Normalizing polydiacetylene colorimetric assays of vesicle binding across lipid systems

Beasley, Maryssa; Stonebraker, Alyssa R.; Legleiter, Justin

doi:10.1016/j.ab.2020.113864

Cited by 9 publications

(13 citation statements)

References 18 publications

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“…By measurement of the absorbance of the blue (640 nm) and red (500 nm) wavelengths of lipid/PDA vesicles upon exposure to htt-exon1(46Q), the % CR was obtained, which directly corresponds to the protein–lipid interaction. In addition, these assays were normalized to an NaOH control, allowing for comparison across lipid systems …”

Section: Resultsmentioning

confidence: 99%

“…In addition, these assays were normalized to an NaOH control, allowing for comparison across lipid systems. 57 Phosphomimetic mutations significantly enhanced the interaction of htt with TBLE, with htt-exon1 T3D and htt-exon1 S13D having the strongest interaction with the membrane [∼2 times the maximum % CR compared to htt-exon1 NT ; p < 0.01 (Figure 5A)]. With TBLE, the enhanced interaction of htt-exon1 S16D compared to htt-exon1 NT was less pronounced and not statistically significant.…”

Section: ■ Materials and Methodsmentioning

confidence: 96%

“…The NaOH control was used to standardize PDA results across experiments. 57 The percent colorimetric response (% CR) was determined every 5 min for 18 h at 37 °C by measuring the blue absorbance (640 nm, A blue ) and the red absorbance (500 nm, A red ) using the following equation:…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…Each plate contained negative controls (neat buffer A) and a positive control (final concentration of 100 μM NaOH). The NaOH control was used to standardize PDA results across experiments . The percent colorimetric response (% CR) was determined every 5 min for 18 h at 37 °C by measuring the blue absorbance (640 nm, A blue ) and the red absorbance (500 nm, A red ) using the following equation: where PB is defined as A blue /( A blue + A red ).…”

Section: Methodsmentioning

confidence: 99%

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Phosphomimetic Mutations Impact Huntingtin Aggregation in the Presence of a Variety of Lipid Systems

et al. 2020

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Huntington’s disease (HD) is a neurodegenerative disorder caused by the abnormal expansion of a polyglutamine (polyQ) tract in the first exon of the htt protein (htt). PolyQ expansion triggers the aggregation of htt into a variety of structures, including oligomers and fibrils. This aggregation is impacted by the first 17 N-terminal amino acids (Nt17) of htt that directly precedes the polyQ domain. Beyond impacting aggregation, Nt17 associates with lipid membranes by forming an amphipathic α-helix. Post-translational modifications within Nt17 are known to modify HD pathology, and in particular, phosphorylation at T3, S13, and/or S16 retards fibrillization and ameliorates the phenotype in HD models. Due to Nt17’s propensity to interact with lipid membranes, the impact of introducing phosphomimetic mutations (T3D, S13D, and S16D) into htt-exon1 on aggregation in the presence of a variety of model lipid membranes (total brain lipid extract, 1-palmitoyl-2-oleoyl-glycero-3-phosphatidylcholine, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-1′-rac-glycerol) was investigated. Phosphomimetic mutations altered htt’s interaction with and aggregation in the presence of lipids; however, this was dependent on the lipid system.

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Section: Resultsmentioning

confidence: 99%

Section: ■ Materials and Methodsmentioning

confidence: 96%

Section: ■ Materials and Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Phosphomimetic Mutations Impact Huntingtin Aggregation in the Presence of a Variety of Lipid Systems

et al. 2020

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“…The applications of PDA vesicles based sensors have also been explored in the other areas such as high-throughput screening of membrane acting antimicrobial peptides, pathogenic/food borne bacteria [11] and viruses (In uenza and latest coronavirus SARS-CoV-2) [41]. The authors have demonstrated great potential in providing a simple, low-cost, accurate and fastspeed detecting method alternative for the current methods which are too expensive and time-consuming for analyzing a large number of samples [44]. The PDA vesicles were used as paper strip sensor for the detection of spores of Bacillus thuringiensis HD-73 [43].…”

Section: Discussionmentioning

confidence: 99%

Polydiacetylene Vesicles Acting as Colorimetric Sensor for the Detection of Plantaricin LD1 Purified From Lactobacillus Plantarum LD1

Yadav

Tiwari

2021

Preprint

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The interaction of antimicrobial peptides with membrane lipids plays a major role in numerous physiological processes. Bacteriocins are antimicrobial peptides known to kill target cells by pore formation and membrane disruption. In this study, polydiacetylene (PDA) vesicles were applied as artificial membrane for detection of plantaricin LD1 purified from Lactobacillus plantarum LD1. Plantaricin LD1 (200 µg/ml) was able to change the color of PDA vesicles from blue to red with colorimetric response CR % 30.26 ± 0.59. Nisin (200 µg/ml), used as control, also changed the color of the vesicles with CR % 50.56 ± 0.98 validating the membrane-acting nature of these bacteriocins. The PDA vesicles treated with nisin and plantaricin LD1 showed increased infrared absorbance at 1411.46 cm-1 and 1000-1150 cm-1 indicated the interaction of bacteriocins with phospholipids and fatty acids, respectively. Further, microscopic examination also suggested the disruption of bacteriocin-treated vesicles indicating the interaction of bacteriocins. These findings suggest that the PDA vesicles may be used as bio-mimetic sensor for the detection of bacteriocins produced by several probiotics in food and therapeutic applications.

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Cholesterol impacts the formation of huntingtin/lipid complexes and subsequent aggregation

et al. 2023

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Huntington's disease (HD) is a neurodegenerative disease resulting from an expansion of the polyglutamine (polyQ) domain within the huntingtin protein (htt). PolyQ expansion triggers toxic aggregation and alters htt/lipid interactions. The first 17 amino acids at the N‐terminus of htt (Nt17) have a propensity to form an amphipathic α‐helix crucial to aggregation and membrane binding. Htt interacts closely with a variety of membrane systems including those of the endoplasmic reticulum, mitochondria, nuclear envelope, and plasma membrane. Membrane composition heavily influences both htt aggregation and lipid interactions, and cholesterol is a crucial membrane component that modulates properties such as fluidity, permeability, and organization. In HD, cholesterol homeostasis is disrupted, and likely plays a role in toxicity. The objective of these studies was to identify the impact of cholesterol on htt aggregation and lipid interactions in various lipid systems. Lipid systems of POPC, DOPC, and POPG with varied levels of exogenously added cholesterol were exposed to htt, and the influences on aggregation, lipid binding, and htt/lipid complexation were evaluated using thioflavin‐T aggregation assays, atomic force microscopy, colorimetric lipid binding assays, and mass spectrometry. The addition of cholesterol to DOPC vesicles enhanced htt aggregation. In the presence of vesicles of either POPC or POPG, the addition of cholesterol reduced htt aggregation. Htt/lipid binding decreased for POPC and increased for both DOPC and POPG with increasing cholesterol content, with observed differences in htt/lipid complexation. Altered cholesterol content influences htt aggregation, lipid binding, and complexation differently depending on overall lipid composition.

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Normalizing polydiacetylene colorimetric assays of vesicle binding across lipid systems

Cited by 9 publications

References 18 publications

Phosphomimetic Mutations Impact Huntingtin Aggregation in the Presence of a Variety of Lipid Systems

Phosphomimetic Mutations Impact Huntingtin Aggregation in the Presence of a Variety of Lipid Systems

Polydiacetylene Vesicles Acting as Colorimetric Sensor for the Detection of Plantaricin LD1 Purified From Lactobacillus Plantarum LD1

Cholesterol impacts the formation of huntingtin/lipid complexes and subsequent aggregation

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