Dynamic Assembly and Disassembly of Functional β‑Endorphin Amyloid Fibrils

Neuropeptides and peptide hormones are stored in the amyloid state in dense-core vesicles of secretory cells. Secreted peptides experience dramatic environmental changes in the secretory pathway, from the endoplasmic reticulum via secretory vesicles to release into the interstitial space or blood. The molecular mechanisms of amyloid formation during packing of peptides into secretory vesicles and amyloid dissociation upon release remain unknown. In the present work, we applied thioflavin T binding, tyrosine intrinsic fluorescence, fluorescence anisotropy measurements, and solid-state NMR spectroscopy to study the influence of physiologically relevant environmental factors on the assembly and disassembly of β-endorphin amyloids in vitro. We found that β-endorphin aggregation and dissociation occur in vitro on relatively short time scales, comparable to times required for protein synthesis and the rise of peptide concentration in the blood, respectively. Both assembly and disassembly of amyloids strongly depend on the presence of salts of polyprotic acids (such as phosphate and sulfate), while salts of monoprotic acids are not effective in promoting aggregation. A steep increase of the peptide aggregation rate constant upon increase of solution pH from 5.0 to 6.0 toward the isoelectric point as well as more rapid dissociation of β-endorphin amyloid fibrils at lower pH indicate the contribution of ion-specific effects into dynamics of the amyloid. Several low-molecular-weight carbohydrates exhibit the same effect on β-endorphin aggregation as phosphate. Moreover, no structural difference was detected between the phosphate- and carbohydrate-induced fibrils by solid-state NMR. In contrast, β-endorphin amyloid fibrils obtained in the presence of heparin demonstrated distinctly different behavior, which we attributed to a dramatic change of the amyloid structure. Overall, the presented results support the hypothesis that packing of peptide hormones/neuropeptides in dense-core vesicles do not necessarily require a specialized cellular machinery

The Mechanism of Toxicity in HET-S/HET-s Prion Incompatibility

<div><p>The HET-s protein from the filamentous fungus <em>Podospora anserina</em> is a prion involved in a cell death reaction termed heterokaryon incompatibility. This reaction is observed at the point of contact between two genetically distinct strains when one harbors a HET-s prion (in the form of amyloid aggregates) and the other expresses a soluble HET-S protein (96% identical to HET-s). How the HET-s prion interaction with HET-S brings about cell death remains unknown; however, it was recently shown that this interaction leads to a relocalization of HET-S from the cytoplasm to the cell periphery and that this change is associated with cell death. Here, we present detailed insights into this mechanism in which a non-toxic HET-s prion converts a soluble HET-S protein into an integral membrane protein that destabilizes membranes. We observed liposomal membrane defects of approximately 10 up to 60 nm in size in transmission electron microscopy images of freeze-fractured proteoliposomes that were formed in mixtures of HET-S and HET-s amyloids. In liposome leakage assays, HET-S has an innate ability to associate with and disrupt lipid membranes and that this activity is greatly enhanced when HET-S is exposed to HET-s amyloids. Solid-state nuclear magnetic resonance (NMR) analyses revealed that HET-s induces the prion-forming domain of HET-S to adopt the β-solenoid fold (previously observed in HET-s) and this change disrupts the globular HeLo domain. These data indicate that upon interaction with a HET-s prion, the HET-S HeLo domain partially unfolds, thereby exposing a previously buried ∼34-residue N-terminal transmembrane segment. The liberation of this segment targets HET-S to the membrane where it further oligomerizes, leading to a loss of membrane integrity. HET-S thus appears to display features that are reminiscent of pore-forming toxins.</p> </div

HeLo domain alignment of the TM segment and HeLo/HET domain architecture comparison.

<p>(A) The sequences in this alignment are the non-redundant output of a PSI-BLAST search with residues 4–33 of HET-S (carried to convergence at an E-value threshold of 0.005). Therefore, these 35 sequences are a subset of HeLo domains that are more similar to HET-S in their TM region. The output score of the TMHMM algorithm (sum of per-residue probability) is plotted to the right of the sequences with a double asterisk to indicate those that are predicted to have a TM helix. When only residues 1–38 are input into the algorithm many more (21 of 35) sequences are predicted to have a TM helix (indicated by a single asterisk). The GI accession numbers in red are for HET-S and HET-s and those in green are for the sequences which have the HET-S domain architecture (HeLo-PFD). (B) Two examples of HeLo domain-containing STAND proteins that have similar architectures to HET domain-containing STAND proteins. The known role of the HET domain as an inducer of cell death suggests that the HeLo domain may have a similar role.</p

HET-S in the presence of HET-s(218–289) amyloid seeds makes holes in liposomes observed by freeze-fracture electron microscopy and liposome leakage assays.

<p>(A) TEM images of the replica of 100-nm diameter extruded liposomes incubated at 4°C in the absence of protein, with a higher magnification on the right. (B) Liposomes incubated in the presence of a mixture of HET-S and HET-s(218–289) fibril seeds display membrane damage: Hole-like structures ranging from a ∼10–∼60-nm width indicated by white arrows are present. In addition, species interpreted as protein aggregates are labeled by a black arrow. (C) The time-dependent release of calcein from <i>E. coli</i> polar liposomes induced by HET-S and HET-s(218–289) 10°C was measured at HET-S concentrations of 0.12–4 µM in the presence (solid lines) or absence (dashed lines) of 0.8 µM HET-s(218–289) amyloid seeds (monomer-equivalent concentration). (D) The calcein leakage measured in the presence (solid lines) or absence (dashed lines) of 8, 0.8, or 0.08 µM HET-S with either 0.008, 0.08, or 0.8 µM HET-s(218–289) amyloid seeds.</p

Proposed mechanism for the generation of toxicity by the HET-s prion/HET-S system.

<p>(1) In the fusion cell, HET-S (in blue with a red TM segment) encounters the β-solenoid structure of the HET-s prion (in brown). (2) HET-S binds to the β-solenoid structure through its own PFD segment, itself adopting the β-solenoid structure. The structural overlap of the HeLo domain and the PFD causes a partial unfolding of the HeLo domain of HET-S, represented here by the transition to a random coil conformation of its three C-terminal helices. (3) The destabilized HeLo domain of HET-S then expels its N-terminal TM segment (residues 1–34, in red). (4) The exposed TM segment targets the activated HET-s/HET-S complex to the membrane where it is able to penetrate the membrane through the formation of a TM helix. The catalytic nature of the activation of HET-S by the HET-s amyloid suggests that activated HET-S may also be released from the co-aggregate before it enters the membrane. The HET-S membrane-disrupting oligomer may form as a direct result of its complex with the HET-s amyloid or only first as it inserts into the membrane. However, the membrane integrity is disrupted by hole-like structures, thus triggering cell death. The model for the HET-s fibril was created from the PFD fibril structure <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001451#pbio.1001451-Wasmer1" target="_blank">[17]</a> and the HeLo domain structure <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001451#pbio.1001451-Greenwald1" target="_blank">[14]</a> with an unwinding the last three helices of the HeLo domain (residues 177–222) to make space for the HeLo domains around the fibril. The HET-s HeLo domains are depicted as dimers between adjacent monomers in the fibril, but these are speculative and it should be emphasized that the structures of the HeLo domains of HET-s and HET-S, in the context of a fibril, are not known except that they lose tertiary structure (more molten globule-like), with a local loss of secondary structure around residues 190–220 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001451#pbio.1001451-Wasmer2" target="_blank">[19]</a>, indicated, in the figure, by the spheres around the HeLo domains.</p

Prion-independent (thermodynamic) activation of HET-S reveals that membrane binding and liposome leakage are distinct activities and that liposome binding is mediated through the N-terminus of HET-S.

<p>(A) TEM image of freeze-fractured liposomes treated with HET-S at 30°C show that many liposomes contain a single large hole, indicated by white arrows similar to those with prion-activated HET-S (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001451#pbio-1001451-g001" target="_blank">Figure 1B</a>). Consistent with the TEM images, is (B) the prion-independent leakage caused by HET-S 30°C. The HET-s-like variant HET-S[E86K] and the HeLo domains of HET-S and HET-s do not cause leakage even at this elevated temperature. (C) SDS-PAGE analysis of 150,000 <i>g</i> supernatant (S) and pellet (P) fractions from a 20 µM HET-S incubation with liposomes reveals that HET-S associates with liposomes at 30°C but not at 4°C. (D) HET-s and HET-s(1–227) do not interact with liposomes and are found in the supernatant. In contrast, HET-S(1–227) and HET-S(E86K)—a HET-S mutant that cannot dimerize in solution and that has a [Het-s]-like phenotype in vivo—bind to liposomes after an extended period of incubation at 30°C. Despite being able to bind liposomes, neither of these two proteins causes leakage from liposomes (B).</p

HET-S oligomerizes in the membrane-like environment of the detergent FC-12.

<p>(A) The time dependence of the oligomerization state of HET-S in 0.4% FC-12 was followed by size-exclusion chromatography with multiple-angle light scattering, UV, and refraction index detection. With time the monomeric species is depleted with a concomitant increase in the oligomeric one (violet = 0 h; blue = 1 h; green = 2.5 h; yellow = 3 h; orange = 3.5 h; red = 16 h). The grey trace is the size exclusion profile of HET-S that was extracted by 0.4% FC-12 from liposomes with which it had been incubated at 37°C for 2 h. The extracted sample is similar in monomer and oligomer content to the 16-h non-liposome measurement. The masses of the HET-S entities (not including the bound FC-12 molecules) as calculated by the Astra V software conjugate analysis are listed on above the chromatograms. The inlay shows that under the same conditions, HET-S 1–227 remains monomeric for 16 h with only a minor accumulation of a high molecular weight aggregate (no intermediate oligomers detected). (B) CD spectra of HET-S in 0.4% FC-12 were independently acquired for the same time course showing a time-dependent loss of alpha-helical content for the first 3 h upon the addition of detergent, after which the sample reaches a stable alpha helical content. Colors are as in (A).</p

HET-S but not HET-s expression is toxic in <i>E. coli</i>.

<p>The growth of <i>E. coli</i> cultures expressing various constructs of (A) HET-S (full-length, 1–227, or N-terminal Histag) or (B) HET-s (full-length, 1–227) was monitored by their OD₆₀₀. After induction of protein expression, the cell growth of cultures with HET-S or HET-S(1–227) but not HET-s or HET-s(1–227) was suppressed, indicating that the HET-S HeLo domain, but not HET-s or its HeLo domain, is toxic to <i>E. coli</i>. Further analysis of the cell pellet showed that HET-S is associated with the inner membrane of <i>E. coli</i>. (C) SDS-PAGE analysis of soluble and insoluble fractions after expression in <i>E. coli</i> at 37°C for 4 h. Lanes 1 and 2 represent the supernatant and pellet of the cell lysate, respectively. The pellet and supernatant fractions after treatment with 1 M urea are in lanes 5 and 6, respectively. HET-S remains in the insoluble fraction after incubation in 1 M Urea (lane 5).The pellet and supernatant fractions after extraction with the detergent N-lauroylsarcosine are shown in lanes 3 and 4, respectively, showing that HET-S is solubilized by this detergent. The identity of the HET-S and OmpF bands were confirmed by trypsin digest followed by MS analysis and are marked with an asterisk. (D) Coomassie stain (top) and Western blot (bottom) visualization of SDS-PAGE fractions from a sucrose density gradient separation of <i>E. coli</i> cell lysates after HET-S expression at 37°C for 4 h (lanes 1–13). Lanes L and M are the cell lysate before density gradient separation and the protein marker (SeeBlue Plus2, Invitrogen), respectively. The relative NADH oxidase activity of each fraction (indicating the location of the inner membrane) is plotted over the Western blot. HET-S is found in fractions that correspond to the inner membrane, whereas the outer membrane protein OmpF moves to a higher density. The majority of HET-S is in the soluble protein fractions while a small amount is also found on the very bottom of the tube (55% sucrose, fraction 1) whose density (>1.25 g/ml) indicates that it likely to be lipid-free (or low-lipid-content) protein aggregates.</p

Correlations between <i>P. anserina</i> heterokaryon incompatibility phenotype and in vitro liposome calcein leakage data.

<p>(A) The TMHMM per-residue predictions (see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001451#pbio.1001451.s003" target="_blank">Figure S3</a>) for HET-s (left) and HET-S (right) are mapped as a color gradient onto a ribbon diagram of the crystal structure of HET-S(1–227). A star indicates the locations of the two critical amino acid residues, 23 and 33, whose identities can interconvert the functionality of HET-s and HET-S. (B) The time course of prion-induced calcein leakage is shown for HET-s, HET-S, and several phenotype-interconverting variants at 4 µM protein concentration with 0.8 µM HET-s(218–289) amyloid seeds. Only HET-S and HET-s[D23A,P33H] show calcein leakage consistent with the HET-S toxic phenotype. The leakage is expressed as fraction based on the positive control experiment using 1% SDS. A comparison of the TM prediction, incompatibility phenotype, and liposome leakage activity of these variants is presented in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001451#pbio.1001451.s005" target="_blank">Table S1</a>. A detailed per-residue TMHMM prediction of these and several other variants of known phenotype is displayed in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001451#pbio.1001451.s003" target="_blank">Figure S3</a>).</p

Megahertz pulse trains enable multi-hit serial femtosecond crystallography experiments at X-ray free electron lasers

The European X-ray Free Electron Laser (XFEL) and Linac Coherent Light Source (LCLS) II are extremely intense sources of X-rays capable of generating Serial Femtosecond Crystallography (SFX) data at megahertz (MHz) repetition rates. Previous work has shown that it is possible to use consecutive X-ray pulses to collect diffraction patterns from individual crystals. Here, we exploit the MHz pulse structure of the European XFEL to obtain two complete datasets from the same lysozyme crystal, first hit and the second hit, before it exits the beam. The two datasets, separated by <1 µs, yield up to 2.1 Å resolution structures. Comparisons between the two structures reveal no indications of radiation damage or significant changes within the active site, consistent with the calculated dose estimates. This demonstrates MHz SFX can be used as a tool for tracking sub-microsecond structural changes in individual single crystals, a technique we refer to as multi-hit SFX