Fibrils Emerging from Droplets: Molecular Guiding Principles behind Phase Transitions of a Short Peptide-Based Condensate Studied by Solid-State NMR

Biochemical reactions occurring in highly crowded cellular environments require different means of control to ensure productivity and specificity. Compartmentalization of reagents by liquid-liquid phase separation is one of these means. However, extremely high local protein concentrations of up to 400 mg/ml can result in pathological aggregation into fibrillar amyloid structures, a phenomenon that has been linked to various neurodegenerative diseases. Despite its relevance, the process of liquid-to-solid transition inside condensates is still not well understood at the molecular level. We thus herein use small peptide derivatives that can undergo both liquid-liquid and subsequent liquid-to-solid phase transition as model systems to study both processes. Using solid-state nuclear magnetic resonance (NMR) and transmission electron microscopy (TEM), we compare the structure of condensed states of leucine, tryptophan and phenylalanine containing derivatives, distinguishing between liquid-like condensates, amorphous aggregates and fibrils, respectively. A structural model for the fibrils formed by the phenylalanine derivative was obtained by an NMR-based structure calculation. The fibrils are stabilised by hydrogen bonds and side-chain π-π interactions, which are likely much less pronounced or absent in the liquid and amorphous state. Such noncovalent interactions are equally important for the liquid-to-solid transition of proteins, particularly those related to neurodegenerative diseases.ISSN:0947-6539ISSN:1521-376

A Short Peptide Synthon for Liquid-Liquid Phase Separation

Liquid-liquid phase separation of disordered proteins has emerged as a ubiquitous route to membraneless compartments in living cells, and similar coacervates may have played a role when the first cells formed. However, existing coacervates are typically made of multiple macromolecular components, and designing short peptide analogues capable of self-coacervation has proven difficult. Here, we present a short peptide synthon for phase separation, made of only two dipeptide stickers linked via a flexible, hydrophilic spacer. These small-molecule compounds self-coacervate into micrometre-sized liquid droplets at sub-mM concentrations, which retain up to 75 weight-% water. The design is general and we derive guidelines for the required sticker hydrophobicity and spacer polarity. To illustrate their potential as protocells, we create a disulphide-linked derivative that undergoes reversible compartmentalisation controlled by redox chemistry. The resulting coacervates sequester and melt nucleic acids, and act as microreactors that catalyse two different anabolic reactions yielding molecules of increasing complexity. This provides a stepping stone for new protocells made of single peptide species.<br /

Fibrils emerging from droplets: Molecular guiding principles behind phase transitions of short peptide-based condensates

Biochemical reactions occurring in highly crowded cellular environments require different means of control to ensure productivity and specificity. Compartmentalization of reagents by liquid-liquid phase separation is one of these means. However, extremely high local protein concentrations of up to 400 mg/ml can result in pathological aggregation into fibrillar amyloid structures, a phenomenon that has been linked to various neurodegenerative diseases. Despite its relevance, the process of liquid-to-solid transition inside condensates is still not well understood at the molecular level. In this work, we use small peptide derivatives that can undergo both liquid-liquid and subsequent liquid-to-solid phase transition as model systems to study both processes at the molecular level. Using solid-state nuclear magnetic resonance (NMR) and transmission electron microscopy, we compare the structure of condensed states of leucine, tryptophan and phenylalanine containing derivatives, distinguishing between liquid-like condensates, amorphous aggregates and fibrils, respectively. A structural model for the fibrils formed by the phenylalanine derivative was obtained by a structure calculation based on NMR distance restraints. Our results show that the fibrils are stabilised by hydrogen bonds and side-chain π-π interactions, which are likely much less pronounced or absent in the liquid and amorphous state. Such noncovalent interactions are equally important for the liquid-to-solid phase transition of proteins, particularly those related to neurodegenerative diseases and our results suggest that aged condensates of these proteins may have partial amyloid-like characteristics

Biomolecular condensates can both accelerate and suppress aggregation of α-synuclein

Biomolecular condensates present in cells can fundamentally affect the aggregation of amyloidogenic proteins and play a role in the regulation of this process. While liquid-liquid phase separation of amyloidogenic proteins by themselves can act as an alternative nucleation pathway, interaction of partly disordered aggregation-prone proteins with preexisting condensates that act as localization centers could be a far more general mechanism of altering their aggregation behavior. Here, we show that so-called host biomolecular condensates can both accelerate and slow down amyloid formation. We study the amyloidogenic protein α-synuclein and two truncated α-synuclein variants in the presence of three types of condensates composed of nonaggregating peptides, RNA, or ATP. Our results demonstrate that condensates can markedly speed up amyloid formation when proteins localize to their interface. However, condensates can also significantly suppress aggregation by sequestering and stabilizing amyloidogenic proteins, thereby providing living cells with a possible protection mechanism against amyloid formation

Induction of HO1 expression and increased iron status in duodenal enterocytes from hemoglobin-supplemented piglets.

<p><b>(A)</b> RT-qPCR analysis of HO1 mRNA expression. The histogram displays HO1 mRNA levels in arbitrary units (means ± S.D., n = 7). <b>(B and C)</b> Western blot analysis of HO1 protein levels in membrane fractions prepared from duodenal scrapings. A representative immunoblot is shown <b>(B)</b>. Immunolabelled HO1 bands from separate blots performed on scrapings isolated from 6 piglets were quantified using a Molecular Imager, and HO1 protein levels (means ± S.D.) are plotted in arbitrary units <b>(C)</b>. <b>(D)</b> Immunofluorescent staining of HO1 in the duodenum. To confirm the specificity of the HO1 detection, duodenum sections of piglets were incubated with only the secondary antibody. No HO1 staining was detected in these negative controls. Counterstaining of nuclei was performed with DAPI. <b>(E and F)</b> Western blot analysis of L-ferritin protein levels in membrane fractions prepared from duodenal scrapings. A representative immunoblot is shown <b>(E)</b>. Immunolabelled L-ferritin bands from separate blots performed on scrapings isolated from 6 piglets were quantified using a Molecular Imager, and L-ferritin protein levels (means ± S.D.) are plotted in arbitrary units <b>(F)</b>. <b>(G)</b> Histological examination of iron loading in duodenum sections. Non-heme iron deposits (indicated by arrows) were detected by staining with Perls’ Prussian blue and counterstained with nuclear red. Duodenum morphology is shown in transmitted light.</p

Increased HRG1 protein levels on the apical membrane of absorptive enterocytes of piglets fed a hemoglobin-enriched diet.

<p><b>(A)</b> Western blot analysis of HRG1 protein levels in membrane fractions prepared from duodenal scrapings as described under Materials and methods. A representative immunoblot is shown. Ponceau Red staining of transferred proteins are shown (bottom panel) to confirm equivalent loading. <b>(B)</b> Immunofluorescent staining of HRG1 in the duodenum. To confirm the specificity of HRG1 detection, duodenum sections of piglets were incubated with only the secondary antibody. No HRG1 staining was detected in these negative controls. Counterstaining of nuclei was performed with DAPI. Duodenum morphology is shown in transmitted light.</p

Reduced expression of DMT1 in piglets supplemented with FeDex and fed a hemoglobin (HGB)-enriched diet.

<p><b>(A)</b> RT-qPCR analysis of DMT1 mRNA expression. The histogram displays DMT1 mRNA levels in arbitrary units (means ± S.D., n = 7). <b>(B and C)</b> Western blot analysis of DMT1 protein levels in membrane fractions prepared from duodenal scrapings. A representative immunoblot is shown <b>(B)</b>. Immunolabelled DMT1 bands from separate blots performed on scrapings isolated from 6 piglets were quantified using a Molecular Imager, and DMT1 protein levels (means ± S.D.) are plotted in arbitrary units <b>(C)</b>. <b>(D)</b> Immunofluorescent staining of DMT1 in the duodenum. To confirm the specificity of DMT1 detection, piglet duodenum sections were incubated with only the secondary antibody. No DMT1 staining was detected in these negative controls. Counterstaining of nuclei was performed with DAPI. Duodenum morphology is shown in transmitted light.</p

Induction of HO1 expression and increased iron status in duodenal enterocytes from hemoglobin-supplemented piglets.

<p><b>(A)</b> RT-qPCR analysis of HO1 mRNA expression. The histogram displays HO1 mRNA levels in arbitrary units (means ± S.D., n = 7). <b>(B and C)</b> Western blot analysis of HO1 protein levels in membrane fractions prepared from duodenal scrapings. A representative immunoblot is shown <b>(B)</b>. Immunolabelled HO1 bands from separate blots performed on scrapings isolated from 6 piglets were quantified using a Molecular Imager, and HO1 protein levels (means ± S.D.) are plotted in arbitrary units <b>(C)</b>. <b>(D)</b> Immunofluorescent staining of HO1 in the duodenum. To confirm the specificity of the HO1 detection, duodenum sections of piglets were incubated with only the secondary antibody. No HO1 staining was detected in these negative controls. Counterstaining of nuclei was performed with DAPI. <b>(E and F)</b> Western blot analysis of L-ferritin protein levels in membrane fractions prepared from duodenal scrapings. A representative immunoblot is shown <b>(E)</b>. Immunolabelled L-ferritin bands from separate blots performed on scrapings isolated from 6 piglets were quantified using a Molecular Imager, and L-ferritin protein levels (means ± S.D.) are plotted in arbitrary units <b>(F)</b>. <b>(G)</b> Histological examination of iron loading in duodenum sections. Non-heme iron deposits (indicated by arrows) were detected by staining with Perls’ Prussian blue and counterstained with nuclear red. Duodenum morphology is shown in transmitted light.</p

Increased FLVCR1 protein levels on the basolateral membrane of duodenal absorptive enterocytes is associated with decreased blood plasma hemopexin (Hpx) levels in hemoglobin-supplemented piglets.

<p><b>(A and B)</b> Western blot analysis of FLVCR1 protein levels in membrane fractions prepared from duodenal scrapings. A representative immunoblot is shown <b>(A)</b>. Immunolabelled FLVCR1 bands from blots performed on scrapings isolated from 6 piglets were quantified using a Molecular Imager (Bio-Rad), and FLVCR1 protein levels (means ± S.D.) are plotted in arbitrary units <b>(B)</b>. <b>(C)</b> Immunofluorescent staining of FLVCR1 in the duodenum. To confirm the specificity of FLVCR1 detection, duodenum sections of piglets were incubated with only the secondary antibody. No FLVCR1 staining was detected in these negative controls. Counterstaining of nuclei was performed with DAPI. Duodenum morphology is shown in transmitted light. <b>(D and E)</b> Western blot analysis of Hpx levels in blood plasma. 7 <i>μ</i>l samples of 20-fold diluted piglet blood plasma were analyzed as described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181117#pone.0181117.ref051" target="_blank">51</a>]. A representative immunoblot is shown <b>(D)</b>. Immunolabelled Hpx bands from separate blots performed with plasma samples collected from 6 piglets were quantified using a Molecular Imager, and Hpx protein levels (means ± S.D.) are plotted in arbitrary units <b>(E)</b>.</p

Increased Fpn expression in the duodenum of hemoglobin-supplemented piglets is associated with reductions in both hepatic hepcidin mRNA levels and plasma hepcidin-25 concentration.

<p><b>(A)</b> RT-qPCR analysis of Fpn mRNA expression. The histogram displays Fpn mRNA levels in arbitrary units (means ± S.D., n = 7). <b>(B and C)</b> Western blot analysis of Fpn protein levels in membrane fractions prepared from duodenal scrapings. A representative immunoblot is shown <b>(B)</b>. Immunolabelled Fpn bands from separate blots performed on scrapings isolated from 8 piglets were quantified using a Molecular Imager, and Fpn protein levels (means ± S.D.) are plotted in arbitrary units <b>(C)</b>. <b>(D)</b> Immunofluorescent staining of Fpn in the duodenum. To confirm the specificity of Fpn detection, duodenum sections of piglets were incubated with only the secondary antibody. No Fpn staining was detected in these negative controls. Counterstaining of nuclei was performed with DAPI. Duodenum morphology is shown in transmitted light. <b>(E)</b> RT-qPCR analysis of hepcidin mRNA expression in the liver. The histogram displays hepcidin mRNA levels in arbitrary units (means ± S.D., n = 7). <b>(F)</b> Hepcidin concentration in the blood plasma of experimental piglets. Values are expressed as the means ± S.D. The plasma hepcidin concentration was determined for 5–7 piglets from each group/day.</p