scholarly journals Quantitating denaturation by formic acid: Imperfect repeats are essential to the stability of the functional amyloid protein FapC

2020 ◽  
Author(s):  
Line Friis Bakmann Christensen ◽  
Jan Stanislaw Nowak ◽  
Thorbjørn Vincent Sønderby ◽  
Signe Andrea Frank ◽  
Daniel Erik Otzen
Keyword(s):  
Formic Acid ◽  
Mechanical Stability ◽  
Complete Removal ◽  
Amyloid Formation ◽  
Amyloid Protein ◽  
Biological Functions ◽  
Functional Amyloid ◽  
High Concentrations ◽  
Functional Amyloids ◽  
The Stability

ABSTRACTBacterial functional amyloids are evolutionarily optimized to aggregate to help them fulfil their biological functions, e.g. to provide mechanical stability to biofilm. Amyloid is formed in Pseudomonas sp. by the protein FapC which contains 3 imperfect repeats connected by long linkers. Stepwise removal of these repeats slows down aggregation and increases the propensity of amyloids to fragment during the fibrillation process, but how these mechanistic properties link to fibril stability is unclear. Here we address this question. The extreme robustness of functional amyloid makes them resistant to conventional chemical denaturants, but they dissolve in formic acid (FA) at high concentrations. To quantify this, we first measured the denaturing potency of FA using 3 small acid-resistant proteins (S6, lysozyme and ubiquitin). This revealed a linear relationship between [FA] and the free energy of unfolding with a slope of mFA, as well as a robust correlation between protein residue size and mFA. We then measured the solubilisation of fibrils formed from different FapC variants (with varying number of repeats) as a function of [FA]. The resulting mFA values revealed a decline in the number of residues driving amyloid formation when at least 2 repeats were deleted. The midpoint of denaturation declined monotonically with progressive removal of repeats and correlated with solubility in SDS. Complete removal of all repeats led to fibrils which were solubilized at FA concentrations 2-3 orders of magnitude lower than the repeat-containing variants, showing that at least one imperfect repeat is required for the stability of functional amyloid.

scholarly journals Quantitating denaturation by formic acid: imperfect repeats are essential to the stability of the functional amyloid protein FapC

2020 ◽  
Vol 295 (37) ◽  
pp. 13031-13046
Author(s):  
Line Friis Bakmann Christensen ◽  
Jan Stanislaw Nowak ◽  
Thorbjørn Vincent Sønderby ◽  
Signe Andrea Frank ◽  
Daniel Erik Otzen
Keyword(s):  
Formic Acid ◽  
Complete Removal ◽  
Systematic Investigation ◽  
Amyloid Formation ◽  
Amyloid Protein ◽  
Functional Amyloid ◽  
Protein Size ◽  
High Concentrations ◽  
Functional Amyloids ◽  
The Stability

Bacterial functional amyloids are evolutionarily optimized to aggregate, so much so that the extreme robustness of functional amyloid makes it very difficult to examine their structure-function relationships in a detailed manner. Previous work has shown that functional amyloids are resistant to conventional chemical denaturants, but they dissolve in formic acid (FA) at high concentrations. However, systematic investigation requires a quantitative analysis of FA's ability to denature proteins. Amyloid formed by Pseudomonas sp. protein FapC provides an excellent model to investigate FA denaturation. It contains three imperfect repeats, and stepwise removal of these repeats slows fibrillation and increases fragmentation during aggregation. However, the link to stability is unclear. We first calibrated FA denaturation using three small, globular, and acid-resistant proteins. This revealed a linear relationship between the concentration of FA and the free energy of unfolding with a slope of mFA+pH (the combined contribution of FA and FA-induced lowering of pH), as well as a robust correlation between protein size and mFA+pH. We then measured the solubilization of fibrils formed from different FapC variants with varying numbers of repeats as a function of the concentration of FA. This revealed a decline in the number of residues driving amyloid formation upon deleting at least two repeats. The midpoint of denaturation declined with the removal of repeats. Complete removal of all repeats led to fibrils that were solubilized at FA concentrations 2–3 orders of magnitude lower than the repeat-containing variants, showing that at least one repeat is required for the stability of functional amyloid.

scholarly journals Modulating functional amyloid formation via alternative splicing of the premelanosomal protein PMEL17

2020 ◽  
Vol 295 (21) ◽  
pp. 7544-7553 ◽  
Author(s):  
Dexter N. Dean ◽  
Jennifer C. Lee
Keyword(s):  
Alternative Splicing ◽  
Amyloid Fibrils ◽  
Limited Proteolysis ◽  
Amyloid Formation ◽  
Functional Amyloid ◽  
Melanin Biosynthesis ◽  
Functional Amyloids ◽  
Fibril Morphology ◽  
Amyloid Assembly ◽  
Β Sheet

The premelanosomal protein (PMEL17) forms functional amyloid fibrils involved in melanin biosynthesis. Multiple PMEL17 isoforms are produced, two of which arise from excision of a cryptic intron within the amyloid-forming repeat (RPT) domain, leading to long (lRPT) and short (sRPT) isoforms with 10 and 7 imperfect repeats, respectively. Both lRPT and sRPT isoforms undergo similar pH-dependent mechanisms of amyloid formation and fibril dissolution. Here, using human PMEL17, we tested the hypothesis that the minor, but more aggregation-prone, sRPT facilitates amyloid formation of lRPT. We observed that cross-seeding by sRPT fibrils accelerates the rate of lRPT aggregation, resulting in propagation of an sRPT-like twisted fibril morphology, unlike the rodlike structure that lRPT normally adopts. This templating was specific, as the reversed reaction inhibited sRPT fibril formation. Despite displaying ultrastructural differences, self- and cross-seeded lRPT fibrils had a similar β-sheet structured core, revealed by Raman spectroscopy, limited-proteolysis, and fibril disaggregation experiments, suggesting the fibril twist is modulated by N-terminal residues outside the amyloid core. Interestingly, bioinformatics analysis of PMEL17 homologs from other mammals uncovered that long and short RPT isoforms are conserved among members of this phylogenetic group. Collectively, our results indicate that the short isoform of RPT serves as a “nucleator” of PMEL17 functional amyloid formation, mirroring how bacterial functional amyloids assemble during biofilm formation. Whereas bacteria regulate amyloid assembly by using individual genes within the same operon, we propose that the modulation of functional amyloid formation in higher organisms can be accomplished through alternative splicing.

scholarly journals Co-aggregation and secondary nucleation in the life cycle of human prolactin/galanin functional amyloids

2021 ◽  
Author(s):  
D. Chatterjee ◽  
R.S. Jacob ◽  
S. Ray ◽  
A. Navalkar ◽  
N. Singh ◽  
...  
Keyword(s):  
Neurological Disorders ◽  
Secretory Granules ◽  
Amyloid Formation ◽  
Female Rat ◽  
Secondary Nucleation ◽  
Functional Amyloid ◽  
Rapid Release ◽  
Efficient Storage ◽  
Human Prolactin ◽  
Functional Amyloids

AbstractSynergistic-aggregation and cross-seeding by two different amyloid proteins/peptides are well evident in various neurological disorders. However, this phenomenon is not well studied in functional amyloid aggregation. Here, we show Prolactin (PRL) is associated with lactation in mammals and neuropeptide galanin (GAL), which are co-stored in the lactotrophs facilitates the synergic aggregation in the absence of secretory granules helper molecules glycosaminoglycans (GAGS). Interestingly, although each partner possesses homotypic seeding ability, a unidirectional cross-seeding of GAL aggregation can be mediated by PRL seeds. The specificity of co-aggregation by PRL and GAL along with unidirectional cross-seeding suggests tight regulation of functional amyloid formation during co-storage of these hormones in secretory granule biogenesis of female rat lactotrophs. Further mixed fibrils release the constituent functional hormone much faster than the corresponding individual amyloid formed in presence of GAGs, suggesting that co-aggregation of functionally distant hormones might have evolved for efficient storage, synergistic and rapid release of both hormones upon stimulation. The co-aggregation and cross seeding by two different hormones of completely different structures and sequences (PRL and GAL) suggest a novel mechanism of heterologous amyloid formation both in disease and functional amyloids.

scholarly journals Functional amyloids in the microbiomes of a rat Parkinson's disease model and wild-type rats

2021 ◽  
Author(s):  
Line F Christensen ◽  
Saeid H Alijanvand ◽  
Michał Burdukiewicz ◽  
Florian Alexander Herbst ◽  
Henrik Kjeldal ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Formic Acid ◽  
Proteinase K ◽  
Amyloid Formation ◽  
Wild Type ◽  
Amyloid Proteins ◽  
Fecal Matter ◽  
Functional Amyloid

Cross-seeding between amyloidogenic proteins in the gut is receiving increasing attention as a possible mechanism for initiation or acceleration of amyloid formation by aggregation-prone proteins such as αSN, which is central in the development of Parkinson's disease. This is particularly pertinent in view of the growing number of functional (i.e. benign and useful) amyloid proteins discovered in bacteria. Here we identify two functional amyloid proteins, Pr12 and Pr17, in fecal matter from Parkinson's disease transgenic rats and their wild type counterparts, based on their stability against dissolution by formic acid. Both proteins show robust aggregation into ThT-positive aggregates that contain higher-order b-sheets and have a fibrillar morphology, indicative of amyloid proteins. In addition, Pr17 aggregates formed in vitro showed significant resistance against formic acid, suggesting an ability to form highly stable amyloid. Treatment with proteinase K revealed a protected core of approx. 9 kDa. Neither Pr12 nor Pr17, however, affected αSN aggregation in vitro. Thus, amyloidogenicity does not per se lead to an ability to cross-seed fibrillation of αSN. Our results support the use of proteomics and formic acid to identify amyloid protein in complex mixtures and indicates the existence of numerous functional amyloid proteins in microbiomes.

Functional amyloid: widespread in Nature, diverse in purpose

2014 ◽  
Vol 56 ◽  
pp. 207-219 ◽  
Author(s):  
Chi L.L. Pham ◽  
Ann H. Kwan ◽  
Margaret Sunde
Keyword(s):  
Spider Silk ◽  
Epigenetic Inheritance ◽  
Fibrillar Structure ◽  
Cytoplasmic Polyadenylation ◽  
Functional Amyloid ◽  
Interacting Protein ◽  
Diverse Range ◽  
Element Binding Protein ◽  
Functional Amyloids ◽  
Micro Organisms

Amyloids are insoluble fibrillar protein deposits with an underlying cross-β structure initially discovered in the context of human diseases. However, it is now clear that the same fibrillar structure is used by many organisms, from bacteria to humans, in order to achieve a diverse range of biological functions. These functions include structure and protection (e.g. curli and chorion proteins, and insect and spider silk proteins), aiding interface transitions and cell–cell recognition (e.g. chaplins, rodlins and hydrophobins), protein control and storage (e.g. Microcin E492, modulins and PMEL), and epigenetic inheritance and memory [e.g. Sup35, Ure2p, HET-s and CPEB (cytoplasmic polyadenylation element-binding protein)]. As more examples of functional amyloid come to light, the list of roles associated with functional amyloids has continued to expand. More recently, amyloids have also been implicated in signal transduction [e.g. RIP1/RIP3 (receptor-interacting protein)] and perhaps in host defence [e.g. aDrs (anionic dermaseptin) peptide]. The present chapter discusses in detail functional amyloids that are used in Nature by micro-organisms, non-mammalian animals and mammals, including the biological roles that they play, their molecular composition and how they assemble, as well as the coping strategies that organisms have evolved to avoid the potential toxicity of functional amyloid.

An analysis of the transport properties and mechanical stability of rapidly solidified Al-Sb alloy

2015 ◽  
Vol 10 (2) ◽  
pp. 2663-2681
Author(s):  
Rizk El- Sayed ◽  
Mustafa Kamal ◽  
Abu-Bakr El-Bediwi ◽  
Qutaiba Rasheed Solaiman
Keyword(s):  
Mechanical Properties ◽  
Thermal Conductivity ◽  
Melt Spinning ◽  
Elastic Moduli ◽  
Mechanical Stability ◽  
Microstructural Analysis ◽  
Alloy System ◽  
Antimony Content ◽  
The Stability ◽  
Rapidly Solidified

The structure of a series of AlSb alloys prepared by melt spinning have been studied in the as melt–spun ribbons  as a function of antimony content .The stability  of these structures has  been  related to that of the transport and mechanical properties of the alloy ribbons. Microstructural analysis was performed and it was found that only Al and AlSb phases formed for different composition.  The electrical, thermal and the stability of the mechanical properties are related indirectly through the influence of the antimony content. The results are interpreted in terms of the phase change occurring to alloy system. Electrical resistivity, thermal conductivity, elastic moduli and the values of microhardness are found to be more sensitive than the internal friction to the phase changes. 

Methods for the Determination of the Purity of Exosomes

2020 ◽  
Vol 25 (42) ◽  
pp. 4464-4485 ◽  
Author(s):  
Katarzyna Kluszczyńska ◽  
Liliana Czernek ◽  
Wojciech Cypryk ◽  
Łukasz Pęczek ◽  
Markus Düchler
Keyword(s):  
Drug Transport ◽  
Biological Fluids ◽  
Drug Carriers ◽  
Biological Functions ◽  
Isolation And Purification ◽  
Pubmed Database ◽  
High Concentrations ◽  
Targeted Release

Background: Exosomes open exciting new opportunities for advanced drug transport and targeted release. Furthermore, exosomes may be used for vaccination, immunosuppression or wound healing. To fully utilize their potential as drug carriers or immune-modulatory agents, the optimal purity of exosome preparations is of crucial importance. Methods: Articles describing the isolation and purification of exosomes were retrieved from the PubMed database. Results: Exosomes are often separated from biological fluids containing high concentrations of proteins, lipids and other molecules that keep vesicle purification challenging. A great number of purification protocols have been published, however, their outcome is difficult to compare because the assessment of purity has not been standardized. In this review, we first give an overview of the generation and composition of exosomes, as well as their multifaceted biological functions that stimulated various medical applications. Finally, we describe various methods that have been used to purify small vesicles and to assess the purity of exosome preparations and critically compare the quality of these evaluation protocols. Conclusion: Combinations of various techniques have to be applied to reach the required purity and quality control of exosome preparations.

scholarly journals Exosome-derived noncoding RNAs in gastric cancer: functions and clinical applications

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Xiao-Huan Tang ◽  
Ting Guo ◽  
Xiang-Yu Gao ◽  
Xiao-Long Wu ◽  
Xiao-Fang Xing ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Noncoding Rnas ◽  
Tumour Microenvironment ◽  
Clinical Applications ◽  
Biological Molecules ◽  
Biological Functions ◽  
Membrane Structures ◽  
Chemical Substances ◽  
Expression Levels ◽  
The Stability

AbstractExosomes are a subpopulation of the tumour microenvironment (TME) that transmit various biological molecules to promote intercellular communication. Exosomes are derived from nearly all types of cells and exist in all body fluids. Noncoding RNAs (ncRNAs) are among the most abundant contents in exosomes, and some ncRNAs with biological functions are specifically packaged into exosomes. Recent studies have revealed that exosome-derived ncRNAs play crucial roles in the tumorigenesis, progression and drug resistance of gastric cancer (GC). In addition, regulating the expression levels of exosomal ncRNAs can promote or suppress GC progression. Moreover, the membrane structures of exosomes protect ncRNAs from degradation by enzymes and other chemical substances, significantly increasing the stability of exosomal ncRNAs. Specific hallmarks within exosomes that can be used for exosome identification, and specific contents can be used to determine their origin. Therefore, exosomal ncRNAs are suitable for use as diagnostic and prognostic biomarkers or therapeutic targets. Regulating the biogenesis of exosomes and the expression levels of exosomal ncRNAs may represent a new way to block or eradicate GC. In this review, we summarized the origins and characteristics of exosomes and analysed the association between exosomal ncRNAs and GC development.

scholarly journals Chaperones mainly suppress primary nucleation during formation of functional amyloid required for bacterial biofilm formation

2022 ◽  
Author(s):  
Madhu Nagaraj ◽  
Zahra Najarzadeh ◽  
Jonathan Pansieri ◽  
Ludmilla A. Morozova-Roche ◽  
Henrik Biverstål ◽  
...  
Keyword(s):  
Neurodegenerative Diseases ◽  
Biofilm Formation ◽  
Bacterial Biofilm ◽  
Functional Amyloid ◽  
E Coli ◽  
Primary Nucleation ◽  
Functional Amyloids

Unlike misfolding in neurodegenerative diseases, aggregation of functional amyloids involved in bacterial biofilm, e.g. CsgA (E. coli) and FapC (Pseudomonas), is carefully regulated. However, it is unclear whether functional aggregation...

scholarly journals The potentional of plants to cleanup metals from an old landfill site

2017 ◽  
Author(s):  
Yahya Jani ◽  
Charlotte Marchand ◽  
William Hogland
Keyword(s):  
Contaminated Soils ◽  
Hazardous Materials ◽  
Average Concentration ◽  
Soil Samples ◽  
Landfill Site ◽  
Group Company ◽  
Timothy Grass ◽  
Xrf Scanning ◽  
High Concentrations ◽  
The Stability

Old landfill sites contain different hazardous materials like heavy metals which have the ability to affects the entire environment. These places are sometimes covered by plants to increase the stability of the soil and to reduce the effects of erosion. 15 soil samples (3 samples from each place) and 5-7 timothy-grass (Phleum pretense) plants from 5 different places were taken from an old landfill place in an active landfill site in Högbytorp /Sweden owned by Ragn-sells Group Company. XRF scanning was used to analyze the metal content of soil samples and of plants. High concentrations of metals were detected in the soil samples like Fe with an average of about 25000 ppm, Mn about 250 ppm and 2800 ppm of Ti. The plants results showed an average concentration of Fe in the shoots about 730 ppm, Mn about 60 ppm and Ti about 1760 ppm. On the other hand, the roots results showed an average concentration of about 10 000 ppm of Fe, about 160 ppm of Mn and 2200 ppm of Ti. These results gave the indication that the Timothy-grass has the ability to extract metals from contaminated soils and can help to cleanup these soils.

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