine X-ray crystallography, native mass spectrometry, single-channel electrical recording, molecular simulations and circular dichroism measurements to provide high-resolution structural insight into the functional amyloid transporter from Pseudomonas, FapF. FapF forms a trimer of gated β-barrel channels in which opening is regulated by a helical plug connected to an extended coil-coiled platform spanning the bacterial periplasm. Although FapF represents a unique type of secretion system, it shares mechanistic features with a diverse range of peptide translocation systems. Our findings highlight alternative strategies for handling and export of amyloid protein sequences...
Biosurfactants are of growing interest as sustainable alternatives to fossil-fuel-derived chemica... more Biosurfactants are of growing interest as sustainable alternatives to fossil-fuel-derived chemical surfactants, particularly for the detergent industry. To realize this potential, it is necessary to understand how they affect proteins which they may encounter in their applications. However, knowledge of such interactions is limited. Here, we present a study of the interactions between the model protein apo-α-lactalbumin (apo-aLA) and the biosurfactant sophorolipid (SL) produced by the yeast Starmerella bombicola. SL occurs both as an acidic and a lactonic form; the lactonic form (lactSL) is sparingly soluble and has a lower critical micelle concentration (cmc) than the acidic form [non-acetylated acidic sophorolipid (acidSL)]. We show that acidSL affects apo-aLA in a similar way to the related glycolipid biosurfactant rhamnolipid (RL), with the important difference that RL is also active below the cmc in contrast to acidSL. Using isothermal titration calorimetry data, we show that a...
The Gal repressor regulates transport and metabolism of D-galactose in Escherichia coli and can m... more The Gal repressor regulates transport and metabolism of D-galactose in Escherichia coli and can mediate DNA loop formation by forming a bridge between adjacent or distant sites. GalR forms insoluble aggregates at lower salt concentrations in vitro, which can be solubilized at higher salt concentrations. Here, we investigate the assembly and disassembly of GalR aggregates. We find that a sharp transition from aggregates to soluble species occurs between 200 and 400 mM NaCl, incompatible with a simple salting-in effect. The aggregates are highly ordered rod-like structures, highlighting a remarkable ability for organized self-assembly. Mutant studies reveal that aggregation is dependent on two separate interfaces of GalR. The highly ordered structures dissociate to smaller aggregates in the presence of D-galactose. We propose that these self-assembled structures may constitute galactose-tolerant polymers for chromosome compaction in stationary phase cells, in effect linking self-assembly with regulatory function. GalR is the main regulator of galactose transport and metabolism in Escherichia coli. The GalR protein can be purified as a homodimer of a 37 kDa subunit at 0.6 M KCl 1. The dimeric repressor protein can bind to specific 16-bp operator sites and regulate transcription of the associated promoters. GalR represses transcription by diverse mechanisms including steric hindrance, contact inhibition, and DNA looping 2-5. GalR can also activate transcription by interacting with the C-terminal domain of the α-subunit of RNA polymerase 6. Repression of the P1 and P2 promoters of the galETKM operon by DNA looping requires assembly of a higher order nucleoprotein complex, the Gal repressosome. Repressosome formation requires (i) binding of two individual GalR dimers to two operator elements separated by 113 base pairs 2,7,8 ; (ii) negatively supercoiled DNA 9 ; (iii) optimal angular orientation of the two operator sites 10 ; (iv) direct interaction of the two DNA-bound GalR dimers, looping out the intervening DNA segment that contains the P1 and P2 promoters 11,12 ; and (v) specific binding of the HU protein to a DNA site (hbs) in the inter-operator region 13. Increased inter-operator distance or enhanced GalR tetramerization alleviates the requirement of supercoiling and HU binding 12,14. Structure-based genetic analysis has defined the GalR surfaces interacting to form a stacked, V-shaped, tetrameric structure 15-17. Finally, binding of D-galactose (D-gal) to GalR inhibits both DNA binding and tetramerization 18,19. GalR can self-associate to the level of octamers that can connect two or more segments of DNA on small synthetic plasmids 20 and to higher order insoluble structures 1. An intriguing feature of GalR self-association is its regulation by salt. At less than 0.2 M salt, GalR forms aggregates that precipitate, a process which is at least partially reversed in the presence of 0.6 M KCl 1. The observed reversibility suggests that the aggregation process could be a salt-dependent thermodynamic equilibrium between soluble and aggregated GalR. The structure of GalR in these aggregates remains unknown. The low ionic strength in the protoplasm of cells grown in typical media (~150 mM) should favor GalR aggregation 21. Indeed, a recent study where GalR was fused to the fluorescent Venus protein showed that GalR likely exists in vivo in an aggregated form when cells are in stationary phase 20. Association of the DNA-bound GalR dimers was suggested both to allow coordination of regulation at distant promoter sites and to contribute to
Proceedings of the National Academy of Sciences, 2016
Emerging evidence strongly suggests that chaperone proteins are cytoprotective in neurodegenerati... more Emerging evidence strongly suggests that chaperone proteins are cytoprotective in neurodegenerative proteinopathies involving protein aggregation; for example, in the accumulation of aggregated α-synuclein into the Lewy bodies present in Parkinson’s disease. Of the various chaperones known to be associated with neurodegenerative disease, the small secretory chaperone known as proSAAS (named after four residues in the amino terminal region) has many attractive properties. We show here that proSAAS, widely expressed in neurons throughout the brain, is associated with aggregated synuclein deposits in the substantia nigra of patients with Parkinson’s disease. Recombinant proSAAS potently inhibits the fibrillation of α-synuclein in an in vitro assay; residues 158–180, containing a largely conserved element, are critical to this bioactivity. ProSAAS also exhibits a neuroprotective function; proSAAS-encoding lentivirus blocks α-synuclein-induced cytotoxicity in primary cultures of nigral d...
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Papers by Daniel Otzen